U.S. patent application number 13/843713 was filed with the patent office on 2014-02-06 for methods for reprogramming cells and uses thereof.
This patent application is currently assigned to New World Laboratories Inc.. The applicant listed for this patent is New World Laboratories Inc.. Invention is credited to Jan-Eric Ahlfors, Rouwayda Elayoubi.
Application Number | 20140038291 13/843713 |
Document ID | / |
Family ID | 50025878 |
Filed Date | 2014-02-06 |
United States Patent
Application |
20140038291 |
Kind Code |
A1 |
Ahlfors; Jan-Eric ; et
al. |
February 6, 2014 |
METHODS FOR REPROGRAMMING CELLS AND USES THEREOF
Abstract
Described herein are reprogrammed cells, and methods for cell
dedifferentiation, transformation and eukaryotic cell
reprogramming. Also descried are cells, cell lines, and tissues
that can be transplanted in a patient after steps of in vitro
dedifferentiation and in vitro reprogramming. In particular
embodiments the cells are Stem-Like Cells (SLCs), including Neural
Stem-Like Cells (NSLCs), Cardiac Stem-Like Cells (CSLC),
Hematopoietic Stem-Like Cells (HSLC), Pancreatic Progenitor-Like
Cells, and Mesendoderm-like Cells. Also described are methods for
generating these cells from human somatic cells and other types of
cells. Also provided are compositions and methods of using of the
cells so generated in human therapy and in other areas.
Inventors: |
Ahlfors; Jan-Eric; (Laval,
CA) ; Elayoubi; Rouwayda; (Laval, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
New World Laboratories Inc. |
Laval |
|
CA |
|
|
Assignee: |
New World Laboratories Inc.
Laval
CA
|
Family ID: |
50025878 |
Appl. No.: |
13/843713 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13464987 |
May 5, 2012 |
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13843713 |
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13504988 |
Apr 30, 2012 |
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PCT/CA10/01727 |
Nov 1, 2010 |
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13464987 |
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61256967 |
Oct 31, 2009 |
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Current U.S.
Class: |
435/441 ;
435/325; 435/455 |
Current CPC
Class: |
C12N 2501/604 20130101;
C12N 5/0623 20130101; C12N 2501/60 20130101; C12N 2506/11 20130101;
C12N 5/0696 20130101; C12N 2506/094 20130101; C12N 2506/1384
20130101; C12N 15/85 20130101; C12N 2501/605 20130101; C12N 2502/99
20130101; C12N 2506/1307 20130101; C12N 5/0678 20130101; C12N
2501/40 20130101; C12N 2501/603 20130101; C12N 5/0662 20130101;
C12N 2501/998 20130101 |
Class at
Publication: |
435/441 ;
435/455; 435/325 |
International
Class: |
C12N 15/85 20060101
C12N015/85 |
Claims
1. A method of transforming a cell of a first type to a desired
cell of a different type, comprising: i) providing a cell of a
first type; ii) transiently increasing in said cell of a first type
intracellular levels of at least one reprogramming agent, whereby
said transient increase induces direct or indirect endogenous
expression of at least one gene regulator; iii) placing the cell in
conditions for supporting the transformation of the desired cell
and maintaining intracellular levels of said at least one
reprogramming agent for a sufficient period of time to allow stable
expression of said at least one gene regulator in absence of the
reprogramming agent; and iv) maintaining the cell in culture
conditions supporting the transformation of the desired cell for a
sufficient period of time to allow a stable expression of a
plurality of secondary genes whose expression is characteristic of
phenotypical and/or functional properties of the desired cell,
wherein at least one of said secondary genes is not characteristic
of phenotypical and functional properties of an embryonic stem
cell, whereby at the end of said period of time the cell of the
first type has been transformed into the desired cell of a
different type.
2. The method of claim 1, wherein said at least one reprogramming
agent is a polynucleotide or a polypeptide, and wherein said
polynucleotide or polypeptide comprises a sequence selected from
the group of GenBank.TM., UniProt.TM./Swiss-Prot.TM. or UniGene.TM.
sequences whose accession numbers is listed in TABLE A.
3. The method of claim 2, wherein the desired cell of a different
type is a Neural Stem-Like Cell and said at least one reprogramming
agent is selected from the group consisting of the following:
Musashi1 (Msi1); Msi1 and Neurogenin 2 (Ngn2); Msi1 and methyl-CpG
binding domain protein 2 (MBD2); Ngn2 and MBD2, and Msi1 Ngn2 and
MBD2.
4. The method of claim 2, wherein the desired cell of a different
type is a Cardiac Stem-Like Cell and said at least one
reprogramming agent is selected from the group consisting of the
following: Brachyury (T) and Mesoderm Posterior 1 (MESP1); T, MESP1
and NK2 Homebox 5 (NKX2.5); T, MESP1 and T-box 5 (TBX5); T, MESP1,
NKX2.5 and TBX5.
5. The method of claim 2, wherein the desired cell of a different
type is a Hematopoietic Stem-Like Cell and said at least one
reprogramming agent is selected from the group consisting of a
combination of the following: Brachyury (T), Caudal Type Homeobox 4
(CDX4), Homeobox B4 (HOXB4), GATA Binding Factor 1 (GATA1), GATA
Binding Factor 2 (GATA2), and/or Kruppel-like Factor 1 (KLF1).
6. The method of claim 2, wherein the desired cell of a different
type is a Pancreatic Progenitor-Like Cell and said at least one
reprogramming agent is selected from the group consisting of the
following: SRY (Sex determining Region Y)-box 17 (SOX17),
Neurogenin 3 (NGN3) and Pancreatic and Duodenal Homebox 1 (PDX1);
SOX17, NGN3, PDX1 and Octamer-binding Transcription Factor 4
(OCT4).
7. The method of claim 2, wherein the desired cell of a different
type is a Mesendoderm-Like Cell and said at least one reprogramming
agent is selected from the group consisting of the following:
Forkhead Box D3 (FOXD3), MIX1 Homeobox-Like Protein 1 (MIXL1),
Neurogenin 3 (NGN3); FOXD3, MIXL1, NGN3 and methyl-CpG binding
domain protein 2 (MBD2).
8. The method of claim 2, wherein the desired cell of a different
type is a Pluripotent-Like Cell and said at least one reprogramming
agent is selected from the group consisting of the following: Zinc
Finger Protein 42 (REX1), Octamer-binding Transcription Factor 4
(OCT4) and Kruppel-like Factor 4 (KLF4); Sal-like 4 (SALL4), OCT4,
KLF4 and Nanog Homeobox (NANOG).
9. The method of claim 1, wherein the desired cell of a different
type is selected from the group consisting of: a neural stem-like
cell expressing one or more of Nestin, Sox2, GFAP and Msi1; a
neural-like cell expressing one or more of .beta.III-tubulin, Map2b
and Synapsin, ACHE; a cardiac stem-like cell expressing one or more
of Gata4, Nkx2.5, CXCR4, and Brachyury; a cardiomyocyte-like cell
expressing one or more of Nkx2.5, Troponin T, Troponin I, and
Connexin-43; a hematopoietic stem-like cell expressing one or more
of CD34, Flt3, Sca-1, HoxB4, and CXCR4; a pancreatic-progenitor
cell expressing one or more of Pdx1 FoxA2, Ngn3, and Isl1; a
pancreatic beta cell expressing one or more of Pdx1 Ngn3, and
Insulin; a myogenic (muscle) stem-like cell expressing one or more
of MyoD, alpha smooth muscle actin, and Mef2c; an ectoderm-like
cell expressing one or more of Sox2, Sox1, Zic1 Nestin, Notch 1,
FoxJ3, Otx2, Cripto1 and Vimentin; a mesendoderm-like cell
expressing one or more of Sox17, FoxA2, CXCR4, GATA4, MixI1,
Eomesodermin; and a pluripotent-like cell expressing one or more of
Oct4, SSEA4, TRA-1-60, TRA-1-81 and AP.
10. The method of claim 1, wherein the desired cell of a different
type obtained is characterized by a stable repression of a
plurality of genes expressed in the first cell type, wherein said
plurality of genes whose expression is stably repressed are
selected from the group of genes listed in TABLE C.
11. The method of claim 1, further comprising contacting the
chromatin and/or DNA of the cell of the first type with an agent
capable of remodeling chromatin and/or DNA of said cell, wherein
the agent capable of remodeling chromatin and/or DNA is selected
from the group consisting of histone acetylators, inhibitors of
histone deacetylation, DNA demethylators, inhibitors of DNA
methylation and combination thereof.
12. The method of claim 1, further comprising treating the cell of
a first type with a cytoskeleton disruptor.
13. The method of claim 1, wherein the cell of a first type is
selected from the group consisting of: germ cells, embryonic stem
cells and derivations thereof, adult stem cells and derivations
thereof, progenitor cells and derivations thereof, cells derived
from mesoderm, endoderm or ectoderm, and a cell of mesoderm,
endoderm or ectoderm lineage, adipose-derived stem cell,
mesenchymal stem cell, hematopoletic stem cell, skin derived
precursor cell, hair follicle cell, fibroblast, keratinocyte,
epidermal cell, endothelial cell, epithelial cell, granulosa
epithelial cell, melanocyte, adipocyte, chondrocyte, hepatocyte, B
lymphocyte, T lymphocyte, granulocyte, macrophage, monocyte,
mononuclear cell, pancreatic islet cell, sertoli cell, neuron,
glial cell, cardiac muscle cell, and other muscle cell.
14. A method of obtaining a Stem-Like Cell (SLC), comprising: i)
providing a cell of a first type; ii) contacting chromatin and/or
DNA of the cell of a first type with a histone acetylator, an
inhibitor of histone deacetylation, a DNA demethylator, and/or an
inhibitor of DNA methylation; and iii) increasing intracellular
levels of at least one gene regulator for that particular stem-like
cell, wherein the gene regulator is capable of driving directly or
indirectly transformation of the cell of the first type into the
particular stem-like cell whereby a SLC is obtained.
15. The method of claim 14, wherein increasing intracellular levels
of at least one stem cell specific polypeptide comprises
transiently transfecting the cell of a first type with an
expression vector allowing expression of one or more reprogramming
agents in Table A.
16. The method of claim 14, wherein the SLC so obtained possesses
one or more of the following characteristics: i) expression of one
or more stem cell marker selected from Table A; ii) decreased
expression of one or more genes specific to the cell that the
stem-like cell was obtained from; iii) capable of being cultured in
suspension (as spheres) or as an adherent culture; iv) capable of
proliferating without the presence of an exogenous reprogramming
agent for over 1 month, preferably over 2 months, over 3 months,
over 5 months or for more than a year; v) positive for telomerase
activity; vi) capable of differentiation into cells according to
the lineage of that stem-like cell; vii) decreased expression of
telomerase and one or more stem cell markers after differentiation;
viii) having one or more morphological features of the stem cells
that the stem-like cell is like; ix) expression of one or more
antigen expressed specifically in the stem cells that the one or
more is like; x) expression of one or more functional markers of
lineage specific differentiated cells after differentiation of the
stem-like cell; xi) negative in a tumor colony forming assay; xii)
negative for tumor growth in SCID mice; xiii) negative for teratoma
growth in SCID mice; xiv) capable of significantly improving one or
more functional measures after placement of an adequate number of
stem-like cells in a model assessing the regenerative potential of
those types of stem cells.
17. The method of claim 14, wherein a plurality of SLCs are
obtained and wherein said plurality of SLCs are organized within a
three-dimensional structure.
18. An isolated stem-like cell (SLC) possessing all of the
following characteristics: i) ability to self-renew for
significantly longer than a somatic cell; ii) is not a cancerous
cell; iii) is stable and not artificially maintained by forced gene
expression or by similar means and may be maintained in standard
stem cell media specific to the SLC; iv) can differentiate to a
progenitor, precursor, somatic cell or to another more
differentiated cell type of the same lineage; v) has the
characteristics of a stem cell and not just certain markers or gene
expression or morphological appearance of a stem cell; and vi) does
not exhibit uncontrolled growth, teratoma formation, and tumor
formation in vivo.
19. An isolated Neural Stem-Like Cell (NSLC), wherein the cell
possesses one or more of the following characteristics: i)
expression of one or more neural stem cell marker selected from the
group consisting of Sox2, Nestin, GFAP, Msi1, and Ngn2; ii)
decreased expression of one or more genes specific of the cell of
the first type; iii) forms neurospheres in the neurosphere colony
formation assay; iv) capable of being cultured in suspension or as
an adherent culture; v) capable of proliferating without the
presence of an exogenous reprogramming agent for over 1 month; vi)
capable of dividing every 36 hours at low passage; vii) positive
for telomerase activity; viii) capable of differentiation into a
neuronal-like cell, astrocyte-like cell, oligodendrocyte-like cell
and combinations thereof; ix) decreased expression of telomerase
and one or more neural stem cell markers upon differentiation; x)
having one or more morphological neurite-like processes (axons
and/or dendrites) greater than one cell diameter in length; xi)
expression of at least one neural-specific antigen selected from
the group consisting of neural-specific tubulin, microtubule
associated protein 2, NCAM, and marker for a neurotransmitter; xii)
expression of one or more functional neural markers upon neuronal
differentiation; xiii) capable of releasing one or more
neurotrophic factors; xiv) negative in a tumor colony forming
assay; xv) negative for tumor growth in SCID mice; xvi) negative
for teratoma growth in SCID mice; xvii) capable of significantly
improving one or more functional measures after placement of an
adequate number of NSLCs into the void in a brain ablation model;
xviii) capable of significantly improving or maintaining one or
more functional measures after injecting an adequate number of
NSLCs into an EAE mouse model; and xix) capable of improving one or
more functional measures more significantly than hNPCs in CNS
injury or neurodegenerative models.
20. A process wherein a cell of a first type is reprogrammed to a
desired cell of a different type, comprising: (i) a transient
increase of intracellular levels of at least one reprogramming
agent, wherein said at least one reprogramming agent induces a
direct or indirect endogenous expression of at least one gene
regulator, wherein said endogenous expression of the at least one
gene regulator is necessary for the existence of the desired cell
of a different type; (ii) a stable expression of said at least one
gene regulator; and (iii) stable expression of a plurality of
secondary genes, wherein the stable expression of said plurality of
secondary genes is the result of the stable expression of the at
least one gene regulator, and wherein: (i) stable expression of
said plurality of secondary genes is characteristic of phenotypical
and/or functional properties of the desired cell, (ii) stable
expression of at least one of said secondary genes is not
characteristic of phenotypical and functional properties of an
embryonic stem cell, and wherein (i) and (ii) are indicative of
successful reprogramming of the cell of the first type to the
desired cell of the different type.
21. The method of claim 2, wherein the desired cell of a different
type is a Cardiac Stem-Like Cell and said at least one
reprogramming agent is selected from the group consisting of the
following: Brachyury (T) and Mesoderm Posterior 1 (MESP1); T, MESP1
and NK2 Homebox 5 (NKX2.5); T, MESP1 and T-box 5 (TBX5); T, MESP1,
NKX2.5 and TBX5.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 13/464,987, filed May 5, 2012, which is a
continuation-in-part of U.S. application Ser. No. 13/504,988, which
is the U.S. National Phase Application of International Appl. No.:
PCT/CA2010/001727, filed Nov. 1, 2010, which claims the benefit of
U.S. Provisional Appl. No. 61/256,967, filed Oct. 31, 2009. The
content of the aforesaid applications are relied upon and are
incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of eukaryotic
cell reprogramming, and particularly to cell dedifferentiation. The
invention is also concerned with methods of generating stable
Reprogrammed Cells and Stem-like Cells (SLCs), including Neural,
Stem-Like Cells (NSLCs) from human somatic cells (and other cells)
and the use of the cells so generated in human therapy.
BACKGROUND OF THE INVENTION
Cell Reprogramming
[0003] There is a desire in the medical, scientific, and diagnostic
fields to reprogram an easily obtainable cell into a cell that is
generally harder to obtain, or to reprogram a cell to have new or
different functionalities, without fusing or exchanging material
with an oocyte or another stem cell.
[0004] Stem cells currently provide a promising venue to treat
diseases or modulate/enhance certain functionalities. However, not
all stem cells are the same and many challenges need to be
overcome, for example: [0005] (i) Embryonic and other pluripotent
stem cells (ex. iPS cells) can turn into any type of cell which
makes them very difficult to control and generally results in the
wrong type of cell to grow in the wrong place, teratoma formation
(a mass of cells containing different types of tissues) and tumors.
Even when their differentiation and growth can be controlled,
pluripotent stem cells will more likely grow a new competing tissue
within the existing tissue rather than integrate into the existing
damaged tissue. [0006] (ii) Multipotent stem cells (somatic stem
cells) are generally lineage-restricted stem cells that regenerate
and grow existing tissues of their specific lineage (for example,
neural stem cells regenerate and grow the nervous system, skin stem
cells regenerate and grow skin, and hematopoietic stem cells
regenerate and grow the hematopoietic tissue (blood cells). Somatic
stem cells are the most effective when the right type of somatic
stem cells is used for its coresposing tissue to be treated (for
example, a hematopoietic or mesenchymal stem cell will have limited
regenerative and growth abilities if implanted into the central
nervous system (CNS), while neural stem cells are the types of
somatic stem cells that have been evolved for this specific purpose
and have significant regenerative and growth abilities in the CNS).
[0007] (iii) For a stem cell to efficiently permanently graft (in a
functional but not competitive manner) into the patient's tissue,
the stem cell generally has to be autologous (the patient's own).
The main exception to this is hematopoietic stem cell grafts (bone
marrow transplants) due to the changes in the immune and other
systems in the body caused by these specific somatic stem
cells.
[0008] Thus there is a need for an efficient method to create safe
and efficacious autologous stem cells for the specific tissue,
organ or condition to be treated, as well as new types of stem
cells with new or unique features such as enhanced potency and/or
safety.
[0009] There are several current "non-reprogramming" mechanisms for
obtaining a certain cell of interest. According to a first
mechanism, a stem cell can naturally divide or differentiate into
another stem cell, progenitor, precursor, or somatic cell.
According to a second mechanism, a somatic cell can sometimes
transiently change its phenotype or express certain markers when
placed in certain conditions, and then revert back when placed back
into the original conditions. According to a third mechanism, the
phenotype of many cells can be changed through forced expression of
certain genes (for example, stably transfecting the c-myc gene into
fibroblasts turns them into immortal cells having neuroprogenitor
characteristics), however once this forced gene expression is
removed, the cells slowly revert back to their original state.
Therefore, none of the three above mechanisms should be considered
true reprogramming: the first is considered natural differentiation
which is part of a cell program that is already in place (going
from a more undifferentiated to a more differentiated state), the
second is a transient phenotypical change, and the third is a
constantly forced cell type. A true stem cell: (i) self-renews
almost `indefinitely` (for significantly longer than a somatic
cell), (ii) is not a cancerous cell, (iii) is not artificially
maintained by forced gene expression or similar means (must also be
able to be maintained in standard stem cell media), (iv) can
differentiate to progenitor, precursor, somatic or other more
differentiated cell type (of the same lineage), and (v) has all the
characteristics of a stem cell and not just certain markers or gene
expression or morphological appearance.
[0010] Despite the numerous scientific and patent publications
claiming successful reprogramming or dedifferentiation, generally
into a stem cell, almost all of these publications do not disclose
true reprogramming because they fall under one of the mechanisms
mentioned above. For instance, Bhasin (WO2010/088735), Cifarelli et
al, (US2010/0003223), Kremer et al. (US2004/0009595), and Winnier
et al. (US2010/0047908) all refer to reprogramming,
dedifferentiation, and/or obtained stem cells (or progenitors) as
phenotypical cell changes based only on a change in cell surface
markers after culture in different media with supplements, with no
evidence of true reprogramming or an actual stem cell
(non-cancerous self-renewal with stem cells markers and no
differentiation markers). The same is true for Benneti
(WO2009/079007) who used increased expression of Oct4 and Sox2.
Others, such as Akamatsu et al. (WO2010/052904) and You et al
(WO2007/097494, US2009/0246870), refer to having made stem cells,
but these came about through constant artificial gene induction
delivered by retrovirus (similar to cMyc) with no evidence of true
stem cells that are not immortal/tumorigenic, and stable instead of
transient. Others, such as Chen et al. (US2005/0176707) and You at
al. (US200910227023), have made "multipotent cells", but not stem
cells. In addition these alleged multipotent cells were not stable
(in the case of You et al. the cells could not even proliferate)
and both used constant media supplements and conditions to force
the phenotypical change. Yet others, such as Oliveri et al.
(WO2009/018832) and Zahner et al. (US2002/0136709), have claimed
the making of pluripotent, totipotent, multipotent, and/or
unipotent cells automatically through genome-wide DNA demethylation
and histone acetylation, but with no evidence of a stable,
non-cancerous, true cell line.
[0011] True reprogramming appears to have been achieved with
induced pluripotent stem cells (iPS cells) created independently by
Yamanaka's group (Takahashi et al., 2007) and Thomson's group (Yu
et al., 2007), and potentially by others before them, and although
many of these cells were later found to be cancerous, some of them
were not. These cells can be induced by true reprogramming since it
was later shown that they can also be induced by non-gene
integrating transient transfection (Soldner at al., 2009; Woltjen
et al., 2009; Yu et al., 2009) as well as by RNA (Warren et al.,
2010) or protein (Kim et al., 2009; Zhou et al., 2009) alone or by
small molecules (Lyssiotis at al., 2009), and by similar methods.
However, these cells are essentially identical to embryonic stem
cells and have the same problems of uncontrolled growth, teratoma
formation, and potential tumor formation. In addition they appear
to be less potent and less safe than embryonic stem cells,
including the differentiated cells (including multipotent/somatic
stem cells) derived using this iPS method.
[0012] A more desirable option is to have multipotent stem cells or
pluripotent-like cells whose lineage and differentiation potential
is more restricted so that they do not readily form teratomas and
uncontrolled growth. There is thus a need for methods of creating
multipotent stem cells, multipotent stem-like cells, and stem-like
cells and method of reprogramming or transforming easily obtainable
cells to highly desirable multipotent stem cells, multipotent
stem-like cells, and stem-like cells.
Neural Stem-Like Cells (NSLC)
[0013] Repairing the central nervous system (CNS) is one of the
frontiers that medical science has yet to conquer. Conditions such
as Alzheimer's disease, Parkinson's disease, and stroke can have
devastating consequences for those who are afflicted. A central
hope for these conditions is to develop cell populations that can
reconstitute the neural network, and bring the functions of the
nervous system back in line. For this reason, there is a great deal
of evolving interest in neural stem and progenitor cells. Up until
the present time, it was generally thought that multipotent neural
progenitor cells commit early in the differentiation pathway to
either neural restricted cells or glia restricted cells.
[0014] Neural stem cells have promise for tissue regeneration from
disease or injury; however, such therapies will require precise
control over cell function to create the necessary cell types.
There is not yet a complete understanding of the mechanisms that
regulate cell proliferation and differentiation, and it is thus
difficult to fully explore the plasticity of neural stem cell
population derived from any given region of the brain or developing
fetus.
[0015] The CNS, traditionally believed to have limited regenerative
capabilities, retains a limited number of neural stem cells in
adulthood, particularly in the dentate gyrus of the hippocampus and
the subventricular zone that replenishes olfactory bulb neurons
(Singec I et al., 2007; Zielton R, 2008). The availability of
precursor cells is a key prerequisite for a transplant-based repair
of defects in the mature nervous system. Thus, donor cells for
neural transplants are largely derived from the fetal brain. This
creates enormous ethical problems, in addition to immuno-rejection,
and it is questionable whether such an approach can be used for the
treatment of a large number of patients since neural stem cells can
lose some of their potency with each cell division.
[0016] Neural stem cells provide promising therapeutic potential
for cell-replacement therapies in neurodegenerative disease
(Mimeault at al., 2007). To date, numerous therapeutic
transplantations have been performed exploiting various types of
human fetal tissue as the source of donor material. However,
ethical and practical considerations and their inaccessibility
limit the availability as a cell source for transplantation
therapies (Ninomiy M et al., 2006).
[0017] To overcome barriers and limitations to the derivation of
patient specific cells, one approach has been to use skin cells and
inducing the trans-differentiation to neural stem cells and/or to
neurons (Levesque at al., 2000). Transdifferentiation has been
receiving increasing attention during the past years, and
trans-differentiation of mammalian cells has been achieved in
co-culture or by manipulation of cell culture conditions.
Alteration of cell fate can be induced artificially in vitro by
treatment of cell cultures with microfilament inhibitors (Shea of
al., 1990), hormones (Yeomans at al., 1976), and Calcium-ionophores
(Shea, 1990; Sato at al., 1991). Mammalian epithelial cells can be
induced to acquire muscle-like shape and function (Paterson and
Rudland, 1985), pancreatic exocrine duct cells can acquire an
insulin-secreting endocrine phenotype (Bouwens, 1998a, b), and bone
marrow stem cells can be differentiated into liver cells (Theise et
al., 2000) and into neuronal cells (Woodbury et al., 2000). Other
such as Page et al. (US 2003/0059939) have transdifferentiated
somatic cells to neuronal cells by culturing somatic cells in the
presence of cytoskeletal, acetylation, and methylation inhibitors,
but after withdrawal of the priming agent, neuron morphology and
established synapses last for not much than a few weeks in vitro,
and complete conversion to a fully functional and stable type of
neuron has never been demonstrated. These are thus transient cell
phenotypes. Complete conversion to a fully functional and stable
type of neuroprogenitor or neural stem cell has also never been
demonstrated. Acquisition of a stable phenotype following
transdifferentiation has been one of the major challenges facing
the field.
[0018] Thus, there is a need in the biomedical field for stable,
potent, and preferably autologous neural stem cells, neural
progenitor cells, as well as neurons and glial cells for use in the
treatment of various neurological disorders and diseases. The same
is true for many other types of cells. Recently, evidence have been
obtained that genes of the basic Helix-Loop-Helix (bHLH) class are
important regulators of several steps in neural lineage
development, and over-expression of several neurogenic bHLH factors
results in conversion of non-determined ectoderm into neuronal
tissue. Proneural bHLH proteins control the differentiation into
progenitor cells and their progression through the neurogenic
program throughout the nervous system (Bertrand et al., 2002).
MASH1, NeuroD, NeuroD2, MATH1-3, and Neurogenin 1-3 are bHLH
transcription factors expressed during mammalian neuronal
determination and differentiation (Johnson et al., 1990; Takebyashi
at al., 1997; McCormick et al., 1996; Akazawa at al., 1995).
Targeted disruptions of MASH1, Ngn1, Ngn2 or NeuroD in mice lead to
the loss of specific subsets of neurons (Guillemot at al., 1993;
Fode of al., 1998; Miyata at al., 1999).
[0019] U.S. Pat. No. 6,087,168 (Levesque et al.,) describes a
method for converting or transdifferentiating epidermal basal cells
into viable neurons. In one example, this method comprises the
transfection of the epidermal cells with one or more expression
vector(s) containing at least one cDNA encoding for a neurogenic
transcription factor responsible for neural differentiation.
Suitable cDNAs include: basic-helix-loop-helix activators, such as
NeuroD1, NeuroD2, ASH1, and zinc-finger type activators, such as
Zic3, and MyT1. The transfection step was followed by adding at
least one antisense oligonucleotide known to suppress neuronal
differentiation to the growth medium, such as the human MSX1 gene
and/or the human HES1 gene (or non-human, homologous counterparts).
Finally, the transfected cells were grown in the presence of a
retinoid and a least one neurotrophin or cytokine, such as brain
derived neurotrophic factor (BDNF), nerve growth factor (NGF),
neurotrophin 3 (NT-3), or neurotrophin 4 (NT-4). This technology
yields 26% of neuronal cells; however, neither functionality nor
stability of these cells was established. In addition, neural stem
cells or neuroprogenitor cells are not produced according to this
method.
[0020] A later process (Levesque et al., 2005; U.S. Pat. No.
6,949,380) mentions the conversion of the epidermal basal cell into
a neural progenitor, neuronal, or glial cell by exposing the
epidermal basal cell to an antagonist of bone morphogenetic protein
(BMP) and growing the cell in the presence of at least one
antisense oligonucleotide comprising a segment of a MSX 1 gene
and/or HES1 gene. However, there is no evidence or examples that
any neural progenitors or glial cells were produced according to
this method, let alone any details or evidence that morphological,
physiological or immunological features of neuronal cells was
achieved. In addition, since there is also no information on
functionality, stability, expansion, and yield about the cells
which may or may not have been produced, it is possible that these
cells actually are skin-derived precursor cells (Fernandes et al.,
2004) that have been differentiated into neuronal cells.
[0021] In view of the above, there is thus a need for stable,
potent, and preferably autologous neural stem cells, neural
progenitor cells, neurons and glial cells, as well as other types
of cells, stem cells and progenitor cells. There is also a need for
methods that could result in true cell dedifferentiation and cell
reprogramming.
[0022] The present invention addresses these needs and provides
various types of stem-like and progenitor-like cells and cells
derived or differentiated from these stem-like or progenitor-like
cells, as well as methods that can result in true cell
dedifferentiation and cell reprogramming.
[0023] Additional features of the invention will be apparent from a
review of the disclosure and description of the invention
herein.
SUMMARY OF THE INVENTION
[0024] The present invention relates to stem-like and
progenitor-like cells and cells derived or differentiated from
these stem-like or progenitor-like cells. The invention further
relates to methods for cell dedifferentiation and cell
reprogramming. The invention further features compositions and
methods that are useful for reprogramming cells and related
therapeutic compositions and methods.
[0025] One particular aspect relates to the development of a
technology to reprogram a somatic cell or non-neuronal cell to a
cell having one or more morphological physiological, and/or
immunological features of a neural stem cell and which possess the
capacity to differentiate along neuronal and glial lineages.
According to some embodiments, the invention is more particularly
concerned with methods of generating stable Neural Stem-Like Cells
(NSLCs) from human somatic cells, human progenitor cells and/or of
human stem cells, as well as cells, cell lines and tissues obtained
by using such methods.
[0026] The invention further relates to compositions and methods to
induce de-differentiation of human somatic cells into Neural
Stem-Like Cells that express neural stem cell specific markers
According to the present invention it is possible to effect the
conversion of cells to various types of differentiated neuronal
cells that can be created from a single cell type taken from an
individual donor and then reprogrammed and transplanted into the
same individual. Upon induction cells according to the invention
express neural stem-cell specific markers and become Neural
Stem-Like cells.
[0027] Other particular aspects relate to the development of
technologies to reprogram a somatic cell to a cell having one or
more morphological physiological, and/or immunological features of
a cardiac stem cell, hematopoietic stem cell, pancreatic progenitor
cell, myogenic (muscle) stem cell, pluripotent stem cell,
ectodermal derived cell and/or mesendodermal derived cell and which
possess the capacity to differentiate along their respective
lineages. According to some embodiments, the invention is more
particularly concerned with methods of generating stable Cardiac
Stem-Like Cells (CSLCs), Hematopoietic Stem-Like Cells (HSLCs),
Pancreatic Progenitor-Like Cells, Myogenic (muscle) Stem-Like
Cells, Pluripotent-Like Cells, Ectoderm-Like Cells and
Mesendoderm-Like Cells from human somatic cells, human progenitor
cells and/or of human stem cells, as well as cells, cell lines and
tissues obtained by using such methods
[0028] The invention further relates to compositions and methods to
induce de-differentiation of human somatic cells into Cardiac
Stem-Like Cells (CSLCs), Hematopoietic Stem-Like Cells (HSLCs),
Pancreatic Progenitor-Like Cells, Myogenic (muscle) Stem-Like
Cells, Pluripotent-Like Cells, Ectoderm-Like Cells and
Mesendoderm-Like Cells that express cardiac stem cell,
hematopoietic stem cell, pancreatic progenitor cell, myogenic
(muscle) stem cell, pluripotent stem cell, ectoderm cell and
mesendoderm cell specific markers, respectively. According to the
present invention it is possible to effect the conversion of cells
to various types of differentiated cardiac, hematopoietic (blood),
pancreatic, myogenic (muscle), pluripotent derived, ectoderm
derived and/or mesendoderm derived cells that can be created from a
single cell type taken from an individual donor and then
reprogrammed and transplanted into the same individual.
[0029] According to one particular aspect, the invention relates to
a method of transforming a cell of a first type to a desired cell
of a different type. This comprises i) obtaining a cell of a first
type; ii) transiently increasing in the cell of a first type
intracellular levels of at least one reprogramming agent, whereby
the transient increase induces direct or indirect endogenous
expression of at least one gene regulator; iii) placing the cell in
conditions for supporting the growth and/or the transformation of
the desired cell and maintaining intracellular levels of the at
least one reprogramming agent for a sufficient period of time to,
allow stable expression of the at least one gene regulator in the
absence of the reprogramming agent; and iv) maintaining the cell in
culture conditions supporting the growth and/or the transformation
of the desired cell. Such conditions are maintained for a
sufficient period of time to allow a stable expression of a
plurality of secondary genes. According to the invention the
expression of one or more of the secondary genes is characteristic
of phenotypical and functional properties of the desired cell while
being not characteristic of phenotypical and functional properties
of an embryonic stem cell. Therefore, at the end of the period of
time, the desired cell of a different type is obtained.
[0030] According to another particular aspect, the invention
relates to a method of transforming a cell of a first type to a
cell of a second different type. The method comprises contacting
the cell of a first type with one or more agents capable of
increasing within said cell levels of at least one reprogramming
agent and directly or indirectly remodeling the chromatin and/or
DNA of the cell. The at least one reprogramming agent is selected
for inducing directly or indirectly the expression of morphological
and functional characteristics of a desired cell of a different
type or different cell lineage.
[0031] According to another aspect, the invention relates to a
method of transforming a cell of a first type to a cell of a second
different type. The method comprises contacting the chromatin
and/or DNA of a cell of a first type with an agent capable of
remodeling chromatin and/or DNA of said cell; and increasing
intracellular levels of at least one reprogramming agent. The at
least one reprogramming agent is selected for inducing directly or
indirectly the expression of morphological and functional
characteristics of a desired cell of a different type or cell
lineage.
[0032] A further aspect of the invention relates to a method of
transforming a cell of a first type to a cell of a desired cell of
a different type, comprising increasing intracellular levels of at
least one reprogramming agent, wherein the at least one
reprogramming agent is selected for inducing directly or indirectly
the expression of morphological and functional characteristics of a
desired second cell type; and maintaining the cell of a first type
in culture conditions for supporting the transformation of the
desired cell for a sufficient period of time to allow stable,
expression of a plurality of secondary genes whose expression is
characteristic of phenotypical and functional properties of the
desired cell, wherein at least one of the secondary genes is not
characteristic of phenotypical and functional properties of an
embryonic stem cell. At the end of the period of time the desired
cell of a different type is obtained and the obtained cell is
further characterized by a stable repression of a plurality of
genes expressed in the first cell type.
[0033] A further aspect of the invention concerns a process wherein
a cell of a first type is reprogrammed to a desired cell of a
different type, the process comprising: [0034] a transient increase
of intracellular levels of at least one reprogramming agent,
wherein the at least one reprogramming agent induces a direct or
indirect endogenous expression of at least one gene regulator, and
wherein the endogenous expression of the said at least one gene
regulator is necessary for the existence of the desired cell of a
different type; [0035] a stable expression of said at least one
gene regulator; [0036] stable expression of a plurality of
secondary genes, wherein the stable expression of the secondary
genes is the result of the stable expression of the at least one
gene regulator, and wherein: (i) stable expression of the plurality
of secondary genes is characteristic of phenotypical and/or
functional properties of the desired cell, (ii) stable expression
of at least one of said secondary genes is not characteristic of
phenotypical and functional properties of an embryonic stem cell,
and wherein (i) and (ii) are indicative of successful reprogramming
of the cell of the first type to the desired cell of the different
type.
[0037] In particular embodiments, the at least one reprogramming
agent is a polypeptide listed in Table A. In additional embodiments
the at least one gene regulator may be one or more of the genes
listed in Table A for the desired cell type (desired cell of a
second different type).
[0038] In particular embodiments, the at least one reprogramming
agent in the process is a Msi1 polypeptide, or a Ngn2 polypeptide
together with a MDB2 polypeptide. In particular embodiments, the at
least one gene regulator is Sox2 Msi1, or both. In additional
embodiments the at least one gene regulator may is one or more of
the genes listed in Table A for Neural Stem-Like Cells.
[0039] According to another aspect, the invention relates to a
method of obtaining a Stem-Like Cell (SLC), comprising: [0040] i)
providing a cell of a first type; [0041] ii) transiently increasing
in the cell intracellular levels of at least one reprogramming
agent, whereby the transient increase induces direct or indirect
endogenous expression of at least one gene regulator; [0042] iii)
placing the cell in conditions for supporting the transformation
into the stem-like cell and maintaining intracellular levels of the
at least one reprogramming agent for a sufficient period of time to
allow stable expression of the at least one gene regulator in
absence of the reprogramming agent; [0043] iv) maintaining the cell
in culture conditions for supporting the transformation into the
stem-like cell for a sufficient period of time to allow stable
expression of a plurality of secondary genes whose expression is
characteristic of phenotypical and/or functional properties of the
stem-like cell, wherein at least one of the secondary genes is not
characteristic of phenotypical and functional properties of an
embryonic stem cell. At the end of said period of time a stem-like
cell is obtained.
[0044] A Stem-Like Cell (SLC) can be any type of stem-like cell
such as a Neural Stem-Like Cell, Cardiac Stem-Like Cell (CSLCs),
Hematopoietic Stem-Like Cell (HSLCs), Pancreatic Progenitor-Like
Cell, Myogenic (muscle) Stem-Like Cell, Pluripotent-Like Cell,
Ectoderm-Like Cell or Mesendoderm-Like Cell.
[0045] According to another aspect, the invention relates to a
method of obtaining a Stem-Like Cell. The method comprises
increasing intracellular levels of at least one polypeptide
specific to the desired stem cell type that is able to drive
directly or indirectly transformation of the cell of the first type
into the Stem-Like Cell. For increasing the yield or type of
Stem-Like Cell, the method may further comprises contacting
chromatin and/or DNA of a cell of a first type with a histone
acetylator, an inhibitor of histone deacetylation, a DNA
demethylator, and/or an inhibitor of DNA methylation; and/or
increasing intracellular levels of at least one other polypeptide
specific to the desired stem cell type that is able to drive
directly or indirectly transformation of the cell of the first type
into a Stem-Like Cell.
[0046] According to another aspect, the invention relates to a
method of obtaining a Stem-Like Cell comprising the use of one or
more compounds that increase intracellular levels of at least one
polypeptide specific to the desired stem cell type that is able to
drive directly or indirectly transformation of the cell of the
first type into the Stem-Like Cell. For increasing the yield or
type of Stem-Like Cell, the method may further comprises contacting
chromatin and/or DNA of a cell of a first type with a histone
acetylator, an inhibitor of histone deacetylation, a DNA
demethylator, and/or an inhibitor of DNA methylation; and/or
increasing intracellular levels of at least one other polypeptide
specific to the desired stem cell type that is able to drive
directly or indirectly transformation of the cell of the first type
into a Stem-Like Cell; and/or disrupting the cell cytoskeleton or
cell integrity with at least one cytoskeleton disruptor.
[0047] According to another aspect, the invention relates to a
method of obtaining a Neural Stem-Like Cell (NSLC). The method
comprises increasing intracellular levels of at least one neural
stem cell specific polypeptide that is able to drive directly or
indirectly transformation of the cell of the first type into a
NSLC. For increasing the yield or type of NSLC, the method further
comprises contacting chromatin and/or DNA of a cell of a first type
with a histone acetylator, an inhibitor of histone deacetylation, a
DNA demethylator, and/or an inhibitor of DNA methylation; and/or
increasing intracellular levels of at least one other neural stem
cell specific polypeptide that is able to drive directly or
indirectly transformation of the cell of the first type into a
NSLC.
[0048] Another aspect of the invention concerns a method of
obtaining a Neural Stem-Like Cell (NSLC). In one embodiment the
method comprises transfecting a skin cell with a polynucleotide
encoding Musashi1, Musashi1 and Neurogenin 2, Musashi1 and
Methyl-CpG Binding Domain Protein 2 (MBD2), or Neurogenin 2 and
Methyl-CpG Binding Domain Protein 2, thereby reprogramming the skin
cell into a NSLC. In another embodiment the method comprises
exposing a skin cell to: (I) an inhibitor of histone deacetylation,
(ii) an inhibitor of DNA methylation, (iii) a histone acetylator,
and/or (iv) a DNA demethylator such as a MBD2 polypeptide and/or
transfecting with a polynucleotide encoding a MBD2 polypeptide; and
further transfecting the cell (either simultaneously, before, or
afterwards) with a polynucleotide encoding MUSASHI1 and/or with a
polynucleotide encoding NGN2, thereby reprogramming the skin cell
into a NSLC. Some other cells, such as keratinocytes and CD34.sup.+
cells, can also be used and reprogrammed. Additionally introducing
into the cells one or more of the following polypeptides (ex. by
transfection of their corresponding polynucleotides) reprograms the
cells to Ectoderm-Like Cells: Zinc Finger Protein 42 (REX1),
Octamer-binding Transcription Factor 4 (OCT4), Kruppel-like Factor
4 (KLF4), Sal-like 4 (SALL4), and Nanog Homeobox (NANOG).
[0049] Another aspect of the invention concerns a method of
obtaining a Cardiac Stem-Like Cell (CSLC). In one embodiment the
method comprises transfecting a mesenchymal stem cell with a
polynucleotide encoding Brachyury (T) and Mesoderm Posterior 1
(MESP1), along with NK2 Homebox 5 (NRx2.5) and/or T-box 5 (TBX5),
thereby reprogramming the mesenchymal stem cell into a CSLC. In
another embodiment the method comprises exposing a mesenchymal stem
cell to compounds that result in histone acetylation and/or DNA
demethylation, and further transfecting the cell (either
simultaneously, before, or afterwards) with polynucleotides
encoding Brachyury (T) and Mesoderm Posterior 1 (MESP1), along with
NK2 Homebox 5 (Nkx2.5) and/or T-box 5 (TBX5), thereby reprogramming
the mesenchymal stem cell into a CSLC.
[0050] Another aspect of the invention concerns a method of
obtaining a Hematopoietic Stem-Like Cell (HSLC). In one embodiment
the method comprises transfecting an adipocyte-derived stem cell
with a combination of polynucleotides encoding Brachyury (T),
Caudal Type Homeobox 4 (CDX4), Homeobox 84 (HOXB4), GATA Binding
Factor 1 (GATA1), GATA Binding Factor 2 (GATA2), and/or
Kruppel-like Factor 1 (KLF1), thereby reprogramming the cell into a
HSLC. In another embodiment the method comprises exposing an
adipocyte-derived stem cell to compounds that result in histone
acetylation and/or DNA demethylation, and further transfecting the
cell (either simultaneously, before, or afterwards) with a
combination of polynucleotides encoding Brachyury (T), Caudal Type
Homeobox 4 (CDX4), Homeobox B4 (HOXB4), GATA Binding Factor 1
(GATA1), GATA Binding Factor 2 (GATA2), and/or Kruppel-like Factor
1 (KLF1), thereby reprogramming the cell into a HSLC.
[0051] Another aspect of the invention concerns a method of
obtaining a Pancreatic Progenitor-Like Cell. In one embodiment the
method comprises transfecting an adipocyte-derived stem cell with a
polynucleotide encoding SRY (Sex determining Region Y)-box 17
(SOX17), Pancreatic and Duodenal Homebox 1 (PDX1) and Neurogenin 3
(NGN3) optionally along with Octamer-binding Transcription Factor 4
(OCT4), thereby reprogramming the cell into a Pancreatic
Progenitor-Like Cell. In another embodiment the method comprises
exposing an adipocyte-derived stem cell to compounds that result in
histone acetylation and/or DNA demethylation; and further
transfecting the cell (either simultaneously, before, or
afterwards) with polynucleotides encoding SRY (Sex determining
Region Y)-box 17 (SOX17), Pancreatic and Duodenal Homebox 1 (PDX1)
and Neurogenin 3 (NGN3) optionally along with Octamer-binding
Transcription Factor 4 (OCT4), thereby reprogramming the cell into
a Pancreatic Progenitor-Like Cell.
[0052] Another aspect of the invention concerns a method of
obtaining a Mesendoderm-Like Cell. In one embodiment the method
comprises transfecting an adipocyte-derived stem cell with a
polynucleotide encoding Forkhead Box D3 (FOXD3), MIX1 Homeobox-Like
Protein 1 (MIXL1) and Neurogenin 3 (NGN3) along with Methyl-CpG
Binding Domain Protein 2 (MBD2), thereby reprogramming the cell
into a Mesendoderm-Like Cell. In another embodiment the method
comprises exposing an adipocyte-derived stem cell to compounds that
result in histone acetylation and/or DNA demethylation; and further
transfecting the cell (either simultaneously, before, or
afterwards) with polynucleotides encoding Forkhead Box D3 (FOXD3),
MIX1 Homeobox-Like Protein 1 (MIXL1) and Neurogenin 3 (NGN3),
thereby reprogramming the cell into a Mesendoderm-Like Cell.
Another aspect of the invention concerns a method of obtaining a
Pluripotent-Like Cell. In one embodiment the method comprises
transfecting an adipocyte-derived stem cell with a polynucleotide
encoding a) Zinc Finger Protein 42 (REX1), Octamer-binding
Transcription Factor 4 (OCT4) and Kruppel-like Factor 4 (KLF4), or
b) Sal-like 4 (SALL4), Octamer-binding Transcription Factor 4
(OCT4), Kruppel-like Factor 4 (KLF4) and Nanog Homeobox (NANOG),
thereby reprogramming the cell into a Pluripotent-Like Cell. In
another embodiment the method comprises exposing an
adipocyte-derived stem cell to compounds that result in histone
acetylation and/or DNA demethylation; and further transfecting the
cell (either simultaneously, before, or afterwards) with
polynucleotides encoding a) Zinc Finger Protein 42 (REX1),
Octamer-binding Transcription Factor 4 (OCT4) and Kruppel-like
Factor 4 (KLF4), or b) Sal-like 4 (SALL4), Octamer-binding
Transcription Factor 4 (OCT4), Kruppel-like Factor 4 (KLF4) and
Nanog Homeobox (NANOG), thereby reprogramming the cell into a
Pluripotent-Like Cell.
[0053] In one particular embodiment, the method of obtaining a
Neural Stem-Like Cell (NSLC), comprises: [0054] providing a cell of
a first type; [0055] introducing into the cell one or more
polynucleotide capable of transient expression of one or more the
following polypeptides: Musashi1 (Msi1); a Musashi1 (Msi1) and a
Neurogenin 2 (Ngn2); a Musashi1 (Msi1) and methyl-CpG binding
domain protein 2 (MBD2); and Neurogenin 2 (Ngn2) and methyl-CpG
binding domain protein 2 (MBD2); and [0056] placing the cell in
culture conditions supporting the transformation into a NSLC for a
sufficient period of time to allow a stable expression of a
plurality of genes whose expression is characteristic of
phenotypical and functional properties of a NSLC.
[0057] At the end of the period of time a NSLC is obtained and the
obtained NSLC is further characterized by a stable repression of a
plurality of genes expressed in the first cell type.
[0058] According to another embodiment, the method of obtaining a
Neural Stem-Like Cell (NSLC), comprises: [0059] providing a cell of
a first type which is not a NSLC; [0060] increasing intracellular
levels of at least one neural stem cell specific polypeptide,
wherein the polypeptide is capable of driving directly or
indirectly transformation of the cell of the first type into a
NSLC; and [0061] contacting the chromatin and/or DNA of the cell of
a first type with a histone acetylator, an inhibitor of histone
deacetylation, a DNA demethylator, and/or a chemical inhibitor of
DNA methylation.
[0062] According to another embodiment, the method of obtaining a
Neural Stem-Like Cell (NSLC), comprises: [0063] obtaining a
non-NSLC; [0064] co-transfecting the non-NSLC with a first
polynucleotide encoding a MBD2 polypeptide and with at least one
second polynucleotide encoding a MUSASHI1 polypeptide and/or
encoding a NGN2 polypeptide; [0065] placing the co-transfected cell
in culture conditions for supporting the transformation of NSLC
until said NSLC is obtained.
[0066] Certain aspects of the invention concerns isolated cells,
cell lines, compositions, 3D assembly of cells, and tissues
comprising cells obtained using the methods described herein.
Additional aspects concerns the use of such isolated cells, cell
lines, compositions, 3D assembly of cells, and tissues of medical
treatment and methods of regenerating a mammalian tissue or
organ.
[0067] Yet, a further aspect concerns a method for repairing or
regenerating a tissue in a subject. In one embodiment the method
comprises the administration of a reprogrammed cell as defined
herein to a subject in need thereof, wherein the administration
provides a dose of reprogrammed cells sufficient to increase or
support a biological function of a given tissue or organ, thereby
ameliorating the subject's condition. In another embodiment the
method comprises the administration of a reprogrammed cell that has
been genetically modified (for therapeutic or other purposes) or
genetically corrected (in cases of a genetic disease or similar
instance) to a subject in need thereof, wherein the administration
provides a dose of reprogrammed genetically-modified/-corrected
cells sufficient to increase, support, replace or correct a
biological function of a given cell, tissue or organ, thereby
ameliorating or correcting the subject's condition or allowing for
a desired condition.
[0068] The benefits of the present invention are significant and
include more potent and/or safer and/or lower cost of cell therapy
by eliminating the need of immuno-suppressive agents, no need for
embryos or fetal tissue, thus eliminating ethical and time
constraints, lower cost of production, and no health risks due to
possible transmission of viruses or other disease, and the
availability of more potent and/or safer stem-like cells and
progenitor-like cells. In addition, since the cells are created
fresh, they tend to be more potent than cells that have been
passaged multiple times.
[0069] Additional aspects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of preferred embodiments and Examples
which are exemplary and should not be interpreted as limiting the
scope of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0070] The present invention relates to reprogrammed cells, methods
for cell dedifferentiation and cell reprogramming, and use of these
cells. A significant aspect of the present invention is that it
permits the use of a patient's own cells to develop different types
of cells either in situ (in the patient) or that can be
transplanted after steps of in vitro dedifferentiation and in vitro
reprogramming. Thus, this technology eliminates the problems
associated with a) lack of the patient's own desired cells; b) lack
of potency or other desired functional features of the patient's
own desired cells; and/or c) problems associated with
transplantation of non-host cells, such as, immunological rejection
and the risk of transmitting disease, and no permanent functional
grafting. In addition, since the cells are "newly created", they
have the potential to be more potent than alternative sources of
natural cells that have already divided multiple times.
DEFINITIONS
[0071] As used herein and in the appended claims, the singular
forms "a," "an", and "the", include plural referents unless the
context clearly indicates otherwise. Thus, for example, reference
to "a cell" includes one or more of such, cells or a cell line
derived from such a cell, reference to "an agent" includes one or
more of such agent, and reference to "the method" includes
reference to equivalent steps and methods known to those of
ordinary skill in the art that could be modified or substituted for
the methods described herein.
[0072] As used herein, the term "polynucleotide" refers to any DNA
or RNA sequence or molecule, comprising encoding nucleotide
sequences. The term is intended to encompass all polynucleotides
whether occurring naturally or non-naturally in a particular cell,
tissue or organism. This includes DNA and fragments thereof, RNA
and fragments thereof, cDNAs and fragments thereof, expressed
sequence tags, artificial sequences including randomized artificial
sequences.
[0073] As used herein, the term "polypeptide" refers to any amino
acid sequence having a desired functional biological activity (e.g.
DNA demethylation). The term is intended to encompass complete
proteins, fragments thereof, fusion proteins and the like,
including carbohydrate or lipid chains or compositions.
[0074] "Trans-differentiation" refers to a direct switch of an
already differentiated cell to another type of differentiated
cell.
[0075] "De-differentiation" refers to the loss of phenotypic
characteristics of a differentiated cell by activating or
deactivating genes or metabolic pathways.
[0076] "Marker" refers to a gene, polypeptide, or biological
function that is characteristic of a particular cell type or
cellular phenotype.
[0077] "Genetically-engineered DNA sequence" is meant a DNA
sequence wherein the component sequence elements of DNA sequence
are organized within the DNA sequence in a manner not found in
nature.
[0078] "Signal sequence" refers to a nucleic acid sequence which,
when incorporated into a nucleic acid sequence encoding a
polypeptide, directs secretion of the translated polypeptide from
cells which express said polypeptide, or allows the polypeptide to
readily cross the cell membrane into a cell. The signal sequence is
preferably located at the 5' end of the nucleic acid sequence
encoding the polypeptide, such that the polypeptide sequence
encoded by the signal sequence is located at the N-terminus of the
translated polypeptide. By "signal peptide" is meant the peptide
sequence resulting from translation of a signal sequence.
[0079] "Ubiquitous promoter" refers to a promoter that drives
expression of a polypeptide or peptides encoded by nucleic acid
sequences to which promoter is operably linked. Preferred
ubiquitous promoters include human cytomegalovirus immediate early
(CMV); simian virus 40 early promter (SV40); Rous sarcoma virus
(RSV); or adenovirus major late promoter.
[0080] "Gene expression profiling" means an assay that measures the
activity of multiple genes at once, creating a global picture of
cellular function. For example, these profiles can distinguish
between human neural stem cells and somatic cells that are actively
dividing or differentiating.
[0081] "Transfection" refers to a method of gene delivery that
introduces a foreign nucleotide sequences (e.g. DNA molecules) into
a cell preferably by a non-viral method. In preferred embodiments
according to the present invention foreign DNA is introduced to a
cell by transient transfection of an expression vector encoding a
polypeptide of interest, whereby the foreign DNA is introduced but
eliminated over time by the cell and during mitosis. By "transient
transfection" is meant a method where the introduced expression
vectors and the polypeptide encoded by the vector, are not
permanently integrated into the genome of the host cell, or
anywhere in the cell, and therefore may be eliminated from the host
cell or its progeny over time. Proteins, polypeptides, or other
compounds can also be delivered into a cell using transfection
methods.
[0082] "Neuroprogenitor Cell" refers to an immature cell of the
nervous system, which can differentiate into neurons and glia
(oligodendrocytes and astrocytes). "Neural Stem Cell" is an
ectoderm germ layer derived multipotent stem cell having, as a
physiological feature, a capacity to form neuroprogenitor cells and
under physiological conditions that favor differentiation to form
neurons and glia. "Neural Stem-Like Cell" or "NSLC" refers to any
cell-derived multipotent stem cell having, as a physiological
feature, a capacity to form other neural stem-like cells and
neuroprogenitor-like cells and under physiological conditions that
favor differentiation to form neuron-like cells and glial-like
cells.
[0083] "Neurosphere" refers to a cellular aggregate of neural stem
cells and neuroprogenitor cells that form a floating sphere formed
as a result of proliferation of the neural stem cells and
neuroprogenitor cells in appropriate proliferation conditions.
NSLCs also form neurospheres consisting of aggregates of NSLCs and
neuroprogenitor-like cells.
[0084] "Cardiosphere" refers to a cellular aggregate of cardaic
stem cells and cardiac progenitor cells that form a floating sphere
formed as a result of proliferation of the cardaic stem cells and
cardiac progenitor cells in appropriate proliferation conditions.
CSLCs also form cardiospheres consisting of aggregates of CSLCs and
cardiac progenitor-like cells. "Reprogrammed cell" refers to a cell
that has undergone stable trans-differentiation,
de-differentiation, or transformation. Some reprogrammed cells can
be subsequently induced to re-differentiate. The reprogrammed cell
stably expresses a cell-specific marker or set of markers,
morphology, and/or biological function that was not characteristic
of the original cell. "Reprogrammed somatic cell" refers to a
process that alters or reverses the differentiation status of a
somatic cell, which can be either complete or partial conversion of
the differentiated state to an either less differentiated state or
a new differentiated state.
[0085] "Regeneration" refers to the capability of contributing to
the repair or do novo construction of a cell, tissue or organ.
[0086] "Differentiation" refers to the developmental process of
lineage commitment of a cell. Differentiation can be assayed by
measuring an increase in one or more cell-differentiation specific
markers relative to the expression of the undifferentiated cell
markers.
[0087] "Lineage" refers to a pathway of cellular development, in
which a more undifferentiated cell undergoes progressive
physiological changes to become a more differentiated cell type
having a characteristic function (e.g., neurons and glia are of a
neuroprogenitor linage, which is of an ectoderm lineage which
formed from blastocysts and embryonic stem (ES) cells).
[0088] "Tissue" refers to an ensemble of cells (identical or not)
and an extracellular matrix (ECM) that together carry out a
specific function or set of functions.
[0089] "CDM" is meant a living tissue equivalent or matrix, a
living scaffold, or cell-derived matrix.
Cell Transformation
[0090] Some aspects of the invention concerns methods and cells to
transform or reprogram a given somatic cell into a pluripotent,
multipotent and/or unipotent cell. Some aspects of the invention
relates to methods for conditioning a somatic cell to reprogramming
into a pluripotent, multipotent or unipotent cell.
[0091] The terms "transform" or "reprogram" are used
interchangeably to refer to the phenomenon in which a cell is
dedifferentiated or transdifferentiated to become pluripotent,
multipotent and/or unipotent. The dedifferentiated cell could
subsequently be redifferentiated into a different type of cell.
Cells can be reprogrammed or converted to varying degrees. For
example, it is possible that only a small portion of cells are
converted or that an individual cell is reprogrammed to be
multipotent but not necessarily pluripotent. Thus, the terms
"transforming" or "reprogramming" methods can refer to methods
wherein it is possible to reprogram a cell such that the "new" cell
shows morphological and functional characteristics of a new or
different specific cell lineage (e.g. the transformation of
fibroblast cells into neuronal cells).
[0092] As used herein, the term "somatic cell" refers to any
differentiated cell forming the body of an organism, apart from
stem cells, progenitor cells, and germline cells (i.e. ovogonies
and spermatogonies) and the cells derived therefrom (e.g. oocyte,
spermatozoa). For instance, internal organs, skin, bones, blood,
and connective tissue are all made up of somatic cells. Somatic
cells according to the invention can be differentiated cells
isolated from adult or can be fetal somatic cells. Somatic cells
are obtained from animals, preferably human subjects, and cultured
according to standard cell culture protocols available to those of
ordinary skill in the art.
[0093] As used herein, "Stem cell" refers to those cells which
retain the ability to renew themselves through mitotic cell
division and which can differentiate into a diverse range of
specialized cell types. It includes both embryonic stem cells that
are found in blastocysts, and adult stem cells that are found in
adult tissues. "Totipotent cells" refers to cells that have the
ability to develop into cells derived from all three embryonic germ
layers (mesoderm, endoderm and ectoderm) and an entire organism
(e.g., human being if placed in a woman's, uterus in the case of
humans). Totipotent cells may give rise to an embryo, the extra
embryonic membranes and all post-embryonic tissues and organs. The
term "pluripotent" as used herein is intended to mean the ability
of a cell to give rise to differentiated cells of all three
embryonic germ layers. "Multipotent cells" refers to cells that can
produce only cells of a closely related family of cells (e.g.
hematopoietic stem cells differentiate into red blood cells, white
blood cells, platelets, etc.). "Unipotent cells" refers to cells
that have the capacity to develop/differentiate into only one type
of tissue/cell type (e.g. skin cells).
[0094] The present invention allows the reprogramming of any cell
to a different type of cell. Although the present application
focuses primarily on the preparation of Stem-Like cells,
especially, Neural Stem-Like Cells (NSLCs), the invention is not so
restricted because many different types of cells can be generated
according to the principles described herein. Similarly, while the
Examples section describes embodiments where fibroblasts,
keratinocytes, CD34.sup.+ cells, adipose-derived stem cells
(ADSCs), neural stem cells (including NSLCs), and cells within a
Cell-Derived Matrix (CDM) are reprogrammed, the invention is not
limited such cells. The invention may be employed for the
reprogramming of virtually any cell of interest.
[0095] Accordingly, a general aspect of the invention relates to a
method of transforming a cell of a first type to a cell of a second
different type. As used herein, examples of cells of a first type
include, but are not limited to germ cells, embryonic stem cells
and derivations thereof, adult stem cells and derivations thereof,
progenitor cells and derivations thereof, cells derived from
mesoderm, endoderm or ectoderm, and a cell of mesoderm, endoderm or
ectoderm lineage such as an adipose-derived stem cell (ADSC),
mesenchymal stem cell, hematopoietic stem cell (CD34.sup.+ cell),
skin derived precursor cell, hair follicle cell, fibroblast,
keratinocyte, epidermal cell, endothelial cell, epithelial cell,
granulosa epithelial cell, melanocyte, adipocyte, chondrocyte,
hepatocyte, lymphocyte (B and T lymphocyte), granulocyte,
macrophage, monocyte, mononuclear cell, pancreatic islet cell,
sertoli cell, neuron, glial cell, cardiac muscle cell, and other
muscle cell.
