U.S. patent application number 15/046885 was filed with the patent office on 2016-12-22 for conversion of somatic cells to induced reprogrammed neural stem cells (irnscs).
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Klaus Christensen, Martin Graf, Roberto Iacone, Ravi Jagasia.
Application Number | 20160369234 15/046885 |
Document ID | / |
Family ID | 43734036 |
Filed Date | 2016-12-22 |
United States Patent
Application |
20160369234 |
Kind Code |
A1 |
Christensen; Klaus ; et
al. |
December 22, 2016 |
CONVERSION OF SOMATIC CELLS TO INDUCED REPROGRAMMED NEURAL STEM
CELLS (IRNSCS)
Abstract
This application relates to a method for converting somatic
cells to Neural Stem Cells (NSCs). Moreover this application
relates to a method for converting human fibroblasts, keratinocytes
or adipocytes to neural stem cells based on linked steps of genes
transduction and chemically defined medium induction.
Inventors: |
Christensen; Klaus; (Overwil
BL, CH) ; Graf; Martin; (Zeiningen, CH) ;
Iacone; Roberto; (Basel, CH) ; Jagasia; Ravi;
(Loerrach, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
43734036 |
Appl. No.: |
15/046885 |
Filed: |
February 18, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13769762 |
Feb 18, 2013 |
9297025 |
|
|
15046885 |
|
|
|
|
PCT/EP2011/064051 |
Aug 16, 2011 |
|
|
|
13769762 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2506/1307 20130101;
C12N 2501/727 20130101; C12N 2501/60 20130101; A61K 35/30 20130101;
C12N 2501/405 20130101; C12N 2501/13 20130101; C12N 2501/119
20130101; C12N 2510/00 20130101; C12N 15/86 20130101; C12N 5/0623
20130101; C12N 2501/115 20130101; C12N 2501/602 20130101; C12N
2501/11 20130101; A61P 25/00 20180101 |
International
Class: |
C12N 5/0797 20060101
C12N005/0797; A61K 35/30 20060101 A61K035/30 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 19, 2010 |
EP |
10173455.6 |
Claims
1. A method of producing Neural Stem Cells (NCS), comprising: a)
providing somatic cells, b) reprogramming said somatic cells to
NSCs by introducing at least two genes selected from the group
consisting of Bmi 1, Sox2, Mash 1, Sox1 1, Emx2, Foxg1 and Pax6;
and c) inducing the reprogramming with growth factors and a small
molecule.
2. The method of claim 1, further comprising d) incubating the
product of steps b) and c) under conditions suitable for
proliferation of the NSCs.
3. The method of claim 1, wherein the somatic cells of step a) are
human cells.
4. The method of claim 3, wherein the somatic cells of step a) are
selected from the group consisting of fibroblasts, keratinocytes
and adipocytes.
5. The method of claim 1, wherein the growth factors and small
molecule of step c) are supplements of a chemically defined
medium.
6. The method of claim 5, wherein the chemically defined medium is
a serum free medium supplemented with insulin, transferrin and
progesterone.
7. The method of claim 1, wherein the at least two genes of step b)
comprise Bmi 1 and Sox2.
8. The method of claim 7, wherein the at least two genes of step b)
additionally comprise at least one gene selected from the group of
Mash 1, Sox1 1, Emx2, Foxg1 and Pax6.
9. The method of claim 7, wherein the at least two genes of step b)
comprise Bmi 1, Sox2 and Mash 1.
10. The method of claim 1, wherein the growth factor of step c) is
selected from the group consisting of FGF2, EGF and BDNF.
11. The method of claim 1, wherein the small molecule of step c)
comprises a ROCK inhibitor.
12. The method of claim 11, wherein the ROCK inhibitor is selected
from the group consisting of 1-(5-Isoquinolinesulfonyl)
homopiperazine,
N-Benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide,
(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)
cyclo-hexanecarboxamide dihydrochloride) and
N-{(3R,4R)-4-[4-(2-Fluoro-6-hydroxy-3-methoxy-benzoyl)-benzoylamino]-azep-
an-3-yl}-4-hydroxy-3,5-dimethyl-benzamide.
13. The method of claim 1, wherein the somatic cells are pretreated
with a histone deacetylase (HDAC) inhibitor.
14. The method of claim 1, wherein reprogramming of said somatic
cells is achieved through delivery of a combination of at least two
genes by a lentivirus.
15. (canceled)
16. (canceled)
17. A therapeutic composition comprising the neural stem cells of
claim 15.
18. The therapeutic composition of claim 17, wherein the neural
stem cells are differentiated into neurons or glia cells.
19. (canceled)
20. (canceled)
21. A method of treating, preventing, or stabilizing a neurological
disease in an individual in need of such treatment comprising
administering a therapeutic composition of claim 17 to the
individual.
22. The method of claim 21, wherein the neurological disease is one
of Alzheimer's disease, Parkinson's disease, Huntington's disease,
or ALS, lysosomal storage diseases, multiple sclerosis, and a
spinal cord injury.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/769,762 having a filing date of Feb. 18, 2013; which is a
continuation of International Application No. PCT/EP2011/064051
having an international filing date of Aug. 16, 2011, the entire
contents of which are incorporated herein by reference and which
claims benefit under 35 U.S.C. .sctn.119 to European Patent
Application No. 10173455.6 filed Aug. 19, 2010.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing
submitted via EFS-Web and is hereby incorporated by reference in
its entirety. Said ASCII copy, created on Feb. 11, 2016, is named
P26845-US-1_SeqList.txt, and is 22,308 bytes in size.
FIELD OF THE INVENTION
[0003] This application relates to a method for converting somatic
cells to Neural Stem Cells (NSCs). Moreover this application
relates to a method for converting human fibroblasts, kerotinocytes
or adipocytes to neural stem cells based on linked steps of genes
transduction and chemically defined medium induction.
BACKGROUND OF THE INVENTION
[0004] The dogma that fully differentiated somatic cells have
absolutely irreversible properties was generally accepted for a
long time. This began to change when a series of pioneering
experiments showed that silent gene expression profiles can be
completely reactivated by the fusion of different pairs of cell
types (Blau, H. M. How fixed is the differentiated state? Lessons
from heterokaryons. Trends Genet. 5, 268-272 (1989)). More recently
it was shown that transfer of nuclei from a somatic cell type into
an enucleated egg cell could lead to the complete reversion of the
somatic cells' gene expression profile, and to the formation of a
pluripotent cell state able to generate new entire animals (see
e.g. Gurdon, J. B. & Melton, D. A. Nuclear reprogramming in
cells. Science 322, 1811-1815 (2008)). Yamanaka and colleagues
(Takahashi, K. & Yamanaka, S. Induction of pluripotent stem
cells from mouse embryonic and adult fibroblast cultures by defined
factors. Cell 126, 663-676 (2006)) demonstrated that somatic cells
can be reprogrammed to induced pluripotent stem cells (iPSCs) by
transduction of four defined factors (Sox2, Oct4, Klf4, c-Myc).
Different types of somatic cells including fibroblasts,
keratynocytes and adipocytes have been reprogrammed to an iPSC
pluripotent state. During the past years the question arose whether
specific somatic cell types could be transdifferentiated to a
completely different somatic cell type such as a neuron. Wernig and
colleagues addressed this question showing the direct conversion of
mouse fibroblasts to functional neurons by transduction of three
crucial genes: Mash1, Brn2 and Myt11 (Wernig at al. Direct
conversion of fibroblasts to functional neurons by defined factors.
Nature 25; 463(7284):1035-41 (2010). However the neurons obtained
are postmitotic cells which are by definition not able to
proliferate and which do not tolerate freezing and thawing
procedures. US2010/0021437 discloses a method for generating
induced pluripotent stem cells from fibroblasts and inducing those
cells to differentiate into neural phenotypes.
[0005] However, direct conversion of differentiated somatic cells
to neural stem cells has not been described so far. Neural stem
cells are multipotent stem cells and are reported to be propagated
under specific conditions. They require a chemically defined
medium, for example N2B27 medium (N2B27 is a 1:1 mixture of
DMEM/F12 (Gibco, Paisley, UK) supplemented with N2 and B27 (both
from Gibco)) supplemented with FGF (fibroblast growth factor 2) and
EGF (epidermal growth factor). They can grow as a monolayer
adherent culture, e.g. on Poly-ornithine/Lamin coated plate or as
floating neurospheres in non-adherent cell culture plates. The two
types of neural stem cell cultures (neurospheres, adherent
cultures) have been reported to be completely inter-convertible.
Neural stem cells can be grown indefinitely and still remain truly
multipotent. Upon special conditions they differentiate into the
cell types that compose the adult brain, including neurons,
astrocytes and oligodendrocytes. Neural stem cells are considered
possible therapeutic agents for treating patients with
neurodegenerative diseases such as Alzheimer's disease, Parkinson's
disease, stroke, and spinal cord injury.
SUMMARY OF THE INVENTION
[0006] It is known that neural stem cells can be generated either
in vitro from Embryonic Stem Cells (ESCs) (Chambers et al. Nature
27; 3 (2009)) or they can be isolated directly from brain samples
(Reynolds B A, Rietze R L (2005) Nat Methods 2:333-336). However
these methods known so far have many major drawbacks as they either
raise a number of highly sensitive ethical considerations and/or
they necessitate complicated and laborious technologies which
suffer from serious troubles with reproducibility. So far no method
has been described wherein neural stem cells can be directly
derived from differentiated somatic cells. In principle, neural
stem cells could be obtained from iPSCs that have been derived from
differentiated cells. However, this would imply culturing of iPSCs.
iPSCs have been reported to expand indefinitely but the culture
conditions are complicated and require huge efforts. In addition
the derivation of neural stem cells from pluripotent stem cells has
been reported to fluctuate due to stochastic mechanisms. A common
obstacle of iPSCs and ESCs is that even a small number of
undifferentiated cells can result in the formation of teratomas
(germ cell tumors comprising several cell types), which pose
serious contaminations that may not be ignored. Somatic stem cells,
such as neural stem cells do not form teratomas. Hence there
remains a need for an easy accessible and reproducible technology
for the generation of neural stem cells. The present invention
provides a method for converting somatic cells directly to neural
stem cells. The new method alleviates the necessity of obtaining
iPS cells and hence removes the risk of teratoma formation. Such
cells without the ability to form teratomas are useful and safe for
regenerative medicine applications. Preferably said somatic cells
are mammalian somatic cells, most preferably human somatic cells.
