U.S. patent application number 12/856043 was filed with the patent office on 2011-06-16 for methods of reprogramming animal somatic cells.
Invention is credited to Karen B. Chapman, Roy Geoffrey Sargent, Michael D. West.
Application Number | 20110143441 12/856043 |
Document ID | / |
Family ID | 37224047 |
Filed Date | 2011-06-16 |
United States Patent
Application |
20110143441 |
Kind Code |
A1 |
West; Michael D. ; et
al. |
June 16, 2011 |
Methods of Reprogramming Animal Somatic Cells
Abstract
This invention generally relates to methods to obtain mammalian
cells and tissues with patterns of gene expression similar to that
of a developing mammalian embryo or fetus, and the use of such
cells and tissues in the treatment of human disease and age-related
conditions. More particularly, the invention relates to methods for
identifying, expanding in culture, and formulating mammalian
pluripotent stem cells and differentiated cells that differ from
cells in the adult human in their pattern of gene expression, and
therefore offer unique characteristics that provide novel
therapeutic strategies in the treatment of degenerative
disease.
Inventors: |
West; Michael D.; (Mill
Valley, CA) ; Chapman; Karen B.; (Mill Valley,
CA) ; Sargent; Roy Geoffrey; (San Lorenzo,
CA) |
Family ID: |
37224047 |
Appl. No.: |
12/856043 |
Filed: |
August 13, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11989988 |
Mar 8, 2010 |
|
|
|
PCT/US2006/030632 |
Aug 3, 2006 |
|
|
|
12856043 |
|
|
|
|
60705625 |
Aug 3, 2005 |
|
|
|
60729173 |
Oct 20, 2005 |
|
|
|
60818813 |
Jul 5, 2006 |
|
|
|
Current U.S.
Class: |
435/455 ;
435/377 |
Current CPC
Class: |
C12N 5/0606 20130101;
C12N 2501/606 20130101; C12N 2501/603 20130101; C12N 2506/00
20130101; C12N 5/16 20130101; C12N 2501/602 20130101; C12N 2501/72
20130101; C12N 15/873 20130101; C12N 5/0676 20130101; C12Q 2600/158
20130101; C12Q 1/6881 20130101; C12N 2501/605 20130101; A01K
67/0271 20130101; C12N 2506/11 20130101 |
Class at
Publication: |
435/455 ;
435/377 |
International
Class: |
C12N 15/87 20060101
C12N015/87; C12N 5/071 20100101 C12N005/071; C12N 5/078 20100101
C12N005/078; C12N 5/0793 20100101 C12N005/0793 |
Claims
1. A method for reprogramming an animal differentiated somatic cell
to an undifferentiated stem cell, comprising the steps of: (a)
injecting one or more animal differentiated somatic cells into an
oocyte, wherein said oocyte is from a species that is different
from said somatic cell, and allowing the remodeling of the nucleus
of said somatic cell, wherein said remodeling results in (i) the
remodeling of the nuclear envelope of the nucleus of said somatic
cell, wherein one or more components of the nuclear envelope of
said nucleus are removed and wherein one or more components from
said oocyte are added to the nuclear envelope of said nucleus of
said somatic cell, and (ii) the reprogramming of the chromatin of
said somatic cell resulting in the expression of genes of an
undifferentiated cell, wherein said reprogramming of said chromatin
comprises the removal of regulatory factors from said chromatin and
the addition of regulatory factors from said oocyte to said
chromatin; and (b) transferring or fusing the resulting remodeled
nucleus from step (a) into the enucleated cytoplasm of an
undifferentiated embryonic cell to form an undifferentiated stem
cell, and allowing the resulting undifferentiated stem cell to
grow.
2-51. (canceled)
52. A method for in vitro reprogramming a differentiated somatic
cell to an undifferentiated stem cell, comprising contacting the
differentiated somatic cell in culture with OCT3/4 (POU5F1) protein
under conditions in which chromatin remodeling and telomere
elongation occurs such that the somatic cell becomes an
undifferentiated stem cell.
53. The method of claim 52, wherein the differentiated somatic cell
is selected from the group consisting of fibroblasts,
keratinocytes, lymphocytes, monocytes, epithelial cells and
hematopoietic cells.
54. The method of claim 52, wherein the differentiated somatic cell
is a fibroblast.
55. The method of claim 52, wherein the differentiated somatic cell
is permeabilized before being contacted with the OCT3/4 protein in
culture.
56. The method of claim 52, wherein the differentiated somatic cell
is further contacted in culture with one or more proteins selected
from the group consisting of SOX2, NANOG, cMYC, DNMT3B, and
embryonic histones.
57. The method of claim 52, wherein the differentiated somatic cell
is further contacted in culture with one or more proteins selected
from the group consisting of SOX2, NANOG, DNMT3B, CROC4, H2AFX,
HIST1H2AB, HIST1H4J, HMGB2, LEFTB, MYBL2, MYC, MYCN, NANOG, OTX2,
SALL4, TERF1, TERT and ZNF206.
58. The method of claim 52, wherein said undifferentiated stem cell
is a pluripotent cell.
59. The method of claim 58, further comprising differentiating said
pluripotent cell.
60. The method of claim 59, wherein said pluripotent cell is
differentiated into a cell type selected from the group consisting
of: cartilage, bone, skeletal muscle, cardiac muscle, renal,
hepatic, blood, blood forming, vascular precursor, vascular
endothelial, pancreatic beta, neuron, glia, retinal, inner ear
follicle, intestinal, and lung.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
11/989,988, filed Mar. 8, 2010, which is a national stage of
PCT/US2006/030632, filed Aug. 3, 2006, which claims the benefit of
priority from U.S. provisional application No. 60/705,625 filed
Aug. 3, 2005, U.S. provisional application No. 60/729,173, filed
Oct. 20, 2005, and U.S. provisional application No. 60/818,813,
filed Jul. 5, 2006, the disclosures of which are all incorporated
in their entirety by reference.
FIELD OF THE INVENTION
[0002] This invention generally relates to methods of reprogramming
an animal somatic cell from a particular differentiated state to
another state, and the use of such cells and tissues in the
treatment of human diseases and age-related conditions. More
particularly, the invention relates to an improved method utilizing
a three-step process whereby the nuclear envelope of the somatic
cell nucleus is first remodeled to that of an undifferentiated cell
or a germ-line cell prior to the second step of transferring the
remodeled nucleus into the cytoplasm of an oocyte or an
undifferentiated cell. This nuclear remodeling step markedly
enhances the efficiency of cellular reconstitution when the
remodeled nucleus is transferred into embryonic or germ-line
cytoplasm for the purpose of stem cell derivation. In addition, the
removal of components of the nuclear envelope specific for
differentiated cells, such as lamin A, and the reprogramming of
chromatin results in a reactivation of telomerase activity, a
lengthening of telomere length, and mechanisms of homologous
recombination that repair tandemly-repeated DNA sequences. When
pluripotent stem cells are derived by the present invention, they
may be utilized in novel therapeutic strategies in the treatment of
cardiac, neurological, endocrinological, vascular, retinal,
dermatological, muscular-skeletal disorders, and other
diseases.
BACKGROUND OF THE INVENTION
[0003] Advances in stem cell technology, such as the isolation and
use of human embryonic stem (hES) cells, have become an important
new subject of medical research. hES cells have a demonstrated
potential to differentiate into any and all of the cell types in
the human body, including complex tissues. This ability of hES
cells has led to the suggestion that many diseases resulting from
the dysfunction of cells may be amenable to treatment by the
administration of hES-derived cells of various differentiated types
(Thomson et al., Science 282:1145-7, (1998)). Nuclear transfer
studies have demonstrated that it is possible to transform a
somatic differentiated cell back to a totipotent state such as that
of ES or ED cells (Cibelli et al., Nature Biotech 16:642-646,
(1998)). The development of technologies to reprogram somatic cells
back to a totipotent ES cell state such as by nuclear transfer
offers a means to deliver ES-derived somatic cella with a nuclear
genotype of the patient (Lanza et al., Nature Medicine 5:975-977,
(1999)). It is expected that such cells and tissues would not be
rejected, despite the presence of allogeneic mitochondria (Lanza et
al., Nature Biotech 20:689-696, (2002)). Nuclear transfer also
allows the rebuilding of telomere repeat length in cells through
the reactivation of the telomerase catalytic component in the early
embryo (Lanza et al., Science 288:665-669, (2000)). Nevertheless,
there remains a need for improvements in methods to reprogram
animal cells that increase the frequency of successful and complete
reprogramming and reduce the dependence on the availability of
human oocytes.
[0004] Because of the relative difficulty of obtaining large
numbers of human oocytes, there has been considerable interest in
determining whether other germ-line cells, such as cultured ES
cells, or cytoplasm from said cells, could be used to reprogram
somatic cells. Such cells would have an important advantage over
oocytes as a means of inducing reprogramming in that they can be
easily expanded in number in vitro. The restoration of expression
of at least some measured embryonic-specific genes has been
observed in somatic cells following fusion with ES cells (Do and
Scholer, Stem Cells 22:941-949, (2004); Do and Scholer, Reprod.
Fertil. Dev. 17:143-149, (2005)). However, the resulting cells are
hybrids, often with a tetraploid genotype, and therefore not suited
as normal or histocompatible cells for transplant purposes. Indeed,
one of the proposed purposes of generating autologous totipotent
cells is to prevent the rejection of ES-derived cells. Using the
techniques described in these published studies, the BS cells used
to reprogram a patient's cell would therefore likely add alleles
that could generate an immune response leading to rejection.
Nevertheless, the evidence that ES cells can reprogram somatic cell
chromosomes has excited researchers and generated a new field of
research called "fusion biology" (Dennis, Nature 426:490-491,
(2003)).
[0005] Another potential source of cells capable of reprogramming
human somatic cells with a greater ease of availability than human
oocytes are oocytes of animal species. The demonstration of the
restoration of totipotency in somatic cells by nuclear transfer
across species (Lanza et al., Cloning 2:79-90, (2000)) opens the
possibility of identifying animal oocytes that can be easily
obtained for use in reprogramming human cells (Byrne et al., Curr
Biol 13:1206-1213, (2003)). However, likely because of molecular
differences between the species, cross species nuclear transfer,
although possible, is often even more inefficient than same-species
nuclear transfer.
[0006] Among the many molecular alterations that occur following
somatic cell nuclear transfer, some of the more critical
alterations are the reprogramming of the chromatin through
poorly-understood mechanisms in the recipient oocyte and remodeling
of the proteins of the nuclear envelope. The nuclear envelope
includes the inner nuclear membrane (INM) and outer nuclear
membrane (ONM), nuclear pore complexes (NPCs), and nuclear lamina.
The proteins of the nuclear envelope, in particular those proteins
of the lamina, differ between somatic and germ-line cells and play
an important role in regulating the cell cycle, monitoring DNA
damage checkpoint pathways, and regulating cell differentiation. In
particular, the protein subunits of the lamina include the type V
intermediate filament proteins, lamin A/C and B, which form a
meshwork internal to the INM (Foisner, J. Cell Sci. 114:3791-3792,
(2001)). Some of these proteins, such as lamin A/C, play an
important role in regulating chromosomal integrity, DNA damage
checkpoints, and telomere status signaling through their
interactions with the WRN helicase, POT1, Tel1, and Tel2. In
germ-line cells that are telomerase positive, or where telomerase
is utilized, the nuclear matrix lacks lamin A/C or otherwise allows
tandemly-repeated sequences of DNA to be repaired and, in the case
of the telomere, to be lengthened by telomerase. Other proteins
associated with the INM include the family of lamina associated
polypeptides (LAPs) including lamina-associated protein 1 (LAP1, of
which there are at least three isoforms (.alpha., .beta., and
.gamma.)), LAP2 (with at least six isoforms) and emerin (which when
mutated leads to abnormal muscle differentiation and Emery-Dreifuss
muscular dystrophy). Other proteins associated with the INM include
the ring finger binding protein (RFBP), otefin, germ cell-less
(GCL) and nurim. The lamins are known to play an important role in
regulating the function of transcriptional regulators such as the
retinoblastoma protein (pRB) and LBR which in turn can bond
heterochromatin protein 1 (HP1). By way of example of the need to
remodel the nuclear envelope in order to reprogram a differentiated
somatic cell to an undifferentiated state, undifferentiated
germ-line cells generally lack the presence of lamin A, while
germ-line cells contain proteins such as germ cell-less (GCL) and
lamin C2, which are often not expressed in differentiated somatic
cells (Furukawa et al., Exp. Cell Res. 212:426-430, 1994).
Incomplete remodeling of the nuclear envelope would contribute to
the inefficiency or incomplete reprogramming of cells using
existing technologies.
[0007] Therefore, each of the technologies to reprogram human
somatic cells known in the art have their own unique difficulties.
SCNT provides a satisfactory level of reprogramming but is limited
by the number of human oocytes available to researchers.
Cross-species nuclear transfer and cell fusion technologies are not
generally limited in the cells used in reprogramming but are
limited by the degree of successful reprogramming or the robustness
of the growth of the resulting reprogrammed cells. Therefore, there
remains a need for improved technologies to both increase the
frequency and quality of reprogramming of differentiated somatic
cells and of producing reprogrammed cells that are capable of
expansion in vitro in order to obtain a useful number of cells for
research, testing for quality control, and for use in cell therapy.
The present invention combines aspects of several existing
technologies already known in the art in a novel and non-obvious
manner to provide a means of reprogramming differentiated cells as
effectively or more effectively than SCNT and to provide a more
acceptable and cost-effective substitute for oocytes as the vehicle
for reprogramming. The present invention achieves these goals in
part by using cells that are easily and inexpensively obtained in
unlimited quantities and a technology that can be scaled such that
thousands or millions of fusions can be performed simultaneously,
thereby increasingly the probability of a successful final outcome.
Additionally, the present invention provides a technique that
facilitates the reactivation of telomerase and an extension of
telomere length, thereby restoring cell replicative lifespan. The
present invention further provides an assay that allows for the
analysis of what components in undifferentiated and germ-line cells
are critical for nuclear reprogramming. The invention also provides
a procedure that can be automated through robotics to reduce cost
and improve quality control.
SUMMARY OF THE INVENTION
[0008] The present invention provides methods for the reprogramming
of animal somatic cells and methods for the derivation,
formulation, and use of the resulting reprogrammed cells and
engineered tissues in modalities of therapy for the prevention and
treatment of disease. More specifically, the invention provides an
improved means of reprogramming differentiated cells to an
undifferentiated state, extending telomere length and therefore
replicative lifespan, and accordingly producing stem cells and
resulting differentiated cells of many kinds with a nuclear
genotype identical to the genotype of the original differentiated
cell. The present invention may be used to analyze the mechanisms
of nuclear reprogramming and or the production of differentiated
cells for use in research and therapy.
[0009] The methods of this invention represent an improvement over
existing techniques, such as human somatic cell nuclear transfer
(SCNT), used to de-differentiate animal somatic cells into an
embryonic state, thereby producing hES cells. The present invention
provides methods to improve such existing techniques by separating
cellular reprogramming into at least two, or preferably three,
separate steps, utilizing in some of those steps cytoplasmic
components from a donor cell source, wherein the donor source is a
differentiated cell from a species different from the species of
the oocyte. Using a donor cell source from a different species than
the species of the oocyte eases access to reprogramming materials,
the degree of successful reprogramming, and the scale-up of the
process of reprogramming differentiated cells.
[0010] In one embodiment of the invention, somatic differentiated
cells are reprogrammed to an undifferentiated state through a novel
reprogramming technique comprised of the following three steps:
[0011] In the first step, designated the nuclear remodeling step,
the degree of reprogramming of the somatic cell genome is increased
and the problem of access to oocytes of the same species as the
somatic cell is alleviated by the use of any or a combination of
several novel reprogramming procedures. In all of these novel
procedures, the somatic cell nucleus is remodeled to replace the
components of the nuclear envelope with the components of an
undifferentiated cell. Simultaneously, or at a point in time early
enough to prevent the incorporation of somatic cell differentiated
components into the nuclear envelope, the chromatin of said cell is
reprogrammed to express genes of an undifferentiated cell. The
first step is advantageous over current SCNT technology in that
oocytes of the same species as the somatic cell are not required;
further, an improved quality of reprogramming can be achieved.
[0012] In the second step, designated herein as the cellular
reconstitution step, the nucleus, containing the remodeled nuclear
envelope of step one, is either transferred to an enucleated
cytoplasm of an undifferentiated embryonic cell, or is fused with a
cytoplasmic bleb containing a requisite mitotic apparatus which is
capable, together with the transferred nucleus, of producing a
population of undifferentiated stem cells such as ES or ED-like
cells capable of proliferation. The second step has the advantage
over SCNT in that a large number of nuclei or chromosome clumps
remodeled in step one may be simultaneously fused with cytoplasmic
blebs in step two to increase the probability of obtaining
reprogrammed cells capable of successfully proliferating in vitro,
resulting in a large number of cultured reprogrammed cells.