[0096] As used herein, examples of cells of a second type include,
but are not limited to germ cells, embryonic stem cells and
derivations thereof, adult stem cells and derivations thereof,
progenitor cells and derivations thereof, cells derived from
mesoderm, endoderm or ectoderm, and a cell of mesoderm, endoderm or
ectoderm lineage such as an adipose-derived stem cell, mesenchymal
stem cell, hematopoietic stem cell, skin derived precursor cell,
hair follicle cell, fibroblast, keratinocyte, epidermal cell,
endothelial cell, epithelial cell, granulosa epithelial cell,
melanocyte, adipocyte, chondrocyte, hepatocyte, lymphocyte (B and T
lymphocyte), granulocyte, macrophage, monocyte, mononuclear cell,
pancreatic islet cell, sertoli cell, neuron, glial cell, cardiac
muscle cell, and other muscle cell. In addition, each of the above
"-like" cell (a cell that has similar but not completely identical
characteristics of the known natural type of the cell) is also
included in the examples of cells of a second type.
[0097] According to one particular aspect, the method of
transforming a cell of a first type into a cell of a second
different type comprises the steps of: [0098] i) providing a cell
of a first type; [0099] ii) transiently increasing in the cell of a
first type intracellular levels of at least one reprogramming
agent, whereby the transient increase induces direct or indirect
endogenous expression of at least one gene regulator; [0100] iii)
placing the cell in conditions for supporting the transformation of
the desired cell and maintaining intracellular levels of the at
least one reprogramming agent for a sufficient period of time to
allow stable expression of the at least one gene regulator in
absence of the reprogramming agent; and [0101] iv) maintaining the
cell in culture conditions supporting the transformation of the
desired cell for a sufficient period of time to allow a stable
expression of a plurality of secondary genes whose expression is
characteristic of phenotypical and functional properties of the
desired cell. At the end of said period of time the cell of the
first type has been transformed into the desired cell of a
different type. Preferably, the cell of a different type obtained
after the transformation is further characterized by a stable
repression of a plurality of genes expressed in the first cell
type.
[0102] According to one particular aspect, the method of
transforming a cell of a first type into a cell of a second
different type comprises the steps of: [0103] i) providing a cell
of a first type; [0104] ii) transiently increasing in the cell of a
first type intracellular levels of at least one reprogramming
agent, whereby the transient increase induces direct or indirect
endogenous expression of at least one gene regulator; [0105] iii)
placing the cell in conditions for supporting the transformation of
the desired cell and maintaining intracellular levels of the at
least one reprogramming agent for a sufficient period of time to
allow stable expression of the at least one gene regulator in
absence of the reprogramming agent; and [0106] iv) maintaining the
cell in culture conditions supporting the transformation of the
desired cell for a sufficient period of time to allow a stable
expression of a plurality of secondary genes whose expression is
characteristic of phenotypical and functional properties of the
desired cell. At least one of the stably expressed secondary genes
is not characteristic of phenotypical and functional properties of
an embryonic stem cell. At the end of said period of time the cell
of the first type has been transformed into the desired cell of a
different type. Preferably, the cell of a different type obtained
after the transformation is further characterized by a stable
repression of a plurality of genes expressed in the first cell
type.
[0107] According to various embodiments, step iii) may be carried
out, consecutively to step ii), simultaneously with step ii), or
before step ii).
[0108] According to a related aspect, the invention relates to a
process wherein a cell of a first type is reprogrammed to a desired
cell of a different type, the process comprising: [0109] a
transient increase of intracellular levels of at least one
reprogramming agent, wherein the at least one reprogramming agent
induces a direct or indirect endogenous expression of at least one
gene regulator, wherein the endogenous expression of the at least
one gene regulator is necessary for the existence of the desired
cell of a different type; [0110] a stable expression of said at
least one gene regulator; [0111] stable expression of a plurality
of secondary genes, wherein the stable expression of the plurality
of secondary genes is the result of the stable expression of the at
least one gene regulator, and wherein: (i) stable expression of the
plurality of secondary genes is characteristic of phenotypical
and/or functional properties of the desired cell, (ii) stable
expression of at least one of the secondary genes is not
characteristic of phenotypical and functional properties of an
embryonic stem cell, and wherein (i) and (ii) are indicative of
successful reprogramming of the cell of the first type to the
desired cell of the different type.
[0112] As used herein, "transiently increasing" refers to an
increase that is not necessarily permanent and therefore, which may
decrease or disappear over time. For instance, when referring to
transiently increasing intracellular levels of at least one
reprogramming agent in a cell, it means that the increase in
present for a sufficient period of time for causing particular
cellular events to occur (e.g. inducing stable endogenous
expression of a gene regulator). Typically a transient increase is
not permanent and is not associated for instance to genome
integration of an expression vector.
[0113] As used herein the term "reprogramming agent" refers to a
compound that is capable of inducing directly or indirectly the
expression of morphological and/or functional characteristics of
the desired cell of a different type. Preferred compounds include
those capable of driving directly or indirectly transformation of
the cell of the first type into the desired cell of a different
type. In preferred embodiment, the reprogramming agent is selected
for inducing a direct or indirect endogenous expression of at least
one gene regulator as defined herein. There are many compounds that
may be helpful in reprogramming a cell according to the invention
and these compounds can be used alone or in combinations. For
example, the compound may be a molecule that induces epigenetic
changes (chromatin remodeling, ex. histone acetylation and/or DNA
demethylation) or a cytoskeleton disruptor that is helpful in
reprogramming a cell according to the invention (or alternatively
the culture conditions can include one or more compounds,
materials, environmental (physical or chemical) effects or
conditions that induce epigenetic changes and/or cytoskeleton
disruptors that support the transformation to the desired cell). In
various embodiments, the reprogramming agent is a polynucleotide or
polypeptide selected according to TABLE A:
TABLE-US-00001 TABLE A Reprogramming agent RefSeq/ UniProt .TM./
UniGene .TM. Examples of GenBank .TM. (NCBI) Swiss-Port Accession
Desired Cell Type Name Accession No. Accession No. No.
Pluripotent-like AGR2 NM_006408.3 O95994 Hs.530009 Cells AGR3
NM_176813.3 Q8TD06 Hs.100686 Markers: BRIX1 NM_018321.3 Q8TDN6
Hs.718510 OCT4 CRABP2 NM_001878.2 P29373 Hs.405662 Nanog DNMT3B,
NM_006892.3 Q9UBC3 Hs.713611 SSEA-4 isoform 1 TRA1-60 DNMT3B,
NM_175848.1 Q9UBC3 Hs.713611 TRA1-81 isoform 2 AP DNMT3B,
NM_175849.1 Q9UBC3 Hs.713611 isoform 3 DNMT3B, NM_175850.1 Q9UBC3
Hs.713611 isoform 6 DPPA2 NM_138815.3 Q7Z7J5 Hs.351113 DPPA3
NM_199286.2 Q6W0C5 Hs.131358 (STELLA) DPPA4 NM_018189.3 Q7L190
Hs.317659 DPPA5 NM_001025290.1 A6NC42 Hs.125331 (ESG1) FOXD3
NM_012183.2 Q9UJU5 Hs.546573 FOXH1 NM_003923.2 O75593 Hs.708365
GABRB3, NM_000814.5 P28472 Hs.302352 isoform 1 GABRB3, NM_021912.4
P28472 Hs.302352 isoform 2 GABRB3, NM_001191320.1 P28472 Hs.302352
isoform 3 GABRB3, NM_001191321.1 P28472 Hs.302352 isoform 4 GBX2
NM_001485.2 P52951 Hs.184945 GDF3 NM_020634.1 Q9NR23 Hs.86232 GJA1
(CX43) NM_000165.3 P17302 Hs.74471 GRB7 NM_005310.2 Q14451 Hs.86859
NM_001030002.1 Q14451 Hs.86859 HESRG NR_027122.1 Q1W209 Hs.720658
IFITM1 NM_003641.3 P13164 Hs.458414 IFITM2 NM_006435.2 Q01629
Hs.709321 KLF2 NM_016270.2 Q9Y5W3 Hs.726356 KLF4 NM_004235.4 O43474
Hs.376206 LEFTY1 NM_020997.2 O75610 Hs.656214 LEFTY2 NM_003240.3
O00292 Hs.520187 (EBAF), isoform 1 LEFTY2 NM_001172425.1 B4E332
Hs.520187 (EBAF), (TrEMBL) isoform 2 LIN28A NM_024674.4 Q9H9Z2
Hs.86154 MYBL2 NM_002466.2 P10244 Hs.179718 NANOG NM_024865.2
Q9H9S0 Hs.635882 NODAL NM_018055.4 Q96S42 Hs.370414 NOG NM_005450.4
Q13253 Hs.248201 NR0B1 NM_000475.4 P51843 Hs.268490 (DAX1) NR5A2,
NM_205860.1 O00482 Hs.33446 isoform 1 NR5A2, NM_003822.3 O00482
Hs.33446 isoform 2 NR6A1, NM_033334.2 Q15406 Hs.586460 isoform 1
NR6A1, NM_001489.3 Q15406 Hs.586460 isoform 2 PHC1 NM_004426.2
P78364 Hs.305985 PITX2, NM_153427.1 Q99697 Hs.643588 isoform a
PITX2, NM_153426.1 Q99697 Hs.643588 isoform b PITX2, NM_000325.5
Q99697 Hs.643588 isoform c PODXL, NM_001018111.2 O00592 Hs.726449
isoform 1 PODXL, NM_005397.3 O00592 Hs.726449 isoform 2 POU5F1
NM_002701.4 Q01860 Hs.249184 (OCT4), isoform 1* POU5F1 NM_203289.4
N/A Hs.249184 (OCT4), NM_001173531.1 isoform 2 PTEN NM_000314.4
P60484 Hs.500466 REST NM_005612.4 Q13127 Hs.307836 NM_001193508.1
Q13127 Hs.307836 REX1 NM_020695.3 Q8N1G1 Hs.192477 SALL4
NM_020436.3 Q9UJQ4 Hs.517113 SEMA3A NM_006080.2 Q14563 Hs.252451
SFRP2 NM_003013.2 Q96HF1 Hs.481022 SOX2 NM_003106.2 P48431
Hs.518438 TDGF1, NM_003212.3 P13385 Hs.385870 isoform 1 TDGF1,
NM_001174136.1 P13385 Hs.385870 isoform 2 TERT, NM_198253.2 O14746
Hs.492203 isoform 1 TERT, NM_001193376.1 O14746 Hs.492203 isoform 2
TPT1 NM_003295.2 P13693 Hs.374596 UTF1 NM_003577.2 Q5T230 Hs.458406
ZFP42 NM_174900.3 Q96MM3 Hs.335787 (REX1) Ectoderm-like ASCL1
NM_004316.3 P50553 Hs.703025 Cells (MASH1) Markers: CDX1
NM_001804.2 P47902 Hs.1545 FoxJ3 DLX3 NM_005220.2 O60479 Hs.134194
Otx2 Nestin NM_006617 P48681 Hs.527971 E-cadherin DLX5 NM_005221.5
P56178 Hs.99348 Nestin FOXD3 NM_012183.2 Q9UJU5 Hs.546573 Sox2 MSI1
NM_002442.2 O43347 Hs.158311 Sox1 NANOG NM_024865.2 Q9H9S0
Hs.635882 ZIC1 POU5F1 NM_002701.4 Q01860 Hs.249184 (OCT4), isoform
1* POU5F1 NM_203289.4 N/A Hs.249184 (OCT4), NM_001173531.1 isoform
2 SOX1 NM_005986.2 O00570 Hs.202526 SOX2 NM_003106.2 P48431
Hs.518438 SP8, NM_182700.4 Q8IXZ3 Hs.195922 isoform 1 SP8,
NM_198956.2 N/A Hs.195922 isoform 2 ZIC1 NM_003412.3 Q15915
Hs.647962 Mesendoderm-like EOMES NM_005442.2 O95936 Hs.591663 Cells
FOXA2, NM_021784.4 Q9Y261 Hs.155651 Markers: isoform 1* FoxA2
FOXA2, NM_153675.2 Q9Y261 Hs.155651 GATA4 isoform 2 Mixl1 FOXD3
NM_012183.2 Q9UJU5 Hs.546573 Eomesodermin GATA4 NM_002052.3 P43694
Hs.243987 Brachyury GATA6 NM_005257.3 Q92908 Hs.514746 MIXL1
NM_031944.1 Q9H2W2 Hs.282079 POU5F1 NM_002701.4 Q01860 Hs.249184
(OCT4), isoform 1* POU5F1 NM_203289.4 N/A Hs.249184 (OCT4),
NM_001173531.1 isoform 2 SOX17 NM_022454.3 Q9H6I2 Hs.98367 T
(Brachyury) NM_003181.2 O15178 Hs.389457 Neural Stem-like ASCL1
NM_004316.3 P50553 Hs.703025 Cells (Mash1) Markers: BMI-1
NM_005180.08 P35226 Hs.731287 Sox2 CALB1 NM_004929.2 P05937
Hs.65425 Nestin DLL1 NM_005618.3 O00548 Hs.379912 GFAP DLX1,
NM_178120.4 P56177 Hs.407015 Msi1 isoform 1 CD133 DLX1,
NM_001038493.1 P56177 Hs.407015 Doublecortin isoform 2 DLX2
NM_004405.3 Q07687 Hs.419 FOXD3 NM_012183.2 Q9UJU5 Hs.546573 GJD2
(CX36) NM_020660.1 Q9UKL4 Hs.283816 HES1 NM_005524.3 Q14469
Hs.250666 HES3 NM_001024598.3 Q5TGS1 Hs.532677 HES5 NM_001010926.3
Q5TA89 Hs.57971 HOXB1 NM_002144.3 P14653 Hs.99992 KLF4 NM_004235.4
O43474 Hs.376206 MBD2 NM_015832.4 Q9UBB5 Hs.25674 MNX1 (HB9),
NM_005515.3 P50219 Hs.37035 isoform 1 MNX1 (HB9), NM_001165255.1
N/A Hs.37035 isoform 2 MSI1 NM_002442.2 O43347 Hs.158311 MSI2
NM_170721.1 Q96DH6 Hs658922 NANOG NM_024865.2 Q9H9S0 Hs.635882
NESTIN NM_006617 P48681 Hs.527971 NEUROD1 NM_002500.2 Q13562
Hs.709709 NEUROD2 NM_006160.3 Q15784 Hs.322431 NEUROG1 NM_006161.2
Q92886 Hs.248149 NEUROG2 NM_024019.2 Q9H2A3 Hs.567563 NEUROG3
NM_020999.3 Q9Y4Z2 Hs.532682 NKX6.1 NM_006168.2 P78426 Hs.546270
Nucleostemin NM_206826.1 Q9BVP2 Hs.313544 Olig2 NM_005806.3 Q13516
Hs.732068 Olig3 NM_175747.2 Q7RTU3 Hs.195398 PAX3 NM_181461.3
P23760 Hs.42146 PAX6, NM_000280.3 P26367 Hs.270303 isoform a* PAX6,
NM_001127612.1 P26367 Hs.270303 isoform a PAX6, NM_001604.4 P26367
Hs.270303 isoform b SFRP2 NM_003013.2 Q96HF1 Hs.481022 SIX3
NM_005413.3 O95343 Hs.567336 SOX1 NM_005986.2 O00570 Hs.202526 SOX2
NM_003106.2 P48431 Hs.518438 SOX3 NM_005634.2 P41225 Hs.157429 SOX9
NM_000346.3 P48436 Hs.647409 SOX10 NM_006941.3 P56693 Hs.376984
SOX11 NM_003108.3 P35716 Hs.432638 SOX21 NM_007084.2 Q9Y651
Hs.187577 ZIC1 NM_003412.3 Q15915 Hs.647962 ZIC3 NM_003413.3 Q60481
Hs.111227 Cardiac Stem-like BAF60C NM_001003802.1 Q6STE5 Hs.647067
Cells (SMARCD3), isoform 1 Markers: BAF60C NM_003078.3 Q6STE5
Hs.647067 CD133 (SMARCD3), MLc2.alpha. isoform 1 Nkx2.5 BAF60C
NM_001003801.1 Q6STE5 Hs.647067 Isl1 (SMARCD3), Brachyury isoform
2* GATA4 FOXD3 NM_012183.2 Q9UJU5 Hs.546573 GATA4 NM_002052.3
P43694 Hs.243987 GATA6 NM_005257.3 Q92908 Hs.514746 HAND1
NM_004821.2 O96004 Hs.152531 HAND2 NM_021973.2 P61296 Hs.388245
ISL1 NM_002202.2 P61371 Hs.505 KDR NM_002253.2 P35968 Hs.479756
MEF2C, NM_002397.4 Q06413 Hs.649965 isoform 1 NM_001193350.1 Q06413
MEF2C, NM_001131005.2 Q06413 Hs.649965 isoform 2 MEF2C,
NM_001193347.1 Q06413 Hs.649965 isoform 3 MEF2C, NM_001193348.1
Q06413 Hs.649965 isoform 4 MEF2C, NM_001193349.1 Q06413 Hs.649965
isoform 5 MESP1 NM_018670.3 Q9BRJ9 Hs.447531 MIXL1 NM_031944.1
Q9H2W2 Hs.282079 MYOCD, NM_001146312.1 Q6N065 Hs.567641 isoform 1
(TrEMBL) MYOCD, NM_153604.2 Q8IZQ8 Hs.567641 isoform 2 MYOCD,
NM_001146313.1 Q8IZQ8 Hs.567641 isoform 3 NKX2.5, NM_004387.3
P52952 Hs.54473 isoform 1* NKX2.5, NM_001166175.1 P52952 Hs.54473
isoform 2* NKX2.5, NM_001166176.1 P52952 Hs.54473 isoform 3*
Snail-1 NM_005985.3 O95863 Hs.48029 T (Brachyury) NM_003181.2
O15178 Hs.389457 TBX5, NM_000192.3 Q99593 Hs.381715 isoform 1*
TBX5, NM_181486.1 Q99593 Hs.381715 isoform 1 TBX5, NM_080718.1
Q99593 Hs.381715 isoform 2 TBX5, NM_080717.2 Q99593 Hs.381715
isoform 3 TBX18 NM_181486.2 Q99593 Hs.381715 SOX17 NM_022454.3
Q9H6I2 Hs.98367 Pancreatic FOXA2, NM_021784.4 Q9Y261 Hs.155651
Progenitor-like isoform 1*
Cells FOXA2, NM_153675.2 Q9Y261 Hs.155651 isoform 2 Markers: FOXD3
NM_012183.2 Q9UJU5 Hs.546573 PDX1 MAFA NM_201589.2 Q8NHW3 Hs.670866
Sox17 MIXL1 NM_031944.1 Q9H2W2 Hs.282079 FoxA2 NEUROG3 NM_020999.3
Q9Y4Z2 Hs.532682 Ngn3 NKX6.1 NM_006168.2 P78426 Hs.546270 Isl1 PAX4
NM_006193.2 O43316 Hs.129706 PDX1 NM_000209.3 P52945 Hs.32938 SOX17
NM_022454.3 Q9H6I2 Hs.98367 Hematopoietic BMI1 NM_005180.8. P35226.
Hs.731287 Stem-like Cells CDX4 NM_005193.1 O14627 Hs.553488 FLI1
NM_001167681.2. Q01543 Hs.504281 Markers: GATA1 NM_002049 P15976
Hs.765 CD34 GATA2 NM_001145661 P23769 Hs.367725 CXCR4 HoxB4
NM_024015 P17483 Hs.664706 Flt3 ISL1 NM_002202.2 P61371 Hs.505
Sca-1 KLF1 NM_006563 Q13351. Hs.37860 HoxB4 LMO2 NM_001142315.1.
P25791 Hs.34560 LMO4 NM_006769.3 P61968 Hs.436792 Runx1
NM_001001890.2. Q01196 Hs.149261 T (Brachyury) NM_003181.2 O15178
Hs.389457 TAL1 NM_003189.2. P17542 Hs.705618 Myogenic (Muscle)
FOXC1 NM_001453.2 Q12948 Hs.348883 Stem-like Cells FOXC2
NM_005251.2 Q99958 Hs.436448 FOXK1 NM_001037165.1 P58037 Hs.487393
Markers: MEF2C, NM_002397.4 Q06413 Hs.649965 .alpha.SM actinin
isoform 1 NM_001193350.1 Q06413 Calponin MEF2C, NM_001131005.2
Q06413 Hs.649965 MyoD isoform 2 MEF2C MEF2C, NM_001193347.1 Q06413
Hs.649965 Pax3 isoform 3 Pax7 MEF2C, NM_001193348.1 Q06413
Hs.649965 isoform 4 MEF2C, NM_001193349.1 Q06413 Hs.649965 isoform
5 MYF3 NM_002478.4 P15172 Hs.181768 (MyoD) MYF4 NM_002479.5 P15173
Hs.2830 (Myogenin) MYF5 NM_005593.2 P13349 Hs178023 Pax3,
NM_181457.3 P23760 Hs.42146 isoform Pax3 Pax3, NM_000438.5 P23760
Hs.42146 isoform Pax3a Pax3, NM_013942.4 P23760 Hs.42146 isoform
Pax3b Pax3, NM_181458.3 Q494Z3, Hs.42146 isoform Q494Z4 Pax3d
(TrEMBL) Pax3, NM_181459.3 Q494Z3, Hs.42146 isoform Q494Z4 Pax3e
(TrEMBL) Pax3, NM_181461.3 Q494Z3, Hs.42146 isoform Q494Z4 Pax3g
(TrEMBL) Pax3, NM_181460.3 Q494Z3, Hs.42146 isoform Q494Z4 Pax3h
(TrEMBL) Pax3, NM_001127366.2 Q494Z4 Hs.42146 isoform Pax3i
(TrEMBL) PAX7, NM_002584.2 P23759 Hs.113253 isoform 1 PAX7,
NM_013945.2 P23759 Hs.113253 isoform 2 PAX7, NM_001135254.1 P23759
Hs.113253 isoform 3
[0114] In some embodiments, the reprogramming agent is a
polypeptide which shares at least 75%, 80%, 85%, 90%, 95%, 97%, 99%
or more of the functionality or sequence identity of any one of the
reprogramming agents in the table hereinbefore.
[0115] Identifying the "sufficient period of time" to allow stable
expression of the at least one gene regulator in absence of the
reprogramming agent and the "sufficient period of time" in which
the cell is to be maintained in culture conditions supporting the
transformation of the desired cell is within the skill of those in
the art. The sufficient or proper time period will vary according
to various factors, including but not limited to, the particular
type and epigenetic status of cells (e.g. the cell of the first
type and the desired cell), the amount of starting material (e.g.
the number of cells to be transformed), the amount and type of
reprogramming agent(s), the gene regulator(s), the culture
conditions, presence of compounds that speed up reprogramming (ex,
compounds that increase cell cycle turnover, modify the epigenetic
status, and/or enhance cell viability), etc. In various embodiments
the sufficient period of time to allow a stable expression of the
at least one gene regulator in absence of the reprogramming agent
is about 1 day, about 2-4 days, about 4-7 days, about 1-2 weeks,
about 2-3 weeks or about 3-4 weeks. In various embodiments the
sufficient period of time in which the cells are to be maintained
in culture conditions supporting the transformation of the desired
cell and allow a stable expression of a plurality of secondary
genes is about 1 day, about 2-4 days, about 4-7 days, or about 1-2
weeks, about 2-3 weeks, about 3-4 weeks, about 4-6 weeks or about
6-8 weeks. In preferred embodiments, at the end of the
transformation period, the number of transformed desired cells is
substantially equivalent or even higher than an amount of cells a
first type provided at the beginning.
[0116] The present invention encompasses various types of compounds
that are suitable for increasing in a cell of a first type the
intracellular levels of at least one reprogramming agent,
Preferably, the compound should also be able to directly or
indirectly remodel the chromatin and/or DNA of the cell, thus
resulting directly or indirectly in the expression of morphological
and functional characteristics of the desired cell of a different
type. Preferred compounds are reprogramming agents as defined
herein or any other compound having a similar activity and having
the ability to activate or enhance the expression of the endogenous
version of genes listed in the table of reprogramming agents
hereinbefore and which are capable of driving directly or
indirectly transformation of the cell of the first type into the
desired cell of a different type.
[0117] As will be explained hereinafter, the increase in
intracellular levels of the at least one reprogramming agent can be
achieved by different means. In preferred embodiments the
reprogramming agent is a polypeptide and increasing intracellular
levels of such polypeptide include transfection (or
co-transferction) of an expression vector having a polynucleotide
(ex. DNA or RNA) encoding the polypeptide(s), or by an
intracellular delivery of polypeptide(s). According to the
invention, transient expression is generally preferable. Additional
suitable compounds may include compounds capable of increasing the
expression of the endogenous version of genes listed in the table
of reprogramming agents and gene regulators including, but not
limited to, reprogramming factors listed in Table B.
TABLE-US-00002 TABLE B Desired cell type Reprogramming Factor
Pluripotent-like Cells Y27632, Butyric acid, Hydrocortisone, Sodium
Selenite, Insulin, TGF.beta.1 (R&D), NODAL, BMP4,
Wnt/B-Catenin, Pluripotin, Glucogen synthetase kinase-3 inhibitor,
CHIR99021, PD0325901, 6- Bromoindirubib-3-oxime (BIO), KSOR,
vitamin A, histone deacetylase inhibitor (HDAC), RG 108,
R(+)BayK8644, SB431542, thiazovivin, BIX01294, Fibroblast growth
factor 2 (FGF2), Activin A, Wnt3a, L-Ascorbic Acid, Cyclic
Pifithrin-.alpha., Tranylcypromine hydrochloride, Kenpaullone,
5-Azacytidine, Valproic Acid (VPA), Theanine. Ectoderm-like Cells a
retinoid compound (ex. ATRA), L-Ascorbic acid, sonic hedgehog
(SHH), Wnt 3a, a neurotrophic factor, FGF2, Epidermal growth factor
(EGF), Transforming growth factor (TGF) alpha, Estrogen, Noggin, 5-
Azacytidine, VPA, BIX01294, R(+)BayK8644, RG108, Butyric acid,
Lithium, Cytosine arabinoside, Chordin, b-Catenin, CHIR99021,
SB431542, rapamycin (mTOR). Mesendoderm-like Cells BIO,
Dorsomorphin, vitamin E, VPA, HAS, Bone morphogenetic protein 4
(BMP4), EGF, FGF2, CHIR99021, Activin A, Insulin-like growth factor
1 (IGF-1), SB431542, PD0325901, Butyric acid, Epidermal growth
factor-Cripto/FRL-1/Cryptic (EGF- CFC) and the TGF.beta.s, Nodal,
SHH, Vg1/GDF1 (growth and differentiation factor-1). Neural
Stem-like cells a retinoid compound (ex. ATRA), a neurotrophic
factor, L-Ascorbic acid, Estrogen, 5-Azacytidine, VPA, BIX01294,
R(+)BayK8644, RG108, Butyric acid, Lithium, Activin A, Noggin, EGF,
FGF2, Wnt3a, TGF, cAMP, Follistatin, SHH, BDNF, IGF-1, CNTF, PDFG,
SDNSF/MCFD2, Neuropeptide Y, Forskolin. Cardiac Stem-like cells
Wnt11, Cardiogenol C, BMP4, FGF2, Activin A, VEGF, DKK1 (dickkopf
homologue 1), Insulin-like growth factor 1 (IGF-1), Oxytocin,
Cardiotropin, Hepatocyte growth factor (HGF), 5-Azacytidine, L-
3,3',5-triiodothyronine, Valproic Acid, BIX01294, R(+)BayK8644,
RG108, Cardiogenol C hydrochloride, Butyric acid, Stem Cell factor.
Pancreatic Progenitor-like Cells Activin A, GLP-1, FGF2, Reg1,
nicotinamide, Betacellulin, SHH, (-)-Indolactam V, a retinoid
compound, Cyclopamine, IDE-1 and 2, 5-Azacytidine, Valproic Acid,
BIX01294, R(+)BayK8644, RG108, Butyric acid. Hematopoietic
Stem-like Cells SCGM, Thrombopoietin, Interleukin-3, Stem Cell
factor (SCF), GM-CSF, Interleukin-6, SHH, Wnt5a, Insulin-like
Growth factor, Angiopoietin factor 2 or 3, butyric acid,
5-azacytidine, estrogen. Myogenic Stem-like Cells HGF, FGF2, IGF,
BMP-2, TGF.beta., TGF.beta.3, Wnt3a, Wnt11, 5-Azacytidine, Valproic
Acid, BIX01294, R(+)BayK8644, RG108, Trichostatin A (TSA), retinoic
acid, Lithium, Butyric acid.
[0118] According to the principles of the invention, increasing
intracellular levels of at least one reprogramming agent should
induce a direct or indirect endogenous expression of at least one
gene regulator. As used herein, "gene regulator" refers to a
polynucleotide or polypeptide whose expression is associated with a
series of intracellular events leading to the transformation of a
given cell of a first type into a pluripotent, multipotent and/or
unipotent cell Typically expression of a gene regulator directly or
indirectly activates genes necessary for the phenotypical and
functional characteristic of pluripotent, multipotent and/or
unipotent cells, while repressing genes of the cell of a first
type. The gene regulator may be the same or be different than the
reprogramming agent. Examples of gene regulators according to the
invention include, but are not limited to, the polynucleotides and
polypeptides listed herein before in TABLE A.
[0119] In some embodiments, the gene regulator is a polypeptide
which shares at least 75%, 80% 85%, 90%, 95%, 97%, 99% or more of
the functionality or sequence identity of any one of the gene
regulators provided in the Table A hereinbefore.
[0120] As used herein, "conditions supporting growth" or
"conditions supporting the transformation" when referring to a
desired cell refers to various suitable, culture conditions
(temperature, pH, O.sub.2 tension, osmolarity, cell media, factors,
compounds, growth substrate (ex. laminin, collagen, fibronectin,
Matrigel.TM., low-bind surface, nanostructured or charged surface,
etc.), 3D environment, etc.) favoring growth of the desired cell
type and/or favoring transformation towards such desired cell type.
Those skilled in the art know that growth or transformation of
particular cell types is stimulated under specific conditions,
while inhibited by others, and it is within their skill to select
suitable conditions (e.g. culture conditions) favoring growth or
transformation of desired cell types.
[0121] The terms "phenotypical and functional properties", when,
referring to a desired cell or to an embryonic stem cell, means the
biological, biochemical, physiological and visual characteristics
of a cell, including expression of certain genes and cell surface
markers, which can be measured or assessed for confirming its
identity or function(s).
[0122] An example of a suitable reprogramming agent according to
preferred embodiments of the invention is MUSASHI1. In some
embodiments this polypeptide is preferred for driving a first cell,
such as a fibroblast, into a Neural Stem-Like Cell (NSLC). In other
embodiments, the at least one reprogramming agent which said
intracellular levels is increased is (are) either Musashi1 (Msi1)
alone; Musashi1 (Msi1) and Neurogenin 2 (Ngn2); Musashi1 (Msi1))
and methyl-CpG binding domain protein 2 (MBD2); or Neurogenin 2
(Ngn2) and methyl-CpG binding domain protein 2 (MBD2). Adequate
intracellular levels of these polypeptides are preferred since they
tend to be expressed throughout an entire cell lineage, from as
early as embryonic stem cells (or even earlier) to pre-somatic
cells (or even later).
[0123] MBD2 is a member of a family of methyl-CpG-binding proteins
that has been reported to be both a transcriptional repressor and a
DNA demethylase (dMTase). As used herein, the term "MBD2" generally
refers to the human methyl-CpG binding domain protein 2. The
GeneBank.TM. (NCBI) accession number of human MBD2 is
NM.sub.--003927.3/AF072242, the UniProt.TM. accession number is
NP-003918/Q9UBB5 and the UniGene.TM. accession number is
Hs.25674.
[0124] As used herein, the term "Msi1" generally refers to the
human musashi homolog 1. The GeneBank.TM. (NCBI) accession number
of human Msi1 is NM.sub.--002442.2/AB012851, the UniProt.TM.
accession number is NP-002433/043347 and the UniGene.TM. accession
number is Hs.158311.
[0125] As used herein, the term "Ngn2" generally refers to the
human neurogenin 2, The GeneBank.TM. (NCBI) accession number of
human Ngn2 is NM.sub.--024019.2/BC036847, the UniProt.TM. accession
number is NP-076924/Q9H2A3 and the UniGene.TM. accession number is
Hs.567563.
[0126] According to additional aspects, the method of transforming
a cell of a first type to a desired cell of a different type
comprises the steps of either: [0127] 1) contacting the cell of a
first type with one or more compounds capable of increasing
intracellular levels of at least one reprogramming agent within the
cell and directly or indirectly remodeling the chromatin and/or DNA
of the cell; or [0128] 2) contacting the chromatin and/or DNA of a
cell of a first type with an agent capable of remodeling the
chromatin and/or DNA of the cell; and increasing intracellular
levels of at least one reprogramming agent.
[0129] According to various embodiments, step 2) may be carried out
consecutively to step 1), simultaneously with step 1), or before
step 1).
[0130] According to a particular aspect, the invention relates to a
method for obtaining a particular stem-like cell, comprising:
[0131] providing a cell of a first type; [0132] contacting
chromatin and/or DNA of a cell of a first type with a histone
acetylator, an inhibitor of histone deacetylation, a DNA
demethylator, and/or a chemical inhibitor of DNA methylation; and
[0133] increasing intracellular levels of at least one gene
regulator for that particular stem-like cells, wherein the gene
regulator is capable of driving directly or indirectly
transformation of the cell of the first type into the particular
stem-like cell.
[0134] According to a particular aspect, the invention relates to a
method for obtaining a Neural Stem-Like Cell (NSLC), comprising:
[0135] providing a cell of a first type which is not a NSLC; [0136]
increasing intracellular levels of at least one neural stem cell
specific polypeptide, [0137] wherein the polypeptide is capable of
driving directly or indirectly transformation of the cell of the
first type into a NSLC; and [0138] contacting chromatin and/or DNA
of a cell of a first type with a histone acetylator, an inhibitor
of histone deacetylation, a DNA demethylator, and/or a chemical
inhibitor of DNA methylation.
[0139] With respect to the second step, the term "remodelling the
chromatin and/or DNA" refers to dynamic structural changes to the
chromatin. These changes can range from local changes necessary for
transcriptional regulation, to global changes necessary for opening
up the chromatin structure or chromosome segregation to allow
transcription of the new set of genes characteristic of the desired
cell of a different type, to closing up of the chromatin structure
or chromosome segregation to prevent transcription of certain genes
that are not characteristic of the desired cell of a different
type. In some embodiments, opening up of the chromatin structure
refers more specifically to acetylation of histones, and
demethylation of DNA, while closing up of the chromatin structure
refers more, specifically to deacetylation of histones, and
methylation of DNA.
[0140] As used herein, "compound" refers to a compound capable of
effecting a desired biological function. The term includes, but is
not limited to, DNA, RNA, protein, polypeptides, and other
compounds including growth factors, cytokines, hormones or small
molecules. As used herein, compounds capable of remodeling
chromatin and/or DNA include, but are not limited to, histone
acetylators, inhibitors of histone deacetylation, DNA
demethylators, inhibitors of DNA methylation and combination
thereof.
[0141] "Inhibitor of DNA methylation" refers to an agent that can
inhibit DNA methylation. DNA methylation inhibitors have
demonstrated the ability to restore suppressed gene expression.
Suitable agents for inhibiting DNA methylation include, but are not
limited to 5-azacytidine, 5-aza-2-deoxycytidine,
1-.beta.-D-arabinofuranosil-5-azacytosine, and
dihydro-5-azacytidine, and zebularine (ZEB), BIX (histone lysine
methyltransferase inhibitor), and RG108.
[0142] "Inhibitor of histone deacetylation" refers to an agent that
prevents the removal of the acetyl groups from the lysine residues
of histones that would otherwise lead to the formation of a
condensed and transcriptionally silenced chromatin. Histone
deacetylase inhibitors fall into several groups, including: (1)
hydroxamic acids such as trichostatin (A), (2) cyclic
tetrapeptides, (3) benzamides, (4) electrophilic ketones, and (5)
aliphatic acid group of compounds such as phenylbutyrate and
valporic acid. Suitable agents to inhibit histone deacetylation
include, but are not limited to, valporic acid (VPA),
phenylbutyrate Trichostatin A (TSA), Na-butyrate, and benzamides.
VPA promotes neuronal fate and inhibits glial fate simultaneously
through the induction of neurogenic transcription factors including
NeuroD.
[0143] "Histone Acetylator" refers to an agent that inserts acetyl
groups to the lysine residues of histones that opens up the
chromatin and turns it into a transcriptionally active state.
Suitable Histone Acetylator agents include, but are not limited to,
Polyamine, CREB (cAMP element binding protein), and BniP3.
[0144] "DNA demethylator" refers to an agent that removes the
methyl groups from DNA and possesses the ability to inhibit
hypermethylation and restore suppressed gene expression. A
demethylase is expected to activate genes by removing the
repressive methyl residues. Suitable DNA demethylators include, but
are not limited to, MBD2 and Gadd45b.
[0145] In some embodiments, the reprogramming agent has one or more
of the following functions: it decrease the expression of one or
more markers of cells of the first type (ex. see Table C), and/or
increase the expression of one or more markers of the desired cell
of the different type (ex. see Table A). Cells that exhibit a
selectable marker for the desired cell of a different type are then
selected and assessed for characteristics of the desired cell of a
different type.
[0146] According to the invention, transformation into the desired
cell results in stable expression of a plurality of secondary genes
whose expression is characteristic of phenotypical and/or
functional properties of the desired cell. Genes whose expression
is characteristic of phenotypical and/or functional properties of
the desired cell include, but is not limited to, those listed in
Table A.
[0147] In some embodiments, expression of secondary genes whose
expression is characteristic of phenotypical and functional
properties of the desired cell results in the expression of markers
defined according to Table A or the following table:
TABLE-US-00003 Desired cell type Markers Neural stem-like cells
Nestin, Sox2, GFAP, Msi1 Neural-like cells .beta.III-tubulin,
Map2b, Synapsin, ACHE Ectoderm-like cells Sox2, Sox1, Zic1, Nestin,
Notch 1, FoxJ3, Otx2, Cripto1, Vimentin Mesendoderm-like cells
Sox17, FoxA2, CXCR4, GATA4, MixI1, Eomesodermin Pluripotent-like
cells Oct4, SSEA4, TRA-1-60, TRA-1-81, AP
[0148] In some embodiments, transformation of a cell of a first
type into the desired cell results in a stable repression of a
plurality of genes typically expressed in the cell of the first
type but not in the desired cell of the different type. Examples of
such suppressed genes include, but are not limited to, those
defined in Table C:
TABLE-US-00004 TABLE C Examples of suppressed genes Cell-type
specific genes typically repressed during Reprogramming RefSeq/
UniProt .TM./ GenBank .TM. Swiss-Prot UniGene .TM. (NCBI) Accession
Accession Accession Cell Type Name No No No Markers Keratinocytes
TP63, NM_003722.4 Q9H3D4 Hs.137569 Keratin 14 isoform 1 TP63,
NM_001114978.1 Q9H3D4 Hs.137569 Basonuclin isoform 2 TP63,
NM_001114979.1 Q9H3D4 Hs.137569 P63 isoform 3 TP63, NM_001114980.1
Q9H3D4 Hs.137569 isoform 4 TP63, NM_001114981.1 Q9H3D4 Hs.137569
isoform 5 TP63, NM_001114982.1 Q9H3D4 Hs.137569 isoform 6 BNC1
NM_001717.3 Q01954 Hs.459153 BCN2 NM_017637.5 Q6ZN30 Hs.656581
KRT14 NM_000526.4 P02533 Hs.654380 Involucrin NM_005547.2 P07476
Hs.516439 Fibroblasts THY1 NM_006288.3 P04216 Hs.724411 CD90 FBN2
NM_001999.3 P35556 Hs.519294 Fibrillin 2 FN1 NM_212482.1 P02751
Hs.203717 Fibronectin VIM NM_003380.3 P08670 Hs.455493 Vimentin
COL5A2 NM_000393.3 P05997 Hs.445827 Collagen 5a2 DNMT1,
NM_001130823.1 P26358 Hs.202672 FSP1 isoform a DNMT1, NM_001379.2
P26358 Hs.202672 isoform b Mesenchymal FN1 NM_212482.1 P02751
Hs.203717 Fibronectin stem cells MCAM NM_006500.2 P43121 Hs.599039
STRO-1 THY1 NM_006288.3 P04216 Hs.724411 CD90 VIM NM_003380.3
P08670 Hs.455493 Vimentin CD34+ Isl1 NM_002202.2 P61371 Hs.505
VEGFR HOXA9 NM_152739.3 P31269 Hs.659350 Cytokeratin HOXB4
NM_0024015.4 P17483 Hs.664706 CD34 Klk-1 NM_002257.2 P06870
Hs.123107 Bry NM_003181.2 O15178 Hs.389457 Adipose- ALCAM
NM_001627.2 Q13740 Hs.591293 ALBO derived stem VCAM-1 NM_001078.2
P19320 Hs.109225 Adiponectin cells (ADSC) VCAM-1, NM_080682.1
P19320 Hs.109225 Leptin isoform b PROM1, NM_006017.2 O43490
Hs.614734 isoform 1 PROM1, NM_001145847.1 O43490 Hs.614734 isoform
2 NM_001145848.1 PROM1, NM_001145852.1 O43490 Hs.614734 isoform 4
PROM1, NM_001145851.1 O43490 Hs.614734 isoform 5 PROM1,
NM_001145850.1 O43490 Hs.614734 isoform 6 PROM1, NM_001145849.1
O43490 Hs.614734 isoform 7 FUT4 NM_002033.3 P22083 Hs.390420
[0149] In preferred embodiments, stable repression of any one or
more of the genes listed in Table C being expressed in the first
cell type is also characterized by a disappearance of the
corresponding markers (see Table C).
[0150] Those skilled in the art will understand that there exist
many alternative steps for facilitating cell reprogramming.
Additional steps for facilitating cell reprogramming include
destabilizing the cell's cytoskeletal structure (for example, by
exposing the cell to cytochalasin B), loosening the chromatin
structure of the cell (for example, by using agents such as 5
azacytidine (5-Aza) and Valproic acid (VPA) or DNA demethylator
agents such as MBD2), transfecting the cell with one or more
expression vector(s) containing at least one cDNA encoding a
neurogenic transcription factor (for example, Msi1 or Ngn2), using
an appropriate medium for the desired cell of a different type and
an appropriate differentiation medium to induce differentiation
commitment of the desired cell of a different type, inhibiting
repressive pathways that negatively affects induction into
commitment into the desired cell of a different type, growing the
cells on an appropriate substrate for the desired cell of a
different type (for example, laminin for NSLCs or a low-bind
surface for culturing floating neurospheres), and growing the cells
in an environment that the desired cell of a different type (or
"-like" cell) would be normally exposed to in viva such as the
proper temperature, pH and low oxygen environment (for example
about 2-5% O.sub.2). In various embodiments, the invention
encompasses these and other related methods and techniques for
facilitating cell reprogramming.
[0151] Accordingly, the method of transforming a cell of a first
type into a cell of a second different type may comprise additional
facultative steps. In one embodiment, the method of transforming a
cell further comprises the step of pretreating the cell of a first
type with a cytoskeleton disruptor. As used herein "cytoskeleton"
refers to the filamentous network of F-actin, Myosin light and
heavy chain, microtubules, and intermediate filaments (IFs)
composed of one of three chemically distinct subunits, actin,
tubulin, or one of several classes of IF protein, as well as the
cell's cytoskeletal structure including the cell membrane and
portions of the cell cytoplasm and integrated organelles and
components. This also refers to other structures and molecules that
are directly or indirectly part of or affected by the cytoskeleton.
Accordingly, the term "cytoskeleton disruptor" refers to any
molecule, compound or process that can inhibit, disrupt, remove or
affect the cell cytoskeleton to destabilize the cell and
consequently remove or change the feedback mechanisms between the
cell's shape and cellular and nuclear function (thus removing or
changing the cell's memory). Suitable cytoskeleton disruptors
according to the invention include, but are not limited to, the
cytochalasin family of actin cytoskeleton inhibitors, such as
Cytochalasin B or D, microtubule inhibitors such as colchicine, and
myosin inhibitors such as 2,3-butanedione monoxime; physical
effects include freezing, low osmolarity of the medium resulting in
osmotic build up within the cell and bursting and disruption (or
removal) of cell membrane and cell cytoskeleton, and other forms of
removal of the cell's physical memory. Such pretreatment may boost
reprogramming. In a preferred embodiment, the cell is cultured in
the presence of at least one cytoskeleton inhibitor one day before,
during, or after introducing a neurogenic transcription
factor(s).
[0152] Placing the cell in conditions for supporting the
transformation of the desired cell, and/or maintaining the cell in
culture conditions supporting the transformation of the desired
cell may comprise culturing the cell in a media comprising one or
more factors appropriate for inducing the expression of the
morphological and functional characteristics of the desired cell of
a different type. In some embodiments the one or more factors are
reprogramming factors helpful in reprogramming a cell and these
reprogramming factors can be used alone or in combinations.
[0153] In other embodiments, the step of culturing the cell in a
media comprising one or more factors appropriate for inducing the
expression of the morphological and functional characteristics of
the desired cell of a different type is carried out subsequently or
simultaneously to steps iii) or iv), or subsequently or
simultaneously to steps 1) or 2), as defined hereinbefore.
[0154] Those skilled in the art know many different types of media
and many reprogramming factors that may be helpful in reprogramming
a cell and these reprogramming factors can be used alone or in
combinations. In various embodiments, the reprogramming factor is
selected according to TABLE B.
[0155] In some embodiments, reprogramming factors have one or more
of the following functions: decrease the expression of one or more
markers of the first type of cell and/or increase the expression of
one or more markers of the desired cell. Cells that exhibit a
selectable marker for the desired cell are then selected and
assessed for unipotency, multipotency, pluripotency, or similar
characteristics (as appropriate).
[0156] In particular embodiments, the cells are cultured in
serum-free medium before, during or after any one of steps i) to
iv) as defined hereinbefore, or during or after steps 1) or 2), as
defined hereinbefore.
[0157] In some embodiments, Mesendoderm-like cells can be created
from cells such as ADSCs by up-regulating the expression of FoxD3,
MixI1, Ngn3 and MBD2. Additionally up-regulating the expression of
Oct4, Sox17, Brachyury, and/or FoxA2 can be used to create
Mesendoderm-like cells. In addition, media compositions detailed in
Table 37 can be used to create mesendoderm-like cells from
adipocyte-derived stem cells (ADSCs) and similar cells, along with
the use of proper cell substrates for mesendoderm cells known in
the art (e.g., gelatin-coated plates).
[0158] In some embodiments, cells such as ADSCs can be reprogrammed
into pancreatic progenitor-like cells and .beta. islet-like cells
by up-regulating the expression of Sox17, Pdx1, Ngn3, and Oct4
(e.g., Oct4+Sox17+Pdx1+Ngn3; Sox17+Pdx1+Ngn3; Oct4+Pdx1+Ngn3).
Additionally up-regulating the expression of FoxA2 and MBD2 can be
used to create these cells. In addition, media compositions
detailed in Table 39 can be used to create pancreatic
progenitor-like cells and .beta. islet-like cells from
adipocyte-derived stem cells (ADSCs) and similar cells, along with
the use of proper cell substrates for growing pancreatic progenitor
cells and .beta. islet-like cells known in the art (e.g.,
fibronectin-coated collagen gels).
[0159] In some embodiments, cells such as ADSCs or mesenchymal stem
cells (MSCs) can be reprogrammed into cardiac stem-like cells and
mesoderm-like cells by up-regulating the expression of a
combination of Mesp1, FoxD3, Tbx5, Brachyury (T), Nkx2.5, Sox17
and/or Gata4 (e.g., Foxd3+Sox17+Mesp1+Tbx5;
Foxd3+Sox17+Mesp1+Nkx2.5; Foxd3+T+Mesp1+Gata4, T+Mesp1+Nkx2.5,
T+Mesp1+Tbx5), which increase the expression of mesoderm and
cardiac stem cell markers. Additionally up-regulating the
expression of Gata6 and Baf60c can be used to create these cells.
In addition, media compositions detailed in Table 44 can be used to
create cardiac stem-like cells from adipocyte-derived stem cells
(ADSCs) or mesenchymal stem cells (MSCs) and similar cells, along
with the use of proper cell substrates for growing cardiac stem
cells known in the art (e.g., matrigel, gelatin, or laminin). In
some embodiments, cells such as ADSCs can be reprogrammed into
pluripotent-tike stem cells up-regulating the expression of Rex1,
Oct4 and Klf4, or SalI4, Oct4, Klf4 and Nanog. Reprogramming by
transient transfection of a combination of the above genes can be
achieved efficiently. These cells can be differentiated into
ectoderm-like cells, endoderm-like cells, or mesendoderm-like cells
by methods known in the art for differentiating pluripotent stem
cells into these lineages. In addition, these pluripotent-like stem
cells have protective and/or therapeutic/regenerative effect on
other cells (e.g., hepatocytes).
[0160] The NSLCs that is a subject of this invention have benefits
over native human neural stem/progenitor cells as well as embryonic
stem cells. The NSLCs do not readily form tumors or teratomas (as
tested in NOD-SCID mice), can be created from easily obtainable
somatic cells (e.g., fibroblasts, CD34+ blood cells, keratinocytes)
and differentiated towards any specific neuronal lineage, express
neurotrophic growth factors (e.g., BDNF and GDNF), express some
neuronal differentiation genes while maintaining a stem cell like
state allowing a higher proportion of neuronal differentiation when
placed in differentiation conditions (compared to native human
neural stem/progenitor cells), form functional gap junctions and
readily form synapses, and are capable of attaching and surviving
on 3D scaffolds and environments.
[0161] The stem-like cells of the present invention have numerous
advantages over the prior art such as efficient reprogramming
without gene integration or constant artificial forced gene
expression, greater potency and safety over native cells (esp.
stem/progenitor cells), and having the capability to be the cell of
interest for a particular application (e.g., a neural stem-like
cell for CNS applications; a cardiac stem-like cell for cardiac
applications, a pancreatic progenitor-like cells or .beta.
islet-like cells for diabetes, an ectoderm-like cell, a
mesoderm-like cells, an endoderm-like cell, etc.). In addition
derived cells of the invention can be a relatively homogeneous
population of autologous cells of a particular phenotype of
interest.
[0162] The methods of the present invention allow the ability to
create the native types of stem cells for particular applications
(e.g., neural stem-like cells for CNS applications; cardiac
stem-like cells for cardiac applications, pancreatic
progenitor-like cells or .beta.-like cells for diabetes, etc.) as
well as creating autologous (from the patient's own cells) versions
of these stem cells that allow them to graft more appropriately
when delivered to the patient as a treatment or for augmenting the
health or functionality of a particular tissue or organ, or for
diagnostic purposes. The cells can also be used for modeling (e.g.,
disease modeling, or testing the effects of particular compounds or
other molecules (e.g., for personalized medicine purposes)). The
cells can also easily be enhanced by specific genes of interest or
a defective gene repaired/replaced before delivery to the patient
for treatment of a genetic disorder or for enhanced therapeutic
value or augmenting the health or functionality of a particular
cell, tissue, organ, system, or organism.
Obtaining Neural Stem-Like Cells (NSLCs)
[0163] According to preferred embodiments for creating Neural
Stem-Like Cells (NSLCs), the methods of the invention are carried
out such that cells are treated with selected agents, compounds and
factors to promote the reprogramming and/or dedifferentiation
towards Stem-Like Cells (SLCs). Such reprogrammed somatic cells can
then be further treated with agents and/or cultured under
conditions suitable for promoting reprogramming towards Neural
Stem-Like Cells (NSLCs), and expansion of the NSLCs for the
long-term. NSLCs according to the invention have the potential to
differentiate to neuronal-like and/or glial-like cells, as well as
neuronal and/or glial cells, for potential treatment of
neurological diseases and injuries such as Parkinson's disease and
spinal cord injury. The methods described herein are also useful
for producing histocompatible cells for cell therapy.
[0164] Accordingly, some aspects of the present invention relates
to generating neurons from an individual patient, thus making
autologous transplantations possible as a treatment modality for
many neurological conditions including neurotrauma, stroke,
neurodegenerative diseases such as Multiple Sclerosis, Parkinson's
disease, Huntington disease, Alzheimer's diseases. Thus, the
invention provides for neurological therapies to treat the disease
or trauma of interest.
[0165] Therefore, another aspect of the invention concerns a method
of obtaining a Neural Stem-Like Cell (NSLC), comprising either:
[0166] 1) contacting the cell of a first type with one or more
neural stem cell regulating polypeptide capable of increasing
intracellular levels of neural stem cell specific polypeptides
within said cell and directly or indirectly remodeling the
chromatin and/or DNA of the cell and driving directly or indirectly
transformation of the cell of the first type into a NSLC; or [0167]
2) contacting the chromatin and/or DNA of a cell of a first type
with a histone acetylator, an inhibitor of histone deacetylation, a
DNA demethylator, and/or an inhibitor of DNA methylation; and
increasing intracellular levels of at least one neural stem cell
specific polypeptide driving directly or indirectly transformation
of the cell of the first type into a NSLC.
[0168] In preferred embodiments, the step 1) comprises increasing
intracellular levels of a MUSASHI1 polypeptide. As it will be
explained hereinafter this can be achieved by different means
including, but not limited to, transient expression of the MUSASHI1
polypeptide, preferably by transfecting an expression vector
encoding the polypeptide.
[0169] In preferred embodiments, the step 2) comprises increasing
intracellular levels of a MBD2 polypeptide or treating the cells
with VPA and 5-AZA. As it will be explained hereinafter this can be
achieved by different means including, but not limited to,
transient expression of the MBD2 polypeptide, preferably by
transfecting an expression vector encoding the polypeptide(s),
and/or pre-treating and/or treating the cells with VPA and
5-AZA.
[0170] In one particular embodiment, reprogramming a cell of a
first type to another type of cell that exhibits at least two
selectable markers for neural stem cells requires transfecting the
cell of a first type with one vector containing a cDNA encoding for
a neurogenic transcription factor and one DNA demethylator. To
enhance the de-differentiation the cells are exposed or pre-exposed
to an agent(s) that inhibits DNA methylation, inhibits histone
deacetylation, and/or disrupts the cell cytoskeleton. For example,
the dedifferentiation can be enhanced by pre treating the cells
with an agent that disrupts the cell cytoskeleton followed by
transfecting the cells with one or more vector(s) containing two
neurogenic transcription factors in the presence of a DNA
demethylator and/or inhibitor of DNA methylation and histone
deacetylation. The histone deacetylator, inhibitor of histone
deacetylation, DNA demethylator, and/or an inhibitor of DNA
methylation are as defined previously.
[0171] As defined previously, the method may further comprise a
preliminary step of pre-treating the cell of a first type with a
cytoskeleton disruptor, as defined previously, and/or culturing the
cell in a media comprising one or more reprogramming factors
appropriate for appearance and maintenance of the morphological and
functional characteristics of NSLCs as defined previously (e.g. a
retinoid compound, a neurotrophic factor, bFGF, EGF, SHH, Wnt 3a,
neuropeptide Y, Estrogen). In some embodiment the method further
comprises inhibiting cellular BMP signaling pathways (e.g. by
NOGGIN, fetuin, or follistatin).
[0172] In preferred embodiments, generation of a NSLC from a first
cell comprises the use of one or more reprogramming agents.
Suitable agents include, but are not limited to, Musashi-1 (Msi1)
and Neurogenin 2 (Ngn2). Other potential agents are listed in Table
A and B.
[0173] The present invention is also directed to the use of DNA
expression vectors encoding a protein or transcript which
upregulates the expression of neurogenesis. The
genetically-engineered DNA sequence, encoding a defined
reprogramming agent such as Msi1 and Ngn2, can be introduced into
cells by using a mono-, bi-, or poly-cistronic vectors. The
expression of an endogenous multipotency gene indicates that the
cDNA encodes a protein whose expression in the cell result directly
or indirectly in the de-differentiation of the cell. The newly
de-differentiated mammalian cells are capable of re-differentiating
to neuronal lineages to regenerate said mammalian cells, tissues,
and organs.
[0174] The present invention is further directed to a method for
generating NSLCs by introducing a genetically-engineered DNA
sequence into human somatic cells via transient transfection. Since
the DNA introduced in the transfection process is not inserted,
into the nuclear genome, the foreign DNA decreases over time and
when the cells undergo mitosis. Nonviral vectors remain in a
non-replicative form, have low immunogenicity, and are easy and
safe to prepare and to use. Furthermore, plasmids may accommodate
large fragments of DNA.
[0175] In one particular embodiment, the method starts with
obtaining cells from the individual, and reprogramming the cells in
vitro to generate NSLCs. The significant aspect of the present
invention is the stable reprogramming of a somatic cell or
non-neuronal cell into a NSLC that can give rise to different types
of, neuronal or glial cells (including neuronal-like or glial-like
cells), as well as neural crest derived cells such as
cardiomyocytes. These can then be implanted back into the same
patient from which the cells were obtained, thus making an
autologous treatment modality for many neurological conditions
including neurotrauma, stroke, and neurodegenerative disease
possible, as well as cardiac conditions such as myocardial
infarction. These can also be implanted into a different individual
from which the cells were obtained. Accordingly, the cells and
methods of the present invention may be helpful to treat, prevent,
or to stabilize a neurological disease such as Alzheimer's disease,
Parkinson's disease, multiple sclerosis, or spinal cord injury, or
a cardiac disease such as myocardial infarction. This technology
provides an ample source of neural stem cells, neuro-progenitor
cells, neurons and glia, as well as neural crest derived cells, for
clinical, treatment, which can be performed by implantation of
NSLCs in vivo or inducing the differentiation in vitro and
implantation of neuro-progenitor cells or specific neurons or glia
in vivo.
[0176] In another embodiment, the method comprises isolating
somatic or non-neuronal cells and exposing the cells to one or more
agents that alter cell morphology and chromatin structure, and
transfecting the cells with one or more genes containing at least
one cDNA encoding for a neurogenic transcription factor. The gene
transfection step may be replaced with alternative agents that
induce the expression of the neurogenic transcription factor(s) in
the cell. Inducing epigenetic modifications to DNA and histones
(especially DNA demethylation and an open chromatic structure)
facilitate true reprogramming of the cells. In another embodiment,
the cells are incubated in a low oxygen environment, for example 5%
O.sub.2, thereby helping in reprogramming the cells.
[0177] This methodology allows the reprogramming of a cell into a
NSLC. The further course of development and the expansion of the
reprogrammed cell depend on the in situ environment cues to which
it is exposed. The embodiments of the invention further include
growing the reprogrammed cell in an appropriate proliferation
medium to expand the generated NSLC, for example Neural Progenitor
proliferation Medium (StemCell Technologies) with the presence of
epidermal growth factor (EGF) and basic fibroblast growth factor
(bFGF), to promote the neural stem cell to proliferate.
[0178] The NSLCs obtained according to the invention can be
differentiated into neuronal, astrocyte, and/or oligodendrocyte
lineages in appropriate differentiation medium, for example NS-A
differentiation medium (StemCell, Technologies) or NbActive medium
(BrainBits.TM.) including a retinoid compound, such as
all-trans-retinoic acid or vitamin A, and BDNF, to induce the
differentiation of NSLCs towards neuronal and/or glial cells.
Neuronal cells include cells that display one or more
neural-specific morphological, physiological, functional and/or
immunological features associated with a neuronal cell type. Useful
criteria features includes: morphological features (e.g., long
processes or neurites), physiological and/or immunological features
such as expression of a set of neuronal-specific markers or
antigens, synthesis of neurotransmitter(s) such as dopamine or
gamma aminobutyric acid (GABA), and functional features such as ion
channels or action potentials characteristic of neurons.
[0179] In accordance with the method, reprogrammed cells can be
selected based on differential adherence properties as compared to
untransfected cells; for example, reprogrammed cells can form
floating neurospheres or grow well on laminin while untransfected
fibroblasts attach and grow well on regular cell culture treated
plates. Reprogrammed cells include cells that exhibit one or more
neural stem specific markers and morphology and the loss of some or
all of the specific markers related to the original cells.
Furthermore, some of the functionality of the neural-like cells
(NLCs) can be assessed at different time points by, for example,
patch-clamping, immunostaining for synaptophysin and MAP2b, and by
immunochemical means such as by enzyme-linked immunosorbent assay
(ELISA).
[0180] In certain embodiments, the present invention provides NSLCs
that are able to initiate and direct central nervous system
regeneration at a site of tissue damage and can be customized for
individual patients using their own cells as the donor or starting
cell. The present invention can be used to generate cells from an
individual patient, thus making autologous transplantations
possible as a treatment modality for many neurological conditions.
