U.S. patent application number 15/826235 was filed with the patent office on 2018-03-22 for human stem cell-derived neural precursors for treatment of autoimmune diseases of the central nervous system.
The applicant listed for this patent is Hadasit Medical Research Services & Development Limited. Invention is credited to Michal Aharonowiz, Tamir Ben-Hur, Ofira Einstein, Benjamin Reubinoff.
Application Number | 20180080007 15/826235 |
Document ID | / |
Family ID | 40591589 |
Filed Date | 2018-03-22 |
United States Patent
Application |
20180080007 |
Kind Code |
A1 |
Aharonowiz; Michal ; et
al. |
March 22, 2018 |
HUMAN STEM CELL-DERIVED NEURAL PRECURSORS FOR TREATMENT OF
AUTOIMMUNE DISEASES OF THE CENTRAL NERVOUS SYSTEM
Abstract
The present invention concerns the use of a population of cells
comprising: (a) neural precursor cells committed to an
oligodendroglial fate; (b) uncommitted neural precursor cells (c)
differentiated oligodendrocytes; or (d) a combination of any one of
(a) to (c) for the treatment of CNS autoimmune diseases, or for the
preparation of a pharmaceutical composition for treating CNS
autoimmune diseases, the population of cells being derived from
human pluripotent stem cells. The invention also provides methods
for obtaining such populations of cells, namely, neural precursor
cells committed to an oligodendroglial fate as well as
differentiated oligodendrocytes which then can be used in the
treatment of CNS autoimmune diseases. A preferred autoimmune
disease in the context of the present invention is multiple
sclerosis where the population of cells is administered to the CNS
for local treatment of the disease.
Inventors: |
Aharonowiz; Michal; (Modiin,
IL) ; Einstein; Ofira; (Modiin, IL) ;
Reubinoff; Benjamin; (Doar Na Haela, IL) ; Ben-Hur;
Tamir; (Jerusalem, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hadasit Medical Research Services & Development
Limited |
Jerusalem |
|
IL |
|
|
Family ID: |
40591589 |
Appl. No.: |
15/826235 |
Filed: |
November 29, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12740496 |
Apr 12, 2011 |
9862925 |
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PCT/IL08/01426 |
Oct 29, 2008 |
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15826235 |
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61000746 |
Oct 29, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/41 20130101;
A61P 25/00 20180101; C12N 2501/135 20130101; C12N 2501/385
20130101; A61P 29/00 20180101; C12N 2501/105 20130101; C12N 2506/02
20130101; A61P 25/28 20180101; C12N 2501/70 20130101; A61K 35/30
20130101; A61K 2035/122 20130101; C12N 5/0623 20130101; A61P 37/00
20180101; C12N 2501/11 20130101; C12N 2501/115 20130101; C12N
2501/395 20130101; C12N 2501/13 20130101 |
International
Class: |
C12N 5/0797 20060101
C12N005/0797; A61K 35/30 20060101 A61K035/30 |
Claims
1. A method for preparing a population of neural precursor cells
committed to an oligodendroglial fate; comprising: (a) incubating
early multipotent uncommitted neural precursor cells derived from
human pluripotent stem cells with RA and an HH agonist at a first
concentration being between about 0.5 .mu.M and about 2.0 .mu.M to
allow the cells to propagate as floating spheres enriched with
oligodendroglial precursors; (b) allowing the floating spheres to
further expand in a medium comprising a second concentration of HH
agonist that is not more than about 0.5 .mu.M to obtain an expanded
population of neural precursor cells committed to an
oligodendroglial fate.
2. The method of claim 1, comprising plating the obtained expanded
population of neural precursor cells committed to an
oligodendroglial fate on an ECM whereby differentiation of the
neural precursor cells committed to an oligodendroglial fate to
terminally differentiated oligodendrocytes is obtained.
3. The method of claim 1, wherein the HH agonist is
purmorphamine.
4. The method of claim 1, wherein incubation with HH agonist is
performed in the presence of at least one mitogen.
5. The method of claim 1, wherein the first concentration of HH
agonist is about 0.5 .mu.M, and the second concentration of HH
agonist is between about 0.2 .mu.M and about 0.5 .mu.M.
6. The method of claim 2, wherein the plating is performed in the
absence of HH agonist and in the absence of a mitogen.
7. A method for treating a subject having a CNS autoimmune disease,
the method comprising administering to the subject a population of
cells derived from human pluripotent stem cells, the population of
cells comprising: (a) neural precursor cells committed to an
oligodendroglial fate; (b) uncommitted neural precursor cells (c)
differentiated oligodendrocytes; or (d) any combination of two or
more of (a) to (c).
8. The method of claim 7, wherein the population of cells
comprising neural precursors committed to an oligodendroglial fate
are obtainable by: (c) incubating early multipotent uncommitted
neural precursor cells derived from human pluripotent stem cells
with RA and an HH agonist at a first concentration being between
about 0.5 .mu.M and about 2.0 .mu.M to allow the cells to propagate
as floating spheres enriched with oligodendroglial progenitors; (d)
allowing the floating spheres to further expand in a medium
comprising a second concentration of HH agonist that is not more
than about 0.5 .mu.M to obtain an expanded population of cells
comprising neural precursor cells committed to an oligodendroglial
fate.
9. The method of claim 8, further comprising plating the thus
expanded population of neural precursor cells committed to an
oligodendroglial fate on an extracellular matrix thereby allowing
differentiation of the committed cells to terminally differentiated
oligodendrocytes.
10. The method of claim 9, wherein the HH agonist is
purmorphamine.
11. The method of claim 9, wherein incubation with HH agonist is
performed in the presence at least one mitogen.
12. The method of claim 9, wherein the first concentration of HH
agonist is about 0.5 .mu.M, and the second concentration of HH
agonist is between about 0.2 .mu.M and about 0.5 .mu.M.
13. The method of claim 9, wherein the extracellular matrix is
fibronectin.
14. The method of claim 7, for the treatment of a CNS autoimmune
disease associated with an inflammatory reaction.
15. The method of claim 14, wherein the autoimmune disease is an
inflammatory demyelinating disease.
16. The method of claim 15, wherein the disease is multiple
sclerosis.
17. The method of claim 7, comprising administration to a subject
in need of two or more populations of cells derived from human
pluripotent stem cells, the population of cells being selected from
a population of neural precursor cells committed to an
oligodendroglial fate; a population of uncommitted neural precursor
cells, a population of differentiated oligodendrocytes; wherein the
two or more populations of cells being administered together or
separately, simultaneously or in sequence.
18. The method of claim 7, comprising local administration of said
population of cells to the CNS.
19. The method of claim 18, wherein the local administration
comprises transplantation of the population of cells to the lateral
ventricles or intrathecally.