Said human somatic cells can be obtained from a healthy individual
or from a patient. Preferably said somatic cells are fibroblast
cells, adipocytes or keratinocytes, most preferably fibroblast
cells. Said fibroblast cells, adipocytes or keratinocytes can be
easily and safely derived from a patient or healthy individual, for
example by non-invasive methods such as skin biopsy or from plucked
hair. The method of this invention allows to convert somatic cells
such as fibroblasts cells, adipocytes or keratinocytes from healthy
or diseased individuals directly to neural stem cells. These
healthy individuals or patients specific neural stem cells can be
expanded indefinitely. Culturing is easy and well characterized. It
is possible to freeze and thaw healthy individuals and patients
specific neural stem cells aliquots reproducibly. In particular,
patient derived neural stem cells represent a disease relevant in
vitro model to study the pathophysiology of CNS diseases.
Conversion of patients' specific somatic cells directly to neural
stem cells represents an easy accessible and reproducible
technology to generate BioBanks of patient specific neural stem
cells. Such BioBanks have great relevance for CNS related diseases,
as a clear pathology has been described in at least one of the
three cell types generated from the neural stem cells: neurons,
oligodentrocytes and astrocytes. Hence the neural stem cells
obtained with the method described herein are valuable disease
models to screen effective and safe drugs.
[0007] A variety of neurodegenerative diseases are characterized by
neuronal cell loss. The regenerative capacity of the adult brain is
rather limited in response to brain injury and neurodegenerative
disease. Further, pharmacological interventions often become
increasingly less effective as the susceptible neuronal populations
are progressively lost. The neural stem cells obtained with the
method described herein can also be used in regenerative medicine
to treat neurodegenerative diseases like Parkinson's disease,
Alzheimer's disease, Huntington's disease, Amyotrophic lateral
sclerosis (ALS/Lou Gehrig's Disease) or spinal cord injury. With
the innovative method described herein it is now possible to
provide sufficient amounts of neuronal precursor cells for use in
cell transplantation therapies. The neural stem cells can either be
obtained from somatic cells isolated from a healthy individual or
from a patient. Patient specific neural stem cells obtained by the
method described herein are an attractive new donor source for
autologous cell transplantation therapies, thereby abrogating any
immune rejection due to immunological incompatibility between
patient and donor. This strategy would eliminate the requirement of
immune suppressants in cell transplantation therapy. Moreover, the
creation of Biobanks of neural stem cells derived from healthy
individuals with various HLA homozygous alleles can be used as
donor banks for treatment of individuals in need. Heterologous
transplantation of neural stem cells with a compatible HLA type
reduces the risk of undesirable immune responses which could lead
to rejection of the transplanted cells.
[0008] To achieve the inventive breakthrough described here, it was
necessary to bypass some of the existing limitations of
reprogramming, as well as to combine genes transduction with the
employment of a step of induction with a specific medium.
[0009] Provided herein is a method for converting somatic cells to
Neural Stem Cells (NSC), said method comprising the steps of:
[0010] a) providing somatic cells
[0011] b) reprogramming said somatic cells to neural stem cells by
introducing at least two genes and
[0012] c) inducing for the reprogramming with growth factors and a
small molecule;
[0013] In a further embodiment, said method additionally
comprises
[0014] d) incubating the product of step b) and c) under conditions
suitable for proliferation of the neural stem cells. Typically the
product of step b) and c) can be easily identified in a cell
culture as neurospheres. Preferably said conditions suitable for
proliferation of the neural stem cells comprise harvesting of said
neurospheres and expanding them in a chemically defined medium.
Preferably, said medium is an expansion medium and the neurospheres
are cultured in non-adherent culturing conditions. Non-limiting
examples of expansion media are described further below.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The term "somatic cell" as used herein refers to any cell
forming the body of an organism that are not germ line cells (e. g.
sperm and ova, the cells from which they are made (gametocytes))
and undifferentiated stem cells. Internal organs, skin, bones,
blood and connective tissue are all made up of somatic cells.
Preferred somatic cells used in the method described herein are
fibroblast cells, adipocytes or keratinocytes and are preferably
obtained from skin biopsy.
[0016] Preferably, the somatic cells used for conversion into
neural stem cells are of mammalian origin, most preferably of human
origin. Said human somatic cells can be obtained from a healthy
individual or from a patient. Preferably said somatic cells are
chosen from the group of fibroblast cells, adipocytes or
keratinocytes. These donor cells can be easily obtained from any
suitable source. Preferred herein are sources that allow isolation
of donor cells without invasive procedures on the human body.
Methods for isolating fibroblast cells are well known in the art.
Fibroblast cells may be obtained from any suitable source, for
example from various organ tissues or skin tissue. Preferred
fibroblasts are lung fibroblasts, foreskin fibroblasts, and adult
dermal fibroblasts. In a special embodiment of this invention, said
human fibroblasts are obtained from a patient, for example by skin
biopsy (e.g. Reprogramming of human somatic cells to pluripotency
with defined factors. George Q. Daley et al. Nature 2008; A method
for the isolation and serial propagation of keratinocytes,
endothelial cells, and fibroblasts from a single punch biopsy of
human skin, Normand et al. In Vitro Cellular & Developmental
Biology--Animal, 1995). Adipocytes and keratinocytes can also be
easily derived by skin biopsy or plucked hair (Isolation and
cultivation of human keratinocytes from skin or plucked hair for
the generation of induced pluripotent stem cells, Belmonte et al.
Nature Protocols 2010) and are also preferred donor cells for the
method of this invention.
[0017] One preferred aspect of the present invention is a method
for generating patient specific neural stem cells. Another aspect
of the present invention is a method for generating neural stem
cells from somatic cells obtained from a healthy individual.
[0018] As used herein, "neural stem cells" refers to a subset of
pluripotent cells which express some neural markers including, for
example, nestin. The neural stem cells obtained by the method
described herein are also referred to as "irNSCs": induced
reprogrammed neural stem cells. Neural stem cells can be expanded
indefinitely and may differentiate into neurons or glial cells
(e.g. astrocytes and oligodendrocytes). The term "patient specific
neural stem cell" refers to neural stem cells obtained from somatic
cells of a patient and are also referred to as autologous neural
stem cells. "Neural stem cells obtained from a healthy individual"
as used herein refers to neural stem cells obtained from somatic
cells of an individual that is not suspected to suffer from any
disorder or disease.
[0019] As used herein, the term "reprogramming" refers to one or
more steps needed to convert a somatic cell to a
less-differentiated cell, for example for converting fibroblast
cells, adipocytes or keratinocytes into neural stem cells.
Reprogramming of a somatic cell to a neural stem cell is achieved
by introducing at least two genes involved in the maintenance of
neural stem cell properties. Genes suitable for reprogramming of
somatic cells to neural stem cells include, but are not limited to
Sox2 (Seq ID No. 1), Brn2 (Seq ID No. 2), Bmi1 (Seq ID No. 3),
Mash1 (Seq ID No. 4), Sox11 (Seq ID No. 5), NCam (Seq ID No. 6),
Kpna1 (Seq ID No. 7), Foxg1 (Seq ID No. 8), Emx2 (Seq ID No. 9) and
Pax6 (Seq ID No. 10). In a preferred embodiment at least two genes
are introduced, in another preferred embodiment three genes are
introduced. A preferred combination of genes to be introduced into
the somatic cells comprises Bmi1 and Sox2. In a further preferred
embodiment this combination of at least two genes additionally
comprises Mash1. In another embodiment this combination of at least
two genes additionally comprises one gene selected from the group
of Mash1, Emx2, Foxg1, Pax6 and Sox11. In a further embodiment the
combination of at least two genes comprises Bmi1 and Sox2 and
Mash1.
[0020] The term "introducing of genes", as used herein, refers to
any method that leads to the stable expression of said gene in a
somatic cell. Said genes are introduced into somatic cells by
methods known in the art, either by delivery into the cell via
reprogramming vectors or by activation of said genes via small
molecules. Examples of reprogramming vectors are retroviruses,
lentiviruses, adenoviruses, plasmids and transposons. Preferred
herein is the use of a lentivirus for the delivery of said genes.
Examples of small molecules suitable for robust activation of said
genes are DNA methylation inhibitors, histone deacytelase
inhibitors, ergolines (e.g. lysergic acid ethylamide), flavones
(e.g. 7' hydroxyflavone), paullones (e.g. Kenpaullone)
(Reprogramming of murine fibroblasts to induced pluripotent stem
cells with chemical complementation of Klf4 PNAS 2009 106 (22)
8912-8917), L-type channel agonists (e.g. BIX01294), BayK8644 and
5' azacytidine (Induction of Pluripotent Stem Cells from Mouse
Embryonic Fibroblasts by Oct4 and Klf4 with Small-Molecule
Compounds Yan Shi et al. Cell Stem Cell--6 Nov. 2008 (Vol. 3, Issue
5, pp. 568-574)). For successful induction of the reprogramming the
somatic cells are grown in a suitable medium supplemented with
growth factors and a small molecule. As used herein, the term
"growth factor" means a biologically active polypeptide which
causes cell proliferation, and includes both growth factors and
their analogues. These include, without limitation, epidermal
growth factor, transforming growth factors, nerve growth factor,
acidic and basic fibroblast growth factor and angiogenesis factor,
platelet-derived growth factor, insulin and insulin-like growth
factors including somatomedins, myxoma and vaccinia virus-derived
growth factors. Preferred growth factors used herein are BDNF
(brain-derived neutrotrophic factor), FGF2 (fibroblast growth
factor 2) and EGF (epidermal growth factor). The growth factors may
be used alone or in pairwise combination, or most preferably all
three factors are used together. In addition the fibroblasts are
cultured in the presence of at least one small molecule. The term
"small molecule", or "small compound" as used herein, refers to
organic or inorganic molecules either synthesized or found in
nature, generally having a molecular weight less than 10,000 grams
per mole, optionally less than 5,000 grams per mole, and optionally
less than 2,000 grams per mole. In one preferred embodiment said
small molecule comprises an inhibitor of the Rho-associated
coiled-coil forming protein serine/threonine kinase (ROCK) family
of protein kinases.
[0021] Non-limiting examples of ROCK inhibitors comprise Fasudil
(1-(5-Isoquinolinesulfonyl)homopiperazine), Thiazovivin
(N-Benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide), Y27632
((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)
cyclo-hexanecarboxamide dihydrochloride) and Balanol-like-324
compound
(N-{(3R,4R)-4-[4-(2-Fluoro-6-hydroxy-3-methoxy-benzoyl)-benzoylamino]-aze-
pan-3-yl}-4-hydroxy-3,5-dimethyl-benzamide). In another embodiment
said small molecule is selected from an inhibitor of one or more of
the kinases AMPK (AMP-activated protein kinase, beta 1
non-catalytic subunit; official symbol: PRKAB1), CHK2 (CHK2
checkpoint homolog (S. pombe), official symbol: CHEK2), MSK1
(ribosomal protein S6 kinase, 90 kDa, polypeptide 5; official
symbol: RPS6KA5), PKA (protein kinase, cAMP-dependent, catalytic,
alpha; official symbol: PRKACA), PKGa (protein kinase,
cGMP-dependent, type I; official symbol: PRKG1) and SGK1
(serum/glucocorticoid regulated kinase 1, official symbol:
SGK1).