Thus, this technology eliminates the problems associated with
transplantations of non-host cells, such as, immunological
rejection and the risk of transmitted disease. The great advantage
of the present invention is that it provides an essentially
limitless supply for autologous grafts suitable for
transplantation. Therefore, it will obviate some significant
problems associated with current source of materials and methods of
transplantation.
Delivery of Polynucleotides
[0181] In certain embodiments, the invention concerns the use of
polynucleotides, e.g. a polynucleotide encoding a MBD2 polypeptide,
a MUSASHI1 polypeptide and/or a Ngn2 polypeptide. Means for
introducing polynucleotides into a cell are well known in the art.
Transfection methods of a cell such as nucleofection and/or
lipofection, or other types of transfection methods may be used.
For instance a polynucleotide encoding a desired polypeptide can be
cloned into intermediate vectors for transfection in eukaryotic
cells for replication and/or expression. Intermediate vectors for
storage or manipulation of the nucleic acid or production of
protein can be prokaryotic vectors, (e.g., plasmids), shuttle
vectors, insect vectors, or viral vectors for example. A desired
polypeptide can also be encoded by a fusion nucleic acid.
[0182] To obtain expression of a cloned nucleic acid, it is
typically subcloned into an expression vector that contains a
promoter to direct transcription. Suitable bacterial and eukaryotic
promoters are well known in the art and described, e.g., in
Sambrook and Russell (Molecular Cloning: a laboratory manual, Cold
Spring Harbor Laboratory Press). The promoter used to direct
expression of a nucleic acid of choice depends on the particular
application. For example, a strong constitutive promoter is
typically used for expression and purification. In contrast, when a
dedifferentiation protein or compound is to be used in vivo, either
a constitutive or an inducible promoter or compound is used,
depending on the particular use of the protein. In addition, a weak
promoter can be used, such as HSV TK or a promoter having similar
activity. The promoter typically can also include elements that are
responsive to transactivation, e.g., hypoxia response elements,
Ga14 response elements, lac repressor response element, and small
molecule control systems such as tet-regulated systems and the
RU-486 system.
[0183] In addition to a promoter, an expression vector typically
contains a transcription unit or expression cassette that contains
additional elements required for the expression of the nucleic acid
in host cells, either prokaryotic or eukaryotic. A typical
expression cassette thus contains a promoter operably linked, e.g.,
to the nucleic acid sequence, and signals required, e.g., for
efficient polyadenylation of the transcript, transcriptional
termination, ribosome binding, and/or translation termination.
Additional elements of the cassette may include, e.g., enhancers,
and heterologous spliced intronic signals.
[0184] Expression vectors containing regulatory elements from
eukaryotic viruses are often used in eukaryotic expression vectors,
e.g., SV40 vectors, papilloma virus vectors, and vectors derived
from Epstein-Barr virus. Other exemplary eukaryotic vectors include
pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any
other vector allowing expression of proteins under the direction of
the SV40 early promoter, SV40 late promoter, metallothionein
promoter, murine mammary tumor virus promoter, Rous sarcoma virus
promoter, polyhedrin promoter, or other promoters shown effective
for expression in eukaryotic cells.
[0185] Standard transfection methods can be used to produce
bacterial, mammalian, yeast, insect, or other cell lines that
express large quantities of dedifferentiation proteins, which can
be purified, if desired, using standard techniques. Transformation
of eukaryotic and prokaryotic cells is performed according to
standard techniques.
[0186] Any procedure for introducing foreign nucleotide sequences
into host cells can be used. These include, but are not limited to,
the use of calcium phosphate transfection, DEAE-dextran-mediated
transfection, polybrene, protoplast fusion, electroporation,
lipid-mediated delivery (e.g., liposomes), microinjection, particle
bombardment, introduction of naked DNA, plasmid vectors, viral
vectors (both episomal and integrative) and any of the other well
known methods for introducing cloned genomic DNA, cDNA, synthetic
DNA or other foreign genetic material into a host cell (see, e.g.,
Sambrook et al, supra). It is only necessary that the particular
genetic engineering procedure used be capable of successfully
introducing at least one gene into the host cell capable of
expressing the protein of choice.
[0187] Conventional viral and non-viral based gene transfer methods
can be used to introduce nucleic acids into mammalian cells or
target tissues. Such methods can be used to administer nucleic
acids encoding reprogramming polypeptides to cells in vitro.
Preferably, nucleic acids are administered for in vivo or ex vivo
gene therapy uses, Non-viral vector delivery systems include DNA
plasmids, naked nucleic acid, and nucleic acid complexed with a
delivery vehicle such as a liposome. Viral vector delivery systems
include DNA and RNA viruses, which have either episomal or
integrated genomes after delivery to the cell.
[0188] Methods of non-viral delivery of nucleic acids include
lipofection, microinjection, ballistics, virosomes, liposomes,
immunoliposomes, polycation or lipid-nucleic acid conjugates, naked
DNA, artificial virions, and agent-enhanced uptake of DNA.
Lipofection reagents are sold commercially (e.g., Transfectam.TM.
and Lipofectin.TM.). Cationic and neutral lipids suitable for
efficient receptor-recognition lipofection of polynucleotides are
known. Nucleic acid can be delivered to cells (ex vivo
administration) or to target tissues (in vivo administration). The
preparation of lipid:nucleic acid complexes, including targeted
liposomes such as immunolipid complexes, is well known to those of
skill in the art.
[0189] The use of RNA or DNA virus-based systems for the delivery
of nucleic acids take advantage of highly evolved processes for
targeting a virus to specific cells in the body and trafficking the
viral payload to the nucleus. Viral vectors can be administered
directly to patients (in vivo) or they can be used to treat cells
in vitro, wherein the modified cells are administered to patients
(ex vivo). Conventional viral based systems for the delivery
include retroviral, lentiviral, poxyiral, adenoviral,
adeno-associated viral, vesicular stomatitis viral and herpesviral
vectors, althoughntegration in the host genome is possible with
certain viral vectors, including the retrovirus, lentivirus, and
adeno-associated virus gene transfer methods, often resulting in
long term expression of the inserted transgene. Additionally, high
transduction efficiencies have been observed in many different cell
types and target tissues.
[0190] pLASN and MFG-S are examples of retroviral vectors that have
been used in clinical trials. In applications for which transient
expression is preferred, adenoviral-based systems are useful.
Adenoviral based vectors are capable of very high transduction
efficiency in many cell types and are capable of infecting, and
hence delivering nucleic acid to, both dividing and non-dividing
cells. With such vectors, high titers and levels of expression have
been obtained. Adenovirus vectors can be produced in large
quantities in a relatively simple system.
[0191] Gene therapy vectors can be delivered in vivo by
administration to an individual patient, typically by systemic
administration (e.g., intravenous, intraperitoneal, intramuscular,
subdermal, or intracranial infusion) or topical application.
Alternatively, vectors can be delivered to cells ex vivo, such as
cells explanted from an individual patient (e.g., lymphocytes, bone
marrow aspirates, tissue biopsy) or universal donor hematopoietic
stem cells, followed by reimplantation of the cells into a patient,
usually after selection for cells which have been reprogrammed.
[0192] Ex vivo cell transfection for diagnostics, research, or for
gene therapy (e.g., via re-infusion of the transfected cells into
the host organism) is well known to those of skill in the art. In a
preferred embodiment, cells are isolated from the subject organism,
transfected with a nucleic acid (gene or cDNA), and re-infused back
into the subject organism (e.g., patient). Various cell types
suitable for ex vivo transfection are well known to those of skill
in the art.
[0193] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)
containing therapeutic nucleic acids can be also administered
directly to the organism for transfection of cells in vivo.
Alternatively, naked DNA can be administered. Administration is by
any of the routes normally used for introducing a molecule into
ultimate contact with blood or tissue cells. Suitable methods of
administering such nucleic acids are available and well known to
those of skill in the art, and, although more than one route can be
used to administer a particular composition, a particular route can
often provide a more immediate and more effective reaction than
another route.
[0194] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions of the present invention.
Delivery of Polypeptides
[0195] In most, if not all the methods described herein, an
alternative possibility consists of bypassing the use of a
polynucleotide and contacting a cell of a first type cell directly
with a compound (e.g. a polypeptide) for which an increased
intracellular level is desired. In other embodiments, for example
in certain in vitro situations, the cells are cultured in a medium
containing one or more functional polypeptides.
[0196] An important factor in the administration of polypeptides is
ensuring that the polypeptide has the ability to traverse the
plasma membrane of a cell, or the membrane of an intra-cellular
compartment such as the nucleus. Cellular membranes are composed of
lipid-protein bilayers that are freely permeable to small, nonionic
lipophilic compounds and are inherently impermeable to polar
compounds, macromolecules, and therapeutic or diagnostic
agents.
[0197] However, proteins, lipids and other compounds, which have
the ability to translocate polypeptides across a cell membrane,
have been described. For example, "membrane translocation
polypeptides" have amphiphilic or hydrophobic amino acid
subsequences that have the ability to act as membrane-translocating
carriers. Polypeptides for which an increased intracellular level
is desired according to the invention can be linked to suitable
peptide sequences for facilitating their uptake into cells. Other
suitable chemical moieties that provide enhanced cellular uptake
can also be linked, either covalently or non-covalently, to the
polypeptides. Other suitable carriers having the ability to
transport polypeptides across cell membranes may also be used.
[0198] A desired polypeptide can also be introduced into an animal
cell, preferably a mammalian cell, via liposomes and liposome
derivatives such as immunoliposomes. The term "liposome" refers to
vesicles comprised of one or more concentrically ordered lipid
bilayers, which encapsulate an aqueous phase. The aqueous phase
typically contains the compound to be delivered to the cell.
[0199] In certain embodiments, it may be desirable to target a
liposome using targeting moieties that are specific to a particular
cell type, tissue, and the like. Targeting of liposomes using a
variety of targeting moieties (e.g., ligands, receptors, and
monoclonal antibodies) has been previously described.
Cells and Cell Lines
[0200] The invention encompasses the cells, cell lines, stem cells
and purified cell preparations derived from any of the methods
described herein. In some embodiments, the cells, cells lines, stem
cells and purified cells preparations of the invention are of
mammalian origins, including but not limited to human, primates,
rodent, dog, cat, horse, cow, or sheep. In preferred embodiments,
they originate from a human.
[0201] Accordingly, another aspect of the invention relates to
modified cells, cell lines, pluripotent, multipotent or unipotent
cells and purified cell preparations, wherein any of these cells
comprise an exogenous polynucleotide encoding Musashi1 (Msi1); Msi1
and Ngn2; Msi1 and MBD2; and Ngn2 and MBD2; Msi1, Ngn2 and MBD2;
Msi1, Ngn2, Nestin and MBD2; and other potential combinations from
Table A preferably including Msi1 and Ngn2 and MBD2. In preferred
embodiments the cell according to the invention is a stem-like
cell, more preferably a Neural Stem-Like Cell (NSLC), the cell
possessing one or more of the following characteristics: [0202]
expression of one or more neural stem cell marker selected from the
group consisting of Sox2, Nestin, GFAP, Msi1, and Ngn2; [0203]
decreased expression of one or more genes specific to the cell that
the NSLC was obtained from (e.g. see Table C); [0204] forms
neurospheres in the neurosphere colony formation assay; [0205]
capable of being cultured in suspension or as an adherent culture;
[0206] capable of proliferating without the presence of an
exogenous reprogramming agent for over 1 month, preferably over 2
months, over 3 months, over 5 months and even for more than a year;
[0207] capable of dividing every 36 hours at low passage; [0208]
positive for telomerase activity; [0209] capable of differentiation
into a neuronal-like cell, astrocyte-like cell,
oligodendrocyte-like cell and combinations thereof; [0210]
decreased expression of telomerase and one or more neural stem cell
markers after differentiation; [0211] having one or more
morphological neurite-like processes (axons and/or dendrites)
greater than one cell diameter in length after differentiation into
a neuronal-like cell; [0212] expression of at least one
neural-specific antigen selected from the group consisting of
neural-specific tubulin, microtubule associated protein 2, NCAM,
and marker for a neurotransmitter after differentiation into a
neuronal-like cell; [0213] expression of one or more functional
neural markers (e.g. synapsin) after differentiation into a
neuronal-like cell; [0214] capable of releasing one or more
neurotrophic factors (e.g. BDNF) after differentiation into a
neuronal-like cell; [0215] negative in a tumor colony forming
assay; [0216] negative for tumor growth in SCID mice; [0217]
negative for teratoma growth in SCID mice; [0218] capable of
significantly improving one or more functional measures after
placement of an adequate number of NSLCs into the void in a brain
ablation model; [0219] capable of significantly improving or
maintaining one or more functional measures after injecting an
adequate number of NSLCs into an EAE model; and [0220] capable of
improving one or more functional measures more significantly than
hNPCs in CNS injury or neurodegenerative models.
[0221] Examples of all of the above items can be found in the
Examples section of this application.
[0222] In preferred embodiments, a NSLC according to the inventions
possesses all of the following characteristics: [0223] ability to
self-renew for significantly longer than a somatic cell; [0224] is
not a cancerous cell; [0225] is stable and not artificially
maintained by forced gene expression or by similar means and may be
maintained in standard neural stem cell media; [0226] can
differentiate to a progenitor, precursor, somatic cell or to
another more differentiated cell type of the same lineage; [0227]
has the characteristics of a stem cell and not just certain markers
or gene expression or morphological appearance; and [0228] does not
exhibit uncontrolled growth, teratoma formation, and tumor
formation in vivo.
[0229] In one particular embodiment, the reprogrammed cells (NSLCs)
according to the invention are capable of proliferating for several
months without losing their neural stem cell markers and their
ability to differentiate towards neuron-like, astrocyte-like, and
oligodendrocyte-like cells. The generation of the neural lineages
is characterized based on morphology, phenotypic changes and
functionality.
[0230] In preferred embodiments the cell according to the invention
is a stem-like cell, the stem-like cell possessing one or more of
the following characteristics: [0231] expression of one or more
stem cell marker selected from Table A; [0232] decreased expression
of one or more genes specific to the cell that the stem-like cell
was obtained from; [0233] capable of being cultured in suspension
(as spheres) or as an adherent culture; [0234] capable of
proliferating without the presence of an exogenous reprogramming
agent for over 1 month, preferably over 2 months, over 3 months,
over 5 months or for more than a year; [0235] positive for
telomerase activity; [0236] capable of differentiation into cells
according to the lineage of that stem-like cell; [0237] decreased
expression of telomerase and one or more stem cell markers after
differentiation; [0238] having one or more morphological features
of the stem cells that the stem-like cell is like; [0239]
expression of one or more antigen expressed specifically in the
stem cells that the one or more is like; [0240] expression of one
or more functional markers of lineage specific differentiated cells
after differentiation of the stem-like cell; [0241] negative in a
tumor colony forming assay; [0242] negative for tumor growth in
SCID mice; [0243] negative for teratoma growth in SCID mice; [0244]
capable of significantly improving one or more functional measures
after placement of an adequate number of stem-like cells in a model
assessing the regenerative potential of those types of stem
cells.
[0245] In some embodiments, the cells of the invention may have one
or more of the following characteristics and properties:
self-renewal, multilineage differentiation in vitro and in vivo,
clonogenicity, a normal karyotype, extensive proliferation in vitro
under well defined culture conditions, and the ability to be frozen
and thawed, as well as any of the commonly known and/or desired
properties or characteristics typical of stem cells. The cells of
the invention may further express molecular markers of multipotent
or pluripotent cells (i.e. gene and surface markers as defined
previously).
[0246] Another aspect of the invention relates to the production of
tissue specific autologous (self) stem and/or progenitor cells.
These stem and/or progenitor cells may be used in cell therapy
applications to treat diseases of cellular degeneration. Diseases
of cellular degeneration include, for example, neurodegenerative
diseases such as stroke, Alzheimer's disease, Parkinson's disease,
multiple sclerosis, Amyotrophic lateral sclerosis, macular
degeneration, osteolytic diseases such as osteoporosis,
osteoarthritis, bone fractures, bone breaks, diabetes, liver
injury, degenerative diseases, myocardial infarct, burns and
cancer. These stem and/or progenitor cells may also be used in cell
therapy applications to treat injury such as spinal cord injury,
traumatic brain injury, burns, and muscle injury as well as other
diseases or other aspects of a disease such as cancer, hepatitis,
HIV/AIDS, leukemia and other blood disorders. It is envisioned that
cells according to the invention may be implanted or transplanted
into a host. An advantage of the invention is that large numbers of
autologous stem cells can be produced for implantation without the
risk of immune system mediated rejection. Those cells can lead to
production of tissue suitable for transplant into the individual.
Since the tissue is derived from the transplant recipient, it
should not stimulate an immune response, as would tissue from an
unrelated donor. Such transplants can constitute tissues (e.g.
vein, artery, skin, muscle), solid organ transplants (e.g., heart,
liver, kidney), neuronal cell transplants, or bone marrow
transplants such as are used in the treatment of various
malignancies such as for example, leukemias and lymphomas. Neural
stem cell, neuroprogenitor, or neuronal cell (as well as NSLCs and
derivations thereof) transplants can also be used in the treatment
of, for example, neurological disorders, stroke, spinal cord
injury, Parkinson's disease, and the like, as well as potentially
some non-neurological disorders such as a cardiac infarct.
[0247] Another aspect of the invention relates to a method of
genetically engineering a cell created according to the present
invention, and using said cell for treating a disease or enhancing
and/or modulating and/or changing the functionality or
characteristics of a cell, tissue, organ or system in a patient. An
example includes genetically correcting the dystrophin or
LAMA.sub.2 gene in a cell obtained from a patient suffering from
muscular dystrophy and creating muscle stem-like cells according to
the methods of the present invention that are then administered to
the patient according to appropriate methods in the art; these
muscle stem-like cells can then help to regenerate the muscles of
the patient as well as potentially gene correcting existing muscle
cells through fusion of the stem-like cells with the patient's
existing muscle cells whereby the corrected gene in the stem-like
cells replaces the defective gene in the muscle cell. The same
process above can be used for treating other genetic diseases
whereby the appropriate type of stem-like cell is created according
to methods of the present invention to regenerate and/or correct
cells, tissues and/or organs in the patient affected by the
disease. The same method can also be used for enhancing and/or
modulating and/or changing the functionality or characteristics of
a cell, tissue, organ or system in a patient. For example, a
patient suffering from HIV/AIDS could be treated by obtaining an
appropriate cell that has not been infected by HIV, knocking out
the CCR5 gene of the cell so that the CCR5 receptor cannot be
expressed (if, for example, the cell was a hematopoletic stem cell
or any differentiated cell of this lineage), reprogramming this
cell into a hematopoletic stem-like cells, and then administering
an appropriate number of these hematopoietic stem-like cells into
the patient (according to methods known in the art) to allow for
proper grafting of these cells into the patient's bone marrow.
Since the HIV cannot enter into the CCR5.sup.- (negative)
hematopoietic stem-like cells and their progeny (differentiated
lineage cells), these cells will over time allow the patient's
blood system to function normally and potentially the HIV may even
be eradicated. It should be noted that the genetic
engineering/modification can take place before, during or after the
creation of the stem-like cell of interest for all the above
methods and processes in this paragraph. It should also be noted
that an allogeneic cell could also be used for all the above
methods and processes in this paragraph.
[0248] Hematopoietic stem-like cells of the present invention can
also be used to repopulate the blood system in patients after
certain types of radiotherapy (ex. for cancer). Other appropriate
stem-like cells (or cells differentiated from these stem-like
cells) can be used to repopulate tissues, organs or a system in a
patient after certain types of therapy, surgical intervention or
injury has left the patient devoid of enough of certain types of
cells that can be provided by the cells, methods and/or uses of the
present invention.
[0249] Another aspect of the invention relates to a method to
produce ex vivo engineered tissues for subsequent implantation or
transplantation into a host, wherein the cellular components of
those engineered tissues comprise cells according to the invention,
or cells derived therefrom. For example, expanded cultures of the
cells of the invention may be differentiated by in vitro treatment
with growth factors and/or morphogens. Populations of
differentiated cells are then implanted into the recipient host
near the site of injury or damage, or cultured in vitro to generate
engineered tissues, as described.
[0250] The methods and cells of the invention described herein can
be used to immortalize (or near-immortalize) cells, for example to
generate a cell line. Using the methods disclosed herein, a somatic
cell can be transformed into one possessing a dedifferentiated
phenotype, thereby facilitating the generation of cell lines from a
variety of tissues. Therefore, the invention encompasses such
immortalized (or near-immortalize) cells.
[0251] In addition, the methods of deriving the cells according to
the invention, may be helpful in scientific and therapeutic
applications including, but not limited to, (a) scientific
discovery and research involving cellular development and genetic
research (e.g. uses in lieu of human stem cells as a model cell
line to study the differentiation, dedifferentiation, or
reprogramming of human cells), (b) drug development and discovery
(e.g., screening for efficacy and toxicity of certain drug
candidates and chemicals, screening for prospective drugs or agents
which mediate the differentiation, dedifferentiation, or
reprogramming of cells), (c) gene therapy (e.g., as a delivery
device for gene therapy), and (d) treatment of injuries, trauma,
diseases and disorders including, but not limited to, Parkinson's,
Alzheimer's, Huntington's, Tay-Sachs, Gauchers, spinal cord injury,
stroke, burns and other skin damage, heart disease, diabetes,
Lupus, osteoarthritis, liver diseases, hormone disorders, kidney
disease, leukemia, lymphoma, infectious diseases, HIV/AIDS,
multiple sclerosis, rheumatoid arthritis, Duchenne's Musclar
Dystrophy, Ontogenesis Imperfecto, birth defects, infertility,
pregnancy loss, and other cancers, degenerative and other diseases
and disorders.
[0252] Additional aspects concern therapeutic methods, methods of
treatment and methods of regenerating a tissue or organ in a mammal
(e.g. a human subject). One particular method concerns a method of
regenerating a mammalian tissue or organ which comprises contacting
the tissue or organ to be regenerated with a SLC, NSLC, or other
desired cell or artificial tissue construct as defined herein. The
SLC, NSLC, desired cell or artificial tissue construct may be
placed in proximity to the tissue or organ to be regenerated by
administering to the subject using any suitable route (e.g.
injecting the cell intrathecally, directly into the tissue or
organ, or into the blood stream).
[0253] Another method for repairing or regenerating a tissue or
organ in a subject in need thereof comprises administering to the
subject a compound inducing a direct or indirect endogenous
expression of at least one gene regulator in cells of the tissue or
organ and/or a compound inducing a direct or indirect endogenous
expression of at least one gene regulator in cells capable of
transformation or dedifferentiation in vivo in the subject.
Accordingly, the expression of the at least one gene regulator
reprograms the cells into desired cells of a different type (e.g.
neural stem-like cells), and these cells of a different type are
effective in repairing or regenerating said tissue or organ.
[0254] Another method comprises obtaining cells or tissue from a
patient (e.g. hematopoietic stem cells, fibroblasts, or
keratinocytes), reprogramming a plurality of such cells or the
tissue, and reintroducing the reprogrammed cells or tissue into the
patient. A related aspect concerns pharmaceutical compositions
comprising a plurality of a desired cell, SLC and/or Neural
Stem-Like Cell (NSLC) or reprogrammed tissue as defined herein.
[0255] The therapeutic methods of the invention may be applicable
to the regeneration or repair of various tissues and organs
including, but not limited to, the brain, the spinal cord, the
heart, the eye, the retina, the cochlea, the skin, muscles,
intestines, pancreas (including beta cells), kidney, liver, lungs,
bone, bone marrow, blood system, cartilage, cartilage discs, hair
follicles, teeth, blood vessels, glands (including endocrine and
exocrine glands), ovaries, reproductive organs, mammary and breast
tissue.
[0256] A related aspect concerns pharmaceutical compositions
comprising a plurality of desired cell, SLC and/or Neural Stem-Like
Cell (NSLC) as defined herein.
Tissues
[0257] Another aspect of the invention relates to a tissue
containing reprogrammed cells as defined herein that can be
implanted into a subject in need thereof.
[0258] In some embodiments the present invention provides for the
reprogramming of cells within a tissue, for example an in vitro
produced 3D tissue construct comprising cells and extracellular
matrix produced by these cells. In addition, transfected cells can
be seeded on top of these 3D tissue constructs that can be made
completely autologously, thus preventing host rejection, making it
completely immunocompatible and as carrier for reprogrammed cells
to be transplanted in vivo. Advantageously, these newly created
cells can be used in their undifferentiated and/or differentiated
state within these tissues for in vitro diagnostic purposes or
transplanted into a patient in need of such a construct in cell
therapy/tissue replacement approaches.
[0259] The invention further encompasses 3D Neuronal-Like
multilayer tissue. Cells within CDM reprogrammed to Neural
Stem-Like Cells according to the invention readily differentiate
into neuronal-like cells, astrocyte-like cells, and
oligodendrocyte-like cells within the CDM. It is thus possible to
use CDM and reprogramming methods of the invention to reprogram the
cells within the CDM to form 3D Neuronal-Like multilayer tissue (up
to >30 cell layers). Such 3D tissue comprises neurons (or
specifically, neuron-like cells), astrocytes (or specifically,
astrocyte-like cells), and oligodendrocytes (or specifically,
oligodendrocyte-like cells) and it can be made completely
autologously, can be manually handled and implanted with relative
ease, or can used as an in vitro CNS tissue model.
[0260] One particular aspect concerns an artificial tissue
construct which comprises a 3D assembly of in vitro cultured cells
and extracellular matrix produced by these cells. The cells may be
desired cells, SLC and/or a plurality of Neural Stem-Like Cell
(NSLC) obtained using any one of the methods described herein.
Screening Methods
[0261] Another aspect of the invention relates to methods for
identifying new compounds (e.g. small molecules, drugs, etc)
capable of transforming a cell of a first type to a desired cell of
a different type, as well as identifying the molecular and cellular
pathways for reprogramming. These new compounds may be useful for
research purposes or as medicaments for use in repairing or
regenerating cells, tissues or organs in a subject; while these
pathways may be useful for research purposes to develop research
tools, diagnostics, and medicaments for use in repairing or
regenerating cells, tissues or organs in a subject and for other
uses. Those skilled in the art will understand that it is
conceivable to screen for compounds that will induce transformation
of a cell of a first type to a stem-like cell by replacing the
"induction" or "biological activity" provided by the transient
increase of the reprogramming agent or gene regulator in the cell
by a candidate compound to be tested (e.g. a library of small
molecules or compounds) and measuring activity or efficacy of the
candidate compound in generating the stem-like cell; individual or
a mixture of active compounds would be selected if they have the
same activity and/or if they can provide the same or similar
effects as these polypeptides or compounds (e.g. cell
transformation and/or appearance of any desirable markers or
desirable characteristics as defined hereinbefore). Another aspect
of the invention relates to methods for identifying the molecular
and cellular pathways for reprogramming.
[0262] The Examples section provides principles, methods and
techniques useful for screening and identifying such desirable
active compounds. For instance, those skilled in the art will
understand that it is conceivable to screen for compounds that will
induce transformation of a cell of a first type to a NSLC by
replacing the "induction" or "biological activity" provided by the
transient increase of Musashi1, NGN2 or MBD2 in the cell by a
candidate compound to be tested (e.g. a library of small molecules
or compounds) and measuring activity or efficacy of the candidate
compound in generating the NSLC. Individual or mixture of active
compounds would be selected if they have the same activity and/or
if they can provide the same or similar effects as these
polypeptides (e.g. cell transformation and/or appearance of any
desirable markers or desirable characteristics, as defined
hereinbefore). For example, a compound or mixture of compounds
capable of transforming a fibroblast into a NSLC could be
identified by: [0263] (i) Setting up, culturing and transforming
the fibroblasts into NSLC as in Example 1; [0264] (ii) Screening a
library of compounds by replacing Msi1, Ngn2 and/or MBD2 with each
candidate compound in a different well; [0265] (iii) Identify a
compound `hit` when the candidate compound is able to transform the
fibroblasts into NSLCs approximately as well as the replaced Msi1,
Ngn2 and/or MBD2; [0266] (iv) If compound from part (iii) did not
replace all of Msi1, Ngn2 and MBD2, and is not able to transform
the fibroblasts into NSLCs by itself, then by including the
compound from (iii) in each well, screening a library of compounds
by replacing the Msi1, Ngn2 and/or MBD2 that was not removed in
part (ii) with each candidate compound in a different well; [0267]
(v) Identify a compound `hit` when the candidate compound is able
to transform, along with the compound from part (iii), the
fibroblasts into NSLCs approximately as well as the replaced Msi1,
Ngn2 and/or MBD2; [0268] (vi) if compound from part (V) did not
replace all of Msi1, Ngn2 and/or MBD2, and is not able to transform
the fibroblasts into NSLCs together with the compound from part
(iii), then by including the compound from (iii) and (v) in each
well, screening a library of compounds by replacing the Msi1, Ngn2
or MBD2 that was not removed in part (ii) and (iv) with each
candidate compound in a different well; [0269] (vii) Identify a
compound `hit` when the candidate compound is able to transform,
along with the compound from part (iii) and (v), the fibroblasts
into NSLCs approximately as well as the replaced Msi1, Ngn2 or
MBD2; [0270] (viii) A combination of the compounds from part (iii),
(v) and (vii) will be able to transform the fibroblasts into NSLC;
modifications to these compounds can be made and further screened
to identify more effective or safe versions of these compounds.
[0271] The same principles are applicable for other desired types
of stem-like cells including pluripotent-like cells,
mesendoderm-like cells, pancreatic progenitor-like cells, etc.
Tables A and B. and the Examples section provides, for each of
these types of cells, a list of potential genes and/or compounds to
be considered in such screening methods.
[0272] Accordingly, the present invention encompasses these and any
equivalent screening methods where candidate compounds are tested
for their efficacy in transforming a cell of a first type to a
desired cell of a different type when compared to the efficacy of
the reprogramming agent, reprogramming factor and/or gene regulator
as defined herein.
Delivery of Neurotrophic Factors
[0273] A related aspect of the invention relates to local delivery
of trophic factors (ex. growth factors) to a tissue or organ of
interest by transplanting stem-like cells and cell lines according
to the invention which can stably express and secrete growth
factors of potential interest after transplantation to said tissue
or organ.
[0274] Local delivery of neurotrophic factors has been suggested as
a method to treat several neurological conditions. Strategies using
neurotrophic molecules focus on preventing the progressive loss of
neurons, maintaining neuronal connections and function
(neuroprotection), and inducing additional regenerative responses
in neurons such as increased neurotransmitter turnover and/or
axonal sprouting (neuroregeneration). Up to date, several
therapeutic strategies to deliver neurotrophic-factors in animal
models have been explored, but so far testing of the effects of
growth factors on the brain and nervous system have been limited to
direct peripheral injection of large doses of these factors, which
carries a significant risk of side effects. Accordingly, a related
aspect of the invention relates to overcoming these problems by
using NSLC cells and cell lines according to the invention which
can stably express and secrete growth factors of potential interest
after transplantation.
[0275] To summarize, the present invention provides a plentiful
source of Stem-Like Cells and their progeny (differentiated cells)
for potential clinical treatments which require transplantation of
specific stem cells to 1) compensate for a loss of host cells or 2)
as vehicles to deliver genetically-based drugs. For example, Neural
Stem-Like Cells, Neuron-Like Cells, Astrocyte-Like Cells or
Oligodendrocyte-Like Cells can be used for potential clinical
treatments which require transplantation of neural stem cells,
neurons, astrocytes or oligodendrocytes 1) to compensate for a loss
of CNS host cells (ex. neurons) or 2) as vehicles to deliver
genetically-based drugs (ex. BDNF). Further, the invention provides
a novel neurological tool for use in basic research and drug
screening.
[0276] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures, embodiments, claims, and
examples described herein. Such equivalents are considered to be
within the scope of this invention and covered by the claims
appended hereto. The invention is further illustrated by the
following examples, which should not be construed as further
limiting.
EXAMPLES
[0277] The examples set forth herein below provide exemplary
methods for obtaining Reprogrammed and Dedifferentiated cells,
including Neural Stem-Like Cells (NSLCs). Also provided are
exemplary protocols, molecular tools, probes, primers and
techniques.
Example I
Preparation of Human Fibroblast Cells
[0278] Human Foreskin fibroblast (HFF) cells were purchased from
American Type Culture Collection (ATCC, Manassas, Va.) and expanded
in cell culture flasks with Dulbecco's Modified Eagle's Medium
(DMEM, Invitrogen), supplemented with 10% heat-inactivated fetal
calf serum (FCS, Hyclone Laboratories), 0.1 mM non-essential amino
acids, and 1.0 mM sodium pyruvate (invitrogen) at 37.degree. C., 5%
CO.sub.2. The medium was changed twice per week. Cells were
trypsinized using Trypsin 0.25% for 4 minutes at 37.degree. C.,
followed by adding trypsin inhibitor solution, pelleting the cells
by centrifugation, washing the cells once with PBS, and plating the
cells at a ratio of 1:2 onto tissue culture flasks until a suitable
number of cells was reached.
[0279] Cells were then trypsinized and plated (8.times.10.sup.4
cells/well) in cell culture plates pre-coated with Laminin (10
.mu.g/ml, Sigma) in two different composition of CDM medium: CDM I
Medium consisting of a 3:1 ratio of Dulbecco's modified Eagle
medium (DMEM, high glucose (4.5 g/L) with L-glutamine and sodium
pyruvate) and Ham's F-12 medium supplemented with the following
components: EGF (4.2.times.10.sup.-10M), bFGF
(2.8.times.10.sup.-1.degree. M), ITS (8.6.times.10.sup.-5 M),
dexamethasone (1.0.times.10.sup.-7M), L-3,3',5-triiodothyronine
(2.0.times.10.sup.-10M), ethanolamine (10.sup.-4 M), GlutaMAX.TM.
(4.times.10.sup.-3M), and glutathione (3.3.times.10.sup.-6M), but
without the presence of L-ascorbic acid.
[0280] CDM II Medium consisting of a 3:1 ratio of Dulbecco's
modified Eagle medium (DMEM, high glucose (4.5 g/L) with
L-glutamine and sodium pyruvate) and Ham's F-12 medium supplemented
with the following components: EGF (2.5 ng/ml), bFGF (10 ng/ml),
ethanolamine (2.03 mg/ml), insulin (10 mg/ml), Selenious acid (2.5
.mu.g/ml), dexamethasone (19.7 .mu.g/ml), L-3,3',5-triiodothyronine
(675 ng/ml), GlutaMAX.TM. (4.times.10.sup.-3M), and glutathione
(3.3.times.10.sup.-6 M).
Transient Transfection of HFF by Lipofectamine Using Constructed
Vectors
[0281] After two days in culture, cells were transfected with
pCMV6-XL5-MBD2 (24 (a DNA demethylator) using lipofectamine reagent
(Invitrogen) as per the manufacturer's protocol. The DNA-lipid
complex was added to cells and incubated for 24 h at 37.degree. C.,
5% CO.sub.2. After 24 hours of transfection with the DNA
demethylator, the medium was changed and cells were transfected by
pCMV6-XL5-Musashi1 (2 .mu.g, Origene) or pCMV6-XL4-Ngn2 (2 .mu.g,
Origene) for 24 h. After 24 hours, the medium was changed to Neural
Progenitor Basal Medium (NPBM, Lonza) supplemented with Noggin (20
ng/ml, Peprotech), EGF (20 ng/ml, Peprotech), and bFGF (20 ng/ml,
Peprotech) and cultured in this Proliferation Medium. Cells were
retransfected after three days and incubated at 37.degree. C., 5%
CO.sub.2 and 5% O.sub.2. After 7 days in proliferation conditions,
50% of the Proliferation Medium was changed to Differentiation
Medium (NbActive, BrainBits.TM.) supplemented with Forskolin (10
.mu.M, Tocris), all-trans-Retinoic Acid (ATRA, 5 .mu.M, Spectrum),
bFGF (20 ng/ml, Peprotech), NGF (20 ng/ml, Peprotech), and BDNF (20
ng/ml, Peprotech); medium was changed every day by increasing the
percentage of Differentiation Medium over Proliferation Medium, and
the cells were cultured for 20 days.
[0282] Visual observation of reprogrammed cells was performed by
light microscopic observation every day following transfection
using bright field at 10.times. magnification. Samples were
collected at different time points (6, 12, and 20 days) to analyze
neuronal gene expression and protein levels by gene array and
immunohistochemistry. Following transfection, reprogramming cells
displayed a rapid change in cellular morphology within 3 days
post-transfection. The cells were more rounded and the cell's
cytoplasm retracted towards the nucleus forming contracted cell
bodies with extended cytoplasmic extensions and exhibiting neuronal
perikaryal appearance at day 6 and 12, which was maintained until
day 20. However, this morphology was not observed in untransfected
cells at day 6 and 12.
Gene Array Analysis
[0283] Characterization of the newly engineering cells after
transfection was performed using a neuronal gene-array containing
48 partial cDNAs coding for these genes and controls.
[0284] RNA was isolated from samples using QIAshredder.TM. (Qiagen)
and RNeasy.TM. Plus mini Kit (Qiagen) as per manufacturer's
instructions. DNase I treatment was performed on the RNeasy.TM.
Column to further remove the transfected plasmid DNA using
Rnase-Free DNase Set (Qiagen). RNA was eluted in 35 .mu.l of
RNase-free water. Before cDNA synthesis, all RNA samples were
quantified using the NanoDrop 1000.TM. (ThermoScientific). cDNA was
prepared using the High Capacity cDNA archive kit (Applied
Biosystems) as per the manufacturer's instructions. 400 ng of RNA
was used in each 50 .mu.l RT reaction. The resulting cDNA samples
were used immediately for TLDA analysis. For each card of the
Taqman.TM. low-density array (TLDA), there are eight separate
loading ports that feed into 48 separate wells for a total of 384
wells per card. Each 2 .mu.l well contains specific, user-defined
primers and probes, capable of detecting a single gene. In this
study, a customized Neuronal Markers 2 TLDA was configured into
eight identical 48-gene sets, i.e. 1 loading port for each 48-gene
set. Genes were chosen based on literature. Each set of 48 genes
also contains three housekeeping genes: ACTIN, GAPDH, and PPIA.
[0285] A sample-specific master mix was made for each sample by
mixing cDNA (160 ng for each loading port), 2.times. Taqman.TM.
Gene Expression Master Mix (Applied Biosystems) and nuclease-free
water (USB) for a total of 100 .mu.l per loading port. After gentle
mixing and centrifugation, the mixture was then transferred into a
loading port on a TLDA card. The array was centrifuged twice for 1
minute each at 1200 rpm to distribute the samples from the loading
port into each well. The card was then sealed and PCR amplification
was performed using Applied Biosystems 7900HT.TM. Fast Real-time
PCR system. Thermal cycler conditions were as follows: 2 minutes at
50.degree. C., 10 minutes at 94.5.degree. C., and 30 seconds at
97.degree. C., 1 minute at 59.7.degree. C. for 40 cycles. 1 TLDA's
was prepared for 8 samples.
[0286] Relative Expression values were calculated using the
Comparative C.sub.T method. Briefly, this technique uses the
formula 2.sup.-.DELTA..DELTA.CT to calculate the expression of
target genes normalized to a calibrator. The threshold cycle
(C.sub.T) indicates the cycle number at which the amount of
amplified target reaches a fixed threshold. C.sub.T values range
from 0 to 40 (the latter representing the default upper limit PCR
cycle number that defines failure to detect a signal).
.DELTA.C.sub.T values [.DELTA.C.sub.T=C.sub.T (target gene)-C.sub.T
(Average of 3 Housekeeping genes)] were calculated for HFF Ctrl,
and subsequently used as the calibrator for the respective samples.
All gene expression values were assigned a relative value of 1.00
for the calibrator, which is used to determine comparative gene
expression such that .DELTA..DELTA.C.sub.T=.DELTA.C.sub.T
(Treated)-.DELTA.C.sub.T (HFF Ctrl). Relative Expression is
calculated using the formula 2.sup.-.DELTA..DELTA.CT.
[0287] Quantitative comparison of astrocyte, neuron, and
oligodendrocyte gene expression allowed identification of the
majority of the genes that are differentially expressed in
reprogrammed cells. Data in Table 1 were analyzed by using a
significance analysis algorithm to identify genes that were
reproducibly found to be enriched in reprogrammed cells compared to
untransfected cells. After the transfection with Msi1 or Ngn2 in
the presence of MBD2, the expression of oligodendrocytes
progenitors such as NKx2.2, olig2, and MAG and two markers for
astrocytes (GFAP and AQP4) were highly increased. Also, several
markers of early neuronal cells were enhanced after the
transfection of HFF. TDLA data revealed a remarkable increase in
specific markers for interneurons, such as somatostatin and
calbindin1. The induction of Doublecortin (DCX), which is expressed
by migrating immature cells during development, and acetylcholine
(ACHE) mRNA, an early marker of neuronal cells, were highly
expressed in the reprogrammed cells (Table 1). Transfection
increased the expression of dihydropyrimidinase-like 3 (DPYSL3), an
early marker of newborn neurons, to fivefold with Msi1 and seven
fold with Ngn2. Expression, of Microtubule-Associated Protein 2
(MAP2), an essential marker for development and maintenance of
early neuronal morphology and neuronal cell adhesion molecule, were
highly expressed with Msi1 and Ngn2 (Table 1). The expression of
enolase-2, a marker of mature neurons, was 20-fold enhanced by Msi1
and Ngn2. Member of the NeuroD family NeuroD1 was highly expressed
after transfection with Msi1 to 84 fold and to 34 by Ngn2.
[0288] Gene expression of growth factors such as IGF-1, IGF2, NPY
and CSF-3 was also enhanced in reprogrammed cells. The expression
of VEGF and GDNF genes were up-regulated to almost five fold and
seven fold by Msi1 and Ngn2, respectively, However, the expression
of BDNF, EGF, and bFGF were not activated and even down-regulated
as compared to untransfected cells. The expression of growth
associated protein (GAP-43), a growth- and regeneration-associated
marker of neurons, and expression of netrin, implicated in neuronal
development and guidance, were highly enriched in reprogrammed
cells. Expression of receptors for growth and neurotrophin factors
was increased, such as type III receptor tyrosine kinase,
neurotrophic tyrosine kinase, and neurotrophic tyrosine kinase
receptor. Vimentin and fibronectin, markers for fibroblasts, were
down-regulated in reprogrammed cells compared to the untransfected
control fibroblast cultures.
TABLE-US-00005 TABLE 1 Gene array of transfected human fibroblast
cells by Msi1/MBD2 and Ngn2/MBD2. Gene array was performed on
samples after two weeks of differentiation. Expression values are
given relative to untransfected fibroblasts. Relative Relative
Company expression to expression Symbol Common name and description
Gene ID Msi1 to Ngn2 Astrocytes and oligodendrocytes markers NKx2-2
Markers for oligodendrocyte NM_002509.2 very high very high
progenitors OLIG2 Oligodendrocyte lineage NM_005806.2 47.511 8.38
transcription factor 2 MAG Myelin-associated glycoprotein
NM-080600.1 212.61 4.51 GFAP Glial fibrillary acidic protein
NM_002055.4 very high very high AQP4 Aquaporin 4 NM_001650.4 83.77
56.86 NC markers SST Somatostatin, specific marker for NM_001048.3
32.73 35.34 interneurons CALB1 Calbindin 1, interneuron marker
NM_004929.2 18.21 13.22 Tubulin1A Are necessary for axonal growth
NM_006009.2 7.45 9.32 NES Precursor neurons (nestin) NM_006617.1
1.61 1.54 DCX An early neuronal marker NM_178151.1 very high very
high (Doublecortin) ACHE Acetylcholinesterase, marker of
NM_015831.2 9.02 13.22 early neuronal development ENO2 A marker for
neurons cells, NM_001975.2 22.62 20.68 enolase NEUROD1 Neural
marker; expression NM_002500.2 84.22 34.27 gradually increased from
neural precursor to fully differentiated neuron DPYSL3
Dihydropyrimidinase-like3, NM_001387.2 5.33 7.02 marker of immature
neurons MAP2 Microtubule-associated protein 2, NM_002374.3 86.38
89.67 essential for development of early neuronal morphology and
maintenance, of adult neuronal morphology NCAM Neural cell adhesion
molecule 1 NM_18135.3 very high very high CEND1 Cell cycle exit
& neuronal NM_016564.3 4.80 5.57 differentiation, early marker
of proliferating precursor cells that will differentiate to neurons
Neuroregeneration and survival genes FGF2 Fibroblast growth factor-
NM_002006.4 0.06 0.11 EGF Epidermal growth factor, Hs00153181_m1
0.99 0.56 IGF-1 Insulin growth factor-1, NM_000618.2 58.92 21.21
IGF-2 Insulin growth factor-2 NM_0000612.3 very high very high CSF3
Granulocyte colony-stimulating NM_2219.1 very high 42.60 BDNF
factor-Brain derived growth NM-199231.1 0.05 0.03 factor,
neurogenesis GDNF Glial derived neurotrophic factor NM-000614.2
4.77 6.89 CNTF Ciliary neurotrophic factor NM_001025366.1 1.86 1.09
VEGF Vascular endothelial growth NM_130850.1 6.67 7.32 factor BMP-4
Bone morphogenetic protein 4 NM_002253.1 5.96 8.57 KDR Type III
receptor tyrosine kinase) NM_006180.3 31.78 6.83 NTRK2 Neurotrophic
tyrosine kinase NM_00905.2 10.31 13.37 receptor (TrkB) NPY
Neuropeptide factors NM_002649.2 very high very high PIK3CG
phosphoinositide-3-kinase, NM_213662.1 very high very high STAT3
Signal transduction transcription 3 NM_002045.2 2.14 3.65 Gap43
Growth associated protein 43 _NM_006180.3 very high very high NTN1
Netrin1, implicated in neuronal NM_024003.1 26.84 2.98 development
and guidance NTRk2 Neurotrophic tyrosine kinase, NM_003061.1 10.31
13.37 receptor, type 2 Slit Axonal guidance molecules Hs00185584
very high very high Vimentin Radial glia and fibroblast marker
NM_212474.1 0.11 0.13 Fibronectin fibronectin is a marker for 0.15
0.23 fibroblasts
Immunohistochemical Analysis
[0289] Cells were fixed with a 4% formaldehyde/PBS solution for 10
min at room temperature and subsequently permeabilized for 5 min
with 0.1% Triton X-100.TM. in 4% formaldehyde/PBS. After two brief
washes with PBS, unspecific antibody binding was blocked by a 30
min incubation with 5% normal goat serum in PBS. Then primary
antibodies were added in 5% normal goat serum/PBS as follows: Mouse
anti-Nestin (1:100, BD) as an intermediate microfilament present in
neural stem cells and mouse anti-NCAM (1:100, Neuromics) as
neuronal adhesion molecule. After a 2 h incubation the cells were
washed 4 times for 5 min each with 0.1% Tween.TM./PBS. Appropriate
fluorescence-tagged secondary antibody was used for visualization;
Goat anti-mouse 546 (1:200, invitrogen) prepared in 5% normal goat
serum/PBS was used. After incubation for one hour, cells were
washed in 0.1% Tween.TM./PBS three times for 5 min each. The DNA
stain Hoechst33342 (Invitrogen) was used as a marker of nuclei
(dilution 1:5000 in PBS, 10 min incubation). Fluorescence images
were taken with a Cellomics.TM. ArrayScan HCS Reader microscopy
system. To determine an estimate of the percentage of cells
adopting neuronal or glial phenotypes, random fields were selected
and for each field the total number of cells (as determined by
counting Hoechst stained nuclei) and the total number of cells
positive for neuronal or glial markers were determined.
[0290] To confirm that these cells exhibited markers of neuronal
lineages, cells were immunostained for nestin and NCAM. This
analysis revealed that reprogrammed cells expressed both proteins.
NCAM was present in cells during the 6 days post-transfection and
increase at day 12 and 20 following differentiation, while the
inverse pattern was observed for the nestin staining.
[0291] This study showed the ability to reprogram HFF cells using
one neurogenic transcription factor with the presence of a DNA
demethylator towards cells that expressed neuronal genes and
proteins specific to neural stem cells and neuronal cells. These
reprogrammed cells were stable in culture for at least 2 weeks.
Example II
Comparison of Reprogramming Efficiency of Three Different
Neurogenic Genes
[0292] HFF cells were cultured as described in Example I and plated
in CDM I medium. Cells were transfected using the Amaxa
Nucleofector.TM. Device (Lanza). The HFFs were harvested with
TrypLE.TM. (Gibco), resuspended in CDM Medium and centrifuged for
10 min at 90.times.g (1.times.10.sup.6 cells/tube). The supernatant
was discarded and gently resuspended in 100 .mu.l of Basic
Nucleofectar.TM. Solution (basic Nucleofector.TM. kit for primary
mammalian fibroblasts, Lanza). Each 100 .mu.l of cell suspension
was combined with a different mix of plasmid DNA (for example,
sample 1 was mixed with 2 .mu.g of pCMV6-XL5-Pax6 and 2 .mu.g
pCMV6-XL5-MBD2). Cell suspension was transferred into an Amaxa
certified cuvette and transfected with the appropriate program
(U023). The sample was transferred without any further resuspension
into a coated culture plate with LAS-Lysine/Alanine (BrainBits.TM.,
50 .mu.g/ml) and the cells were incubated at 37.degree. C., 5%
CO.sub.2. These steps were repeated for each sample that was
transfected. After 24 hours, the medium was changed to
Proliferation Medium. After two days, cells were retransfected
using lipofectamine as described in Example I and incubated at
37.degree. C., 5% CO.sub.2 and 5% O. After 6 days, differentiation
was induced with Differentiation Medium that gradually replaced the
Proliferation Medium over several days. Cells were collected at day
14 for RT-PCR and immunohistochemistry analysis.
Gene Expression Analysis
[0293] RNA isolation and quantification was performed as previously
described in Example I. cDNA was prepared using the High Capacity
cDNA RT kit (Applied Biosystems) as per the manufacturer's
instructions with a final cDNA concentration of 2 ng/.mu.l.
Real-time PCR was then performed for each gene of interest using
the FAST PCR master mix (Applied Biosystems) and the
Taqman.TM..RTM. Gene Expression Assays (Applied Biosystems) listed
below:
TABLE-US-00006 Gene Name Assay ID ACHE Hs00241307_m1 NES
Hs00707120_s1 TUBB3 Hs00964962_g1 GFAP Hs00157674_m1 PAX6
Hs00240871_m1 MSI1 Hs01045894_m1 NGN2 Hs00702774_s1 MAP2
Hs00258900_m1 GAPDH (housekeeping gene) Hs99999905_m1 PPIA
(housekeeping gene) Hs99999904_m1
[0294] The FAST 96-well reaction was performed with 8 ng cDNA per
well in a 10 .mu.l reaction with 40 cycles. Thermal cycler
conditions were as follows: 20 seconds at 95.degree. C., and 1
second at 95.degree. C., 20 seconds at 60.degree. C. for 40
cycles.
[0295] Relative Expression values were calculated as previously
described in Example I, except the Average of 2 Housekeeping genes
(GAPDH & PPIA) was used for normalization instead of the
Average of 3 Housekeeping genes. Identification of neuronal lineage
genes was investigated following the transfection with three
independent vectors containing Msi1, Ngn2, and Pax6.
[0296] As shown in Table 2, after 14 days following transfection,
relative expression of mRNA of neuronal lineage was undetectable in
untransfected cells (HFF), while the cells transfected with Msi1 or
Ngn2 in the presence of MBD2 expressed neural stem cell markers
(Nestin and Sox2), however the expression of Sox2 was much more
highly expressed than nestin following transfection with Ngn2 or
Msi1. Neuronal and astrocyte specific genes (.beta.III-Tubulin,
MAP2b, GFAP, and ACHE) was increased as well. mRNAs level of the
tripotent-associated genes .beta.III-tubulin, MAP2b, acetycholine,
and GFAP were undetectable in Pax6 transfected cells, indicating
that Pax6 alone was not implicated in the reprogramming process
toward neuronal lineage.
TABLE-US-00007 TABLE 2 Relative expression of gene expression of
different neuronal lineage performed by RT-PCR following the
transfection of HFF by Msi1, Ngn2, or Pax6 in the presence of MBD2
and cultured for 14 days. MSI1 NGN2 PAX6 NES TUBB3 Rel. Std. Std.
Std. Rel. Std. Rel. Std. Exp. Dev. Rel. Exp. Dev. Rel. Exp. Dev.
Exp. Dev. Exp. Dev. #1 Control 1.00 0.07 1.08 0.57 1.11 0.67 1.00
0.02 1.00 0.01 Untransfect #2 4077.82 248.02 1.18 0.66 487.09 69.58
8.62 0.00 6.58 0.11 MSI1/MBD2 #3 14.16 0.63 47803.26 192.78 624.31
91.27 8.62 0.02 8.33 0.02 NGN2/MBD2 #4 1.70 0.36 0.27 0.01 29564.43
357.89 0.46 0.00 0.49 0.02 PAX6/MBD2 ACHE GFAP MAP2 SOX2 Rel. Std.
Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp.
Dev. #1 Control 1.02 0.29 1.00 0.06 1.00 0.01 1.00 0.09 Untransfect
#2 6.58 0.64 215.71 20.65 5.50 0.46 3499.53 184.85 MSI1/MBD2 #3
8.33 0.97 365.60 5.11 5.42 0.00 4039.03 8.65 NGN2/MBD2 #4 1.98 0.48
1.15 0.13 0.55 0.04 1.00 0.03 PAX6/MBD2
Immunohistochemical Analysis
[0297] Fluorescent immunohistochemical staining was performed as
previously described in Example I. In agreement with the RT-PCR
data, immunohistochemical analysis of these cultures revealed that
reprogrammed cells (with Msi1 or Ngn2) generated morphologically
complex neurons that were positive for MAP2b, indicating the
differentiation of NSLCs to neuron-like cells (NLCs). However, the
positive staining for these markers was undetectable after
transfection with Pax6/MBD2. Moreover, the newly formed neurons
expressed the markers for and developed long neurites with growth
cones at their ends, expressed neural specific genes, and ceased to
proliferate when they were exposed to differentiation
conditions.
Example III
Transfection of HFF by Various Combinations of Vectors and
Disruption of Cell Cytoskeleton
[0298] Various combinations of neurogenic regulators and cytokines
for epigenetic modifications were tested to ascertain their effect
on reprogramming efficiency. Starting one day before transfection,
cells were treated with or without cytochalasin B (Calbiochem),
with the concentration decreased every day over five days during
media changes (starting with 10 .mu.g/ml Cytochalasin B on day 1 to
7.5 .mu.g/ml, 5 .mu.g/ml, 2.5 .mu.g/ml, and 0 .mu.g/ml over the
subsequent four days) in order to investigate the effect of
disrupting the cell cytoskeleton on the process of reprogramming.
Cells were transiently transfected as described in Example H with
one or two vectors containing one neurogenic transcription factors
by nucleofection. Cells were co-transfected with either of two DNA
demethylators, MBD2 or GAdd45B, (e.g. 2.times.10.sup.6 cells were
transfected with pCMV6-XL5Msi1 (2 .mu.g) and pCMV6-XL5-MBD2 (2
.mu.g)). After 24 hours, the medium was changed to Neural
Proliferation Medium (NeuroCult.TM. proliferation Kit, StemCell
Technologies) consisting of DMEM/F12 (1:1), glucose (0.6%), sodium
bicarbonate (0.1%), glutamine (20 mM), HEPES (5 mM), insulin (230
.mu.g/ml), transferrin (100 .mu.g/ml), progesterone (200 nM),
putrescine (90 .mu.g/ml), and sodium selenite (300 nM) and
supplemented with Noggin (20 ng/ml, Peprotech), recombinant hFGF
(20 ng/ml, Peprotech), and recombinant hEGF (20 ng/ml, Peprotech)
and cells were cultured for two weeks at 37.degree. C., 5% CO.sub.2
and 5% O.sub.2. Cells were then analyzed for neural stem cell
markers.
Gene Expression Analysis
[0299] Gene expression analysis was performed for neural
stem-specific markers (Sox2, Nestin, GFAP) and a
fibroblast-specific marker (Col5A2) by RT-PCR as previously
described in Example I. RT-PCR analysis showed that the relative
expression of Sox2, nestin and GFAP was enhanced after transfecting
the cells with the neurogenic transcription factors. As shown in
Table 3, transfecting the cells with one transcription factor Msi1
in the presence of Gadd45b was associated with up-regulation of
relative expression of Sox2 (22.3.+-.5.26) and GFAP (10.14.+-.0.15)
and the expression of the these genes was highly increase when
transfecting the cells with Ngn2 by 20 fold and 10 fold
respectively. Combining the two neurogenic factors (Msi1 and Ngn2)
with Gadd45b enhanced further the expression of Sox2 and GFAP.
Transfecting the cells with one transcription factor (Msi1 or Ngn2)
in the presence of MBD2 was associated with up-regulation of
relative expression of Sox2, Nestin, and GFAP and down-regulation
of Col5A2, while co-transfection with Gadd45b did not increased the
expression of nestin and the expression of Col5A2 was not
regulated. The enhancement of neural stem cells relative expression
was observed when transfecting the cells with two neurogenic genes
in combination with MBD2; a small increase in the expression was
noticed in the presence of cytochalasin B under certain conditions.
An increase in the relative expression of the neural stem-specific
markers (Sox2, Nestin, GFAP) and a decrease in the
fibroblast-specific gene (COL5A2) was observed after transfection
with Msi1/Ngn2/MBD2, Msi1/Ngn2/Gadd45b, Msi1/MBD2 or Ngn2/MBD2
(Table 3). This study demonstrated that MBD2 increased more
reprogramming efficiency then GDA45b and showed that cytochalasin B
had no effect of its own in the control cultures.
TABLE-US-00008 TABLE 3 RT-PCR analysis of relative expression of
neuronal precursor cell markers such as nestin, Sox2, and GFAP
after transfection of fibroblast cells with different combinations
with or without the co-treatment with cytochalasin B. Relative
expression of Sox2, nestin, and GFAP in NSLCs was increased after
transfection with both transcription factors (Ngn2 and Msi1) with
MBD2 as the DNA demethyaltor. As demonstrated, this upregulation of
neural stem cell gene expression was associated with a decrease of
CoL5A2, a specific gene for fibroblast cells. COL5A2 FBN2 NES MAP2
TUBB3 SOX2 ACHE GFAP Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel.
Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev.
Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. #1, +CytoB, 1.00
0.07 1.00 0.01 1.00 0.04 1.00 0.05 1.00 0.05 1.00 0.05 1.00 0.10
1.00 0.11 Control #2, -CytoB, 1.00 0.03 1.00 0.08 1.00 0.00 1.00
0.09 1.00 0.09 1.15 0.80 1.01 0.18 1.00 0.01 Control #3, +CytoB,
0.85 0.04 0.75 0.02 0.60 0.01 0.29 0.01 0.44 0.00 22.39 5.26 0.81
0.19 10.14 0.15 Msi1, GAD45b #4, -CytoB, 0.87 0.03 1.81 0.09 1.84
0.04 2.31 0.00 2.09 0.03 20.28 5.33 1.99 0.74 6.03 0.05 Msi1,
GAD45b #5, +CytoB, 0.84 0.04 0.77 0.03 0.44 0.00 0.24 0.00 0.36
0.01 470.84 13.43 0.63 0.05 103.22 0.80 Ngn2, GAD45b #6, -CytoB,
0.75 0.07 1.97 0.02 1.83 0.00 4.40 0.16 2.02 0.10 789.33 60.35 1.70
0.13 110.48 4.90 Ngn2, GAD45b #7, +CytoB, 0.74 0.12 1.08 0.00 0.89
0.01 0.51 0.00 0.63 0.04 1.64 0.98 0.86 0.12 2.49 0.21 Pax6, GAD45b
#8, -CytoB, 0.66 0.04 2.41 0.09 2.70 0.03 4.96 0.30 3.48 0.07 0.46
0.33 2.97 1.04 0.43 0.09 Pax6, GAD45b #9, +CytoB, 0.14 0.01 0.28
0.01 1.30 0.03 4.07 0.11 0.84 0.00 54768.27 6709.56 0.81 0.24
3391.96 64.63 Msi1, Ngn2, GAD45b #10, -CytoB, 0.12 0.00 0.73 0.03
5.28 0.21 50.84 1.23 4.93 0.28 17400.66 822.88 3.58 0.10 1255.76
5.27 Msi1, Ngn2 GAD45b #11, +CytoB, 0.10 0.00 0.26 0.01 1.11 0.01
3.69 0.09 0.76 0.00 55588.41 1331.20 0.55 0.14 2849.96 261.51 Msi1,
Ngn2 MBD2 #12, -CytoB, 0.44 0.01 1.47 0.06 5.49 0.14 47.30 0.11
5.50 0.31 14587.46 789.19 3.90 0.13 1424.04 39.29 Msi1, Ngn2 MBD2
#13, +CytoB, 1.11 0.04 1.09 0.06 0.92 0.08 0.68 0.01 0.82 0.03
63.93 2.81 1.19 0.17 17.43 1.86 GAD45b #14, -CytoB, 0.94 0.01 2.22
0.00 2.82 0.02 6.49 0.30 4.01 0.05 6.12 0.61 2.34 0.17 1.42 0.10
GAD45b #15, +CytoB, 0.83 0.00 0.83 0.05 0.36 0.01 0.16 0.01 0.36
0.00 3.42 3.74 0.63 0.37 2.18 0.12 MBD2 #16, -CytoB, 0.68 0.02 1.55
0.04 1.57 0.05 1.47 0.01 2.00 0.00 0.52 0.29 1.45 0.15 0.55 0.04
MBD2 #17, +CytoB, 1.10 0.01 1.16 0.03 1.37 0.01 1.12 0.06 0.86 0.06
5.59 1.48 1.07 0.27 1.70 0.46 Msi1, Ngn2 #18, -CytoB, 0.93 0.04
2.52 0.10 3.48 0.01 9.01 0.02 4.55 0.18 1.78 1.46 3.83 0.42 0.59
0.01 Msi1, Ngn2 #19, +CytoB, 0.20 0.03 0.36 0.01 1.25 0.05 6.68
0.31 0.72 0.02 66592.29 3481.89 2.57 0.03 4450.08 131.85 Msi1, MBD2
#20, -CytoB, 0.12 0.00 0.64 0.03 4.70 0.22 77.51 0.11 4.12 0.11
19128.03 1542.00 8.14 0.13 999.22 24.75 Msi1, MBD2 #21, +CytoB,
0.17 0.01 0.28 0.00 1.16 0.04 5.73 0.06 0.62 0.00 67945.51 3000.74
2.15 0.04 4736.83 11.92 Ngn2, MBD2 #22, -CytoB, 0.17 0.00 0.78 0.03
4.32 0.08 68.89 5.26 4.01 0.04 16570.91 92.96 7.04 0.53 1427.13
13.19 Ngn2, MBD2 #23, +CytoB, 0.71 0.05 0.79 0.06 0.87 0.01 0.63
0.06 0.67 0.04 2.86 0.70 1.08 0.08 2.08 0.11 Msi1 #24, -CytoB, 0.66
0.04 1.92 0.17 2.03 0.02 2.77 0.02 2.68 0.02 0.32 0.12 1.85 0.65
0.58 0.04 Msi1
Immunohistochemical Analysis
[0300] Fluorescent immunohistochemical staining was performed as
previously described in Example I. Table 4 shows the percentage of
Nestin and Sox2 in each condition, with the highest percentage of
Sox2 (38.18.+-.1.75%) and nestin (28.18.+-.2.77%) positive cells
observed after transfecting the cells simultaneously with both
neurogenic transcription factors and in the presence of a DNA
demethylator and cytochalasin B. A slight increase of Sox2 positive
cells (10.42.+-.10.27%) and nestin positive cells (4.85.+-.1.10%)
was detected following transfection with one transcription factor
Msi1 and MBD2. Same tendency of nestin and Sox2 positive cells was
observed following transfection with Ngn2 and MBD2. Disrupting the
cell cytoskeleton with Cytochalasin B significantly enhanced
reprogramming, but had no reprogramming effect on its own (Table
4).
TABLE-US-00009 TABLE 4 Percentage of positive cells for Sox2 and
nestin after transfection of fibroblast cells with different
expression vectors with or without the presence of cytochalasin B.
After transfection the cells were cultured in proliferation medium
(StemCell Technologies) supplemented by EGF (20 ng/ml, Peprotech)
and FGF (20 ng/ml, Peprotech) for two weeks at 37.degree. C./5%
CO.sub.2/5% O.sub.2. The percentage of immunopositive cells was
determined by Cellomics .TM. and represented as mean .+-. SD (n =
3-5). % of Sox2 positive cells % of Nestin positive cells +CytoB
-Cyto B +CytoB -CytoB Untransfected cells 0.02 .+-. 0.01 0.01 .+-.
0.00 0.14 .+-. 0.04 0.11 .+-. 0.09 Ngn2 0.35 .+-. 0.36 0.15 .+-.
0.05 2.34 .+-. 0.99 1.04 .+-. 0.21 Msi1 0.23 .+-. 0.15 0.12 .+-.
0.09 1.95 .+-. 0.11 1.11 .+-. 0.18 Gadd45b 0.30 .+-. 0.17 0.29 .+-.
0.11 4.94 .+-. 0.25 2.33 .+-. 0.42 MBD2 0.22 .+-. 0.13 0.22 .+-.
0.11 2.8 .+-. 0.11 1.53 .+-. 0.6 Msil/Ngn2 0.19 .+-. 0.13 0.32 .+-.
0.05 1.91 .+-. 0.56 2.59 .+-. 1.28 Msi1/MBD2 10.42 .+-. 10.27 8.84
.+-. 11.63 4.85 .+-. 1.10 2.06 .+-. 0.08 Msi1/Gadd45b 0.06 .+-.
0.01 0.14 .+-. 0.17 0.55 .+-. 0.06 0.24 .+-. 0.11 Ngn2/MBD2 11.17
.+-. 0.08 9.07 .+-. 11.31 5.7 .+-. 0.10 2.18 .+-. 0.23 Ngn2/GAdd45b
0.29 .+-. 0.11 0.95 .+-. 0.17 1.17 .+-. 0.54 0.98 .+-. 0.25
Msi1/Ngn2/MBD2 38.18 .+-. 1.75 22.03 .+-. 1.90 28.18 .+-. 2.77
14.54 .+-. 0.45 Msi1/Ngn2/Gadd45b 22.65 .+-. 5.03 18.54 .+-. 9.40
18.72 .+-. 6.26 8.70 .+-. 4.51
[0301] Various DNA demethylators were tested as well for their
effect on reprogramming efficiency. Cells were co-transfected with
one vector (MSI1/NGN2) containing two neurogenic pCMV6-Msi1-Ngn2
factors with various DNA demethylators. Simultaneously another
neurogenic factor was tested for its effect on cells
de-differentiation towards NSCs, pCMV-XL-Nestin individually or in
combination with pCMV-Msi1-Ngn2, pCMV-XL5-Msi1, or pCMV-XL4-Ngn2 in
the presence of MBD2 as previously described in Example II.
[0302] Cells were co-transfected pCMV-Msi1-Ngn2 with different DNA
demethylators (MBD1, MBD2, MBD3, MBD4, MeCP2, AICDA). Another assay
was performed to assess the effect of nestin on the reprogramming
efficiency; therefore cells were transfected with nestin
individually or in combination with one vector containing one
neurogenic factor (Msi1 or Ngn2) or both neurogenic factors in the
presence of MBD2. Cells were cultured following transfection in the
presence of proliferation medium supplemented with EGF (20 ng/ml),
FGF (20 ng/ml), and Noggin (20 ng/ml) with and without VPA (1 mM)
treatment for 12 days at 37.degree. C., 5% CO.sub.2 and 5%
O.sub.2.
[0303] Gene expression analysis and immunohistochemistry was
performed to analyse neural specific gene and protein expression
(.beta.III-tubulin, GFAP, Sox2, Nestin) as described in Example II.
Transfecting cells with Msi1 and Ngn2 in the presence of various
DNA demethylators revealed and confirm previous data showing that
the among various DNA demethylators used in this study, MBD2
promotes the expression of neural stem genes (Sox2, GFAP, Nestin)
as shown in Table 5. Furthermore, transfecting cells with nestin
with and without the presence of one neurogenic factor had no
effect on the reprogramming efficiency into neural stem-like cells.
However co-transfection with nestin and Msi1/Ngn2/MBD2 enhanced the
expression of neural stem cells genes and this increase was more
pronounced in the presence of VPA.
TABLE-US-00010 TABLE 5 RT-PCR analysis of relative expression of
neuronal precursor cell markers such as nestin, Sox2,
.beta.III-tubulin, and GFAP after transfection of fibroblast cells
with various combinations of pCMV-Msi1-Ngn2 (MSI1/NGN2),
pCMV-XL5-Msi1, pCMV-XL4-Ngn2, pCMV-XL-Nestin with different
combinations of DNA demethylators, with and without the
co-treatment with VPA. TUBB3 GFAP SOX2 NES Rel. Std. Rel. Std. Rel.
Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Day 12,
Untransfected 1.00 0.11 1.00 0.05 1.01 0.16 1.00 0.13 (-VPA) Day
12, Untransfected 1.00 0.03 1.00 0.06 1.00 0.00 1.00 0.02 (+VPA)
Day 12, MSI1/NGN2/MBD1 0.96 0.06 2.69 0.13 1.15 0.49 0.46 0.02
(-VPA) Day 12, MSI1/NGN2/MBD1 1.10 0.06 2.22 0.06 0.80 0.01 0.84
0.02 (+VPA) Day 12, MSI1/NGN2/MBD2 123.52 0.06 1638.53 99.86
61467.29 1487.21 31.77 0.17 (-VPA) Day 12, MSI1/NGN2/MBD2 232.00
0.08 1889.30 42.39 72022.15 7894.41 42.69 0.14 (+VPA) Day 12,
MSI1/NGN2/MBD3 0.92 0.07 3.98 0.59 28.05 4.67 0.56 0.01 (-VPA) Day
12, MSI1/NGN2/MBD3 1.23 0.05 1.66 0.18 11.31 2.35 0.87 0.02 (+VPA)
Day 12, MSI1/NGN2/MBD4 0.85 0.01 4.80 0.23 5.42 5.20 0.62 0.00
(-VPA) Day 12, MSI1/NGN2/MBD4 0.95 0.01 1.57 0.16 2.27 0.04 0.79
0.03 (+VPA) Day 12, 1.11 0.06 3.80 0.38 6.54 6.45 0.69 0.01
MSI1/NGN2/MeCP2 (-VPA) Day 12, 1.37 0.09 1.63 0.45 10.53 10.49 1.07
0.01 MSI1/NGN2/MeCP2 (+VPA) Day 12, 1.07 0.04 4.59 0.02 0.65 0.01
0.74 0.02 MSI1/NGN2/AICDA (-VPA) Day 12, 1.10 0.01 2.37 0.29 1.21
0.16 0.91 0.04 MSI1/NGN2/AICDA (+VPA) Day 12, Msi1/MBD2 (-VPA) 1.31
0.17 3.78 0.49 0.70 0.02 0.78 0.00 Day 12, Msi1/MBD2 1.36 0.07 1.75
0.31 1.26 0.03 1.15 0.03 (+VPA) Day 12, Ngn2/MBD2 (-VPA) 0.85 0.06
2.93 0.51 0.79 0.05 0.58 0.02 Day 12, Ngn2/MBD2 1.41 0.05 1.60 0.11
2.30 0.06 1.03 0.03 (+VPA) Day 12, Nes/Msi1 (-VPA) 0.84 0.03 3.21
0.72 0.76 0.01 0.51 0.01 Day 12, Nes/Msi1 (+VPA) 0.86 0.09 1.82
0.30 2.14 1.02 0.94 0.01 Day 12, Nes/Ngn2 (-VPA) 0.69 0.05 2.88
0.32 0.99 0.10 0.57 0.02 Day 12, Nes/Ngn2 (+VPA) 0.88 0.01 1.53
0.19 2.71 0.02 0.83 0.03 Day 12,Nes/MSI1/NGN2/MBD2 111.58 0.04
1423.56 82.87 72069.27 624.51 51.52 0.12 (-VPA) Day
12,Nes/MSI1/NGN2/MBD2 321.00 0.04 2600.14 1.90 88932.00 708.72
82.74 0.18 (+VPA) Day 12, Nes/MSI1/NGN2 (-VPA) 0.74 0.11 2.60 0.28
1.98 0.97 0.55 0.01 Day 12, Nes/MSI1/NGN2 0.86 0.00 1.70 0.49 1.70
0.04 0.88 0.05 (+VPA) Day 12, Nes/MBD2 (-VPA) 0.76 0.12 3.15 0.17
0.87 0.03 0.44 0.00 Day 12, Nes/MBD2 (+VPA) 0.87 0.03 2.05 0.07
2.66 1.64 0.91 0.00 Day 12, Nes/Msi1/MBD2 (-VPA) 0.81 0.05 3.41
0.66 1.11 0.01 0.58 0.01 Day 12, Nes/Msi1/MBD2 1.01 0.13 2.43 0.07
3.27 0.26 0.93 0.02 (+VPA) Day 12, Nes/Ngn2/MBD2 (-VPA) 1.19 0.07
5.71 1.30 4.11 0.07 0.91 0.04 Day 12, Nes/Ngn2/MBD2 1.29 0.03 2.98
0.66 21.20 0.42 1.65 0.02 (+VPA)
[0304] Immunohistochemistry analysis performed in parallel with
RT-PCR data indicated that positive Sox2 cells were undetectable
when transfecting the cells with Msi1/Ngn2 in the presence of MBD1,
MBD3, MBD4, MeCP1, or AICADA (Table 6) and that among the different
types of DNA demethylator genes tested only MBD2 plays a
significant positive role in the reprogramming efficiency of HFF
towards NSLCs when using the above neurogenic genes.
Immunohistochemistry analysis revealed a small increase of
immunopositive Sox2 cells (89.49.+-.3.18) after co-transfecting the
cells with nestin and Msi1/Ngn2 in the presence of MBD2 (Table
6).
TABLE-US-00011 TABLE 6 Percentage of positive cells for Sox2 after
transfection of fibroblast cells with different expression vectors
with or without the presence the various DNA demethylators. After
transfection the cells were cultured in proliferation medium
(StemCell Technologies) supplemented by EGF (20 ng/ml, Peprotech)
and FGF (20 ng/ml, Peprotech) for two weeks at 37.degree. C./5%
CO.sub.2/5% O.sub.2. The percentage of immunopositive cells was
determined by Cellomics .TM. and represented as mean .+-. SD (n =
3-5). % Sox2 positive .+-. stdv HFF untransfected 0.13 .+-. 0.12
Msi-Ngn2 + MBD1 0.92 .+-. 0.13 Msi-Ngn2 + MBD2 79.44 .+-. 9.86
Msi-Ngn2 + MBD3 1.22 .+-. 0.82 Msi-Ngn2 + MBD4 0.59 .+-. 0.03
Msi-Ngn2 + MeCP2 1.10 .+-. 0.25 Msi-Ngn2 + AICDA 0.69 .+-. 0.28 Msi
+ MBD2 0.79 .+-. 0.28 Ngn2 + MBD2 1.74 .+-. 1.01 Nestin + Msi 0.91
.+-. 0.01 Nestin + Ngn2 2.16 .+-. 1.44 Nestin + MSI1/NGN2 + MBD2
89.49 .+-. 3.18 Nestin + MSI1/NGN2 10.20 .+-. 0.21 Nestin .+-. MBD2
0.00 .+-. 0.00 Nestin + Msi + MBD2 8.45 .+-. 0.08 Nestin + Ngn2 +
MBD2 5.71 .+-. 0.66
[0305] Another study was designed to test the effect of various
neurogenic genes on the reprogramming efficiency towards neural
stem-like cells. HFF cells were cultured as described in Example I,
and transfected using the Nucleofector.TM..RTM. 96-well
Shuttle.RTM. Device (Lonza) following procedure described in
Example IV, except for the untreated HFF control and the
untransfected HFF control (for determining the effect of the
complete media & compound treatments on the cells). The cells
that had been pre-treated with VPA and 5-Aza and the untreated
cells were transfected with the mixes of DNA as described in Table
7. The cells were plated on Laminin-coated plates and incubated at
37.degree. C., 5% CO.sub.2. Media was changed daily according to
Table 7. Cells were analysed at day 3, 7, 12 by
immunohistochemistry analysis and at Day 9 by gene array for
multipotent and pluripotent gene expression.
Gene Array Analysis
[0306] An additional batch of cells treated according to 0a and 1a
in Table 7 was analyzed at Day 9, along with HFFs, hNPCs, and
passage 5 NSLCs (frozen from previous experiments from Example III)
by the Pluripotency Gene Array (ABI) (Tables 8a and b) and a set of
genes (Table 8c) to determine the gene expression profile of select
pluripotency, ectoderm, endoderm, mesoderm, and neural lineage
genes in passage 1 and, passage 5 NSLCs compared to HFFs (from
which they were created) and normal human neuroprogenitor cells
(hNPCs). The results in Table 8 indicate that all the genes related
to neural stem cells (some of the significantly expressed
pluripotency markers and mesendoderm markers are also expressed in
neural stem cells) and the neural lineage were significantly
expressed in NSLCS as opposed to HFFs, and the expression pattern
was a bit different from hNPCs indicating that NSLCS are similar
to, but not identical, to the hNPCs tested. Passage 5 NSLCs 5 had a
higher expression of stemness genes than Passage 1 NSLCs. hNPCs had
a higher expression of neuronally committed genes than NSLCS,
indicting their neuroprogenitor status versus the greater stemness
status of NSLCs.
TABLE-US-00012 11 Untreated pCMV6-XL5-Sox2 + Neural proliferation
medium + Egf + Neural proliferation medium + Neural proliferation
medium + Egf + pCMV6-XL4-Ngn2 + Fgf-2 Egf + Fgf-2 Fgf-2
pCMV6-XL5-MBD2 12 Untreated pCMV6-XL5-Nanog + Neural proliferation
medium + Egf + Neural proliferation medium + Neural proliferation
medium + Egf + pCMV6-XL4-Ngn2 + Fgf-2 Egf + Fgf-2 Fgf-2
pCMV6-XL5-MBD2 13 Untreated pCMV6-XL4-Oct4 + Neural proliferation
medium + Egf + Neural proliferation medium + Neural proliferation
medium + Egf + pCMV6-XL4-Ngn2 + Fgf-2 Egf + Fgf-2 Fgf-2
pCMV6-XL5-MBD2 14 VPA + Msi1/Ngn2 Neural proliferation medium + Egf
+ Neural proliferation medium + Neural proliferation medium + Egf +
5-Aza Fgf-2 Egf + Fgf-2 Fgf-2 pre-treated 15 VPA +
pCMV6-XL5-Musashi Neural proliferation medium + Egf + Neural
proliferation medium + Neural proliferation medium + Egf + 5-Aza
Fgf-2 + VPA + 5-Aza Egf + Fgf-2 Fgf-2 pre-treated 16 VPA +
pCMV6-XL5-Musashi Neural proliferation medium + Egf + Neural
proliferation medium + Neural proliferation medium + Egf + 5-Aza
Fgf-2 + Noggin + VPA + 5-Aza Egf +Fgf-2 + Noggin Fgf-2 + Noggin +
Forskolin pre-treated 17 VPA + pCMV6-XL5-Musashi + Neural
proliferation medium + Egf + Neural proliferation medium + Neural
proliferation medium + Egf + 5-Aza pCMV6-XL5-MBD2 Fgf-2 + Noggin +
VPA + 5-Aza Egf + Fgf-2 + Noggin Fgf-2 + Noggin + Forskolin
pre-treated 18 VPA + pCMV6-XL4-Ngn2 Neural proliferation medium +
Egf + Neural proliferation medium + Neural proliferation medium +
Egf + 5-Aza Fgf-2 + Noggin + VPA + 5-Aza Egf + Fgf-2 + Noggin Fgf-2
+ Noggin + Forskolin pre-treated 19 VPA + pCMV6-XL5-MBD2 Neural
proliferation medium + Egf + Neural proliferation medium + Neural
proliferation medium + Egf + 5-Aza Fgf-2 + Noggin + VPA + 5-Aza Egf
+ Fgf-2 + Noggin Fgf-2 + Noggin + Forskolin pre-treated 20 VPA +
Ngn2 + pCMV6-XL5- Neural proliferation medium + Egf + Neural
proliferation medium + Neural proliferation medium + Egf + 5-Aza
MBD2 Fgf-2 +Noggin + VPA + 5-Aza Egf + Fgf-2 + Noggin Fgf-2 +
Noggin + Forskolin pre-treated 21 VPA + No plasmid Neural
proliferation medium + Egf + Neural proliferation medium + Neural
proliferation medium + Egf + 5-Aza Fgf-2 + Noggin + VPA + 5-Aza Egf
+ Fgf-2 + Noggin Fgf-2 + Noggin + Forskolin pre-treated *
Immunohistochemistry analysis performed in parallel with RT-PCR
data indicated among all the combinations in this experiment where
no cytochalasin B was used, positive Sox2 cells were detectable
only in cells transfected with Msi1/Ngn2 with and without MBD2.
indicates data missing or illegible when filed
TABLE-US-00013 TABLE 8a Results for Human Stem Cell Pluripotency
Array (n = 4 for each sample) - Embryonic Stem Cell Markers, Germ
Cell Markers and Trophoblast Markers. For Relative Expression
calculations, each sample was normalized to the average Ct of the 6
housekeeping genes (ACTB, 18S, CTNNB1, EEF1A1, GAPD, RAF1), and
calibrated to the Untreated HFF (Passage 8) control. Relative
Expression values with asterisk (*) indicate values with
significant up or down-regulation (>2-fold or <0.5-fold). For
these samples, for Ct values <35 is considered that the
expression of the gene is adequate for quantification. For the
Relative Expression values that are >2-fold or <0.5-fold but
without asterisk, the values could have significant error due to
the low expression of the gene (Ct > 35), and thus the up or
down-regulation could be merely a result of the high standard
deviation of the high Ct values of the genes, or fluctuations in
the housekeeping genes. As for the Relative Expression values that
are between 0.5-fold and 2-fold, it indicates no significant change
in the expression of the gene for these samples. MSI1/NGN2/MBD2-
transfected HFF Neural stem-like cells, Untreated HFF Untransfected
HFF hNPC neurospheres (Day 9) NSLC (Passage 8) (Day 9) (Passage 4)
(NSLC, Passage 1) (Passage 5) Gene Rel. Exp. Std. Dev. Rel. Exp.
Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std.
Dev. Embryonic Stem cell markers BRIX 1.03 0.30 0.47 0.10 0.78 0.22
0.78 0.25 0.83 0.10 CD9 1.01 0.18 2.46* 0.62 1.86 0.29 2.24* 0.19
1.00 0.39 COMMD3 1.08 0.53 0.94 0.36 0.94 0.40 0.98 0.40 1.05 0.59
DNMT3B 1.07 0.50 0.34* 0.14 2.96* 0.84 1.90 0.41 0.35 0.34
EBAF/LEFTY2 1.00 0.00 2.10 0.00 7.95 4.60 7.79 4.88 70.56* 26.12
FGF4 1.00 0.00 2.10 0.00 1.44 0.00 1.54 0.00 1.37 0.00 FOXD3 1.00
0.00 2.10 0.00 1.44 0.00 7.13 11.18 222.41* 63.43 GABRB3 1.06 0.38
4.22* 0.71 66.65* 12.52 40.01* 4.54 1.62 0.98 GAL 1.00 0.04 9.73*
0.32 0.03* 0.01 4.25* 0.46 2.89* 0.83 GBX2 1.00 0.09 0.04 0.05
45.28* 4.59 90.92* 12.14 55.22* 2.36 GDF3 1.00 0.00 2.10 0.00 1.44
0.00 1.54 0.00 1.37 0.00 GRB7 1.02 0.24 0.30* 0.16 0.05* 0.04 0.29*
0.08 0.06* 0.08 IFITM1 1.01 0.17 63.96* 6.04 0.04* 0.01 21.80* 4.31
3.35* 0.63 IFITM2 1.00 0.12 3.84* 0.89 0.02* 0.00 0.65 0.11 0.43*
0.09 IL6ST 1.01 0.21 2.19* 0.39 0.85 0.14 1.59 0.26 0.75 0.06 IMP2
1.11 0.66 1.65 0.92 1.06 0.48 0.78 0.26 1.96 0.97 KIT 1.02 0.26
1.15 0.30 0.02* 0.00 0.31* 0.09 0.00* 0.00 LEFTB 1.61 1.15 12.28*
7.84 5.45 3.15 5.58 2.65 8.96* 4.12 LIFR 2.29 3.57 13.51 16.55 6.31
7.24 12.98 9.81 2.85 4.31 LIN28 4.69 8.62 5.25 8.88 28.38* 19.25
26.97* 8.68 32.13* 14.32 NANOG 1.71 1.97 18.61 16.43 64.94* 28.32
70.87* 9.88 5.87 3.52 NOG 1.03 0.27 0.18* 0.08 0.18* 0.06 0.22*
0.06 0.02* 0.00 NR5A2 2.04 2.05 6.85 8.80 0.38 0.00 3.89 4.36 0.36
0.00 NR6A1 1.11 0.66 1.37 0.31 5.08* 0.37 2.71* 0.63 2.04* 0.17
PODXL 1.00 0.07 0.01* 0.01 0.80 0.11 2.09* 0.04 6.49* 0.64 POU5F1
1.01 0.13 0.27* 0.17 0.89 0.09 0.71 0.09 0.19* 0.06 PTEN 1.00 0.02
2.68* 0.29 0.87 0.04 1.07 0.12 0.80 0.14 RESET 1.01 0.12 1.53 0.17
0.94 0.18 1.04 0.21 1.10 0.24 SEMA3A 1.00 0.11 1.99 0.19 0.66 0.05
1.05 0.11 0.90 0.16 SFRP2 1.11 0.56 122.57* 14.57 3480.98* 702.37
1500.84* 272.46 2.75 2.85 SOX2 1.00 0.00 2.45 0.70 127594.46*
11326.91 88615.76* 15003.70 137424.37* 26622.02 TDGF1 1.41 1.28
2.92 0.68 6.13 1.52 5.46 1.95 2.20 1.51 TERT 1.00 0.00 2.10 0.00
10.81 18.75 10.74 18.41 6506.88* 893.84 TFCP2L1 1.00 0.00 2.10 0.00
7.84 12.80 32.49 10.01 1.37 0.00 UTF1 1.00 0.00 8.21 12.23 27.86
19.24 1.54 0.00 30.68 25.94 XIST 1.00 0.00 2.10 0.00 24609.46*
4337.83 22637.95* 3988.10 1.37 0.00 ZFP42 1.24 1.06 12.38 12.58
1.41 0.78 2.01 1.85 1.76 0.93 Germ cell markers DDX4 1.00 0.00 2.10
0.00 1.44 0.00 5.84 8.60 19.11 20.49 SYCP3 1.58 1.95 11.97 8.01
11.12 3.46 15.46 11.65 2.25 2.85 Trophoblast markers CDX2 1.00 0.00
2.10 0.00 1.44 0.00 1.54 0.00 1.37 0.00 CGB 1.02 0.24 2.08* 0.74
0.15* 0.16 0.57 0.41 0.09* 0.17 EOMES 1.51 1.14 0.33 0.00 0.71 0.97
0.24 0.00 0.77 1.12 GCM1 2.61 2.80 0.42 0.00 3.25 5.92 5.68 1.44
1.47 2.38 KRT1 1.00 0.00 2.10 0.00 1.44 0.00 1.54 0.00 1.37
0.00
TABLE-US-00014 TABLE 8b Results for Human Stem Cell Pluripotency
Array (n = 4 for each sample) - Ectoderm, Endoderm and Mesoderm
Markers. For Relative Expression calculations, each sample was
normalized to the average Ct of the 6 housekeeping genes (ACTB,
18S, CTNNB1, EEF1A1, GAPD, RAF1), and calibrated to the Untreated
HFF (Passage 8) control. Relative Expression values with asterisk
(*) indicate values with significant up or down-regulation
(>2-fold or <0.5-fold). For these samples, for Ct values
<35 is considered that the expression of the gene is adequate
for quantification. For the Relative Expression values that are
>2-fold or <0.5-fold but without asterisk, the values could
have significant error due to the low expression of the gene (Ct
> 35), and thus the up or down-regulation could be merely a
result of the high standard deviation of the high Ct values of the
genes, or fluctuations in the housekeeping genes. As for the
Relative Expression values that are between 0.5-fold and 2-fold, it
indicates no significant change in the expression of the gene for
these samples. MSI1/NGN2/MBD2- transfected HFF Neural stem-like
Untreated HFF Untransfected hNPC neurospheres (Day 9) cells, NSLC
(Passage 8) HFF (Day 9) (Passage 4) (NSLC, Passage 1) (Passage 5)
Gene Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev.
Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Ectoderm markers CRABP2
1.04 0.35 26.14* 4.28 0.01* 0.01 21.11* 2.80 0.21* 0.05 FGF5 1.01
0.15 0.21* 0.07 0.00* 0.00 0.10* 0.02 0.00* 0.00 GFAP 1.22 0.84
9.89* 5.46 798.04* 162.37 487.99* 79.84 12052.09* 2984.71 ISL1 1.01
0.12 2.19* 0.27 0.02* 0.02 0.42* 0.08 0.00* 0.00 NES 1.10 0.58
3.19* 0.95 6.78* 0.95 3.84* 0.19 7.47* 0.54 NEUROD1 1.00 0.00 2.10
0.00 1.44 0.00 2.32 1.57 25.54 6.42 OLIG2 1.00 0.00 2.10 0.00
124181.50* 14735.13 80826.42* 27820.32 36172.45* 3145.67 PAX6 1.11
0.48 0.06* 0.00 533.31* 120.59 326.02* 33.14 371.42* 77.50 SYP 1.02
0.25 5.22* 2.10 229.40* 22.54 143.94* 17.41 16.48* 4.47 TH 1.00
0.00 9.52 14.86 1218.08* 186.74 217.79* 45.71 348.31* 150.50
Endoderm markers AFP 1.00 0.00 2.10 0.00 1.44 0.00 1.54 0.00 1.37
0.00 FN1 1.00 0.06 1.41 0.10 0.02* 0.00 1.96 0.19 0.00* 0.00 FOXA2
1.00 0.00 150.00* 55.92 1.44 0.00 1.54 0.00 1.37 0.00 GATA4 1.00
0.00 11.93 19.67 7.22 11.56 9.14 12.35 1.37 0.00 GATA6 1.00 0.09
0.37* 0.17 0.00* 0.00 0.44* 0.04 0.02* 0.01 GCG 1.00 0.00 7.96
11.74 1.44 0.00 33.59* 22.17 1.37 0.00 IAPP 1.00 0.00 2.10 0.00
1.44 0.00 1.54 0.00 1.37 0.00 INS 1.00 0.00 2.10 0.00 1.44 0.00
12.67 22.26 1.37 0.00 IPF1 1.00 0.00 2.10 0.00 1.44 0.00 1.54 0.00
1.37 0.00 LAMA1 1.00 0.11 4.42* 0.86 78.49* 6.82 43.99* 2.79 46.49*
16.59 LAMB1 1.02 0.26 12.51* 2.40 0.29* 0.09 2.27* 0.77 3.89* 1.12
LAMC1 1.00 0.10 2.82* 0.10 1.54 0.33 3.01* 0.94 1.31 0.30 NODAL
1.00 0.00 12.16 11.62 16.27 11.25 1.54 0.00 1.37 0.00 PAX4 1.00
0.00 6.77 9.35 1.44 0.00 1.54 0.00 1.37 0.00 PTF1A 1.00 0.00 2.10
0.00 1.44 0.00 1.54 0.00 1.37 0.00 SERPINA1 1.03 0.30 0.79 0.53
0.24 0.00 1.52 1.17 0.99 0.68 SOX17 1.00 0.00 2.10 0.00 1.44 0.00
1.35 5.63 1.37 0.00 SST 1.25 1.00 52.58* 10.67 0.55 0.36 48.97*
8.70 0.92 0.42 TAT 1.00 0.00 2.10 0.00 255.86* 84.52 106.04* 45.87
1.37 0.00 Mesoderm markers ACTC 1.04 0.35 0.01* 0.00 0.02* 0.01
0.05* 0.01 0.01* 0.01 CD34 1.67 1.69 501.85* 61.88 45.17* 27.01
113.96* 39.39 13203.40* 5385.80 CDH5 1.00 0.00 4.12 4.06 16.69 8.07
32.41* 20.31 13447.65* 3220.80 COL1A1 1.01 0.12 2.28* 0.41 0.00*
0.00 0.50* 0.05 0.02* 0.00 COL2A1 3.56 6.27 103.52* 37.78 1813.86*
236.76 873.19* 259.80 3815.72* 839.02 DES 1.00 0.07 1.94 0.33 1.09
0.33 0.87 0.07 0.22* 0.08 FLT1 1.01 0.15 0.68 0.29 0.00 0.00 0.46*
0.05 0.00* 0.00 HBB 3.08 4.01 0.39 0.00 0.27 0.00 0.29 0.00 0.26
0.00 HBZ 1.14 0.63 3.53 1.32 0.25 0.22 0.61 0.63 2.88 1.20 HLXB9
1.00 0.00 2.10 0.00 59.80* 16.35 24.94 3.14 35.12 40.50 MYF5 1.77
1.87 0.69 0.00 0.47 0.00 0.51 0.00 0.45 0.00 MYOD1 1.71 2.27 1.22
0.00 0.83 0.00 0.89 0.00 0.80 0.00 NPPA 1.00 0.00 2.10 0.00 96.60*
76.23 18.97 26.98 32.37 10.96 PECAM1 1.00 0.00 1041.24* 150.95
31.30* 24.22 964.70* 200.82 7305.03* 1127.69 RUNX2 1.01 0.12 1.76
0.37 0.09* 0.02 0.78 0.23 1.18 0.27 T 1.00 0.00 2.10 0.00 1.44 0.00
1.54 0.00 1.37 0.00 WT1 2.09 3.13 1.11 0.00 0.76 0.00 2.72 3.82
4.24 4.21
TABLE-US-00015 TABLE 8c Results for relative expression of
Embryonic Stem Cell, Ectoderm, Endoderm/mesoderm, and neuronal
markers in untransfected and transfected HFF with Msi1/Ngn2/MBD2
calibrated to untreated HFF (passage 8). Genes with asterisk (*)
indicate that the Ct values of the test samples are within the
quantifiable range (Ct < 35), suggesting the expression of the
gene in the test sample is adequate for quantification. For genes
without asterisk, the values may be inaccurate due to the low
expression of the gene (Ct > 35) and thus the up or down-
regulation is merely a result of the high standard deviation of the
high Ct values of the genes, or fluctuation of the housekeeping
genes; the trend for these samples may be correct, but the absolute
relative expression values may not. Expression of NGN3 and LIN28
were also tested but these two genes were not expressed in any of
the test samples (data not shown). RT-PCR revealed a significant
increase of ectoderm and neuronal markers. MSI1/NGN2/MBD2- Neural
stem-like cells, Untreated HFF Untransfected hNPC neurospheres
transfected HFF (Day 9) NSLC (Passage 8) HFF (Day 9) (Passage 4)
(NSLC, Passage 1) (Passage 5) Gene Rel. Exp. Std. Dev. Rel. Exp.
Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std.
Dev. Embryonic Stem Cell Markers OCT4* 1.04 0.38 7.27 0.81 6.26
0.05 6.63 0.51 3.15 0.58 OCT4 (5'UTR) 1.04 0.41 0.08 0.00 2.07 0.11
1.82 0.53 0.55 0.59 NANOG (5'UTR) 1.02 0.32 19.29 2.23 11.27 0.89
16.73 6.86 9.94 6.32 FBXO15* 1.05 0.46 2.58 0.45 3.57 0.23 5.89
1.22 1.13 0.39 ALPL* 1.03 0.33 0.57 0.73 652.20 46.60 194.23 10.82
13.04 4.04 SALL4* 1.02 0.25 9.20 1.35 9.76 0.62 15.84 0.92 2.35
0.55 NR0B1 (DAX1)* 1.01 0.19 18.62 4.70 2.64 0.11 11.59 3.17 0.06
0.00 Ectoderm Markers ZIC1* 1.01 0.24 2.01 0.25 1889.80 93.48
1158.21 80.43 156.40 12.64 SOX1* 1.00 0.01 2.05 0.06 1776.83 128.63
1052.75 243.07 47.98 2.12 CDH1 (E-cadherin)* 1.00 0.01 2.05 0.06
264.59 6.22 59.14 7.57 18.20 3.73 p63 1.00 0.01 68.37 72.49 18.01
5.33 39.72 12.76 37.83 6.76 MSX1 1.00 0.05 4.19 0.56 0.10 0.01 1.53
0.35 0.09 0.00 NOTCH1* 1.00 0.07 1.26 0.08 7.38 1.20 4.51 0.54 4.75
0.26 SOX2* 1.00 0.01 2.50 0.57 340909.59 5659.15 194495.82 17929.15
219269.76 31399.68 SOX2 (3'UTR)* 1.00 0.01 7.74 8.11 864191.09
60204.44 452684.80 26457.70 618245.01 7107.48 Mesoderm/Endoderm
Markers CXCR4* 1.05 0.46 12.45 5.64 5048.23 172.14 2763.82 30.29
3773.11 78.89 Neuronal markers MAP2* 1.01 0.17 2.98 0.20 155.33
9.08 88.82 6.48 27.38 0.13 TUBB3* 1.00 0.04 0.38 0.02 1.15 0.05
0.89 0.05 0.98 0.09 ASCL1 (MASH1)* 1.29 1.16 11.19 0.22 42618.46
68.52 23554.16 1588.45 31358.79 2301.26 NGN2* 1.00 0.01 2.05 0.06
19.45 6.64 247883.48 16409.80 968.11 191.73 NGN2 (3'UTR)* 1.83 2.17
1.17 0.76 13.39 5.10 8.45 1.75 539.02 59.72 MSI1* 1.00 0.01 263.87
70.10 100376.36 81.45 479098.05 2281.62 116105.29 2745.03 MSI1
(3'UTR)* 1.01 0.20 13.61 2.00 3601.96 345.79 2163.87 59.84 3698.14
160.78 ACHE* 1.00 0.00 2.00 0.26 25.00 3.71 12.84 0.84 21.30 0.30
Glia markers CNP* 1.01 0.18 1.43 0.10 3.48 0.58 2.69 0.12 1.93 0.07
SOX9* 1.00 0.04 3.54 0.06 88.25 9.71 41.11 2.70 26.96 0.53 Note
that custom primers (5'UTR) for detecting endogenous gene
expression are generally not as sensitive and/or effective as
standard primers (from the supplier's (Origene) catalog) that dtect
overall gene expression (both endogenous and exogenous) of a
particular gene.
[0307] In another part of the experiment, another batch of cells
that were transfected with Msi1/Ngn2 pCMV6-XL5-MBD2 were plated on
Poly-Ornithine (30 min at RT) and Laminin (1 h at RT) coated plates
in CDM II medium in 5 different wells. On day 1, medium in two of
the wells was switched to the same medium as in condition 1a (Table
7) until day 12. Medium was changed daily until day 12, at which
point it was switched to either NS-A Differentiation Medium
(StemCell Technologies) or NbActive4 (BrainBits.TM.) medium that
were both supplemented with BDNF (20 ng/ml), NT-3 (20 ng/ml), NGF
(20 ng/ml), Retinoic acid (5 .mu.M), Noggin (20 ng/ml) and
Forskolin (10 .mu.M). These cells showed a typical neural stem-like
cell morphology by day 7, and proliferated until day 12. During the
exposure to either of the two differentiation media, these NSLC
changed to a more neuronal and glial phenotype as shown in the
bright field pictures, but only expressed GFAP by Day 17.
[0308] For the other three wells, on day 1 medium was switched to
either NS-A Differentiation Medium (StemCell Technologies),
NbActive4 (BrainBits), or CDM II medium; these first two were
supplemented with the same cytokines as previously described but
with the addition of Fgf-2 (20 ng/ml). On day 12, Fgf-2 was removed
from the first two differentiation media while cells in the CDM II
medium were switched to the NS-A Differentiation Medium (StemCell
Technologies) supplemented with cytokines without Fgf-2. Between
day 12 and day 17, media was changed every two to three days.
During the first 12 days of culture, cells in all 3 media developed
into a mix of more spindle shaped cells compared to untransfected
fibroblasts and some into cells with a NSLC morphology; upon
removal of Fgf-2 cell morphology turned into a very pronounced
neuronal shape as well as glial cells with a network established
between cells as shown in the bright field pictures that expressed
GFAP and Bill-tubulin by Day 17.
[0309] An additional study was designed to assess the effect of
Msi1, Ngn2 and MBD2 on their endogenous proteins levels in
reprogrammed cells. Cells were transfected with the MSI1/NGN2
vector and MBD2 as previously described and cultured in
proliferation condition at 37.degree. C., 5% CO.sub.2 and 5%
O.sub.2. Samples were collected at various time points from Day
2-10 and analyzed by RT-PCR to investigate the expression of
endogenous genes and the expression of neural stem cell and
neuronal genes at different time points. RT-PCR revealed a gradual
loss of total Msi1 Ngn2 and MBD2 gene expression starting from Day
2 to Day 10, with the increase in MBD2 expression relative to
control having been almost completely lost by Day 5. This decrease
was associated with a significant activation of endogenous Msi1 and
Ngn2 on Day 5, with another jump in endogenous gene expression at
Day 9 (Table 9). A significant increase in Sox2 expression was
detected at Day 4, and the expression of this ectoderm/neural stem
cell I neuronal gene continued to increase with each subsequent
timepoint (Table 10). GFAP (a neural stem cell and astrocyte
marker) was slightly elevated already from Day 2 onwards, but
significantly increased on Day 5 with a large jump in gene
expression at Day 7 analysis timepoint and stayed at this
expression level for the rest of the study period. Expression of
the neural stem cell marker Nestin also started to slowly increase
from Day 5 onwards. Expression of the neuronal genes
.beta.III-tubulin (TUBB3) and Map2b were slightly elevated already
from Day 2 onwards, but significantly increased on Day 5 onwards.
Expression of a marker for acetylcholine receptors (found in
neurons), acetylcholine esterase (ACHE), was also slightly elevated
from Day 2 onwards, but did not significantly increased until Day 7
onwards. It should be noted that among the neural stem cell markers
that were analyzed, the relative expression of Sox2 was highly and
early expressed which could then be directly or indirectly interact
with the exogenous Msi/Ngn2 and/or other genes in the activation of
Nestin, GFAP, and endogenous Msi1 and Ngn2 and other genes that
promote the reprogramming and cell fate change, as well as the
activation of neuronal genes like 3111-tubulin (TUBB3), Map2b, and
ACHE.
TABLE-US-00016 TABLE 9 RT-PCR analysis of exogenous and endogenous
relative expression of Msi1, Ngn2 and MBD2 from Day 2-10 after
transfection of fibroblast cells with pCMV-Msi1-Ngn2(Msi1/Ngn2) and
MBD2 and cultured for 10 days in proliferation medium. Cells were
collected at different time point to analyse endogenous gene
expression. Endogenous Endogenous Endogenous MSI1 MSI1 NGN2 NGN2
MBD2 MBD2 Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel.
Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. #1
Day12 1.01 0.18 1.04 0.38 1.01 0.15 1.01 0.15 1.01 0.21 1.00 0.14
Untransfected HFF #2 Day12 HFF 1102.17 91.80 620.56 19.49 2208.36
375.09 51.09 14.69 1.09 0.00 0.83 0.06 Msi1/Ngn2 + MBD2 #3 Day18
HFF 1470.36 164.35 950.07 152.50 71.57 52.59 122.66 39.63 1.21 0.02
0.73 0.08 Msi1/Ngn2 + MBD2 #4 Untransfected 1.49 N/A 1.01 N/A 1.00
N/A 1.00 N/A 1.02 N/A 1.00 N/A Keratinocytes #5 Day 12 4142.78
872.87 364.20 60.90 4656.42 232.63 102.01 3.18 0.40 0.14 0.74 0.30
Keratinocytes Msi1/Ngn2 + MBD2 #6 Day 18 4830.20 291.17 486.38
19.59 50.01 6.99 43.08 13.78 0.40 0.01 0.67 0.01 Keratinocytes
Msi1/Ngn2 + MBD2 #7 Untransfected CD34+ 1.01 0.19 1.00 0.01 1.01
0.16 1.17 0.87 1.00 0.02 1.00 0.07 #8 Day 18 CD34+ 3969.52 286.36
147.99 7.08 2.03 0.55 3.72 1.23 0.43 0.06 0.90 0.18 Msi1/Ngn2 +
MBD2 hNPC (14-Oct-09, 7574.57 234.74 1141.14 49.15 8.18 5.64 6.27
5.19 0.58 0.00 2.35 0.03 EXP0067)
TABLE-US-00017 TABLE 10 RT-PCR analysis of relative expression of
Nestin, Map2b, TUBB3, ACHE, GFAP, and Sox2 from Day 2-10 after
transfection of fibroblast cells with pCMV-Msi1-Ngn2 (Msi1/Ngn2)
and MBD2 and cultured for 10 days in proliferation medium. Cells
were collected at different time point to analyse endogenous gene
expression. NES MAP2 TUBB3 ACHE GFAP SOX2 Rel. Std. Rel. Std. Rel.
Std. Rel. Std. Rel. Std. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp.
Dev. Exp. Dev. Rel. Exp. Dev. #1 Untransfected Day2 1.00 0.04 1.00
0.01 1.00 0.03 1.00 0.08 1.01 0.23 1.17 0.87 #2 Msi1/Ngn2 +
MBD2/+Noggin 0.88 0.01 8.59 0.18 1.38 0.03 5.71 1.06 4.56 0.08 1.26
0.82 Day2 #3 Untransfected Day3 1.38 0.07 0.66 0.03 0.40 0.02 1.36
0.06 1.95 0.38 2.34 2.29 #4 Msi1-Ngn2 + MBD2/+Noggin 1.39 0.08 4.31
0.24 0.79 0.09 6.03 0.60 4.66 0.02 0.96 0.10 Day3 #5 Untransfected
Day4 2.43 0.23 1.78 0.11 0.44 0.01 2.70 0.02 3.76 0.86 0.93 0.01 #6
Msi1/Ngn2 + MBD2/+Noggin 1.91 0.06 2.81 0.20 0.64 0.02 6.76 0.64
8.67 1.06 5.37 6.06 Day4 #7 Untransfected Day5 1.40 0.05 1.13 0.04
0.41 0.03 1.17 0.37 5.44 0.02 15.03 8.77 #8 Msi1-Ngn2 +
MBD2/+Noggin 4.31 0.08 71.60 6.43 1.34 0.01 7.60 0.18 42.28 2.94
66377.25 4089.77 Day5 #9 Untransfected Day7 2.24 0.00 4.02 0.15
1.22 0.05 1.10 0.48 7.61 1.24 1.34 0.02 #10 Msi1/Ngn2 +
MBD2/+Noggin 3.07 0.11 48.10 2.85 2.70 0.05 13.11 1.30 3271.10
149.81 44255.59 2004.08 Day7 #11 Untransfected Day9 4.37 0.23 14.55
0.96 1.75 0.14 3.35 0.36 15.95 0.23 429.09 119.98 #12 Msi1-Ngn2 +
MBD2/+Noggin 7.97 0.16 123.55 3.27 2.79 0.12 16.59 0.03 3152.25
3.31 114149.70 3372.20 Day9 #13 Untransfected Day10 3.48 0.44 10.03
0.37 1.63 0.21 3.20 0.81 5.64 1.92 14.66 5.03 #14 Msi1/Ngn2 +
MBD2/+Noggin 7.48 0.22 100.25 6.66 2.87 0.03 17.49 1.35 3374.03
22.47 101105.49 3996.44 Day10
Example IV
[0310] Comparison of the Nucleofector.TM..RTM. II Device and the
Nucleofector.TM..RTM. 98-Well Shuttle.RTM. Device in the
Reprogramming of HFF into NSLC in Adherent and Floating
Conditions
[0311] HFF cells were cultured as described in Example 1, and
transfected using the Nucleofector.TM..RTM. II Device (Lonza) as
previously described in Example II or the Nucleofector.TM..RTM.
96-well Shuttle.RTM. Device (Lonza). The HFFs were harvested with
TrypLE.TM. (Gibco), and 1.times.10.sup.6 cells/transfection with
the Nucleofector.TM..RTM. II Device for 10 min at 90 g and
6.times.10.sup.5 cells transfection with the Nucleofector.TM..RTM.
96-well Shuttle.RTM. Device for 5 min at 80.times.g. After
centrifugation, the cell pellet was gently resuspended in either
100 .mu.l of Basic Nucleofector.TM. Solution for the
Nucleofector.TM..RTM. II or 20 .mu.l of SE Solution (Cell line kit.
SE, Lonza) for the Nucleofector.TM..RTM. 96-well Shuttle.RTM.. For
the Nucleofector.TM..RTM. II Device, each 100 .mu.l of cell
suspension was combined with 2 different mixes of plasmid DNA
(sample 1 was mixed with 2 .mu.g of pCMV6-XL5-Msi1 and 2 .mu.g
pCMV6-XL5-MBD2, and sample 2 with 2 .mu.g of Msi1/Ngn2 and 2 .mu.g
pCMV6-XL5-MBD2). Each cell suspension was transferred into an Amaxa
certified cuvette and transfected with the appropriate program
(U-023). Right after transfection, 900 .mu.l of warm CDM1 medium
was added to each cuvette and the sample was transferred into a
culture plate coated with Laminin (Stemgent, 10 .mu.g/ml) at a cell
density of 1.times.10.sup.5 to 1.5.times.10.sup.5 cells per
cm.sup.2 or into non-cell culture treated Petri dishes for
neurosphere formation. The cells were incubated at 37.degree. C.,
5% CO.sub.2 overnight. However for the Nucleofector.TM..RTM.
96-well Shuttle.RTM. Device, the steps described before were
similar with the following exceptions: the cell suspension was
mixed with 0.6 .mu.g of each DNA of the same 2 DNA mixes, the cell
suspension was transferred to a well of a 96-well Nucleoplate.TM.
(Lonza) and transfected with the program FF-130.TM.. After
transfection, 80 .mu.l of warm CDM1 medium was added to each well
and the samples were left for 10 min in the incubator prior to
being transferred into a laminin coated plate or non-cell culture
treated Petri dishes at the same cell density as previously
mentioned. For both devices, these steps were repeated for each
sample that was transfected. Prior to transfection cells were
cultured in CDM1 as described in Example I. After 24 hours, the
medium was switched to a mix of 75% CDM medium and 25%
Proliferation Medium which was supplemented with EGF (20 ng/ml),
FGF-2 (20 ng/ml), Noggin (20 ng/ml) and Cytochalasin B (10
.mu.g/ml) and the cells were incubated at 37.degree. C., 5%
CO.sub.2 and 5% O.sub.2. The medium was changed daily with an
increased proportion of Neural proliferation medium up to 100% by
Day 4 and a decreased proportion of Cytochalasin B that was
completely omitted by Day 5. Forskolin (10 .mu.M) was added to the
medium from Day 4 onwards. The cells in floating conditions were
pelleted by centrifugation and their medium changed daily as
described for the adherent condition. Cells were collected at Day
3, 7, and 12 for immunohistochemistry analysis.
[0312] Fluorescence images were taken with a Cellomics.TM.
ArrayScan HCS Reader.TM. microscopy system to determine an estimate
of the percentage of cells positive for Sox2, a neural stem cell
marker. This analysis revealed that in untransfected controls and
at 3 days after transfection, no nuclear Sox2 staining was
detectable. However, at Day 7 and Day 12 the percentage of Sox2
positive cells increased progressively under all transfection
conditions except the pCMV6-XL5-Musashi and pCMV6-XL5-MBD2
Nucleofector.TM..RTM. II condition. The highest percentage at Day
1.2 was obtained with Msi1/Ngn2 and pCMV6-XL5-MBD2 transfected with
the Nucleofector.TM..RTM. 96-well Shuttle.RTM. Device (.about.80%).
The same combination transfected with the Nucleofector.TM..RTM. II
yielded only .about.35% positive cells. The pCMV6-XL5-Musashi and
pCMV6-XL5-MBD2 with the Shuttle.RTM. produced .about.20% positive
cells, while generally none were observed with the
Nucleofector.TM..RTM. II. The percentage of positive cells varied
strongly between wells. The staining indicated that the cell
population was not homogenous, since fields of densely arranged
Sox2 positive cells and complete fields with only negative cells
could be found in all cases. In general the Shuttle.RTM. was
initially more toxic to cells than the Nucleofector.TM..RTM.
however at least in the case of Msi1/Ngn2 and pCMV6-XL5-MBD2
shuttle, the Sox2 positive population rapidly expanded from Day 7
to Day 12 to have twice as many Sox2 positive cells as compared to
the Nucleofector.TM..RTM. II. The cells in floating conditions did
not form spheres during the 12 day experiment in any of the
conditions, suggesting that the formation of neurospheres requires
either the generation of neural stem-like cells in adherent
conditions first or more time.
[0313] Table 11 shows the percentage of Sox2 positive cells with a
typical neural stem cell morphology using both the
Nucleofector.TM..RTM. II Device and the Nucleofector.TM..RTM.
96-well Shuttle.RTM. Device. The latter had the advantages of
requiring a smaller starting material (less cells and less DNA
required) and in addition gave rise to a higher number of Sox2
positive cells. Moreover a very small population of Sox2 positive
cells was observed with the Shuttle.RTM. Device only upon
transfection with only one neurogenic transcription factor (Msi) in
the presence of the DNA demethylator MBD2.
TABLE-US-00018 TABLE 11 Percentage of positive cells for Sox2 after
transfection of fibroblast cells with different expression vectors.
After transfection the cells were cultured in proliferation medium
(StemCell Technologies) supplemented by EGF (20 ng/ml, Peprotech)
and FGF (20 ng/ml, Peprotech) for two weeks at 37.degree. C./5%
CO.sub.2/5% O.sub.2. The percentage of immunopositive cells was
determined by Cellomics .TM. and represented as mean .+-. SD (n =
3-5). Day3 Day7 Day12 Total Total Total Cell Cell Cell Sox2 count
Sox2 count Sox2 count Shuttle MSI1/ 1.34 .+-. 0.10 6430 .+-. 566 31
.+-. 20 .+-. 8.03 10683 .+-. 1112 78.17 .+-. 3.10 29341 .+-. 2527
NGN2 + MBD2 Msi + MBD2 1.08 .+-. 0.61 8253 .+-. 399 3.19 .+-. 3.57
8953 .+-. 672 19.05 .+-. 17.88 11082 .+-. 2999 Nucleofector .TM.
MSI1/ 0.87 .+-. 0.30 21870 .+-. 4476 14.30 .+-. 1.83 37321 .+-.
6877 35.93 .+-. 7.10 33009 .+-. 1567 NGN2 + MBD2 Msi + MBD2 0.64
.+-. 0.07 46793 .+-. 8808 0.35 .+-. 0.16 34854 .+-. 2186 0.51 .+-.
0.25 32095 .+-. 3236
Example V
Neurosphere Formation Assay and Cell Differentiation Analysis
[0314] Based on previous studies showing that greater proportional
reprogramming is achieved: by transfecting two neurogenic genes,
this study was designed to evaluate the number of reprogramming
cells by using, the vector Msi1/Ngn2, containing two neurogenic
transcription factors (Msi1 and Ngn2) and the role of DNA
demethylator or DNA methylation inhibitor (5-azacytidine) and
histone deacetylation inhibitor (VPA) in the reprogramming
process.
[0315] HFFs were cultured and treated with cytochalasin B as
described in Example 111, and treated simultaneously with VPA (1
mM) and 5-Azacytidine (0.5 .mu.M). After two days of treatment,
cells were transfected by Nucleofection as described in Example II
with the constructed vector Msi1/Ngn2. After preparing the cells,
they were mixed with 2 .mu.g of total DNA (Msi1/Ngn2) and cells
that had not been treated with chemical inhibitors (VPA and 5-Aza)
were co-transfect with MBD2 (2 .mu.g), using the appropriate
program (UO23). The samples were transferred into a coated culture
plate with Laminin (10 .mu.g/ml, Sigma) and incubated in a
humidified 37.degree. C./5% O.sub.2/5% CO.sub.2 incubator. The
medium was changed to the proliferation basal media, Neural
Proliferation Medium (NeuroCult.TM. proliferation Kit, StemCell
Technologies), with the presence of Noggin (20 ng/ml, Peprotech),
recombinant hFGF (20 ng/ml, Peprotech), and recombinant hEGF (20
ng/ml, Peprotech). Following 6 days of transfection, cells were
harvested using Accutase.TM. (Millipore), centrifuged (300.times.g,
5 min, RT) and plated in uncoated cell culture dishes in
NeuroCult.TM. NSC Proliferation medium to investigate the capacity
to grow cells in suspension as neurospheres or on Laminin
coated-plates for adherent culture. To prevent loss of floating
spheres during media changes, cells were sedimented by
centrifugation at 150.times.g for 3 min at room temperature (RT).
The pellet was then resuspended in fresh medium and plated into new
uncoated, low-bind cell culture dishes. Cultures were incubated at
37.degree. C., 5% CO.sub.2, 5% O.sub.2 and were fed daily for at
least two months.
[0316] To investigate whether a single cell from human neural
precursor cells (hNPCs) and human NSLCs was able to generate a
neurosphere (a standard test for proving that a cell is a neural
stem cell), neurospheres were dissociated into single cells and
these single cells were isolated and cultured in proliferation
medium in suspension, and neurosphere formation was monitored by
taking bright field images using light microscope (Nikon,
10.times.) and by Cellomics.TM.. These cells started to proliferate
and grew as spheres starting day 6 to day 10. Immunohistochemistry
analysis of these spheres on Day 20, revealed immunopositive
staining for the neural stem cells markers Sox2, Musashi, CD133,
Nestin, and GFAP. Cells also stained positive for .beta.III-tubulin
(a marker for neurons), O4 (a marker for oligodendrocytes), and
GFAP (a marker for astrocytes), indicating the tri-potent
differentiation potential of both sets of cells (NSLC and hNPC),
and negative for NGFrec and NeuN (markers for differentiated
neurons) indicating that the cells were not terminally
differentiated.
TABLE-US-00019 TABLE 12 Percentage of positive cells for neural
stem cells, and neuronal, astrocyte and oligodendrocyte lineage
markers in neurospheres formed from single NSLCs and hNPCs cultured
in proliferation medium (StemCell Technologies) supplemented by EGF
(20 ng/ml, Peprotech) and FGF (20 ng/ml, Peprotech) for 20 days at
37.degree. C./5% CO.sub.2/5% O.sub.2. The percentage of positive
cells was determined by Cellomics .TM. and represented as mean .+-.
SD. % of positive cells NSLCs hNPCs Musashi 91.8 .+-. 6.8 88.6 .+-.
7.9 Nestin 78.6 .+-. 5.7 75.4 .+-. 12.0 GFAP 69.2 .+-. 7.4 78.6
.+-. 8.4 .beta.III-tubulin 85.6 .+-. 6.4 76.6 .+-. 8.4 P75 0 0 NeuN
0 0 O4 65.4 .+-. 6.6 71.4 .+-. 7.5 CD133 0 0
[0317] HFF cells were cultured as described in Example 1, and
transfected using the Nucleofector.TM. II device (Lanza) as
described in Example II. Cells were co-transfected with
pCMV6-XL5-Msi/pCMV6-XL4-Ngn2, pCMV-Msi1-Ngn2 with MBD2 or
pre-treated with VPA/5aza, Cells were cultured in proliferation
medium as suspension or adherent cultures. Gene expression analysis
on 8 samples was performed as previously described in Example I
with the customized Neuronal Markers 2 TLDA (Table 13) which
profiled the expression of 48 genes (including three housekeeping
genes: ACTIN, GAPDH and PPIA) in four major categories; 1)
fibroblast specific genes; 2) neuronal lineage specific genes; 3)
Neural stem cell marker specific genes; and 4) Genes for growth
factors and their receptors.