20. A method for producing a population of differentiating neural
precursor cells committed towards oligodendroglial fate, the method
comprising: (a) incubating early multipotent uncommitted neural
precursor cells derived from human pluripotent stem cells with
retinoic acid (RA) and an hedgehog (HH) agonist at a first
concentration between about 0.5 .mu.M and about 2.0 .mu.M to allow
the cells to propagate as floating spheres enriched with
oligodendroglial precursors; and (b) allowing the floating spheres
to further expand in a medium comprising a second concentration of
HH agonist that is not more than about 0.5 .mu.M to obtain an
expanded population of neural precursor cells committed to an
oligodendroglial fate.
21. A method for producing a population of differentiated
oligodendrocyte cells the method comprising: (a) incubating early
multipotent uncommitted neural precursor cells derived from human
pluripotent stem cells with retinoic acid (RA) and an hedgehog (HH)
agonist at a first concentration between about 0.5 .mu.M and about
2.0 .mu.M to allow said early multipotent NPs to propagate as
floating spheres being enriched with oligodendroglial precursors;
(b) allowing the floating spheres to further expand in a medium
comprising a second concentration of HH agonist that is not more
than about 0.5 .mu.M to obtain an expanded population of neural
precursor cells committed to an oligodendroglial fate; and (c)
plating expanded population of neural precursor cells committed to
an oligodendroglial fate on an extracellular matrix thereby
allowing differentiation into oligodendrocytes.
22. The method of claim 20, wherein the HH agonist is
purmorphamine.
23. The method of any one of claim 22, wherein incubation with HH
agonist is performed in the presence of at least one mitogen.
24. The method of claim 23, wherein the first concentration of HH
agonist is about 0.5 .mu.M, and the second concentration of HH
agonist is between about 0.2 .mu.M and about 0.5 .mu.M.
25. The method of claim 21, wherein the extracellular matrix is
fibronectin.
26. The method of any one of claim 21, for obtaining neural
precursor cells committed to an oligodendroglial fate, the cells
expressing at least one of the following markers: Olig1, Olig2,
NG2, PDGFR.alpha., GD3, O4, GalC and MBP, wherein at least Olig2 is
co expressed with one or more of a marker selected from NG2,
PDGFR.alpha. and GD3.
27. The method of claim 21, for obtaining differentiated
oligodendrocytes expressing at least one of the following markers:
Olig1, Olig2, NG2, PDGFR.alpha., GD3, O4, GalC and MBP, where at
least Olig2 is co expressed with one or more of a marker selected
from 04, GalC and MBP.
28. The method of claim 21, wherein the cells thus obtained are
expandable.
29. The method of claim 21, wherein the plating is in the absence
of one or more of an HH agonist and a mitogen.
30. A population of oligodendroglial committed precursor cells
obtainable by incubating floating spheres of early multipotent
neural precursor cells in a medium comprising a concentration of HH
agonist that is not more than about 0.5 .mu.M; the oligodendroglial
committed progenitor cells expressing one or more of the markers
selected from Olig1, Olig2, NG2, PDGFR.alpha., GD3, where at least
Olig2 is co expressed with one or more of a marker selected from
NG2, PDGFR.alpha., GD3.
31. The oligodendroglial committed precursor cells of claim 30,
wherein the oligodendroglial committed precursor cells
are-expandable.
32. A method for promoting differentiation of early multipotent
neural precursor cells towards oligodendroglial fate, the method
comprising propagating floating spheres comprising early
multipotent neural precursors in a medium comprising purmorphamine.
Description
FIELD OF THE INVENTION
[0001] This invention relates to cell therapy and in particular to
the use of human stem cells (hESC) for the production of neural
precursors for treatment of autoimmune diseases.
PRIOR ART
[0002] The following is a list of prior art which is considered to
be pertinent for describing the state of the art in the field of
the invention. Acknowledgement of these references herein will be
made by indicating the number from their list below within
brackets. [0003] Reubinoff, B. E., et al., Embryonic stem cell
lines from human blastocysts: somatic differentiation in vitro. Nat
Biotechnol, 2000. 18(4): p. 399-404. [0004] Thomson, J. A., et al.,
Embryonic stem cell lines derived from human blastocysts. Science,
1998. 282(5391): p. 1145-7. [0005] Brustle, O., et al., Embryonic
stem cell-derived glial precursors: a source of myelinating
transplants. Science, 1999. 285(5428): p. 754-6. [0006] Keirstead,
H. S., et al., Human embryonic stem cell-derived oligodendrocyte
progenitor cell transplants remyelinate and restore locomotion
after spinal cord injury. J Neurosci, 2005. 25(19): p. 4694-705.
[0007] Liu, S., et al., Embryonic stem cells differentiate into
oligodendrocytes and myelinate in culture and after spinal cord
transplantation. Proc Natl Acad Sci USA, 2000. 97(11): p. 6126-31.
[0008] Zhang, P. L., et al., Increased myelinating capacity of
embryonic stem cell derived oligodendrocyte precursors after
treatment by interleukin-6/soluble interleukin-6 receptor fusion
protein. Mol Cell Neurosci, 2006. 31(3): p. 387-98. [0009] Kang, S.
M., et al., Efficient Induction of Oligodendrocytes from Human
Embryonic Stem Cells. Stem Cells, 2006. 25(2) p: 419-24 [0010]
Einstein, O., et al., Intraventricular transplantation of neural
precursor cell spheres attenuates acute experimental allergic
encephalomyelitis. Mol Cell Neurosci, 2003. 24(4): p. 1074-82.
[0011] Einstein, O., et al., Transplanted neural precursor cells
reduce brain inflammation to attenuate chronic experimental
autoimmune encephalomyelitis. Exp Neurol, 2006. 198(2): p. 275-84.
[0012] Pluchino, S., et al., Neurosphere-derived multipotent
precursors promote neuroprotection by an immunomodulatory
mechanism. Nature, 2005. 436(7048): p. 266-71. [0013] Itsykson, P.,
et al., Derivation of neural precursors from human embryonic stem
cells in the presence of noggin. Mol Cell Neurosci, 2005. 30(1): p.
24-36. [0014] Billon, N., et al., Normal timing of oligodendrocyte
development from genetically engineered, lineage-selectable mouse
ES cells. J Cell Sci, 2002. 115(Pt 18): p. 3657-65. [0015] Nistor,
G. I., et al., Human embryonic stem cells differentiate into
oligodendrocytes in high purity and myelinate after spinal cord
transplantation. Glia, 2005. 49(3): p. 385-96.