[0022] A "suitable medium for induction of reprogramming", also
depicted as "induction medium", as used herein refers to any
chemically defined medium useful for induction of reprogramming of
the somatic cells. Preferred herein is a serum free medium
supplemented with insulin, transferrin and progesterone. Preferred
media used herein contain 10-50 .mu.g/ml insulin, 10-100 .mu.g/ml
transferrin and 10-50 nM progesterone. Examples of serum-free media
suitable for induction of reprogramming are N2B27 medium (N2B27 is
a 1:1 mixture of DMEM/F12 (Gibco, Paisley, UK) supplemented with N2
and B27 (both from Gibco)), N3 medium (composed of DMEM/F12 (Gibco,
Paisley, UK), 25 .mu.g/ml insulin, 50 .mu.g/ml transferrin, 30 nM
sodium selenite, 20 nM progesterone (Sigma), 100 nM putrescine
(Sigma)), or NeuroCult.RTM. NS-A Proliferation medium (Stemcell
Technologies). Most preferred herein is a serum free medium as
described above which is additionally supplemented with FGF2, EGF,
BDNF and a ROCK inhibitor. Preferably, said ROCK inhibitor
comprises Fasudil or Balanol-like-324 compound. In a preferred
embodiment, the medium is supplemented with 10-50 ng/ml FGF2, 10-50
ng/ml EGF, 1-20 ng/ml BDNF and 1-50 .mu.M Fasudil or 1-10 .mu.M
Balanol-like-324 compound. After introduction of at least two genes
the somatic cells to be reprogrammed are preferably grown in said
induction medium for at least 1 day, preferably for 1 to 7 days,
most preferably for 2 to 3 days.
[0023] In one embodiment the somatic cells of step a) are
pretreated with a Histone Deacetylase (HDAC) inhibitor.
"Pretreating" or "pretreatment" as used herein means incubation of
the somatic cells in a suitable medium supplemented with said HDAC
inhibitor for 4 to 60 hours, preferably 48 hours. HDAC inhibitors
useful herein are selected from the group comprising sodium
butyrate (butanoic acid, sodium salt) Trichostatin A (TSA,
7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxohepta-2,4-d-
ienamide) and Valproic Acid (2-propyl-pentanoic acid). In one
embodiment the somatic cells of step a) are pretreated with
Valproic Acid. In another embodiment the somatic cells of step a)
are pretreated with Valproic Acid for 48 hours.
[0024] For propagating proliferation of the neural stem cells as
neurospheres cultures, the induced neural stem cells are grown in
an expansion medium comprising a serum free medium supplemented
with insulin, transferrin and progesterone and growth factors as
described above. Preferably said growth factors comprise FGF2, BDNF
and EGF. In another embodiment said expansion medium additionally
comprises one or more supplements selected from the group of
Heparin, Ascorbic Acid, SHH (Recombinant Human Sonic Hedgehog),
FGF8 (Recombinant Human FGF8a Isoform), DLL4 (Recombinant Human
DLL4), Jagged1 (Recombinant Human Jagged 1 Fc Chimera), Fasudil and
Balanol-like-324 compound.
[0025] In another embodiment of the invention, the neural stem
cells obtained by the method described herein are in a next step
stimulated for differentiation by omission of at least one of the
growth factors of the reprogramming medium. Preferably said growth
factors to be withdrawn comprise EGF and FGF.
[0026] In another preferred embodiment of the invention, a marker
gene is employed to facilitate screening and quantification of
successfully reprogrammed neural stem cells. For example, a gene
encoding for a fluorescent marker protein is introduced into the
target somatic cells by lentivirus transduction. Examples of
fluorescent marker proteins are GFP, YFP, EGFP or DsRed. Preferably
said marker gene is operably linked to a nestin promoter. Nestin is
specifically expressed in neural stem cells, therefore the marker
gene under the control of a nestin promoter allows rapid screening
and identification of induced reprogrammed neural stem cells.
Thereafter, those cells are screened to identify a cell exhibiting
the desired phenotype, i.e. neurospheres. Neurospheres bigger than
20 .mu.m, preferably bigger than 50 .mu.m, are selected and
harvested for further expansion.
[0027] In another aspect of the invention, a population of neural
stem cells produced by any of the foregoing methods is provided.
Preferably, the population of neural stem cells is patient
specific, i.e. derived from somatic cells obtained from diseased
individuals. In another embodiment said population of stem cells is
obtained from a healthy individual. The neural stem cells can be
expanded indefinitely. Culturing is easy and well characterized. It
is possible to freeze and thaw neural stem cells aliquots
reproducibly. Patient derived neural stem cells represent a disease
relevant in vitro model to study the pathophysiology of CNS
diseases. Conversion of patients specific somatic cells directly to
neural stem cells represents an easy accessible and reproducible
technology to generate BioBanks of patient specific neural stem
cells. Hence in a further preferred aspect of the invention a
BioBank comprising patient specific neural stem cells is envisaged.
In another embodiment, a BioBank comprising different populations
of neural stem cells obtained from healthy individuals is
generated. The term "BioBank" as used herein means a library of
biological samples taken from different individuals or species. The
archived collection of specimen and associated data is intended for
research purposes with the aim of addressing neural diseases like
neurodegenerative diseases such as Alzheimer's disease, Parkinson's
disease, Huntington's disease, Amyotrophic lateral sclerosis
(ALS/Lou Gehrig's Disease) stroke, and spinal cord injury or for
therapy of said neurological diseases.
[0028] Another aspect of the invention is the use of neural stem
cells obtained by this method. In a preferred embodiment the neural
stem cells obtained by this method are used as in vitro model to
study the pathophysiology of CNS diseases. For example, the neural
stem cells obtained by the method of the invention can be used for
screening for compounds that reverse, inhibit or prevent
neurological diseases. In addition they can be used for screening
for compounds that reverse, inhibit or prevent neural side effects
of medicaments, for example diabetes medicaments. Preferably, said
neural stem cells obtained by the method of the invention described
herein are derived from diseased subjects.
[0029] In another aspect, the invention provides a therapeutic
composition containing cells produced by any of the foregoing
methods or containing any of the foregoing cell populations.
Preferably, the therapeutic compositions further comprise a
physiologically compatible solution including, for example,
artificial cerebrospinal fluid or phosphate-buffered saline. Said
therapeutic composition can be used to treat, prevent, or stabilize
a neurological disease such as for example, Alzheimer's disease,
Parkinson's disease, Huntington's disease, or ALS, lysosomal
storage diseases, multiple sclerosis, or a spinal cord injury. For
example, fibroblast cells, keratinocytes or adipocytes may be
obtained by skin biopsy from the individual in need of treatment or
from a healthy individual and reprogrammed to neural stem cells by
the method of the invention. In one embodiment of the invention the
neural stem cells are harvested and introduced into the individual
to treat the condition. In another embodiment said neural stem
cells are cultured under conditions suitable for differentiation
into neurons, oligodendrocytes or astrocytes prior to introduction
into the individual, and may be used to replace or assist the
normal function of diseased or damaged tissue. The great advantage
of the present invention is that it provides an essentially
limitless supply of patient specific human neural cells or
compatible neural stem cells from healthy individuals with the same
HLA type suitable for transplantation. The use of autologous and/or
compatible cells in cell therapy offers a major advantage over the
use of non-autologous cells, which are likely to be subject to
immunological rejection. In contrast, autologous cells are unlikely
to elicit significant immunological responses.
[0030] Another embodiment of the invention is the use of biobanks
of neural stem cells for therapy of neurological diseases. The
biobanks preferably comprise neural stem cells obtained from
patients or healthy individuals with several HLA types.
Transplanting cells obtained from a healthy donor to an individual
in need of treatment with a compatible HLA type obviates the
significant problem of rejection reactions normally associated with
heterologous cell transplants. Conventionally, rejection is
prevented or reduced by the administration of immunosuppressants or
anti-rejection drugs such as cyclosporin. However, such drugs have
significant adverse side-effects, e.g., immunosuppression,
carcinogenic properties, kidney toxicity as well as being very
expensive. The present invention should eliminate, or at least
greatly reduce, the need for anti-rejection drugs, such as
cyclosporine, imulan, FK-506, glucocorticoids, and rapamycin, and
derivatives thereof.
[0031] With respect to the therapeutic methods of the invention, it
is not intended that the administration of neural stem cells to a
mammal be limited to a particular mode of administration, dosage,
or frequency of dosing; the present invention contemplates all
modes of administration, including intramuscular, intravenous,
intraarticular, intralesional, subcutaneous, or any other route
sufficient to provide a dose adequate to prevent or treat a
disease. The neural stem cells may be administered to the mammal in
a single dose or multiple doses. When multiple doses are
administered, the doses may be separated from one another by, for
example, one week, one month, one year, or ten years. One or more
growth factors, hormones, interleukins, cytokines, small molecules
or other cells may also be administered before, during, or after
administration of the cells to further bias them towards a
particular cell type.
SHORT DESCRIPTION OF THE FIGURES
[0032] FIG. 1: Schematic representation of the method for
converting human fibroblast to irNSCs. Day 0: human fibroblasts
were trypsinized and transfected in a small volume with a
combination of genes and the nestin GFP reporter using the
induction medium (N2B27 with FGF, EGF 30 ng/ml; BDNF 20 ng/ml;
Fasudil 10 .mu.M, Polybrene 4 .mu.g/ml). Fibroblasts were plated in
a normal tissue culture plate at a concentration of 10000-30000
cells/cm.sup.2. Day 1: Media change with fresh induction medium.
GFP/nestin positive (GFP+) irNSCs started to appear with very low
frequency (.about.50 irNSC GFP+ out of 100000). Day 2: The GFP+
irNSCs were increasing in number and they started to move together
forming cell clusters. Day 3: The cell clusters are organized in a
clear spheroid structure that lifts off and starts floating as a
GFP+ neurospheres. The neurospheres bigger than 20 .mu.m were
counted and harvested for further expansion.
[0033] FIG. 2: Schematic representation of the human nestin GFP
reporter lentivirus. The fluorescent protein copGFP and the zeocin
selectable marker were cloned under the expression control of a 1.8
kb enhancer fragment from the human nestin intron 2 linked to a
minimal CMV promoter.