TABLE-US-00020 TABLE 13 Neuronal Markers 2 TLDA Layout (Applied
Biosystems) Neuronal Markers 2 TLDA Layout (Applied Biosystems)
Gene Symbols 1 2 3 4 5 6 7 8 1 ACTB PPIA COL3A1 LOX SI00A4 SYT1
SNAP25 NEUROD1 2 VIM SOX3 SOX9 PROM1 SOX1 SOX2 KLF4 POU5F1 3 ACTB
PPIA COL3A1 LOX SI00A4 SYT1 SNAP25 NEUROD1 4 VIM SOX3 SOX9 PROM1
SOX1 SOX2 KLF4 POU5F1 5 ACTB PPIA COL3A1 LOX SI00A4 SYT1 SNAP25
NEUROD1 6 VIM SOX3 SOX9 PROM1 SOX1 SOX2 KLF4 POU5F1 7 ACTB PPIA
COL3A1 LOX SI00A4 SYT1 SNAP25 NEUROD1 8 VIM SOX3 SOX9 PROM1 SOX1
SOX2 KLF4 POU5F1 9 ACTB PPIA COL3A1 LOX SI00A4 SYT1 SNAP25 NEUROD1
10 VIM SOX3 SOX9 PROM1 SOX1 SOX2 KLF4 POU5F1 11 ACTB PPIA COL3A1
LOX SI00A4 SYT1 SNAP25 NEUROD1 12 VIM SOX3 SOX9 PROM1 SOX1 SOX2
KLF4 POU5F1 13 ACTB PPIA COL3A1 LOX SI00A4 SYT1 SNAP25 NEUROD1 14
VIM SOX3 SOX9 PROM1 SOX1 SOX2 KLF4 POU5F1 15 ACTB PPIA COL3A1 LOX
SI00A4 SYT1 SNAP25 NEUROD1 16 VIM SOX3 SOX9 PROM1 SOX1 SOX2 KLF4
POU5F1 9 10 11 12 13 14 15 16 1 MBP NKX-2 GAPDH OLIG2 ALDHIL1 DIO2
GFAP NCAM1 2 STAT3 PIK3CG GDNF NGF BDNF CNTFZFP91- GAP43 NRG1 CNTF
3 MBP NKX-2 GAPDH OLIG2 ALDHIL1 DIO2 GFAP NCAM1 4 STAT3 PIK3CG GDNF
NGF BDNF CNTFZFP91- GAP43 NRG1 CNTF 5 MBP NKX-2 GAPDH OLIG2 ALDHIL1
DIO2 GFAP NCAM1 6 STAT3 PIK3CG GDNF NGF BDNF CNTFZFP91- GAP43 NRG1
CNTF 7 MBP NKX-2 GAPDH OLIG2 ALDHIL1 DIO2 GFAP NCAM1 8 STAT3 PIK3CG
GDNF NGF BDNF CNTFZFP91- GAP43 NRG1 CNTF 9 MBP NKX-2 GAPDH OLIG2
ALDHIL1 DIO2 GFAP NCAM1 10 STAT3 PIK3CG GDNF NGF BDNF CNTELFP91-
GAP43 NRG1 CNTF 11 MBP NKX-2 GAPDH OLIG2 ALDHIL1 DIO2 GFAP NCAM1 12
STAT3 PIK3CG GDNF NGF BDNF CNTFZFP91- GAP43 NRG1 CNTF 13 MBP NKX-2
GAPDH OLIG2 ALDHLLI DIO2 GFAP NCAM1 14 STAT3 PIK3CG GDNF NGF BDNF
CNTFZFP91- GAP43 NRG1 CNTF 15 MBP NKX-2 GAPDH OLIG2 ALDHIL1 DIO2
GFAP NCAM1 16 STAT3 PIK3CG GDNF NGF BDNF CNTFZFP91- GAP43 NRG1 CNTF
17 18 19 20 21 22 23 24 1 FOXJ1 PDGFRA MK167 NES CSPG4 DLX2 MSI1
CROCC 2 NPY CSF3 BMP4 TGFB1 VEGFA NGFR EGFR KDR 3 FOXJ1 PDGFRA
MK167 NES CSPG4 DLX2 MSI1 CROCC 4 NPY CSF3 BMP4 TGFB1 VEGFA NGFR
EGFR KDR 5 FOXJ1 PDGFRA MK167 NES CSPG4 DLX2 MSI1 CROCC 6 NPY CSF3
BMP4 TGFB1 VEGFA NGFR EGFR KDR 7 FOXJ1 PDGFRA MK167 NES CSPG4 DLX2
MSI1 CROCC 8 NPY CSF3 BMP4 TGFB1 VEGFA NGFR EGFR KDR 9 FOXJ1 PDGFRA
MK167 NES CSPG4 DLX2 MSI1 CROCC 10 NPY CSF3 BMP4 TGFB1 VEGFA NGFR
EGFR KDR 11 FOXJ1 PDGFRA MK167 NES CSPG4 DLX2 MSI1 CROCC 12 NPY
CSF3 BMP4 TGFB1 VEGFA NGFR EGFR KDR 13 FOXJ1 PDGFRA MK167 NES CSPG4
DLX2 MSI1 CROCC 14 NPY CSF3 BMP4 TGFB1 VEGFA NGFR EGFR KDR 15 FOXJ1
PDGFRA MK167 NES CSPG4 DLX2 MSI1 CROCC 16 NPY CSF3 BMP4 TGFB1 VEGFA
NGFR EGFR KDR
Sample Information
TABLE-US-00021 [0318] Sample ID Sample Name TLDA Port 1 HFF Ctrl 1
2 ReNcell Undifferentiated Ctrl 2 3 Msi1-Ngn2/MBD2 3 4
Msi1-Ngn2/MBD2 4 5 Msi1-Ngn2/VPA + AZA 5 6 Msi1-Ngn2 6 7
Msi1-Ngn2/MBD2, neurospheres 7 8 Msi1-Ngn2/MBD2, neurospheres 8
[0319] As shown in Table 14, fibroblast-specific genes (Col3A1,
Lox, S100A4) were down-regulated in reprogrammed cells, indicating
the loss of fibroblast-specific genes following transfection (note
that not all cells got transfected and reprogrammed, so the
presence of fibroblast-specific gene expression in the cultures is
mostly from the un-programmed fibroblasts left in the culture). The
expression of these genes is observed to increase when HFFs were
transfected in the absence of DNA demethylator or the DNA
methylation inhibitor, indicating that down-regulation of
differentiated markers of fibroblast cells requires DNA
demethylation. The expression of ectoderm genes such as Msi1, Sox2,
and Nestin was remarkably increased following transfection in
conjunction with DNA demethylation. The expression of neuronal
markers, such as synaptoga mini (a synaptic vesicle protein) and
NeuroD1 was up-regulated in transfected cells with Msi1/Ngn2/MBD2,
and slightly increased in transfected cells with Msi1/Ngn2/VPA and
5-AZA. The selected three markers of oligodendrocytes were detected
in the transfected cells with a strong increase of Olig2. Two
markers for astrocytes, GFAP and ALDH1L1, were enhanced following
transfection. The results support the idea that neurospheres are
composed of heterogeneous progenitor subtypes.
[0320] Among the neurotrophic factors, expression of CNTF was
slightly increased in the reprogrammed cells. The expression of
GAP-43 and neuropeptide Y (NPY) were the most annotated genes.
GAP-43 has long been acknowledged to play a pivotal role in axonal
plasticity and is used as a marker of regenerating neurite
outgrowth and synaptogenesis, both in embryonic development and in
neuronal regeneration in injured brain and spinal cord. Expression
of receptors for growth and neurotrophic factors was increased,
such as neurotrophic receptor tyrosine kinase expression.
TABLE-US-00022 TABLE 14 Gene array analysis was performed after one
month of transfection of human fibroblast cells with Msi1/Ngn2, in
the presence MBD2 or VPA and 5-Aza. Cells were cultured on coated
culture plates as adherent cells or on untreated culture plates as
neurospheres in proliferation medium (StemCell Technologies)
supplemented with EGF (20 ng/ml) and FGF (20 ng/ml). Untransfected
cells were considered as negative control and ReNcell (Millipore)
as positive control. Relative expression to #1 HFF Ctrl #3 #4 #7 #8
#2 Msi1- Msi1- #5 #6 Msi1-Ngn2/ Msi1-Ngn2/ ReNcell Ngn2/ Ngn2/
Msi1-Ngn2/ Msi1- MBD2, VPA + AZA, Symbol Common name and
description Undiff MBD2 MBD2 VPA + AZA Ngn2 neurospheres
neurospheres Fibroblast/ ECM component COL3A1 Collagen, type III,
alpha 1, fibroblast 0.00 0.03 0.02 0.02 11.92 0.00 0.00 marker LOX
Lysyl oxidase, ECM component 0.01 0.03 0.01 0.01 2.38 0.00 0.00
FSP1 Fibroblast transcription site-1, 0.04 0.04 0.06 0.05 3.22 0.05
0.05 enzyme for ECM remodeling Neuron markers SYT1 Synaptotagmin1,
a synaptic vesicle 106.49 108.40 78.66 26.72 22.42 37.61 16.80
protein in neurons SNAP25 SNAP25, mature neuron marker 4.72 6.10
7.89 3.11 3.19 6.47 4.00 NEUROD1* Neurogenic differentiation 1,
neuron 2.32 93.35 100.84 2.02 3.11 271.11 10.23 marker Oligoden-
drocyte markers MBP* Myelin Basic Protein, mature 2.32 48.53 18.11
6.94 667.56 16.67 1.67 oligodendrocyte marker NKX2-2* NK2 homeobox
2, remyelination 2.32 75.31 54.65 1.66 3.11 1.67 1.74 OLIG2*
Oligodendrocyte lineage transcription 2856.4 15594 67369 38733 3.11
92420 101733 factor 2, oligodendrocyte progenitor Astrocyte markers
ALDH1L1* Aldehyde dehydrogenase 1 family 6.20 3.77 4.65 1.66 0.02
5.87 9.59 member L1, astrocyte DIO2* Deiodinase iodothyronine type
II, 23.20 0.00 0.00 0.00 0.51 0.00 0.00 astrocyte marker GFAP Glial
fibrillary acidic protein, 3342.1 6899.0 6291.0 4800.9 1.27 3118.7
3222.0 astrocyte marker NSCS markers NCAM1 NCAM1, neuroblast marker
23.21 43.90 24.45 12.72 1.13 31.93 36.70 PDGFRA Plate-derived
growth factor receptor 0.05 0.01 0.01 0.00 4.42 0.00 0.01 alpha,
oligodendrocyte progenitor cells NES Nestin, neural progenitor 5.76
19.84 19.56 3.46 4.23 16.57 8.36 MSI1*,** Musashi I, neuroblast
marker 5120.3 5985.2 5262.7 5645.1 204.34 3179.6 4113.6 SOX1* Sox1,
neural progenitor 679.21 223.59 373.14 361.67 3.11 287.82 323.23
SOX2* Sox2, NSCs 1924084 2265299 1889166 1014816 3.11 1313765
1103212 Neuro- trophic/ Growth Factor GDNF* Glial cell derived
neurotrophic factor 0.01 0.02 0.02 0.00 1.69 0.00 0.00 NGF* Nerve
growth factor 0.00 0.00 0.00 0.00 1.48 0.00 0.00 BDNF Brain derived
neurotrophic factor 0.03 0.09 0.09 0.05 0.82 0.02 0.01 CNTF*
Ciliary neurotrophic factor 9.25 4.32 3.11 2.90 64.05 2.31 3.39
GAP43 Growth associated protein 43, neural 917.52 3506.5 1530.8
452.75 584.00 746.25 578.52 regeneration NRG1* Neuregulin 1, neural
regeneration 0.01 0.00 0.00 0.00 0.40 0.00 0.00 NPY* Neuropeptide
Y, interneuron 2.32 675.69 465.04 153.54 3.11 1244.0 130.38 CSF3*
Colony stimulating factor 3, neural 0.50 0.03 0.02 0.58 18.62 0.02
0.02 regeneration BMP4 Bone morphogenetic protein 4, 0.83 0.26 0.74
0.45 11.03 0.09 0.07 remyelination marker TGFB1 Transforming growth
factor, beta 1 0.85 2.39 0.92 0.83 0.65 0.45 0.58 Angio- genesis
VEGFA Vascular endothelial growth factor 2.77 14.93 15.01 2.67 3.82
2.80 3.21 Neuro- trophin/ Growth Factor Receptors NGFR/P75 NGFR,
neurotrophin receptor 5.35 3.29 5.78 9.10 7.53 7.26 17.51 EGFR
Epidermal growth factor receptor 0.89 0.77 0.86 0.79 1.63 1.44 1.25
KDR* Kinase insert domain receptor, 210.87 259.42 263.45 51.85 0.07
11.23 17.50 growth factor receptor
[0321] Further analysis and quantification of the adherent
population of NSLCs showed that cells were positively stained for
Sox2 (93.43.+-.1.9%), nestin (60.76.+-.5.7%), and GABA
(37.48.+-.4.9), while these markers were undetectable in
untransfected cells. Furthermore, these cells stained positive for
p75NTR (31.15.+-.1.6), .beta.III-tubulin (37.55.+-.0.6%) and GFAP
(16.47.+-.0.9). However, untransfected HFFs only stained positive
for HFF markers, such as fibronectin and fibroblast protein marker,
while these markers were undetectable in reprogrammed cells,
demonstrating that the reprogrammed cells lost markers of the
original cells and adopted morphology and markers of neural stem
cells and a neuronal lineage.
TABLE-US-00023 TABLE 15 The percentage of cells stained positive
for neural stem cell markers and fibroblast markers in
untransfected cells and transfected cells with pMsi1/Ngn2/MBD2.
Transfected cells (NSLCs) possess a high percentage of neural stem
markers but a very low percentage of fibroblast markers as compared
to untransfected cells. The percentage of immunopositive cells was
determined by Cellomics .TM. and represented as mean .+-. SD (n =
5). Untransfected Transfected fibroblast fibroblast cells (% of
average cells (% of average Marker protein positive cells .+-.
stdv) positive cells .+-. stdv) Sox2 93.43 .+-. 1.9 1.90 .+-. 0.5
Nestin 60.76 .+-. 5.7 0.84 .+-. 0.2 p75NTR 31.15 .+-. 1.6 3.95 .+-.
1.7 NCAM 26.84 .+-. 3.8 0.87 .+-. 0.2 S100 41.80 .+-. 0.6 1.60 .+-.
0.3 GFAP 16.47 .+-. 0.9 3.84 .+-. 0.9 .beta.III-Tubulin 37.55 .+-.
0.6 1.90 .+-. 0.9 GABA 37.48 .+-. 4.9 2.54 .+-. 0.5 Fibronectin
1.05 .+-. 0.7 94.19 .+-. 0.9 Fibroblast marker protein 4.81 .+-.
1.0 50.30 .+-. 7.8
[0322] This study showed as well that NSLCs have the capacity to
proliferate in culture and exhibit stable morphology, gene and
protein expression that were maintained for the entire study
period, which was for over five month in culture (Table 16).
TABLE-US-00024 TABLE 16 Doubling time of NSLCs over serial
passages. NSLCs were maintained in proliferation conditions for 35
passages in a 37.degree. C., 5% CO.sub.2 and 5% O.sub.2 incubator.
The time required for the cell population to double (g) was
calculated for each passage, and was defined as g = (In2)/k, where
k was the number of generations that occured per unit time (t)
defined as, k = (In N.sub.f - In N.sub.0)/t, where N.sub.f was the
final cell number and N.sub.0 the initial seeded cell number. The
average generation time was 25.4 h over 35 passages. Passage number
Time (h) LN N.sub.0 LN N.sub.f k (h.sup.-1) g (h) 2 168 11.513
15.577 0.024 38.655 3 216 11.513 16.195 0.022 31.977 4 192 11.513
18.258 0.035 39.730 5 144 11.513 16.258 0.033 21.036 6 144 11.513
16.258 0.033 21.036 7 144 11.513 15.702 0.029 33.824 8 168 11.513
15.870 0.026 26.729 9 120 11.513 16.811 0.031 32.548 10 144 11.513
15.415 0.027 35.580 11 120 13.122 15.895 0.023 30 12 120 11.513
15.747 0.035 19.645 13 168 11.513 15.870 0.026 26.729 14 168 12.429
15.870 0.020 23.847 15 168 11.513 15.520 0.024 29.059 16 192 11.513
16.167 0.024 28.596 17 144 11.513 15.239 0.026 36.791 18 168 11.513
15.790 0.025 37.229 19 120 13.122 15.870 0.023 30.276 20 144 13.122
16.249 0.022 31.922 21 96 13.122 15.761 0.027 25.214 22 120 13.122
15.870 0.023 30.276 23 120 13.122 15.761 0.022 31.518 24 96 13.122
15.687 0.027 25.943 25 96 13.122 16.013 0.030 23.022 26 96 13.122
16.067 0.031 22.599 27 96 13.122 16.300 0.033 20.938 28 120 13.122
16.482 0.028 24.752 29 96 13.122 16.380 0.034 20.424 30 96 13.122
16.300 0.033 19.938 31 120 13.122 16.483 0.028 22.752 32 96 13.122
16.062 0.031 20.640 33 96 13.122 16.300 0.033 20.938 34 96 13.122
16.077 0.031 15.519 35 96 13.122 16.077 0.031 15.519
Gene Expression Microarray
[0323] Microarray expression analysis was performed to get a global
overview to compare the gene expression profile of passage 7 NSLC
to both HFF (the cells that the NSLC were created from) and hNPCs.
NSLC (n=3), HFF (n=2), and hNPC (n=3) were resuspended in
RNAlater.TM. (Qiagen) and shipped to Genotypics (India) where the
samples were processed and the Gene Expression Microarray was
performed.
[0324] In brief, Genotypics extracted RNA from the samples and
performed Quality Control using an Agilent Bioanalyzer.TM..
Labelling was done using Agilent's Quick Amp.TM. kit (cDNA
synthesis and in vitro transcription), followed by Labelling QC.
Hybridization was then performed using the 8.times.60K array, and
scanning was done using high throughput Agilent scanner with
SureScan.TM. technology. The Agilent Feature Extraction software
was used for automated feature extraction, followed by Raw Data QC
and Image QC. Advanced Data Analysis was then performed, including
Pathway and Gene Ontology analyisis using Agilent's GeneSpring
GX.TM. v10.0 and Genotypic's Biointerpreter Software. The NSLC
samples were compared to the HFF samples (Set 1) and hNPC samples
(Set 2) The NSLC samples had a global gene expression pattern that
was much closer to the hNPCs than the HFFs from which the NSLCs
were created. Pearson correlation analysis revealed that NSLCs are
closely related to hNPCs, including in terms of neuronal lineage
markers, regenerative genes and migration genes. These data confirm
that NSLCs are similar, but not identical, to hNPCs.
[0325] Microarray analysis revealed an up-regulation of neural
precursor genes in the NSLC samples as compared to the HFF samples.
ACTL6A and PHF10, which both belong to the neural
progenitors-specific chromatin remodelling complex (npbaf complex)
and are required for the proliferation of neural progenitors, were
up-regulated by 2.9-fold and 2.3 fold respectively. MSI2, which
plays a role in the proliferation and maintenance of stem cells in
the central nervous system, was up-regulated by 6-fold (Table X1).
Glia genes were up-regulated in the NSLC samples as compared to the
HFF samples. GFAP, is a neural stem cell- and astrocyte-specific
marker that, during the development of the central nervous system,
distinguishes astrocytes from other glial cells, is highly
up-regulated in the NSLC sample as compared to HFF (690-fold).
OLIG1, which promotes formation and maturation of oligodendrocytes,
especially within the brain, is also highly up-regulated in NSLC
sample as compared to HFF (370-fold) (Table X2).
[0326] Table X3 lists a subset of regenerative genes that are
up-regulated in the NSLC samples as compared to the HFF samples.
SOX2, a gene critical for early embryogenesis and for embryonic
stem cell pluripotency as well as neural stem cells, is highly
up-regulated in the NSLC samples as compared to the HFF samples
(5000-fold). CCND2, which is essential for the control of the cell
cycle at the G1/S (start) transition, is also up-regulated in NSLC
samples (70-fold as compared to HFF samples). As shown in Table X4,
numerous fibroblast genes were down-regulated in the NSLC samples
as compared to the HFF samples. This shows that the NSLC lose the
expression of numerous fibroblast genes as it gets reprogrammed
from HFF to NSLC.
[0327] Table X5 show that neural precursor genes were also
up-regulated in the NSLC samples as compared to the hNPC samples.
BDNF, which promotes the survival and differentiation of selected
neuronal populations of the peripheral and central nervous systems
during development, is even more highly expressed in NSLC samples
than in hNPC samples (34-fold up-regulation). Table X6 shows that a
subset of Glia genes are also up-regulated in the NSLC samples as
compared to the hNPC samples. GFAP, a neural stem cell- and
astrocyte-specific marker that, during the development of the
central nervous system, distinguishes astrocytes from other glial
cells, is more highly expressed in NSLC samples than hNPC samples
(13-fold). PLP1, the major myelin protein of the central nervous
system which plays an important role in the formation or
maintenance of the multilamellar structure of myelin, is also more
highly expressed in NSLC samples than in hNPC samples
(20-fold).
[0328] Regenerative genes were also up-regulated in the NSLC
samples as compared to the hNPC samples (Table X7). BMP2, a neural
crest marker, but which induces growth especially of cartilage and
bone formation and BMP4, which in turn induces cartilage and bone
formation and acts in mesoderm induction, tooth development, limb
formation and fracture repair, but also in neural stem cells, were
both more highly expressed in NSLC samples than in hNPC samples
(18-fold and 20-fold respectively). GAP43, which is a major
component of the motile growth cones that form the tips of
elongating axons was more highly expressed in NSLC samples than
hNPC samples (4-fold). This suggests the regenerative potential of
NSLC. HOXB4, a transcription factor that is involved in development
and also in the expansion of neural stem cells as well as
hematopoietic stem and progenitor cells in vivo and in vitro making
it a potential candidate for therapeutic stem cell expansion, was
also more highly expressed in NSLCs than in hNPCs. This data
indicates that NSLCs are more `stem-like` or have more `stemness`
than hNPCs.
TABLE-US-00025 TABLE X1 Up-regulated Neural Precursor genes (NSLC
vs. HFF) Fold change of NSLC GeneSymbol Accession Number compared
to HFF.sup.1 p-value ACTL6A NM_178042 2.90 0.000 ADAM9 NM_001005845
2.64 0.004 AIFM1 NM_004208 2.45 0.000 BCAT1 NM_005504 3.23 0.000
BMP2 NM_001200 17.49 0.000 DLL1 NM_005618 40.32 0.000 EDNRB
NM_003991 933.03 0.000 ERBB4 NM_005235 53.22 0.006 GMNN NM_015895
4.42 0.000 HES5 BC087840 102.33 0.000 KIF1B NM_015074 9.45 0.002
LIMK1 NM_002314 2.44 0.002 MAPK8IP1 NM_005456 5.88 0.001 MCHR1
NM_005297 68.19 0.001 MEF2C NM_002397 2.91 0.000 MSI2 NM_170721
6.76 0.000 NMB NM_021077 3.65 0.000 NOS2A NM_000625 279.45 0.000
NOTCH1 NM_017617 6.75 0.000 NPAS3 NM_022123 187.85 0.000 PHF10
NM_018288 2.28 0.001 PHLPP NM_194449 8.84 0.000 SMAD1 NM_005900
4.74 0.000 SNTG1 AL161971 34.05 0.000 SP8 NM_198956 1392.67 0.000
STAU2 AK002152 3.35 0.000 STIL NM_003035 4.94 0.003 .sup.1Fold
change represents the up-regulation of the gene in the NSLC samples
as compared to the HFF samples. (n = 2 for HFF samples, n = 3 for
NSLC samples).
TABLE-US-00026 TABLE X2 Up-regulated Glia genes (NSLC vs. HFF) Fold
change of NSLC compared to GeneSymbol Accession Number HFF.sup.1
p-value ASTN1 NM_004319 51.44 0.000 ATP1B2 NM_001678 186.64 0.000
B3GAT1 NM_018644 1784.49 0.000 BCL2 NM_000633 2.65 0.002 BMP7
NM_001719 41.35 0.000 CA14 NM_012113 43.44 0.000 CLCN2 NM_004366
4.18 0.000 CNDP1 NM_032649 4.39 0.010 CP NM_000096 93.08 0.002
CXCR4 NM_001008540 4124.29 0.000 ERBB4 NM_005235 53.22 0.006 FABP7
NM_001446 18702.36 0.000 GAB1 NM_207123 2.44 0.001 GFAP NM_002055
696.51 0.000 GJB2 NM_004004 13.89 0.001 ITGB8 NM_002214 8.48 0.005
KCNJ10 NM_002241 263.42 0.000 LMO3 NM_018640 194.32 0.000 MAP6D1
NM_024871 3.99 0.000 MAPT NM_016835 2.38 0.001 NDE1 NM_017668 2.21
0.002 NEFL NM_006158 10.30 0.001 NKX6-2 NM_177400 10.83 0.026 NOVA2
NM_002516 7.51 0.000 NTN1 NM_004822 5.29 0.015 NTRK3 NM_001012338
15.32 0.000 OLIG1 NM_138983 372.11 0.000 OLIG2 NM_005806 163.20
0.000 PARD6A NM_016948 4.12 0.001 PASK NM_015148 3.89 0.001 PAX6
NM_001604 28.53 0.001 PDCD11 ENST00000369797 2.23 0.001 PDE6B
NM_000283 5.55 0.001 PER1 NM_002616 2.43 0.001 PLP1 M54927 351.09
0.000 PTK2 NM_153831 4.22 0.000 QKI NM_206855 8.75 0.003 S100B
NM_006272 456.00 0.000 SLC1A3 NM_004172 49.49 0.000 SORL1 NM_003105
27.61 0.000 SOX9 NM_000346 27.82 0.000 SPRY2 NM_005842 15.83 0.000
TARDBP NM_007375 2.69 0.005 TSPAN12 NM_012338 259.78 0.000
TABLE-US-00027 TABLE X3 Up-regulated Regenerative genes (NSLC vs.
HFF) Fold change of NSLC GeneSymbol Accession Number compared to
HFF.sup.1 p-value BMP2 NM_001200 17.49 0.000 CCND2 NM_001759 72.79
0.000 DLL1 NM_005618 40.32 0.000 EGR1 NM_001964 2.19 0.000 GAL
NM_015973 25.93 0.000 GAP43 NM_002045 1297.42 0.000 HOXB4 NM_024015
102.34 0.000 NFE2L2 AF323119 2.80 0.004 NOTCH1 NM_017617 6.75 0.000
PRPH NM_006262 6.44 0.000 SEMA3A NM_006080 3.03 0.004 SEMA6A
NM_020796 23.58 0.000 SOX2 NM_003106 5165.92 0.000
TABLE-US-00028 TABLE X4 Down-regulated Fibroblast genes (NSLC vs.
HFF) Fold change of NSLC GeneSymbol Accession Number compared to
HFF.sup.1 p-value ACOT2 NM_006821 0.30 0.000 AEBP1 NM_001129 0.16
0.001 AGA NM_000027 0.35 0.000 ANXA2 NM_001002857 0.26 0.029 AP4E1
NM_007347 0.30 0.008 APOE NM_000041 0.08 0.000 ARHGDIB NM_001175
0.24 0.009 ASAH1 NM_004315 0.31 0.000 BDKRB1 NM_000710 0.00 0.001
BDKRB2 NM_000623 0.00 0.000 BDNF NM_170735 0.12 0.000 BMP4
NM_001202 0.28 0.001 C3 NM_000064 0.25 0.001 C5orf13 NM_004772 0.18
0.000 CACNA1C NM_000719 0.03 0.000 CASP4 NM_033306 0.00 0.000 CASP5
NM_004347 0.00 0.001 CCL2 NM_002982 0.20 0.000 CD36 NM_001001547
0.07 0.023 CDC42EP2 NM_006779 0.06 0.000 CDC42EP3 NM_006449 0.41
0.000 CDC42EP5 NM_145057 0.41 0.040 CDH11 NM_001797 0.00 0.000
CEMP1 AL833099 0.30 0.001 CFH NM_001014975 0.01 0.010 CITED2
NM_006079 0.14 0.000 COL12A1 NM_004370 0.00 0.001 COL1A1 NM_000088
0.01 0.000 COL1A2 NM_000089 0.00 0.001 COL3A1 NM_000090 0.00 0.001
COL5A1 NM_000093 0.00 0.000 CPT1A NM_001876 0.16 0.002 CROT
NM_021151 0.27 0.002 CTSA NM_000308 0.10 0.000 CTSB NM_147780 0.11
0.001 CXCL1 NM_001511 0.01 0.003 CXCL12 NM_000609 0.00 0.001
CYP27A1 NM_000784 0.28 0.011 CYR61 NM_001554 0.10 0.000 DCHS1
NM_003737 0.29 0.000 DMPK NM_004409 0.36 0.000 DPT NM_001937 0.05
0.006 EFEMP1 NM_004105 0.00 0.000 ELN NM_000501 0.13 0.001 EMX2
NM_004098 0.00 0.001 EPS8 NM_004447 0.18 0.000 ETS1 NM_005238 0.15
0.003 FAH NM_000137 0.17 0.000 FAM14A NM_032036 0.22 0.001 FAP
NM_004460 0.00 0.000 FBLN2 NM_001004019 0.18 0.000 FBN1 NM_000138
0.01 0.002 FGF1 NM_000800 0.20 0.004 FGF13 NM_004114 0.04 0.006
FGF2 NM_002006 0.06 0.000 FGF5 NM_004464 0.01 0.003 FGF7 NM_002009
0.04 0.001 FGF9 NM_002010 0.01 0.000 FGFR1 NM_023110 0.34 0.026
FHL2 NM_201555 0.11 0.000 FN1 NM_212482 0.00 0.001 FSTL1 NM_007085
0.09 0.000 GADD45B NM_015675 0.09 0.001 GALNT6 NM_007210 0.13 0.001
GAS6 NM_000820 0.02 0.000 GBA NM_001005749 0.22 0.002 GBAP
NR_002188 0.19 0.000 GCH1 NM_000161 0.22 0.001 GGTA1 NR_003191 0.28
0.013 GIT2 NM_057169 0.37 0.003 GJA1 NM_000165 0.46 0.001 GLIS1
NM_147193 0.02 0.000 GM2A AK127910 0.25 0.010 GNS NM_002076 0.29
0.000 GPC3 NM_004484 0.22 0.038 GREM1 NM_013372 0.00 0.011 GSTM1
NM_146421 0.27 0.001 HAAO NM_012205 0.43 0.001 HERPUD1 NM_014685
0.19 0.000 HEXA NM_000520 0.24 0.000 HEXB NM_000521 0.36 0.000 HGF
NM_001010932 0.09 0.028 HGS NM_004712 0.26 0.029 HIF1A NM_181054
0.36 0.005 HLA-A NM_002116 0.31 0.002 HLA-H NR_001434 0.19 0.001
HOXB13 NM_006361 0.03 0.004 HR NM_005144 0.18 0.002 HSPG2 NM_005529
0.19 0.004 IDUA NM_000203 0.16 0.000 IGF1 NM_000618 0.10 0.004
IGFBP7 NM_001553 0.28 0.040 IKBKG NM_003639 0.42 0.001 IRF1
NM_002198 0.28 0.002 ITGA1 NM_181501 0.00 0.001 ITGB3 NM_000212
0.05 0.000 KLF4 NM_004235 0.05 0.002 LEP NM_000230 0.07 0.001
LEPRE1 NM_022356 0.24 0.000 LMNA NM_005572 0.42 0.000 LOX NM_002317
0.01 0.000 LOXL4 NM_032211 0.10 0.003 LRRC8C NM_032270 0.15 0.013
MAGEL2 AJ243531 0.31 0.002 MAN2B1 NM_000528 0.45 0.006 MAP3K8
NM_005204 0.27 0.001 MEIS2 NM_170677 0.00 0.001 MKNK1 NM_003684
0.37 0.005 MMP1 NM_002421 0.00 0.000 MMP14 NM_004995 0.07 0.001
MMP2 NM_004530 0.04 0.000 MMP3 NM_002422 0.00 0.001 MOXD1 NM_015529
0.24 0.000 MRAS NM_012219 0.15 0.001 MSX2 NM_002449 0.15 0.031
MTHFR NM_005957 0.27 0.014 MYC NM_002467 0.05 0.000 MYL6 NM_079423
0.33 0.001 MYL9 NM_181526 0.01 0.000 NAGLU NM_000263 0.23 0.000
NBL1 NM_182744 0.11 0.000 NEK9 NM_033116 0.41 0.001 NF2 NM_181831
0.46 0.000 NPC1 NM_000271 0.34 0.000 OPTN NM_001008211 0.04 0.000
P4HB NM_000918 0.37 0.001 PALLD NM_016081 0.29 0.001 PAPPA
NM_002581 0.05 0.000 PCDHGB4 NM_032098 0.28 0.001 PCK2 NM_004563
0.04 0.000 PCOLCE NM_002593 0.00 0.000 PDGFRA NM_006206 0.02 0.010
PEX14 BC017848 0.48 0.000 PFKL NM_001002021 0.35 0.004 PPARG
NM_138711 0.01 0.000 PPFIBP2 NM_003621 0.08 0.000 PRR5 NM_015366
0.23 0.022 PSEN2 NM_012486 0.34 0.002 PTGS1 NM_000962 0.29 0.000
PXDN AF200348 0.12 0.000 PYCARD NM_013258 0.03 0.000 QSOX1
NM_002826 0.09 0.000 RASSF1 NM_170713 0.30 0.001 RBMS1 NM_002897
0.14 0.001 RECK NM_021111 0.07 0.000 RET NM_020975 0.35 0.015
RFPL1S NR_002727 0.22 0.039 ROD1 NM_005156 0.37 0.001 RSU1
NM_012425 0.41 0.002 S100A4 NM_002961 0.03 0.000 SAMD9 NM_017654
0.07 0.007 SCARB2 NM_005506 0.42 0.001 SDC2 NM_002998 0.38 0.000
SDPR NM_004657 0.03 0.005 SENP2 AF151697 0.44 0.006 SEPP1
NM_001085486 0.00 0.005 SFRP1 NM_003012 0.37 0.000 SHOC2 NM_007373
0.39 0.000 SIGIRR NM_021805 0.47 0.000 SLC17A5 NM_012434 0.14 0.001
SLC22A5 NM_003060 0.21 0.001 SLC9A3R2 NM_004785 0.29 0.000 SMPD1
NM_000543 0.17 0.000 STAT1 NM_139266 0.19 0.000 STAT6 NM_003153
0.00 0.000 STS NM_000351 0.10 0.007 STYK1 NM_018423 0.05 0.013
SUMF1 NM_182760 0.28 0.000 TAGLN NM_001001522 0.01 0.000 TFAP2A
NM_003220 0.03 0.005 THBS2 NM_003247 0.02 0.000 THRA NM_199334 0.31
0.000 THRB NM_000461 0.10 0.014 TNXB NM_019105 0.26 0.043 TPM2
NM_213674 0.12 0.000 TRIOBP NM_007032 0.15 0.003 TRIP11 NM_004239
0.45 0.001 TSC22D3 NM_004089 0.14 0.000 TWIST1 NM_000474 0.01 0.003
VCAN NM_004385 0.04 0.000 VCL NM_014000 0.28 0.000 VLDLR NM_003383
0.15 0.000 WISP1 NM_003882 0.05 0.013 WNT5A NM_003392 0.01 0.000
YAP1 NM_006106 0.41 0.007 ZBTB7B NM_015872 0.44 0.000
TABLE-US-00029 TABLE X5 Up-regulated Neural Precursor genes (NSLC
vs. hNPC) Fold change of NSLC compared to GeneSymbol Accession
Number hNPC.sup.2 p-value ACTL6A NM_178042 2.33 0.000 BCAT1
NM_005504 9.92 0.000 BDNF NM_170735 33.90 0.000 BMP2 NM_001200
17.71 0.000 CDKN2A NM_058197 5.57 0.000 COL18A1 NM_030582 7.22
0.001 DIAPH1 NM_005219 2.33 0.001 EDNRB NM_003991 2.78 0.000 IDE
NM_004969 2.74 0.000 LIMK1 NM_002314 3.61 0.000 MAPK8IP1 NM_005456
2.77 0.000 MCHR1 NM_005297 4.02 0.000 MYLIP NM_013262 4.22 0.000
NEDD4 NM_006154 2.23 0.000 NOS2A NM_000625 267.58 0.000 PCSK9
NM_174936 9.65 0.000 PSEN2 NM_000447 2.07 0.000 SMAD1 NM_005900
3.09 0.000 TBX1 NM_080647 3.65 0.028 TGFB1 NM_000660 6.66 0.000
.sup.2Fold change represents the up-regulation of the gene in the
NSLC samples as compared to the hNPC samples. (n = 3 for hNPC
samples, n = 3 for NSLC samples).
TABLE-US-00030 TABLE X6 Up-regulated Glia genes (NSLC vs. hNPC)
Fold change of NSLC GeneSymbol Accession Number compared to
hNPC.sup.1 p-value ACSL4 NM_004458 2.10 0.000 BDNF NM_170735 33.90
0.000 BMP4 NM_001202 20.55 0.001 CP NM_000096 159.46 0.000 CSPG4
NM_001897 4.94 0.000 FOXC1 NM_001453 5.12 0.000 GFAP NM_002055
13.67 0.000 GJB2 NM_004004 7.25 0.000 GLIPR1 NM_006851 5.58 0.000
ITGA3 NM_002204 24.64 0.000 LMO3 NM_018640 129.25 0.000 NEFL
NM_006158 7.14 0.000 NKX6-2 NM_177400 11.50 0.000 NRTN NM_004558
3.39 0.001 PDCD11 NM_014976 2.48 0.000 PLP1 NM_000533 20.64 0.000
TGFB1 NM_000660 6.66 0.000 TSPAN12 NM_012338 2.58 0.006
TABLE-US-00031 TABLE X7 Up-regulated Regenerative genes (NSLC vs.
hNPC) Fold change of NSLC GeneSymbol Accession Number compared to
hNPC.sup.1 p-value ATR NM_001184 2.57 0.000 BMP2 NM_001200 17.71
0.000 BMP4 NM_001202 20.55 0.001 CAV3 NM_001234 26.23 0.000 CCND1
NM_053056 10.34 0.000 CDKN2A NM_058197 5.57 0.000 CEBPB NM_005194
2.58 0.000 GAL NM_015973 12.21 0.000 GAP43 NM_002045 4.27 0.000
HOXB4 NM_024015 133.37 0.000 SMAD3 NM_005902 2.27 0.000
[0329] In order to investigate the differentiation potential of
NSLCs to neuronal lineages (Neurons, astrocytes, and
oligodendocytes), neurospheres were dissociated and plated in
laminin/poly-D-Lysine (10 .mu.g/ml; Sigma) in differentiation
medium for two weeks. The differentiation towards neuronal lineage
was performed using two different mediums: NbActive medium
(BrainBits.TM.) supplemented with Brain Derived Neurotrophin Factor
(BDNF, 20 ng/ml, Peprotech), all-trans-retinoic acid (ATRA, 5
.mu.M, Spectrum), and bFGF (40 ng/ml, Peprotech) or NeuroCult.TM.
differentiation medium (NeuroCult.TM. Differentiation kit, StemCell
Technologies), supplemented with BDNF (20 ng/ml, Peprotech) and
bFGF (40 ng/ml, Peprotech). After two weeks in culture, the cells
were stained with the neuronal marker .beta.III-tubulin, astrocyte
markers GFAP and 510013, andoligodendrocyte marker CNPase. The
cells were fixed with 4% formaldehyde and the primary antibodies
were added in 5% normal goat serum/PBS as follows: Mouse antibody
.beta.III-tubulin (1:200, Abcam), rabbit antibody S100.beta.
(1:100, Abcam), and Chicken antibody CNPase (1:50, Abcam).
Secondary antibodies are added in 5% normal goat serum/PBS as
follows: Goat anti mouse Alexa546.TM. (1:200, Invitrogen), Goat
anti rabbit Alexa488.TM. (1:200, Invitrogen), and Goat anti-chicken
cy5 (1:100, Jackson ImmunoResearch Labs).
[0330] Immunohistochemistry analysis showed that NbActive medium
promoted the differentiation equally to neuronal (48.66.+-.14.07%,
.beta.III-tubulin) and potential early oligodendrocyte lineages
(50.01.+-.4.04%, CNPase) and to a lower percentage of astrocyte
cells (2.68.+-.1.13%, S100.beta.), while NS-A differentiation
medium induced the differentiation mainly to neurons
(64.89.+-.4.11%, .beta.III-tubulin) and astrocytes (35.94.+-.4.04%,
S100beta), and a low percentage of potential early oligodendrocytes
cells (8.68.+-.2.71%, CNPase). The NSC-A medium was selected over
NbActive for further differentiation studies. Differentiation of
cells in NS-A differentiation medium promote the differentiation of
hNPC and NSLC similarly as shown in Table 17 by the decrease of the
percentage of sox2, musashi and nestin positive cells. NSLCs were
differentiated to neuronal (74.3.+-.0.1, GABA), astrocyte lineage
(65.6.+-.0.0, S100beta) and to a lower percentage of
oligodendrocyte cells (5.2.+-.0.6, CNPase). The same pattern of
tripotent lineage differentiation was observed with hNPCs (Table
17).
TABLE-US-00032 TABLE 17 The percentage of cells stained positive
for neural stem cell and neuronal lineage markers in transfected
and untransfected cells. NSLCs and hNPCs were cultured in
NS-A-differentiation medium supplemented with BDNF (20 ng/ml) and
FGF (40 ng/ml), cultures were incubated at 37.degree. C., 5%
CO.sub.2, 5% O.sub.2 for three weeks. The percentage of
immunopositive cells was determined by Cellomics .TM. and
represented as mean .+-. SD (n = 5). Sox2 Nestin Musashi S100 O4
GABA Tripotent hNPC 73.8 .+-. 0.5 46.1 .+-. 5.2 22.1 .+-. 7.0 20.8
.+-. 1.3 6.4 .+-. 2.9 68.5 .+-. 1.6 medium NSLC 68.6 .+-. 3.9 41.0
.+-. 5.4 26.7 .+-. 5.0 65.6 .+-. 0.0 8.2 .+-. 0.6 74.3 .+-. 0.1
[0331] Several additional antibodies to neuronal antigens were used
to characterize, in more detail, the nature of differentiated
cells. Antibodies against microtubule-associated protein (MAP2b),
NCAM, and synaptophysin were used as recommended by the antibody
manufacturer. After three weeks in differentiation medium, there
was a differentiation-induced reduction in markers of precursors
cells and an increase in mature neuronal markers. The percentage of
neural precursor markers such as Sox2 were decreased during
differentiation, while p75NTR, .beta.III-tubulin and GABA were
increased with lengthening differentiation time; however, O4
positive cells were very low after 3 weeks of differentiation of
hNPCs (6.4.+-.2.9) and NSLCs (8.2.+-.0.6). Synaptophysin, an
antibody used to identify functional neuronal cells, was increased
following 2 and 3 weeks of differentiation, indicating maturity of
the neuronal cells. GABA and acetycholine markers were increased
following 2 weeks of differentiation and decreased at week 3.
[0332] The morphological changes and expression of a number of
neuronal antigens and genes show that the above method results in
normal and viable neuronal cells. Additionally, the newly formed
neuronal cells have the morphological criteria of neurons. In
addition to the above markers, the differentiated cells were
evaluated by characterizing morphological markers of neurite
differentiation. Neuron type cells (cells strongly expressing
.beta.III-tubulin) showed neurite formation after differentiation,
including an increase in the average number of neurites per neuron
(from e.g. 1.38.+-.0.1) The same pattern was observed in
.beta.III-tubulin positive cells. Accordingly, the average neurite
length (118.3.+-.3.5 .mu.m) and the number of branch points
(3.28.+-.0.3) per neuron also increased. The differentiated
neuron-like cells developed long neurites that were greater than
three cell diameters in length with a growth cone at the end,
expressed neuron-specific genes, and stopped proliferating after
the induction of differentiation.
[0333] Further differentiation was performed using an optimised
medium that promoted the differentiation towards oligodendrocyte
lineage. NSLCs and hNPCs were cultured in NS-A differentiation
medium as described previously supplemented with FGF-2 (10 ng/ml,
Peprotech) and sonic hedgehog (SHH, 100 ng/ml, Peprotech) for 4
days. After 4 days medium was changed to NS-A differentiation
medium supplemented by T3 (60 ng/ml, Peprotech), IGF1 (10 ng/ml,
Peprotech), NT-3 (10 ng/ml, Peprotech), and PDGF (10 ng/ml,
Peprotech). Cells were cultured for 20 days at 37.degree. C., 5%
CO.sub.2.
TABLE-US-00033 TABLE 18 The percentage of cells stained positive
for neural stem cell and neuronal lineage markers in transfected
and untransfected cells. NSLCs and hNPCs were cultured in
differentiation medium supplemented with SHH (100 ng/ml,
Peprotech), T3 (60 ng/ml, Peprotech), IGF1 (10 ng/ml, Peprotech),
NT-3 (10 ng/ml, Peprotech), and PDGF (10 ng/ml, Peprotech) to
induce different- iation towards oligodendrocytes. The percentage
of immunopositive cells was determined by Cellomics .TM. and
represented as mean .+-. SD (n = 5). % of positive cells Sox2
Nestin Musashi S100 O4 GABA hNPC 84.3 .+-. 3.7 26.9 .+-. 4.4 51.8
.+-. 2.9 33.4 .+-. 1.9 40.1 .+-. 6.4 89.6 .+-. 0.8 NSLC 69.3 .+-.
4.4 24.3 .+-. 2.5 45.1 .+-. 11.1 51.6 .+-. 9.5 8.5 .+-. 0.6 76.9
.+-. 1.4
[0334] Quantification of the differentiation of hNPCs and NSLCs
revealed a population of cells that were positively stained for O4.
As shown in Table 18, the percentage of O4 positive cells was more
pronounced in differentiated hNPC (40.1.+-.6.4%) as compared to
differentiated NSLCs (8.5.+-.0.6%) when using the above
differentiation protocol.
[0335] This study showed that transfecting the cells with one or
two neurogenic transcription factors in the presence of a DNA
demethylator or small molecules for epigenetic modification
achieves stable reprogrammed cells (NSLCs). Like a DNA
demethylator, epigenetic modification (inhibition of acetylation
and methylation) are sometimes useful in boosting the reprogramming
process. These cells possess and retain neural stem cell properties
as determined by: (1) the expression of neural stem cell genes and
proteins, (2) the capacity to generate and grow as neurospheres
starting from a single cell, and (3) to differentiate to neuronal
lineages in differentiation conditions. When differentiated to
neurons, cells display one or more neural-specific morphological,
physiological and/or immunological features associated with a
neuronal cell type. Useful criteria include morphological features
(long processes or neurites), physiological, and/or immunological
features such as expression of a set of neuronal-specific markers
or antigens. Furthermore, NSLCs readily turn into a tripotent-like
precursor cell with differentiation potential to a high percentage
of neuronal, astrocytes and lower percentage of oligodendrocyte
populations.
Example VI
Implication of BMP Signaling Pathway in the Reprogramming of
HFFs
[0336] This study was designed to evaluate the role of Noggin in
the process of de-differentiation of HFFs towards NSLCs. HFFs were
cultured and treated with cytochalasin B as described in Example
III. After two days of treatment, cells were transfected by
Nucleofection as described in Example II with the constructed
vector Msi1/Ngn2. Briefly, after preparing the cells, they were
mixed with 2 .mu.g of total DNA (Msi1/Ngn2) and were co-transfect
with MBD2 (24), by the Amaxa's Nucleofector.TM. according to the
manufacturers protocol. The samples were then transferred into a
Laminin (10 .mu.g/ml, Sigma) coated culture plate and cultured in
the presence of Neural Proliferation Medium (NeuroCult.TM.
proliferation Kit, StemCell Technologies) with recombinant hFGF (20
ng/ml, Peprotech), recombinant hEGF (20 ng/ml, Peprotech), and with
or without the presence of Noggin (20 ng/ml, Peprotech). Samples
were collected at different time points (1, 3, 4, 6, and 8 days) to
analyze neuronal gene expression by RT-PCR and protein expression
levels by immunohistochemistry.
[0337] Fluorescent immunohistochemical staining was performed on
samples after 4 days of transfection as previously described in
Example I. Transfected cells were stained and analyzed for
expression of Sox2, the percentage of Sox2 was 33.3.+-.1.00% in the
presence of Noggin compared to 27.5.+-.0.50% without the presence
of noggin at day 4. RT-PCR analysis of relative expression of
neuronal precursor cell markers such as nestin and Sox2 after
transfection of HFFs with pCMV-Msi1-2A-Ngn2 and pCMV6-XL5-MBD2 with
or without the presence of Noggin (20 ng/ml) was associated with an
increase in nestin and Sox2 starting at day 3 and maintained until
day 8 (Table 19). No difference in the expression was noticed in
the absence of Noggin. Inhibiting the BMP signaling pathway by
Noggin thus enhanced reprogramming, but had no reprogramming effect
on its own.
TABLE-US-00034 TABLE 19 RT-PCR analysis of relative expression of
neuronal precursor cell markers such as nestin and Sox2 after
transfection of HFF with pCMV-Msi1-2A-Ngn2 and pCMV6-XL5- MBD2 with
or without Noggin (20 ng/ml). Relative expression of Sox2, and
nestin was increased after transfection with and without Noggin.
ACHE GFAP NES SOX2 TUBB3 Rel. Std. Rel. Std. Rel. Std. Rel. Std.
Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. #1
Msi1/Ngn2 + 7.08 1.70 2.97 0.42 1.33 0.10 0.93 0.91 1.37 0.10
MBD2/+Noggin Day1 #2 Msi1/Ngn2 + 7.34 1.03 2.01 0.08 1.28 0.18 0.60
0.10 0.98 0.05 MBD2/+Noggin Day2 #3 Msi1/Ngn2 + 9.67 2.41 15.13
1.66 1.98 0.20 6333.63 277.87 0.95 0.07 MBD2/+Noggin Day3 #4
Msi1/Ngn2 + 11.68 2.65 194.07 25.22 4.19 0.52 20231.33 1034.29 1.90
0.45 MBD2/+Noggin Day4 #5 Msi1/Ngn2 + 3.58 0.66 227.99 16.83 1.68
0.09 6298.51 289.84 0.96 0.17 MBD2/+Noggin Day6 #6 Msi1/Ngn2 +
10.89 0.57 650.34 22.92 4.42 0.03 18134.90 63.93 1.81 0.06
MBD2/+Noggin Day8 #7 Ctrl 1.01 0.19 1.00 0.05 1.00 0.02 1.12 0.70
1.00 0.09 Untransfected + Noggin Day1 #8 Msi1/Ngn2 + 2.79 0.83 1.62
0.19 0.99 0.08 1.28 0.25 0.75 0.01 MBD2/ -Noggin Day1 #9 Msi1/Ngn2
+ 3.79 0.91 1.47 0.08 1.23 0.08 1.36 0.08 0.72 0.07 MBD2/ -Noggin
Day2 #10 Msi1/Ngn2 + 6.18 0.59 14.60 1.85 2.62 0.30 10949.28 448.28
0.90 0.01 MBD2/ -Noggin Day3 #11 Msi1/Ngn2 + 5.63 0.74 74.56 16.56
2.97 0.21 19623.99 3109.69 0.75 0.11 MBD2/ -Noggin Day4 #12
Msi1/Ngn2 + 3.21 0.96 232.42 5.47 1.47 0.07 15311.64 1909.23 0.86
0.03 MBD2/ -Noggin Day6 #13 Msi1/Ngn2 + 3.82 0.52 496.99 75.81 3.32
0.32 26892.31 1817.05 2.05 0.10 MBD2/ -Noggin Day8 #14 Ctrl 1.08
0.57 1.01 0.14 1.00 0.04 1.15 0.81 1.00 0.00 Untransfected - Noggin
Day1
Example VII
[0338] NSLCs Created from HFF Cells are not Skin-Derived Precursors
(SKPs)
[0339] It's known that cells termed skin-derived precursors (SKPs)
may reside in adult human skin (Fernandes et al., 2004). These
cells are capable of proliferating in response to EGF and bFGF and
express nestin, versican and fibronectin, and can differentiate
into both neuronal and mesodermal progeny. In order to verify that
NSLCs are distinct from SKPs, differentiation towards adipocyte
cells was performed. Adipose derived stem cells (ADSC) were
maintained in StemPro.TM. MSC serum free medium (Invitrogen) on
flasks coated with CellStart.TM. (Invitrogen). CellStart.TM. was
diluted 1:100 in dPBS/Ca.sup.2+/Mg.sup.2+ and the flask incubated
for 2 hours at 37.degree. C. Cells are passaged every 3 to 4 days
using Accutase.TM. and medium was changed every 2 days. Three to
four days before initiating differentiation, ADSCs and NSLCs were
seeded in 6-wellplates in CellStart.TM. (1:100 in
dPBS/Ca.sup.2+/Mg.sup.2+/2 hours at 37.degree. C.) coated tissue
culture plates. When cells reached confluence (after 3 to 4 days),
proliferation media were replaced by differentiation medium
consisting in DMEM/F12 (50:50), ITS (1:100), HEPES (1:100),
GlutaMAX.TM. (1:100), T3 (0.2 nM), Rosiglitasone (0.5 .mu.g/ml),
IBMX (100 .mu.M) and Dexamethasone (1 .mu.M). Three days after,
IBMX and dexamethasone were withdrawn from the differentiation
medium. At day 10, cells were fixed with a 4% formaldehyde solution
for 10 min and stained with Oil Red O (invitrogen) staining
solution for 15 min. Staining was removed and cells washed twice
with PBS. Adipose cells appeared red with lipid droplets
specifically stained with Oil Red O, however NSLCs were stained
negative, with no presence of lipid droplets in the cells, and the
cells adopted neuronal cell morphology.
[0340] Immunohistochemistry analysis confirmed that NSLCs are
distinct from SKPs: NSLCs stained positive for p75NTR and negative
for fibronectin and versican, while SKPs express fibronectin and
versican and do not express p75NTR (Fernandes et al., 2004). This
study indicates that NSLCs represent a tripotent-like precursor
cell and they are not a subpopulation of SKPs.
Example VIII
[0341] BDNF Release from Neural-Like Cells (NLCs)
[0342] Neural Stem-Like Cells (NSLCs) differentiated into neuronal
and glial cells were kept in culture for 55 days, and BDNF released
in the conditioned medium was measured by antigen-capture ELISA at
different time points and compared to the release in mature neurons
(ScienCell), undifferentiated Neural Human Normal Precursor cells
(NHNP, Lonza) as well as to undifferentiated NSLCs and
untransfected cells (HFF). Conditioned medium from each group was
collected, centrifuged, and then stored at -80.degree. C. until
assaying. BDNF concentrations were measured by ELISA kits (BDNF
E.sub.max Immunoassay System, Promega Corporation, USA), according
to the manufacturers instructions. Briefly, 96-well ELISA
immunoplates were coated with Anti-BDNF (CatNb#G700B) diluted
1/1000 in carbonate buffer (pH 9.7) and incubated at 4.degree. C.
overnight. The following day, all wells were washed with
TBS-Tween.TM. 0.5% before incubation with Block/Sample buffer
1.times. at room temperature for one hour without shaking. After
blocking, standards and samples were added to the plates and
incubated and shaken (450.+-.100 rpm) for 2 h at room temperature.
Subsequently, after washing with TBS-Tween.TM. wash buffer, plates
were incubated for 2 h with Anti-Human BDNF pAb (1:500 dilution in
Block & Sample 1.times. Buffer) at 4.degree. C. After
incubation, plates were washed five times with TBS-Tween.TM. 0.5%
wash buffer and 100 .mu.l of diluted Anti-IgYHRP Conjugate was
added to each well (1:200 dilution in Block & Sample 1.times.
Buffer) and incubated for 1 hour at room temperature with shaking
(450.+-.100 rpm). Then, plates were washed five times with
TBS-Tween.TM. 0.5% wash buffer and 100 .mu.l of TMB One Solution
was added to each well. Following 10 minutes incubation at room
temperature with shaking (450.+-.100 rpm) for the BDNF plate, a
blue color formed in the wells. After stopping the reaction by
adding 100 .mu.l of 1N hydrochloric acid, the absorbance was read
at 450 nm on a microplate reader (Synergy 4.TM.) within 30 minutes
of stopping the reactions. Concentration of released BDNF in the
supernatants was determined according to the standard curves.
[0343] ELISA results revealed that BDNF was released at the same
concentration from differentiated Neuron-Like Cells (NLCs
differentiated from NSLCs) and normal Human neuron cells starting
at day 11 and remained until day 55 (Table 20), while no BDNF
(except for tiny amounts in the untransfected HFF group) was
released in the other groups.
TABLE-US-00035 TABLE 20 Quantification of BDNF release by
Neural-Like Cells (NLCs) that had been differentiated for 55 days
from Neural Stem-Like Cells (NSLCs) that had been created from
transfected HFFs. BDNF release from NLCs into the medium, at
different time points, was measured by antigen- capture ELISA and
compared to BDNF release of normal mature human neurons
(ScienCell). Control medium Neurons NLC day 0 day 11 1.55 30.25
22.99 day 18 0.33 29.49 25.15 day 24 0.33 22.01 26.39 day 34 0.23
25.53 32.21 day 41 0.27 19.02 22.43 day 55 0.02 20.73 30.01
[0344] In addition to adopting neuronal morphology criteria, the
NLCs were functional and possessed the capacity to release
neurotrophic factor (BDNF). Generating reprogrammed neuronal-like
cell lines that can locally deliver these neurotrophic factors
could be used as a method to treat several neurological conditions
and may offer crucial benefits in regeneration and functional
recovery from brain and other injuries.
Example IX
[0345] Reprogramming of Different Cell Types Towards NSLCs:
[0346] This study was performed to investigate the capacity of
keratinocytes (Invitrogen), human Adipocytes Derived Stem Cells
(ADSCs, Invitrogen) and human hematopoietic stem cells (CD34.sup.+,
Invitrogen) cells into neural stem-like cells.
[0347] Preparation of Human CD34.sup.+ Cells, Human ADSC and Human
Keratinocytes:
[0348] Human mobilized peripheral blood CD34.sup.+ cells were
purchased from StemCell Technologies and expanded as a floating
culture in Petri Dishes in complete StemPro.TM..RTM.-34 Serum-free
Medium (Invitrogen) supplemented with Stem Cell Factor (SCF, 150
g/ml, Peprotech), Granulocyte Colony-Stimulating Factor (GM-CSF,
37.5 ng/ml, Peprotech) and IL-3 (75 ng/ml, Peprotech). Medium
supplemented with cytokines was changed everyday 2-3 days after
centrifugation of the cell suspension at 300.times.g for 10 min.
Every other day the cytokines were added directly to the culture
without changing the media. Cells were incubated at 37.degree. C.,
5% CO.sub.2. For their passaging, cells were centrifugated,
resuspended in the above medium plus cytokines and placed into the
adequate number of Petri dishes.
[0349] Human Adipose-Derived Stem Cells (ADSC) were purchased from
Invitrogen and expanded in complete StemPro.TM. MSC Serum-free
medium (Invitrogen) on CellStart.TM. (Invitrogen) coated flasks
(diluted 1:100 in PBS containing Ca.sup.2+/Mg.sup.2+) at a cell
density of 1.times.10.sup.4 cells/cm.sup.2. Medium was replaced
every two days with fresh pre-warmed complete StemPro.TM. MSC SFM.
Cells were incubated at 37.degree. C., 5% CO.sub.2. Cells were
sub-passaged when 80% confluent by incubation for 3-5 min in
pre-warmed TrypLE.TM..TM. (Invitrogen) and then collected in
StemPro.TM. MSC medium. After centrifugation at 1500 rpm for 5 min,
cells were seeded on CellStart.TM. coated flasks as described
above.
[0350] Primary human keratinocytes were purchased from Invitrogen
and expanded in Defined Keratinocyte Serum-free medium on Coating
matrix (Invitrogen) coated flasks (Invitrogen) at a cell density of
5.times.10.sup.3 cells/cm.sup.2. The cells were incubated at
37.degree. C., 5% CO.sub.2. Media was replaced with fresh, complete
growth media every two to three days until subculture. Once the
cells had reached 70-80% confluency, media was removed and the
cells were incubated in Versene.TM. (Invitrogen) for 3-5 min at
room temperature. Versene.TM. was removed, and pre-warmed 0.05%
trypsin-EDTA (Invitrogen) was added to the flasks. After 5-10 min
incubation, growth medium containing Soybean Trypsin inhibitor
(Invitrogen) was added to the flasks and the cells gently
triturated. After centrifugation at 100.times.g for 10 min, cells
were resuspended in the desired volume of pre-warmed, complete
growth medium on coated flasks as described above.
[0351] Prior to transfection, cells were trypsinized and
transiently co-transfected with pCMV-Msi1-Ngn2 and pCMV6-XL5-MBD2
as previously described in Example IV using the Shuttle and plated
into a culture plate coated with laminin (Sigma, 10 .mu.g/ml).
Starting one day after transfection, cells were treated with VPA (1
mM) for 4 days and the medium was changed gradually to
proliferation medium supplemented with FGF (20 ng/ml) and EGF (20
ng/ml) and were cultured for 18 days at 37.degree. C., 5% CO.sub.2
and 5% O.sub.2. Cells were then analyzed for neural stem cell
markers by RT-PCR and Immunohistochemistry.
[0352] Further analysis and quantification of the reprogrammed
cells revealed a population of NSLCs engendered from keratinocyte
and CD34.sup.+ cells. RT-PCR Analysis revealed an increase of
relative expression of neural stem cell markers such as Sox2,
nestin, GFAP, and .beta.III-tubulin after transfecting Keratinocyte
and CD34.sup.+ by Msi1 and Ngn2. Relative expression of nestin and
GFAP was enhanced in NSLCs created from keratinocytes and
CD34.sup.+ cells as compared to NSLCs from HFFs; however, the
reverse was true for Sox2 and ACHE expression. .beta.III-tubulin
(TUBB3) and Map2b expression was highest in NSLCs created from
CD34.sup.+ cells, followed by NSLCs created from HFF (Table 21).