BACKGROUND OF THE INVENTION
[0016] Multiple sclerosis (MS) is a chronic immune mediated disease
of the central nervous system (CNS), which is the leading cause for
neurological disability in young adults. The pathological process
of MS includes immune cell infiltrations, oligodendrocyte death,
demyelination and axonal damage. Several pathological and imaging
studies indicate that the chronic disability is attributed mainly
to axonal damage. Axonal damage in MS occurs in the early phase of
the disease, in actively demyelinating lesions. In later stages of
the disease, however, an ongoing, low grade axonal degeneration
occurs in silent inactive plaques but not in remyelinated
axons.
[0017] Spontaneous remyelination is a regular feature at early
stages of lesion formation in some MS cases. Nevertheless, the
remyelination process eventually fails due to environmental factors
and intrinsic properties of progenitor cells.
[0018] The potential of Human embryonic stem cells (hESC) [1, 2] to
differentiate into oligodendroglial cells was demonstrated both
with mouse and human ES cells [3-7]. Moreover, the potential of ES
cells-derived neural progeny to remyelinate in genetic models of
hypo/dysmyelination and in models of focal demyelination was shown
[8-11].
[0019] Transplanted cells may have a therapeutic effect in CNS
autoimmune disorders not only by serving as a source of cells for
regeneration, but also by immunomodulation and
attenuation/abolishment of the inflammatory process. Einstein et al
show that rodent fetal brain-derived neural precursor cells (NPC)
transplanted into the ventricles decrease brain inflammation [12].
Similarly, peripherally injected rodent brain-derived NPC migrate
into white matter and decrease brain inflammation [14]. Pluchino et
al. show that intravenously injected, adult rodent brain-derived
NPC, promote functional recovery in a chronic model of MS
(Experimental Autoimmune Encephalomyelitis, EAE) [15]. In a later
publication, Pluchino et al [14] show that adult rodent
brain-derived NPC promote neuroprotection using immune-like
functions, e.g. induce apoptosis of encephalitogenic T cells,
exerting their effect within the CNS. Einstein et al [13] show that
intravenous injection of rodent-fetal-derived NPC attenuates EAE by
interacting with the peripheral immune system.
[0020] Also describes is a system for regulating the immune
response in the context of regenerative medicine or treatment of
autoimmune disease, e.g. multiple sclerosis. The inventors propose
administering undifferentiated human ES cells at the site of the
pathology in an attempt to inhibit an immune response. However,
since one of the inherent properties of undifferentiated ES cells
is to generate tumors, this approach is probably not suitable for
use in vivo, and hence immune modulation by cell therapy requires a
different approach [23].
SUMMARY OF THE INVENTION
[0021] It is an object of the present invention to provide for
various uses of neural precursors derived from human pluripotent
stem cells for treating CNS inflammatory conditions, such as CNS
autoimmune disorders.
[0022] The present disclosure is based on the finding that
transplantation of hESC-derived neural precursors attenuates the
clinical and pathological features of myelin oligodendrocyte
glycoprotein (MOG) EAE at least by an immunosuppressive mechanism.
Further it was found that after transplantation into the site of
inflammation which is the CNS in EAE mice, hESC-derived neural
precursors were capable of migrating and integrating in the host
CNS, and differentiating towards an oligodendroglial lineage. The
unique combination of therapeutic advantages of hESC-derived neural
precursor cells (committed or uncommitted) and/or of differentiated
oligodendrocytes propagated therefrom underlies their use as a
novel form of cell therapy.
[0023] Thus, in accordance with one aspect, there is provided the
use of a population of cells comprising: (a) neural precursor cells
committed to an oligodendroglial fate; (b) uncommitted neural
precursor cells (c) differentiated oligodendrocytes; or (d) any
combinations of two or more of (a) to (c) for the treatment of CNS
autoimmune diseases, said population of cells being derived from
human pluripotent stem cells.
[0024] There is also provided the use of a population of cells
comprising: (a) neural precursor cells committed to an
oligodendroglial fate; (b) uncommitted neural precursor cells (c)
differentiated oligodendrocytes; or (d) any combinations of two or
more of same for the preparation of a pharmaceutical composition
for the treatment of CNS autoimmune diseases, said population of
cells being derived from human pluripotent stem cells.
[0025] In another aspect there is provided a method for preparing a
population of neural precursor cells committed to an
oligodendroglial fate; comprising: [0026] (a) incubating early
multipotent uncommitted neural precursor cells derived from human
pluripotent stem cells with RA and an HH agonist at a first
concentration being between about 0.5 .mu.M and about 2.0 .mu.M to
allow the cells to propagate as floating spheres enriched with
oligodendroglial precursors; [0027] (b) allowing the floating
spheres to further expand in a medium comprising a second
concentration of HH agonist that is not more than about 0.5 .mu.M
to obtain an expanded population of neural precursor cells
committed to an oligodendroglial fate.
[0028] An feature of the invention includes the use of a second
concentration of HH being not more than 0.5 .mu.M.
[0029] For the preparation of a population of cells comprising
differentiated oligodendrocytes, the thus obtained expanded
population of neural precursor cells committed to an
oligodendroglial fate is plated on an ECM thereby allowing
differentiation into said differentiated oligodendrocytes. A
preferred method includes plating in the absence of HH agonist and
in the absence of mitogens. A cocktail of survival an maturation
factors is preferentially used, such as that detailed in the
Materials and Methods.
[0030] The invention also concerns a method for treating a subject
having a CNS autoimmune disease, the method comprising
administering to said subject a population of cells derived from
human pluripotent cells, the population of cells comprising: (a)
neural precursor cells committed to an oligodendroglial fate; (b)
uncommitted neural precursor cells (c) differentiated
oligodendrocytes; or (d) any combination of two or more of (a) to
(c).
[0031] A feature of the method of treatment concerns the local
administration of the population of cells, namely, the
transplantation of the cells in the CNS, specifically, to the
lateral ventricles and/or intrathecally.
[0032] The invention also provides a pharmaceutical composition for
the treatment of a CNS autoimmune disease, comprising a population
of cells comprising: (a) neural precursor cells committed to an
oligodendroglial fate; (b) uncommitted neural precursor cells (c)
differentiated oligodendrocytes; or (d) a combination of any one of
(a) to (c), said population of cells being derived from human
pluripotent stem cells.
[0033] Yet, the invention provides a method for producing a
population of differentiating neural precursor cells committed
towards oligodendroglial fate, the method comprising: [0034] (a)
incubating early multipotent uncommitted neural precursor cells
derived from human pluripotent stem cells with retinoic acid (RA)
and an hedgehog (HH) agonist at a first concentration between about
0.5 .mu.M and about 2.0 .mu.M to allow the cells to propagate as
floating spheres enriched with oligodendroglial precursors; [0035]
(b) allowing the floating spheres to further expand in a medium
comprising a second concentration of HH agonist that is not more
than about 0.5 .mu.M to obtain an expanded population of neural
precursor cells committed to an oligodendroglial fate.