[0013] In the third step, colonies of cells arising from one or a
number of cells resulting from step two are characterized for the
extent of reprogramming and for the normality of the karyotype and
colonies of a high quality are selected. While this third step is
not required to successfully reprogram cells and is not necessary
in some applications of the present invention, such as in analyzing
the molecular mechanisms of reprogramming, for many uses, such as
when reprogramming cells for use in human transplantation, the
inclusion of the third quality control step is preferred. Colonies
of reprogrammed cells that have a normal karyotype but not a
sufficient degree of reprogramming may be recycled by repeating
steps 1-2 or 1-3.
[0014] In another embodiment of the invention, the nucleus is
remodeled in step one by the transfer of one or numerous
permeabilized or nonpermeabilized somatic cells into an oocyte of
another species. The resulting remodeled nucleus or nuclei are then
removed and further processed in steps two and three.
[0015] In another embodiment of the invention, the genome of a
somatic cell is remodeled in step one by condensation to a
chromosome clump through the exposure of isolated somatic cell
nuclei to an extract from mitotic cells, such as metaphase II
oocytes, metaphase germ-line cells such as the EC cell line
NTera-2, or of mitotic somatic cells of the same or different
species. Said chromosome clumps are then further processed in steps
two and three and the previous steps repeated if the cells do not
show an acceptable degree of reprogramming.
[0016] In another embodiment of the invention, the genome of a
somatic cell is remodeled in step one by condensation to a
chromosome clump through the exposure of isolated somatic cell
nuclei to an extract from mitotic cells, such as metaphase II
oocytes, metaphase germ-line cells such as the EC cell line
NTera-2, or of mitotic somatic cells of the same or different
species. Said chromosome clumps are then subsequently encapsulated
in a new nuclear envelope in vitro using extracts from
undifferentiated cells. The resulting remodeled nuclei are then
further processed in steps two and three and the previous steps
repeated if the cells do not show an acceptable degree of
reprogramming. Additionally, the remodeled nuclei and cells may be
used in assays to analyze the mechanisms of reprogramming.
[0017] In another embodiment of the invention, one or more factors
expressed in undifferentiated cells (e.g., EC cells, ES cells,
etc.) are transiently expressed or overexpressed in the
undifferentiated cell extracts or cells of step 1 and/or step 2 or
are added as proteins to said cell extracts. Expression of these
factors may confer characteristics of an undifferentiated cell to
the somatic cell and facilitate reprogramming of the somatic cell.
Such factors include, for example, NANOG, SOX2, DNMT3B, CROC4,
H2AFX, HIST1H2AB, HIST1H4J, HMGB2, LEFTB, MYBL2, MYC, MYON, NANOG,
OCT3/4 (POUSF1), OTX2, SALL4, TERF1, TERT, ZNF206, or any other
factors (such as transcriptional regulators) that confer
characteristics of an undifferentiated cell state. Further, any
number or combinations of the above-mentioned factors may be
used.
[0018] In another embodiment of the invention, the various kinds of
in vitro reprogramming of step one of the present invention are
utilized as an in vitro model of nuclear reprogramming useful in
analyzing the molecular mechanisms of reprogramming. For example,
particular molecular components may be added or deleted from the
extract to determine the role of certain components in
reprogramming.
[0019] In another embodiment of the invention, the various
components determined to play an important role in reprogramming
identified in the above assay or by other means are then
correspondingly incorporated or deleted from the reprogramming
extract to increase the efficiency of reprogramming in the same or
cross species reprogramming protocol. Such molecules include but
are not limited to human protein components, purified RNA,
including miRNA from oocytes, blastomeres, morulae, ICMs, embryonic
disc, ES cells, EG cells, EC cells, or other germ-line cells. The
components may be added or deleted during any of steps 1-3.
Particular components may be deleted by methods such as, for
example, immunoprecipitation.
[0020] In another embodiment of the invention, steps 1-2 are
repeated as step one followed by step two, followed by step one,
followed by step two, until characterization in step three
demonstrates successful reprogramming of the somatic cells.
[0021] In another aspect of the invention, cytoplasts from
undifferentiated or germ-line cells are depleted of mitochondria to
make cell lines into which donor cell mitochondria may be added
before, during, or after step two to result in reprogrammed cells
wherein the mitochondrial genotype as well as the nuclear genotype
is identical to the donor differentiated cell.
[0022] In another embodiment of the invention, undifferentiated
cell factors such as, for example, SOX2, NANOG, DNMT3B, CROC4,
H2AFX, HIST1H2AB, HIST1H4J, HMGB2, LEFTB, MYBL2, MYC, MYCN, NANOG,
OCT3/4 (POU5F1), OTX2, SALL4, TERF1, TERT, ZNF206, are added to the
cytoplasts or cytoplasmic blebs from undifferentiated or germ-line
cells of step 2. In particular embodiments, two, three, four, or
five of the factors are added to the cytoplasts. In other
embodiments, six or more of the factors are added to the
cytoplasts.
[0023] In another aspect of the invention, reprogrammed cells
resulting from the use of steps 1-2 or 1-3 are differentiated in a
variety of in vitro, in vivo, or in ova differentiation conditions
to yield cells of any or a combination of the three primary germ
layers endoderm, mesoderm, or ectoderm, including complex tissues
such as tissues formed in teratomas. In certain embodiments,
differentiated cell types are derived from the reprogrammed cells
of the present invention without the generation of an ES cell line.
For example, differentiated cells may be obtained by culturing
undifferentiated reprogrammed cells in the presence of at least one
differentiation factor and selecting differentiated cells from the
culture. Selection of differentiated cells may be based on
phenotype, such as the expression of certain cell markers present
on differentiated cells, or by functional assays (e.g., the ability
to perform one or more functions of a particular differentiated
cell type). Differentiated cells derived by the methods of the
present invention include, but are not limited to, pancreatic beta
cells and pancreatic precursor cells.
[0024] In another embodiment, the cells reprogrammed according to
the present invention are genetically modified through the
addition, deletion, or modification of their DNA sequence(s). Such
modifications can be made by the random incorporation of an
exogenous vector, by gene targeting, or through the use of
artificial chromosomes.
[0025] In another embodiment of the present invention, the nucleus
being remodeled in step one is modified by the addition of extracts
from cells such as, for example, DT40, known to have a high level
of homologous recombination. The addition of DNA targeting
constructs and the extracts from cells permissive for a high level
of homologous recombination will then yield cells after
reconstitution in step 2 and screening in step 3 that have a
desired genetic modification.
[0026] Another embodiment of the invention is a business model for
commercializing cells produced from the use of said invention. The
business model includes the transfer of human somatic
differentiated cells to regional centers where the reprogramming
steps 1, 2, 1-2 or 1-3 are performed.
[0027] In another embodiment of the invention, the differentiated
somatic cells or the reprogrammed cells resulting from the
application of steps 1, 2, 1-2 or 1-3 are cryopreserved and banked
for future use.
[0028] In another embodiment of the present invention, the
reprogrammed cells resulting from the application of steps 1, 2,
1-2, or 1-3 are shipped to health care facilities where they are
differentiated into medically useful cell types for use in research
and transplantation.
[0029] In another embodiment of the present invention, kits
containing ingredients useful in performing the activities of steps
1, 2, or 3 are shipped to research, biomedical, or health care
facilities where they are used to reprogram differentiated cells
into cell types for use in research and transplantation.
[0030] In another embodiment of the present invention, the
reprogrammed cells resulting from the application of steps 1, 2,
1-2, or 1-3 are shipped to health care facilities after having been
differentiated into a useful composition of cell types.
[0031] Other features and advantages of the invention will be
apparent from the following description and from the claims.
DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 shows the remodeling of multiple somatic cell nuclei
within one oocyte, the subsequent lysing of the oocyte to retrieve
remodeled nuclei, and their fusion with ES cell cytoplasmic blebs
to yield ES cell lines.
[0033] FIG. 2 shows a diagram displaying the modification of
isolated chromosomes, chromatin, or nuclei in vitro. Purified
recombinase or cell free extract is shown as spheres.
DESCRIPTION OF THE INVENTION
Table of Abbreviations
[0034] CT--Chromatin Transfer
[0035] CyT--Cytoplasmic Transfer
[0036] DMAP--Dimethylaminopurine
[0037] SC Cells--Embryonal Carcinoma Cells
[0038] ED Cells--Embryo-derived cells are cells derived from a
zygote, blastomeres, morula or blastocyst-staged mammalian embryo
produced by the fusion of a sperm and egg cell, nuclear transfer,
parthenogenesis, or the reprogramming of chromatin and subsequent
incorporation of the reprogrammed chromatin into a plasma membrane
of an oocyte or blastomere to produce a cell line. The resulting
cell line may be either a differentiated cell line or the cells may
be maintained as undifferentiated ES cells. Therefore ED cells are
inclusive of ES cells and cells derived by directly differentiating
cells from a mammalian preimplantation embryo. hED Cells are human
embryo-derived cells derived from, for example, human
pre-implantation embryos. Human embryo-derived cells may refer to
morula-derived cells, blastocyst-derived cells including those of
the inner cell mass, embryonic shield, or epiblast, or other
totipotent or pluripotent stem cells of the early embryo, including
primitive endoderm, ectoderm, and mesoderm and their derivatives,
but excluding hES cells that have been passaged as cell lines. The
hED cells may be derived from fertilization of an egg cell with
sperm or DNA, nuclear transfer, parthenogenesis, or by means to
generate hES cells with homozygosity in the HLA region.
[0039] ES Cell--Embryonic stem cells derived from a zygote,
blastomeres, morula or blastocyst-staged mammalian embryo produced
by the fusion of a sperm and egg cell, nuclear transfer,
parthenogenesis, or the reprogramming of chromatin and subsequent
incorporation of the reprogrammed chromatin into a plasma membrane
to produce a cell. hES Cells are human embryonic stem cells,
derived from, for example, human pre-implantation embryos. hES
Cells may be derived from the inner cell mass of human blastocysts
or morulae that have been serially passaged as cell lines. The hES
cells may be derived from fertilization of an egg cell with sperm
or DNA, nuclear transfer, parthenogenesis, or by means to generate
hES cells with homozygosity in the HLA region.
[0040] GCL--Germ cell-less
[0041] HSE--Human skin equivalents are mixtures of cells and
biological or synthetic matrices manufactured for testing purposes
or for therapeutic application in promoting wound repair.
[0042] ICM--Inner cell mass of the mammalian blastocyst-stage
embryo.
[0043] INM--Inner nuclear membrane
[0044] MBS--Magnesium buffered saline
[0045] MiRNA--Micro RNA
[0046] NPC--Nuclear Pore Complex
[0047] NT--Nuclear Transfer
[0048] ONM--Outer nuclear membrane
[0049] PEG--Polyethylene glycol
[0050] PS fibroblasts--Pre-scarring fibroblasts are fibroblasts
derived from the skin of early gestational skin or derived from ED
cells that display a prenatal pattern of gene expression with that
they promote the rapid healing of dermal wounds without scar
formation.
[0051] SCNT--Somatic Cell Nuclear Transfer
[0052] SLO--Streptolysin O
[0053] SPF--Specific Pathogen-Free
[0054] The present invention provides improved methods for the
reprogramming of differentiated cells to a more pluripotent state
by utilizing a multiple-step procedure that includes a distinct
nuclear remodeling step and a cellular reconstitution step.
[0055] The term "cellular reconstitution" refers to the transfer of
a nucleus or chromatin to cellular cytoplasm so as to obtain a
functional cell.
[0056] The term "chromatin transfer" (CT) refers to the cellular
reconstitution of condensed chromatin.
[0057] The term "condensed chromatin" refers to DNA not enclosed by
a nuclear envelope. Condensed chromatin my result, for example, by
exposing a nucleus to a mitotic extract such as from an M1 or an
MII oocyte or other mitotic cell extract, by transferring a nucleus
into an M1 or an MII oocyte or other mitotic cell and retrieving
the resulting condensed chromatin following the breakdown of the
nuclear envelope. Condensed chromatin refers to chromosomes that
are in a greater degree of compaction than the degree of compaction
that occurs in any phase of the cell cycle other than
metaphase.
[0058] The term "cytoplasmic bleb" refers to the cytoplasm of a
cell bound by an intact, or permeabilized, but otherwise intact
plasma membrane but lacking a nucleus. It is used interchangeably
and synonymously with the term "enucleate cytoplast" and
"enucleate(d) cytoplasm", unless the term "enucleate cytoplasm" is
explicitly used in the context of an extract in which the plasma
membrane has been removed.
[0059] The term "cytoplasmic transfer" (CyT) refers to any number
of techniques known in the art for juxtaposing the nucleus of a
somatic cell with the cytoplasm of an undifferentiated cell. Such
techniques include, but are not limited to, the direct transfer of
said undifferentiated cytoplasm into the cytoplasm of a
differentiated cell, the permeabilization of a somatic cell to
allow the diffusion of undifferentiated cell cytoplasm into the
somatic cell, or the transfer of the somatic cell nucleus into a
cytoplasmic bleb of an undifferentiated cell.
[0060] The term "cytoplasmic transfer" refers to the juxtaposition
of the genome of a differentiated cell with the cytoplasm of an
undifferentiated cell. This can be accomplished in a number of ways
including the permeabilization of a somatic cell and exposure of
the permeabilized cell(s) to extracts of undifferentiated cells,
the microinjection of the cytoplasm of undifferentiated cells into
the cytoplasm of
[0061] The term "differentiated cell" refers to any cell from any
vertebrate species in the process of differentiating into a somatic
cell lineage or having terminally differentiated into the type of
cell it will be in the adult organism.
[0062] The term "pluripotent stem cells" refers to animal cells
capable of differentiating into more than one differentiated cell
type. Such cells include ES cells, EG cells, EDCs, ED-like cells,
and adult-derived cells including mesenchymal stem cells, neuronal
stem cells, and bone marrow-derived stem cells. Pluripotent stem
cells may be genetically modified or not genetically modified.
Genetically modified cells may include markers such as fluorescent
proteins to facilitate their identification within the egg.
[0063] The term "embryonic stem cells" (ES cells) refers to, for
example, cells derived from the inner cell mass of blastocysts or
morulae that have been serially passaged as cell lines. The ES
cells may be derived from fertilization of an egg cell with sperm
or DNA, nuclear transfer, parthenogenesis or by means to generate
ES cells with homozygosity in the MEC region. hES cells are human
ES Cells.
[0064] The term "fusigenic compound" refers to a compound that
increases the likelihood that a condensed chromatin or nucleus is
fused with and incorporated into a recipient cytoplasmic bleb
resulting in a viable cell capable of subsequent cell division.
Such fusigenic compounds may, by way of nonlimiting example,
increase the affinity of a condensed chromatin or a nucleus with
the plasma membrane. Alternatively, the fusigenic compound may
increase the likelihood of the joining of the lipid bilayer of the
target cytoplasmic bleb with the condensed chromatin, nuclear
envelope of an isolated nucleus, or the plasma membrane of a donor
cell.
[0065] The term "heteroplasmon" refers to a cell resulting from the
fusion of a cell containing a nucleus and cytoplasm with the
cytoplast of another cell.
[0066] The term "human embryo-derived cells" (hEDC) refer to
blastomeres, morula-derived cells, blastocyst-derived cells
including those of the inner cell mass, embryonic shield, or
epiblast, or other totipotent or pluripotent stem cells of the
early embryo, including primitive endoderm, ectoderm, and mesoderm
and their derivatives, but excluding hES cells that have been
passaged as cell lines. The hEDC cells may be derived from
fertilization of an egg cell with sperm or DNA, nuclear transfer,
parthenogenesis, or by means to generate hES cells with
homozygosity in the HLA region.
[0067] The term "human embryo-derived-like cells" (hED-like) refer
to pluripotent stem cells produced by the present invention that
are not cultured so as to retain the characteristics of ES cells,
but like morula-derived cells, blastocyst-derived cells including
those of the inner cell mass, embryonic shield, or epiblast, or
other totipotent or pluripotent stem cells of the early embryo,
including primitive endoderm, ectoderm, and mesoderm and their
derivatives that have not been cultured so as to maintain stable
hES lines, are capable of differentiating into any of the somatic
cell differentiated types. The hED-like cells may be derived with
genetic modifications, including modified so as to lack genes of
the MHC region, to be hemizygous or homozygous in this region.
[0068] The term "nuclear remodeling" refers to the artificial
alteration of the molecular composition of the nuclear lamina or
the chromatin of a cell.
[0069] The term "permeabilization" refers to the modification of
the plasma membrane of a cell such that there is a formation of
pores enlarged or generated in it or a partial or complete removal
of the plasma membrane.
[0070] The term "pluripotent" refers to the characteristic of a
stem cell that said stem cell is capable of differentiating into a
multitude of differentiated cell types.
[0071] The term "totipotent" refers to the characteristic of a stem
cell that said stem cell is capable of differentiating into any
cell type in the body.
[0072] The term "undifferentiated cell" refers to an oocyte, an
undifferentiated cell such as an ES, EG, ICM, ED, EC,
teratocarcinoma cell, blastomere, morula, or germ-line cell.