This data shows that different types of NSLCs with different gene
expression profiles (and characteristics) can be created from
different types of starting/source cells (and the same has been
observed for creating some other types of stem-like cells discussed
in this application). The data is also intriguing since it was not
expected that keratinocytes (which are derived from the ectoderm
just as endogenous neural stem cells) would have a lower expression
than HFFs for all the genes analyzed except for Nestin (it was
expected that keratinocytes would be the easiest to reprogram into
NSLCs since they are derived from the ectoderm).
TABLE-US-00036 TABLE 21 RT-PCR analysis was performed after one
month of transfection of human fibroblasts (HFF), Keratinocytes,
and CD34.sup.+ cells with Msi1/Ngn2 (MSI1/NGN2), in the presence
MBD2 with VPA treatment. Cells were cultured on coated culture
plates in proliferation medium (StemCell Technologies) supplemented
with EGF (20 ng/ml) and FGF (20 ng/ml) for 18 days. Untransfected
cells were considered as negative control. NES MAP2 TUBB3 ACHE GFAP
SOX2 Rel. Rel. Std. Rel. Std. Rel. Std. Std. Rel. Exp. Std. Dev.
Exp. Dev. Exp. Dev. Exp. Dev. Rel. Exp. Dev. Exp. Std. Dev. #1
Day12 1.00 0.07 1.00 0.05 1.00 0.01 1.01 0.15 1.00 0.02 1.08 0.59
Untransfected HFF #2 Day12 HFF 2.25 0.03 21.48 2.09 3.41 0.45 12.92
1.88 558.69 80.08 71513.12 14146.80 Msi1/Ngn2 + MBD2 #3 Day18 HFF
2.56 0.15 17.12 0.14 2.65 0.02 4.13 0.64 75.96 8.82 84794.40 318.54
Msi1/Ngn2 + MBD2 #4 1.07 0.54 1.00 0.07 1.00 0.02 1.01 0.19 1.06
0.48 1.00 0.01 Untransfected Keratinocytes #5 Day 12 11452.65
1137.13 0.96 0.11 6.78 0.28 1.09 0.05 5815.54 510.91 975.81 7.47
Keratinocytes Msi1/Ngn2 + MBD2 #6 Day 18 12593.79 431.06 0.93 0.04
6.41 0.27 0.48 0.03 1295.15 32.05 1047.17 139.48 Keratinocytes
Msi1/Ngn2 + MBD2 #7 1.00 0.04 1.01 0.16 1.00 0.00 1.00 0.01 1.10
0.66 1.01 0.21 Untransfected CD34+ #8 Day 18 839.57 134.51 346.61
33.97 33.91 4.38 0.28 0.00 2790.18 304.43 25080.35 35.93 CD34+
Msi1/Ngn2 + MBD2 hNPC 4.56 0.07 278.36 11.50 0.81 0.06 72.65 1.83
1285.73 5.27 565552.30 41717.72
[0353] Immunohistochemistry revealed positive staining for GFAP,
Sox2, and nestin. NSLCs developed from HFF yield a higher
percentage of positive staining for Sox2 and GFAP (55.8.+-.3.8 and
78.1.+-.2.4) as compared to CD34.sup.+ cells (42.8.+-.2.7 and
24.2.+-.4.4), and keratinocytes (47.1.+-.2.1 and 43.4.+-.8.9). The
percentage of nestin positive cells was high in Keratinocytes
(77.8.+-.10.7) and HFF (88.45.+-.12.9) and lower in CD34.sup.+
cells (15.5.+-.2.7) (Table 22). Sox2 and Nestin positive staining
was undetectable in ADSCs.
TABLE-US-00037 TABLE 22 The percentage of Sox2 and nestin positive
cells for neural stem cell markers after transfecting fibroblast,
keratinocyte, and CD34.sup.+ cells with pCMV-Msi1-Ngn2 in the
presence of MBD2 and VPA. Cells were cultured on coated culture
plates in proliferation medium (StemCell Technologies) supplemented
with EGF (20 ng/ml) and FGF (20 ng/ml) for 18 days. Untransfected
cells were considered as negative control. The percentage of
immunopositive cells was determined by Cellomics .TM. and
represented as mean .+-. SD (n = 5). % positive Untransfected cells
cells Fibroblasts Keratinocytes CD34.sup.+ Sox2 1.5 .+-. 1.7 55.8
.+-. 3.8 47.1 .+-. 2.1 42.8 .+-. 2.7 GFAP 0.04 +/- 0.2 78.1 .+-.
2.4 43.4 .+-. 8.9 24.2 .+-. 4.4 Nestin 0.3 +/- 0.3 68.45 .+-. 12.9
77.6 .+-. 10.7 15.5 .+-. 2.7
[0354] NSLCs generated from keratinocytes and CD34.sup.+ cells were
tested for tripotent capacity. Further differentiation studies were
performed to induce differentiation of these NSLCs towards neuronal
lineage, using NeuroCult.TM. differentiation medium (NeuroCult.TM.
differentiation Kit, StemCell Technologies) supplemented with BDNF
(20 ng/ml, Peprotech) and bFGF (40 ng/ml, Peprotech) as described
in Example V. NSLCs generated from HFFs and hNPCs were used as
controls, cultures were incubated at 37.degree. C., 5% CO.sub.2, 5%
O.sub.2 for three weeks. Samples were collected or fixed at Day 14
and 28 following differentiation for further analysis. RT-PCR
analysis revealed decrease of undifferentiated genes (Nestin and
Sox2) and increased of differentiated genes (Map2,
.beta.III-tubulin, CNPase, and GFAP) as shown in Tables 23A, 23B,
23C and 23D.
TABLE-US-00038 TABLE 23A RT-PCR analysis was performed on NSLCs
generated from human fibroblasts (HFF), keratinocytes, and
CD34.sup.+ cells that were cultured on Poly-D-Lysin/Laminin coated
culture plates in differentiation medium for 28 days (StemCell
Technologies) supplemented with BDNF (20 ng/ml) and FGF (40 ng/ml).
hNPCs (Lonza) were considered as a positive control. hNPCs had a
much lower increase in ACHE, GFAP, and MAP2b (which actually
decreased in hNPCs), but an increase in Nestin, compared to NSLCs
under differentiation conditions. NES MAP2 TUBB3 ACHE GFAP SOX2
SOX9 CNP Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel.
Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev.
Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. hNPC Control 1.00 0.08 1.00
0.10 1.00 0.08 1.01 0.16 1.00 0.09 1.01 0.16 1.00 0.12 1.00 0.09
Diff. hNPC Day 3.86 0.20 0.65 0.05 4.87 0.57 0.74 0.52 97.26 7.13
1.85 0.21 0.50 0.04 1.43 0.05 14 Diff. hNPC Day 1.86 0.06 0.68 0.02
3.67 0.13 1.33 0.09 102.74 1.89 1.29 0.01 0.73 0.05 1.37 0.02 28
NSLC Control 1.00 0.04 1.00 0.04 1.00 0.04 1.00 0.03 1.00 0.01 1.00
0.01 1.00 0.02 1.00 0.05 Diff. NSLC Day 1.38 0.01 1.00 0.09 2.06
0.02 1.57 0.24 1.79 0.12 0.73 0.01 0.56 0.01 1.31 0.05 14 Diff.
NSLC Day 0.62 0.02 0.90 0.08 5.14 0.21 6.47 0.78 5.70 0.15 1.30
0.02 0.79 0.03 1.41 0.01 28 HFF-NS 1.00 0.00 1.00 0.05 1.00 0.01
1.00 0.07 1.00 0.00 1.00 0.07 1.00 0.01 1.00 0.02 Control Diff.
HFF-NS 2.70 0.08 3.08 0.12 3.24 0.14 59.93 5.85 478.97 0.27 2.90
0.32 0.81 0.03 4.02 0.35 Day 14 Diff. HFF-NS 1.27 0.05 1.48 0.11
1.59 0.03 24.62 1.00 576.80 20.98 1.52 0.00 0.86 0.08 2.74 0.23 Day
28 Kerat-NS 1.00 0.06 1.00 0.02 1.00 0.03 1.00 0.11 1.00 0.01 1.00
0.07 1.00 0.02 1.00 0.01 Control Diff. Kerat-NS 2.43 0.06 3.48 0.08
2.82 0.11 56.22 5.58 665.91 10.52 3.09 0.29 1.01 0.14 3.72 0.17 Day
14 Diff. Kerat-NS 0.81 0.03 1.72 0.00 1.61 0.18 26.09 1.12 673.65
11.34 1.29 0.03 1.12 0.03 2.02 0.05 Day 28 CD34+-NS 1.00 0.05 1.00
0.07 1.00 0.04 1.00 0.08 1.00 0.00 1.00 0.08 1.00 0.02 1.00 0.07
Control Diff. CD34+-NS 2.21 0.04 3.47 0.07 2.75 0.04 57.87 6.68
407.54 52.07 2.90 0.18 1.10 0.05 3.54 0.02 Day 14 Diff. CD34+-NS
0.79 0.04 1.48 0.01 1.83 0.37 26.92 3.73 485.51 10.66 1.02 0.04
1.20 0.09 2.34 0.05 Day 28
TABLE-US-00039 TABLE 23B RT-PCR analysis was performed on
undifferentiated NSLCs generated from human fibroblasts (HFF),
keratinocytes, and CD34.sup.+ cells that were cultured on Laminin
coated culture plates in Proliferation medium for 4 days (StemCell
Technologies) supplemented with EGF (20 ng/ml) and FGF (20 ng/ml).
Relative expression calibrated to undifferentiated hNPCs. NES MAP2
TUBB3 ACHE GFAP SOX2 SOX9 CNP Rel. Std. Rel. Std. Rel. Std. Rel.
Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev.
Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev.
Undifferentiated 1.00 0.08 1.00 0.10 1.00 0.08 1.01 0.16 1.00 0.09
1.01 0.16 1.00 0.12 1.00 0.09 hNPC Control Day 4 Undifferentiated
1.23 0.05 0.12 0.00 1.12 0.04 0.09 0.00 21.45 0.26 0.65 0.01 0.28
0.01 0.37 0.02 NSLC Control Day 4 Undifferentiated 0.94 0.00 0.12
0.01 0.92 0.01 0.03 0.00 0.38 0.00 0.37 0.02 0.32 0.00 0.31 0.00
HFF-NS Control Day 4 Undifferentiated 1.00 0.06 0.09 0.00 0.97 0.03
0.03 0.00 0.23 0.00 0.38 0.03 0.26 0.00 0.30 0.00 Kerat-NS Control
Day 4 Undifferentiated 1.10 0.05 0.12 0.01 0.95 0.04 0.04 0.00 0.33
0.00 0.44 0.04 0.26 0.00 0.30 0.02 CD34+-NS Control Day 4
TABLE-US-00040 TABLE 23C RT-PCR analysis was performed on
differentiated NSLCs generated from human fibroblasts (HFF),
keratinocytes, and CD34.sup.+ cells that were cultured on
Poly-D-Lysin/Laminin coated culture plates in differentiation
medium for 14 days (StemCell Technologies) supplemented BDNF (20
ng/ml) and FGF (40 ng/ml). Relative expression calibrated to Day 14
differentiated hNPCs. NES MAP2 TUBB3 ACHE GFAP SOX2 SOX9 CNP Rel.
Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std.
Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp.
Dev. Exp. Dev. Exp. Dev. Diff. hNPC Day 1.00 0.05 1.00 0.07 1.00
0.12 1.15 0.80 1.00 0.07 1.00 0.11 1.00 0.08 1.00 0.03 14 Diff.
NSLC Day 0.44 0.00 0.18 0.02 0.47 0.00 0.22 0.03 0.40 0.03 0.26
0.00 0.31 0.00 0.34 0.01 14 Diff. HFF-NS 0.66 0.02 0.56 0.02 0.62
0.03 2.96 0.29 1.86 0.00 0.58 0.06 0.52 0.02 0.87 0.08 Day 14 Diff.
Kerat-NS 0.63 0.02 0.51 0.01 0.56 0.02 2.78 0.28 1.56 0.02 0.64
0.06 0.54 0.08 0.79 0.04 Day 14 Diff. CD34+-NS 0.63 0.01 0.62 0.01
0.54 0.01 3.77 0.43 1.39 0.18 0.69 0.04 0.58 0.03 0.76 0.00 Day
14
TABLE-US-00041 TABLE 23D RT-PCR analysis was performed on
differentiated NSLCs generated from human fibroblasts (HFF),
keratinocytes, and CD34.sup.+ cells that were cultured on Poly
D-Lysin/Laminin coated culture plates in differentiation medium for
28 days (StemCell Technologies) supplemented with BDNF (20 ng/ml)
and FGF (40 ng/ml). Relative expression calibrated to Day 28
differentiated hNPCs. NES MAP2 TUBB3 ACHE GFAP SOX2 SOX9 CNP Rel.
Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std.
Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp.
Dev. Exp. Dev. Exp. Dev. Diff. hNPC Day 1.00 0.03 1.00 0.02 1.00
0.04 1.00 0.07 1.00 0.02 1.00 0.01 1.00 0.07 1.00 0.02 28 Diff.
NSLC Day 0.41 0.01 0.15 0.01 1.56 0.06 0.44 0.05 1.19 0.03 0.66
0.01 0.30 0.01 0.38 0.00 28 Diff. HFF-NS 0.64 0.03 0.26 0.02 0.40
0.01 0.59 0.02 2.12 0.08 0.43 0.00 0.38 0.04 0.62 0.05 Day 28 Diff.
Kerat-NS 0.44 0.02 0.24 0.00 0.42 0.05 0.62 0.03 1.50 0.03 0.38
0.01 0.40 0.01 0.44 0.01 Day 28 Diff. CD34+-NS 0.47 0.03 0.25 0.00
0.47 0.10 0.85 0.12 1.57 0.03 0.35 0.01 0.43 0.03 0.52 0.01 Day
28
[0355] Fluorescent immunohistochemical staining was performed on
samples after 14 days and 28 days of differentiation. The
expression of Sox2 and Nestin was decreased time dependently in
differentiated cells (HFF, keratinocyte, and CD34.sup.+). This
decrease was associated with an increase of differentiated markers
at day 28 such as GFAP (68.51.+-.11.87 for HFF-NC, 59.55.+-.9.12
for Keratinocyte NC, and 61.70.+-.1.48 for CD34.sup.+-NC). A high
percentage for .beta.III-tubulin positive cells was generated from
differentiated NSLCs generated from HFF (57.83.+-.4.49) as compared
to .beta.III-tubulin positive cells generated from Keratinocytes
(23.27.+-.2.91) and CD34.sup.+ cells (39.15.+-.7.99) (Table 24)
TABLE-US-00042 TABLE 24 The percentage of cells stained positive
for neural stem cell markers and neuronal lineage markers in hNPCs
(Lonza) and transfected keratinocytes, HFF, and CD34.sup.+ cells
with pMsi1/Ngn2/MBD2. Transfected cells (NSLCs) were cultured in
Proliferation medium or differentiation medium for 28 days at
37.degree. C., 5% CO.sub.2, 5% O.sub.2. The percentage of
immunopositive cells (Sox2, Nestin, GFAP, S100beta, and
.beta.III-tubulin) was determined by Cellomics .TM. and represented
as mean .+-. SD (n = 5). Proliferation 14 days 28 days % positive
cells conditions differentiation differentiation hNPC Sox2 96.23
.+-. 0.51 59.05 .+-. 3.01 41.43 .+-. 6.05 Nestin 41.47 .+-. 0.23
10.77 .+-. 4.78 16.14 .+-. 7.41 S100.beta. 37.38 .+-. 7.85 49.51
.+-. 2.39 n.d. .beta.III-tubulin 2.34 .+-. 0.43 11.54 .+-. 4.03
23.34 .+-. 4.77 GFAP 1.16 .+-. 0.14 23.42 .+-. 2.51 48.04 .+-. 8.30
HFF-NC Sox2 93.28 .+-. 0.53 79.48 .+-. 0.54 52.06 .+-. 9.07 Nestin
29.29 .+-. 4.72 1.15 .+-. 0.46 2.18 .+-. 1.96 S100.beta. 13.51 .+-.
0.28 80.75 .+-. 3.50 79.38 .+-. 10.62 .beta.III-tubulin 3.91 .+-.
0.33 42.16 .+-. 15.07 57.83 .+-. 4.49 GFAP 8.41 .+-. 0.73 59.66
.+-. 11.48 68.51 .+-. 11.87 Keratinocyte-NC Sox2 96.55 .+-. 1.01
76.93 .+-. 5.13 63.11 .+-. 8.54 Nestin 40.10 .+-. 8.41 2.67 .+-.
1.61 3.57 .+-. 0.48 S100.beta. 13.58 .+-. 4.97 76.6 .+-. 9.72 74.75
.+-. 11.21 .beta.III-tubulin 6.42 .+-. 2.94 20.58 .+-. 8.34 23.27
.+-. 2.91 GFAP 9.36 .+-. 0.34 43.43 .+-. 2.44 59.55 .+-. 9.12
CD34.sup.+-NC Sox2 95.49 .+-. 2.6 81.18 .+-. 1.24 63.46 .+-. 5.14
Nestin 51.68 .+-. 14.27 12.64 .+-. 1.27 8.46 .+-. 4.6 S100.beta.
30.1 .+-. 1.03 72.40 .+-. 4.5 79.57 .+-. 8.52 .beta.III-tubulin
5.82 .+-. 2.08 25.04 .+-. 19.95 39.15 .+-. 7.99 GFAP 13.99 .+-.
5.48 51.79 .+-. 13.68 61.70 .+-. 1.48 n.d. = not determined; .+-. =
standard deviation CD34.sup.+-NC: neuronal cells generated after
differentiation of NSLCs generated from CD34.sup.+ cells. Each data
point represents the analysis of at least 1000 cells from at least
8 images.
[0356] The % of Sox2 positive cells decreased faster, the % of
Nestin positive cells generally decreased slower, and the % of
cells expressing one of the differentiation markers (S100.beta.,
.beta.III-tubulin, GFAP) generally increased slower in hNPCs than
in the NSLCs during differentiation. Out of the three types of
created NSLC lines, the % of cells expressing one of the
differentiation markers (S100.beta., .beta.III-tubulin, GFAP)
generally increased slowest in NSLCs created from keratinocytes and
fastest in NSLCs created from HFFs.
[0357] This study indicates that NSLCs can be created from
keratinocytes and CD34.sup.+ blood cells, and these cells share
morphology and markers similarly to NSLCs generated from HFF.
Similarly to hNPCs, NSLCs created from keratinocytes, CD34.sup.+
cells, and HFFs had a tendency to differentiate more towards an
astrocyte lineage than a neuronal lineage (except NSLCs created
from HFFs had an almost similar number of .beta.III-tubulin
positive and GFAP positive cells) as shown by the high percentage
of GFAP positive cells during differentiation, which was confirmed
by S100beta staining. However, the proportion of astrocyte and
neuronal cells generated from hNPCs was lower in same culture
conditions, indicating that NSLCs generated from HFF,
Keratinocytes, and CD34 cells can give rise to a higher number of
neuronal and astrocyte cells as compared to hNPCs. NSLCs, whether
created from HFFs, Keratinocytes or CD34.sup.+ cells (or
potentially even some other cell), are tripotent cells and possess
the capacity to differentiate to neurons, astrocytes, and
oligodendrocytes similarly to hNPCs. However, RT-PCR and
immunohistochemistry analysis of transfected ADSCs did not reveal
any significant expression of neural stem cell genes, indicating a
need to optimize conditions for turning ADSCs to NSLCs or to
investigate the effect of others neurogenic factors that could turn
these into NSLCs.
Example X
Fabrication 3D Extracellular Matrix (CDM)
[0358] Fibroblast cells were cultured in DMEM medium in the
presence of 10% FCS as described in Example I, followed by seeding
onto 12-well plates pre-coated with laminin (10 .mu.g/ml) at a
concentration of 2.times.10.sup.6 cells/ml in defined CDM Medium
consisting of a 3:1 ratio of Dulbecco's modified Eagle medium
(DMEM, high glucose (4.5 g/L) with L-glutamine and sodium pyruvate)
and Ham's F-12 medium supplemented with the following components:
EGF (4.2.times.10.sup.-10 M), bFGF (2.8.times.10.sup.-10M), ITS
(8.6.times.10.sup.-6M), dexamethasone (1.0.times.10.sup.-7M),
L-ascorbic acid phosphate magnesium salt n-hydrate
(3.2.times.10.sup.4M), L-3,3',5-triiodothyronine
(2.0.times.10.sup.-10 M), ethanolamine (10.sup.-4M), GlutaMAX.TM.
(4.times.10.sup.-3M), glutathione (3.3.times.10.sup.-6M), and 1%
penicillin/streptomycin/amphotericin B. By culturing the fibroblast
cells at hyperconfluent density in this completely chemically
defined medium causes them to enter a high synthetic phase with a
slow-down in proliferation, leading to the production of a living
tissue equivalent (LTE) consisting of multiple layers of
fibroblasts within de novo 3D extracellular matrix (CDM) that is
completely synthesized by the fibroblasts themselves.
Trans-Differentiation and Reprogramming of Cells within CDM
[0359] Day 14 CDM samples were treated with cytochalsin B (100/ml,
Caibiochem), with the concentration of cytochalsin B reduced from
10 .mu.g/ml to 0 .mu.g/ml (none) over 5 days while at the same time
switching the medium from CDM Medium to NbActive medium. Samples
were cultured for another 12 days at 37.degree. C., 5% CO.sub.2,
and the medium was changed every day. Samples were fixed to perform
immunohistochemistry as described previously to detect Neuronal
markers. The following antibodies were used: mouse anti-nestin 647
(1:100, BD) and anti-.beta.III-tubulin (1:200, Neuromics). No clear
morphology change of the cells was observed within the CDM and the
immunohistochemical analysis failed to detect .beta.III-tubulin
positive cells. Thus, inducing the trans-differentiation of cells
using only cytochalasin B and chemically-defined neural medium was
not sufficient to reprogram the cells.
[0360] Next, Day 6 CDM samples grown in LAS pre-coated plates at
37.degree. C. and 5% CO.sub.2, were exposed simultaneously to
cytocahlasin B (10 .mu.g/ml) over 5 days, histone deacetylation
inhibitor (VPA, 4 mM, Calbiochem) and inhibitor of DNA methylation
(5-Azacytidine, 5 .mu.M, Sigma). Four days later, the medium was
changed to differentiation medium consisting of a 3:1 ratio of CDM
medium without the presence of EGFand NbActive medium
(BrainBits.TM.) supplemented with NT-3 (20 ng/ml, Peprotech) and
BDNF (20 ng/ml, Peprotech). The ratio of the differentiation medium
was increased gradually day after day until reaching 100% of
complete differentiation medium. After two weeks of treatment,
cells were fixed for immunohistochemical analysis to investigate
the identity of the cells. Immunostained cells with fill-tubulin at
day 7, indicating the de-differentiation of fibroblast cells to
neurons. However, one week later, these trans-differentiated cells
reverted back to fibroblast cells and .beta.III-tubulin expression
was lost. The loss of morphology and .beta.III-tubulin expression
after withdrawal of the priming agents indicate that complete
conversion to functional and stable reprogrammed cells did not
occur.
[0361] Next CDM was treated with VPA (4 mM), 5-Aza (5 .mu.M) and
cytochalasin B (10 .mu.g/ml) as above. After 2 days of chemical
treatment, fibroblast cells within the CDM were transfected with
DNA using Lipofectamine reagent (Invitrogen) as per the
manufacturer's protocol. 15 .mu.g of the eukaryotic DNA expression
vectors pCMV6-XL5-Pax6, pCMV6-XL5-Msi1 and pCMV6-XL4-Ngn2 (Origene)
were used to transfect the cells. 24 hours later, the media was
changed to Neural Progenitor Basal Medium (Lonza) supplemented with
Noggin (50 ng/ml), EGF (20 ng/ml), and bFGF (20 ng/ml), and the
cells were cultured at 37.degree. C., 5% CO.sub.2 and 5% O.sub.2,
and the medium was changed every day. At day 6, differentiation was
initiated by adding gradually NBActive medium (BrainBits.TM.)
supplemented with NT-3 (20 ng/ml, Peprotech), all-trans-retinoic
acid (ATRA, 5 .mu.M, Spectrum), BDNF (20 ng/ml, Peprotech), and
bFGF (40 ng/ml, Peprotech). To characterize the reprogrammed cells,
immunohistochemical analysis and RT-PCR was performed at various
time points according to the methods described in Example II using
primers for nestin, GFAP, MAP2b, and ACHE. In agreement with
previous studies, un-transfected cells and cells transfected with
Pax6 did not expressed genes specific for neuronal lineages (Table
25). On the other hand, following transfection with Msi1, levels of
nestin and ACHE were increased to 4-fold and 8-fold, respectively,
and this expression was maintained over the 12-day period. Also
levels of GFAP mRNA was enhanced time dependently by approximately
14 times. Likewise, the same pattern was observed in Ngn2
transfected cells. While expression of .beta.III-tubulin and MAP2b
were modestly increased following transfection with one neurogenic
transcription factors the regulation of gene expression after
transfecting the cells with two neurogenic factors, Msi1 or Ngn2
with Pax6, did not further increase the expression of neuronal
genes. Expression of these genes was enhanced when the cells were
transfected with Msi1 and Ngn2, with .beta.III-tubulin enhanced to
almost 6-fold at day 12.
TABLE-US-00043 TABLE 25 RT-PCR analysis of relative expression of
neuronal precursor cell markers such as nestin, .beta.III-tubulin,
MAP2b, ACHE, and GFAP after transfection of fibroblast cells with
pCMV6-XL5-Msi1, pCMV6-XL4-Ngn2, pCMV6-XL5-Pax6, and pCMV6-XL5-MBD2.
After 24 h following transfection, CDM I Medium was changed and
cells were cultured in proliferation medium (NPBM, Lonza)
supplemented with EGF (20 ng/ml. Peprotech) and bFGF (20 ng/ml,
Peprotech) for one week. Differentiation was induced by changing
the medium to NbActive (BrainBits .TM.) supplemented with NT-3 (20
ng/ml), bFGF (20 ng/ml), ATRA (5 .mu.M) and Forskolin (10 .mu.M).
Cells were incubated at 37.degree. C., 5% CO.sub.2, 5% O.sub.2 for
12 days. Relative expression of Msi1, Ngn2, Pax6, nestin,
.beta.III-tubulin, ACHE, MAP2b and GFAP in NSLCs and NLCs was
increased after transfection with both transcription factors Ngn2
and Msi1 with MBD2 as the DNA demethylator. COL5A2 FBN2 NES MAP2
TUBB3 SOX2 ACHE GFAP Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel.
Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev.
Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. #1, +CytoB,
Control 1.00 0.07 1.00 0.01 1.00 0.04 1.00 0.05 1.00 0.05 1.00 0.05
1.00 0.10 1.00 0.11 #2, -CytoB, 1.00 0.03 1.00 0.08 1.00 0.00 1.00
0.09 1.00 0.09 1.15 0.80 1.01 0.18 1.00 0.01 Control #3, +CytoB,
0.85 0.04 0.75 0.02 0.60 0.01 0.29 0.01 0.44 0.00 22.39 5.26 0.81
0.19 10.14 0.15 Msi1, GAD45b #4, -CytoB, 0.87 0.03 1.81 0.09 1.84
0.04 2.31 0.00 2.09 0.03 20.28 5.33 1.99 0.74 6.03 0.05 Msi1,
GAD45b #5, +CytoB, 0.84 0.04 0.77 0.03 0.44 0.00 0.24 0.00 0.36
0.01 470.84 13.43 0.63 0.05 103.22 0.80 Ngn2, GAD45b #6, -CytoB,
0.75 0.07 1.97 0.02 1.83 0.00 4.40 0.16 2.02 0.10 789.33 60.35 1.70
0.13 110.48 4.90 Ngn2, GAD45b #7, +CytoB, 0.74 0.12 1.08 0.00 0.89
0.01 0.51 0.00 0.63 0.04 1.64 0.98 0.86 0.12 2.49 0.21 Pax6, GAD45b
#8, -CytoB, 0.66 0.04 2.41 0.09 2.70 0.03 4.96 0.30 3.48 0.07 0.46
0.33 2.97 1.04 0.43 0.09 Pax6, GAD45b #9, +CytoB, 0.14 0.01 0.28
0.01 1.30 0.03 4.07 0.11 0.84 0.00 54768.27 6709.56 0.81 0.24
3391.96 64.63 Msi1, Ngn2, GAD45b #10, -CytoB, 0.12 0.00 0.73 0.03
5.28 0.21 50.84 1.23 4.93 0.28 17400.66 822.88 3.58 0.10 1255.76
5.27 Msi1, Ngn2 GAD45b #11, +CytoB, 0.10 0.00 0.26 0.01 1.11 0.01
3.69 0.09 0.76 0.00 55588.41 1331.20 0.55 0.14 2849.96 261.51 Msi1,
Ngn2 MBD2 #12, -CytoB, 0.44 0.01 1.47 0.06 5.49 0.14 47.30 0.11
5.50 0.31 14587.46 789.19 3.90 0.13 1424.04 39.29 Msi1, Ngn2 MBD2
#13, +CytoB, 1.11 0.04 1.09 0.06 0.92 0.08 0.68 0.01 0.82 0.03
63.93 2.81 1.19 0.17 17.43 1.86 GAD45b #14, -CytoB, 0.94 0.01 2.22
0.00 2.82 0.02 6.49 0.30 4.01 0.05 6.12 0.61 2.34 0.17 1.42 0.10
GAD45b #15, +CytoB, 0.83 0.00 0.83 0.05 0.36 0.01 0.16 0.01 0.36
0.00 3.42 3.74 0.63 0.37 2.18 0.12 MBD2 #16, -CytoB, 0.68 0.02 1.55
0.04 1.57 0.05 1.47 0.01 2.00 0.00 0.52 0.29 1.45 0.15 0.55 0.04
MBD2 #17, +CytoB, 1.10 0.01 1.16 0.03 1.37 0.01 1.12 0.06 0.86 0.06
5.59 1.48 1.07 0.27 1.70 0.46 Msi1, Ngn2 #18, -CytoB, 0.93 0.04
2.52 0.10 3.48 0.01 9.01 0.02 4.55 0.18 1.78 1.46 3.83 0.42 0.59
0.01 Msi1, Ngn2 #19, +CytoB, 0.20 0.03 0.36 0.01 1.25 0.05 6.68
0.31 0.72 0.02 66592.29 3481.89 2.57 0.03 4450.08 131.85 Msi1, MBD2
#20, -CytoB, 0.12 0.00 0.64 0.03 4.70 0.22 77.51 0.11 4.12 0.11
19128.03 1542.00 8.14 0.13 999.22 24.75 Msi1, MBD2 #21, +CytoB,
0.17 0.01 0.28 0.00 1.16 0.04 5.73 0.06 0.62 0.00 67945.51 3000.74
2.15 0.04 4736.83 11.92 Ngn2, MBD2 #22, -CytoB, 0.17 0.00 0.78 0.03
4.32 0.08 68.89 5.26 4.01 0.04 16570.91 92.96 7.04 0.53 1427.13
13.19 Ngn2, MBD2 #23, +CytoB, 0.71 0.05 0.79 0.06 0.87 0.01 0.63
0.06 0.67 0.04 2.86 0.70 1.08 0.08 2.08 0.11 Msi1 #24, -CytoB, 0.66
0.04 1.92 0.17 2.03 0.02 2.77 0.02 2.68 0.02 0.32 0.12 1.85 0.65
0.58 0.04 Msi1
[0362] Same pattern of gene expression was observed when
transfecting the cells with three transcription factors (Msi1,
Ngn2, and Pax6), but the expression was less pronounced than in
cells transfecting with just Msi1 and Ngn2. In terms of
immunohistochemical analysis after the 12 days of the transfection,
cells displayed neuronal markers after transfection with Msi1 or
Ngn2, as indicated by the expression of nestin and MAP2b. Cells
transfected with pCMV-XL-PAx6 did not stain for Nestin and
MAP2b.
[0363] This study shows that transfecting cells within CDM with
only one neurogenic factor (Msi1 or Ngn2) induces morphological
changes and expression of one or more markers of neural stem cells
and neuronal cells. Since the reprogrammed cells expressed a key
neurogenic factor, a neuronal precursor marker, and a mature
neuronal marker at low percentage (10%), this suggests that cells
within the CDM were transformed to NSLCs and then started to
differentiated through the various phases of the neuronal
determination and differentiation program induced in neural stem
cells.
Example XI
[0364] Gene Expression Analysis of Reprogrammed Cells within
COM
[0365] This study was designed to test the effect of transfecting
cells with Msi1 and Ngn2 in the presence of MBD2 in the
reprogramming process. Cells were transfected after two days of
pre-treatment with cytocahlasin B with the DNA expression vectors
using Lipofectamine reagent as described in Example X. 15 .mu.g of
eukaryotic DNA expression vectors pCMV6-XL5-Musashi or
pCMV6-XL4-Ngn2, and pCMV6-XL5-MBD2 (Origene), were used to
co-transfect cells. After 24 hours, the media was changed to
CDM:Neural Progenitor Maintenance Medium (1:1) supplemented with
Noggin (50 ng/ml), EGF (20 ng/ml), and bFGF (20 ng/ml). Medium was
changed every day by increasing the percentage of NPBM and
decreasing CDM medium. Cells were cultured for 6 days at 37.degree.
C., 5% CO.sub.2 and 5% O.sub.2. After one week, differentiation was
initiated by gradually supplementing the NPBM Medium with NT-3 (20
ng/ml, Peprotech), all-trans-retinoic acid (ATRA, 5 .mu.M,
Spectrum), BDNF (20 ng/ml, Peprotech), and bFGF (40 ng/ml,
Peprotech). Samples were collected at the end of the study (day 14)
and data were analyzed by gene array to identify genes that were
reproducibly found to be specific for neuronal lineages,
Gene Expression Analysis
[0366] Gene expression analysis on 8 samples was performed as
previously described in Example I with the customized Neuronal
Markers 2 TLDA In order to identify the expression of genes related
to neural stem cells, neuronal cells and glial cells, and growth
factors expressed by the cells after transfection. The expression
of oligodendrocyte genes, such as NKx2.2, olig2, and MAG was
increased by Msi1 and Ngn2; however, the increased was more
pronounced by Msi1 as compared to Ngn2 (Table 26). Two markers for
astrocytes (GFAP and AQP4) were highly expressed after transfection
with Msi1 and Ngn2 in the presence of the DNA demethylator MBD2.
Interestingly, several markers of early neuronal cells were
enhanced; 12 days after transfection, TDLA data revealed increases
in specific markers for interneurons, such as somatostatin and
calbindin1. Doublecortin (DCX), which is expressed by migrating
immature cells during development, and acetylcholine (ACHE), an
early marker of neuronal cells, were highly expressed in
reprogrammed cells (Table 26). Transfection with Msi1 or Ngn2
increased the expression of dihydropyrimidinase-like 3 (DPYSL3), an
early marker of newborn neurons to five-fold with Msi1 and
seven-fold with Ngn2. Expression of microtubule-associated protein
2 (MAP2), an essential marker for development and maintenance of
early neuronal morphology, and neuronal cell adhesion molecule
(NCAM) were highly expressed with Msi1 and Ngn2. The expression of
enolase-2, a marker of mature neurons, was 20-fold enhanced by Msi1
and Ngn2. Member of the NeuroD family NeuroD1 was highly expressed
after transfection with Mail to 84.22 fold and to 34.27 by Ngn2.
Gene expression of growth factors such as IGF-1, IGF-2, NPY and
CSF-3 was enhanced following transfection with Msi1 or Ngn2. The
expression of VEGF and GDNF genes were increased to almost
five-fold and seven-fold by Msi1 and Ngn2, respectively. However in
transfected cells, the expression of BDNF, EGF, and bFGF were not
activated and even down-regulated as compared to untransfected
cells. The expression of growth associated protein (GAP-43), a
growth- and regeneration-associated marker of neurite extension,
and expression of netrin, implicated in neuronal development and
guidance, were highly expressed in transfected cells (Table 26).
Expression of receptors for growth and neurotrophic factors was
increased, such as type III receptor tyrosine kinase, Neurotrophic
tyrosine kinase receptor, and neurotrophic tyrosine kinase. The
fibroblast-specific markers vimentin and fibronectin were
down-regulated in the reprogrammed cells.
[0367] Transfection of HFF with only Mail and Ngn2 in the presence
of MBD2 increased the expression of glial cells and neuronal cells
markers.
TABLE-US-00044 TABLE 26 Gene array of CDM transfected with pMsi1
and pNgn2 following the pre-treatment with cytochalasin B (10
.mu.g/ml), VPA (4 mM) and 5-Azacytidine (5 .mu.M). Transfected
cells were cultured in differentiation medium (NbActive, BrainBits
.TM.) supplemented by ATRA (5 .mu.M), bFGF (40 ng/ml) and BDNF (20
ng/ml). Gene array of CDM transfected with pMsi1 and pNgn2
following the pre-treatment with cytochalasin B (10 .mu.g/ml), VPA
(4 mM) and 5-Azacytidine (5 .mu.M) Relative Relative Expression
Symbol Common name and description Company Gene ID Expression Msi 1
Ngn 2 Astrocytes and oligodendrocytes markers NKx2-2 Markers for
oligodendrocyte progenitors NM_002509.2 1.72 10.19 OLIG2
Oligodendrocyte lineage transcription factor 2 NM_005806.2 1.72
1.52 MBP Myelin-basic protein NM-001025090.1 1.72 1.52 GFAP Glial
fibrillary acidic protein NM_002055.4 6.04 2.41 AQP4 Aquaporin 4
NM_001650.4 1.72 1.52 DIO2 Deiodinase iodothyronine NM_013989.3
8.29 10.61 NC markers SST Somatostatin, specific marker for
NM_001048.3 Very high Very high interneurons CALB1 Clabindin 1,
interneuron marker NM_004929.2 1.72 1.52 Tubulin1A Are necessary
for axonal growth NM_006009.2 0.63 0.76 NES Precursor neurons
(nestin) NM_006617.1 2.42 2.86 DCX An early neuronal marker
(Doublecortin) NM_178151.1 1.72 1.52 ACHE Acetylcholinesterase,
marker of early NM_016831.2 10.68 20.37 neuronal development ENO2 A
marker for neurons cells, enolase NM_001975.2 0.55 0.54 NEUROD1
Neural marker; expression gradually NM_002500.2 1.72 1.50 increased
from neural precursor to fully differentiated neuron DPYSL3
Dihydropyrimidinase-like 3, marker of NKM_001387.2 0.62 0.71
immature neurons MAP2 Microtubule-associated protein 2, essential
NM_002374.3 1.99 1.70 for development of early neuronal morphology
and maintenance of adult neuronal morphology NCAM Neural cell
adhesion molecule 1 NM_18135.3 3.11 5.72 CEND1 Cell cycle exit
& neuronal differentiation, NM_016564.3 6.68 8.28 early marker
of proliferating precursor cells that will differentiate into
neurons Neuroregeneration and survival genes FGF2 Fibroblast growth
factor NM_002006.4 1.19 1.26 EGF Epidermal growth factor
Hs00153181_ml 28.37 52.13 IGF-1 Insulin growth factor-1 NM_000618.2
0.82 1.03 IGF-2 Insulin growth factor-2 NM_0000612.3 0.99 1.21 CSF3
Granulocyte colony stimulating factor NM_2219.1 Very high Very high
BDNF Brain derived growth factor, neurogenesis NM-199231.1 8.54
7.84 GDNF Glial derived neurotrophic factor NM-000614.2 0.63 0.91
CNTF Ciliary neurotrophic factor NM_001025366.1 3.80 14.92 VEGF
Vascular endothelial growth factor NM_130850.1 6.28 7.22 BMP-4 Bone
morphogenetic protein 4 NM_002253.1 1.17 1.34 KDR Type III receptor
tryrosine kinase NM_006180.3 113.85 43.87 NTRK2 Neurotrophic
tyrosine kinase receptor NM_000905.2 0.02 0.02 (TrkB) NPY
Neuropeptide Y NM_00905.2 33.39 1.52 NTF-5 Neuortrophin 5
NM_006179.3 4.43 5.93 PIK3CG Phosphinositide-3-kinase NM_002649.2
1.70 1.50 STAT3 Signal transduction transcription 3 NM_213662.1
3.15 2.24 Gap43 Growth associated protein 43 NM_002045.2 1.82 2.98
NTN1 Netrin1, implicated in neuronal NM_004822.2 0.50 0.29
development and guidance NTRk2 Neurotrophic tyrosine kinase,
receptor, type 2 NM_006180.3 0.02 0.02 L1CAM L! cell adhesion
molecule, associated with NM_024003.1 0.08 0.11 regenerating axons
LIMK1 LIM domain kinase 1 NM_002314.2 2.88 2.96 Vimentin Radial
glia and fibroblast marker NM-003380.2 0.21 0.20 Fibronectin
Fibronectin is a marker for fibroblasts NM_212474.1 0.15 0.14
Transfected cells were cultured in differentiation medium
(NbActive, BrainBits .TM.) supplemented by ATRA (5 .mu.M), bFGF (40
ng/ml) and BDNF (20 ng/ml).
Example XII
[0368] Reprogramming of Cells within CDM by Lipofectamine and
Nucleofection
[0369] This study was designed to improve transfection of CDM by
combining lipofectamine and nucleofection and using two vectors
pCMV6-XL5-Msi1 and pCMV6-XL4-Ngn2 individually or in combination
together with pCMV-XL5-MBD2. Cells within Day 4 CDM were
lipotransfected for 6 hours with Msi1/MBD2, Ngn2/MBD2 or
Msi/Ngn2/MBD2 after 2 days of pre-treatment with or without
cytochalasin B. In parallel, transfection was performed on fresh
HFFs after the 6 hours using Nucleofection as described in Example
II, and transferred on top of the CDM when the lipofectamine media
was changed to fresh CDM medium. After 24 hours, the medium was
changed to Neural Progenitor Basal Medium (NPBM, Lonza) with the
presence of Noggin (50 ng/ml, Peprotech), recombinant hFGF (20
ng/ml, Peprotech), and recombinant hEGF (20 ng/ml, Peprotech).
Differentiation was induced at day 7, by adding NSA-A
differentiation medium (StemCell Technologies) for 21 days.
Gene Expression Analysis
[0370] Samples were collected at 8, 15, and 21 days to evaluate the
nature of newly formed cells by analyzing the expression of several
neuronal marker genes using RT-PCR according to the methods
previously described in Example I. As shown in Table 27, cells
transfected with one neurogenic transcription factor (Msi1 or Ngn2)
express high levels of nestin and .beta.III-tubulin at day 8. The
same pattern of expression was observed at day 15 and 21, while the
expression was slightly decreased in the absence of cytochalasin B
in cells transfected with Ngn2. The expression of all genes, except
the mature neuronal marker MAP2b, were remarkably increased in
cells transfected with both neurogenic transcription factors. The
upregulation of these genes was slightly reduced in the absence of
cytochalasin B, indicating its role in enhancing
TABLE-US-00045 TABLE 27 RT-PCR analysis of relative expression of
neuronal stem cell markers such as nestin, Sox2, and GFAP after
transfection of fibroblast cells within the CDM with different
combinations with or without the co-treatment with cytochalasin B.
Relative expression of Sox2, nestin, and GFAP in NSLCs was
increased after transfection with both transcription factors Ngn2
and Msi1 with MBD2 as the DNA demethylator. MSI1 NGN2 TUBB3 GFAP
NES MAP2 Rel. Std. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std.
Exp. Dev. Rel. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. #1
Day8 CDM - CytoB Control 1.11 0.21 1.33 0.20 1.10 0.02 0.91 0.02
1.18 0.09 0.91 0.02 #2 Day8 CDM - CytoB Control 1.11 0.17 0.65 0.08
0.92 0.06 0.91 0.11 0.82 0.01 0.91 0.11 #3 Day8 CDM - CytoB Control
0.83 0.01 0.71 0.86 0.99 0.04 1.21 0.00 1.03 0.00 1.21 0.00 #4 Day8
CDM + CytoB Control 7.42 0.35 1.52 0.53 1.32 0.16 0.44 0.06 1.04
0.02 0.44 0.06 #5 Day8 CDM + CytoB Control 7.01 0.42 2.14 0.58 1.23
0.07 0.62 0.05 1.02 0.06 0.62 0.05 #6 Day8 CDM + CytoB Control 9.15
0.48 0.76 0.08 0.40 0.05 0.59 0.14 0.34 0.16 0.59 0.14 #7 Day15 CDM
- CytoB Control 1.45 0.07 1.53 0.33 1.32 0.01 0.90 0.07 1.31 0.03
0.90 0.07 #8 Day15 CDM - CytoB Control 0.79 0.02 2.01 1.49 0.91
0.03 1.14 0.16 0.91 0.01 1.14 0.16 #9 Day15 CDM - CytoB Control
0.87 0.04 0.64 0.72 0.84 0.08 0.98 0.15 0.84 0.01 0.98 0.15 #10
Day15 CDM + CytoB 1.27 0.14 0.99 0.66 1.70 0.21 0.36 0.02 1.08 0.08
0.36 0.02 Control #11 Day15 CDM + CytoB 1.39 0.04 0.97 0.65 2.65
0.38 0.44 0.06 1.97 0.30 0.44 0.06 Control #12 Day15 CDM + CytoB
1.09 0.21 0.49 0.46 1.32 0.14 0.47 0.15 2.45 0.15 0.47 0.15 Control
#13 Day21 CDM - CytoB 1.21 0.00 1.06 0.06 1.10 0.01 0.86 0.16 1.07
0.01 0.86 0.16 Control #14 Day21 CDM - CytoB 0.97 0.09 2.16 0.77
0.96 0.01 1.11 0.10 0.94 0.01 1.11 0.10 Control #15 Day21 CDM -
CytoB 0.86 0.02 1.01 1.27 0.94 0.00 1.08 0.26 0.99 0.04 1.08 0.26
Control #16 Day21 CDM + CytoB 1.41 0.21 1.29 1.64 2.46 0.07 0.88
0.22 1.58 0.05 0.88 0.22 Control #17 Day21 CDM + CytoB 2.24 0.00
0.35 0.01 2.23 0.03 0.55 0.16 1.57 0.02 0.55 0.16 Control #18 Day21
CDM + CytoB 2.18 0.14 0.77 0.06 2.29 0.12 0.54 0.04 1.47 0.04 0.54
0.04 Control #19 Day8 CDM - CytoB 694.16 18.10 0.51 0.05 1.46 0.04
2.18 0.13 1.02 0.03 2.18 0.13 Msi1/MBD2 #20 Day8 CDM - CytoB 2.38
0.29 4106.88 48.57 0.46 0.02 1.88 0.14 0.99 0.02 1.88 0.14
Ngn2/MBD2 #21 Day8 CDM - CytoB 365.04 6.71 2702.81 55.69 4.44 0.02
2.95 0.38 5.11 0.05 2.95 0.38 Msi1/Ngn2/MBD2 #22 Day8 CDM + CytoB
1262.00 63.21 0.75 0.91 0.54 0.03 2.48 0.11 1.16 0.05 2.48 0.11
Msi1/MBD2 #23 Day8 CDM + CytoB 2.34 0.20 10963.51 19.89 0.53 0.00
2.27 0.26 1.00 0.06 2.27 0.26 Ngn2/MBD2 #24 Day8 CDM + CytoB 869.15
65.33 6401.28 87.12 4.58 0.01 3.65 0.13 3.15 0.00 3.65 0.13
Msi1/Ngn2/MBD2 #25 Day15 CDM - CytoB 41.07 1.74 2.58 0.36 1.43 0.05
0.58 0.06 1.34 0.07 0.58 0.06 Msi1/MBD2 #26 Day15 CDM - CytoB 0.73
0.02 2192.64 15.74 0.95 0.08 1.01 0.09 0.99 0.03 1.01 0.09
Ngn2/MBD2 #27 Day15 CDM - CytoB 45.59 2.33 3318.42 51.51 5.32 0.08
3.80 0.01 4.32 0.01 4.80 0.01 Msi1/Ngn2/MBD2 #28 Day15 CDM + CytoB
106.34 4.43 4.90 1.70 1.47 0.01 0.57 0.10 1.19 0.03 0.57 0.10
Msi1/MBD2 #29 Day15 CDM + CytoB 1.09 0.11 6715.95 505.86 1.30 0.05
0.70 0.17 1.18 0.07 0.70 0.17 Ngn2/MBD2 #30 Day15 CDM + CytoB 46.77
0.76 2816.33 90.83 5.76 0.02 4.52 0.09 3.60 0.03 5.52 0.09
Msi1/Ngn2/MBD2 #31 Day21 CDM - CytoB 22.94 1.09 10.09 2.72 1.08
0.07 0.58 0.08 1.17 0.02 0.58 0.08 Msi1/MBD2 #32 Day21 CDM - CytoB
0.78 0.02 4450.56 255.75 1.00 0.03 0.75 0.21 1.09 0.03 0.75 0.21
Ngn2/MBD2 #33 Day21 CDM - CytoB 24.02 0.86 2509.95 64.00 5.18 0.05
4.74 0.16 4.37 0.06 3.74 0.16 Msi1/Ngn2/MBD2 #34 Day21 CDM + CytoB
54.17 1.41 8.31 3.32 1.42 0.05 0.70 0.22 1.71 0.02 0.70 0.22
Msi1/MBD2 #35 Day21 CDM + CytoB 1.19 0.15 1180.19 27.29 1.21 0.06
1.03 0.34 1.31 0.04 1.03 0.34 Ngn2/MBD2 #36 Day21 CDM + CytoB 81.66
1.34 7789.96 345.72 5.24 0.05 5.84 0.10 4.37 0.05 5.84 0.10
Msi1/Ngn2/MBD2
Immunohistochemical Analysis
[0371] Samples were collected at 4, 8, 14, and 21 days to evaluate
the nature of any reprogrammed cells by analyzing the expression of
several neuronal markers using immunohistochemical analysis
according to the methods previously described in Example I. The
immunohistochemical analysis at various time points revealed that
within the first 8 days the expression of nestin was induced in a
large proportion of cells and decreased time-dependently after
inducing the differentiation.
[0372] This study indicates that upon transfecting the cells with
one or two neurogenic genes in the presence of cytochalasin B and
MBD2, reprogrammed cells were stable in culture, responded to
environmental changes (proliferation vs differentiation), and
expressed neuronal markers for at least 24 days in culture.
Example XIII
Telomerase Activity of NSLCs
[0373] Telomerase is active in neural precursor cells and suggest
that its regulation is an important parameter for cellular
proliferation to occur in the mammalian brain (Caporaso G L et,
2003). This study was performed to evaluate telomerase activity in
cell extracts of adherent NSLCs (NSLCs cultured on laminin-coated
plates) as well as NSLCs in floating neurospheres (NSLCs cultured
in plates with a low-bind surface) at early (P7) and late passage
(P27). The telomerase activity of the 4 samples was measured by the
PCR-based telomere repeat amplification protocol (TRAP) using the
TRAPeze.RTM. Telomerase Detection Kit (Chemicon). Briefly, the
cells were grown in 24-well plates, washed in PBS, and homogenized
for 30 min on ice in buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM
MgCl.sub.2, 1 mM EGTA, 0.1 mM Benzamidine, 5 mM
.beta.-mercapthethanol, 0.5% CHAPS and 10% Glycerol (1.times. CHAPS
Lysis Buffer, provided in kit) and RNase Inhibitor. The samples
were spun down and the protein concentration of the supernatant was
determined using the BCA Assay. 900 ng of protein from each cell
extract was added directly to the TRAP reaction mixture containing
TRAP reaction buffer, dNTPs, template substrate (TS) primer, TRAP
primer mix and Tag polymerase. The reaction mixtures were incubated
at 30.degree. C. for 30 minutes for template synthesis, followed by
a PCR procedure (95.degree. C./15 min for initial denaturation,
94.degree. C./30 sec, 59.degree. C./30 sec, 72.degree. C./1 min for
32 cycles) for amplification of the extended telomerase products.
To detect telomerase activity, polyacrylamide gel electrophoresis
(PAGE) was performed for the reaction products on a 10%
non-denaturing TBE gel. After electrophoresis, the gel was stained
with SYBR.RTM. Green I Nucleic Acid Gel Stain for 30 minutes,
followed by image capture using a Gel-Documentation System (Alpha
Innotech).
[0374] All 4 samples were telomerase positive (as indicated by the
TRAP product ladder). As expected, the Heat-treated control
(.DELTA.H) showed no Telomerase activity (Negative Control). A 36
bp internal control band (S-IC) is used to monitor PCR
amplification (to distinguish false-negative results). This S-IC
band was observed for all samples except for the test samples. This
may have been due to the excessively high telomerase activity in
the test samples; amplification of the TRAP products and the S-IC
control band are semi-competitive. All controls gave expected
results (No TRAP products for CHAPS ctrl, and TRAP ladder of
products for the positive control cells and the TSR8 control).
Example XIV
Tumor Formation Assay
[0375] Malignantly transformed cells show reduced requirements for
extracellular growth promoting factors, are not restricted by
cell-cell contact, and are often immortal. Anchorage-independent
growth and proliferation is one of the hallmarks of malignant
transformation, which is considered the most accurate and stringent
in vitro assay for detecting malignant transformation of cells.
[0376] Adherent and neurosphere NSLCs at early and late passage (P7
and P25), as well as normal human neuroprogenitor cells (hNPCs),
were investigated for the anchorage-independent growth. HFFs were
used as a negative control and cervical carcinoma HeLa cells were
used as a positive control. Cells were sedimented by centrifugation
at 150.times.g for 3 min at room temperature (RT). The assay was
performed using the CytoSelect.TM.96-well cell transformation assay
(CellBiolabs). The base agar layer (1.2%) was dissolved in
2.times.DMEM/20% PBS solution and 50 .mu.l of the agar solution was
added to the plate and incubated for 30 min at 4.degree. C. to
solidify. Prior to adding the cell agar layer, the plate was
allowed to warm up for 15 minutes at 37.degree. C. The cells were
resuspended at different density (20.000 and 5000 cells/well),
except the hNPCs were resuspended only at 5000 cells/well due to a
lack of enough cells. The cells were mixed with the 1.2% agar
solution, 2.times.DMEM/20% PBS, and cell suspension (1:1:1), and 75
.mu.l of the mixture was transferred to wells already containing
the solidified base agar layer, and was then placed in 4.degree. C.
for 15 minutes to allow the cell agar layer to solidify. 100 .mu.l
of proliferation medium (StemCell Technologies) was added and the
plate was incubated for 8 days at 37.degree. C. and 5% CO.sub.2
before being solubilized, lysed and detected by the CyQuant.TM. GR
dye in a fluorescence plate reader. The fluorescence measurement
was performed using the Flexstation.TM. (Molecular Devices) with a
485/538 nm filter.
TABLE-US-00046 TABLE 28 Fluorescence measurement (Relative
Fluorescence Unit, RFU) indicate that under the same conditions
only carcinoma HeLa cells grow as an anchorage-independent colony,
while both hNPCs and NSLCs (adherent and floating neurospheres)
were negative for tumor growth in the standard agar plate tumor
formation assay (CytoSelect .TM. cell transformation kit, Cell
Biolabs Inc.). Cell density/Cell types Hela HFF NSLCs HNPCs 20.000
60.05 .+-. 8.70 14.82 .+-. 1.57 19.22 .+-. 1.85 19.00 .+-. 2.71
10.000 39.03 .+-. 3.97 13.73 .+-. 1.05 14.99 .+-. 1.12 21.61 .+-.
9.95 5000 24.70 .+-. 3.89 11.65 .+-. 0.57 12.29 .+-. 0.79 12.45
.+-. 0.73
[0377] As shown in Table 28, fluorescence measurement indicated
that under the same conditions only carcinoma HeLa cells
significantly grew and proliferated as anchorage-independent
colonies, while both hNPCs and NSLCs (adherent and floating
neurospheres) were negative for tumor growth (same value as HFFs
(negative control) for 5,000 and 10,000 cells) in the standard agar
plate tumor formation assay by visual observation of cells by light
microscopic observation using bright field at 10.times. confirm
Fluorescence measurement. Thus the transient transfection method
and genes used allows the reprogramming of cells without the
neoplastic transformation that generally occurs with stable
transfection or certain genes via a series of genetic and
epigenetic alterations that yield a cell population that is capable
of proliferating independently of both external and internal
signals that normally restrain growth.
Example XVI
[0378] No Genomic Integration of Plasmid DNA in NSLCs from
Transient Transfection
[0379] The DNA plasmid Msi1/Ngn2 (designed and constructed in
house) was used in transient transfection for generation of NSLCs
along with MBD2 (for sample 1), or 5-Aza and VPA (for sample 2).
Two weeks after transfection, Southern blot was performed to test
for possible genomic integration of the plasmid DNA. 3 .mu.g of
genomic DNA extracted from the NSLC samples, as well as from HFF (a
human fibroblast cell line) used as a negative control, was
digested with several restriction enzymes including BgIII, PstI and
StuI, subjected to electrophoresis on a 1% agarose gel and
transferred to a positively charged nylon membrane (Roche). The
membrane was hybridized in the DIG Easy Hyb.TM. buffer (Roche) at
42.degree. C. overnight with a 1.2 kb Dig-labeled PCR probe
amplified from the plasmid DNA using a set of primers. The membrane
was washed twice at room temperature with 2.times.SSC, 0.1% SDS for
5 min per wash, twice with 0.5.times.SSC, 0.1% SDS at 65.degree. C.
for 15 min per wash. Hybridization signals of the membrane were
detected using the CDP-Star.TM. substrate (Roche). The membrane was
exposed to an X-ray film for analysis. The signals were stripped
from the membrane using stripping buffer (0.2 M NaOH, 0.1% SDS).
The membrane was re-hybridized with a 0.9 kb Dig-labeled PCR probe
amplified from the plasmid DNA using a set of primers.
[0380] The Southern blot analysis with the 1.2 kb. Dig-labeled PCR
probe revealed distinct signals in the positive control samples
where the Msi1/Ngn2 plasmid DNA was spiked into HFF genomic DNA for
the equivalence of 1, 10 or 100 integrations per genome. There were
a few weak and identical bands that appeared in the restriction
enzyme digested genomic DNA from HFF, NSLC samples #1 and #2,
suggesting that there is no plasmid DNA integration in the genomic
DNA of NSLCs. These bands may represent the endogenous Ngn2 gene
since the 1.2 kb Dig-labeled PCR probe contains a small part of the
Ngn2 gene. This data shows that no, or only a tiny number of, NSLCs
had plasmid integration into the host genome after transient
transfection, and that the transfected genes are only present in
the cell for a short period of time (less than two weeks).
Example XVII
[0381] Neuroprotective Effect of Transplanted hNSLCs in:
1) Animal Model of Multiple Sclerosis.
[0382] Multiple Sclerosis (MS) is an incurable inflammatory
demyelinating disease of the central nervous system (CNS) (Frohman
E M et al 2006). Therapies for MS rely on manipulation of the
immune system, but with often modest effectiveness on reducing
clinical episodes or permanent neurological disability, requiring
frequent injections, and with sometimes-significant side effects
(Langer-Gould A et al 2004). Experimental Allergic
Encephalomyelitis (EAE) is an animal model of MS commonly used for
studying disease mechanisms and testing potential therapies. EAE
can be induced in a variety of species and strains of animals
[mice, Rat, marmoset monkey, rhesus macaques] using various CNS
antigens [Myelin Oligodendrocyte Glycoprotein (MOG), proteolipid
protein (PLP) and myelin basic protein (MBP)].
[0383] After obtaining all appropriate animal approvals for the
experiments, Female 7 to 8 weeks old C57BL/6 mice were purchased
from Charles Rivers, and housed at MISPRO animal facility for one
week before experimentation for adaption to the new environment.
C57BL/6 mice were injected s.c. with 100 .mu.g MOG 35-55 in CFA
(Sheldon Biotechnology, McGill University) containing 5 mg/ml
Mycobacterium tuberculosis H37Ra (Difco, inc), at 2 sites on the
back. All mice received 200 ng pertussis toxin (List Biological
Laboratories, Inc) i.p. on day 0 and 2, while clinical scores were
calculated blindly daily during a 43 day period, according to the
0-5 scale as follows: 1, limp tail or waddling gait with tail
tonicity; 2, waddling gait with limp tail (ataxia); 2.5, ataxia
with partial limb paralysis; 3, full paralysis of 1 limb; 3.5, full
paralysis of 1 limb with partial paralysis of second limb; 4, full
paralysis of 2 limbs; 4.5, moribund; and 5, death.
Treatment of EAE Animal Model with and without the Cells:
[0384] hNSLC and hNPCs (1.5.times.10.sup.6 cells in 200 .mu.l
PBS/each mouse) were given by single injection i.v. via the tail
vein when the animals started to show symptoms of EAE (day 13 i.v).