[0036] Also provided is a method for producing a population of
differentiated oligodendrocyte cells the method comprising the
above steps followed by plating expanded population of neural
precursor cells committed to an oligodendroglial fate on an
extracellular matrix (in the absence of HH agonist or mitogens)
thereby allowing differentiation into oligodendrocytes.
[0037] The invention also provides a population of oligodendroglial
committed precursor cells obtainable, and preferably obtained, by
incubating floating spheres of early multipotent uncommitted neural
precursor cells in a medium comprising a concentration of HH
agonist that is not more than about 0.5 .mu.M; said
oligodendroglial committed progenitor cells expressing one or more
of the markers selected from Olig1, Olig2, NG2, PDGFR.alpha., GD3,
where at least Olig2 is co expressed with one or more of a marker
selected from NG2, PDGFR.alpha., GD3. A unique feature of these
cells is that they are expandable.
[0038] Finally, there is provided a method for promoting
differentiation of early multipotent uncommitted neural precursor
cells towards oligodendroglial fate, the method comprising
propagating floating spheres comprising early multipotent
uncommitted neural precursors in a medium comprising
purmorphamine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] In order to understand the invention and to see how it may
be carried out in practice, embodiments will now be described, by
way of non-limiting example only, with reference to the
accompanying drawings, in which:
[0040] FIGS. 1A-1F are fluorescent images showing characteristics
of hESC-derived neural precursors generated in accordance with the
present disclosure for transplantation, the characteristics
including expression of A2B5 (FIG. 1A), Musashi (FIG. 1B), Nestin
(FIG. 1C) and PSA-NCAM (FIG. 1D) by over 90% of the cells, and
characteristics of these cells following seven days of
differentiation showing that the neural precursors differentiated
mainly into .beta.III tubulin expressing neurons (FIG. 1E) and GFAP
expressing astrocytes (FIG. 1F). Cells expressing oligodendroglial
markers were not detected.
[0041] FIG. 2 is a graph showing a significant inhibition of the
clinical parameters in transplanted animals (.box-solid.) in
comparison to control animals (.tangle-solidup.) following
transplantation of hESC-derived neural precursors.
[0042] FIGS. 3A-3J: are images showing Immunofluorescence stainings
of brain sections demonstrating the survival and differentiation of
transplanted neural precursors, which were identified by the
expression of human mitochondria (FIGS. 3A, 3C-3G), human nuclei
(FIG. 3B) and GFP (inset in FIG. 3A, 2H). (A): The neural
precursors migrated extensively into white matter areas of the CNS
such as the corpus callosum (CC) and were not observed in grey
areas such as subcortical grey matter (SGM). Costaining against the
oligodendroglial marker, O4 (red) was used to identify the white
matter. Also shown are images from transverse semi-thin sections
cut from resin embedded spinal cords of transplanted (FIG. 3J) and
control (FIG. 3I) animals. Arrows indicate demyelinated axons.
[0043] FIG. 4 provides linear regression analysis of neural
precursors transplanted and control mice at 13 and 20 days post EAE
induction showing in both groups in the acute phase of EAE a strong
correlation between the numbers of T cells and macrophages per
mm.sup.2 and the percentage of axonal loss. (r.sup.2=0.86,
P=0.00002).
[0044] FIGS. 5A-50 show evolution of inflammation, demyelination
and axonal damage in the CNS of hESC-derived neural
precursors-transplanted versus control EAE mice exhibited by
immune-cell infiltrates (FIG. 5A), T cells (FIG. 5D) and
macrophages/activated microglia (FIG. 5G) Kluver Barrera staining
indicative of demylination (FIG. 5J) and Bielschowsky staining
indicative of axonal damage (FIG. 5M), as well as representative
images of H&E staining (FIG. 5B-5C), CD3 (FIG. 5E-5F) and Mac3
immunostaining (FIG. 5H-5I), Kluver Barrera staining (FIG. 5K-5L)
and Bielschowsky silver staining (FIG. 5N-5O)
[0045] FIG. 6A-6E show suppressive effects of hESC-derived neural
precursors on lymph node cells (LNCs) and T cells derived from
naive C57BL mice as exhibited by neural precursors suppression of
.sup.3H-thymidine incorporation into the activated LNCs (FIG. 6A),
as well as by FACS analysis of interleukin-2 receptor .alpha.
(IL-2R.alpha.; CD25) expression after 24 hours of ConA-stimulation
showing the inhibitory effect of human neural precursors on Thy1.2+
T cells as determined by the fraction of labeled cells and by mean
fluorescent intensity (MFI) (FIGS. 6B-6C); and FACS analysis of
CFSE labeled LNCs after 72 hours of ConA stimulation showing the
inhibitory effect of human neural precursors on proliferation of
Thy1.2+ T cells (FIGS. 6D-6E).
[0046] FIGS. 7A-7I are fluorescent images showing oligodendroglial
differentiation of hESCs in vitro following retinoic acid
treatment, exhibited by expression of the (oligodendrocyte
precursor cells) OPCs markers NG2 (12%) (FIGS. 7A,7C,7D,7F) GD3
(20%) (FIGS. 7B, 7C) and PDGFR.alpha. (15%) (FIGS. 7E, 7F), as well
as of the mature oligodendrocyte markers, O4 (FIG. 7G, 7I) and GalC
(FIG. 7H, 7I). Nuclei in FIGS. 7C, 7F, 7G-7I are counter stained
with DAPI.
[0047] FIG. 8A-8H are fluorescence images of oligodendroglial
differentiation of hESCs in vitro following treatment with retinoic
acid and purmorphamine and propagation in low purmorphamine
concentrations, as exhibited by expression of the OPCs markers
Olig2 (30%) (FIG. 8A), PDGFR.alpha. (20%) (FIG. 8B) and NG2 (20%)
(FIG. 8C) as well as markers of mature oligodendrocytes O4 (20%)
(FIG. 8D), GalC (15%) (FIG. 8E) and MBP (3%) (FIG. 8F), as well as
the expression of Olig2 (FIG. 8G) and O4 (FIG. 8H) in the absence
of low purmorphamine concentrations
DETAILED DESCRIPTION OF EMBODIMENTS
[0048] The present invention stems from a sequence of empiric
findings by the inventors which led to the development of novel
methods and products as detailed below. Specifically, empiric data
was collected concerning the effect of neural precursors derived
from human embryonic stem cells (hESCs) on a chronic model of
multiple sclerosis (MS), namely, EAE.