[0034] FIG. 3: irNSCs at day 1 of the reprogramming induction
method. Upper panel: human untransformed fetal lung fibroblasts
IMR90 (phase contrast). Lower panel: generation of irNSC GFP+ cells
(phase contrast and GFP channel).
[0035] FIG. 4: irNSCs at day 2 of the reprogramming induction
method. The cells tend to migrate close together and start to form
a spheroid structure with a core of irNSCs GFP+ (phase contrast and
GFP channel).
[0036] FIG. 5: irNSCs at day 3 of the reprogramming induction
method. The spheroid structures formed at day 2 are now completely
mature appear as neurospheres floating in the medium. The
neurospheres have a dimension that ranges from 20-100 .mu.m in
diameter with a high density of cells. The irNSCs are labeled by
the nestin GFP expression and can be indentified in almost all the
neurospheres, although not all the neurospheres have the same
proportion of irNSC GFP+ (phase contrast and GFP channel).
[0037] FIG. 6: Number of neurospheres generated with different
combinations of genes.
[0038] FIG. 7: Attached neurospheres after transduction with
Sox2-Bmi1. The attached neurospheres show a characteristic
morphology of elongated bipolar cells. Lower panel: higher
magnification of the irNSC GFP+ neuropsheres.
[0039] FIG. 8: Differentiated cells after 1 week EGF and FGF
withdrawal. The irNSCs upon withdrawal of the proliferative growth
factors give rise to cells with very thin protrusions stained
positive for the neuronal marker tuj1.
[0040] FIG. 9: Generation of a batch of irNSC neurospheres for
expansion and characterization. 3.6 million human fibroblasts IMR90
were trypsinized and infected in a small volume with: Sox2, Bmi1,
nestin GFP reporter using the induction medium (N2B27 with FGF, EGF
30 ng/ml BDNF 20 ng/ml) supplemented with Fasudil 10 .mu.M and
Polybrene 4 .mu.g/ml. From Day 4 to Day 8: The GFP+ neurospheres
bigger than 50 .mu.m were harvested and further used for expansion.
Half of the neurospheres have been expanded using the expansion
medium (N2B27 with FGF, EGF 30 ng/ml BDNF 20 ng/ml) with Fasudil
and the other half without Fasudil. Day15: Neurospheres grown in
the expansion medium with Fasudil have a better morphology and
clear and sharp borders (a hallmark of well-formed neurospheres,
panel B); without Fasudil the neurospheres have bleary borders
(panel A).
[0041] FIG. 10: Immunocytochemistry characterization of irNSC
neurospheres for the expression of the NSCs markers Sox2 and
Nestin. Day15 irNSC neurospheres expanded with Fasudil have been
plated on PO/Lam coated plates and after 48 h stained for Sox2 and
Nestin expression. The irNSC neurospheres attached and irNSCs
spread from the spheres. The irNSCs have a typical NSC morphology
and were Sox2 and Nestin positive. Panel A: Merge and single
channels DAPI, Sox2, Nestin; 20.times. magnification; Panel B:
Merge channels DAPI, Sox2, Nestin; 10.times. magnification.
[0042] FIG. 11: Comparison of Fasudil versus Balanol-like-324
compound stimulation to generate irNSC neurospheres. Human
fibroblasts IMR90 were trypsinized and infected in a small volume
with: Sox2, Bmi1, nestin GFP reporter using the induction medium
(NeuroCult.RTM. NS-A Proliferation Kit (Human, StemCells
Technologies) with FGF, EGF BDNF 20 ng/ml; Heparin 2 .mu.g/ml;
Polybrene 4 .mu.g/ml) supplemented with Fasudil 10 .mu.M (hatched
graph) or Balanol-like-324 compound 2 .mu.M (black graph).
Fibroblasts were plated in a normal tissue culture plate at a
concentration of 10000-30000 cells/cm.sup.2. Day 1: Media change
with fresh induction medium. Day 4: The GFP+ neurospheres bigger
than 50 .mu.m were counted. The Balanol-like-324 small compound
increased the efficiency of the neurospheres generation
approximately twofold (1.9) and has a better reproducibility
(STDEV, n=3).
[0043] FIG. 12: Pre-treatment of human fibroblasts with Valproic
Acid (VPA) improves the yield of GFP+ irNSC neurospheres. Human
fibroblasts IMR90 were pre-treated for 48 hours with or without the
HDAC inhibitor Valproic Acid (2-propyl-pentanoic acid, monosodium
salt) (1 mM) prior infection with: Sox2, Bmi1, nestin GFP reporter.
Induction medium (NeuroCult.RTM. NS-A Proliferation Kit (Human,
StemCells Technologies) with FGF, EGF BDNF 20 ng/ml; Heparin 2
.mu.g/ml; Balanol-like-324 2 .mu.M). Day 7: The Neurospheres bigger
than 50 .mu.m were counted (Panel A) and the average number of GFP+
irNSC per neurosphere is reported (Panel B). Representative
pictures for the irNSCs neuropheres generated with the VPA
pre-treatment (Panel C). The VPA pre-treatment did not
significantly affect the number of neurospheres at day 7; although
the VPA treatment increased (2.1 fold) the number of GFP+ irNSCs
(STDEV, n=3).
[0044] FIG. 13: Defining a minimal pool of genes in combination
with Sox2 and Bmi1 for efficient induction of irNSCs neurospheres.
Human fibroblasts IMR90 were pre-treated for 48 hours with VPA (1
mM) prior infection with: Sox2, Bmi1, nestin GFP reporter plus
different candidate genes to address their synergism. Induction
medium: NeuroCult.RTM. NS-A Proliferation Kit (Human, StemCells
Technologies) with FGF, EGF BDNF 20 ng/ml; Heparin 2 .mu.g/ml and
Balanol-like-324 compound 2 .mu.M. Quantification at Day 7 of
irNSCs neurospheres bigger than 50 .mu.m. Mash1, Emx2, Foxg1, Pax6
and Sox11 synergize with Bmi1 and Sox2 to generate irNSC
neuropheres.
[0045] FIG. 14: Generation of irNSC neurospheres from adult human
dermal fibroblasts (HDFa). The adult human dermal fibroblasts are
provided by the GIBCO (Cat. Number: C-013-5C). The adult human
dermal fibroblasts were trypsinized and infected in a small volume
with: Sox2, Bmi1, nestin GFP reporter using the induction medium
(NeuroCult.RTM. NS-A Proliferation Kit (Human, StemCells
Technologies) with FGF, EGF BDNF 20 ng/ml; Heparin 2 .mu.g/ml)
supplemented with Fasudil 10 .mu.M. Day 8: irNSCs neurospheres are
detected (representative pictures 2.5 and 10.times.
magnification).
[0046] FIG. 15: Expansion of irNSC neurospheres using a combination
of Ascorbic Acid, Sonic Hedgehog (Shh), Jagged1, DLL4 and FGF8 to
obtain a monolayer culture of irNSCs GFP+. Human fibroblasts IMR90
were infected with: Sox2, Bmi1, Mash1 and nestin GFP reporter using
the induction medium (NeuroCult.RTM. NS-A Proliferation Kit (Human,
StemCells Technologies) with FGF, EGF BDNF 20 ng/ml; Heparin 2
.mu.g/ml; Balanol-like-324 2 .mu.M). Day 7: The neurospheres bigger
than 50 .mu.m were harvested and further expanded with the
expansion medium (NeuroCult.RTM. NS-A Proliferation Kit (Human,
StemCells Technologies) with FGF, EGF BDNF 20 ng/ml; Heparin 2
.mu.g/ml; Balanol-like-324 2 .mu.M; Ascorbic Acid 0.2 mM, SHH
(Recombinant Human Sonic Hedgehog, Catalog Number: 1845SH) 500
ng/ml, FGF8 (Recombinant Human FGF8a Isoform, Catalog Number:
4745F8) 100 ng/ml, DLL4 (Recombinant Human DLL4, Catalog Number:
1506D4) 500 ng/ml, Jagged1 (Recombinant Human Jagged 1 Fc Chimera,
Catalog Number: 1277JG) 500 ng/ml, conditioned media 1/10 from the
hESC-derived NSCs cultured for two days in NeuroCult.RTM. NS-A
Proliferation Kit (Human, StemCells Technologies) with FGF, EGF
BDNF 20 ng/ml; Heparin 2 .mu.g/ml. Representative pictures for the
irNSCs neuropheres at day14 expanded with expansion medium reported
above (Panel A). The neuropheres have defined borders and it is
possible to observe the protrusion of spines from the neurospheres
(Panel B, zoom-in). At day 21 the expanded irNSC neurospheres were
dissociated and plated on PO/Lam coated plates to obtain a
homogenous monolayer culture of irNSCs GFP+ (Panel C, phase
contrast and GFP channel of the irNSCs monolayer after 4 days in
culture on the monolayer).
[0047] FIG. 16: Immunocytochemistry characterization of irNSC
neurospheres for the expression of the NSC markers Nestin and the
early neuronal marker Tuj1. Day21 irNSC neurospheres generated as
described in FIG. 15 were dissociated and plated in NSC
self-renewal conditions (NeuroCult.RTM. NS-A Proliferation Kit
(Human, StemCells Technologies) with FGF, EGF BDNF 20 ng/ml;
Heparin 2 .mu.g/ml) to test the expression of the Nestin marker
(Panel A after 48 h, all the cells are Nestin+ and Tuj1-) or plated
in differentiation conditions (NeuroCult.RTM. NS-A differentiation
Kit (Human, StemCells Technologies) with BDNF 20 ng/ml) and stained
for Tuj1 and Nestin at day7 (Panel B, all the cells are Tuj1+ and
few cells are Nestin+).
EXAMPLES
[0048] The method can be illustrated by reference to FIG. 1 herein,
which depicts a method according to the invention being used for
converting human fibroblasts to neural stem cells (NSCs). In this
method human fibroblast were trypsinized at day 0, counted and
their viability determined. Between
1.0.times.10.sup.5-3.0.times.10.sup.5 trypsinized fibroblasts were
then resuspended in the induction medium and the combination of
genes delivered as lentiviruses. At the induction medium polybrene
(hexadimethrine bromide) was added to increase the efficiency of
the lentiviruses transduction. The infection was performed for 15
minutes in an eppendorf tube. In combination with the genes, a
human Nestin GFP reporter was used. Nestin is a well known marker
expressed specifically in NSCs. In the nestin reporter the
fluorescent protein GFP is under expression of the human nestin
promoter (FIG. 2), therefore it allows an easy screen for induced
reprogrammed neural stem cells (irNSCs) GFP+.