Both animals groups received cyclosporine (10 mg/kg/day) one day
before the injection of cells and daily from the day of
transplantation to avoid any rejection of the human cells.
Sham-treated age-, sex-, and strain-matched mice, injected i.p.
with PBS alone, were used as controls. All groups of animals were
observed for 43 days. Animals were sacrificed at 43 days p.t.,
brains and spiral cord were harvested in 30% sucrose in PBS.
Statistical analysis of the clinical scores revealed that the
clinical signs of EAE were significantly attenuated in
NSLC-injected animals as compared to control and hNPCs-injected
animals. Cumulative scores was significantly reduced in the NSLC
transplanted animals and the treatment has no effect on body
weight.
2) Hemiplegic Animal Model (Unilateral Ablation of the Left
Sensorimotor Cortex in Adult Rats).
[0385] After obtaining all appropriate animal approvals for the
experiments, 8 rats per group (Sprague-Dawley, 250-300 g, Charles
River) were anaesthetized using ketamine (Bimeda-MTC)/xylazine
(50/10 mg/kg, Novopharm) and placed onto a, stereotaxic frame. A
midline cranial incision was performed with a sterile surgical
scalpel blade, the cranial vault exposed and the bregma identified.
The skull above the sensorimotor cortex was opened and the
sensorimotor cortex area [0.5-4.0 mm caudal to bregma and 1.8-3.8
mm lateral to the midline (Paxinos and Watson 1986)] was carefully
aspirated. After ablation, the treatments (Alginate.TM.,
Alginate+hNPC, Alginate+NSLCs, RM.sub.x+NSLCs, RM.sub.x Only,
Fibrin Gel, or Saline) were applied directly on the brain after
ablation. The opening in the skull was then filled with Bone Wax.
In case of a bleeding, small pieces of sterile homeostatic tissue
were inserted into the lesion in order to stop the bleeding. The
sutures were performed using Ethicon.TM. monofilament suture 1/2
circle needle shape. Surgeries were performed in sterile clean
rooms, and topical antibiotics (Cicatrin.RTM., GlaxoSmithKline)
were applied to the exposed skull and scalp to limit local
infection. Rats were immunosuppressed by daily injection i.p. of
cyclosporine A (10 mg/kg/day) starting the day before the surgery
until the end of the study period. The purpose of the cyclosporine
A injection was to reduce the rat's immune reaction to the
treatment. The immune-suppression was sustained until the end of
the study to ensure that any potential failure of regeneration (if
taken place) was not due to the immune reaction against the
treatment. Functional scores were performed weekly, in all groups,
sensorimotor impairment was evaluated based on the behavioural
tests as described below.
[0386] Rotarod Test:
[0387] The rotarod speed was manually calibrated for the 10 and 20
RPM speed before all procedures. Animals were required to perch on
the stationary rod for 30 sec to acclimate themselves to the
environment. During this time, if any animal fell, it was placed
back on the rod until it had achieved stationary capabilities for a
period of 30 seconds. The animals were allowed 3 trials. The
animals that were comfortable staying on the stationary rod for 30
sec were allowed to run with a constant speed of 10 and 20 RPM for
60 sec, and the number of falls were electronically recorded.
[0388] Beam Walking:
[0389] Beam walking measures hindlimb coordination by means of
distance traveled across 100 cm beam (2.3 cm in diameter, 48 cm off
the floor). Rats were systematically trained to walk along the
elevated beam from start to finish with the aim of completing the
task. A safe location, i.e, a flat box, is placed at the end of the
beam so that the rat is motivated to complete the task.
TABLE-US-00047 Scale used for evaluation of beam-walking
performance Scale Performance characteristic 1 Animals fail to
traverse the beam and do not place the hindlimb on the horizontal
surface of the beam 2 Animals fail to traverse the beam, but place
the hindlimb on the horizontal surface of the beam and maintain
balance 3 Animals traverse the beam while dragging the hindlimb 4
Animals traverse the beam and place the hindlimb at least once
during the traverse 5 Animals traverse the beam using the hindlimb
to aid less than 50% of its steps on the beam 6 Animals traverse
the beam using the left hindlimb to aid more than 50% of its steps
on the beam 7 Animals traverse the beam with no more than two foot
slips 8 Normal animals
[0390] Before the surgery, all the animals fell at least once from
the rotarod, not because they had a walking or coordination
problem, but because the speed was high. After the surgery (2
days), all the animals showed signs of significant walking and
coordination problems leading to an increase in the number of falls
from the rotarod. Three weeks after the surgery, the number of
falls was clearly reduced for the animals receiving NSLCs as
treatment compared to controls.
[0391] Animals passed the beam-walking test before the surgery
without any difficulty. The rats crossed the 100 cm beam and got to
the safe spot without failing off the beam. Two days after surgery,
all groups completely failed to pass the test, and the animals were
not able to stay in balance on the beam. One week after the
surgery, all the animals showed some improvement in their walking
capacity, but no significant difference was noticeable between the
different treated groups. From week 4 until week 26, the animals
treated with NSLCs as well as RM.sub.x showed significant
improvements in their walking capacity compared to the
controls.
Example XVIII
[0392] Transfection of HFF by Various Combinations of Genes Using
the Shuttle.RTM. Device and Treatment with Different Small
Molecules for Reprogramming to Mesendoderm-Like Cells
[0393] HFF cells were cultured as described in CDM II medium as
described in Example I with only modifying EGF (5 ng/ml) and FGF
(10 ng/ml), and transfecting using the Nucleofector.TM..RTM.
96-well Shuttle.RTM. Device (Lonza) following the procedure
described in Example IV. The cells were transfected with various
combinations of cDNA clones as described in Table 29. After
transfection, the cells were plated on 0.1% Gelatin-coated plates
and incubated at 37.degree. C., 5% CO.sub.2, 5% O.sub.2. Medium was
changed every other day according to Table 30. Cells were analyzed
at Day 4 by Quantitative Real-time PCR.
TABLE-US-00048 TABLE 29 Various combinations of plasmids with
potential to transfect the cells towards mesendoderm lineage. Da -2
to Day 0 Plasmids transfected at Day 0.sup.1 1 Untreated Oct4,
FoxD3, MBD2 2 Oct4, T, MBD2 3 Oct4, Mixl1, MBD2 4 Oct4, Sox17, MBD2
5 FoxD3, T, MBD2 6 FoxD3, Mixl1, MBD2 7 FoxD3, Sox17, MBD2 8 T,
Mixl1, MBD2 9 T, Sox17 MBD2 10 Mixl1, Sox17, MBD2 13 Pre-treated
with Oct4, FoxD3 14 VPA & 5-Aza FoxD3, T 15 FoxD3, Mixl1 16
FoxD3, Sox17 17 Oct4, FoxD3, T 18 Mixl1, Sox17, FoxA2 19 Oct4,
FoxD3, T 20 Mixl1, Sox17, FoxA2 .sup.1where Oct4 = pCMV6-XL4-Oct4,
FoxD3 = pCMV6-XL5-FoxD3, MBD2 = pCMV6-AC-MBD2, T = pCMV6-XL5-T,
Mixl1 = pCMV6-XL5-MIXL1, Sox17 = pCMV6-XL4-SOX17, FoxA2 =
pCMV6-XL5-FOXA2. All clones were purchased from Origene and
prepared using the EndoFree Plasmid Maxi Kit (Qiagen).
TABLE-US-00049 TABLE 30 Medium composition from Day -2 to Day 10
Media Composition.sup.2 Day 0 Day 1 Day 2 to Day 3 Day 4 to Day 7
Day 8 to Day 10 CDM II (3:1 of CDM II IMDM/F12 + IMDM/F12 +
IMDM/F12 + DMEM: F12; (50%) + NEAA + ITS + NEAA + ITS + NEAA + ITS
+ GlutaMAX .TM. IMDM/F12 HSA + HSA + bFGF + HSA + bFGF + 100x,
(50%) + bFGF + EGF + EGF + EGF + BMP4 Dexthamesone, NEAA + ITS +
VPA + Activin A + 19.7 .mu.g/ml, HSA + Activin A + CHIR99021 +
Glutathione (500 .mu.g/ml, bFGF + EGF + CHIR99021 + BMP4 L-Ascorbic
VPA + BMP4 75 mg/ml, Activin A + Selenious acid CHIR99021 2.5
.mu.g/ml, Insulin solution 10 mg/ml, T3 675 ng/ml, ethanolamine
500X, bFGF 2.5 ug/ml, and Egf (1.25 ug/ml) + Activin A + HSA
.sup.2Supplements added to media at the following concentrations:
Activin A (Peprotech, 30 ng/ml), HSA (Baxter, 0.5%), NEAA (Gibco,
1X), ITS (Gibco, 1X), EGF (Peprotech, 5 ng/ml), bFGF (Peprotech, 10
ng/ml), CHIR99021 (Stemgent, 2 uM), VPA (Stemgent, 1 mM), 5-Aza
(Sigma, 0.5 uM), BMP4 (Peprotech, 10 ng/ml)
[0394] Cells were collected on Day 4 by detaching with TrypLE.TM.,
followed by centrifugation at 80.times.g for 5 minutes. Supernatant
was aspirated and the cell pellet was frozen at -86.degree. C.
until ready for RNA Isolation. RNA isolation and quantification was
performed as previously described in Example I. cDNA was prepared
and quantitative real-time PCR was performed as previously
described in Example II, except the following Taqman.TM..RTM. Gene
Expression Assays (Applied Biosystems) were used:
TABLE-US-00050 Gene Taqman .TM..RTM. Assay ID GAPDH (housekeeper)
Hs99999905_m1 PPIA (housekeeper) Hs99999904_m1 FOXA2 Hs00232764_m1
SOX17 Hs00751752_s1 Endogenous T Hs00610073_g1 GSC Hs00418279_m1
CXCR4 Hs00607978_s1 GATA4 Hs00171403_m1 CER1 Hs00193796_m1 CDH1
(E-cadherin) Hs01023894_m1 p63 Hs00978340_m1 SOX2 SOX2_1078-ANY
TABLE-US-00051 TABLE 31 Relative Expression FoxA2, Sox17, and Cxcr4
after transfecting HFFs once with various gene combinations with
potential to reprogram cells into mesoendoderm-like cells. The
exact values are not significantly accurate due to low RNA yield,
however a trend of increasing gene expression was detected for
FoxA2, Sox17, and CXCR4. FOXA2 SOX17 CXCR4 Std. Std. Rel. Std. Rel.
Exp. Dev. Rel. Exp. Dev. Exp. Dev. Untreated HFF 1.00 0.04 1.00
0.04 1.00 0.04 Day4 HFF untransf. 1.01 0.06 1.01 0.06 4.77 2.51
(+G.F), Day4 HFF untransf. (-G.F), 1.38 0.11 1.38 0.11 1.38 0.11
Day4 HFF Untransf. 0.98 0.02 0.98 0.02 3.32 3.31 (+G.F.), Day4 HFF
Untransf. (-G.F.), 4.12 4.07 1.28 0.06 1.28 0.06 Day4 4.67 4.60
3.19 2.78 76.43 7.91 Oct4/FoxD3/MBD2 Day4 Oct4/T/MBD2 3.91 3.55
4.33 2.36 15.18 2.52 Day4 Oct4/Mixl1/MBD2 2.66 1.77 10.33 0.43 7.31
3.21 Day4 Oct4/Sox17/MBD2 14.19 4.85 413533.31 127089.61 56.04 0.71
Day4 FoxD3/T/MBD2 38.62 38.00 3.12 1.32 42.41 5.23 Day4 7.76 5.29
2.41 0.30 137.17 27.74 FoxD3/Mixl1/MBD2 Day4 26.02 1.95 50904.45
1523.33 131.03 17.53 FoxD3/Sox17/MBD2 Day4 T/Mixl1/MBD2 3.67 3.26
5.64 4.15 14.04 2.89 Day4 T/Sox17/MBD2 9.76 9.70 209797.21 24533.81
111.35 16.40 Day4 3.60 3.10 237310.10 57448.60 36.76 1.07
Mixl1/Sox17/MBD2 Day4 Oct4/FoxD3 13.87 0.16 13.87 0.16 35.44 14.57
Day4 FoxD3/T 60.93 60.18 19.45 1.51 19.45 1.51 Day4 FoxD3/Mixl1
21.20 2.31 28.96 8.66 62.31 55.82 Day4 FoxD3/Sox17 96.88 3.60
54177.20 3313.15 44.57 41.51 Day4 Oct4/FoxD3/T 25.99 18.15 12.27
1.26 21.17 11.33 Day4 1850864.68 98259.84 112641.65 15923.21 23.18
23.10 Mixl1/Sox17/FoxA2 Day4 Oct4/FoxD3/T 9.52 5.61 1.52 0.02 35.74
4.36 (IMDM/F12) Day4 486705.82 19101.53 57060.09 1262.81 13.44 2.36
Mixl1/Sox17/FoxA2 (IMDM/F12)
TABLE-US-00052 TABLE 32 Expression of GATA4, CDH1 (E-cadherin),
p63, and SOX2 relative to untreated HFF control 4 days after
transfecting HFF cells with various gene combinations with
potential to reprogram cells into mesoendoderm-like cells. CDH1 (E-
GATA4 cadherin) p63 SOX2 Std. Rel. Std. Rel. Std. Rel. Std. Rel.
Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Untreated HFF 1.00 0.04
1.00 0.04 1.00 0.04 1.00 0.04 Day4 HFF untransf. 12.13 0.70 1.01
0.06 3.09 1.45 1.11 0.21 (+G.F), Day4 HFF untransf. 4.48 0.85 1.38
0.11 3.11 2.54 1.38 0.11 (-G.F), Day4 HFF Untransf. 2.37 2.00 0.98
0.02 4.41 4.40 1.94 1.34 (+G.F.), Day4 HFF Untransf. 6.12 3.33 1.28
0.06 13.23 7.43 1.28 0.06 (-G.F.), Day4 95.23 27.44 98.90 21.58
1.81 0.86 12.72 1.53 Oct4/FoxD3/MBD2 Day4 Oct4/T/MBD2 33.66 10.30
1.42 0.02 2.05 0.87 2.62 1.67 Day4 106.33 5.70 1.43 0.03 8.68 0.99
25.68 2.18 Oct4/Mixl1/MBD2 Day4 23.50 5.39 4.65 4.43 95.23 13.86
18.77 6.94 Oct4/Sox17/MBD2 Day4 FoxD3/T/MBD2 121.36 11.68 26.85
0.02 2.22 0.04 16.99 4.74 Day4 130.21 21.04 69.19 22.84 4.05 3.56
1.52 0.01 FoxD3/Mixl1/MBD2 Day4 99.49 30.30 6.89 3.69 1.78 0.01
15.19 9.08 FoxD3/Sox17/MBD2 Day4 T/Mixl1/MBD2 110.30 3.55 1.36 0.00
1.36 0.00 6.64 2.25 Day4 T/Sox17/MBD2 53.19 4.02 2.69 1.86 18.01
0.54 14.21 5.21 Day4 16.53 16.50 2.91 2.13 13.44 6.68 10.55 3.27
Mixl1/Sox17/MBD2 Day4 Oct4/FoxD3 66.45 26.34 47.31 47.30 13.87 0.16
23.87 14.31 Day4 FoxD3/T 68.25 68.00 39.08 29.27 19.45 1.51 19.45
1.51 Day4 FoxD3/Mixl1 78.18 78.00 21.20 2.31 21.20 2.31 25.10 3.20
Day4 FoxD3/Sox17 176.45 93.54 15.64 0.60 15.64 0.60 26.78 16.35
Day4 Oct4/FoxD3/T 12.27 1.26 12.27 1.26 12.27 1.26 12.27 1.26 Day4
85.89 64.52 20.06 20.00 3.67 0.13 13.66 0.66 Mixl1/Sox17/FoxA2 Day4
Oct4/FoxD3/T 89.05 50.00 10.40 8.14 1.52 0.02 1.52 0.02 Day4 6.16
6.10 1.23 0.04 1.23 0.04 1.23 0.04 Mixl1/Sox17/FoxA2
[0395] Identification of gene combinations that may induce the
formation of Mesendoderm-like cells was investigated by
transfection with combinations of Oct4, Sox17, FoxD3, T, MixI1,
FoxA2, and MBD2. As shown in Table 25 and 26, the Relative
Expression of CXCR4 and GATA4, both Mesendoderm/Endoderm/Mesoderm
markers, appear to be up-regulated in various combinations, most
noticeably in FoxD3/MixI1/MBD2 and FoxD3/Sox17/MBD2. Similarly,
FOXA2, a marker for Endoderm and Mesoderm, was up-regulated
FoxD3/Sox17-transfected sample, although the expression is still
very low. Four days following transfection, SOX17 is still highly
expressed in the SOX17-transfected samples (50,000 to 400,000-fold
as compared to the untreated HFF sample). The SOX17 gene expression
represents leftover plasmid DNA (exogenous SOX17) that still
remains. 4 days post-transfection, and any endogenous SOX17
expression that may have been induced. Ectoderm markers CDH1, p63
and Sox2 were also up-regulated in some samples (e.g.
Oct4/FoxD3/MBD2, Oct4/Sox17/MBD2).
[0396] Reprogramming HFFs into Pancreatic Progenitor-Like
Cells:
[0397] HFF cells were cultured as described in Example I, and
transfected using the Nucleofector.TM..RTM. 96-well Shuttle.RTM.
Device (Lonza) following the procedure described in Example IV. The
cells were transfected with various combinations of cDNA clones as
described in Table 27. After transfection, the cells were plated on
Fibronectin-coated collagen gels and incubated at 37.degree. C., 5%
CO.sub.2, 5% O.sub.2. Fibronectin-coated Collagen gel plates were
prepared prior to transfection. Rat Tail Collagen I (Gibco) was
diluted to 1.125 mg/ml using 10.times.PBS and distilled water,
where 125 .mu.l was added to each well of a 24-well plate and
incubated in 37.degree. C. for 40 minutes. After rinsing with
1.times.PBS, Fibronectin (BD Biosciences) was added on top of the
gel at a concentration of 1.9 ug/well. Media was changed every
other day according to Table 33. Cells were analyzed at Day 7 by
Quantitative Real-time PCR.
TABLE-US-00053 TABLE 33 Plasmids and media composition from Day 0
to Day 14 Plasmids Media Composition.sup.2 transfected at Day
0.sup.1 Day 0 Day 1 to Day 3 Day 4 to Day 14 1 FoxD3, Sox17, Pdx1,
CDM II + DMEM/F12 + DMEM/F12 + NEAA + ITS + MBD2 Activin A + NEAA +
ITS + HSA + B27 + EGF + 2 FoxD3, Sox17, Ngn3, HSA HSA + B27 + bFGF
+ Retinoic Acid + MBD2 EGF + bFGF + FGF10 + Cyclopamine + 3 FoxD3,
Mixl1, Pdx1, Activin A + Noggin MBD2 CHIR99021 + Na 4 FoxD3, Mixl1,
Ngn3, Butyrate MBD2 5 Sox17, Mixl1, Pdx1, MBD2 6 Sox17, Mixl1,
Ngn3, MBD2 7 FoxD3, Sox17, Mixl1 DMEM/F12 + Pdx1 NEAA + ITS + 8
FoxD3, Sox17, Mixl1, HSA + B27 + Ngn3 EGF + bFGF + 9 FoxD3, Sox17,
Pdx1, Activin A + Ngn3 CHIR99021 + Na 10 FoxD3, Mixl1, Pdx1,
Butyrate + VPA + Ngn3 5-Aza 11 Sox17, Mixl1, Pdx1, Ngn3 .sup.1where
FoxD3 = pCMV6-XL5-FoxD3, Sox17 = pCMV6-XL4-SOX17, Mixl1 =
pCMV6-XL5-MIXL1, Pdx1 = pCMV6-XL5-Pdx1, and Ngn3 = pCMV6-XL5-Ngn3.
All clones were purchased from Origene and prepared using the
EndoFree Plasmid Maxi Kit (Qiagen). .sup.2Supplements added to
media at the following concentrations: Activin A (Peprotech, 30
ng/ml), HSA (Baxter, 0.5%), NEAA (Gibco, 1X), ITS (Gibco, 1X), B27
(Gibco, 1%), EGF (Peprotech, 5 ng/ml), bFGF (Peprotech, 10 ng/ml),
CHIR99021 (Stemgent, 2 uM), Na Butyrate (Stemgent, 1 mM), VPA
(Stemgent, 1 mM), 5-Aza (Sigma, 0.5 uM), Retinoic Acid (Sigma, 2
uM), FGF10 (Peprotech, 50 ng/ml), Cyclopamine (Stemgent, 2.5 uM),
Noggin (Peprotech, 50 ng/ml)
[0398] Cells were collected on Day 7 and RNA isolation and
quantification was performed as previously described in Example I.
cDNA was prepared and quantitative real-time PCR was performed as
previously described in Example 11, except the following
Taqman.TM..RTM. Gene Expression Assays (Applied Biosystems) were
used:
TABLE-US-00054 Gene Taqman .TM..RTM. Assay ID GAPDH (housekeeper)
Hs99999905_m1 PPIA (housekeeper) Hs99999904_m1 FOXA2 Hs00232764_m1
SOX17 Hs00751752_s1 GATA4 Hs00171403_m1 Endo PDX1 PDX1_1201 SOX9
Hs00165814_m1 NGN3 Hs01875204_s1 NKX2-2 Hs00159616_m1 PAX4
Hs00173014_m1 INS Hs02741908_m1 CXCR4 Hs00607978_s1
[0399] Identification of gene combinations that may induce the
formation of Pancreatic Progenitor-like cells was investigated by
transfection with combinations of FoxD3, Sox17, Pdx1 Ngn3, MixI1,
and MBD2. FoxA2, a marker for Endoderm and Mesoderm, was slightly
up-regulated for the FoxD3/Sox17/Ngn31MBD2-transfected sample as
compared to the GFP mock-transfected control sample. Similarly,
CXCR4, also a marker for both endoderm and mesoderm, was slightly
up-regulated (3-fold compared to GFP-ctrl) for the
FoxD3/MixI1/Ngn3/MBD2-transfected sample. 7 days following
transfection, SOX17 can still be detected for the samples
transfected with SOX17 at varying levels (4 to 570-fold
up-regulation as compared to the GFP-ctrl). The highest SOX17
expression up-regulation is detected for the sample transfected
with Sox17/MixI1/Pdx1/Ngn3 (570-fold as compared to GFP-ctrl),
which may suggest that this gene combination may increase the
amount of SOX17 RNA in cells.
Example XIX
[0400] Reprogramming Human Adipocytes Derived Stem Cells (ADSC) to
Pluripotent-Like Stem Cells (PLSC):
[0401] ADSCs (Invitrogen Corporation) were cultured in cell culture
flasks with complete StemPro.TM.-43 medium (Invitrogen) at
37.degree. C., 5% CO.sub.2 and the medium was changed 3 times per
week. After 3 days in culture cells (passage 5) were trypsinized
and counted to be transfected. Cells were transiently transfected
with one plasmid: pCMV6-Oct4-2A-Klf4-2A-Nanog,
pCMV-SalI4-2A-Oct4-2A-Klf4-2A-Nanog, pCMV-Dax1-2A-Oct4-2A-klf4,
pCMV-FoxD3-2A-Oct4-2A-klf4, pCMV-Oct4-2A-Klf4-2A-SalI4,
pCMV-MBD2-2A-Oct4-2A-Klf4-2A, pCMV-AGR2-2A-Oct4-2A-Klf4-2A, or
Rex1-EF-Oct4-2A-Klf4 (2 .mu.g); or by two plasmids:
pEF-Oct4nuc-IRES2-MBD2 with pCMV-Sox2nuc-IREC-Lin28 or
pCMV-Klf4nuc-IRES2-Tpt1nuc or pEF-Stella-IRES2-NPM2, using
Nucleofector.TM. as described in Example II. Following the
transfection cells were cultured in 6-well plates in suspension
with 50:50 ratio of adipocytes complete medium (StemPro.TM.-43) and
embryonic stem cells medium (mTeSR1). After two days in culture,
cells were re-transfected with the same plasmids listed above and
cells were plated in 96 well-plates coated with Matrigel.TM. (BD
Biosciences) in the presence of mTesR complete medium supplemented
with thiazovivin (0.5 .mu.M), an ALK-5 inhibitor (SB 341542,
Stemgent, 2 .mu.M), and inhibitor of MEK (PD0325901, Stemgent, 0.5
.mu.M). Medium was changed every day and cells were cultured for 22
days at 37.degree. C., 5% CO.sub.2, 5% O.sub.2. Alkaline
Phosphatase Detection Kit (AP, Millipore) and immunohistochemistry
were performed to analyse the expression of pluripotency markers.
ALP staining was performed using AP detection kit (Millipore)
according to manufacturer's instructions.
[0402] Visual observation of reprogrammed cells was performed by
Cellomics.TM. using a live staining for SSEA-4.sub.647 (BD
Biosciences) and TRA-1-81.sub.555 (BD Biosciences) starting on Day
6 after transfection and every 5 days thereafter. Reprogrammed
colonies of PLSCs, positively stained with SSEA-4 and TRA1-81, was
observed only with Plasmid pCMV-SalI4-2A-Oct4-2A-Klf4-2A-Nanog,
pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP, pEF-Oct4nuc-IRES1-MBD2 with
pCMV-Sox2nuc-IRES1-Lin28, and pEF-Oct4nuc-IRES1-MBD2 with
pCMV-Klf4nuc-IRES2-Tpt1nuc. These colonies emerged around Day 6 and
maintained in culture up to the end of the study period (Pay 22)
with a stable morphology. Among the plasmids cited above,
pCMV-SalI4-2A-Oct4-2A-Klf4-Nanog and
pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP gave the highest number of
colonies. Live staining showed that these colonies express typical
pluripotency markers, including SSEA-4 and TRA1-81, and further
analysis of these colonies showed that the colonies also expressed
other ESC markers such as alkaline phosphatase and Oct4. When the
cultures were treated with PD0325901 and SB431542 for up to 22
days, a 4-fold improvement in efficiency over the conventional
method was obtained following the transfection of ADSCs with
pCMV-SalI4-2A-Oct4-2A-Klf4-Nanog and
pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP.
[0403] Based on the previous study, the highest reprogramming
efficiency was observed using pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP and
pCMV-SalI4-2A-Oct4-2A-Klf4-2A-Nanog. Another study was designed to
ascertain the effect of pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP on the
reprogramming efficiency and to investigate the effect of
individual pluripotent genes Rex1, Oct4, and Klf4 in different
combinations. ADSCs were transfected as above with
pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP, pCMV6-XL5-Rex1,
pCMV6-XL4-Oct4/pCMV6-XL5-Klf4, pCMV6-XL5-Rex1/pCMV6-XL4-Oct4, or
pCMV6-XL5-Rex1/pCMV6-XL5-Klf4. After the second transfection, ADSC
were cultured in 96-well plates coated with Matrigel.TM. for 24
days in the presence of mTeSR1 medium supplemented with SB341542
and PD. 0.325901 at 37.degree. C., 5% CO.sub.2, 5%02. In order to
characterize subpopulations of cells after transfection, live
staining, immunohistochemistry, and AP staining was used to follow
the change in pluripotent markers. 1-5% of total cells transfected
with Rex1/Oct4 or Rex1/Klf4 showed a SSEA4.sup.+ and TRA-1-81.sup.+
phenotype, and this pattern was stable until the end of the study
period (Day 22). The observation over time showed that the
phenotype of these colonies moved from an early SSEA-4.sup.+
phenotype to a late Oct4.sup.+/Sox2/Nanog.sup.+ phenotype by Day
22, which is closer to the final reprogrammed state of a
pluripotent-like stem cell.
[0404] Various genes were tested for their effect on reprogramming
efficiency towards pluripotent-like cells. ADSC cells were cultured
as described in Example IX with 2 days VPA and 5-AZA pre-treatment
(1 mM and 0.5 .mu.M respectively) in StemPro.TM. MSC SFM medium.
Cells were transfected using the Nucleofector.TM..RTM. 96-well
Shuttle.RTM. Device (Lonza) following procedure described in
Example IV and using the transfection program EW-104 with the DNA
mixes described in Table 34. Following transfection the cells were
plated in StemPro.TM. MSC SFM medium described in example A on
Matrigel.TM. (BD Biosciences) coated 24 well plates and incubated
at 37.degree. C., 5% CO.sub.2, 5% O.sub.2. On Day 1, media was
changed to a mix of 75% StemPro.TM. MSC and 25% hES cell medium;
the percentage of StemPro.TM. MSC was decreased every day over four
days to have 100% hES cell medium by Day 4. From then onwards the
medium was changed every two days. The hES cell medium consisted in
Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) supplemented
with 20% Knockout.TM. Serum Replacement (KSR, Invitrogen), 1 mM
GluthMAXT.TM., 100 .mu.M Non-essential Amino acids, 100 .mu.M
.beta.-mercaptoethanol and 10 ng/ml Fgf-2. Different inhibitors and
growth factors were added through the course of the experiment;
these are listed in Table 34. Cells were analysed at Day 7 and Day
14 by immunohistochemistry analysis and at Day 7 by RT-PCR.
TABLE-US-00055 TABLE 34 Plasmids and media composition from Day 1
to Day 14. From day 7 From day Plasmids to -2 transfected at From
day 1 to From day 3 to day to day 0 day 0 day 3 day 7 14 1 VPA + 5-
pCMV6-XL4- StemPro .TM./hES StemPro .TM./hES hES medium Aza pre-
Oct4 + medium + medium + treated pCMV6-XL5- ActivinA (30 ng/ml) +
ActivinA (30 ng/ml) + Sox2 + CHIR99021 (3 .mu.M) CHIR99021 (3
.mu.M) pCMV6-XL5- MBD2 2 VPA + 5- pCMV6-XL4- StemPro .TM./hES
StemPro .TM./hES hES Aza pre- Oct4 + medium + medium + medium
treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30 ng/ml) +
FoxD3 + CHIR99021 (3 .mu.M) CHIR99021 (3 .mu.M) pCMV6-XL5- MBD2 3
VPA + 5- pCMV6-XL4- StemPro .TM./hES StemPro .TM./hES hES medium
Aza pre- Oct4 + medium + medium + treated pCMV6-XL5- ActivinA (30
ng/ml) + ActivinA (30 ng/ml) + UTF1 + CHIR99021 (3 .mu.M) CHIR99021
(3 .mu.M) pCMV6-XL5- MBD2 4 VPA + 5- pCMV6-XL4- StemPro .TM./hES
StemPro .TM./hES hES medium Aza pre- Oct4 + medium + medium +
treated pCMV6-XL4- ActivinA (30 ng/ml) + ActivinA (30 ng/ml) +
DPPA4 + CHIR99021 (3 .mu.M) CHIR99021 (3 .mu.M) pCMV6-XL5- MBD2 5
VPA + 5- pCMV6-XL5- StemPro .TM./hES StemPro .TM./hES hES medium
Aza pre- Sox2 + medium + medium + treated pCMV6-XL5- ActivinA (30
ng/ml) + ActivinA (30 ng/ml) + FoxD3 + CHIR99021 (3 .mu.M)
CHIR99021 (3 .mu.M) pCMV6-XL5- MBD2 6 VPA + 5- pCMV6-XL5- StemPro
.TM./hES StemPro .TM./hES hES medium Aza pre- Sox2 + medium +
medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30
ng/ml) + UTF1 + CHIR99021 (3 .mu.M) CHIR99021 (3 .mu.M) pCMV6-XL5-
MBD2 7 VPA + 5- pCMV6-XL5- StemPro .TM./hES StemPro .TM./hES hES
medium Aza pre- Sox2 + medium + medium + treated pCMV6-XL4-
ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + DPPA4 + CHIR99021 (3
.mu.M) CHIR99021 (3 .mu.M) pCMV6-XL5- MBD2 8 VPA + 5- pCMV6-XL5-
StemPro .TM./hES StemPro .TM./hES hES medium Aza pre- FoxD3 +
medium + medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA
(30 ng/ml) + UTF1 + CHIR99021 (3 .mu.M) CHIR99021 (3 .mu.M)
pCMV6-XL5- MBD2 9 VPA + 5- pCMV6-XL5- StemPro .TM./hES StemPro
.TM./hES hES medium Aza pre- FoxD3 + medium + medium + treated
pCMV6-XL4- ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + DPPA4 +
CHIR99021 (3 .mu.M) CHIR99021 (3 .mu.M) pCMV6-XL5- MBD2 10 VPA + 5-
pCMV6-XL5- StemPro .TM./hES StemPro .TM./hES hES medium Aza pre-
UTF1 + medium + medium + treated pCMV6-XL4- ActivinA (30 ng/ml) +
ActivinA (30 ng/ml) + DPPA4 + CHIR99021 (3 .mu.M) CHIR99021 (3
.mu.M) pCMV6-XL5- MBD2 11 VPA + 5- pCMV6-XL4- StemPro .TM./hES
StemPro .TM./hES hES medium Aza pre- Oct4 + medium + medium +
treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + Sox2
+ CHIR99021 (3 .mu.M) + CHIR99021 (3 .mu.M) pCMV6-XL5- VPA + 5-
FoxD3 Aza 12 VPA + 5- pCMV6-XL4- StemPro .TM./hES StemPro .TM./hES
hES medium Aza pre- Oct4 + medium + medium + treated pCMV6-XL5-
ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + Sox2 + CHIR99021 (3
.mu.M) + CHIR99021 (3 .mu.M) pCMV6-XL5- VPA + 5- UTF1 Aza 13 VPA +
5- pCMV6-XL4- StemPro .TM./hES StemPro .TM./hES hES medium Aza pre-
Oct4 + medium + medium + treated pCMV6-XL5- ActivinA (30 ng/ml) +
ActivinA (30 ng/ml) + Sox2 + CHIR99021 (3 .mu.M) + CHIR99021 (3
.mu.M) pCMV6-XL4- VPA + 5- DPPA4 Aza 14 VPA + 5- pCMV6-XL4- StemPro
.TM./hES StemPro .TM./hES hES medium Aza pre- Oct4 + medium +
medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30
ng/ml) + FoxD3 + CHIR99021 (3 .mu.M) + CHIR99021 (3 .mu.M)
pCMV6-XL5- VPA + 5- UTF1 Aza 15 VPA + 5- pCMV6-XL4- StemPro
.TM./hES StemPro .TM./hES hES medium Aza pre- Oct4 + medium +
medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30
ng/ml) + FoxD3 + CHIR99021 (3 .mu.M) + CHIR99021 (3 .mu.M)
pCMV6-XL4- VPA + 5- DPPA4 Aza 16 VPA + 5- pCMV6-XL4- StemPro
.TM./hES StemPro .TM./hES hES medium Aza pre- Oct4 + medium +
medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30
ng/ml) + UTF1 + CHIR99021 (3 .mu.M) + CHIR99021 (3 .mu.M)
pCMV6-XL4- VPA + 5- DPPA4 Aza 17 VPA + 5- pCMV6-XL5- StemPro
.TM./hES StemPro .TM./hES hES medium Aza pre- Sox2 + medium +
medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30
ng/ml) + FoxD3 + CHIR99021 (3 .mu.M) + CHIR99021 (3 .mu.M)
pCMV6-XL5- VPA + 5- UTF1 Aza 18 VPA + 5- pCMV6-XL5- StemPro
.TM./hES StemPro .TM./hES hES medium Aza pre- Sox2 + medium +
medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30
ng/ml) + FoxD3 + CHIR99021 (3 .mu.M) + CHIR99021 (3 .mu.M)
pCMV6-XL4- VPA + 5- DPPA4 Aza 19 VPA + 5- pCMV6-XL5- StemPro
.TM./hES StemPro .TM./hES hES medium Aza pre- Sox2 + medium +
medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30
ng/ml) + UTF1 + CHIR99021 (3 .mu.M) + CHIR99021 (3 .mu.M)
pCMV6-XL4- VPA + 5- DPPA4 Aza 20 VPA + 5- pCMV6-XL5- StemPro
.TM./hES StemPro .TM./hES hES medium Aza pre- FoxD3 + medium +
medium + treated pCMV6-XL5- ActivinA (30 ng/ml) + ActivinA (30
ng/ml) + UTF1 + CHIR99021 (3 .mu.M) + CHIR99021 (3 .mu.M)
pCMV6-XL4- VPA + 5- DPPA4 Aza 21 VPA + 5- GFP StemPro .TM./hES
StemPro .TM./hES hES medium Aza pre- medium + medium + treated
ActivinA (30 ng/ml) + ActivinA (30 ng/ml) + CHIR99021 (3 .mu.M)
+/or- CHIR99021 (3 .mu.M) VPA + 5- Aza
[0405] In order to characterize subpopulations of cells after
transfection, live staining, immunohistochemistry, and AP staining
was performed to follow the change in pluripotent markers. Cells
transfected with either Oct4/UTF1/MBD2, Oct4/Dppa4/MBD2,
FoxD3/Dppa4/MBD2, Oct41FoxD31Dppa4, or Sox2/FoxD3/UTF1 showed
positive colonies for TRA1-60, TRA1-81, and SSEA4. This observation
indicated that MBD2 generally had no effect by itself on
reprogramming towards pluripotent-like cells, except in the case of
Oct4/FoxD3/MBD2 transfection. Colonies started to form on Day 7 and
continued to form until Day 14 (the end of the study period). These
colonies were positive for AP as well.
[0406] These results were confirmed by RT-PCR analysis showing
up-regulation of Oct4 expression as shown in Table 35. Relative
expression for SOX2 was also slightly up-regulation in Day 7 after
transfecting cells with Oct4/Foxd3/MBD2. There is also a trend of
Sox2 up-regulation following transfection with Oct41Sox2/Foxd3 and
Oct4/Foxd3/Utf1.
TABLE-US-00056 TABLE 35 Relative expression of Pluripotent genes
after transfecting ADSCs with various combinations of vectors as
described in Table 34. OCT4 Endogenous SOX2 Rel. Exp. Std. Dev.
Rel. Exp. Std. Dev. #1 Day 7, Oct4/Sox2/MBD2 25.20 1.89 3.89 2.06
#2 Day 7, Oct4/Foxd3/MBD2 1.28 0.13 18.79 0.03 #3 Day 7,
Oct4/Utf1/MBD2 2.01 0.20 2.93 1.73 #4 Day 7, Oct4/Dppa4/MBD2 9.68
1.36 1.18 0.15 #5 Day 7, Sox2/Foxd3/MBD2 1.06 0.55 2.68 2.90 #6 Day
7, Sox2/Utf1/MBD2 0.66 0.10 3.36 0.68 #7 Day 7, Sox2/Dppa4/MBD2
0.74 0.00 5.03 4.73 #8 Day 7, Foxd3/Utf1/MBD2 4.6 0.61 4.15 2.92 #9
Day 7, Foxd3/Dppa4/MBD2 0.63 0.02 3.90 2.17 #10 Day 7,
Utf1/Dppa4/MBD2 0.96 0.04 4.97 1.92 #11 Day 7, Oct4/Sox2/Foxd3
48.17 1.89 7.68 1.79 #12 Day 7, Oct4/Sox2/Utf1 48.97 6.93 3.71 0.39
#13 Day 7, Oct4/Sox2/Dppa4 32.40 2.74 4.61 2.37 #14 Day 7,
Oct4/Foxd3/Utf1 4.30 0.91 9.83 3.03 #15 Day 7, Oct4/Foxd3/Dppa4
4.21 0.11 4.57 0.85 #16 Day 7, Oct4/Utf1/Dppa4 10.29 3.70 3.53 1.63
#17 Day 7, Sox2/Foxd3/Utf1 1.42 0.83 3.32 2.12 #18 Day 7,
Sox2/Foxd3/Dppa4 1.19 0.14 3.37 1.23 #19 Day 7, Sox2/Utf1/Dppa4
1.34 0.09 2.33 2.91 #20 Day 7, Foxd3/Utf1/Dppa4 0.72 0.07 2.45 0.27
#21 Day 7, GFP (-VPA/-5aza) 1.02 0.29 1.01 0.17 #22 Day 7,
Untransf. ADSC (-VPA/-5aza) 1.25 N/A 0.30 N/A #23 Day 7, GFP
(+VPA/+5aza) 0.14 0.20 1.87 2.23 #24 Day 7, Untransf. ADSC 1.45 N/A
0.27 N/A (+VPA/+5aza)
Reprogramming Efficiency of Defined Pluripotency Factors on FIFE
after Triple Transfection (One Transfection Every 3 Days)
[0407] HFF cells were cultured as described in Example I with the
exception of the concentrations of VPA and 5-AZA that were
respectively 2 mM and 2.5 .mu.M. Cells were transfected using the
Nucleofector.TM..RTM. II Device (Lonza) following procedure
described in Example 11 with the exception of the DNA quantity: 1
.mu.g of each of the 3 plasmids DNA was used. The cells that had
been pre-treated with VPA and 5-Aza and the untreated cells were
both transfected with a mix of pCMV-Oct4nuc-IRES2-Sox2nuc,
pCMV-Klf4nuc-IRES2-Cmycnuc or pCMV-Nanognuc-IRES2-Lin28. Following
transfection the cells were plated in the fibroblasts medium
described in, Example 1, supplemented with or without VPA and 5-AZA
on Matrigel.TM. (BDBiosciences) coated on 6-well plates and
incubated at 37.degree. C., 5% CO.sub.2. On Day 1 and 2, media was
changed to 100% mTeSR1 medium (StemCell Technologies) supplemented
with or without VPA and 5-AZA. On Day 3 and Day 6, cells from each
condition were detached by incubation in TrypLE.TM. for 5 min,
counted and centrifuged. Cells were retransfected as above and
plated on Matrigel.TM. coated plates in mTeSR1 medium supplemented
with or without VPA and 5-AZA. Media was changed daily as described
for day 1 and 2. Medium was supplemented in Y27632 (Stemgent, 10
.mu.M) from day 7 to day 14 to promote viability and clonal
expansion of potential reprogrammed cells. Cells were analysed at
Day 20 using the Alkaline Phosphatase Detection Kit (Millipore) and
by immunohistochemistry analysis.
[0408] This analysis revealed that after three transfections, three
clones were found to be positive for alkaline phosphatase activity
and showed a rounded cell/colony morphology. Staining with
antibodies against the embryonic stem (ES) cell markers SSEA-4 and
TRA-1-81 confirmed that these clones were pluripotent-like.
Surrounding HFF cells were, negative for these markers. These
clones were obtained only in the condition that did not contain
inhibitors (i.e.: VPA and 5-AZA). Unexpectedly, no clones were
observed for the condition treated with these inhibitors.
Reprogramming of NSLCs into Pluripotency
[0409] NSLC and neuronal stem cells derived from BG-01, a human ES
cell line that expresses markers that are characteristic of ES
cells including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and OCT-314,
were reprogrammed into pluripotency. BG-01 cells had previously
been cultured in conditions to induce the differentiation towards
neural stem cells as described by Chambers S M et al., 2009. NSLCs
and BG-01-NSC were cultured in proliferation medium supplemented
with FGF (20 ng/ml) and EGF (20 ng/ml). NSLCs and BG-01-NSCs were
transfected as previously described in Example II by two episomal
vectors, pEF-Oct4nuc-IRES2-MBD2 (NC1) or pCMV-FoxD3-2A-Oct4-2A-Klf4
(F72). Following transfection cells were collected and plated onto
uncoated petri-dishes in the presence of Proliferation medium and
mTeSR1 medium (50:50) in proliferation conditions at 37.degree. C.,
5% CO2. After 48 hours, cells were re-transfected by the same
plasmid and plated in 96-well plates coated with Matrigel.TM. and
cultured in the presence of mTeSR1 medium supplemented by the small
molecules BIX01294 (Stemgent, 2 .mu.M) and BayK8644 (Stemgent, 2
.mu.M) at 37.degree. C., 5% O.sub.2 for 22 days. Live staining and
immunohistochemistry were performed to characterize subpopulations
of cells for pluripotency markers.
[0410] NSLCs and BG-01-NSCs were positively stained with SSEA-4
starting on Day 7 and maintained until 22 days in culture (the end
of the study). Within ten days, cells that were morphologically
similar to ESCs were observed and they expressed a wide panel of
pluripotency markers, including SSEA-4, TRA1-81, Nanog and Oct4.
This study identified another way to get pluripotent-like cells
from somatic cells via Neural Stem-Like Cells (NSLCs). The utility
of NSLCs could offer multiple advantages for reprogramming towards
pluripotent-like cells. For example, obviating the requirement for
tumorigenic genes like c-Myc reduces the risk of inducing cancerous
cells. For neuroregenerative and neurodegenerative applications
these cells could represent an invaluable source of cells to
investigate furthermore human pluripotent cell induction, and also
represent a potential source of cells for deriving patient-specific
multipotent and pluripotent stem cells for modeling human
disease.
Example XX
Teratoma Formation Assay in SCID Mice
[0411] Transplantation of human pluripotent stem cells (SC) into
"severly compromised immuno-deficient" (SCID) mice leads to the
formation of differentiated tumors comprising all three germ layers
for pluripotent stem cells, resembling spontaneous human teratomas,
and specialized tissue for multipotent stem cells. These assays are
considered the standards in the literature for demonstrating
differentiation potential of pluripotent stem cells and hold
promise as a standard for assessing safety among SC-derived cell
populations intended for therapeutic applications
[0412] After all appropriate animal approvals for the experiment
has been obtained, 24 mice were purchased from Charles Rivers, and
housed at MISPRO animal facility for one week without any
experimentation for adaption to the new environment. One million
human NSLCs, normal human neuroprogenitor cells (hNPCs), or human
embryonic stem (ES) cells in 100 .mu.l Phosphate buffered saline,
calcium- and magnesium-free (CMF-PBS) were injected with a 21-G
needle intramuscularly into the right hind limb of the 4-week-old
male SCID-beige mice under anesthesia with Ketamine/xylazine (8
mice per group). Following injection, the syringe was aspirated up
and down a couple of times in a culture dish containing medium to
verify that the cells were injected and not stuck inside the
syringe.
[0413] The mice were maintained for 12 weeks and monitored for
clinical signs and any tumor growth regularly. Any specialized
tissue or teratoma growth was monitored by external examination and
an increase in the size of the muscle relative to the same muscle
on the left hind limb. When a specialized tissue or teratoma was
identified, the location and size of the growth was measured (using
measuring calipers) and recorded. The specialized tissue or
teratoma is usually first identified as a small growth of the
muscle size compared to the left control muscle. Animals were
monitored weekly until onset of any tumor growth, and daily after
tumors appeared. After 12 weeks, the mice were sacrificed by
CO.sub.2 euthanasia. Each entire animal was observed for any tumor
growth anywhere on the animal, and the injected muscle and the
comparable left muscle control were measured (with measuring
calipers)(see results table below) and then removed and stored in
4% paraformaldehyde solution for histological analysis. The sizes
of the muscles were as follows:
TABLE-US-00057 Left leg (control) Right leg (treated) Dorso- Dorso-
ventral ventral Lateral Treatment width Lateral width width width
Human 6.44 .+-. 0.11 5.03 .+-. 0.17 6.91 .+-. 0.15 5.3 .+-. 0.14
Embryonic Stem Cells Human 6.60 .+-. 0.17 5.43 .+-. 0.15 7.01 .+-.
0.23 5.58 .+-. 0.13 Neuro- progenitor Cells Human NSLC 6.85 .+-.
0.2 5.32 .+-. 0.14 6.86 .+-. 0.21 5.33 .+-. 0.11 Values represent
the Average of 8 mice .+-. the standard error
[0414] Measurement of the size of the muscles revealed that all the
human embryonic stem cell injected muscles were bigger than the
comparable left muscle controls, indicating teratoma growth in the
ES cell injected muscles. About half of all the human
neuroprogenitor cell injected muscles were bigger than the
comparable left muscle controls, while the mice injected with NSLC
did not show any difference between the muscles (treated with the
cells or not). The mice injected with NSLC did not show any
evidence of tumor or teratoma growth.
Example XXI
Transfection of ADSCs by Various Combinations of Genes and
Treatment with Small Molecules for Reprogramming to
Mesendoderm-Like Cells
[0415] Human Adipose-Derived Stem Cells (ADSCs) were purchased from
Invitrogen and expanded in complete StemPro.TM. MSC serum-free
medium (Invitrogen on, CellStart.TM. coated flasks (diluted 1:100
in PBS containing Ca.sup.2+/Mg.sup.2+) at a cell density of
1.times.10.sup.4 cells/cm.sup.2. ADSCs were cultured as described
in complete StemPro medium (Invitrogen) and were pretreated for 2
days prior to transfection with VPA (1 mM) and 5-Aza (0.5 .mu.M).
Cells were transfected using the Nucleofector.RTM. 96-well
Shuttle.RTM. Device (Lonza) following the procedure described
previously. The cells were transfected with various combinations of
cDNA clones summarized in Table 36. After transfection, the cells
were plated on 0.1% Gelatin-coated plates and incubated at
37.degree. C., 5% CO.sub.2, 5% O.sub.2. Medium was changed every
other day according to Table 37. Cells were analyzed at Day 10 by
Quantitative Real-time PCR.
TABLE-US-00058 TABLE 36 Various combinations of plasmids with
potential to transfect the cells towards mesendoderm lineage. Day
-2 to Day 0 Plasmids transfected at Day 0.sup.1 1 Untreated FoxD3,
Ngn3, Sox17, MBD2 2 FoxD3, Eomes, Gata6, Mixl1, MBD2 3 Pre-treated
with Eomes, FoxD3, Gata6, Mixl1 4 VPA, & 5-Aza Eomes, FoxD3,
Gata6, Sox17 5 Eomes, Gata6, Mixl1, T 6 FoxD3, Gata4, Mixl1, Sox17
7 Gata4, Gata 6, Mixl1, Oct4 8 Gata4, Mixl1, Sox17, T 9 Eomes,
Mixl1, Sox17 10 Eomes, Gata6, Sox17 11 Gata4, Mixl1, Sox17 12
Gata6, Mixl1, Sox17 13 GFP .sup.1where FoxD3 = pCMV6-XL5-FoxD3,
MBD2 = pCMV6-AC-MBD2, T = pCMV6-XL5-T, Mixl1 = pCMV6-XL5-MLXL1,
Sox17 = pCMV6-XL4-SOX17, Gata4 = pCMV6-XL5-Gata4, Eomes =
pCMV6-XL4-Eomes, and Gata6 = pCMV6-XL6-Gata6. All clones were
purchased from Origene and prepared using the EndoFree Plasmid Maxi
Kit (Qiagen).
TABLE-US-00059 TABLE 37 Medium composition from Day -2 to Day 20
Day 2 to Day 4 to Day 8 to Day 11 to Day -2 to 0 Day 1 day 3 day 7
day 10 day 20 StemPro CDM II (50%) + IMDM/F12 + IMDM/F12 + IMDM/F12
IMDM/F12 + medium + VPA + IMDM/F12 NEAA + NEAA + NEAA + NEAA + 5Aza
(50%) + ITS + HSA + ITS + HSA + ITS + HSA + ITS + HSA + NEAA + ITS
+ bFGF + bFGF + bFGF + Exendin-4, HSA + EGF + EGF + EGF +
nicotinamide bFGF + EGF + VPA + Activin A + BMP4 VPA + Activin
Activin A + 5-Aza + A + 5-Aza 5-Aza + BMP4 BMP4 .sup.2Supplements
added to media at the following concentrations: Activin A
(Peprotech, 30 ng/ml), HSA (Baxter, 0.5%), NEAA (Gibco, 1X), ITS
(Gibco, 1X), EGF (Peprotech, 5 ng/ml), bFGF (Peprotech, 10 ng/ml),
VPA (Stemgent, 1 mM), 5-Aza (Sigma, 0.5uM), BMP4 (Peprotech, 10
ng/ml), Exendin-4 (50 ng/ml, Cat Nb: 24463, Anaspec), and
Nicotinamide (10 mM final, Cat Nb: N5535, Sigma).
[0416] Cells were collected on Day 10 by detaching with TrypLE,
followed by centrifugation at 80.times.g for 5 minutes. Supernatant
was aspirated and the cell pellet was frozen at -86.degree. C.
until ready for RNA Isolation. RNA isolation and quantification was
performed as previously described: RNA isolation and cDNA was
prepared using the High Capacity cDNA RT kit (Applied Biosystems)
as per the manufacturer's instructions with a final cDNA
concentration of 2 ng/.mu.l. Real-time PCR was then performed for
each gene of interest using the FAST PCR master mix (Applied
Biosystems) and the Taqman.RTM. Gene Expression Assays (Applied
Biosystems) listed below:
TABLE-US-00060 Forward Reverse Taqman .RTM. Primer Primer Probe
Gene Assay ID Sequence Sequence Sequence GAPDH (housekeeper)
Hs99999905_m1 PPIA (housekeeper) Hs99999904_m1 FOXA2 Hs00232764_m1
SOX17 Hs00751752_s1 Endogenous T Hs00610073_g1 GSC Hs00418279_m1
CXCR4 Hs00607978_s1 GATA4 Hs00171403_m1 CERT Hs00193796_m1 CDH1
(E-cadherin) Hs01023894_m1 p63 Hs00978340_m1 SOX2 SOX2_1078-ANY
CCACCTACA GACCACC CTGGCAT GCATGTCCT GAACCCA GGCTCTTG ACTC TGGA
[0417] Ten days following the transfection with various
combinations of Oct4, Sox17, FoxD3, T, MixI1, FoxA2, and MBD2 cDNA,
analysis by RT-PCR to investigate the expression of Mesoendoderm
and mesendoderm differentiation genes was performed. As summarized
in Table 38, the Relative Expression of Sox17 and FoxA2, the
markers for Endoderm and Mesoderm, was up-regulated noticeably in
the FoxD3/MixI1/Ngn3/MBD2-transfected sample, while the expression
of mesendoderm differentiation (ex. pancreatic lineage) genes are
low indicating an early mesendoderm phenotype. Ten days following
transfection, SOX17 is still highly expressed in the
SOX17-transfected samples as compared to the untreated ADCs sample
that represents leftover plasmid DNA (exogenous SOX17) that still
remains 10 days post-transfection, and any endogenous SOX17
expression that may have been induced.
TABLE-US-00061 TABLE 38 Relative Expression of the mesendoderm
genes FoxA2 and Sox17 after transfecting ADSCs once with various
gene combinations with potential to reprogram cells into
mesoendoderm-like cells. FoxA2 Sox17 Gata4 EndoPdX1 Sox9 Ngn3
Nkx2.2 Insulin Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std.
Rel. Std. Rel. Std. Rel. Std. Day 10 Exp. Dev. Exp. Dev. Exp. Dev.
Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Foxd3/Sox17/Ngn3/
1.13 0.06 44.41 0.51 0.16 0.11 1.13 0.06 0.98 0.05 1.13 0.06 1.13
0.06 1.13 0.06 MBD2 Foxd3/Mixl1/Pdx1/MBD2 2.36 1.68 0.33 0.07 0.08
0.00 1.19 0.03 1.24 0.08 1.19 0.03 1.19 0.03 1.66 0.63
Eomes/FoxD3/Gata6/ 3.12 0.06 0.05 0.00 0.22 0.00 3.12 0.06 1.87
0.02 3.12 0.06 3.12 0.06 3.12 0.06 Mixl1 Eomes/FoxD3/Gata6/ 2.64
0.01 137.24 12.01 0.71 0.70 2.64 0.01 1.73 0.02 2.64 0.01 2.64 0.01
2.64 0.01 Sox17 Eomes/Gata6/Mixl1/T 1.51 0.02 1.37 0.05 0.11 0.00
1.51 0.02 1.46 0.03 1.51 0.02 1.51 0.02 1.51 0.02
FoxD3/Gata4/Mixl1/ 0.78 0.00 84.72 3.22 0.21 0.00 0.78 0.00 1.19
0.12 0.55 0.00 0.78 0.00 0.26 0.00 Sox17 Gata4/Gata6/Mixl1/ 1.35
0.04 3.20 1.20 0.36 0.01 1.35 0.04 1.33 0.10 0.95 0.03 1.35 0.04
0.45 0.01 oct4 Gata/Mixl1/Sox17/T 1.20 0.21 228.37 5.78 1.48 0.93
0.41 0.00 0.62 0.02 0.29 0.00 0.41 0.00 0.14 0.00 Eomes/Mixl1/Sox17
1.72 1.41 139.00 7.36 0.20 0.01 0.76 0.05 1.21 0.03 0.54 0.04 0.76
0.05 0.44 0.28 Eomes/Gata6/Sox17 0.57 0.03 573.14 65.66 0.15 0.01
0.93 0.49 0.97 0.05 0.40 0.02 1.55 1.38 0.19 0.01 Gata4/Mixl1/Sox17
1.23 0.02 253.21 45.51 123.32 62.32 1.52 0.21 2.32 0.04 0.58 0.01
2.25 0.65 0.98 0.01 Gata6/Mixl1/Sox17 1.20 0.32 354.21 7.65 0.32
0.01 1.52 0.52 1.23 0.05 0.98 0.12 2.32 0.54 0.78 0.08 ADCs
untransf. 0.49 0.02 0.93 0.19 0.49 0.48 0.49 0.02 1.16 0.08 0.34
0.01 0.73 0.33 0.16 0.01
Example XXII
Transfection of ADSCs by Various Combinations of Genes and
Treatment with Small Molecules for Reprogramming to Pancreatic
Progenitor-Like Cells
[0418] Reprogramming ADSCs into Pancreatic Progenitor-Like Cells:
Human ADSCs were purchased from Invitrogen and expanded in complete
StemPro.TM. MSC serum-free medium (Invitrogen on CellStart.TM.
coated flasks (diluted 1:100 in PBS containing Ca.sup.2+/Mg.sup.2+)
at a cell density of 1.times.10.sup.4 cells/cm.sup.2. Cells were
transfected using the Nucleofector.RTM. 96-well Shuttle.RTM. Device
(Lonza) following the procedure described previously. The cells
were transfected with various combinations of cDNA clones as
described in Table 4. After transfection, the cells were plated on
Fibronectin-coated collagen gels and incubated at 37.degree. C., 5%
CO.sub.2, 5% O.sub.2. Plates were coated with fibronectin (BD
Biosciences) at a concentration of 1.9 .mu.g/well. Media was
changed every other day according to Table 39. Following
transfection cells will be cultured using high concentration of
activin A (50 ng/ml) and BMP (30 ng/ml) to push the reprogramming
towards the mesendoderm. To initiate the transition of definitive
endoderm to Primitive Gut tube, the medium was supplemented by FGF
10 (Peprotech) and cyclopamine (Stemgent). Thereafter, the gut tube
was exposed to Retinoic acid, cyclopamine, and FGF10. In order to
stimulate the expression of NGN3 and NKx2.2, the medium was
supplemented by exendin 4 and hepatocyte growth factor.
TABLE-US-00062 TABLE 39 Plasmids and media composition from Day 0
to Day 20 Media Composition.sup.1 Plasmids transfected at Day -2 to
Day 15 and Day 0 Day 0 Day 1 to 3 Day 3 to 6 Day 7 to 9 Day 10 to
14 over 1 - MBD2/Oct4/Sox17 StemPro StemPro/ RPMI, B27 DMEM free
DMEM free DMEM/F12 2 - MBD2/Oct4/Pdx1 medium RPMI with 1%. With of
of nicotinamide 3 - Sox17/PDX1/Ngn3 17.5 mM 1 mM glucose/F12
glucose/F12 (10 nM), + 4 - MBD2/Ngn3/Sox17 glucose, glutamine,
(1:1) + B27 (1:1) + B27 Hepatocyte 5 - MBD2/FoxA2/NGN3 ITS, 1 mM 1%
HSA + 1%, 1% 1%, 1% growth factor 6 - MBD2/PDX1/Ngn3 glutamine, FGF
10 HSA, HSA, (20 ng/ml) & 7 - Oct4/Sox17/ 1% HSA + (10 ng/ml)
Retinoic acid Exendin4 exendin4 Pdx1/Ngn3 Activin A (2 nM) (10 nM)
& (10 nM) 8 - Oct4/Pdx1/Ngn3 (50 ng/ml) + Cyclopamine
Cyclopamine 9 - FoxA2/Pdx1/Sox17 BMP4 (10 mM), (10 mM), 10 - GFP
(30 ng/ml) FGF10 (10 ng/ml) .sup.1Supplements added to media at the
following concentrations: Activin A (Peprotech, 30 ng/ml), HSA
(Baxter, 0.5%), NEAA (Gibco, 1X), ITS (Gibco, 1X), B27 (Gibco, 1%),
bFGF (Peprotech, 10 ng/ml),,, VPA (Stemgent, 1 mM), 5-Aza (Sigma,
0.5 uM), Retinoic Acid (Sigma, 2 uM), FGF10 (Peprotech, 50 ng/ml),
Cyclopamine (Stemgent, 2.5 uM), Exendin-4 (50 ng/ml, Cat Nb: 24463,
Anaspec).