[0049] MS is the prototype of several related immune-mediated CNS
diseases that are relevant to the present disclosure. The
pathological process of many CNS-associated immune diseases, such
as MS, involves immune cell infiltrations, oligodendrocyte death,
demyelination and axonal damage. Thus, it has been envisages by the
inventors that there would be a therapeutic benefit if conditions
that contribute to both the process of remyelination and
immunosuppression are provided. Moreover, it has been envisaged by
the inventors that it would be especially advantageous to deliver
the effect in a targeted fashion to the involved tissue, namely,
local treatment as opposed to systemic administration.
[0050] Transplantation of a population of cells derived from human
pluripotent stem cells and comprising neural precursors that
include oligodendroglia-committed cells and/or oligodenrocytes may
be used both for suppression of the inflammatory process and thus,
halting the disease progression, as well as for oligodendrocytes
and myelin regeneration. The integration of such cells in host
tissue and their selective migration to inflamed sites may be
utilized to deliver the beneficial effect specifically to the
inflamed sites of disease.
[0051] The present disclosure generally concerns the use of cells
derived from human pluripotent stem cells, e.g. human embryonic
stem cells (hESC) or induced pluripotent stem cells (iPS cells) for
targeted (tissue-specific) suppression of inflammatory processes
associated with CNS-autoimmune diseases, such as MS, as well as for
in situ regeneration of oligodendrocyte population, by
transplantation of the cells to the lateral ventricle or
intrathecally. Thus immunosuppressive effects are combined with
remyelination capabilities.
[0052] Accordingly, by a first of its aspects, the present
disclosure provides the use of a population of cells comprising:
(a) neural precursor cells committed to an oligodendroglial fate;
(b) uncommitted neural precursor cells (c) differentiated
oligodendrocytes; or (d) a combination of any one of (a) to (c) for
the treatment of autoimmune diseases of the central nervous system
(CNS), or for the preparation of a pharmaceutical composition for
said treatment, the population of cells being derived from human
pluripotent stem cells.
[0053] In the context of the present invention the human
pluripotent stem cells include, without being limited thereto,
human embryonic stem cells (hESC), human induced pluripotent stem
cells (iPS cells) or any other "reprogrammed" human cell being
capable of differentiating towards a desired fate, i.e. towards
oligodendroglial fate
[0054] The population of cells comprises uncommitted neural
precursors having well defined characteristics. Such
characteristics include, without being limited thereto, expression
of one or more of the following markers: A2B5 (FIG. 1A), Musashi
(FIG. 1B), Nestin (FIG. 1C) and PSA-NCAM (FIG. 1D) as well as the
potential to differentiate into .beta.III tubulin expressing
neurons (FIG. 1E) and GFAP expressing astrocytes. As known in the
art these uncommitted neural precursors differ from the
stem/progenitor cells derived from the brain of fetal tissues (16).
Conditions suitable for inducing hESCs differentiation towards
neural precursors are known in the art, such as from Itsykson, P.,
et al., (17). The minimal conditions for obtaining uncommitted
neural precursors from hESCs are culturing small clusters of hESCs
in suspension in a chemically defined medium that promote the
culture of neural precursors. Such chemically defined mediums are
known in the art. The process of neutralization may be augmented by
supplementation with noggin. The process of neutralization is also
promoted by supplementation with FGF2 or FGF2+EGF.
[0055] When referring to a population of neural precursors derived
from human pluripotent stem cells it is to be construed that at
least 40%, preferably 70% and more preferably above 90% of the
cells exhibit at least one characteristic of neural precursors as
provided above.
[0056] The population of cells may include committed as well as
uncommitted neural precursors. When referring to committed cells it
is meant cells committed to an oligodendroglial fate. At times,
neural precursor cells committed to an oligodendroglial fate will
be referred to as oligodendroglial progenitors. The population of
cells may also include (in addition or alternatively) terminally
differentiated oligodendrocytes, derived from human pluripotent
stem cells, as will be explained below.
[0057] In the context of the present disclosure, "cells committed
to an oligodendroglial fate" are to be construed as human
pluripotent stem cells that under appropriate conditions will
differentiate into oligodendrocytes. Cells committed to an
oligodendroglial fate have characteristics that distinguish them
from uncommitted neural precursors. Such characteristics include,
without being limited thereto, the oligodendroglial markers Olig1,
Olig2, NG2, PDGFR.alpha., GD3, O4, GalC and MBP.
[0058] The population of cells may be employed for the treatment of
various acute and chronic CNS-autoimmune diseases that exhibits at
least one inflammatory component. The diseases may include, without
being limited thereto, stroke and ischemic damage to the nervous
system; neural trauma (e.g., closed and penetrating injuries to the
brain and spinal cord); multiple sclerosis and its variants such as
neuromyelitis optica, acute disseminated encephalomyelitis (ADEM)
and transverse myelitis; Guillain-Barre syndrome and its variants,
acute motor axonal neuropathy, Fisher Syndrome and other
immune-mediated neuropathies (e.g.,); Amyotropic Lateral Sclerosis
(ALS).
[0059] The term "central nervous system" refers to all structures
within the dura mater. Such structures include, but are not limited
to, the brain and spinal cord.
[0060] The term "treatment" as used herein is to be construed as
referring to protective treatment (i.e. prophylactice, in terms of
preventing or partially preventing the CNS autoimmune disease) as
well as therapeutic in terms of a partial or complete cure of the
disease or adverse effect attributed to the disease. The term
"treatment", as used herein, includes: (a) preventing the disease
from occurring in a subject which may be predisposed to the disease
but has not yet been diagnosed as having it, i.e., causing the
clinical symptoms of the disease not to develop in a subject that
may be predisposed to the disease but does not yet experience or
display symptoms of the disease; (b) inhibiting the disease, i.e.,
arresting or reducing the development of the disease or its
clinical symptoms; or (c) relieving the disease, i.e., causing
regression of the disease and/or its symptoms or conditions.
[0061] More specifically, "treatment" in the context of the present
invention is such that it would result in one or both of
immunosuppressive effect and remyelination in the site of
inflammation so as to prevent damage to an inflammed tissue or
improve the condition of the damaged tissue. Improvement may result
in the inhibition or cesation of damage caused to the damaged
tissue as well as regeneration of damage already existing.
[0062] In one embodiment, the CNS autoimmune disease is associated
with an inflammatory reaction, such as an inflammatory
demyelinating disease. One particular disease in accordance with
this embodiment is multiple sclerosis (MS).
[0063] In one embodiment, the population of cells is enriched with
cells committed to an oligodendroglial fate. In the same or another
embodiment, the population of cells comprises uncommitted neural
precursors. In yet the same or different embodiment, the population
of cells comprises cells terminally differentiated
oligodendrocytes. In all embodiments, a common inventive feature is
that all cells are derived from human pluripotent stem cells,
preferably from human embryonic stem cells.