[0073] Throughout this specification and claims, the word
"comprise," or variations such as "comprises" or "comprising", will
be understood to imply the inclusion of a stated integer or group
of integers but not the exclusion of any other integer or group of
integers.
[0074] Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular.
[0075] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. In case of conflict, the present
specification, including definitions, will control.
[0076] All publications, patents, patent publications and other
references mentioned herein are incorporated by reference in their
entirety.
Step 1: Nuclear Remodeling
[0077] The present invention utilizes a three-step process to
improve the efficiency of reprogramming differentiated cells to an
undifferentiated state.
[0078] In the first step, designated the nuclear remodeling step,
the nuclear envelope and the chromatin of a differentiated cell are
remodeled to more closely resemble the molecular composition of the
nuclear envelope and chromatin, respectively, of an
undifferentiated or a germ-line cell. This remodeling step can be
performed in numerous ways, but the unique and nonobvious feature
of this invention is that this remodeling step is performed in a
separate step from the transfer of the remodeled genome into a
cytoplast; further, the cytoplast is a cytoplast that is readily
available, such as nonhuman animal oocyte cytoplasts or cytoplasts
prepared from embryonal carcinoma (EC) cell lines, including EC
cell lines genetically modified to make extracts and cytoplasts
with improved capacity to reprogram under the present invention and
that will then yield the final proliferating cell types. The
remodeling of the somatic cell nucleus could be performed by
transferring the nucleus into an oocyte of the same species (though
differing in genotype from that somatic cell) or into an oocyte of
a different species such as fish or amphibian (e.g. Xenopus) oocyte
or egg, or in dispersed extracts from cells capable of
reconstituting an undifferentiated or germ-line nuclear envelope
around what was originally a genome from a differentiated cell.
[0079] Separating the nuclear remodeling step from the cellular
reconstitution step solves problems inherent in existing
reprogramming technologies. If nuclear remodeling is performed in
one step separate from the step of cellular reconstitution to
generate cells capable of proliferation, then it is possible to
eliminate a dependence on oocytes of the same species as the
differentiated cell and increase efficiency.
[0080] In the case of SCNT, the oocyte is a relatively large cell
and as a result when a differentiated cell is transferred into a
metaphase II oocyte, the ensuing breakdown of the nuclear envelope
and chromosome condensation, and reassembly of the nuclear envelope
largely from egg cell-derived components, results in the formation
of a remodeled nuclear envelope as well as the impartation of
nuclear regulatory factors, such as transcription factors, useful
in reprogramming the chromatin. If the egg cell is activated at
about the time of nuclear transfer, cell division may also occur,
resulting in an embryo capable of giving rise to a culture of ES
cells. The problems inherent in nuclear transfer, however, are that
despite the relatively large volume of the oocyte and the
incorporation of oocyte cell nuclear components into the
reconstructed cell, nuclear transfer requires micromanipulation,
which is a highly-skilled procedure, as well as serial production
using one cell at a time. Further, nuclear transfer is limited by
the number of oocytes available. In the present invention, these
difficulties are addressed by utilizing alternative nuclear
remodeling technologies that, although requiring more than one step
to obtain intact cells capable of cell division, nevertheless allow
easy access to cytoplasm and are capable of remodeling a nucleus.
Furthermore, these alternative techniques allow the simultaneous
remodeling of many nuclei or genomes.
[0081] One modality for performing the first step of nuclear
remodeling is through the use of fish or amphibian oocytes. The
oocytes or eggs from the species Xenopus laevis have the advantage
that they are widely studied, though most other oocytes or eggs
from vertebrate species will function in a similar manner with the
exception of egg cells with a large amount of yolk. While Xenopus
oocytes are only marginally useful in reprogramming the chromatin
of mammalian differentiated cell nuclei (Byrne et al., Curr Biol
13:1206-1213, (2003)), they can be used to nearly completely
reassemble a germ-line nuclear envelope around a large number of
differentiated somatic cells. Using Xenopus oocytes or Xenopus
oocyte extract, the nuclear envelope and chromatin of the somatic
cell is remodeled in the presence of such undifferentiated or
germ-line proteins through a variety of means, including the
injection of one or more intact or permeabilized differentiated
cells into the oocyte, or the injection of isolated nuclei from
said cells, into an oocyte. Further, other undifferentiated protein
or other factors may be added to the oocytes or oocyte extract, or
oocytes may be modified to express such additional factors that
facilitate nuclear remodeling.
[0082] The differentiated cell that is reprogrammed may be any
differentiated cell of a vertebrate species such as human, canine,
equine, or feline somatic cells including fibroblasts,
keratinocytes, lymphocytes, monocytes, epithelial cells,
hematopoietic cells, or other cells.
[0083] One protocol for remodeling the nuclear envelope of these
differentiated cells using oocytes from another species, such as
Xenopus oocytes, is to inject permeabilized differentiated cells
into interphase Xenopus oocytes, thereby allowing multiple
differentiated cell nuclear envelopes to be remodeled over a period
of several days. Xenopus oocytes from anesthetized mature females
are surgically removed in MBS (magnesium buffered saline) and
inspected for quality as is well-known in the art (Gurdon, Methods
Cell Biol 16:125-139, (1977)). The oocytes are then washed twice in
MBS and stored overnight at 14.degree. C. in MBS. The next day,
good quality stage V or VI oocytes are selected (Dumont, J.
Morphol. 136:153-179, (1972)) and follicular cells are removed
under a dissecting microscope in MBS. After defolliculation, the
oocytes are stored again at 14.degree. C. overnight in MBS with 1
.mu.g/mL gentamycin (Sigma). The next day, oocytes with a healthy
morphology are washed again in MBS and stored in MBS at 14.degree.
C. until use that day. The differentiated cells are then
permeabilized by a permeabilization agent, such as Streptolysin 0
(SLO) or digitonin (Chan & Gurdon, Int. J. Dev. Biol.
40:441-451, (1996); Adam et al., Methods Enzymol. 219:97-110,
(1992)). Approximately 1.times.10.sup.4 differentiated cells are
permeabilized, and suspended in ice-cold lysis buffer
[1.times.Ca.sup.2+-free MBS containing 10 mM BGTA (Gurdon, (1977)].
SLO (Wellcome diagnostics) is added at a final concentration of 0.5
units/mL. The suspension is maintained on ice for 7 minutes, then
four volumes of 1.times.Ca.sup.2+-free MBS containing 1% bovine
serum albumin (Sigma) is added. Aliquots of the cells may then be
removed, diluted 1.times. in 1.times.Ca.sup.2+-free MBS containing
1% bovine serum albumin, and incubated at room temperature for five
minutes to activate permeabilization. The cells are then placed
back on ice for transfer into the Xenopus oocytes. The
permeabilized cells are then transferred into Xenopus oocytes as is
well known in the art (Gurdon, J. Embryol. Exp. Morphol.
36:523-540, (1976). Briefly, oocytes prepared as described above
are placed on agar in high salt MBS (Gurdon, J. Embryol. Exp.
Morphol. 36:523-540, (1976)). The DNA in the egg cells is
inactivated by UV as described (Gurdon, Methods in Cell Biol
16:125-139, 1977) with the exception that the second exposure to
the Hanovia UV source is not performed. Briefly, egg cells are
placed on a glass slide with the animal pole facing up and are
exposed to a Mineralite UV lamp for 1 minute to inactivate the
female germinal vesicle. The permeabilized differentiated cells are
taken up serially into a transplantation pipette 3-5 times the
diameter of the cells and injected into the oocyte, preferably
aiming toward the inactivated pronucleus. The egg containing the
nuclei are incubated for one hour to 7 days and the nuclei are then
removed and cryopreserved or used immediately in step two to
reconstitute cells capable of proliferation.
[0084] Another manner in which the nuclear envelope and chromatin
are remodeled is in cell-free extracts capable of forming nuclear
envelopes from naked DNA or chromatin. Techniques for assembling
nuclear envelopes around DNA or chromatin are known in the art
(Marshall & Wilson, Trends in Cell Biol 7:69-74, (1997)). Such
extracts may be isolated, for example, from Xenopus oocytes as is
well-known in the art (Lohka, Cell Biol Int. Rep. 12:833-848
(1988)). Alternatively, extracts from undifferentiated cells of the
same species may be used such as MII oocytes, oocytes at other
stages of development, ES cells, EC cells, EG cells, or other cells
in a relatively undifferentiated state. EC cells provide the
advantage that they can be easily propagated in large quantities
and human rather than nonhuman EC cells lessen concerns over the
transmission of uncharacterized pathogens. Nonlimiting examples of
such human EC cells include NTera-2, NTera-2 Cl. D1, NCCIT,
Cates-1B, Tera-1, AND TBRA-2 and nonlimiting examples of murine EC
lines include MPRO, BML, F9, F19, D1 ORL UVA, NFPE, NF-1, and
PFHR9. EC lines are readily obtained from sources such as the
American Type Culture Collection and are grown at 37.degree. C. in
monolayer culture in medium characterized for that cell type and
readily available on the internet, (http://stemcells.atcc.org)
(complete medium).
[0085] In certain embodiments of the invention, the genome of the
remodeled nucleus may be modified. Such modifications include, but
are not limited to, the correction of mutations affecting disease,
and other genetic modifications that alleviate disease symptoms or
causes (e.g., in genes that would otherwise be targeted or used in
gene-therapy). The nucleus being remodeled in step one may be
modified by the addition of extracts from cells such as DT40 known
to have a high level of homologous recombination. The addition of
DNA targeting constructs and the extracts from cells permissive for
a high level of homologous recombination will then yield cells
after reconstitution in step 2 and screening in step 3 that have a
desired genetic modification. For example, in certain embodiments,
reprogrammed cells may be used to generate cells or tissues for
cell-based therapies and/or transplantation.
[0086] In other embodiments of the invention, one or more factors
are expressed or overexpressed in the undifferentiated cells (for
example, in EC cells) used to obtain the nuclear remodeling extract
or one or more factors may be added to the undifferentiated cells.
Such factors include, for example, 50.times.2, NANOG, cMYC, OCT4,
DNMT3B, embryonic histones, as well as other factors listed in
Table 1 and their non-human counterparts. Increased expression of
these factors may confer characteristics of an undifferentiated
cell to the somatic cell nuclei and/or remove differentiated cell
factors, thereby improving the frequency of reprogramming.
Accordingly the invention also includes adding, expressing or
over-expressing any other proteins that confer characteristics of
an undifferentiated cell. In addition to the proteins mentioned
above, the present invention includes other factors (such as
transcriptional regulators and regulatory RNA) that induce or
increase the expression of proteins expressed in undifferentiated
cells and that improve the frequency of reprogramming. Further, any
combinations of the above-mentioned factors may be used. For
example, undifferentiated cells of the present invention may be
modified to have increased expression of two, three, four, or more
of any of the factors listed in Table 1. Likewise, two, three,
four, or more of any of the factors listed in Table 1 may be added
to the remodeling extract.
[0087] In other embodiments of the invention, the level of one or
more factors in the undifferentiated cells used to obtain the
nuclear remodeling extract is decreased relative to the levels
found in unmodified cells. Such decreases in the level of a cell
factor may be achieved by known methods, such as, for example, by
use of transcriptional regulators, regulatory RNA, or antibodies
specific for the cell factor.
[0088] In certain embodiments, gene constructs encoding the
proteins listed in Table 1 or other factors, or regulatory proteins
or RNAs that induce expression of these factors, are transfected
into the cells by standard techniques. Such techniques include
viral infection (e.g., lentivirus, papilloma virus, adenovirus,
etc.) and transfection of plasmid and other vectors by chemical
transfection (e.g., via calcium phosphate, lipids, dendrimers,
etc.), electroporation, and microinjection. Alternatively,
constructs that target the factors' endogenous promoters may be
used to induce or increase expression of the factors. Other
embodiments may use artificial chromosomes comprising one or more
of these factors. In additional embodiments, chromosome mediated
gene transfer or cell fusion/microcell fusion are used to introduce
these factors into an undifferentiated cell. In other embodiments,
homologous recombination to modify gene regulatory sequences can
achieve increased expression of one or more of these factors.
[0089] In some embodiments, a transgene encoding the cell factor of
interest may be delivered to the cell by pronuclear microinjection
of DNA that is coated with recombinase. See, for example, Maga et
al., Transgenic Research 12:485-496 (2003). Other known methods to
improve the efficiency of generating transgenic cells may likewise
be useful for purposes of this invention. Alternatively, the
oocytes and/or undifferentiated cell extracts of the present
invention may be obtained from transgenic animals that express
human reprogramming factors (such as the factors listed in Table
1). For example, transgenic animals are generated using expression
constructs carrying one or more of the genes listed in Table 1.
[0090] In some embodiments, the cell factors, or agents that alter
the intracellular levels of the cell factors, may be introduced
into undifferentiated cells by direct intracellular delivery. For
example, the factors may be delivered using protein transduction
domains or cell penetrating peptides, such as, for example,
polyarginine. See Noguchi et al., Acta Med. Okayama 60:1-11 (2006).
Cells into which the factors have been introduced may thus be
useful in the above methods for nuclear remodeling.
[0091] In alternative embodiments, undifferentiated cell factors
(such as the proteins and protein equivalents listed in Table 1),
or agents that affect the levels of the cell factors, are
introduced directly to the nuclear remodeling extract. In certain
embodiments, recombinant proteins are added to the extract to
improve the reprogramming efficiency.
[0092] The differentiated cells that may be effectively
reprogrammed using the present invention include differentiated
cells of any kind from any vertebrate (including human), including
without limitation skin fibroblasts, keratinocytes, mucosal
epithelial cells, or peripheral nucleated blood cells, using the
following steps.
Preparation of Nuclear Remodeling Extract
[0093] Extracts from germ-line cells, such as ES, EG, or EC cells
including but not limited to NTera-2 cells, are prepared in the
prometaphase as is known in the art (Burke & Gerace, Cell 44:
639-652, (1986)). Briefly, after two days and while still in a log
growth state, the medium is replaced with 100 mL of complete medium
containing 2 mM thymidine (which sequesters the cells in S phase).
After 11 hours, the cells are rinsed once with 25 mL of complete
medium, then incubated with 75 mL of complete medium for four
hours, at which point nocodazole is added to a final concentration
of 600 ng/mL from 10,000.times. stock solution in DMSO. After one
hour, loosely-attached cells are removed by mitotic shakeoff (Tobey
et al., J. Cell Physiol. 70:63-68, (1967)). This first collection
of removed cells is discarded, the medium is replaced with 50 mL of
complete medium also containing 600 ng/mL of nocodazole.
Prometaphase cells are then collected by shakeoff 2-2.5 hours
later. The collected cells are then incubated at 37.degree. C. for
45 minutes in 20 mL of complete medium containing 600 ng/mL
nocodazole and 20 .mu.M cytochalasin B. Following this incubation,
the cells are washed twice with ice-cold Dulbecco's PBS, then once
in KHM (78 mM KCl, 50 mM Hepes-KOH [pH 7.0], 4.0 mM MgCl.sub.2, 10
mM EGTA, 8.37 mM CaCl.sub.2, 1 mM DTT, 20 .mu.M cytochlasin B). The
cells are then centrifuged at 1000 g for five minutes, the
supernatant discarded, and the cells are resuspended in the
original volume of KHM. The cells are then homogenized in a dounce
homogenizer on ice with about 25 strokes and progress determined by
microscopic observation. When at least 95% of the cells are
homogenized extracts held on ice for use in envelope reassembly or
cryopreserved as is well known in the art.
Preparation of Condensed Chromatin from Differentiated Cells
[0094] Donor differentiated cells are exposed to conditions that
remove the plasma membrane, resulting in the isolation of nuclei.
These nuclei, in turn, are exposed to cell extracts that result in
nuclear envelope dissolution and chromatin condensation. This
dissolution and condensation results in the release of chromatin
factors such as RNA, nuclear envelope proteins, and transcriptional
regulators such as transcription factors that are deleterious to
the reprogramming process. Differentiated cells are cultured in
appropriate culture medium until they reach confluence.
1.times.10.sup.6 cells are then harvested by trypsinization as is
well known in the art, the trypsin is inactivated, and the cells
are suspended in 50 mL of phosphate buffered saline (PBS), pelleted
by centrifuging the cells at 500 g for 10 minutes at 4.degree. C.,
the PBS is discarded, and the cells are placed in 50.times. the
volume of the pellet in ice-cold PBS, and centrifuged as above.