[0049] The infected cells were plated in tissue culture plates
using a concentration of 10000-30000 cells/cm.sup.2 in the
appropriate volume of induction medium. At day 1 the total
induction medium was renewed. It was possible to identify some
irNSC GFP+ (FIG. 3) with a clear change in morphology compared to
the human fibroblasts. The irNSCs GFP+ acquired a bipolar and
elongated morphology with a more condensed cytoplasm, typical of
NSCs. Moreover, the irNSCs are growing in a packed monolayer
culture that resembles the typical cell-to-cell interaction
acquired in traditional NSC cultures for activating the
pro-proliferative signal of the Notch pathway. At day 2 the irNSCs
GFP+ were in a more mature state and started to form very packed
clusters of cells. These clusters of irNSCs started to form a
spheroid structure with a dense core containing irNSCs GFP+ (FIG.
4). At day 3 the spheroid structures were completely formed and
started to lift off from the tissue culture plate floating as
neurospheres in the medium. The neurospheres have a dimension of
approximately 20-100 .mu.m with clear borders and a core with a
high density of cells, where it is possible to identify irNSCs GFP+
(FIG. 5).
[0050] To achieve the inventive breakthrough, it was necessary to
use a specific combination of genes. The following list of genes
involved in the maintenance of the NSC property in vivo and in
vitro were retrieved from literature knowledge: Sox2 (Sox
transcription factor and important marker for NSC), Brn2 (POU
domain protein known to bind to Sox proteins. Reported binding of
Sox2 and Brn2 on the nestin promoter. Brn2 KO mice have impairment
of CNS development), Bmi1 (Protein involved in the regulation of
the cell cycle, reported to increase expression of the p21 and p27
inhibitors of the cyclinE/cdk2 complex. CyclinE/cdk2 inhibition
determines the lost of the retinoblastoma protein control on the
cell cycle that results in a fast cell cycle during the
self-renewal state of NSCs), Mash1 (described to be an important
regulator for the proliferation of neural precursors in vivo),
Sox11 (Sox protein reported to be expressed in SGZ in vivo), NCam
(NSC marker in Flow Cytometry and expressed in different regions of
the CNS), Kpna1 (better known as importin alpha5 responsible
together with importin beta for the protein nuclear import in
ectoderm derived tissues).
[0051] All genes were cloned as cDNAs into lentiviruses plasmids,
and subsequently packaged into lentiviruses. The lentiviruses
packaged particles for Sox2, Bmi1, Mash1, Sox11, NCam, Kpna1,
nestin GFP reporter were transduced directly into human
fibroblasts. Different combinations of genes were tested in the
method described above. At day 3 it was possible to evaluate the
success of the production of the irNSCs by counting the
neurospheres generated. Only neurospheres bigger than 50 .mu.m were
taken into account.
[0052] As represented in FIG. 6 the transduction of the nestin
reporter lentivirus without addition of Fasudil to the induction
medium did not reprogram the human fibroblasts to irNSCs. With the
addition of Fasudil to the induction medium the generation of
neurospheres (around 50 .mu.m) and some smaller (around 20 .mu.m,
not counted) were reported.
[0053] Neurospheres generated with our innovative method using the
genes combination: Sox2-Bmi1 were harvested at day 3 and expanded
for further 14 days. Expansion of the irNSCs neurospheres was a
critical step. The neurospheres were cultured using the N2B27
medium supplemented with FGF, EGF, BDNF in special ultra-low non
adherent plates (Corning). In order to achieve a homogenous
population of irNSCs GFP+ neurospheres a cleaning procedure every
2-3 days was applied. During 14 days of expansion some neurospheres
with low density of irNSCs GFP+ were not able to proliferate
properly, most probably due to a contamination by not converted
fibroblasts. Such kind of contaminated neurospheres were fallen
apart in single cells that needed to be removed. At day 14, the
neurospheres were tested for: attachment on poly-ornithine/laminin
coated plates and generation of neuronal-like cells. For the
attachment, 20-40 neurospheres/cm.sup.2 were plated on
poly-ornithine/laminin coated plates in the expansion medium
supplemented just for the first day with Fasudil 10 .mu.M, in order
to improve cell attachment and spreading. At day 1 of culture was
possible to show the attachment and spreading of the neurospheres
(FIG. 7). At the centre of the spreading neurospheres we identified
irNSCs GFP+ with a typical NSC morphology. The neurospheres were
grown for additional three days and then just BDNF was added to the
N2B27 (neuronal differentiating conditions). Upon EGF and FGF
withdrawal the irNSCs changed morphology. They became more
elongated and started to form neurite-like cellular protrusions. At
day 7 of the differentiating conditions cells were fixed and
stained for the neuronal marker tuj1 (FIG. 8).
[0054] Neurospheres expanded with Fasudil have a better morphology
and clear and sharp borders (a hallmark of well-formed
neurospheres, FIG. 9, panel B); without Fasudil the neurospheres
have bleary borders (FIG. 9, panel A). The irNSCs have a typical
NSC morphology and were Sox2 and Nestin positive (FIG. 10).
[0055] FIG. 11 shows that Rock kinase inhibitor Balanol-like-324
compound increases the yield of GFP+ neurospheres.
[0056] These evidences show that the method was able to convert
human fibroblasts to irNSCs based on linked steps of genes
transduction (best combinations: Sox2-Bmi1, Sox2-Bmi1-Mash1,
Sox2-Bmi1-Sox11, Sox2-Bmi1-Emx2, Sox2-Bmi1-Foxg1 and
Sox2-Bmi1-Pax6, see also FIG. 13) and chemically defined medium
induction.
[0057] To increase the yield of irNSCs, human fibroblasts were
pretreated with or without the HDAC inhibitor Valproic Acid (VPA,
2-propyl-pentanoic acid, monosodium salt). Towards this end, the
human fibroblasts were incubated in DMEM/F12 supplemented with FBS
10% and L-glutamine supplemented with 1 mM VPA prior to infection
(FIG. 12).
[0058] FIG. 14 shows the generation of irNSC Neurospheres from
adult human dermal fibroblasts (HDFa).
[0059] FIG. 15 shows the expansion irNSC Neurospheres using a
combination of Ascorbic Acid, Sonic Hedgehog (Shh), Jagged1, DLL4
and FGF8. FIG. 16 shows Immunocytochemistry characterization of
irNSC Neurospheres for the expression of the NSCs markers Nestin
and the early neuronal marker Tuj1.
[0060] Materials and Methods
[0061] Cell Culture:
[0062] Induction Medium: N2B27 (N2B27 is a 1:1 mixture of DMEM/F12
(Gibco, Paisley, UK) supplemented with N2 and B27 (both from Gibco)
supplemented with human EGF (Peprotech) 30 ng/ml, human FGF2 30
ng/ml (Peprotech), human BDNF (Roche) 20 ng/ml and Fasudil
(Calbiochem) 10 .mu.M or Balanol-like-324 compound
(N-{(3R,4R)-4-[4-(2-Fluoro-6-hydroxy-3-methoxy-benzoyl)-benzoylamino]-aze-
pan-3-yl}-4-hydroxy-3,5-dimethyl-benzamide) 2 .mu.M.
[0063] Expansion Medium: N2B27 supplemented with human EGF
(Peprotech) 30 ng/ml, human FGF2 30 ng/ml (Peprotech), human BDNF
(Roche) 20 ng/ml, or
[0064] NeuroCult.RTM. NS-A Proliferation Kit (Human, StemCells
Technologies) with FGF, EGF BDNF 20 ng/ml; Heparin 2 .mu.g/ml;
Balanol-like-324 2 .mu.M; Ascorbic Acid 0.2 mM, SHH (Recombinant
Human Sonic Hedgehog, Catalog Number: 1845SH) 500 ng/ml, FGF8
(Recombinant Human FGF8a Isoform, Catalog Number: 4745F8) 100
ng/ml, DLL4 (Recombinant Human DLL4, Catalog Number: 1506D4) 500
ng/ml, Jagged1 (Recombinant Human Jagged 1 Fc Chimera, Catalog
Number: 1277JG) 500 ng/ml.
[0065] Differentiation Medium: N2B27 supplemented with human BDNF
(Roche) 20 ng/ml, Laminin 2 .mu.g/ml (Invitrogen).
[0066] Human fibroblasts: IMR90 foetal lung fibroblasts (ATCC Lot.
Num. 580229699) or adult human dermal fibroblasts (GIBCO, Cat.
Number: C-013-5C).
[0067] Lentiviruses: Prepackaged, ready-to use lentivirus particles
were obtained from Sigma (Stemgent Reprogramming Lentivirus human
Sox2, Catalog No. ST070012), Genecopeia (human Bmi1 Lentifect
Lentiviral Particles, Catalog Nr. LP-B0015-Lv105; Sox11 Lentifect
Lentiviral Particles, Catalog Nr. LP-MO425-LV105; Mash1 Lentifect
Lentiviral Particles, Catalog Nr. LP-Z0740-LV105; human
Kpna1Lentifect Lentiviral Particles, Catalog Nr. LP-U1286-Lv105;
NCam1 Lentifect Lentiviral Particles, Catalog Nr. LP-Z2645-Lv105)
and SBI Systems Biosciences (Nestin GFP Reporter:
pGreenZeo.TM.-hNestin Transcriptional Reporter Virus,
SR10035VA-1)
[0068] Titers Nestin GFP 1.45*10.sup.5/.mu.l, BMI1
4.3*10.sup.5/.mu.l, Sox2 1.07*10.sup.4/.mu.l, Sox11
3.2*10.sup.6/.mu.l, Mash1 4.7*10.sup.6/.mu.l, NCam
3.3*10.sup.4/.mu.l, Kpna1 1.8*10.sup.5/.mu.l.
[0069] Protocols:
[0070] 1. Generation of the irNSCs: [0071] 200.000 IMR90 human
fibroblasts infected with the lentiviruses for different genes
combination (multiplicity of infection (M.O.I.) used for each
single lentivirus 30) and the reporter nestin GFP lentivirus
(M.O.I. used 10) in an eppendorf with 300 .mu.l induction medium
with polybrene (hexadimethrine bromide, Sigma) 4 .mu.g/ml. [0072]
Incubate at room temperature for 15 min. [0073] Plate the 300 .mu.l
in 1.7 ml induction medium in a well of a 6-well-plate tissue
treated [0074] Day 1, renew the 2 ml of induction medium/each well
[0075] Day 3, harvest of the neurospheres collecting carefully the
2 ml with the floating spheres [0076] Expand the neurospheres2.