[0064] The population of cells comprising neural precursors
committed to an oligodendroglial fate are obtainable, preferably
obtained, without being limited thereto, by the following
procedure: [0065] (a) incubating early multipotent uncommitted
neural precursor cells derived from human pluripotent stem cells
(which, as indicated above, may be obtained as described by
Itsykson, P., et al., (18) or by any other method known in the art,
some of which is referred to above), with retinoic acid (RA, at a
concentration of between 0.5 to 20 .mu.M, preferably at about 1
.mu.M) and an hedgehog (HH) agonist at an HH concentration between
0.5 .mu.M and 2.0 .mu.M preferably 0.5 .mu.M, to allow said early
multipotent NPs to propagate as floating spheres being enriched
with oligodendroglial precursors; [0066] (b) allowing the floating
spheres to further expand in a medium comprising a second
concentration of HH agonist that is not more than about 0.5 .mu.M,
preferably between about 0.2 .mu.M and 0.5 .mu.M, to obtain an
expanded population of neural precursorcells committed to an
oligodendroglial fate.
[0067] As indicated above, the population of cells may in addition
or alternatively comprise differentiated oligodendrocytes. Such
differentiated oligodendrocytes may be obtained by: plating the
thus expanded population of cells on a culture matrix, namely,
extracellular matrix (ECM), such as laminin or fibronectin, the
latter being preferably, in the absence of HH agonist and mitogens,
thereby allowing differentiation into said differentiated
oligodendrocytes.
[0068] In one embodiment, the HH agonist, being preferably Sonic HH
agonist, is selected from purmorphamine, Hh-Ag1.3 (Curis Company)
and others.
[0069] In one embodiment, the incubation with HH agonist is
performed in the presence of at least one mitogen. The term
"mitogen" is known in the art as any chemical substance that
induces cell division, i.e. triggers mitosis. Non-limiting mitogens
to be used in accordance with the method disclosed herein, are bFGF
and EGF or PDGF.
[0070] A surprising finding underlying the present invention is
based on empiric data where hESC-derived neural precursors were
administered to the target site, namely, to the lateral ventricle
and the cells selectively migrated to the inflamed/damaged area. In
other words, in difference with hitherto described or proposed
treatments of CNS-autoimune diseases making use of stem cell
derived neural precursors being administered systemically, the
present disclosure has established an effective local treatment for
CNS-autoimmune conditions. Those versed in the art of medicine
would readily appreciate the advantages of local treatment vs.
systemic treatment, at least in terms of side effects. Essentially,
systemic immunosuppressive treatments render the entire body to be
susceptible to infections that invaded the body or rose from the
microbial flora that resides constantly in the body, as well as to
malignant tumors. Here, the anti-inflammatory effect of the
population of cells as defined herein are targeted specifically to
the disease sites, thus avoiding any systemic complications.
Targeting of cell therapy is achieved by both the direct delivery
of cells to the central nervous system, as well as the nature of
cells in the defined population to be attracted and to migrate
towards the inflamed CNS tissue.
[0071] The present disclosure also pertains to a method for
preparing a population of neural precursor cells committed to an
oligodendroglial fate comprising [0072] (a) incubating early
multipotent uncommitted neural precursor cells derived from human
pluripotent stem cells with RA and an HH agonist at a first
concentration being between about 0.5 .mu.M and about 2.0 .mu.M to
allow the cells to propagate as floating spheres enriched with
oligodendroglial precursors; [0073] (b) allowing the floating
spheres to further expand in a medium comprising a second
concentration of HH agonist that is not more than about 0.5 .mu.M
to obtain an expanded population of neural precursor cells
committed to an oligodendroglial fate
[0074] For preparing a population of cells comprising
differentiated oligodendrocytes, a further step is required, which
includes plating the thus obtained expanded population of neural
precursor cells committed to an oligodendroglial fate on an ECM, in
the absence of HH agonist and mitogens, thereby allowing
differentiation into said differentiated oligodendrocytes.
[0075] Further provided herein is a method for treating a subject
having a CNS autoimmune disease, preferably those associated with
an inflammatory reaction, e.g. inflammatory demyelinating disease,
the method comprising administering to said subject a population of
cells derived from human pluripotent stem cells as described above.
The method in accordance with the present disclosure is preferably
for the treatment of multiple sclerosis (MS).
[0076] The method of treatment may involve administration to a
subject having a CNS-autoimmune disease with more than one
population of cells derived from human pluripotent stem cells, the
populations may be selected from a population of cells comprising
uncommitted cells, another population comprising neural precursor
cells committed to an oligodendroglial fate; and yet a further
population comprising differentiated oligodendrocytes; the
populations may be administered together or separately,
simultaneously or in sequence (with an interval of minutes, hours
or even days or weeks). The method may also include administration
of a population of cells comprising a mixture of cells derived from
human pluripotent stem cells selected from precursor cells
committed to an oligodendroglial fate, uncommitted neural
precursors, and terminally differentiated oligodendrocytes.
[0077] Treatment in accordance with the present disclosure
involves, preferably, local administration (transplantation) of the
population of cells in accordance with the invention to the CNS. To
this end, the treatment cells may be formulated in a form suitable
so or intrathecally. It has been established by empiric data, such
as that presented for the first time herein, that cells derived
from human pluripotent stem cells, specifically, from hESC and
transplanted to the lateral ventricle migrated essentially
exclusively to the inflamed site.
[0078] It is noted that the neural precursors migrate to the target
site whereby they may be retained as uncommitted precursors, or
committed into common bipotential neuronal/oligodendroglial
precursors, or further differentiated into neuronal pogenitors,
oligodendroglial-commited precursors, astrocytes, and/or mature
oligodendrocytes.
[0079] The cells may be administered by various techniques known in
the art of cell transplantation. These include intrathecal
injection into the spinal subarachnoid space and intraventricualr
injection, as performed for insertion of an Omaya reservoir or a
ventriculostomy, and similar methods,
[0080] Treatment in accordance with the invention may include a
single administration or several administrations of the populations
of cells in intervals of days, weeks as well as of months. The
several administrations may also include administrations of the
same or different populations. For instance, one or more
administrations may include a population enriched with neural
precursors committed to an oligodendroglial fate, and other
administrations may include terminally differentiated
oligodendrocytes.
[0081] In accordance the present disclosure the amount of cells in
the population to be transplanted is determined by methods known in
the art of cell transplantation. The amount must be effective to at
least attenuate the inflammatory response, namely to at least
achieve an immunosuppressive effect on the CNS inflammatory
reaction, thereby achieving improvement in the condition of the
subject undergoing the treatment.