Following this centrifugation, the supernatant is discarded and the
pellet is resuspended in 50.times. the volume of the pellet of
hypotonic buffer (10 mM HEPES, pH 7.5, 2 mM MgCl.sub.2, 25 mM KCl,
1 mM DTT, 10 .mu.M aprotinin, 10 .mu.M leupeptin, 10 .mu.M
pepstatin A, 10 .mu.M soybean trypsin inhibitor, and 100 .mu.M
PMSF) and again centrifuged at 500 g for 10 min at 4.degree. C. The
supernatant is discarded and 20.times. the volume of the pellet of
hypotonic buffer is added and the cells are carefully resuspended
and incubated on ice for an hour. The cells are then physically
lysed using procedures well-known in the art. Briefly, 5 ml of the
cell suspension is placed in a glass bounce homogenizer and
homogenized with 20 strokes. Cell lysis is monitored
microscopically to observe the point where isolated and yet
undamaged nuclei result. Sucrose is added to make a final
concentration of 250 mM sucrose (1/8 volume of 2 M stock solution
in hypotonic buffer). The solution is carefully mixed by gentle
inversion and then centrifuged at 400 g at 4.degree. C. for 30
minutes. The supernatant is discarded and the nuclei are then
gently resuspended in 20 volumes of nuclear buffer (10 mM
[0095] HEPES, pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 25 mM KCl, 1
mM DTT, 10 .mu.M aprotinin, 10 .mu.M leupeptin, 10 .mu.M pepstatin
A, 10 .mu.M soybean trypsin inhibitor, and 100 .mu.M PMSF). The
nuclei are re-centrifuged as above and resuspended in 2.times. the
volume of the pellet in nuclear buffer. The resulting nuclei may
then be used directly in nuclear remodeling as described below or
cryopreserved for future use.
Preparation of Condensation Extract
[0096] The condensation extract, when added to the isolated
differentiated cell nuclei, will result in nuclear envelope
breakdown and the condensation of chromatin. Because the purpose of
step 1 is to remodel the nuclear components of a somatic
differentiated cell with that of an undifferentiated cell, the
condensation extract used is from undifferentiated cells which may
or may not be also be the cells used to derive the extract for
nuclear envelope reconstitution above. This results in a dilution
of the components from the differentiated cell in extracts which
contain the corresponding components desirable in reprogramming
cells to an undifferentiated state. Germ-line cells such as ES, EG,
or EC cells such as NTera-2 cl. D1 cells are easily obtained from
sources such as the American Type Culture Collection and are grown
at 37.degree. C. in monolayer culture in appropriate medium
(complete medium). While in a log growth state, the cells are
plated at 5.times.10.sup.6 cells per sq cm tissue culture flask in
200 mL of complete medium. Methods of obtaining extracts capable of
inducing nuclear envelope breakdown and chromosome condensation are
well known in the art (Collas et al., J. Cell Biol. 147:1167-1180,
(1999)). Briefly, the germ-line cells in log growth as described
above are synchronized in mitosis by incubation in 1 .mu.g/ml
nocodazole for 20 hours. The cells that are in the mitotic phase of
the cell cycle are detached by mitotic shakeoff. The harvested
detached cells are centrifuged at 500 g for 10 minutes at 4.degree.
C. Cells are resuspended in 50 ml of cold PBS, and centrifuged at
500 g for an additional 10 min. at 4.degree. C. This PBS washing
step is repeated once more. The cell pellet is then resuspended in
20 volumes of ice-cold cell lysis buffer (20 mM HEPES, pH 8.2, 5 mM
MgCl.sub.2, 10 mM EDTA, 1 mM DTT, 10 .mu.M aprotinin, 10 .mu.M
leupeptin, 10 .mu.M pepstatin A, 10 .mu.M soybean trypsin
inhibitor, 100 .mu.M PMSF, and 20 .mu.g/ml cytochalasin B, and the
cells are centrifuged at 800 g for 10 minutes at 4.degree. C. The
supernatant is discarded, and the cell pellet is carefully
resuspended in one volume of cell lysis buffer. The cells are
placed on ice for one hour then lysed with a Dounce homogenizer.
Progress is monitored by microscopic analysis until over 90% of
cells and cell nuclei are lysed. The resulting lysate is
centrifuged at 15,000 g for 15 minutes at 4.degree. C. The tubes
are then removed and immediately placed on ice. The supernatant is
gently removed using a small caliber pipette tip, and the
supernatant from several tubes is pooled on ice. If not used
immediately, the extracts are immediately flash-frozen on liquid
nitrogen and stored at -80.degree. C. until use. The cell extract
is then placed in an ultracentrifuge tube and centrifuged at
200,000 g for three hours at 4.degree. C. to sediment nuclear
membrane vesicles. The supernatant is then gently removed and
placed in a tube on ice and used immediately to prepare condensed
chromatin or cryopreserved as described above.
Methods of Use of Condensation Extract
[0097] If beginning with a frozen aliquot on condensation extract,
the frozen extract is thawed on ice. Then an ATP-generating system
is added to the extract such that the final concentrations are 1 mM
ATP, 10 mM creatine phosphate, and 25 .mu.g/ml creatine kinase. The
nuclei isolated from the differentiated cells as described above
are then added to the extract at 2,000 nuclei per 10 .mu.l of
extract, mixed gently, the incubated in a 37.degree. C. water bath.
The tube is removed occasionally to gently resuspend the cells by
tapping on the tube. Extracts and cell sources vary in times for
nuclear envelope breakdown and chromosome condensation. The
progress is therefore monitored by periodic monitoring samples
microscopically. When the majority of cells have lost their nuclear
envelope and there is evidence of the beginning of chromosome
condensation, the extract containing the condensing chromosome
masses is placed in a centrifuge tube with an equal volume of 1 M
sucrose solution in nuclear buffer. The chromatin masses are
sedimented by centrifugation at 1,000 g for 20 minutes at 4.degree.
C. The supernatant is discarded, and the chromatin masses are
gently resuspended in nuclear remodeling extract derived above. The
sample is then incubated in a water bath at 33.degree. C. for up to
two hours and periodically monitored microscopically for formation
of remodeled nuclear envelopes around the condensed and remodeled
chromatin as described (Burke & Gerace, Cell 44:639-652,
(1986). Once a large percentage of chromatin has been encapsulated
in nuclear envelopes, the remodeled nuclei may be used in cellular
reconstitution using any of the techniques described below in step
2.
Step 2-Cellular Reconstitution
[0098] Step 2, also referred to as "cellular reconstitution" in the
present invention, is carried out using nuclei or chromatin
remodeled by any of the techniques described in the present
invention, such as in Examples 1 and 2 or combinations of the
techniques described in Examples 1 and 2 as described more fully in
the present invention.
[0099] One manner of performing step 2 using nuclei remodeled in
step 1 of the present invention is to fuse the remodeled nuclei
with enucleated cytoplasts of germ-line cells such as blastomeres,
morula cells, inner cell mass cells, ES cells (including hES cells,
EG cells, and EC cells) as is known in the art (Do & Scholer,
Stem Cells 22:941-949 (2004)). Briefly, the human ES Cells are
cultured under standard conditions (Klimanskaya et al. Lancet 365:
4997 (1995)). The cytoplasmic volume of the cells is increased by
adding 10 .mu.M cytochalasin B for 20 hours prior to manipulation.
Cytoplasts are prepared by centrifuging trypsinized cells through a
Ficoll density gradient using a stock solution of autoclaved 50%
(wt/vol) Ficoll-400 solution in water. The stock Ficoll 400
solution is diluted in DMEM and with a final concentration of 10
.mu.M cytochalasin B. The cells are centrifuged through a gradient
of 30%, 25%, 22%, 18%, and 15% Ficoll-400 solution at 36.degree. C.
Layered on top is 0.5 mL of 12.5% Ficoll-400 solution with
10.times.10.sup.6 ES cells. The cells are centrifuged at 40,000 rpm
at 36.degree. C. in an MLS-50 rotor for 30 minutes. The cytoplasts
are collected from the 15% and 18% gradient regions marked on the
tubes, rinsed in PBS, and mixed on a 1:1 ratio with remodeled
nuclei from step one of the present invention or cryopreserved.
Fusion of the cytoplasts with the nuclei is performed using a
number of techniques known in the art, including polyethylene
glycol (see Pontecorvo "Polyethylene Glycol (PEG) in the Production
of Mammalian Somatic Cell Hybrids" Cytogenet Cell Genet.
16(1-5):399-400 (1976)), the direct injection of nuclei, sedai
viral-mediated fusion, or other techniques known in the art. The
cytoplasts and the nuclei are placed briefly in 1 mL of prewarmed
50% polyethylene glycol 1500 (Roche) for one minute. 20 mL of DMEM
was then added over a five minute period to slowly remove the
polyethylene glycol. The cells are centrifuged once at 130 g for
five minutes and then taken back up in 50 .mu.L of ES cell culture
medium and placed beneath a feeder layer of fibroblasts under
conditions to promote the outgrowth of an ES cell colony.
[0100] Another technique for performing step 2, also referred to as
"cellular reconstitution" in the present invention, is to fuse the
remodeled nuclei with anucleate cytoplasmic blebs of germ-line
cells, such as hES cells, attached to a physical substrate as is
well known in the art (Wright & Hayflick, Exp. Cell Res.
96:113-121, (1975); & Wright & Hayflick, Proc. Natl. Acad.
Sci., USA, 72:1812-1816, (1975). Briefly, the cytoplasmic volume of
the germ-line cells is increased by adding 10 .mu.M cytochalasin B
for 20 hours prior to manipulation. The cells are then trypsinized
and replated on sterile 18 mm coverslips, cylinders, or other
physical substrate coated with material promoting attachment. The
cells are plated at a density such that after an overnight
incubation at 37.degree. C. and one gentle wash with medium, the
cells cover a portion, preferably about 90%, of the surface area of
the coverslip or other substrate. The substrates are then placed in
a centrifuge tube in a position such that centrifugation will
result in the removal of the nuclei from the cytoplast containing 8
mL of 10% Ficoll-400 solution and centrifuged at 20,000 g at
36.degree. C. for 60 minutes. Remodeled nuclei resulting from step
one of the present invention are then spread onto the coverslip or
substrate with a density of at least that of the cytoplasts,
preferable at least five times the density of the cytoplasts.
Fusion of the cytoplasts with the nuclei is performed using
polyethylene glycol (see Pontecorvo "Polyethylene Glycol (PEG) in
the Production of Mammalian Somatic Cell Hybrids" Cytogenet Cell
Genet. 16(1-5):399-400 (1976). Briefly, in 1 mL of prewarmed 50%
polyethylene glycol 1500 (Roche) in culture medium is placed over
the coverslip or substrate for one minute. 20 mL of culture medium
is then added drip-wise over a five minute period to slowly remove
the polyethylene glycol. The entire media is then aspirated and
replaced with culture medium. Techniques other than centrifugation
such as vibration or physical removal of the nuclei using a
micropipette may also be used.
[0101] In certain embodiments of the present invention, the
undifferentiated cells used in step 2 may first be manipulated to
express or overexpress factors such as, for example, SOX2, NANOG,
cMYC, OCT4, DNMT3B, any other factors listed in Table 1 and their
non-human homologues, and/or other factors (e.g., regulatory RNA or
constructs targeting the promoters of the genes listed in Table 1
and their non-human homologues) that confer undifferentiated cell
behavior and facilitate reprogramming. Constructs encoding such
factors may be transfected into undifferentiated cells, such as
germ-line cells (e.g., blastomeres, morula cells, inner cell mass
cells, ES cells, including hES cells, EG cells, or EC cells), by
standard techniques known in the art. Examples of manipulating
undifferentiated cells to express cellular factors are described
above in Step 1. In alternative embodiments, such factors are
introduced into the undifferentiated cells by injection or other
methods. Examples of such methods to manipulate undifferentiated
cells are likewise described above in Step 1.
[0102] In alternative embodiments of the present invention, nuclear
envelope reconstitution occurs following homologous recombination
reactions that have modified target chromosomes. Thus, in one
embodiment, as an optional step following nuclear envelope
breakdown and chromatin condensation but before nuclear envelope
reconstitution, DT40 extracts, or other recombination-proficient
extracts or protein preparations, are added to the condensed
chromosomes along with DNA targeting constructs such that
recombination will result in the replacement of one or more genomic
DNA sequences with the sequence(s) provided in the constructs.
Exemplary embodiments of such methods are provided in Examples 3,
4, 5, and 6.
Step 3-Analysis of the Karyotype and Extent of Reprogramming
[0103] Cells reconstituted following steps 1 and 2 of the present
invention can be characterized to determine the pattern of gene
expression and whether the reprogrammed cells display a pattern of
gene expression similar to the expression pattern expected of
undifferentiated cells such as ES cell lines using techniques well
known in the art including transcriptomics (Klimanskaya et al.,
Cloning and Stem Cells, 6(3): 217-245 (2004)). Karyotypic analysis
may be performed by means of chromosome spreads from mitotic cells,
spectral karyotyping, assays of telomere length, total genomic
hybridization, or other techniques well known in the art. In the
case where the karyotype is normal, but telomere length or the
extent of reprogramming is not complete, the cells may be used as
nuclear donors and steps 1 and 2 repeated any number of times.
[0104] For example, the gene expression pattern of the reprogrammed
cells may be compared to the gene expression pattern of embryonic
stem cells or other undifferentiated cells. If the gene expression
patterns are not similar, then the reprogrammed cell may be used in
subsequent reprogramming steps until its gene expression is similar
to the expression pattern of an undifferentiated cell (e.g.,
embryonic stem cell). The undifferentiated or embryonic stem cell
to which the reprogrammed cell is compared may be from the same
species as the donor differentiated somatic cell; alternatively,
the undifferentiated or embryonic stem cell to which the
reprogrammed cell is compared may be from the same species as the
cytoplast or cytoplasmic bleb used in step 2. In some embodiments,
a similarity in gene expression pattern exists between a
reprogrammed cell and an undifferentiated cell (e.g., embryonic
stem cell) if certain genes expressed in an undifferentiated cell
are also expressed in the reprogrammed cell. For example, certain
genes (e.g., telomerase) that are typically undetectable in
differentiated somatic cells may be used to monitor the extent of
reprogramming. Likewise, for certain genes, the absence of
expression may be used to assess the extent of reprogramming. In
certain embodiments, a cell may be considered reprogrammed if it
expresses (1) E-cadherin (for human cells, CDH1; Accession No.
NM.sub.--004360.2) mRNA at levels of at least 5% of the expression
level of the housekeeping gene GAPD (for human cells,
NM.sub.--002046.2) (data not shown); (2) detectable telomerase
reverse transcriptase mRNA or exhibits telomerase activity as
assessed by the TRAP assay (TRAPeze); and (2) LIN28
(NM.sub.--024674.3; or its non-human equivalent for non-human
cells) at levels of at least 5% of the housekeeping gene GAPD (for
human cells, NM.sub.--002046.2) (data not shown).
[0105] Other examples of the ways the different means of performing
steps 1 and 2 of the present invention can be combined include
permeabilizing somatic cells by SLO, resealing the cells, and
isolating the resulting partially-remodeled nuclei and then using
the nuclei in the cellular reconstitution of step two. Also, the
remodeled chromatin obtained by isolating differentiated cell
nuclei, then exposing the nuclei to extracts from cells in the
mitotic phase of the cell cycle to cause nuclear envelope breakdown
and chromatin condensation, may then be transferred into the
cytoplast of an ES cell, EC cell, or EG cell without reforming the
nuclear envelope prior to cellular reconstitution. In addition, the
somatic differentiated cell may be permeabilized as described above
and exposed to extracts from oocytes or germ-line cells. The
condensed chromatin from such cells may then be obtained, and then
that chromatin may be fused with the recipient cytoplasts to yield
reprogrammed cells. The fusion of chromatin with the cytoplasts is
achieved by microinjection or is aided by fusigenic compounds as is
known in the art (see, for example, U.S. Pat. Nos. 4,994,384 and
5,945,577). The fusigenic reagents include, but are not limited to,
polyethylene glycol (PEG), lipophilic compounds such as
Lipofectin.RTM., Lipofectamin.RTM., DOTAP.RTM., DOSPA.RTM., or
DOPE.RTM.. For insertion of the chromatin into the cytoplasts, the
coated chromatin is placed next to the cytoplast membrane and the
complexes are maintained at a temperature of 20-30.degree. C. and
monitored using a microscope. Once fusion has occurred, the medium
is replaced with culture medium for the cultivation of
undifferentiated cells and in culture conditions that promote the
growth of said undifferentiated cells.
[0106] The cellular factors and methods of use listed herein may be
used in alternative reprogramming techniques, such as in the
methods disclosed by Collas and Robl, U.S. patent application Ser.
No. 10/910,156, which is incorporated herein by reference in its
entirety. The factors may, for example, be added to media (or
alternatively expressed in cells used to obtain extract media) used
to incubate a nucleus or chromatin mass from a donor cell under
conditions that allow nuclear or cytoplasmic components from an
undifferentiated cell to be added to the donor nucleus or chromatin
mass.
[0107] The in vitro remodeling of somatic cell-derived DNA in step
one of the present invention is utilized as a model of
reprogramming of a somatic cell and an assay useful in analyzing
the molecular mechanisms of reprogramming. The selective addition,
alteration, removal, or sequestration of particular molecular
components, and the subsequent scoring of the extent of
reprogramming or the extent of activation of telomerase and
extension of telomere length allow the characterization of the role
of particular molecules in the reprogramming that occurs during
SCNT. The critical molecules characterized in this application of
the present invention are then used by their corresponding addition
or deletion (e.g., by their addition if they facilitate
reprogramming, or by their deletion if they inhibit reprogramming).