Expansion of the neurospheres: [0077] Collect the medium with the
floating neurospheres in 15 ml tubes from 3 wells of a 6-well-plate
[0078] Let the spheres seed down for 10 min [0079] Remove the
supernatant very carefully (single cells will not seed down and are
aspirated with the supernatant) [0080] Resuspend the spheres in 4
ml final volume expansion medium [0081] Plate in a B6 plate ultra
low adherent plate (Corning) [0082] Incubate 2-3 days [0083] Repeat
the expansion procedure every 2-3 days till day 14 from the
generation irNSCs
[0084] 3. Differentiation Neurospheres: [0085] Day 14 generation of
the irNSCs and after the expansion procedure select under the
stereo microscope round neurospheres with clear borders and rich in
irNSCs GFP+ [0086] Plate 40 spheres in a well 24-well-plate
previously coated with poly-ornhitine/laminin using the expansion
medium with addiction of Fasudil 10 .mu.M or Balanol-like-324
compound 2 .mu.M. [0087] The day after renew the expansion medium
without Fasudil/Balanol-like-324 compound 2 .mu.M. [0088] Incubate
for three days [0089] Remove expansion medium and add
differentiating medium [0090] Renew differentiating medium after
3-4 days [0091] Incubate for 3-4 days [0092] Fix cells with PFA 4%
and perform immunostainings
[0093] Protocol Staining irNSCs Neurospheres:
[0094] Neurospheres at day 15 were stained for characterization
with the following antibodies: Mouse Nestin and Rabbit Sox2 O/N and
then the secondary anti-mouse 488 and anti-rabbit 555 for one
hour.
[0095] Primary Antibodies:
[0096] Nestin Mouse, monoclonal, 1/500 dilution (MAB5326
Millipore)
[0097] Sox2 Rabbit, polyclonal, 1/500 dilution (AB5603MI
Millipore)
[0098] Secondary Antibodies:
[0099] Alexa fluor 488, IgG, 1/1000 dilution, Goat anti mouse
(A11029 Invitrogen)
[0100] Alexa fluor 555, IgG 1/1000 dilution, Goat anti rabbit
(A21429 Invitrogen)
Sequence CWU 1
1
101954DNAHomo sapiens 1atgtacaaca tgatggagac ggagctgaag ccgccgggcc
cgcagcaaac ttcggggggc 60ggcggcggca actccaccgc ggcggcggcc ggcggcaacc
agaaaaacag cccggaccgc 120gtcaagcggc ccatgaatgc cttcatggtg
tggtcccgcg ggcagcggcg caagatggcc 180caggagaacc ccaagatgca
caactcggag atcagcaagc gcctgggcgc cgagtggaaa 240cttttgtcgg
agacggagaa gcggccgttc atcgacgagg ctaagcggct gcgagcgctg
300cacatgaagg agcacccgga ttataaatac cggccccggc ggaaaaccaa
gacgctcatg 360aagaaggata agtacacgct gcccggcggg ctgctggccc
ccggcggcaa tagcatggcg 420agcggggtcg gggtgggcgc cggcctgggc
gcgggcgtga accagcgcat ggacagttac 480gcgcacatga acggctggag
caacggcagc tacagcatga tgcaggacca gctgggctac 540ccgcagcacc
cgggcctcaa tgcgcacggc gcagcgcaga tgcagcccat gcaccgctac
600gacgtgagcg ccctgcagta caactccatg accagctcgc agacctacat
gaacggctcg 660cccacctaca gcatgtccta ctcgcagcag ggcacccctg
gcatggctct tggctccatg 720ggttcggtgg tcaagtccga ggccagctcc
agcccccctg tggttacctc ttcctcccac 780tccagggcgc cctgccaggc
cggggacctc cgggacatga tcagcatgta tctccccggc 840gccgaggtgc
cggaacccgc cgcccccagc agacttcaca tgtcccagca ctaccagagc
900ggcccggtgc ccggcacggc cattaacggc acactgcccc tctcacacat gtga
95421332DNAHomo sapiens 2atggcgaccg cagcgtctaa ccactacagc
ctgctcacct ccagcgcctc catcgtgcac 60gccgagccgc ccggcggcat gcagcagggc
gcggggggct accgcgaagc gcagagcctg 120gtgcagggcg actacggcgc
tctgcagagc aacggacacc cgctcagcca cgctcaccag 180tggatcaccg
cgctgtccca cggcggcggc ggcgggggcg gtggcggcgg cggggggggc
240gggggcggcg gcgggggcgg cggcgacggc tccccgtggt ccaccagccc
cctgggccag 300ccggacatca agccctcggt ggtggtgcag cagggcggcc
gcggagacga gctgcacggg 360ccaggcgccc tgcagcagca gcatcagcag
cagcaacagc aacagcagca gcaacagcag 420caacagcagc agcagcagca
gcaacagcgg ccgccgcatc tggtgcacca cgccgctaac 480caccacccgg
gacccggggc atggcggagc gcggcggctg cagcgcacct cccaccctcc
540atgggagcgt ccaacggcgg cttgctctac tcgcagccca gcttcacggt
gaacggcatg 600ctgggcgccg gcgggcagcc ggccggtctg caccaccacg
gcctgcggga cgcgcacgac 660gagccacacc atgccgacca ccacccgcac
ccgcactcgc acccacacca gcagccgccg 720cccccgccgc ccccgcaggg
tccgcctggc cacccaggcg cgcaccacga cccgcactcg 780gacgaggaca
cgccgacctc ggacgacctg gagcagttcg ccaagcagtt caagcagcgg
840cggatcaaac tgggatttac ccaagcggac gtggggctgg ctctgggcac
cctgtatggc 900aacgtgttct cgcagaccac catctgcagg tttgaggccc
tgcagctgag cttcaagaac 960atgtgcaagc tgaagccttt gttgaacaag
tggttggagg aggcggactc gtcctcgggc 1020agccccacga gcatagacaa
gatcgcagcg caagggcgca agcggaaaaa gcggacctcc 1080atcgaggtga
gcgtcaaggg ggctctggag agccatttcc tcaaatgccc caagccctcg
1140gcccaggaga tcacctccct cgcggacagc ttacagctgg agaaggaggt
ggtgagagtt 1200tggttttgta acaggagaca gaaagagaaa aggatgaccc
ctcccggagg gactctgccg 1260ggcgccgagg atgtgtacgg ggggagtagg
gacactccac cacaccacgg ggtgcagacg 1320cccgtccagt ga 13323981DNAHomo
sapiens 3atgcatcgaa caacgagaat caagatcact gagctaaatc cccacctgat
gtgtgtgctt 60tgtggagggt acttcattga tgccacaacc ataatagaat gtctacattc
cttctgtaaa 120acgtgtattg ttcgttacct ggagaccagc aagtattgtc
ctatttgtga tgtccaagtt 180cacaagacca gaccactact gaatataagg
tcagataaaa ctctccaaga tattgtatac 240aaattagttc cagggctttt
caaaaatgaa atgaagagaa gaagggattt ttatgcagct 300catccttctg
ctgatgctgc caatggctct aatgaagata gaggagaggt tgcagatgaa
360gataagagaa ttataactga tgatgagata ataagcttat ccattgaatt
ctttgaccag 420aacagattgg atcggaaagt aaacaaagac aaagagaaat
ctaaggagga ggtgaatgat 480aaaagatact tacgatgccc agcagcaatg
actgtgatgc acttaagaaa gtttctcaga 540agtaaaatgg acatacctaa
tactttccag attgatgtca tgtatgagga ggaaccttta 600aaggattatt
atacactaat ggatattgcc tacatttata cctggagaag gaatggtcca
660cttccattga aatacagagt tcgacctact tgtaaaagaa tgaagatcag
tcaccagaga 720gatggactga caaatgctgg agaactggaa agtgactctg
ggagtgacaa ggccaacagc 780ccagcaggag gtattccctc cacctcttct
tgtttgccta gccccagtac tccagtgcag 840tctcctcatc cacagtttcc
tcacatttcc agtactatga atggaaccag caacagcccc 900agcggtaacc
accaatcttc ttttgccaat agacctcgaa aatcatcagt aaatgggtca
960tcagcaactt cttctggttg a 9814711DNAHomo sapiens 4atggaaagct
ctgccaagat ggagagcggc ggcgccggcc agcagcccca gccgcagccc 60cagcagccct
tcctgccgcc cgcagcctgt ttctttgcca cggccgcagc cgcggcggcc
120gcagccgccg cagcggcagc gcagagcgcg cagcagcagc agcagcagca
gcagcagcag 180cagcaggcgc cgcagctgag accggcggcc gacggccagc
cctcaggggg cggtcacaag 240tcagcgccca agcaagtcaa gcgacagcgc
tcgtcttcgc ccgaactgat gcgctgcaaa 300cgccggctca acttcagcgg
ctttggctac agcctgccgc agcagcagcc ggccgccgtg 360gcgcgccgca
acgagcgcga gcgcaaccgc gtcaagttgg tcaacctggg ctttgccacc
420cttcgggagc acgtccccaa cggcgcggcc aacaagaaga tgagtaaggt
ggagacactg 480cgctcggcgg tcgagtacat ccgcgcgctg cagcagctgc
tggacgagca tgacgcggtg 540agcgccgcct tccaggcagg cgtcctgtcg
cccaccatct cccccaacta ctccaacgac 600ttgaactcca tggccggctc
gccggtctca tcctactcgt cggacgaggg ctcttacgac 660ccgctcagcc
ccgaggagca ggagcttctc gacttcacca actggttctg a 71151325DNAHomo
sapiens 5atggtgcagc aggcggagag cttggaagcg gagagcaacc tgccccggga
ggcgctggac 60acggaggagg gcgaattcat ggcttgcagc ccggtggccc tggacgagag
cgacccagac 120tggtgcaaga cggcgtcggg ccacatcaag cggccgatga
acgcgttcat ggtatggtcc 180aagatcgaac gcaggaagat catggagcag
tctccggaca tgcacaacgc cgagatctcc 240aagaggctgg gcaagcgctg