[0082] The amount will depend, inter alia, on the type and severity
of the autoimmune disease to be, the site and method of
administration, scheduling of administration, patient age, sex,
body weight and other factors known to medical practitioners. The
effective amount is typically determined in appropriately designed
clinical trials (dose range studies) and the person versed in the
art will know how to properly conduct such trials in order to
determine the required amount.
[0083] Also provided by the present disclosure are pharmaceutical
compositions for the treatment of a CNS autoimmune disease,
comprising a pharmaceutically acceptable carrier and the population
of NP cells derived from hESC as disclosed herein. The
pharmaceutically acceptable carrier may include an ECM, such as
fibronectin, laminin or any other medium required for the viability
of the cells during the process of transplantation.
[0084] Also provided herein is a method for producing a population
of differentiating neural precursor cells committed towards
oligodendroglial fate, the method comprising: [0085] (a) incubating
early multipotent uncommitted neural precursor cells derived from
human pluripotent stem cells with retinoic acid (RA) and an
hedgehog (HH), the HH agonist at a first concentration between
about 0.5 .mu.M and about 2.0 .mu.M to allow the cells to propagate
as floating spheres being enriched with oligodendroglial
precursors; [0086] (b) allowing the floating spheres to further
expand in a medium comprising a second concentration of HH agonist
that is not more than about 0.5 .mu.M to obtain an expanded
population of neural precursor cells committed to an
oligodendroglial fate.
[0087] Also provided is a method for producing a population of
differentiated oligodendrocyte, comprising: [0088] (a) incubating
early multipotent uncommitted neural precursor cells derived from
human pluripotent stem cells with retinoic acid (RA) and an
hedgehog (HH), the HH agonist at a first concentration between
about 0.5 .mu.M and about 2.0 .mu.M to allow the cells to propagate
as floating spheres being enriched with oligodendroglial
precursors; [0089] (b) allowing the floating spheres to further
expand in a medium comprising a second concentration of HH agonist
that is not more than about 0.5 .mu.M to obtain an expanded
population of neural precursor cells committed to an
oligodendroglial fate; and [0090] (c) plating expanded population
of neural precursor cells committed to an oligodendroglial fate on
an extracellular matrix, in the absence of HH agonist and mitogens,
thereby allowing differentiation into oligodendrocytes.
[0091] As used herein "differentiating cells" denotes cells that
are capable of differentiating into other cell types having a
particular, specialized function.
[0092] As used herein "differentiated oligodendrocytes" denotes
mature cells that have fully differentiated into such cells
(terminally differentiated), exhibiting the specialized function
and characteristics of oligodendrocytes.
[0093] The method of producing a population of differentiating
cells committed towards oligodendroglial fate or differentiated
oligodendrocytes preferably makes use of the following components:
[0094] the use of HH or its agonist; where the HH agonist is
preferably purmorphamine; [0095] incubation with HH agonist is
preferably, although not exclusively, performed in the presence of
at least one mitogen; [0096] the first concentration of HH agonist
is preferably, although not exclusively about 0.5 .mu.M, and the
second concentration of HH agonist is between about 0.2 .mu.M and
about 0.5 .mu.M; [0097] the second concentration of HH agonist
allows expansion of the population of oligodendroglial committed
precursors. [0098] to obtain differentiated oligodendrocytes, the
committed precursor cells are plated on an ECM being preferably,
but not exclusively, fibronectin in the absence of HH agonist and
mitogens;
[0099] The cells obtained may be characterized by the expression of
at least one of the following markers: Olig1, Olig2, NG2,
PDGFR.alpha., GD3, O4, GalC and MBP. Committed NPs to
oligodendroglial fate may be characterized by the expression of
Olig2 and at least one of NG2, PDGFR.alpha., and GD3. Further, the
neural precursors committed to oligodendroglial fate may be
characterized by their capability to expand in the presence of low
(not more than 0.5 .mu.M) HH agonist. Differentiated
oligodendrocytes are characterized by the expression of Olig2 and
at least one of 04, GalC and MBP.
[0100] Also provided by the present invention is a population of
oligodendroglial committed precursor cells obtainable by incubating
floating spheres of early multipotent uncommitted neural precursors
in a medium comprising a concentration of HH agonist that is not
more than about 0.5 .mu.M; said oligodendroglial committed
precursor cells expressing one or more of the markers selected from
Olig1, Olig2, NG2, PDGFR.alpha., GD3, where at least Olig2 is
co-expressed with one or more of NG2, PDGFR.alpha., GD3. The
oligodendroglial committed precursor cells can expand, under
suitable conditions.
[0101] Finally, there is provided by the present invention a method
for promoting differentiation of early multipotent uncommitted
neural precursors towards oligodendroglial fate, the method
comprising propagating for a period of at least 3 weeks, 5 weeks, 8
weeks, 12 weeks, and even 3 months, of multipotent NPs in a medium
comprising purmorphamine, the cells being preferably, although not
only exclusively, cultured as floating spheres. It is noted that
for the first time purmorphamine is used for oligodendroglial
differentiation, which allows the propagation of the committed
precursor cells for such a long period of time, thus increasing the
probability that the committed cells will successfully terminally
differentiate into oligodendrocytes.
DESCRIPTION OF SOME NON-LIMITING EXAMPLES
Materials and Methods:
[0102] hESC Culture:
[0103] hESC (HES-1 cell line) with a stable normal (46XX) karyotype
were cultured on human foreskin feeders in serum free medium as
described (18) and were passaged weekly by treatment with
collagenase IV (1 mg/ml for 20 min at 37'C).
[0104] Generation of Highly Enriched Populations of Uncommitted
Neural Precursors for Transplantation:
[0105] wild type and cloned genetically modified hESCs that were
infected by a lentiviral vector expressing eGFP under the human
EF1.alpha. promoter [19] were used for derivation of uncommitted
neural precursors for transplantation into EAE mice.
[0106] Colonies of undifferentiated hESCs were removed from the
feeders by treatment with collagenase IV (1 mg/ml for 20 min at
37.degree. C.), transferred to 24-well culture dishes (Costar;
Corning, Inc., Corning, N.Y., USA), and cultured in suspension in a
chemically defined neural precursor medium (NPM) consisting of
DMEM/F12 (1:1), B27 supplement (1:50), 2 mM glutamine, 50 units/ml
penicillin, 50 .mu.g/ml streptomycin (Gibco), and 20 ng/ml rh-FGF-2
(R&D Systems Inc., Minneapolis, Minn.). Recombinant mouse
noggin (700 ng/ml; R&D Systems Inc.) was added to the NPM to
promote neural differentiation as described [18]. After three weeks
under these culture conditions the neural spheres that developed
[38] were further expanded in NPM and bFGF in the absence of
noggin, for 5 more weeks before transplantation.