Deletion can be achieved by, for instance, immune depletion, in
oocytes or reprogramming extracts used in step one. Specific
molecular alterations can be introduced by techniques well known in
the art, including but not limited to, the addition of protein
components, the removal of protein components such as by
immunoprecipitation, the addition of other cellular components such
as lipids, ions, DNA, or RNA. RNA may be prepared from oocytes,
blastomeres, morula cells, ICM cells, ED cells or germ-line cells
such as ES, EG, or EC cells. Total or fractions of the RNA such as
microRNA are prepared as is well known in the art. This "germ-line
RNA" is then introduced into the permeabilized cells of Example 1
at the point of incubating the cells at room temperature in order
to allow the RNA to diffuse into the cells and improve the
reprogramming of the somatic cells to an embryonic state once
transplanted into the oocyte.
[0108] A common feature of the present invention is that,
regardless of which techniques are used to remodel the nuclear
envelope and chromatin of a differentiated cell, at least two, and
in some embodiments three, steps are used: one step wherein the
chromatin and/or nuclear envelope are remodeled, a second step
wherein the remodeled chromatin and/or nuclear envelope are
reconstituted into a cytoplast to make a cell capable of cell
division, and a third step wherein the resulting proliferating
reprogrammed cells are analyzed to determine the degree of
reprogramming and karyotype. If there is not a sufficient degree of
reprogramming, the cells are cycled back to step one.
[0109] Somatic cells reprogrammed as described herein may be used
to generate ES cells or ES cell lines including, but not limited to
human ES cell lines. Since isolated human ES cells have a poor
efficiency in generating cell lines, the reprogrammed cells of the
present invention may be aggregated together to facilitate the
generation of stable ES cell lines. Such aggregation may include
plating the cells at high density, placing the cells in a
depression in the culture dish such that gravity brings the cells
into close proximity, or the cells can be co-cultured with feeder
cells or with existing ES cell lines.
[0110] Human embryonic cells, e.g., human ES cells may be cultured
on feeder cells, e.g., mouse embryonic fibroblasts, or human feeder
cells such as fibroblasts (e.g., human foreskin fibroblasts, human
skin fibroblasts, human endometrial fibroblasts, human oviductal
fibroblasts) and placental cells. In one embodiment, the human
feeder cells may be autologous feeder cells derived from the same
culture of reprogrammed cells by direct differentiation and the
clonal isolation of cells useful in ES cell derivation. The human
embryonic cells are grown in ES cell medium or any medium that
supports growth of the embryonic cells, e.g., Knockout DMEM
(Invitrogen Cat # 10829-018).
[0111] Alternatively, the reprogrammed cells obtained from the
methods of the present invention may be co-cultured in
juxtaposition with exiting ES cell lines. Exemplary human embryonic
cells include, but are not limited to, embryonic stem cells, such
as from already established lines, embryo carcinoma cells, murine
embryonic fibroblasts, other embryo-like cells, cells of embryonic
origin or cells derived from embryos, many of which are known in
the art and available from the American Type Culture Collection,
Manassas, Va. 20110-2209, USA, and other sources.
[0112] The embryonic cells may be added directly to the cultured
reprogrammed cells or may be grown in close proximity to, but not
in direct contact with, the cultured reprogrammed cells. The method
comprises the step of directly or indirectly contacting the
cultured reprogrammed cells with embryonic cells. Alternatively,
the culture of reprogrammed cells and the culture of, embryonic
cells are indirectly connected or merged. This can be achieved by
any method known in the art including, for example, using light
mineral oil such as Cooper Surgical ACT# ART4008, paraffin oil or
Squibb's oil. The connections can be made by using a glass
capillary or similar device. Such indirect connections between the
cultured reprogrammed cells and the embryonic cells allows gradual
mixing of the embryo medium (in which the reprogrammed cells are
cultured) and the ES cell medium (in which the human embryonic
cells are grown).
[0113] In another embodiment, the reprogrammed cells may be
co-cultured with a human embryo. For example, the reprogrammed
cells are co-cultured with the embryo in a microdroplet culture
system or other culture system known in the art, but which does not
permit cell-cell contact but could permit cell-secreted factor
and/or cell-matrix contact. The volume of the microdrop may be
reduced, e.g., from 50 microliters to about 5 microliters to
intensify the signal. In another embodiment the embryonic cells may
be from a species other than human, e.g., non-human primate or
mouse.
[0114] After about 3-4 days, the reprogrammed cells exhibit
properties of SS cells. While not wishing to be bound by any
particular theory, it is believed that over a period of days or
weeks the cultured reprogrammed cells exhibit facilitated ES cell
growth perhaps as a result of factors secreted by the human
embryonic cells or by the extracellular matrix. The above-described
methods for producing ES cells are described in application
PCT/US05/39776, U.S. Ser. No. 11/267,555 and 60/831,698, which are
incorporated herein in their entirety. Properties of ES cells or an
ES cell line may include, without limitation, the expression of
telomerase and/or telomerase activity, and the expression of one or
more known ES cell markers.
[0115] In certain embodiments, the reprogrammed cell culture
conditions may include contacting the cells with factors that can
inhibit or otherwise potentiate the differentiation of the cells,
e.g., prevent the differentiation of the cells into non-ES cells,
trophectoderm or other cell types. Such conditions can include
contacting the cultured cells with heparin or introducing
reprogramming factors into the cells or extracts as described
herein. In yet another embodiment, expression of cdx-2 is prevented
by any means known in the art including, without limitation,
introducing CDX-2 RNAi into the reprogrammed cells, thereby
inhibiting differentiation of the reprogrammed cells into TS cells,
thereby insuring that said cells could not lead to a competent
embryo.
[0116] In another embodiment of the present invention, the
reprogrammed cells resulting from steps 1 and 2 of the methods of
this invention are directly used to produce differentiated progeny
without the production of an ES cell line. Thus, in one aspect, the
present invention provides a method for producing differentiated
progenitor cells, comprising: [0117] (i) obtaining reprogrammed
cells using steps 1-2 or 1-3 of the methods of this invention; and
[0118] (ii) inducing differentiation of the reprogrammed cells to
produce differentiated progenitor cells without producing an
embryonic stem cell line. The differentiated progenitor cells can
be used to derive cells, tissues and/or organs which are
advantageously used in the area of cell, tissue, and/or organ
transplantation which include all of the cells and applications
described herein for ES-derived cells and tissues.
[0119] In the past, long-term culture of inner cell mass cells was
used to produce embryonic stem cell lines. Subsequently, the
embryonic stem cells were cultured and conditionally
genetically-modified, and induced to differentiate in order to
produce cells to make cells for therapy. Co-owned pending U.S.
pending application 2005/0265976A1 describes a method of producing
differentiated progenitor cells from inner cell mass cells or
morula-derived cells by directly inducing the differentiation of
those cells without producing an embryonic stem cell line. The
application also describes the use of said differentiated cells,
tissues, and organs in transplantation therapy. In the method of
the present invention, reprogrammed cells derived from steps 1-2 or
1-3 as described herein are induced to directly differentiate into
differentiated progenitor cells which are then used for cell
therapy and for the generation of cells, tissues, and organs for
transplantation. If desired, genetic modifications can be
introduced, for example, into somatic cells prior to reprogramming
or into the chromatin in the extracts as described herein. Thus,
the differentiated progenitor cells of the present invention do not
possess the pluripotency of an embryonic stem cell, or an embryonic
germ cell, and are, in essence, tissue-specific partially or fully
differentiated cells. These differentiated progenitor cells may
give rise to cells from any of three embryonic germ layers, i.e.,
endoderm, mesoderm, and ectoderm. For example, the differentiated
progenitor cells may differentiate into bone, cartilage, smooth
muscle, dermis with a prenatal pattern of gene expression and
capable of promoting scarless wound repair, and hematopoietic or
hemangioblast cells (mesoderm); definitive endoderm, liver,
primitive gut, pancreatic beta cells, progenitors of pancreatic
beta cells, and respiratory epithelium (endoderm); or neurons,
glial cells, hair follicles, or eye cells including retinal neurons
and retinal pigment epithelium using techniques known in the art,
or using techniques described in the pending applications
PCT/US2006/013573 filed Apr. 11, 2006, and U.S. Application No.
60/811,908, filed Jun. 7, 2006, which are both incorporated in
their entirety by reference.
[0120] One advantage of the methods of the present invention is
that the cells obtained by steps 1-2 or steps 1-3 can be
differentiated without prior purification or establishment of a
cell line. The cells obtained by the methods disclosed herein can
be differentiated without the selection or purification of the
cells. Accordingly in certain embodiments, a heterogeneous
population of cells comprising reprogrammed cells are
differentiated into a desired cell type. In one embodiment, a
mixture cells obtained from steps 1-2 as described herein are
exposed to one or more differentiation factors and cultured in
vitro. Thus in certain embodiments, there is no need to purify the
reprogrammed cells or to establish an ES or other cell line before
differentiation. In one embodiment, a heterogeneous population of
cells comprising reprogrammed cells is permeabilized to facilitate
access to differentiation factors and subsequent
differentiation.
[0121] Furthermore, it is not necessary for the differentiated
progenitor cells of the present invention to express the catalytic
component of telomerase (TERT) and be immortal, or that the
progenitor cells express cell surface markers found on embryonic
stem cells such as the cell surface markers characteristic of
primate embryonic stem cells: positive for SSEA-3, SSEA-4,
TRA-1-60, TRA-1-81, alkaline phosphatase activity, and negative for
SSEA-1. Moreover, the differentiated progenitor cells of the
present invention may be distinct from embryoid bodies, i.e.,
embryoid bodies are derived from embryonic stem cells whereas the
differentiated stem cells of the present invention may be derived
from reprogrammed cells without the production of ES cell
lines.
Applications
[0122] The cells resulting from steps 1 and 2 of the methods of
this invention are plated in conditions that promote the growth of
ES cells, such as hES cells, as is well known in the art. Briefly,
the cells may be left on the substrate in which the enucleated
cytoplasts are prepared, or they may be trypsinized and centrifuged
at 700.times.g for 3 minutes and taken up into a sterile Pasteur
pipette and placed under a feeder monolayer to concentrate and to
co-localize the cells. The cells may be co-cultured with other
vigorously-growing ES cell lines that can be easily removed by
means such as suicide induction after encouraging the growth of the
reprogrammed stem cells. The reprogrammed cells may also be
concentrated into a small surface area of the growth surface by
plating in a small cloning cylinder as well as be cultured by other
techniques well known in the art.
[0123] In another aspect of the invention, the method comprises the
utilization of cells derived from the reprogrammed cells of the
present invention in research and in therapy. Such reprogrammed
pluripotent or totipotent cells may be differentiated into any of
the cells in the body including, without limitation, skin,
cartilage, bone skeletal muscle, cardiac muscle, renal, hepatic,
blood and blood forming, vascular precursor and vascular
endothelial, pancreatic beta, neurons, glia, retinal, inner ear
follicle, intestinal, lung, cells.
[0124] In a particular embodiment, the reprogrammed cells may be
differentiated into cella with a dermatological prenatal pattern of
gene expression that is highly elastogenic or capable of
regeneration without causing scar formation. Dermal fibroblasts of
mammalian fetal skin, especially corresponding to areas where the
integument benefits from a high level of elasticity, such as in
regions surrounding the joints, are responsible for synthesizing de
novo the intricate architecture of elastic fibrils that function
for many years without turnover. In addition, early embryonic skin
is capable of regenerating without scar formation. Cells from this
point in embryonic development made from the reprogrammed cells of
the present invention are useful in promoting scarless regeneration
of the skin including forming normal elastin architecture. This is
particularly useful in treating the symptoms of the course of
normal human aging, or in actinic skin damage, where there can be a
profound elastolysis of the akin resulting in an aged appearance
including sagging and wrinkling of the akin.
[0125] In another embodiment of the invention, the reprogrammed
cells are exposed to inducers of differentiation to yield other
therapeutically-useful cells such as retinal pigment epithelium,
definitive endoderm, pancreatic beta cella and precursors to
pancreatic beta cells, hematopoietic precursors and hemangioblastic
progenitors, neurons, respiratory cells, muscle progenitors,
cartilage and bone-forming cells, cells of the inner ear, neural
crest cells and their derivatives, gastrointestinal cells, liver
cells, kidney cells, smooth and cardiac muscle cells, dermal
progenitors including those with a prenatal pattern of gene
expression useful in promoting scarless wound repair, as well as
many other useful cell types of the endoderm, mesoderm, and
endoderm. Such inducers include but are not limited to: cytokines
such as interleukin-alpha A, interferon-alpha A/D, interferon-beta,
interferon-gamma, interferon-gamma-inducible protein-10,
interleukin-1-17, keratinocyte growth factor, leptin, leukemia
inhibitory factor, macrophage colony-stimulating factor, and
macrophage inflammatory protein-1 alpha, 1-beta, 2, 3 alpha, 3
beta, and monocyte chemotactic protein 1-3, 6kine, activin A,
amphiregulin, angiogenin, 3-endothelial cell growth factor, beta
cellulin, brain-derived neurotrophic factor, C10, cardiotrophin-1,
ciliary neurotrophic factor, cytokine-induced neutrophil
chemoattractant-1, eotaxin, epidermal growth factor, epithelial
neutrophil activating peptide-78, erythropoietin, estrogen
receptor-alpha, estrogen receptor-beta, fibroblast growth factor
(acidic and basic), heparin, FLT-3/FLK-2 ligand, glial cell
line-derived neurotrophic factor, Gly-His-Lys, granulocyte colony
stimulating factor, granulocytemacrophage colony stimulating
factor, GRO-alpha/MGSA, GRO-beta, GRO-gamma, HCC-1, heparin-binding
epidermal growth factor, hepatocyte growth factor, heregulin-alpha,
insulin, insulin growth factor binding protein-1, insulin-like
growth factor binding protein-1, insulin-like growth factor,
insulin-like growth factor II, nerve growth factor,
neurotophin-3,4, oncostatin M, placenta growth factor,
pleiotrophin, rantes, stem cell factor, stromal cell-derived factor
18, thromopoietin, transforming growth factor-(alpha,
beta1,2,3,4,5), tumor necrosis factor (alpha and beta), vascular
endothelial growth factors, and bone morphogenic proteins, enzymes
that alter the expression of hormones and hormone antagonists such
as 17B-estradiol, adrenocorticotropic hormone, adrenomedullin,
alpha-melanocyte stimulating hormone, chorionic gonadotropin,
corticosteroid-binding globulin, corticosterone, dexamethasone,
estriol, follicle stimulating hormone, gastrin 1, glucagons,
gonadotropin, L-3,3',5'-triiodothyronine, leutinizing hormone,
L-thyroxine, melatonin, MZ-4, oxytocin, parathyroid hormone,
PEC-60, pituitary growth hormone, progesterone, prolactin,
secretin, sex hormone binding globulin, thyroid stimulating
hormone, thyrotropin releasing factor, thyroxin-binding globulin,
and vasopressin, extracellular matrix components such as
fibronectin, proteolytic fragments of fibronectin, laminin,
tenascin, thrombospondin, and proteoglycans such as aggrecan,
heparan sulphate proteoglycan, chontroitin sulphate proteoglycan,
and syndecan. Other inducers include cells or components derived
from cells from defined tissues used to provide inductive signals
to the differentiating cells derived from the reprogrammed cells of
the present invention. Such inducer cells may derive from human,
nonhuman mammal, or avian, such as specific pathogen-free (SPF)
embryonic or adult cells.
[0126] Differentiated progeny may also be derived from reprogrammed
ES cell lines or directly differentiated from reprogrammed cells
using clonal isolation procedures as described in the pending
application PCT/US2006/013573 filed Apr. 11, 2006, and U.S.
Application No. 60/811,908, filed Jun. 7, 2006, which are
incorporated my means of reference. Methods of differentiating
reprogrammed cells obtained by the methods disclosed herein may
comprise a step of permeabilization of the reprogrammed cell. For
example, ES cell lines generated by the reprogramming techniques
described herein, or alternatively a heterogeneous mixture of cells
comprising reprogrammed cells, may be permeabilized before exposure
to one or more differentiation factors or cell extract or other
preparation comprising differentiation factors. Permeabilization
techniques include, for example, incubation of cell(s) with a
detergent, such as digitonin, or a bacterial toxin, such as
Streptolysin O, or by methods as described in PCT/US2006/013573
filed Apr. 11, 2006, and U.S. Application No. 60/811,908, filed
Jun. 7, 2006, which are incorporated my means of reference. In
certain embodiments, reprogrammed cells are permeabilized and then
exposed to extract from beta cells (e.g., bovine beta cells).
[0127] The methods of the present invention also enable the
generation of cell lines homozygous or hemizygous for MHC antigens.
Hemizygous or homozygous HLA cell lines may be generated in
differentiated cell lines that are dedifferentiated to generate a
totipotent or pluripotent stem cell line that is homozygous at the
HLA locus. See for example U.S. Patent Publication No. US
2004/0091936, filed May 14, 2004, the disclosure of which is
incorporated by reference herein. For instance, differentiated
cells can be dedifferentiated using the reprogramming methods
disclosed herein to generate a totipotent or pluripotent stem cell.