[0419] Cells were collected on Day 20 to be analyzed by Qualitative
Real-time PCR and RNA isolation and quantification was performed as
described previously. cDNA was prepared and quantitative real-time
PCR was performed with the following Taqman.RTM. Gene Expression
Assays (Applied Biosystems) used:
TABLE-US-00063 Taqman .RTM. Forward Primer Reverse Primer Probe
Gene Assay ID Sequence Seguence Sequence GAPDH Hs99999905_m1
(housekeeper) PPIA Hs99999904_m1 (housekeeper) FOXA2 Hs00232764_m1
SOX17 Hs00751752_s1 GATA4 Hs00171403_m1 Endogenous PDX1_1201
GGCCCTCTTTT GTAGGAGGGC ACAGCCA PDX1 AGTGATACTG AGGGATGTG CAAACAA
GATT CG SOX9 Hs00165814_m1 NGN3 Hs01875204_s1 NKX2-2 Hs00159616_m1
PAX4 Hs00173014_m1 INS Hs02741908_m1 CXCR4 Hs00607978_s1
[0420] RT-PCR was used to evaluate the expression of
pancreatic-related genes from cells after 20 days post-transfection
with different combinations of genes (Table 40). This study
revealed that FoxA2 (13.12.+-.0.06), Nkx2.2 (23.12.+-.0.06) and
Gata4 (5.21.+-.2.36), markers for Endoderm and Mesoderm, were
up-regulated for the Sox17/Ngn3/Pdx1-transfected sample as compared
to the GFP mock-transfected control sample. Addition of Oct4 to
this gene combination increased Gata4 expression as well as Sox17
and endogenous Pdx1 expression, while both gene combinations
expressed insulin indicative of islet .beta.-like cells.
TABLE-US-00064 TABLE 40 Relative Expression of mesendoderm,
pancreatic progenitor, and islet .beta.-cell genes after
transfecting ADSCs once with various gene combinations with
potential to reprogram cells into pancreatic progenitor like cells
(including mesoendoderm-like cells and islet .beta.-like cells).
FoxA2 Sox17 Gata4 EndoPdx1 Rel. Std. Rel. Std. Rel. Std. Std. Day
20 Exp. Dev. Exp. Dev. Exp. Dev. Rel. Exp. Dev. MBD2/Oct4/Sox17
1.13 0.06 62.97 27.53 0.16 0.11 1.13 0.06 MBD2/Oct4/Pdx1 4.72 3.36
10.59 0.29 0.08 0.00 15.91 0.03 MBD2/FoxA2/PDX1 43.16 6.34 0.33
0.07 0.08 0.00 14.69 2.26 Sox17/PDX1/Ngn3 13.17 6.06 72.37 27.67
5.21 2.36 32.08 14.55 MBD2/Ngn3/Sox17 2.64 0.01 137.24 12.01 0.71
0.07 2.64 0.01 MBD2/FoxA2/NGN3 72.59 0.02 10.22 0.14 0.11 0.00 1.51
0.02 MBD2/PDX1/Ngn3 0.78 0.00 42.36 1.61 0.21 0.00 20.07 6.32
Oct4/Sox17/Pdx1Ngn3 11.35 0.04 139.00 7.36 34.53 121.8 116.68 41.77
Oct4/Pdx1/Ngn3 1.72 1.41 1.33 0.36 9.85 5.24 67.42 34.45
FoxA2/Pdx1/Sox17 100.57 0.03 143.29 16.42 0.15 0.01 13.19 4.24 ADSC
untransf. 0.04 0.01 0.21 0.08 0.08 0.00 1.16 0.01 Sox9 Ngn3 Nkx2.2
Insulin Rel. Std. Rel. Std. Rel. Std. Rel. Std. Day 20 Exp. Dev.
Exp. Dev. Exp. Dev. Exp. Dev. MBD2/Oct4/Sox17 0.98 0.05 1.13 0.06
1.13 0.06 1.13 0.06 MBD2/Oct4/Pdx1 0.97 0.08 1.13 0.04 1.51 0.51
1.13 0.04 MBD2/FoxA2/PDX1 1.24 0.08 1.19 0.03 1.19 0.03 1.66 0.63
Sox17/PDX1/Ngn3 1.87 0.02 97.04 18.77 23.12 0.06 6.12 0.06
MBD2/Ngn3/Sox17 1.73 0.02 12.58 3.08 2.64 0.01 2.64 0.01
MBD2/FoxA2/NGN3 1.46 0.03 20.33 5.17 1.51 0.02 1.51 0.02
MBD2/PDX1/Ngn3 1.19 0.12 59.24 14.07 0.78 0.00 0.26 0.00
Oct4/Sox17/Pdx1Ngn3 1.33 0.10 121.30 4.76 5.35 0.04 6.07 0.06
Oct4/Pdx1/Ngn3 1.21 0.03 34.50 2.38 0.76 0.05 1.44 0.28
FoxA2/Pdx1/Sox17 0.97 0.05 0.40 0.02 1.55 1.38 0.19 0.01 ADSC
untransf. 1.02 0.13 1.16 0.01 1.71 0.77 1.16 0.01
[0421] Next immunohistochemical analysis was performed to evaluate
protein expression in cells transfecting with the different gene
combinations. On day 20, the cells were immunostained and examined
using the Cellomics.TM. ArrayScan VTI. Cells were fixed with a 4%
formaldehyde/PBS solution for 10 min at room temperature and
subsequently permeabilized for 5 min with 0.1% Triton X-100 in 4%
formaldehyde/PBS. After two brief washes with PBS, unspecific
antibody binding was blocked by a 30 min incubation with 5% normal
goat serum in PBS. Then primary antibodies were added in 5% normal
goat serum/PBS as follows: Mouse anti-Gata4 (1:100, BD) and mouse
anti-FoxA2 (1:200, BD), a marker for endoderm layer. After a 2 h
incubation the cells were washed 4 times for 5 min each with 0.1%
Tween/PBS. Appropriate fluorescence-tagged secondary antibody was
used for visualization; Goat anti-mouse 546 (1:200, invitrogen)
prepared in 5% normal goat serum f PBS was used. After incubation
for one hour, cells were washed in 0.1% Tween/PBS three times for 5
min each. The DNA stain Hoechst33342 (Invitrogen) was used as a
marker of nuclei (dilution 1:5000 in PBS, 10 min incubation).
Fluorescence images were taken with the Cellomics.TM. ArrayScan HCS
Reader microscopy system. To determine an estimate of the
percentage of cells adopting endodermal phenotypes, random fields
were selected and for each field the total number of cells (as
determined by counting Hoechst stained nuclei) and the total number
of cells positive were determined. The cells in the Sox17/Pdx1/Ngn3
and Oct4/Sox17/Pdx1/Ngn3 transfected groups significantly expressed
both Gata4 and FoxA2: the number of positive cells was enhanced in
the presence of Oct4 as shown in Table 41.
TABLE-US-00065 TABLE 41 Percentage of positive cells for Gata4 and
FoxA2 at day 20 after transfection of ADSCs with different
expression vectors. After transfection cells were cultured in
IMDM/F12 supplemented with different type of growth factors and
small molecules (as described in Table 4). The percentage of
immunopositive cells was determined by the Cellomics .TM. ArrayScan
HCS Reader. Day 20 cell count Gata4 % FoxA2 % GFP 4825 0.21 0.23
untransfected 18646 0.01 0.03 MBD2/Oct4/Sox17 12563 0.17 0.35
MBD2/Oct4/Pdx1 13481 2.61 0.2 MBD2/FoxA2/Pdx1 13418 0.17 0.16
Sox17/Pdx1/Ngn3 15306 11.15 16.9 MBD2/Ngn3/Sox17 19867 1.32 0.31
MBD2/Ngn3/FoxA2 14984 0.33 0.42 MBD2/Ngn3/Pdx1 18613 0.31 10.29
Oct4/Sox17/Pdx1/Ngn3 19515 21.07 30.32 Oct4/Pdx1/Ngn3 6103 0.28
0.49 FoxA2/Pdx1/Sox17 10001 0.65 8.82
[0422] Transfection with Pdx1, Ngn3, Sox17 (and Oct4) increased
significantly selected pancreas-related genes (Pdx1, Ngn3, NKX2.2,
Gata4, FoxA2) indicating a transition from endoderm to a pancreatic
fate during this time period. To evaluate pancreatic hormone
secretion, pancreatic-like cells were cultured for 50 days, during
which time the medium was replaced with fresh medium every two
days. We then assayed the supernatant for insulin in the
conditioned medium by antigen-capture ELISA kit (Abnova, KA0921) at
different time points (day 25 and day 55) and compared to the
release of insulin in control cultures. According to the
manufacturer's instructions, briefly, 96-well ELISA immunoplates
were coated with Anti-insulin (CatNb#) diluted 1/1000 in carbonate
buffer (pH 9.7) and incubated at 4.degree. C. overnight. The
following day, all wells were washed with TBS-Tween 0.5% before
incubation with Block/Sample buffer 1.times. at room temperature
for one hour without shaking. After blocking, standards and samples
were added to the plates and incubated and shaken (450.+-.100 rpm)
for 2 h at room temperature. Subsequently, after washing with
TBS-Tween wash buffer, plates were incubated for 2 h with
Anti-Human insulin pAb (1:500 dilution in Block & Sample
1.times. Buffer) at 4.degree. C. After incubation, plates were
washed five times with TBS-Tween 0.5% wash buffer and 100 .mu.l of
standard and sample was added in the plates precoated with
anti-insulin and incubated for 1 hour at room temperature with
shaking (450.+-.100 rpm). Then, plates were washed five times with
TBS-Tween 0.5% wash buffer and 100 .mu.l of TMB One Solution was
added to each well. Following 10 minutes incubation at room
temperature with shaking (450.+-.100 rpm) for the insulin plate, a
blue color formed in the wells. After stopping the reaction by
adding 100 .mu.l of 1N hydrochloric acid, the absorbance was read
at 450 nm on a microplate reader (Synergy 4) within 30 minutes of
stopping the reactions. Concentration of released insulin in the
supernatants was determined according to the standard curves. ELISA
results revealed that insulin was released from cells transfected
with Oct4/Sox17/Pdx1/Ngn3, and to a lesser extent in the other
groups as summarized in Table 42.
TABLE-US-00066 TABLE 42 Quantification of insulin release by
pancreatic progenitor/.beta.-like cells that had been cultured for
25 and 55 days after transfecting ADSCs. Insulin release into the
medium, at days 25 and 55, was measured by antigen-capture ELISA
(Abnova). Concentration of insulin Concentration of insulin Insulin
release (.mu.lU/ml) at day 25 (.mu.lU/ml) at day 50 MBD2/Oct4/Sox17
0.05 0.07 MBD2/Oct4/Pdx1 1.02 1.87 MBD2/FoxA2/PDX1 1.13 1.11
Sox17/PDX1/Ngn3 2.3 4.32 MBD2/Ngn3/Sox17 0.07 1.03 MBD2/FoxA2/NGN3
0.09 1.30 MBD2/PDX1/Ngn3 1.23 1.03 Oct4/Sox17/Pdx1/Ngn3 4.32 7.85
Oct4/Pdx1/Ngn3 2.45 3.21 FoxA2/Pdx1/Sox17 1.02 1.23 ADSC
untransfected 0.01 0.03
[0423] In addition to increase of gene expression, the transfecting
cells were able to release insulin even in low concentrations.
Generating reprogrammed B-like cell lines that can locally deliver
insulin could be used as a method to treat and allow functional
recovery from diabetes.
Example XXIII
Transfection of ADSCs by Various Combinations of Genes and
Treatment with Different Small Molecules for Reprogramming to
Cardiac Progenitor-like Cells
[0424] ADSCs were cultured in StemPro.TM. MSC serum-free medium
(Invitrogen) as previously described, and then transfected with
different combinations of cDNA clones as described in Table 43
using the Nucleofector.RTM. 96-well Shuttle.RTM. Device (Lonza) as
described previously. After transfection, the cells were plated on
Matrigel-coated plates and incubated at 37.degree. C., 5% CO.sub.2,
5% O.sub.2. Medium was changed every other day according to Table
44. Cells were analyzed at Day 20 by Quantitative Real-time PCR and
by Immunohistochemistry.
TABLE-US-00067 TABLE 43 Various combinations of plasmids with
potential to transfect the cells towards Cardiac Progenitor-like
cells lineage. Day -3 to Day 0 Plasmids transfected at Day 0.sup.1
1 Pre-treated with T, FoxD3, Sox17, Mesp1 2 VPA & 5-Aza Foxd3,
Sox17, Mesp1, Nkx2.5 3 Foxd3, Tbx5, Baf, Nkx2.5 4 Foxd3, T, Mesp1,
Gata6 5 Sox17, Tbx5, Baf, Nkx2.5 6 Foxd3, T, Mesp1, Gata4 7 T,
Tbx5, Baf, Nkx 2.5 8 Foxd3, T, Mesp1, Tbx5 9 Mesp1, Tbx5, Baf,
Nkx2.5 10 Foxd3, T, Mesp1, Tbx5 11 Foxd3, Sox17, Mesp1, Gata6 12
Foxd3, Sox17, Mesp1, Gata4 13 FoxD3, Sox17, Mesp1, Tbx5 GFP
TABLE-US-00068 TABLE 44 media composition from day -3 to day 20.
Media Composition.sup.1 Day -3 to 0 Day 1 Day 2 to Day 3 Day 4 to
Day 6 Day 7 to Day 20 StemPRo IMDM/F12 IMDM/F12 + IMDM/F12 +
IMDM/F12 + medium + (50%) + NEAA + ITS + NEAA + ITS + NEAA + ITS +
VPA + 5-Aza NEAA (1X) + HSA + FGF8 HSA + FGF8 HSA + FGF ITS + HAS
(10 ng/ml) + (10 ng/ml) + (50 ng/ml) + BMP4 (5 mg/ml) + 5Aza + VPA
+ FGF (50 ng/ml) + (50 ng/ml) VPA + Activin CHIR99021 5Aza + A (30
ng/ml) + (2 nM) + Wnt 11 CHIR99021 CHIR99021 (50 ng/ml) + (2 nM) +
Wnt (2 nM) BMP4 (50 ng/ml) 11 (50 ng/ml) + BMP4 (50 ng/ml)
.sup.1Supplements added to media at the following concentrations:
Activin A (Peprotech, 30 ng/ml), HSA (Baxter, 0.5%), NEAA (Gibco,
1X), ITS (Gibco, 1X), FGF8 (Peprotech, 10 ng/ml), bFGF (Peprotech,
50 ng/ml), CHIR99021 (Stemgent, 2 uM), VPA (Stemgent, 1 mM), 5-Aza
(Sigma, 0.5 uM), BMP4 (Peprotech, 50 ng/ml)
[0425] Cells were collected on Day 20 by detaching with TrypLE,
followed by centrifugation at 80.times.g for 5 minutes. Supernatant
was aspirated and the cell pellet, was frozen at -86.degree. C.
until ready for RNA Isolation. RNA isolation, cDNA preparation and
quantitative real-time PCR was performed as previously
described.
[0426] Gene expression analysis on day 20 following transfection
revealed an increased of several Mesoderm markers. Interestingly,
three markers for cardiac progenitors cells (Nkx2.5, MyoCD, Tbx5)
and three markers of early mesoderm lineage (Gata4, Mesp1, and
Meox1) were highly increased after transfecting cells with
brachyury (T)/Tbx5/Nkx2.2/Mesp1. Similarly, expression of Mesp1,
Tbx5, Nkx2.5, MyoCd, and Meox1 was increased after transfecting the
cells with Foxd3/Sox17/Mesp1/Thx5; while expression of Mesp1,
Gata4, Nkx2.5, MyoCd, and Meox1 was increased after transfecting
with Foxd3/Sox17/Mesp1/Nkx2.5 and Foxd3/T/Mesp1/Gata4. As shown in
Table 45, the Relative Expression of mesoderm and cardiac
progenitor cells markers are especially up-regulated in the
combination of Mesp1/Tbx5/Nkx2.5/T, Foxd3/Sox17/Mesp1/Nkx2.5 or
/Tbx5, and Foxd3/T/Mesp1/Gata4. In general, transfection of ADSCs
with a combination Mesp1, FoxD3, Tbx5, Brachyury, Nkx2.5, Sox17
and/or Gata4 increased the expression of mesoderm and cardiac
progenitors markers. In addition, samples were collected at day 20
to evaluate the nature of reprogrammed cells by analyzing the
expression of mesoderm markers using Immunohistochemistry analysis
for Brachyury and Nkx2.5 according to the methods previously
described. Brachyury was significantly expressed in the
Mesp1/Tbx5/Nkx2.5/T, Foxd3/Sox17/Mesp1/Nkx2.5 or /Tbx5, and
Foxd3/T/Mesp1/Gata4 groups, while all groups expressed Nkx2.5.
These cells could represent and invaluable source of cells to
investigate furthermore human cardiac cells and also represent a
potential source of cells for deriving patient-specific multipotent
stem cells for modeling, regenerating, or treating human
cardiovascular diseases.
TABLE-US-00069 TABLE 45 Relative Expression Gata4, Mesp1, Tbx5, Nkx
2.5, MyoCD, and Meox1, after transfecting ADCs once with various
gene combinations with potential to reprogram ADSCs cells into
Cardiac Progenitor-like cells. Relative Expression Gata4, Mesp1,
Tbx5, Nkx 2.5, MyoCD, and Meox1, after transfecting ADCs once with
various gene combinations with potential to reprogram ADSCs cells
into Cardiac Progenitor-like cells Gata4 MESP1 TBX5 NKX2.5 MYOCD
MEOX1 Std. Std. Std. Rel. Std. Rel. Std. Std. Day 20 Rel. Exp. Dev.
Rel. Exp. Dev. Rel. Exp. Dev. Exp. Dev. Exp. Dev. Rel. Exp. Dev.
GFP- 1.01 0.16 1.01 0.19 1.00 0.05 1.02 0.27 1.00 0.07 1.02 0.27
transfected ADCs Foxd3/Sox17/ 0.02 0.01 6.70 1.18 1.52 0.12 0.94
0.07 1.98 0.13 1.37 0.10 T/Mesp1 Foxd3/Tbx5/ 0.08 0.03 1.86 0.20
1.59 0.08 24.94 2.97 2.11 0.11 1.17 0.15 Baf60c/Nkx2.5 Sox17/Tbx5/
0.62 0.09 3.89 0.10 1.45 0.00 41.72 2.35 0.90 0.02 1.19 0.02
Baf60c/Nkx2.5 T/Tbx5/Baf60c/ 0.93 0.12 7.59 0.61 1.76 0.12 27.54
2.36 0.94 0.05 1.23 0.19 Nkx2.5 Mesp1/Tbx5/ 1.12 0.13 31.55 0.91
4.26 0.07 154.76 3.50 1.54 0.06 5.96 1.60 Baf60c/Nkx2.5
Foxd3/Sox17/ 0.71 0.02 19.59 0.76 1.77 0.15 31.38 1.23 1.44 0.09
3.30 0.68 Mesp1/Gata6 Foxd3/Sox17/ 0.02 0.00 2.66 0.44 1.45 0.24
0.81 0.24 0.75 0.03 0.65 0.17 Mesp1/Gata4 Foxd3/Sox17/ 1.75 3.18
138.28 2.20 15.61 0.87 45.20 14.88 11.06 1.02 4.36 3.45 Mesp1/Tbx5
Foxd3/Sox17/ 17.76 4.34 22.03 0.08 3.03 0.01 95.73 11.68 10.25 2.32
10.54 2.39 Mesp1/Nkx2.5 Foxd3/T/Mesp1/ 27.10 6.47 3.59 0.42 2.58
0.29 5.11 0.85 21.03 1.02 12.99 1.31 Gata6 Foxd3/T/Mesp1/ 15.87
6.13 50.20 6.41 2.37 0.18 55.43 6.95 19.86 2.10 4.32 0.15 Gata4
Foxd3/T/Mesp1/ 0.83 0.02 8.20 0.48 2.40 0.37 29.25 1.28 0.76 0.08
1.40 0.46 Tbx5 Foxd3/T/Mesp1/ 19.79 2.03 111.35 15.32 2.56 0.35
32.52 12.53 0.29 0.03 24.70 3.12 Nkx2.5 T/Tbx5/Nkx2.5/ 18.46 1.06
103.09 22.28 102.53 15.02 96.32 16.60 15.32 5.01 15.73 2.83
Mesp1
Example XXIV
Reprogramming Human ADSCs to Pluripotent-Like Stem Cells (PLSC)
[0427] Based on previous results in Example XIX, the highest
reprogramming efficiency was observed using
pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP(NF10) and
pCMV-SalI4-2A-Oct4-2A-Klf4-2A-Nanog (S71). ADSCs (Invitrogen
Corporation) were cultured in cell culture flasks with complete
StemPro-43 medium (Invitrogen) at 37.degree. C., 5% CO.sub.2 and
the medium was changed 3 times per week. After 3 days in culture
cells (passage 5) were trypsinized and counted to be transfected.
Cells were transiently transfected with one plasmid:
pCMV-SalI4-2A-Oct4-2A-Klf4-2A-Nanog or Rex1-EP-Oct4-2A-Klf4-2A-RFP
(2 .mu.g) using nucleofector as described in Example II. Following
the transfection cells were cultured in 6-well plates in suspension
with 50:50 ratio of adipocyte complete medium (StemPro-43) and
embryonic stem cell medium (mTesR1). After two days in culture,
cells were re-transfected with the same plasmids listed above and
cells were plated in 96 well-plates coated with Matrigel (BD
Biosciences) in the presence of mTesR complete medium supplemeneted
with thiazovivin (0.5 .mu.M), an ALK-5 inhibitor (SB 341542,
Stemgent, 2 .mu.M), and inhibitor of MEK (PD0325901, Stemgent, 0.5
.mu.M). Medium was changed every day and cells were cultured for 22
days at 37.degree. C., 5% CO.sub.2, 5% O.sub.2. Alkaline
Phosphatase Detection Kit (AP, Millipore) and immunohistochemistry
were performed to analyze the expression of pluripotency markers.
ALP staining was performed using AP detection kit (Millipore)
according to manufacturer's instructions. Colonies emerged around
Day 15 and maintained in culture up to the end of the study period
(up to 2 months) with a stable morphology. Live staining showed
that these colonies express typical pluripotency markers, including
SSEA-4 and TRA1-81, TRA1-60, Oct4, Nanog, Sox2, and E-cadherin.
Further analysis of these colonies showed that the colonies present
as well other ESC markers such as alkaline phosphatase (ALP, not
shown). Supplementing the small molecules PD0325901 and SB431542
with the KSOR medium treated cultures, a 6 fold improvement in
efficiency over the conventional method was obtained following the
transfection of ADSCs with pCMV-pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP.
This pattern did not change up to the 2 month culture period and
same positive colonies were observed after transfecting ADSCs with
SalI4-2A-Oct4-2A-Klf4-Nanog (not shown). The observation over time
showed that the phenotype of these colonies moves from an early
SSEA+ phenotype to a late Oct4+/Sox2+/Nanog+ phenotype, which is
closer to the final reprogrammed state.
[0428] Following the reprogramming of ADSCs cells to
Pluripotent-like cells using the two vectors
pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP or
pCMV-SalI4-2A-Oct4-2A-Klf4-2A-Nanog, a study was performed to
examine the differentiation capacity of these cells. Beginning on
Day 30, reprogrammed cells were cultured in conditions in order to
stimulate the differentiation to embryonic bodies (EBs). These
differentiation conditions consisted of placing the cells on
uncoated petri-dish in EB Differentiation medium consisting of KSOR
medium with FBS 5% without FGF for 45 days. To further
differentiate the embryonic bodies, Day 30 EBs were cultured in
three different media in order to drive cell differentiation to
either ectoderm, mesoderm or endoderm lineages in CDM medium
consisting of DMEM/F12 supplemented with 2 mM Glutamine, 0.11 mM
2-mercaptoethanol, 1 mM nonessential amino acids, and 0.5 mg/ml HAS
(Yao S et al., PNAS 2006). This CDM medium was further supplemented
with different concentrations of cytokines to induce the
differentiation towards the three lineages. Ectoderm media
consisted of N2/B27-CDM in the presence of Noggin (100 ng/ml) and
after 10 days NGF (20 ng/ml) was added to the medium and the cells
were grown on laminin-coated plates. Endoderm media consisted of
N2/B27 CDM with the presence of Activin A (100 ng/ml) and the cells
were grown on Gelatin-coated plates. Mesoderm media consisted of
N2/B28-CDM in the presence of Activin A (50 ng/ml) and BMP-4 (50
ng/ml) and the cells were grown on Gelatin-coated plates. Cells
that had originally been reprogrammed with
pCMV-SalI4-2A-Oct4-2A-Klf4-2A-Nanog differentiated better than
cells originally reprogrammed with pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP
which detached from the coated plates. Visual observation of the
differentiation of the cells into the three lineages was performed
by RT-PCR and Cellomics using the Ectoderm markers (Nestin, GFAP,
Beta-tubulin, Sox2), Endoderm markers (GATA4, Brachyury) and
Mesoderm markers (GATA4, Brachyury, and Nkx2.5). RT-PCR analysis
revealed the Identification of endoderm, mesoderm and ectoderm
markers for all 3 embryonic germ layers (Table 46).
TABLE-US-00070 TABLE 46 RT-PCR analysis of three embryonic germ
layers following the differentiation of PLSCs. CDH1 ASCL1
(E-cadherin) (MASH1) NOTCH1 Std. Rel. Std. TP63 Rel. Std. A -
Ectoderm Rel. Exp. Dev. Exp. Dev. Rel. Exp. Std. Dev. Exp. Dev.
S71-transf. ADSC 59180.71 2036.56 70.28 12.35 123114.61 17452.81
5.47 0.19 NF10-transf. ADSC 77944.51 1747.33 48.20 3.27 166929.89
6007.52 6.47 0.12 Mel-2 Undifferentiated, P17 84021.24 178.93 9.80
4.30 10.72 2.96 5.90 0.48 ADSC Untreated Ctrl 1.20 0.93 1.00 0.00
1.00 0.00 1.00 0.03 SOX17 CDX2 B - Endoderm Rel. Exp. Std. Dev.
Rel. Exp. Std. Dev. S71-transf. ADSC 9.39 2.21 37.77 5.11
NF10-transf. ADSC 2.33 0.94 21.05 18.59 Mel-2 Undifferentiated, P17
0.77 0.00 4.34 5.05 ADSC Untreated Ctrl 1.00 0.00 1.00 0.00 FOXA2
GSC CXCR4 C - Mesoderm Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel.
Exp. Std. Dev. S71-transf. ADSC 1193.70 51.50 0.85 0.11 512.33
45.62 NF10-transf. ADSC 1662.76 160.28 0.57 0.01 352.65 39.26 Mel-2
Undifferentiated, P17 191.53 18.61 0.01 0.01 105.05 10.62 ADSC
Untreated Ctrl 1.00 0.00 1.00 0.06 1.19 0.91
[0429] The Expression of TP63 (Ectoderm/Surface Ectoderm) was
up-regulated in both the Day 71 S71-transfected sample
(123,000-fold) and the Day 75 NF O-transfected sample
(167,000-fold) as compared to the ADSC untreated control. The
expression of NOTCH1, a marker for the neuroectoderm, was slightly
up-regulated in the Day 71 S71-transfected sample (5-fold) and the
Day 75 NF10-transfected sample (6-fold) as compared to the ADSC
untreated control. Expression of CDH1/E-cadherin, surface ectoderm
marker, was up-regulated in both the Day 71 S71-transfected sample
(59,000-fold) and the Day 75 NF10-transfected sample (78,000-fold)
as compared to the ADSC untreated control. Furthermore, the
expression of ASCL1/MASH1, a neural crest marker, was up-regulated
in both the Day 71 S71-transfected sample (70-fold) and the Day 75
NF10-transfected sample (48-fold) as compared to the ADSC untreated
control.
[0430] The expression of definitive endoderm markers were analyzed,
such as SOX17, CDX2, and NKX2.1. The expression of SOX17 was
up-regulated in the Day 71 S7'-transfected sample (9-fold) as
compared to the ADSC untreated control. CDX2 gene expression is
up-regulated in both the Day 71 S71-transfected sample (38-fold)
and the Day 75 NF10-transfected sample (27-fold) as compared to the
ADSC untreated control. However, NKX2-1 (Endoderm-Primitive Gut
Derivative) was not expressed in any of the 4 test samples at the
time points tested.
[0431] Markers of Mesoderm were analyzed as well: the expression of
T (Mesoderm) was up-regulated in the Day 71 S71-transfected sample
(16-fold) as compared to the ADSC untreated control. However the
expression of MEOX1, OSR1 (intermediate mesoderm), and FOXF1
(lateral mesoderm) was decreased in both the S71-transfected and
the NF10-transfected samples as compared to the ADSC Untreated
Ctrl. The expression of GATA4 is up-regulated in both the Day 71
S71-transfected sample (150-fold) and the Day 75 NF10-transfected
sample (110-fold) as compared to the ADSC untreated control. The
expression of FOXA2 (Mesendoderm, Mesoderm, Endoderm) was
up-regulated in both the Day 71 S71-transfected sample (1,100-fold)
and the Day 75 NF10-transfected sample (1,600-fold) as compared to
the ADSC untreated control. No significant change in GSC
(mesoendoderm, Endoderm) expression for both the S71-transfected
and the NF10-transfected samples as compared to the ADSC Untreated
Ctrl. The expression of CXCR4, a marker for Mesoderm/Endoderm, was
up-regulated in both the Day 71 S7'-transfected sample (500-fold)
and the Day 75 NF10-transfected sample (350-fold) as compared to
the ADSC untreated control.
[0432] Using immunohistochemistry, the potential of PLSCs to
express and differentiate into the three embryonic layers markers
was analyzed. Consistent with their hES-like morphology, PLSCs were
able to differentiation towards the three lineages: Ectodermal
differentiation: Nestin is strongly and widely expressed throughout
the experiment. The neural markers OFAP and .beta.III-tubulin are
also expressed, while Sox2 as a neural precursor marker was not
detectable at the time points tested, indicating that cells had
already differentiated into the neural pathway. At day 12 and 18,
many cells co-expressed GFAP and .beta.III-tubulin. At day 28,
however, GFAP expressing cells have almost completely disappeared,
while .beta.III-tubulin positive cells with well formed neurites
become detectable. The medium supported neural differentiation, but
less astrocyte differentiation, since, especially at day 12 and 18,
there are high amounts of GFAP-positive collapsed cells detectable,
indicating massive cell death of this type of cells. The
omnipresence of the neural markers suggest that differentiation
occurs along the neural pathway in this media; although different
differentiation lineages could be achieved with different media
compositions and growth substrates/growth conditions known in the
art. By Day 12 of differentiation, the expression of the stem cell
markers Oct4 and Nanog are completely suppressed.
[0433] Endodermal differentiation: Large fields of cells that are
positive for FoxA2 are detectable indicating endodermal
differentiation.
[0434] Mesodermal differentiation: Cells treated for mesodermal
differentiation cease to express Oct4 and nanog (not shown),
indicating that they indeed started to differentiate. The strong
and ubiquitous expression of GATA4 and the later appearance of
nuclear Nkx2.5 stain indicate that many cells might develop a
cardiac profile in the conditions tested. There are some cell
clusters that express brachyury staining together with GATA4.
[0435] As for the Ectoderm and Endoderm differentiation, different
media compositions and growth substrates/growth conditions known in
the art would promote the acquisition of other differentiation
lineages.
Example XXV
Cytotoxic Effects of Human PLSCs
[0436] Human Hepatocellular Carcinoma cell line (HepG2, CRL-10741,
ATCC) were seeded in a 24 well plate (1.times.10.sup.4
cells/cm.sup.2) in DMEM with 10% Fetal bovine serum (FBS). PLSCs
were harvested using dispase and seeded (1.times.10.sup.4
cells/cm.sup.2) in transwells (Corning, 0.4 .mu.m) above the HepG2
containing wells. The HepG2 cells were analyzed for cytotoxicity
after 24 hours by high-content analysis staining by Propodium
Iodide, Yopro1, and Ethidium Heterodimer (cell-impermeable DNA
stains that stain only cells with a compromised plasma membrane as
occurs in unhealthy cells) along with Calcein (a live stain,
marking enzymatic activity in the cytoplasm), Mitotracker Red
(marks active, healthy mitochondria) and Cleaved Caspase 3 (a
marker for apoptotie or stressed cells). HepG2 cells in the
presence of PLSCs had significantly less Propodium Iodide, Yopro1
and Cleaved Caspase 3 staining indicating that the PLSCs had a
protective and/or therapeutic/regenerative effect on these cells
(Table 47).
TABLE-US-00071 TABLE 47 The percentage of Yopro1, Propidium,
ethidium Heterodimer, Calcein AM, Mitotracker Red, and Cleaved
Caspase-3 positive cells for untreated and PLSCs treated
hepatocytes. Average % positive cells intensity Number of Propidium
Ethidium Cleaved Mitotracker cells Yopro1 Iodide Heterodimer
CalceinAM Caspase3 Red HepG2 14539 .+-. 1359 5.15 .+-. 0.47 4.57
.+-. 0.21 5.66 .+-. 0.02 69.41 .+-. 1.44 0.95 .+-. 0.05 26.88 .+-.
3.52 untreated HepG2, 12498 .+-. 1454 3.41 .+-. 0.37 3.85 .+-. 0.36
5.81 .+-. 0.33 71.67 .+-. 1.55 0.63 .+-. 0.03 27.99 .+-. 3.74 PSLC
treated
Example XXVI
Teratoma Formation Assay in Immunodeficient NOD-SCID Mice
[0437] Fifty million human NSLCs derived from fibroblast cells
(HFF) and fifty millions NSLCs derived from blood cells (CD34+)
(according to Examples IV and IX), as well as twenty-five million
Human embryonic stem cells (Mel2) were prepared as units of five
million cells each embedded with 30% Matrigel (Invitrogen) in 200
.mu.l phosphate buffered saline which were then injected
subcutaneously into the back of individual NOD/SCID mice using a
21-G needle within 20 minutes of preparation. Each cell line had
been in culture for approximately 1 month prior to the study. Thus
10 mice received an injection of 5 million NSLCs derived from HFF,
10 mice received an injection of 5 million NSLCs derived from CD34+
cells, and 5 mice received an injection of 5 million Mel2 cells.
All standard and appropriate animal approval and ethical committee
approvals were obtained prior to the commencement of the study.
Although the study was for 3 months, within 1 month of injection
one of the mice injected with the Mel2 cells had to be sacrificed
due to the size of the teratoma that had formed according to the
animal approval protocol. Less than 3 weeks later, the rest of the
Mel2 injected mice had to also be sacrificed due to the size of the
teratoma that had formed. None of the 10 mice injected with NSLCs
derived from fibroblast cells or the 10 mice injected with NSLCs
derived from CD34+ blood cells formed any tumors or teratomas,
indicating that these NSLCs are safe multipotent stem cells.
Example XXVII
Differentiation of NSLC Derived from HFF, CD34+ Cells, and
Keratinocytes to Different Neuronal Lineages
[0438] To investigate the differentiation potential of NSLCs to
different types of neurons, NSLC Neurospheres were dissociated and
plated onto laminin/poly-D-Lysine (10 .mu.g/ml; Sigma) coated
plates in NeuroCult differentiation medium (NeuroCult
Differentiation basal, StemCell Technologies) supplemented with
NeuroCult.RTM. SM1 Neuronal Supplement and BDNF (20 ng/ml,
Peprotech), bFGF (40 ng/ml, Peprotech), FGF 8 (20 ng/ml, Peprotech)
and SHH (20 ng/ml, Peprotech) for 30 days. NeuroCult.RTM. SM1
(StemCell Technologies) is a standardized serum-free supplement
containing antioxidants and retinoic acid; its formulation was
developed based on the published supplement formulation identified
as B27 but has been optimized to give reproducibly high numbers of
functional neurons with minimal glial cell contamination (<1%
GFAP+). After one month in culture, the cells were stained with the
neuronal marker tyrosine hydroxylase, acetycholine, GABA, and
Dopamine. All cell lines stained positive for each of these
markers. Immunohistochemistry analysis showed that differentiation
medium supplemented with Neurocult SM1 and neurotrophic factors
promoted the differentiation of NSLCs derived from HFF, CD34+
cells, and Keratinocytes to dopaminergic, adrenergic, and
Gabanergic neurons.
[0439] This study showed that NSLCs derived from fibroblast,
keratinocyte, or blood cells are capable to differentiate towards
different types of neurons in the appropriate differentiation
conditions. The specific neurons could be used to treat diseases
where such neurons are affected or have been lost, for example,
dopaminergic neruons in Parkinson's disease. Other types of
multipotent stem cells and progenitor cells prepared according to
the methods in the previous examples would be expected to reprogram
or differentiate more along certain pathways with the appropriate
media and supplements, growth substrate and growth conditions known
in the art.
Example XXVIII
Comparison of Expression of Specific Genes in Human HFFs, NSLCs
Created from the HFFs, and Human Primary Neuroprogenitor Cells
[0440] The expression of selected genes and proteins in NSLCs
created from HFFs according to Example IV were determined. Total
RNA was extracted from cells, using Trizol following manufacturer's
recommendation. Briefly, cells were examined for the expression of
different genes associated with pluripotency: SOX2 (460 bp), OCT4a
(172 bp), OCT4b (169 bp) and NANOG (276 bp); early neural markers:
SOX2 (460 bp), NES (327 bp), CD133 (200 bp), PAX6 (431 bp) and ASCL
(220 bp); Notch signaling: NOTCH1 (126 bp), NOTCH2 (475 bp), HES1
(314 bp) and HESS (265 bp); neurotrophic factors: GDNF (389 bp) and
BDNF (743 bp) and astrocyte marker GFAP (650 bp) and markers of
neurons: NFL (284 bp), NFM (333 bp), NFH (316 bp), SYN (289 bp),
NSE (292 bp) and MAP2 (321 bp). Positive controls used were NT2
cells (human NT2/D1 teratomacarcinoma cell line (ATCC), NT2-derived
neurons (NT2-N) and astrocytes (NT2-A) and SH-SY5Y cells were
appropriate. No template control was used as negative control for
every gene examined.
[0441] NSLCs had the same expression profile as human primary
neuroprogenitor cells (NP; Lonza), namely in terms of expression of
Sox2, Oct4b, Notch1, Notch2, Hes1, Hes5, Nestin, CD133, Pax6, Ascl1
(Mash1), NFL, NFM, NSE and Map2, but additionally the of the
neurotrophic factors GDNF and BDNF. The HFF cells that the NSLCs
were created from also expressed Oct4b (Oct4b is a spicing variant
of Oct4a (a stem cell marker) which expression is not limited to
stem cells), Nestin, some of the notch signaling genes and neuronal
markers, making it significantly easier to reprogram than adult
dermal fibroblasts that do not express this genes (thus, for
example, Nestin is important to add to the reprogramming cocktail
when reprogramming adult fibroblasts to NSLCs).
[0442] Next immunohistochemistry was used to confirm that the
encoded protein of some of the above expressed genes was present in
the cells. Cells were fixed with 65% Ethanol and 0.15M NaCl for 20
minutes and stained for the following proteins: SOX2, NES, GFAP,
MAP2, NCAM and RBPJ (green). Phase and Hoechst (blue) counter stain
was also preformed. NT2 and NP cells were used as comparison and
positive controls. NSLCs expressed intense staining for Sox2,
Nestin, GFAP, Map2, and RBPJ with only a small amount of NCAM
detected. NP cells had an identical staining pattern to the NSLCs
except slightly less intense staining especially for Sox2 and
Nestin. The NT2 cells only expressed Sox2, Nestin and RBPJ at
appreciable levels. The HFFs from which the NSLCs were created from
only expressed Nestin and RBPJ at appreciable levels.
Example XXIX
Potential of NSLCs for CNS Therapeutic Applications
[0443] NSLCs prepared from HFFs according to Example IV were tested
for their ability to form functional cell-cell communication
through gap junctions, which is an important characteristic for
therapeutic potential when implanted into the CNS. Dye coupling
experiments showed functional cell-cell communication in NSLC cells
through gap junctions. A single NSCL cell was pre-loaded with two
dyes [Dil (red) and calcein AM (green)] and plated on an unlabeled
layer of NSCL cells. Calcein readily transferred from the donor
cells to a large population of receiving cells after 3 hours in
vitro, confirming gap junctional intercellular communication among
NSCL cells.
[0444] Next NSLCs were tested for the expression of synaptotagmin
(a synaptic vesicle protein) and MAP2 (a neural differentiation
associated marker) since NSLCs have some unique characteristics
over native neural stem/progenitor cells including expression of
some growth factors as well as a more significant expression of
some neural differentiation markers while maintaining the ability
to remain in a stem cell like state. NSLCs were found to readily
express synaptotagmin and Map2.
[0445] Next NSLCs were tested for their tolerance to glutamate and
NMDA to determine their potential robustness in areas of
significant ischemic, trauma, and neurodegeneration in the CNS.
NSLCs maintained a healthy neuronal state in the presence of
glutamate (200 mM), a characteristic not observed in the presence
of NMDA (25 mM), indicating an ability to form synapses upon
neuronal differentiation.
[0446] Next the NSLCs were tested for their ability to attach and
survive on 3D scaffolds; in this case PGA scaffolds. NSLCs were
found to readily attach to the PGA scaffolds designed for
implantation into the brain. Carboxyfluourodiacetate (CFDA)
confirms optimal cell survival (>93%) 24 hours after cells were
plated on the scaffold. NSLCs showed robust attachment and survival
to the surface for at least one week in vitro. Furthermore,
Neurites grew along the length of each PGA fiber, following the
pattern of the scaffold, indicating that NSLCs can be grown on 3D
scaffold and structures useful for implantation into humans or
animals, or for in vitro modeling.
Example XXX
Neural Stem-Like Chromatin State of Neural Stem-Like Cells
[0447] Human keratinocytes (Invitrogen, Cat. #12332-011),
CD34.sup.+ hematopoietic cells (StemCell Technologies, Cat.
#MPB015F), and neonatal foreskin fibroblasts (HFF, American Type
Culture Collection, Cat. #CRL-2097)) were transfected with
pCMV-MSI1-2A-Ngn2 and pCMV6-XL5-MBD2 and grown in neural stem cell
medium supplemented with EGF and FGF2 (NeuroCult Proliferation Kit
Cat. #5751, StemCell Technologies) on CELLStart.TM. (Invitrogen,
Cat. #A10142-01) coated plates at 37.degree. C., 10% CO.sub.2, 5%
O.sub.2 and supplemented with VPA and Noggin for the first 6 days.
Neural stem-like cells (NSLC) expressing Nestin, Sox2 and GFAP were
obtained within 1-2 weeks from each cell line and allowed to
proliferate for at least 7 passages.
[0448] Since one of the most widely used markers for neural stem
cells is Nestin expression and all the NSLC expressed Nestin,
Bisulfite genomic sequencing was performed on the second intron
region of Nestin to determine its methylation status in each of the
3 NSLC lines to determine if this gene becomes unmethylated in
NSLCs thus allowing stable endogenous expression of this neural
stem cell gene. The original keratinocyte, CD34.sup.+ hematopoietic
cells, and HFF cell were used as comparison controls, and normal
human fetal neuroprogenitor cells (NPC) as a positive control.
Nestin is methylated in most other cells, and thus not
significantly expressed, since methylation in the 5-position of a
cytosine in a cytosine-phosphate-guanosine (CpG) dinucleotide
results in long-term gene silencing and thus no gene expression. To
determine the Nestin gene methylation status of each of these
cells, genomic DNA was isolated with the DNeasy kit (Qiagen,
Toronto, Ontario) and bisulfite treatment was carried out using EZ
DNA Methylation-Gold Kit (ZYMO Research, Irvine, Calif.). The
second intron region of the human Nestin gene was amplified by PCR
(40 cycles of denaturation at 94.degree. C. for 30 s, annealing at
55.degree. C. for 30 s, extension at 72.degree. C. for 30s with a
1st denaturation at 94.degree. C. for 5 min for Hot-Start TAQ
activation, and a final extension at 72.degree. C. for 10 min),
then purified (PCR Purification Kit, Qiagen), subcloned using the
TOPO TA PCR Cloning Kit (Invitrogen) and 5 randomly selected clones
were sequenced (IRIC Genomics platform, Universite de Montreal).
The promoter in the starting cells was hypermethylated as expected
for these cells, but was demethylated in all NSLC similarly to NPC.
Thus during the reprogramming process NSLC gain a neural stem-like
chromatin state as shown by the DNA demethylation status of
specific loci, indicating genomic stability of the reprogramming
event, but also epigenetic resetting of the fibroblast,
keratinocyte, and CD34.sup.+ cell genomes to a neural stem-like
state.
Example XXXI
Normal Karyotype (Chromosomes) in Stem-Like Cells
[0449] A karyotype analysis was performed to confirm that normal
chromosomes were maintained in neural stem-like cells (NSLC). NSLC
derived from either CD34.sup.+ cells or HFF (from Example 1) were
arrested with colchicine (10 .mu.g/ml) for 30 min. Chromosome
preparations were made by incubating the cell suspension in 0.075M
KCl at 37.degree. C. for 13 min, followed by a fixation step in a
freshly prepared mixture of 3:1 methanol:acetic acid at -20.degree.
C. GTG banding was performed by incubating the glass slides in a
0.05% trypsing solution (Difco) at 37.degree. C. for 15 sec,
followed by rinsing the slides in phosphate-buffered saline and
staining in a 5% Giemsa stain for 8 min. The slides were rinsed
with water and air-dried. The slides were stained with DAPI, washed
in distilled water, and dried at ambient temperature. The
preparations were examined under imaging fluorescent microscope
(Zeiss) and analysis of biologic chromosome structures was
performed after GTG banding.
[0450] No karyotypic abnormalities were observed in NSLC derived
from CD34.sup.+ and HFF. The light and dark bands are clearly
detectable and all chromosomes could be identified.
Example XXXII
[0451] Stem-Like Cells do not Form Tumors or Teratomas
[0452] NSLC derived from either CD34.sup.+ cells or HFF (from
Example 1) were injected subcutaneously into Fox-Chase SCID Beige
mice at a concentration of approximately 250 million NSLC per
kilogram of body weight. This cell concentration would represent an
equivalent injection of approximately 15 billion NSLCs into an
average 60 kg adult human. During the 120-day follow-up, no tumors
or teratomas were observed by gross necropsy nor by
histopathological analysis in any of the NSLC-implanted animals. In
comparison all animals that were injected with human embryonic stem
cells (MEL-2 hES cells) had to be euthanized before the end of the
study (Days 36-46) due to the formation of massive tumors teratomas
at the site of injection, hence confirming the sensitivity of this
study in being able to detect any tumor/teratoma forming cells.
Example XXXIII
Creation of Stem-Like Cells Using RNA
[0453] Human neonatal foreskin fibroblasts (HFF, American Type
Culture Collection, Cat. #CRL-2097)) were transfected with
synthetic messenger RNAs (mRNAs) of MSi1, NGN2 and MBD2. The mRNAs
were synthetized as standard RNAs using T7 mScript Standard mRNA
Production sSstem from CellScript (Cat#C-MSC100625). To promote
efficient translation, the mRNAs were modified by enzymatic capping
and A-tailing as described in the mScript Standard mRNA production
system.
[0454] For the transfection method, 20,000 cells were seeded per
well in a 96-well laminin-coated plate in neural stem cell medium
supplemented with EGF and FGF2 (NeuroCult Proliferation Kit Cat.
#5751, StemCell Technologies) at 37.degree. C., 10% CO.sub.2, 5%
O.sub.2 and supplemented with VPA and Noggin for the first 8 days.
The cells were transfected on Day 2 with the MSi1, NGN2 and MBD2
mRNA using TransiT mRNA transfection reagent (Mirus, Cat #MIR
2250): the mRNA was diluted in 100 .mu.l OptiMEM and incubated 2-5
min with 2 .mu.l Boost and 2 .mu.l transfection reagent for each
.mu.g of RNA. A total of 100 ng mRNAs was administered to each
well. The transfections were repeated with MSi1 NGN2 on Days 4, 6
and 8.
[0455] Neural stem-like cells (NSLC) expressing Nestin and Sox2
were obtained within 1-2 weeks, along with .beta.III-tubulin
expressing cells (about half of all cells) with early neuronal-like
phenotype after 14 days of differentiation.
[0456] It is thus expected that a similar methodology of
transfecting/introducing the MSi1, NGN2 and MBD2 proteins into
isolated starting cells can be used to create neural stem-like
cells. It is also expected that the same methodology can be used to
create other types of stem-like cells using the appropriate
reprogramming agents.
Example XXXiV
Creation of Hematopoietic Stem-tike Cells
[0457] Human adipocyte-derived stem cells (ADSC, Invitrogen) and
HFF (ATCC, Cat. #CRL-2097) were transfected every other day for a
total of three times with combinations of pCMV6-AC-CDX4,
pCMV-AC-Gata1, pCMV-AC-Gata2, pCMV-XL5-HoxB4, pCMV-XL5-T, and
pCMV-AC-KLF1 (all from Origene) and grown in hematopoietic stem
cell medium (complete StemPro-43 medium, Invitrogen) on Lamini-411
(Biolamina) coated plates at 37.degree. C., 10% CO.sub.2, 5%
O.sub.2 and supplemented with Stem Cell Factor (100 ng/ml, R&D
Systems), IL-3 (50 ng/ml, R&D Systems), GM-CSF (25 ng/ml,
R&D Systems), and human TPO (50 ng/ml, R&D Systems). After
2 weeks, the cells were transferred to a low-bind petri-dish
(Corning) and grown in complete StemPro-43 medium supplemented with
3 U/ml erythropoietin, 50 ng/ml SCF, 20 ng/ml GM-SCF, 20 ng/ml
IL-3, 20 ng/ml IL-6. Formed colonies were analyzed for the
expression of CD34.sup.+ and Sca-1 indicating the presence of
hematopoietic stem-like cells (HSLC).
Example XXXV
[0458] Creation of Cardiac Stem-like Cells
[0459] Human Mensenchymal Stem Cells (MSCs, Lanza) were seeded on
cellbind flasks (Corning) at 2.1.times.10.sup.4 per cm.sup.2 of
tissue culture surface area and expanded in cell culture flasks
with complete mesenchymal stem cell media (MSCGM, Lonza) containing
mesenchymal basal medium and MSC growth supplements. The medium was
changed twice per week. Cells were trypsinized using Tryple
(Invitrogen) for 4 minutes at 37.degree. C., pelleting the cells by
centrifugation, washing the cells once with PBS, and plating the
cells at a ratio of 1:2 onto tissue culture flasks until a suitable
number of cells was reached.
[0460] Cells were then transfected using the Amaxa Nucleofector.TM.
96-well Shuttle Device (Lonza). The MSCs were harvested with Tryple
(Invitrogen), resuspended in MSCGM Medium and centrifuged for 10
min at 200.times.g (1.times.10.sup.6 cells/tube). The supernatant
was discarded and gently resuspended in 80 .mu.l of Basic
Nucleofector Solution (Basic Nucleofector kit for primary Mammalian
MSC, Lonza). Each 80 .mu.l of cell suspension was combined with a
different mix of plasmid DNA (pCMV-XL4-Tbx5, pCMV-XL5-Mesp1,
pOMV-XL5-Brachyury; or pCMV-XL4-Nkx2.5, pCMV-XL5-Mesp1,
pCMV-XL5-Brachyury). The cell suspension was transferred into an
Amaxa certified shuttle 96 well-plate and transfected with the
appropriate program (V4SP-1096). The sample was transferred into a
Laminin-211 (Biolamina) or Gelatin 0.1% coated culture plate, and
the cells were incubated at 37.degree. C., 5% CO.sub.2. After 24
hours, the medium was changed to maintenance medium consisting of
RPMI 1640, with L-glutamine and sodium pyruvate supplemented with
the following components: bFGF (20 ng/ml), ITS (1.times.), Activin
A (30 ng/ml), BMP4 (20 ng/ml), NRG (10 ng/ml), Valproic acid (VPA)
(0.5 mM), and 5-azacytidine (5-Aza) (0.5 .mu.M). After two days,
cells were retransfected using lipofectamine with 2 .mu.g of
pCMV6-XL5-T, pCMV-XL4-TBX5, pCMV-XL-Mesp1 or pCMV-XL5-Mesp1,
pCMV6-XL5-T, pCMV-XL4-NKX2.5 using lipofectamine reagent
(Invitrogen) as per the manufacturer's protocol. The DNA-lipid
complex was added to cells and incubated for 24 h at 37.degree. C.,
5% CO.sub.2.
[0461] After 15 days some of the cells were fixed for analysis with
a 4% formaldehyde/PBS solution for 10 min at room temperature and
subsequently permeabilized for 5 min with 0.1% Triton X-100 in 4%
formaldehyde/PBS. After two brief washes with PBS, unspecific
antibody binding was blocked by a 30 min incubation with 5% normal
goat serum in PBS. Then primary antibodies were added in 5% normal
goat serum/PBS as follows: Mouse anti-Nkx2.5 (1:50), anti-Brachyury
(1:50), anti-Gata4 (1:20), anti-CXCR4 (1:100), and anti-troponin 1
(1:100). After a 2 h incubation the cells were washed 4 times for 5
min each with 0.1% Tween/PBS. Appropriate fluorescence-tagged
secondary antibody was used for visualization; Goat anti-mouse 546
(1:200, invitrogen), anti-rabbit Alexa 488 (1:200), and anti-goat
Alexa Fluor 546 (1:200) prepared in 5% normal goat serum/PBS was
used. After incubation for one hour, cells were washed in 0.1%
Tween/PBS three times for 5 min each. The DNA stain Hoechst33342
(Invitrogen) was used as a marker of nuclei (dilution 1:5000 in
PBS, 10 min incubation). Fluorescence images were taken with a
Cellomics ArrayScan HCS Reader microscopy system. To determine the
morphology and certain markers of cardiac stem cells, random fields
were selected and analyzed by the Cellomics ArrayScan HCS software
(the number of cells was determined by counting Hoechst stained
nuclei). The `pCMV6-XL4-Nkx-2.5, pCMV6-XL5-Mesp1,
pCMV6-XL5-brachyury` transfected cells showed better expression of
the Cardiac stem cell lineage markers Gata4, Nkx2.5, CXCR4,
Brachyury, and Troponin1, as well as cells grown on the Laminin-211
coated plates. Similar results were obtained for cells transfected
with pCMV-MSI1-2A-Ngn2 and pCMV6-XL5-MBD2 using the identical
methods/protocol of this Example 6 showing that CSLC can be
obtained through the NSLC pathway by differentiation towards neural
crest-like cells and then into CSLC by the appropriate conditions
and reprogramming factors.
[0462] Characterization of the cardiac stem-like cells was
performed using a cardiomyocyte gene-array containing 48 partial
cDNAs coding for these genes and controls. RNA was isolated from
samples using QIAshredder (Qiagen) and RNeasy Plus mini Kit
(Qiagen) as per manufacturer's instructions. DNase I treatment was
performed on the RNeasy Column to further remove the transfected
plasmid DNA using Rnase-Free DNase Set (Qiagen). RNA was eluted in
35 .mu.l of RNase-free water. Before cDNA synthesis, all RNA
samples were quantified using the NanoDrop 1000 (ThermoScientific).
cDNA was prepared using the High Capacity cDNA archive kit (Applied
Biosystems) as per the manufacturer's instructions. 400 ng of RNA
was used in each 50 .mu.l RT reaction. The resulting cDNA samples
were used immediately for TLDA analysis (1 TLDA was prepared for 4
samples) in an Applied Biosystems 7900HT Fast Real-time PCR system.
Thermal cycler conditions were as follows: 2 minutes at 50.degree.
C., 10 minutes at 94.5.degree. C., and 30 seconds at 97.degree. C.,
1 minute at 59.7.degree. C. for 40 cycles. Relative Expression
values were calculated using the Comparative C.sub.T method using
the formula 2.sup.-.DELTA..DELTA.CT and shown in Table 1.
TABLE-US-00072 TABLE 1 Gene array results comparing relative
expression levels of select genes between cardiac stem-like cells,
floating cardiosphere stem-like cells, and fetal cardiomyocytes.
Gene array on the stem-like cells was performed on samples after
four weeks of transfection with Mesp1/Tbx5/brachyury. Expression
values given relative to the starting untransfected human
Mesenchymal Stem Cells (MSC). Relative expression to MSC Control
Adherent Cardiosphere Common name Company Cardiac Stem- Stem-Like
Human fetal Symbol and description Gene ID Like Cells Cells
cardiomyocytes ACTN2 Actinn, alpah2 NM_001103 38.96 27.91 274.14
MYH7 Myosin, heavy NM_000257 102.30 24.38 99.96 chain 7 cardiac
muscle beta SLC8A1 Sodium/Calcium NM_021097 1.77 6.17 3.81
exchanger ADRB1 Adrenergic NM_000684 311.55 285.19 1205.91 beta-1
receptor MYL2 Myosin light NM_000432 3.73 2.55 29.07 chain 2 TNN3
Troponin I type NM_000363 2.79 2.70 9.65 3 (cardiac) CKM Creatine
kinase NM_001824 0.41 0.75 0.97 muscle MYL3 Myosin light NM_000258
15.17 2.91 9.95 chain 3 ventricular TNNT2 Troponin T type NM_000364
5.20 4.03 9.72 2 (cardiac) DES Desmin NM_001927 9.58 31.06 114.15
GATA4 GATA binding NM_002052 68.86 2.64 532.67 protein 4 NKX2-5 NK2
homeobox 5 NM_004387 9.48 3.20 23.14 NPPA Natriuretic NM_006172
2.78 14.61 85.94 peptide A HAND2 Heart and NM_021973 10.08 3.58
43.81 neural crest derivatives expressed 2
[0463] Quantitative comparison of cardiomyocytes gene expression
was analyzed by using a significance analysis algorithm to identify
genes that were reproducibly found to be enriched in reprogrammed
cells compared to untransfected starting cells (the MSC; see Table
1). The TLDA data (Table 1) revealed a remarkable increase in
specific markers for cardiac lineage in reprogrammed adherent or
floating cells, such as ACTN2, MYH7, ADRB1, MYL3, Desmin, GATA4,
and HAND2. There was also a difference in the gene expression
pattern between adherent and floating reprogrammed cells. After the
transfection with Mesp1/Tbx5/Brachyury, the expression of Myosin
heavy chain 7 (MYH7, 102.30 fold), Myosin light chain 3 (MYL3,
15.17 fold), Gate 4 (68.86 fold), HAND2 (10.08 fold) and Nkx2.5
(9.48 fold) was markedly more increased in adherent cardiac
stem-like cells as compared to cells cultured in suspension as
cardiospheres (Table 1), indicating a potentially more
differentiated state of the adherent cardiac stem-like cells
compared to the floating cardiospheres. Natriuretic peptide A
(NPPA) was more highly expressed in the floating cardiospheres than
adherent cardiac stem-like cells.
Example XXXVI
Formation of floating Colonies and Cell Differentiation
[0464] To investigate whether a single Cardiac Stem-Like Cell
(CSLC) is able to generate a cardiosphere, CSLC were dissociated
into single cells and cultured in cardiac stem cell medium (RPMI
1640 Glutamax 100.times. supplemented with Activin A (R&D
Systems, 30 ng/ml), NEAA (Invitrogen, 1.times.), ITS (Invitrogen,
1.times.), bFGF (Invitrogen, 20 ng/ml), BMP4 (R&D Systems, 20
ng/ml), NRG (Peprotech.TM., 10 ng/ml)) in a low-bind cell culture
dish (Corning), and cardiosphere formation was monitored by taking
bright field images using light microscope (Nikon, 10.times.) over
several days. The CSLC started to proliferate and grew into a
cardiosphere that continuously grew in size and volume.
[0465] In order to determine the differentiation potential of CSLC
to differentiate into Cardiomyocyte-Like Cells (CLC), the medium
was additionally supplemented with Oxytocin (5 .mu.M, R&D
Systems), T3 (10.sup.-4 M), and Cardiotropin (20 ng/ml) for two
weeks. After two weeks in culture, the cells were stained for early
cardiac markers such as Brachyury, Nkx2.5 and late markers of
cardiac cells such as Troponin T, Troponin I, and Connexin-43.
Immunohistochemistry analysis on Day 30 revealed that the cells
stained positive for all 5 markers indicating the cardiac phenotype
of these cells. CLC differentiated from cardiosphere-CSLC generally
had higher expression levels of all the markers than CLC
differentiated from adherent CSLC.
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[0505] Headings are included herein for reference and to aid in
locating certain sections These headings are not intended to limit
the scope of the concepts described therein under, and these
concepts may have applicability in other sections throughout the
entire specification Thus, the present invention is not intended to
be limited to the embodiments shown herein but is to be accorded
the widest scope consistent with the principles and novel features
disclosed herein.
[0506] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the
present invention and scope of the appended claims.
* * * * *