gaaaatgctg aaggacagcg agaagatccc gttcatccgg 300gaggcggagc
ggctgcggct caagcacatg gccgactacc ccgactacaa gtaccggccc
360cggaaaaagc ccaaaatgga cccctcggcc aagcccagcg ccagccagag
cccagagaag 420agcgcggccg gcggcggcgg cgggagcgcg ggcggaggcg
cgggcggtgc caagacctcc 480aagggctcca gcaagaaatg cggcaagctc
aaggcccccg cggccgcggg cgccaaggcg 540ggcgcgggca aggcggccca
gtccggggac tacgggggcg cgggcgacga ctacgtgctg 600ggcagcctgc
gcgtgagcgg ctcgggcggc ggcggcgcgg gcaagacggt caagtgcgtg
660tttctggatg aggacgacga cgacgacgac gacgacgacg agctgcagct
gcagatcaaa 720caggagccgg acgaggagga cgaggaacca ccgcaccagc
agctcctgca gccgccgggg 780cagcagccgt cgcagctgct gagacgctac
aacgtcgcca aagtgcccgc cagccctacg 840ctgagcagct cggcggagtc
ccccgaggga gcgagcctct acgacgaggt gcgggccggc 900gcgacctcgg
gcgccggggg cggcagccgc ctctactaca gcttcaagaa catcaccaag
960cagcacccgc cgccgctcgc gcagcccgcg ctgtcgcccg cgtcctcgcg
ctcggtgtcc 1020acctcctcgt ccagcagcag cggcagcagc agcggcagca
gcggcgagga cgccgacgac 1080ctgatgttcg acctgagctt gaatttctct
caaagcgcgc acagcgccag cgagcagcag 1140ctggggggcg gcgcggcggc
cgggaacctg tccctgtcgc tggtggataa ggatttggat 1200tcgttcagcg
agggcagcct gggctcccac ttcgagttcc ccgactactg cacgccggag
1260ctgagcgaga tgatcgcggg ggactggctg gaggcgaact tctccgacct
ggtgttcaca 1320tattg 132562547DNAHomo sapiens 6atgctgcaaa
ctaaggatct catctggact ttgtttttcc tgggaactgc agtttctctg 60caggtggata
ttgttcccag ccagggggag atcagcgttg gagagtccaa attcttctta
120tgccaagtgg caggagatgc caaagataaa gacatctcct ggttctcccc
caatggagaa 180aagctcaccc caaaccagca gcggatctca gtggtgtgga
atgatgattc ctcctccacc 240ctcaccatct ataacgccaa catcgacgac
gccggcattt acaagtgtgt ggttacaggc 300gaggatggca gtgagtcaga
ggccaccgtc aacgtgaaga tctttcagaa gctcatgttc 360aagaatgcgc
caaccccaca ggagttccgg gagggggaag atgccgtgat tgtgtgtgat
420gtggtcagct ccctcccacc aaccatcatc tggaaacaca aaggccgaga
tgtcatcctg 480aaaaaagatg tccgattcat agtcctgtcc aacaactacc
tgcagatccg gggcatcaag 540aaaacagatg agggcactta tcgctgtgag
ggcagaatcc tggcacgggg ggagatcaac 600ttcaaggaca ttcaggtcat
tgtgaatgtg ccacctacca tccaggccag gcagaatatt 660gtgaatgcca
ccgccaacct cggccagtcc gtcaccctgg tgtgcgatgc cgaaggcttc
720ccagagccca ccatgagctg gacaaaggat ggggaacaga tagagcaaga
ggaagacgat 780gagaagtaca tcttcagcga cgatagttcc cagctgacca
tcaaaaaggt ggataagaac 840gacgaggctg agtacatctg cattgctgag
aacaaggctg gcgagcagga tgcgaccatc 900cacctcaaag tctttgcaaa
acccaaaatc acatatgtag agaaccagac tgccatggaa 960ttagaggagc
aggtcactct tacctgtgaa gcctccggag accccattcc ctccatcacc
1020tggaggactt ctacccggaa catcagcagc gaagaaaaga ctctggatgg
gcacatggtg 1080gtgcgtagcc atgcccgtgt gtcgtcgctg accctgaaga
gcatccagta cactgatgcc 1140ggagagtaca tctgcaccgc cagcaacacc
atcggccagg actcccagtc catgtacctt 1200gaagtgcaat atgccccaaa
gctacagggc cctgtggctg tgtacacttg ggaggggaac 1260caggtgaaca
tcacctgcga ggtatttgcc tatcccagtg ccacgatctc atggtttcgg
1320gatggccagc tgctgccaag ctccaattac agcaatatca agatctacaa
caccccctct 1380gccagctatc tggaggtgac cccagactct gagaatgatt
ttgggaacta caactgtact 1440gcagtgaacc gcattgggca ggagtccttg
gaattcatcc ttgttcaagc agacaccccc 1500tcttcaccat ccatcgacca
ggtggagcca tactccagca cagcccaggt gcagtttgat 1560gaaccagagg
ccacaggtgg ggtgcccatc ctcaaataca aagctgagtg gagagcagtt
1620ggtgaagaag tatggcattc caagtggtat gatgccaagg aagccagcat
ggagggcatc 1680gtcaccatcg tgggcctgaa gcccgaaaca acgtacgccg
taaggctggc ggcgctcaat 1740ggcaaagggc tgggtgagat cagcgcggcc
tccgagttca agacgcagcc agtccaaggg 1800gaacccagtg cacctaagct
cgaagggcag atgggagagg atggaaactc tattaaagtg 1860aacctgatca
agcaggatga cggcggctcc cccatcagac actatctggt caggtaccga
1920gcgctctcct ccgagtggaa accagagatc aggctcccgt ctggcagtga
ccacgtcatg 1980ctgaagtccc tggactggaa tgctgagtat gaggtctacg
tggtggctga gaaccagcaa 2040ggaaaatcca aggcggctca ttttgtgttc
aggacctcgg cccagcccac agccatccca 2100gccaacggca gccccacctc
aggcctgagc accggggcca tcgtgggcat cctcatcgtc 2160atcttcgtcc
tgctcctggt ggttgtggac atcacctgct acttcctgaa caagtgtggc
2220ctgttcatgt gcattgcggt caacctgtgt ggaaaagccg ggcccggggc
caagggcaag 2280gacatggagg agggcaaggc cgccttctcg aaagatgagt
ccaaggagcc catcgtggag 2340gttcgaacgg aggaggagag gaccccaaac
catgatggag ggaaacacac agagcccaac 2400gagaccacgc cactgacgga
gcccgagaag ggccccgtag aagcaaagcc agagtgccag 2460gagacagaaa
cgaagccagc gccagccgaa gtcaagacgg tccccaatga cgccacacag
2520acaaaggaga acgagagcaa agcatga 254771617DNAHomo sapiens
7atgaccaccc caggaaaaga gaactttcgc ctgaaaagtt acaagaacaa atctctgaat
60cccgatgaga tgcgcaggag gagggaggaa gaaggactgc agttacgaaa gcagaaaaga
120gaagagcagt tattcaagcg gagaaatgtt gctacagcag aagaagaaac
agaagaagaa 180gttatgtcag atggaggctt tcatgaggct cagattagta
acatggagat ggcaccaggt 240ggtgtcatca cttctgacat gattgaaatg
atattttcca aaagcccaga gcaacagctt 300tcagcaacac agaaattcag
gaagctgctt tcaaaagaac ctaaccctcc tattgatgaa 360gttatcagca
caccaggagt agtggccagg tttgtggagt tcctcaaacg aaaagagaat
420tgtacactgc agtttgaatc agcttgggta ctgacaaata ttgcttcagg
aaattctctt 480cagacccgaa ttgtgattca ggcaggagct gtgcccatct
tcatagagtt gctcagctca 540gagtttgaag atgtccagga acaggcagtc
tgggctcttg gcaacattgc tggagatagt 600accatgtgca gggactatgt
cttagactgc aatatccttc cccctctttt gcagttattt 660tcaaagcaaa
accgcctgac catgacccgg aatgcagtat gggctttgtc taatctctgt
720agagggaaaa gtccacctcc agaatttgca aaggtttctc catgtctgaa
tgtgctttcc 780tggttgctgt ttgtcagtga cactgatgta ctggctgatg
cctgctgggc cctctcatat 840ctatcagatg gacccaatga taaaattcaa
gcggtcatcg atgcgggagt atgtaggaga 900cttgtggaac tgctgatgca
taatgattat aaagtggttt ctcctgcttt gcgagctgtg 960ggaaacattg
tcacagggga tgatattcag acacaggtaa ttctgaattg ctcagctctg
1020cagagtttat tgcatttgct gagtagccca aaggaatcta tcaaaaagga
agcatgttgg 1080acgatatcta atattacagc tggaaatagg gcacagatcc
agactgtgat agatgccaac 1140attttcccag ccctcattag tattttacaa
actgctgaat ttcggacaag aaaagaagca 1200gcttgggcca tcacaaatgc
aacttctgga ggatcagctg aacagatcaa gtacctagta 1260gaactgggtt
gtatcaagcc gctctgtgat ctcctcacgg tcatggactc taagattgta
1320caggttgccc taaatggctt ggaaaatatc ctgaggcttg gagaacagga
agccaaaagg 1380aatggcactg gcattaaccc ttactgtgct ttgattgaag
aagcttatgg tctggataaa 1440attgagttct tacagagtca tgaaaaccag
gagatctacc aaaaggcctt tgatcttatt 1500gagcattact tcgggaccga
agatgaagac agcagcattg caccccaggt tgaccttaac 1560cagcagcagt
acatcttcca acagtgtgag gctcctatgg aaggtttcca gctttga
161781470DNAHomo sapiens 8atgctggaca tgggagatag gaaagaggtg
aaaatgatcc ccaagtcctc gttcagcatc 60aacagcctgg tgcccgaggc ggtccagaac
gacaaccacc acgcgagcca cggccaccac 120aacagccacc acccccagca
ccaccaccac caccaccacc atcaccacca cccgccgccg 180cccgccccgc
aaccgccgcc gccgccgcag cagcagcagc cgccgccgcc gccgcccccg
240gcaccgcagc ccccccagac gcggggcgcc ccggccgccg acgacgacaa
gggcccccag 300cagctgctgc tcccgccgcc gccaccgcca ccaccggccg
ccgccctgga cggggctaaa 360gcggacgggc tgggcggcaa gggcgagccg
ggcggcgggc cgggggagct ggcgcccgtc 420gggccggacg agaaggagaa
gggcgccggc gccggggggg aggagaagaa gggggcgggc 480gagggcggca
aggacgggga ggggggcaag gagggcgaga agaagaacgg caagtacgag
540aagccgccgt tcagctacaa cgcgctcatc atgatggcca tccggcagag
ccccgagaag 600cggctcacgc tcaacggcat ctacgagttc atcatgaaga
acttccctta ctaccgcgag 660aacaagcagg gctggcagaa ctccatccgc