Totipotent and pluripotent stem cells homozygous for
histocompatibility antigens, e.g., MHC antigens, can be produced by
remodeling the nucleus of a somatic cell homozygous for the
antigens and then reconstituting the remodeled nucleus as described
in the present disclosure. Cytoplasm from an undifferentiated cell
may be added to isolated nuclei or chromatin from differentiated
cells, or differentiated cells that are permeabilized. Following
reprogramming of the somatic cell, the resulting de-differentiated,
pluripotent, stem or stem-like homozygous cell may be
differentiated into a desired cell type. Methods for inducing
re-differentiation into a cell type other than that of the initial
differentiated cells are described, for example, in co-owned and
co-pending U.S. publication 20030027330, filed Apr. 2, 2002, the
disclosure of which is incorporated herein by reference in its
entirety. Further, during step 1 of de-differentiation, the nucleus
remodeled in step one may be modified by homologous recombination.
The addition of extracts from cells such as DT40 known to have a
high level of homologous recombination along with DNA targeting
constructs will then yield cells after reconstitution in step 2 and
screening in step 3 that have a desired genetic modification and
that are homozygous for MHC antigen.
[0128] Many of the steps in the present invention are time
intensive and require skilled technicians to perform the steps at a
high level of quality. To decrease cost and increase quality and
reproducibility, many of the steps described above can be automated
through the use of robotics. Robotic platforms can, for example,
culture cells, introduce buffers and other reagents, thaw and
introduce extracts, and reconstitute cells in step 2.
[0129] The present invention is commercialized by regional centers
that receive differentiated cells from animals or humans in need of
cell therapy and perform steps 1-2 or 1-3 of the methods of the
present invention, and return either the reprogrammed pluripotent
stem cells to a clinical center where they are differentiated into
a therapeutically-useful cell type, or the differentiation is
performed in the regional center and the cells ready for
transplantation are shipped in live cultures or in a cryopreserved
state to the health care provider.
EXAMPLES
Example 1
Nuclear Remodeling
[0130] The first step (also referred to herein as the "nuclear
reprogramming step") is performed using human peripheral blood
mononuclear cells which are purified from blood using Ficoll
gradient centrifugation to yield a buffy coat comprised primarily
of lymphocytes and monocytes as is well known in the art. The use
of lymphocytes with a rearranged immunoglobulin locus as donors in
the present invention will result in stem cells with the same
rearranged loci. In the case where the desired outcome of the
experiment is not cells with a preformed rearrangement in
immunoglobulin genes, the monocytes are purified from the
lymphocytes by flow cytometry as is well known in the art and
stored at room temperature in Dulbecco's minimal essential medium
(MEM) or cryopreserved until use. Xenopus oocytes from MS222
anesthetized mature females are surgically removed in MBS buffer
and inspected for quality as is well-known in the art (Gurdon,
Methods Cell Biol 16:125-139, (1977)). The oocytes are then washed
twice in MBS and stored overnight at 14.degree. C. in MBS. The next
day, good quality stage V or VI oocytes are selected (Dumont, J.
Morphol. 136:153-179, (1972)) and follicular cells are removed
under a dissecting microscope in MBS. After defolliculation, the
oocytes are stored again at 14.degree. C. overnight in MBS with 1
.mu.g/mL gentamycin (Sigma). The next day, oocytes with a healthy
morphology are washed again in MBS and stored in MBS at 14.degree.
C. until use that day. Approximately 1.times.10.sup.4 monocytes are
permeabilized by SLO treatment as described by Chan & Gurdon,
Int. J. Dev. Biol. 40:441-451, (1996). Briefly, the cells are
suspended in ice-cold lysis buffer [1.times.Ca.sup.2+-free MBS
containing 10 mM EGTA (Gurdon, (1977)]. SLO (Wellcome diagnostics)
is added at a final concentration of 0.5 units/mL. The suspension
is maintained on ice for 7 minutes, then four volumes of
1.times.Ca.sup.2+-free MBS containing 1% bovine serum albumin
(Sigma) is added. Aliquots of the cells may then be removed,
diluted 1.times. in 1.times.Ca.sup.2+-free MBS containing 1% bovine
serum albumin, and incubated at room temperature for five minutes
to activate permeabilization. The cells are then placed back on ice
for transfer into the Xenopus oocytes. The permeabilized cella are
then transferred into Xenopus oocytes as is well known in the art
(Gurdon, J. Embryol. Exp. Morphol. 36:523-540, (1976). Briefly,
oocytes prepared as described above are placed on agar in high salt
MBS (Gurdon, J. Embryol. Exp. Morphol. 36:523-540, (1976)). The DNA
in the egg cells is inactivated by UV as described (Gurdon, Methods
in Cell Biol 16:125-139, 1977) with the exception that the second
exposure to the Hanovia UV source is not performed. Briefly, egg
cells are placed on a glass slide with the animal pole facing up
and are exposed to a Mineralite UV lamp for 1 minute to inactivate
the female germinal vesicle. The permeabilized monocytes are taken
up serially into a transplantation pipette 3-5 times the diameter
of the monocytes and injected into the oocyte, preferably aiming
toward the inactivated pronucleus. The egg containing the nuclei
are incubated for one hour to 7 days, preferably 7 days, then
removed and used in step 2. The oocytes may, if desired, be
manipulated prior to use to alter the levels of one or more cell
factors as described above.
Example 2
Nuclear Remodeling
[0131] In this example, step one of nuclear remodeling is carried
out in an extract from undifferentiated cells of the same species
as the differentiated cell; human dermal fibroblasts nuclei are
remodeled in vitro using mitotic cell extracts from the human
embryonal carcinoma cell line NTera-2. However, extracts from cells
of a different species may alternatively be used.
Preparation of Nuclear Remodeling Extract NTera-2 cl. D1 cells are
easily obtained from sources such as the American Type Culture
Collection (CRL-1973) and are grown at 37.degree. C. in monolayer
culture in DMEM with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate
and 4.5 g/L glucose, 10% fetal bovine serum (complete medium).
While in a log growth state, the cells are plated at
5.times.10.sup.6 cells per eq cm tissue culture flask in 200 mL of
complete medium. Extracts from cells in the prometaphase are
prepared as is known in the art (Burke & Gerace, Cell 44:
639-652, (1986)). Briefly, after two days and while still in a log
growth state, the medium is replaced with 100 mL of complete medium
containing 2 mM thymidine (which sequesters the cells in S phase).
After 11 hours, the cells are rinsed once with 25 mL of complete
medium, then the cells are incubated with 75 mL of complete medium
for four hours, at which point nocodazole is added to a final
concentration of 600 ng/mL from 10,000.times. stock solution in
DMSO. After one hour, loosely-attached cells are removed by mitotic
shakeoff (Tobey et al., J. Cell Physiol. 70:63-68, (1967)). This
first collection of removed cells is discarded, the medium is
replaced with 50 mL of complete medium also containing 600 ng/mL of
nocodazole. Prometaphase cells are then collected by shakeoff 2-2.5
hours later. The collected cells are then incubated at 37.degree.
C. for 45 minutes in 20 mL of complete medium containing 600 ng/mL
nocodazole and 20 .mu.M cytochalasin B. Following this incubation,
the cells are washed twice with ice-cold Dulbecco's PBS, then once
in KHM (78 mM KCl, 50 mM Hepes-KOH [pH 7.0], 4.0 mM MgCl.sub.2, 10
mM EGTA, 8.37 mM CaCl.sub.2, 1 mM DTT, 20 .mu.M cytochlasin B). The
cells are the centrifuged at 1000 g for five minutes, the
supernatant discarded, and the cells are resuspended in the
original volume of KHM. The cells are then homogenized in a dounce
homogenizer on ice with about 25 strokes and progress determined by
microscopic observation. When at least 95% of the cells are
homogenized extracts held on ice for use in envelope reassembly or
cryopreserved as is well known in the art. Preparation of Condensed
Chromatin from Differentiated Cells
[0132] Donor dermal fibroblasts will be exposed to conditions that
remove the plasma membrane, resulting in the isolation of nuclei.
These nuclei, in turn, will be exposed to cell extracts that result
in nuclear envelope dissolution and chromatin condensation. This
results in the release of chromatin factors such as RNA, nuclear
envelope proteins, and transcriptional regulators such as
transcription factors that are deleterious to the reprogramming
process. Dermal fibroblasts are cultured in DMEM with 10% fetal
calf serum until the cells reach confluence. 1.times.10.sup.6 cells
are then harvested by trypsinization as is well known in the art,
the trypsin is inactivated, and the cells are suspended in 50 mL of
phosphate buffered saline (PBS), pelleted by centrifuging the cells
at 500 g for 10 minutes at 4.degree. C., the PBS is discarded, and
the cells are suspended in 50.times. the volume of the pellet in
ice-cold PBS, and centrifuged as above. Following this
centrifugation, the supernatant is discarded and the pellet is
resuspended in 50.times. the volume of the pellet of hypotonic
buffer (10 mM HEPES, pH 7.5, 2 mM MgCl.sub.2, 25 mM KCl, 1 mM DTT,
10 .mu.M aprotinin, 10 .mu.M leupeptin, 10 .mu.M pepstatin A, 10
.mu.M soybean trypsin inhibitor, and 100 .mu.M PMSF) and again
centrifuged at 500 g for 10 min at 4.degree. C. The supernatant is
discarded and 20.times. the volume of the pellet of hypotonic
buffer is added and the cells are carefully resuspended and
incubated on ice for an hour. The cells are then physically lysed
using procedures well-known in the art. Briefly, 5 ml of the cell
suspension is placed in a glass Dounce homogenizer and homogenized
with 20 strokes. Cell lysis is monitored microscopically to observe
the point where isolated and yet undamaged nuclei result. Sucrose
is added to make a final concentration of 250 mM sucrose (1/8
volume of 2 M stock solution in hypotonic buffer). The solution is
carefully mixed by gentle inversion and then centrifuged at 400 g
at 4.degree. C. for 30 minutes. The supernatant is discarded and
the nuclei are then gently resuspended in 20 volumes of nuclear
buffer (10 mM HEPES, pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 25 mM
KCl, 1 mM DTT, 10 .mu.M aprotinin, 10 .mu.M leupeptin, 10 .mu.M
pepstatin A, 10 .mu.M soybean trypsin inhibitor, and 100 .mu.M
PMSF). The nuclei are re-centrifuged as above and resuspended in
2.times. the volume of the pellet in nuclear buffer. The resulting
nuclei may then be used directly in nuclear remodeling as described
below or cryopreserved for future use.
Preparation of Condensation Extract
[0133] The condensation extract, when added to the isolated
differentiated cell nuclei, will result in nuclear envelope
breakdown and the condensation of chromatin. Since the purpose of
step 1 is to remodel the nuclear components of a somatic
differentiated cell with that of an undifferentiated cell, the
condensation extract used in this example is obtained from NTera-2
cells which are also the cells used to derive the extract for
nuclear envelope reconstitution above. This results in a dilution
of the components from the differentiated cell in extracts which
contain the corresponding components desirable in reprogramming
cells to an undifferentiated state. NTera-2 cl. D1 cells are easily
obtained from sources such as the American Type Culture Collection
(CRL-1973) and are grown at 37.degree. C. in monolayer culture in
DMEM with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate and 4.5 g/L
glucose, 10% fetal bovine serum (complete medium). While in a log
growth state, the cells are plated at 5.times.10.sup.6 cells per sq
cm tissue culture flask in 200 mL of complete medium. Methods of
obtaining extracts capable of inducing nuclear envelope breakdown
and chromosome condensation are well known in the art (Collas et
al., J. Cell Biol. 147:1167-1180, (1999)). Briefly, NTera-2 cells
in log growth as described above are synchronized in mitosis by
incubation in 1 .mu.g/ml nocodazole for 20 hours. The cells that
are in the mitotic phase of the cell cycle are detached by mitotic
shakeoff. The harvested detached cells are centrifuged at 500 g for
10 minutes at 4.degree. C. Cells are resuspended in 50 ml of cold
PBS, and centrifuged at 500 g for an additional 10 min. at
4.degree. C. This PBS washing step is repeated once more. The cell
pellet is then resuspended in 20 volumes of ice-cold cell lysis
buffer (20 mM HEPES, pH 8.2, 5 mM MgCl.sub.2, 10 mM EDTA, 1 mM DTT,
10 .mu.M aprotinin, 10 .mu.M leupeptin, 10 .mu.M pepstatin A, 10
.mu.M soybean trypsin inhibitor, 100 .mu.M PMSF, and 20 .mu.g/ml
cytochalasin B, and the cells are centrifuged at 800 g for 10
minutes at 4.degree. C. The supernatant is discarded, and the cell
pellet is carefully resuspended in one volume of cell lysis buffer.
The cells are placed on ice for one hour then lysed with a Dounce
homogenizer. Progress is monitored by microscopic analysis until
over 90% of cells and cell nuclei are lysed. The resulting lysate
is centrifuged at 15,000 g for 15 minutes at 4.degree. C., the
tubes are then removed and immediately placed on ice. The
supernatant is gently removed using a small caliber pipette tip,
and the supernatant from several tubes is pooled on ice. If not
used immediately, the extracts are immediately flash-frozen on
liquid nitrogen and stored at -80EC until use. The cell extract is
then placed in an ultracentrifuge tube and centrifuged at 200,000 g
for three hours at 4.degree. C. to sediment nuclear membrane
vesicles. The supernatant is then gently removed and placed in a
tube on ice and used immediately to prepare condensed chromatin or
cryopreserved as described above.
Methods of Use of Condensation Extract
[0134] If beginning with a frozen aliquot on condensation extract,
the frozen extract is thawed on ice. Then an ATP-generating system
is added to the extract such that the final concentrations are 1 mM
ATP, 10 mM creatine phosphate, and 25 .mu.g/ml creatine kinase. The
nuclei isolated from the differentiated cells as described above
are then added to the extract at 2,000 nuclei per 10 .mu.l of
extract, mixed gently, the incubated in a 37.degree. C. water bath.
The tube is removed occasionally to gently resuspend the cells
tapping on the tube. Extracts and cell sources vary in times for
nuclear envelope breakdown and chromosome condensation. The
progress is therefore monitored by periodic monitoring samples
microscopically. When the majority of cells have lost their nuclear
envelope and there is evidence of the beginning of chromosome
condensation, the extract containing the condensing chromosome
masses is placed in a centrifuge tube with an equal volume of 1 M
sucrose solution in nuclear buffer. The chromatin masses are
sedimented by centrifugation at 1,000 g for 20 minutes at 4.degree.
C. The supernatant is discarded, and the chromatin masses are
gently resuspended in nuclear remodeling extract derived above. The
sample is then incubated in a water bath at 33.degree. C. for up to
two hours and periodically monitored microscopically for formation
of remodeled nuclear envelopes around the condensed and remodeled
chromatin as described (Burke & Gerace, Cell 44:639-652,
(1986). Once a large percentage of chromatin has been encapsulated
in nuclear envelopes, the remodeled nuclei may be used in cellular
reconstitution using any of the techniques described in the present
invention.
Modification of Cell Extracts
[0135] As an optional modification to the methods disclosed herein,
one or more factors are expressed or overexpressed in the
undifferentiated cells (for example, in EC or other cells) used to
obtain the nuclear remodeling and/or condensation extracts. Such
factors include, for example, SOX2, NANOG, cMYC, OCT4, DNMT3B,
embryonic histones, as well as other factors listed in Table 1
below and regulatory RNA that induce or increase the expression of
proteins expressed in undifferentiated cells and that improve the
frequency of reprogramming. Any combinations of the above-mentioned
factors may be used. For example, undifferentiated cells of the
present invention may be modified to have increased expression of
two, three, four, or more of any of the factors listed in Table 1.
Alternatively, the level of one or more factors in the
undifferentiated cells used to obtain the nuclear remodeling
extract may be decreased relative to the levels found in unmodified
cells. Such decreases in the level of a cell factor may be achieved
by known methods, such as, for example, by use of transcriptional
regulators, regulatory RNA, or antibodies specific for the cell
factor.
[0136] Gene constructs encoding the proteins listed in Table 1 or
their non-human homologues, or regulatory proteins or RNAs that
alter expression of these factors, are transfected into the cells
by standard techniques. Alternatively, recombinant proteins or
other agents are directly added to the extracts.
Example 3
Genetic Modification of Remodeled Nuclei or Chromatin
[0137] The isolated nuclei or condensed chromatin may optionally be
modified by methods involving recombinase treated targeting vectors
or oligonucleotides. The DNA from cell free chromosomes and
chromatin can be genetically modified enzymatically with targeting
vectors or oligonucleotides, using purified recombinases, purified
DNA repair proteins, or protein or cell extract preparations
comprising such proteins. The targeting DNAs may have tens of
kilobasepairs to oligonucleotides of at least 50 basepairs of
homology to the chromosomal target. Recombinase catalyzed
recombination intermediates formed between target chromosomes and
vector DNA can be enzymatically resolved in cell free extracts with
other purified recombination or DNA repair proteins to produce
genetically modified chromosomes. These modified chromosomes can be
reintroduced into cells or used in the formation of nuclei in vitro
prior to introduction into cells; modified condensed chromatin can
be used in nuclear envelope reconstitution (see step 2 below).
Recombinase treated vector or oligonucleotides can also be directly
introduced into isolated nuclei by microinjection or by diffusion
into permeabilized nuclei to allow in situ formation of
recombination intermediates that can be resolved in vitro, upon
nuclear transfer into intact cells, or upon fusion with recipient
cells.
[0138] In this approach, enyzmatically active nucleoprotein
filaments are first formed between targeting vector, or
oligonucleotides, and recombinase proteins. Recombinase proteins
are cellular proteins that catalyze the formation of heteroduplex
recombination intermediates intracellularly and can form similar
intermediates in cell free systems. Well studied, prototype
recombinases are the RecA protein from E. coli and Rad51 protein
from eukaryotic organisms. Recombinase proteins cooperatively bind
single stranded DNA and actively catalyze the search for homologous
DNA sequences on other target chromosomal DNAs. Heteroduplex
structures may also be formed and resolved using cell free extracts
from cells with recombinogenic phenotypes (e.g., DT40 extracts). In
a second step, heteroduplex intermediates may be resolved in cell
free extracts by treatment with purified recombination and DNA
repair proteins to recombine the donor targeting vector DNA or
oligonucleotide into the target chromosomal DNA (FIG. 2).
Resolution may also be accomplished using cell free extracts from
normal cells or extracts from cells with a recombinogenic
phenotype. Finally, the nuclear membrane is reformed around
modified chromosomes and the remaining unmodified cellular
chromosomal complement for introduction into recipient cells or
oocytes.
[0139] Several techniques are available that can be used in gene
modification of the reprogrammed cell. One technique is the Cre-Lox
targeting system. Cre recombinase has been used to efficiently
delete hundreds of basepairs to megabasepairs of DNA in mammalian
cells. The LoxP and FRT recombinase recognition sequences allow
recombinase mediated gene modifications of homologous recombinant
cells.
Forming Recombinase Coated Nucleoprotein Filaments
[0140] Circular DNA targeting vectors are first linearized by
treatment with restriction endonucleases, or alternatively linear
DNA molecules are produced by PCR from genomic DNA or vector DNA.
All DNA targeting vectors and traditional DNA constructs are
removed from vector sequences by agarose gel electrophoresis and
purified with Elutip-D columns (Schleicher & Schuell, Keene, N.
H.). For RecA protein coating of DNA, linear, double-stranded DNA
(200 ng) is heat denatured at 98.degree. C. for 5 minutes, cooled
on ice for 1 minute and added to protein coating mix containing
Tris-acetate buffer, 2 mM magnesium acetate and 2.4 mM ATP.gamma.S.
RecA protein (8.4 .mu.g) is immediately added, the reaction
incubated at 37.degree. C. for 15 minutes, and magnesium acetate
concentration increased to a final concentration of 11 mM. The RecA
protein coating of DNA is monitored by agarose gel electrophoresis
with uncoated double-stranded DNA as control. The electrophoretic
mobility of RecA-DNA is significantly retarded as compared with
non-coated double stranded DNA.
[0141] Isolation of cell free chromosomes and chromatin is achieved
as described above. The condensation extract, when added to the
isolated differentiated cell nuclei, will result in nuclear
envelope breakdown and the condensation of chromatin. The resulting
nuclei may then be used directly for gene modifications as just
described, nuclear remodeling, or cryopreserved for future use. A
separate extract is used for nuclear envelope reconstitution after
cell free homologous recombination reactions have modified target
chromosomes. Extract for nuclear envelope breakdown and chromatin
condensation, and for nuclear envelope reconstitution may be
prepared from any proficient mammalian cell line. However, extracts
from the human embryonal carcinoma cell line NTera-2 can be
potentially used for the condensation extract and for nuclear
envelope reconstitution extract as well as for remodeling
differentiated chromatin to an undifferentiated state, thus
enhancing production of genetically modified human ES cells
starting from differentiated human dermal cells.
Forming Heteroduplex Recombination Intermediates Between Preformed
Recombinase Coated Nucleoprotein Targeting Vectors and
Oligonucleotides and Cell Free Chromosomes and Chromatin
[0142] Formation of targeting vector/chromosome heteroduplexes is
performed by adding approximately 1-3 .mu.g of double-stranded
chromosomal DNA or chromatin masses to the RecA coated
nucleoprotein filaments described above, and incubated at
37.degree. C. for 20 minutes. If the nucleoprotein heteroduplex
structures are to be deproteinized prior to additional in vitro
recombination steps, they are treated by with the addition of SDS
to a final concentration of 1.2%, or by addition of proteinase K to
10 mg/ml with incubation for 15 to 20 minutes at 37.degree. C.,
followed by addition of SDS to a final concentration of 0.5 to 1.2%
(wt/vol). Residual SDS is removed prior to subsequent steps by
microdialysis against 100 to 1000 volumes of protein coating
mix.
Resolving Recombination Intermediates with Cell Free Extracts
[0143] Cell free extracts may be prepared from normal fibroblast or
hES cell lines, or may be prepared from cells demonstrated to have
recombinogenic phenotypes. Cell lines exhibiting high levels of
recombination in vivo are the chicken pre-B cell line DT40 and the
human lymphoid DG75 cell line.
[0144] Preparation of cell free extracts is performed at 4.degree.
C. About 108 actively growing cells are harvested from either
dishes or suspension cultures. The cells are washed three times
with phosphate-buffered saline (PBS; 140 mM NaCl, 3 mM KCl, 8 mM
NaH.sub.2PO.sub.4, 1 mM K.sub.2HPO.sub.4, 1 mM MgCl.sub.2, 1 mM
CaCl.sub.2), resuspended in 2 to 3 ml of hypotonic buffer A (10 mM
Tris hydrochloride [pH 7.4], 10 mM MgCl.sub.2, 10 mM KCl, 1 mM
dithiothreitol), and kept on ice for 10 to 15 minutes.
Phenylmethylsulfonyl fluoride is added to a concentration of 1 mM,
and the cells are broken by 5 to 10 strokes in a Dounce
homogenizer, pestle B. The released nuclei are centrifuged at 2,600
rpm in a Beckman TJ-6 centrifuge for 8 min. The supernatant is
removed carefully and stored in 10% glycerol-100 mM NaCl at
-70.degree. C. (cytoplasmic fraction). The nuclei are resuspended
in 2 ml of buffer A containing 350 mM NaCl, and the following
proteinase inhibitors are added: pepstatin to a concentration of
0.25 .mu.g/ml, leupeptin to a concentration of 0.1 .mu.g/ml,
aprotinin to a concentration of 0.1 .mu.g/ml, and
phenylmethylsulfonyl fluoride to a concentration of 1 mM (all from
Sigma Chemicals). After 1 h of incubation at 0.degree. C., the
extracted nuclei are centrifuged at 70,000 rpm in a Beckman
TL-100/3 rotor at 2.degree. C. The clear supernatant is adjusted to
10% glycerol, 10 mM .beta.-mercaptoethanol and frozen immediately
in liquid nitrogen prior to storage at -70.degree. C. (fraction
1).
[0145] To resolve recombination intermediates in vitro, chromosomal
heteroduplex intermediates are incubated with 3 to 5 .mu.g of
extract protein in a reaction mixture containing 60 mM NaCl, 2 mM
2-mercaptoethanol, 2 mM KCl, 12 mM Tris hydrochloride (pH 7.4), 1
mM ATP, 0.1 mM each deoxyribonucleoside triphosphate (dNTP), 2.5 mM
creatine phosphate, 12 mM MgCl.sub.2, 0.1 mM spermidine, 2%
glycerol, and 0.2 mM dithiothreitol. After 30 minutes at 37.degree.
C., the reaction is stopped by the addition of EDTA to a
concentration of 25 .mu.M, sodium dodecyl sulfate (SDS) to a
concentration of 0.5%, and 20 .mu.g of proteinase K and incubated
for 1 hour at 37.degree. C. SDS is removed prior to subsequent
steps by microdialysis. An equal volume of 1 M sucrose is added to
the treated chromatin masses and sedimented by centrifugation at
1,000.times.g for 20 minutes at 4.degree. C.
Reforming Nuclear Envelopes Around Recombinant Chromosomes and
Chromatin
[0146] The supernatant is discarded, and the chromatin masses are
gently resuspended in nuclear remodeling extract described above.
The sample is then incubated in a water bath at 33.degree. C. for
up to two hours and periodically monitored microscopically for the
formation of remodeled nuclear envelopes around the condensed and
remodeled chromatin as described (Burke & Gerace, Cell
44:639-652, (1986). Once a large percentage of chromatin has been
encapsulated in nuclear envelopes, the remodeled nuclei may be used
for cellular reconstitution using any of the techniques described
in the present invention.
Detection of Cells Containing Genetically Modified Chromosomes
[0147] Reconstituted cells are grown for 7 to 14 days and screened
for recombinants using PCR and Southern hybridization.
Example 4
Modification of Chromosomes and Chromatin in Isolated Nuclei with
Targeting Vectors or Oligonucleotides to Engineer Cells
[0148] Chromosomes and chromatin may be genetically modified in
isolated nuclei from cells. In this approach, intact nuclei are
isolated from growing cells, and reversibly permeabilized to allow
diffusion of nucleprotein targeting vectors and oligonucleotides
into the nucleus interior. Heteroduplex intermediates formed
between nucleoprotein targeting vectors and oligonucleotides and
chromosomal DNA may be resolved by treatment with recombination
proficient cell extracts, purified recombination and DNA repair
proteins, or by cellular reconstitution with the nuclei into
recombination proficient cells.
Isolation and Permeabilization of Nuclei
[0149] Preparation of Synchronous Populations of nuclei cell
culture and synchronization are carried out as previously described
(Leno et al., Cell 69:151-158 (1992)). Nuclei are prepared as
described except that all incubations are carried out in HE buffer
(50 mM Hepes-KOH, pH 7.4, 50 mM KCl, 5 mM MgCl.sub.2, 1 mM EGTA, 1
mM DTT, 1 .mu.g/ml aprotinin, pepstatin, leupeptin,
chymostatin).
[0150] Nuclear Membrane Permeablization Streptolysin O
(SLO)-prepared nuclei (Leno et al., Cell 69:151-158 (1992)) are
incubated with 20 .mu.g/ml lysolecithin (Sigma Immunochemicals) and
10 .mu.g/ml cytochalasin B in HE at a concentration of
.about.1.5.times.10.sup.6 nuclei/ml for 10 min at 23.degree. C.
with occasional gentle mixing. Reactions are stopped by the
addition of 1% nuclease free BSA (Sigma Immunochemicals). Nuclei
are gently pelleted by centrifugation in a RC5B rotor (Sorvall
Instruments, Newton, Conn.) at 500 rpm for 5 min and then washed
three times by dilution in 1 ml HE. Pelleted nuclei are recovered
in a small volume of buffer and resuspended to -1.times.10.sup.4
nuclei/.mu.l.
Forming Heteroduplex Recombination Intermediates Between Preformed
Recombinase Coated Nucleoprotein Targeting Vectors and
Oligonucleotides and Cell Free Chromosomes and Chromatin
[0151] Formation of targeting vector/chromosome heteroduplexes is
performed by adding approximately 1.times.10.sup.5 to
1.times.10.sup.6 permeabilized nuclei to the RecA coated
nucleoprotein filaments described above, and incubated at
37.degree. C. for 20 minutes.
Resolving Recombination Intermediates with Cell Free Extracts
[0152] Cell free extracts may be prepared from normal fibroblast or
hES cell lines, or may be prepared from cells demonstrated to have
recombinogenic phenotypes. Cell lines exhibiting high levels of
recombination in vivo are the chicken pre-B cell line DT40 and the
human lymphoid DG75 cell line. Preparation of cell free extracts
are performed at 4.degree. C. About 10.sup.8 actively growing cells
are harvested from either dishes or suspension cultures. The cells
are washed three times with phosphate-buffered saline (PBS; 140 mM
NaCl, 3 mM KCl, 8 mM NaH.sub.2PO.sub.4, 1 mM K.sub.2HPO.sub.4, 1 mM
MgCl.sub.2, 1 mM CaCl.sub.2), resuspended in 2 to 3 ml of hypotonic
buffer A (10 mM Tris hydrochloride [pH 7.4], 10 mM MgCl.sub.2, 10
mM KCl, 1 mM dithiothreitol), and kept on ice for 10 to 15 minutes.
Phenylmethylsulfonyl fluoride is added to 1 mM, and the cells are
broken by 5 to 10 strokes in a Dounce homogenizer, pestle B. The
released nuclei are centrifuged at 2,600 rpm in a Beckman TJ-6
centrifuge for 8 min. The supernatant is removed carefully and
stored in 10% glycerol-100 mM NaCl at -70.degree. C. (cytoplasmic
fraction). The nuclei are resuspended in 2 ml of buffer A
containing 350 mM NaCl, and the following proteinase inhibitors are
added: pepstatin to 0.25 .mu.g/ml, leupeptin to 0.1 .mu.g/ml,
aprotinin to 0.1 .mu.g/ml, and phenylmethylsulfonyl fluoride to 1
mM (all from Sigma Chemicals). After 1 h of incubation at 0.degree.
C., the extracted nuclei are centrifuged at 70,000 rpm in a Beckman
TL-100/3 rotor at 2.degree. C. The clear supernatant is adjusted to
10% glycerol, 10 mM .beta.-mercaptoethanol and frozen immediately
in liquid nitrogen prior to storage at -70.degree. C. (fraction
1).
[0153] To resolve recombination intermediates in permeabilized
nuclei, nuclei containing chromosomal heteroduplex intermediates
are incubated with 3 to 5 .mu.g of extract protein in a reaction
mixture containing 60 mM NaCl, 2 mM 3-mercaptoethanol, 2 mM KCl, 12
mM Tris hydrochloride (pH 7.4), 1 mM ATP, 0.1 mM each
deoxyribonucleoside triphosphate (dNTP), 2.5 mM creatine phosphate,
12 mM MgCl.sub.2, 0.1 mM spermidine, 2% glycerol, and 0.2 mM
dithiothreitol. After 30 minutes at 37.degree. C., the reaction is
stopped.
Nuclear Envelope Repair
Preparation and Fractionation of Nuclear Repair Extract
[0154] Low-speed Xenopus egg extracts (LSS) 1 are prepared
essentially according to the procedure described by Blow and Laskey
Cell 21; 47:577-87 (1986)). Extraction buffer (50 mM Hepes-KOH, pH
7.4, 50 mM KCl, 5 mM MgCl.sub.2) is thawed and supplemented with 1
mM DTT, 1 .mu.g/ml leupeptin, pepstatin A, chymostatin, aprotinin,
and 10 .mu.g/ml cytochalasin B (Sigma Immunochemicals, St. Louis,
Mo.) immediately before use. Extracts are supplemented with 2%
glycerol and snap-frozen as 10-20 .mu.l beads in liquid nitrogen or
subjected to further fractionation. High speed supernatant (HSS)
and membrane fractious are prepared from low-speed egg extract as
described (Sheehan et al., J Cell Biol. 106:1-12 (1988)).
Membranous material, isolated by centrifugation of 1-2 ml of
low-speed extract, is washed at least two times by dilution in 5 ml
extraction buffer. Diluted membranes are centrifuged for 10 minutes
at 10 k rpm in an SW50 rotor (SW50; Beckman Instruments, Inc., Palo
Alto, Calif.) to yield vesicle fraction 1. The supernatant is then
centrifuged for a further 30 min at 30 k rpm to yield vesicle
fraction 2. Washed membranes are supplemented with 5% glycerol and
snap-frozen in 5 .mu.l beads in liquid nitrogen. Vesicle fractions
1 and 2 are mixed in equal proportions before use in nuclear
membrane repair reactions.
Treatment for Nuclear Envelope Repair
[0155] Lysolecithin-permeabilized nuclei are repaired by incubation
with membrane components prepared from Xenopus egg extracts. Nuclei
at a concentration of approximately 5000/.mu.l are mixed with an
equal volume of pooled vesicular fractions 1 and 2 and supplemented
with 1 mM GTP and ATP. 10-20 .mu.l reactions are incubated at
23.degree. C. for up to 90 min with occasional gentle mixing.
Aliquots are taken at intervals and assayed for nuclear
permeability.
[0156] Once a large percentage of chromatin is encapsulated in
nuclear envelopes, the remodeled nuclei may be used for cellular
reconstitution using any of the techniques described in the present
invention.
Detection of Cells Containing Genetically Modified Chromosomes
[0157] Reconstituted cells are grown for 7 to 14 days and screened
for recombinants using. PCR and Southern hybridization.