cacaatctgt ccctcaacaa gtgcttcgtg 720aaggtgccgc gccactacga
cgacccgggc aagggcaact actggatgct ggacccgtcg 780agcgacgacg
tgttcatcgg cggcaccacg ggcaagctgc ggcgccgctc caccacctcg
840cgggccaagc tggccttcaa gcgcggtgcg cgcctcacct ccaccggcct
caccttcatg 900gaccgcgccg gctccctcta ctggcccatg tcgcccttcc
tgtccctgca ccacccccgc 960gccagcagca ctttgagtta caacggcacc
acgtcggcct accccagcca ccccatgccc 1020tacagctccg tgttgactca
gaactcgctg ggcaacaacc actccttctc caccgccaac 1080ggcctgagcg
tggaccggct ggtcaacggg gagatcccgt acgccacgca ccacctcacg
1140gccgccgcgc tagccgcctc ggtgccctgc ggcctgtcgg tgccctgctc
tgggacctac 1200tccctcaacc cctgctccgt caacctgctc gcgggccaga
ccagttactt tttcccccac 1260gtcccgcacc cgtcaatgac ttcgcagagc
agcacgtcca tgagcgccag ggccgcgtcc 1320tcctccacgt cgccgcaggc
cccctcgacc ctgccctgtg agtctttaag accctctttg 1380ccaagtttta
cgacgggact gtctggggga ctgtctgatt atttcacaca tcaaaatcag
1440gggtcttctt ccaacccttt aatacattaa 147092908DNAHomo sapiens
9cgggcgccgc aggagcgagt gagctgggag cgaggggcga aggcgcggag aagcccggcc
60gcccggtggg cggcagaagg ctcagccgag gcggcggcgc cgactccgtt ccactctcgg
120cccggatcca ggcctccggg ttcccaggcg ctcacctccc tctgacgcac
tttaaagagt 180ctcccccctt ccacctcagg gcgagtaata gcgaccaatc
atcaagccat ttaccaggct 240tcggaggaag ctgtttatgt gatccccgca
ctaattaggc tcatgaacta acaaatcgtt 300tgcacaactt gtgaagaagc
gaacacttcc atggattgtc cttggactta gggcgccctg 360cccgcctttt
gcagaggaga aaaaactttt tttttttttt gcctcccccg agaactttcc
420ccccttctcc tccctgcctc taactccgat ccccccacgc catctcgcca
aaaaaaaaaa 480aaaaaaaaaa aaagaaaaaa aaagaaaaaa aaagaaaaaa
aattacccca atccacgcct 540gcaaattctt ctggaaggat tttcccccct
ctcttcaggt tgggcgcgtt tggtgcaaga 600ttctcgggat cctcggcttt
gcctctccct ctccctcccc cctcctttcc tttttccttt 660cctttccttt
ctttcttcct ttccttcccc ccacccccac ccccacccca aacaaacgag
720tccccaattc tcgtccgtcc tcgccgcggg cagcgggcgg cggaggcagc
gtgcggcggt 780cgccaggagc tgggagccca gggcgcccgc tcctcggcgc
agcatgttcc agccggcgcc 840caagcgctgc ttcaccatcg agtcgctggt
ggccaaggac agtcccctgc ccgcctcgcg 900ctccgaggac cccatccgtc
ccgcggcact cagctacgct aactccagcc ccataaatcc 960gttcctcaac
ggcttccact cggccgccgc cgccgccgcc ggtaggggcg tctactccaa
1020cccggacttg gtgttcgccg aggcggtctc gcacccgccc aaccccgccg
tgccagtgca 1080cccggtgccg ccgccgcacg ccctggccgc ccacccccta
ccctcctcgc actcgccaca 1140ccccctattc gcctcgcagc agcgggatcc
gtccaccttc tacccctggc tcatccaccg 1200ctaccgatat ctgggtcatc
gcttccaagg gaacgacact agccccgaga gtttcctttt 1260gcacaacgcg
ctggcccgaa agcccaagcg gatccgaacc gccttctccc cgtcccagct
1320tctaaggctg gaacacgcct ttgagaagaa tcactacgtg gtgggcgccg
aaaggaagca 1380gctggcacac agcctcagcc tcacggaaac tcaggtaaaa
gtatggtttc agaaccgaag 1440aacaaagttc aaaaggcaga agctggagga
agaaggctca gattcgcaac aaaagaaaaa 1500agggacgcac catattaacc
ggtggagaat cgccaccaag caggcgagtc cggaggaaat 1560agacgtgacc
tcagatgatt aaaaacataa acctaacccc acagaaacgg acaacatgga
1620gcaaaagaga cagggagagg tggagaagga aaaaacccta caaaacaaaa
acaaaccgca 1680tacacgttca ccgagaaagg gagagggaat cggagggagc
agcggaatgc ggcgaagact 1740ctggacagcg agggcacagg gtcccaaacc
gaggccgcgc caagatggca gaggatggag 1800gctccttcat caacaagcga
ccctcgtcta aagaggcagc tgagtgagag acacagagag 1860aaggagaaag
agggagggag agagagaaag agagagaaag agagagagag agagagagag
1920agaaagctga acgtgcactc tgacaagggg agctgtcaat caaacaccaa
accggggaga 1980caagatgatt ggcaggtatt ccgtttatca cagtccactt
aaaaaatgat gatgatgata 2040aaaaccacga cccaaccagg cacaggactt
ttttgttttt tgcacttcgc tgtgtttccc 2100ccccatcttt aaaaataatt
agtaataaaa aacaaaaatt ccatatctag ccccatccca 2160cacctgtttc
aaatccttga aatgcatgta gcagttgttg ggcgaatggt gtttaaagac
2220cgaaaatgaa ttgtaatttt cttttccttt taaagacagg ttctgtgtgc
tttttatttt 2280gatttttttt cccaagaaat gtgcagtctg taaacacttt
ttgatacctt ctgatgtcaa 2340agtgattgtg caagctaaat gaagtaggct
cagcgatagt ggtcctctta cagagaaacg 2400gggagcagga cgacgggggg
gctgggggtg gcgggggagg gtgcccacaa aaagaatcag 2460gacttgtact
gggaaaaaaa cccctaaatt aattatattt cttggacatt ccctttccta
2520acatcctgag gcttaaaacc ctgatgcaaa cttctccttt cagtggttgg
agaaattggc 2580cgagttcaac cattcactgc aatgcctatt ccaaacttta
aatctatcta ttgcaaaacc 2640tgaaggactg tagttagcgg ggatgatgtt
aagtgtggcc aagcgcacgg cggcaagttt 2700tcaagcactg agtttctatt
ccaagatcat agacttacta aagagagtga caaatgcttc 2760cttaatgtct
tctataccag aatgtaaata tttttgtgtt ttgtgttaat ttgttagaat
2820tctaacacac tatatacttc caagaagtat gtcaatgtca atattttgtc
aataaagatt 2880tatcaatatg ccctcaaaaa aaaaaaaa 2908102191DNAHomo
sapiens 10gcgacgaaag agaggatgcc tcttaaaggc agaagacttt aactaggggc
gggcgagcag 60atgtgtgaga tcttctattc aaagagtgga catatagccc agttttcaga
gccacgtatt 120cgagccccgt gggatccgga ggctgccaac cagctccagc
atgcagaaca gtcacagcgg 180agtgaatcag cttggtggtg tctttgtcaa
cgggcggcca ctgccggact ccacccggca 240gaagatcgta gagctagctc
acagcggggc ccggccgtgc gacatttccc gaattctgca 300ggtgtccaac
ggatgtgtga gtaaaattct gggcaggtat tacgagactg gctccatcag
360acccagggca atcggaggca gtaagccaag agtggcgact ccagaagttg
taagcaaaat 420agcccagtat aaacgggagt gcccgtccat ctttgcttgg
gaaatccgag acagattact 480ctccgagggg gtctgtacca acgacaatat
acccagtgtg tcatcaataa acagagttct 540tcgcaacctg gctagcgaaa
agcaacagat gggcgcagac ggcatgtatg ataaactaag 600gatgctgaac
ggacagaccg gaagctgggg cacccgccct ggttggtatc ccgggacgtc
660agtaccaggg caacccacgc aagacggctg ccagcaacag gaaggacagg
gagaaaacac 720caactccatc agctccaatg gagaagactc ggatgaggct
caaatgcggc tgcagctgaa 780gcggaagctg cagagaaata gaacatcttt
tacccaggag cagattgagg ctctggagaa 840agagtttgag aggacccatt
atccagatgt gtttgcccgg gaaagactag cagccaaaat 900agatctacct
gaagcaagga tacaggtgtg gttttctaac cgaagggcca agtggagaag
960agaagaaaaa ctgaggaacc agagaagaca ggccagcaac accccgagtc
acatccctat 1020cagcagcagt ttcagtacca gtgtctacca gccaattcca
cagcccacca cacctgtctc 1080ctcctttaca tcgggttcca tgttgggccg
cacagacacc gccctcacca acacgtacag 1140tgctttgccg ccgatgccca
gcttcaccat ggcaaataac ctgcctatgc aacccccagt 1200ccccagtcag
acctcctcgt actcctgcat gctgcccacc agcccttcag tgaatgggcg
1260gagttatgat acctacaccc ctccgcacat gcaaacacac atgaacagtc
agcccatggg 1320cacctcggga accacttcaa caggactcat ttcacctgga
gtgtcagttc ccgtccaagt 1380tcccggaagt gaacctgaca tgtctcagta
ctggcctcga ttacagtaaa gagagagaaa 1440gagagagaat gtgatcgaga
gggggattgt gttcactcag ccaatgacta tgtggacaca 1500gcggttgggt
attcaggaaa gaaagagaaa tggctgttag aagcacttca ctttacaact
1560gtgtcctata ctggagcccg ggaatggact agaaaccagg acctttgcgt
acagaaggca 1620cggtatcagt tggaacaaat cttcattttg gtatccaaac
ttttattcat tttggtgtat 1680tatttgtaaa tgggcatttg tatgttataa
tgaagaaaag aacaacacag gctgttggat 1740cttggatctg tgttggctca
tgtggttgtt taaaggaaac catgatcgac aagatttgcc 1800atggatttaa
gagttttatc aagatatatc gaatacttct acccatctgt tcatagttta
1860tggactgatg ttccaagttt gtatcattcc tttgcatata attaaacatg
gaacaacata 1920cactagatat atgtaaaaaa tatctgttgg tttttccaaa
ggttgttaac agataaagtt 1980tatgtgcaaa aaagggtaag atatgaattc
gaggagaagt tgatagctaa aaggtagagt 2040gtgtcttcga tataatccaa
tttgttttat gtcaaaatgt aagtatttgt cttccctaga 2100aatcctcaga
atgatttcta taataaagtt aatttcattt atatttgaaa aaaaaaaaaa
2160aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 2191
* * * * *