Example 5
Modification of Isolated Chromosomes, Chromatin, and Nuclei Using
Cell Free Extracts to Engineer Cells with Exogenous Genetic
Material
[0158] In this approach, targeting vectors or oligonucleotides and
the target chromosomal DNA are directly treated with recombination
proficient cell free extracts from cells with recombinogenic
phenotypes such as the chicken pre-B cell line DT40 and the human
lymphoid cell line DG75. These cell free extracts may be used on
isolated chromosome and chromatin or on isolated permeabilized
nuclei. Essentially, targeting vector/oligonucleotides are
incubated with isolated chromosomes, chromatin, or nuclei and cell
free recombination extract. The nuclear envelope is reconstituted
around recombinant chromosomes or chromatin, or the nuclear
envelope of recombinant, permeabilized, nuclei are repaired prior
to cell reconstitution with the reconstituted or repaired
nuclei.
Preparation of Cell Free Extracts
[0159] Cell free extracts from DT40 or DG75 cells are prepared as
described above.
Preparation of Chromosomes, Chromatin, or Nuclei
[0160] Isolated chromosomes, chromatin, and permeabilized nuclei
from fibroblasts, hES cell lines, or germ cell lines are as
described above.
Recombination Between Targeting Vectors and Oligonucleotides, and
Cell Free Chromosomes and Chromatin Using Cell Free Extracts from
Recombinogenic Cells
[0161] Circular DNA targeting vectors are first linearized by
treatment with restriction endonucleases, or alternatively linear
DNA molecules are produced by PCR from genomic DNA or vector DNA.
All DNA targeting vectors and traditional DNA constructs are
removed from vector sequences by agarose gel electrophoresis and
purified with Elutip-D columns (Schleicher & Schuell, Keene, N
H). Double-stranded DNA (200 ng) is heat denatured at 98.degree. C.
for 5 minutes, cooled on ice for 1 minute and added to
approximately 1-3 .mu.g of double-stranded chromosomal DNA or
chromatin masses, or approximately 1.times.10.sup.5 to
1.times.10.sup.6 permeabilized nuclei, and 3 to 5 .mu.g of extract
protein in a reaction mixture containing 60 mM NaCl, 2 mM
3-mercaptoethanol, 2 mM KCl, 12 mM Tris hydrochloride (pH 7.4), 1
mM ATP, 0.1 mM each deoxyribonucleoside triphosphate (dNTP), 2.5 mM
creatine phosphate, 12 mM MgCl.sub.2, 0.1 mM spermidine, 2%
glycerol, and 0.2 mM dithiothreitol. The reaction mixtures are
incubated at 37.degree. C. for at least 30 minutes are processed as
describe above prior to reconstituting cellular envelopes or
repairing permeabilized nuclei.
Reforming Nuclear Envelopes Around Recombinant Chromosomes and
Chromatin
[0162] Nuclear envelopes are reconstituted around recombinant
chromosomes and chromatin and reconstituted nuclei used for
cellular reconstitution as describe above.
Nuclear Envelope Repair
[0163] Recombinant, permeabilized nuclei are repaired and repaired
recombinant nuclei used for cellular reconstitution as described
above.
Detection of Cells Containing Genetically Modified Chromosomes
[0164] Reconstituted cells are grown for 7 to 14 days and screened
for recombinants using PCR and Southern hybridization.
Example 6
Modification of Chromosomes and Chromatin in Intact Cells with
Recombinase Treated Targeting Vectors or Oligonucleotides to
Engineer Cells with Exogenous Genetic Material
[0165] In this approach, double stranded targeting vectors,
targeting DNA fragments, or oligonucleotides are coated with
bacterial or eukaryotic recombinase and introduced into mammalian
cells or oocytes. The activated nucleoprotein filament forms
heteroduplex recombination intermediates with the chromosomal
target DNA that is subsequently resolved to a homologous
recombinant structure by the cellular homologous recombination or
DNA repair pathways. While the most direct delivery of
nucleoprotein filaments is by direct nuclear/pronuclear
microinjection, other delivery technologies can be used including
electroporation, chemical transfection, and single cell
electroporation.
[0166] To form human Rad51 nucleoprotein filaments, linear,
double-stranded DNA (200 ng) is heat denatured at 98.degree. C. for
5 minutes, cooled on ice for 1 minute and added to a protein
coating mix containing 25 mM Tris acetate (pH 7.5), 100 .mu.g/ml
BSA, 1 mM DTT, 20 mM KCl (added with the protein stock), 1 mM ATP
and 5 mM CaCl.sub.2, or AMP-PNP and 5 mM MgCl.sub.2. hRad51 protein
(1 .mu.M) is immediately added and the reaction incubated for 10
minutes at 37.degree. C. The hRad51 protein coating of the DNA is
monitored by agarose gel electrophoresis with uncoated
double-stranded DNA as control. The electrophoretic mobility of
hRad51-DNA nucleoprotein filament is significantly retarded as
compared with non-coated double stranded DNA. hRad51-DNA
nucleoprotein filaments are diluted to a concentration of 5
ng/.mu.l and used for nuclear microinjection of human fibroblasts
or somatic cells, or used for pronuclear microinjection of
activated oocytes created by somatic cell nuclear transfer or in
vitro fertilization.
Detection of Cells Containing Genetically Modified Chromosomes
[0167] Injected cells or oocytes are grown for 7 to 14 days and
screened for recombinants using PCR and Southern hybridization.
Example 7
Cellular Reconstitution
[0168] Step 2, also referred to as "cellular reconstitution" in the
present invention is carried out using nuclei or chromatin
remodeled by any of the techniques described in the present
invention, such as in Examples 1 and 2 above or combinations of the
techniques described in Examples 1 and 2 as described more fully in
the present invention. During cellular reconstitution in this
example, the remodeled nuclei are fused with enucleated cytoplasts
of hES cells as is known in the art (Do & Scholer, Stem Cells
22:941-949 (2004)). Briefly, the human ES Cell line H9 is cultured
under standard conditions (Klimanskaya et al., Lancet 365: 4997
(1995)). The cytoplasmic volume of the cells is increased by adding
10 .mu.m cytochalasin B for 20 hours prior to manipulation.
Cytoplasts are prepared by centrifuging trypsinzed cells through a
Ficoll density gradient using a stock solution of autoclaved 50%
(wt/vol) Ficoll-400 solution in water. The stock Ficoll 400
solution is diluted in DMEM and with a final concentration of 10
.mu.m cytochalasin B. The cells are centrifuged through a gradient
of 30%, 25%, 22%, 18%, and 15% Ficoll-400 solution at 36.degree. C.
Layered on top is 0.5 mL of 12.5% Ficoll-400 solution with
10.times.10.sup.6 ES cells. The cells are centrifuged at 40,000 rpm
at 36.degree. C. in an MLS-50 rotor for 30 minutes. The cytoplasts
are collected from the 15% and 18% gradient regions marked on the
tubes, rinsed in PBS, and mixed on a 1:1 ratio with remodeled
nuclei from step one of the present invention or cryopreserved.
Fusion of the cytoplasts with the nuclei is performed using
polyethylene glycol (see Pontecorvo "Polyethylene Glycol (PEG) in
the Production of Mammalian Somatic Cell Hybrids" Cytogenet Cell
Genet. 16(1-5):399-400 (1976), briefly in 1 mL of prewarmed 50%
polyethylene glycol 1500 (Roche) for one minute. 20 mL of DMEM was
then added over a five minute period to slowly remove the
polyethylene glycol. The cells were centrifuged once at 130 g for
five minutes and then taken back up in 50 .mu.L of ES cell culture
medium and placed beneath a feeder layer of fibroblasts under
conditions to promote the outgrowth of an ES cell colony.
Example 8
Cellular Reconstitution
[0169] Step 2, also referred to as "cellular reconstitution" in the
present invention is also carried out using nuclei remodeled by any
of the techniques described in the present invention, in this
example as in Example 2 above and the cellular reconstitution step
that follows. The nuclei are fused with anucleate cytoplasmic blebs
of hES cells as is well known in the art (Wright & Hayflick,
Exp. Cell Res. 96:113-121, (1975); & Wright & Hayflick,
Proc. Natl. Acad. Sci., USA, 72:1812-1816, (1975). Briefly, the
cytoplasmic volume of the hES cells is increased by adding 10 .mu.M
cytochalasin B for 20 hours prior to manipulation. The cells are
trypsinized and replated on sterile 18 mm coverslips coated with
mouse embryonic fibroblast feeder extracellular matrix as described
(Klimanskaya et al., Lancet 365: 4997 (2005). The cells are plated
at a density such that after an overnight incubation at 37.degree.
C. and one gentle wash with medium, the cells cover about 90% of
the surface area of the coverslip. The coverslips are then placed
face down in a centrifuge tube containing 8 mL of 10% Ficoll-400
solution and centrifuged at 20,000 g at 36.degree. C. for 60
minutes. Remodeled nuclei resulting from step one of the present
invention are then spread onto the coverslip with a density of at
least that of the cytoplasts, preferable at least five times the
density of the cytoplasts. Fusion of the cytoplasts with the nuclei
is performed using polyethylene glycol (see Pontecorvo
"Polyethylene Glycol (PEG) in the Production of Mammalian Somatic
Cell Hybrids" Cytogenet Cell Genet. 16(1-5):399-400 (1976).
Briefly, in 1 mL of prewarmed 50% polyethylene glycol 1500 (Roche)
in culture medium is placed over the coverslip for one minute. 20
mL of culture medium is then added drip-wise over a five minute
period to slowly remove the polyethylene glycol. The entire media
is then aspirated and replaced with culture medium.
Example 9
Analysis of the Molecular Mechanisms of Reprogramming
[0170] The in vitro remodeling of somatic cell-derived DNA as
described in example 2 of the present invention is utilized as a
model of the reprogramming of a somatic cell and an assay useful in
analyzing the molecular mechanisms of reprogramming. The protocol
of example 2 is followed to the time immediately preceding that
when extracts from mitotic NTera2 cells are added. Prior to the
addition of mitotic NTera2 cell extract, purified lamin A protein
from human skin fibroblasts is added in amounts corresponding to
10.sup.-6, 10.sup.-4, 10.sup.-3, 10.sup.-2, 10.sup.-1, 1.times. and
10.times. the concentration in human fibroblast mitotic cell
extract. The lamin A reduces the extent of successful reprogramming
following step 2 cellular reconstitution, and the use of this assay
system determines the extent of lamin A interference in successful
reprogramming.
Example 10
Reprogramming Factors
[0171] The frequency of obtaining reprogrammed cells may be
improved by increasing the expression of undifferentiated cell
factors in the undifferentiated cells or cell extracts of steps 1
and 2 of the present invention. These factors may be introduced
into the extracts of step 1, or into the enucleated cytoplasts of
step 2 using techniques well known in the art and described herein.
The final concentration of said factor should be at least the
concentration observed in cultures of human ES cells grown under
standard conditions, or preferably 2-50-fold higher in
concentration than that observed in said standard hES cell
cultures. Table 1 provides a list of exemplary undifferentiated
cell factors. The table provides the names and accession names for
the human genes; however homologues found in other species may also
be used:
TABLE-US-00001 TABLE 1 Gene Name Accession Number BARX1 NM_021570.2
CROC4 NM_006365.1 DNMT3B NM_175849.1 H2AFX NM_002105.1 HHEX
NM_002729.2 HIST1H2AB NM_003513.2 HIST1H4J NM_021968.3 HMGB2
NM_002129.2 hsa-miR-18a M10000072 hsa-miR-18b M10001518 hsa-miR-20b
M10001519 hsa-miR-96 M10000098 hsa-miR-106a M10000113 hsa-miR-107
M10000114 hsa-miR-141 M10000457 hsa-miR-183 M10000273 hsa-miR-187
M10000274 hsa-miR-203 M10000283 hsa-miR-211 M10000287 hsa-miR-217
M10000293 hsa-miR-218-1 M10000294 hsa-miR-218-2 M10000295
hsa-miR-302a M10000738 hsa-miR-302c M10000773 hsa-miR-302d
M10000774 hsa-miR-330 M10000803 hsa-miR-363 M10000764 hsa-miR-367
M10000775 hsa-miR-371 M10000779 hsa-miR-372 M10000780 hsa-miR-373
M10000781 hsa-miR-496 M10003136 hsa-miR-508 M10003195
hsa-miR-512-3p hsa-miR-512-5p hsa-miR-515-3p hsa-miR-515-5p
hsa-miR-516-5p hsa-miR-517 hsa-miR-517a M10003161 hsa-miR-518b
M10003156 hsa-miR-518c M10003159 hsa-miR-518e M10003169
hsa-miR-519e M10003145 hsa-miR-520a M10003149 hsa-miR-520b
M10003155 hsa-miR-520e M10003143 hsa-miR-520g M10003166
hsa-miR-520h M10003175 hsa-miR-523 M10003153 hsa-miR-524 M10003160
hsa-miR-525 M10003152 hsa-miR-526a-1 M10003157 hsa-miR-526a-2
M10003168 LEFTB NM_020997.2 LHX1 NM_005568.2 LHX6 NM_014368.2 LIN28
NM_024674.3 MYBL2 NM_002466.2 MYC NM_002467.2 MYCN NM_005378.3
NANOG NM_024865.1 NFIX NM_002501.1 OCT3/4 (POU5F1) NM_002701.2 OCT6
(POU3F1) NM_002699.2 OTX2 NM_172337.1 PHC1 NM_004426.1 SALL4
NM_020436.2 SOX2 NM_003106.2 TERF1 NM_003218.2 TERT NM_198254.1
TGIF NM_003244.2 VENTX2 NM_014468.2 ZIC2 NM_007129.2 ZIC3
NM_003413.2 ZIC5 NM_033132.2 ZNF206 NM_032805.1
[0172] Methods for expressing proteins or regulatory RNA that
increases expression of these proteins within cells or means of
introducing these factors into cellular extracts are well-known in
the art and include a variety of techniques including without
limitation:
[0173] Viral infection for stable and transient expression of
proteins and regulatory RNAs, such viruses including without
limitation: lentivirus bovine papilloma and other papilloma
viruses, adenoviruses and adeno-associated viruses. In addition,
the genes or RNAs may be introduced by transfection for transient
and stable expression of proteins and regulatory RNAs through the
use of plasmid vectors, mammalian artificial chromosomes BACS/PACS
the direct addition of the proteins encoded in the listed genes,
the miRNA or mRNA listed, using CaPO4 precipitate-mediated
endocytosis, dendrimers, lipids, electroporation, microinjection,
homologous recombination to modify the gene or its promoters or
enhancers, chromosome-mediated gene transfer, cell fusion,
microcell fusion, or the addition of cell extracts containing said
useful factors, all of such techniques are well-known in the art
and protocols for carrying out said techniques to administer said
factors are readily available to researchers in the literature and
Internet.
Example 11
Induction Beta Cell Differentiation from Reprogrammed Cells without
the Generation of ES Cell Lines
[0174] Peripheral blood nucleated cells are obtained from a patient
in need of pancreatic beta cells. The cells are purified using flow
cytometry to obtain monocytes using techniques well-known in the
art. Nuclei from the monocytes are then prepared by placing the
cells in hypotonic buffer and dounce homogenizing the cells as is
described in the art. The isolated monocyte nuclei from the patient
are then exposed to a mitotic extract from the human EC cell line
Tera-2 and incubated while monitoring samples of the extract to
observed nuclear envelope breakdown and subsequent reformation of
the nuclear envelope as described herein. The resulting
reprogrammed cell nuclei are then fused with EC cell cytoplasts
from the EC cell line Tera-2 that have been transfected with
plasmids to overexpress the genes OCT4, SOX2, and NANOG as
described herein. The resulting reconstituted cells in a
heterogeneous mixture of reprogrammed and non-reprogrammed cells
are then permeabilized and exposed to extracts of beta cells
isolated from bovine pancreas as described herein and then directly
differentiated into endodermal lineages without the production of
an ES cell line. One million of the heterogeneous mixture of cells
are then added onto mitotically-inactivated feeder cells that
express high levels of NODAL or cell lines that express members of
the TGF beta family that activate the same receptor as NODAL such
as CM02 cells that express relatively high levels of Activin-A, but
low levels of Inhibins or follistatin. The cells are then incubated
for a period of five days in DMEM medium with 0.5% human serum.
After five days, the resulting cells which include definitive
endodermal cells are purified by flow cytometry or other
affinity-based cell separation techniques such as magnetic bead
sorting using antibody specific to the CXCR4 receptor and then
cloned using techniques described in the pending patent
applications PCT/US2006/013573 filed Apr. 11, 2006; and U.S.
Application No. 60/811,908, filed Jun. 7, 2006, which are
incorporated by means of reference. These cells are then directly
differentiated into pancreatic beta cells or beta cell precursors
using techniques known in the art for differentiating said cells
from human embryonic stem cell lines or by culturing the cells on
inducer cell mesodermal cell lines as described in
PCT/US2006/013573 filed Apr. 11, 2006, and U.S. Application No.
60/811,908, filed Jun. 7, 2006, which are both incorporated by
means of reference.
[0175] It is envisioned that the disclosed improved methods for the
reprogramming of animal somatic cells are generally useful in
mammalian and human cell therapy, such as human cells useful in
treating dermatological, cardiovascular, neurological,
endocrinological, skeletal, and blood cell disorders.
[0176] From the foregoing description, it will be apparent that
variations and modifications may be made to the invention described
herein to adopt it to various usages and conditions.
* * * * *
References