[0107] Animals:
[0108] For MOG EAE induction and transplantation experiments, 6-7
weeks old C57BL female mice were supplied by Harlan laboratories
and were maintained in a specific pathogen free (SPF) unit.
[0109] MOG EAE Induction:
[0110] EAE was induced in 6-7 weeks old female C57B/6 mice by
immunization with an emulsion containing 300 .mu.g of purified
myelin oligodendrocyte glycoprotein (MOG) peptide
(MEVGWYRSPFSRVVHLYRNGK, the amino acid sequence of the MOG peptide
corresponding to residues 35-55) in PBS and an equal volume of
complete Freund's adjuvant containing 5 mg H37RA (Difco). 0.2 ml of
the inoculum was injected subcutaneously at day of induction (day
0) and at day 7. In addition, 300 ng of Bordetella pertusis toxin
(Sigma) in 0.2 ml PBS was injected intraperitoneally at day of
induction and at day 2.
[0111] Clinical Evaluations of CEAE:
[0112] After CEAE induction, mice were scored daily for CEAE
clinical signs, according to the following score: 0, asymptomatic;
1, partial loss of tail tonicity; 2, atonic tail; 3, hind leg
weakness and/or difficulty to roll over; 4, hind leg paralysis; 5,
four leg paralysis; 6, death due to EAE.
[0113] At the end of the follow-up period, the maximal score and
the cumulative score of each animal were calculated. Maximal
clinical score was calculated as the mean of the maximal clinical
scores during the experimental period. Cumulative clinical score
was calculated as the mean of the sum of the daily clinical scores
during the experimental period.
[0114] Transplantation of Uncommitted Neural Precursors:
[0115] Seven days post EAE induction (day 7) the mice were
anesthetized with intraperitoneal injection of pentobarbital (0.6
mg/10 gr) and were fixed in a stereotactic device. Quantities of
5.times.10.sup.5 cells or NPM in a volume of 7.5 .mu.l were
injected into each lateral ventricle.
[0116] Tissue Fixation and Histological Preparation:
[0117] For analysis of the in-vivo localization and differentiation
of the transplanted cells, EAE animals were sacrificed at the end
of the follow-up period (50 days post-EAE induction). For
histopathological analysis of the progression of inflammation and
tissue damage in the time course experiment animals were sacrificed
at 10, 13, 20 and 50 days post EAE induction (n=4-5 per group on
each time point). Animals were anesthetized with a lethal dose of
pentobarbital and brains and spinal cords were perfused via the
ascending aorta with ice-cold PBS followed by cold 4%
paraformaldehyde in PBS. The tissues were dissected and post-fixed
by immersion in the same fixative for 24 h at 4.degree. C. Brains
were deep frozen in liquid nitrogen and cut to serial 6-8 .mu.M
axial and longitudinal sections and spinal cords were embedded in
paraffin for pathological analysis.
[0118] Pathological Analysis:
[0119] Analysis of inflammation, demyelination and axonal damage
was performed on 5 .mu.m paraffin-embedded serial transverse
sections in three different rostrocaudal levels of the spinal cord.
For histochemical analysis, sections were stained with hematoxylin
and eosin, Luxol fast blue/periodic-acid Schiff staining, and
Bielschowsky silver impregnation to assess inflammation,
demyelination, and axonal pathology, respectively. In adjacent
serial sections, immunohistochemistry was performed with antibodies
against macrophages/activated microglia (rat anti-mouse Mac3,
01781D, clone M3/84; 1:200; Pharmingen, San Diego, Calif.) and T
cells (rat anti-human CD3, MCA 1477; 1:400, Serotec, Bicester,
United Kingdom). Primary antibodies were detected by the
avidin-biotin technique using biotin conjugated secondary
antibodies. The total average number of positive cells per square
millimeter, in spinal cord cross sections, was counted using a grid
overlay.
[0120] Apoptosis of T cells in the CNS was determined
morphologically by the appearance of condensed and fragmented
nuclei in CD3+ cells. The percentage of apoptotic cells was
determined in transplanted and control animals (n=3 in each group)
by morphological analysis of 250 CD3+ cells in random CNS
sections.
[0121] Demyelination and axonal damage were assessed in spinal cord
sections by calculating the area of Luxol fast blue and
Bielschowsky silver staining loss, representing areas of myelin
destruction and axonal loss, respectively. The percentage of
demyelinated and axonal damage areas was determined by counting
intersections of the grid over the demyelinated lesions and the
areas of axonal loss.
[0122] For the evaluation of remyelination, animals were perfused
with 4% gluteraldehyde. The fixed spinal cords were cut into 1 mm
transverse blocks from the cervical, thoracic and lumbar areas. The
blocks were osmicated, dehydrated through an ascending series of
ethanols and embedded in TAAB resin. One .mu.m sections were cut
from each block, stained with toluidine blue (Sigma), and examined
by light microscopy.
[0123] To determine whether transplantation had an effect on
remyelination axons from toluidine blue stained spinal cord
semi-thin sections were measured, and their G ratios were
calculated (G=axon diameter/(axon+myelin sheath diameter)). The G
ratio of intact axons is 0.5-0.8. Since the myelin sheath is
thinner in remyelinated axons, an axon with a G ratio >0.8 was
considered remyelinated.
[0124] Immunofluorescent Staining of Uncommitted Neural Precursors
In Vitro and In Vivo:
[0125] The following primary antibodies were used: Rabbit IgG anti
GFP (1:100, Chemicon), mouse IgG anti human specific mitochondria
(1:200, Chemicon), mouse IgM anti-A2B5 (1:1, ATCC), mouse IgM anti
PSA-NCAM (1:200, Chemicon), rabbit IgG anti-nestin (1:50,
Chemicon), rabbit anti musashi (1:100, Chemicon), Anti human NUC,
rabbit IgG anti-NG2 (1:50, Chemicon), mouse IgM anti-PDGFR.alpha.
(1:20, R&D), rabbit IgG anti NGN2 (1:300, Chemicon), rabbit
anti-galactocerebroside (GalC, 1:20, Chemicon), mouse IgM anti-O4
(1:20, Chemicon), rabbit IgG anti MAP2 (1:200, Chemicon), mouse IgG
anti .beta. tubulin III (1:2000, Sigma) rabbit anti-glial
fibrillary acidic protein (GFAP, 1:100, Dako), rabbit IgG anti
olig1 (1:20, Chemicon) and goat anti olig2 (1:30, R&D). Texas
red or Alexa 488-conjugated goat anti-mouse IgM (1:100, Jackson,
West Grove, PN), goat anti-rabbit IgG (1:100, Molecular Probes),
goat anti-mouse IgG (1:100, Molecular Probes) or donkey anti-goat
IgG (1:200, Jackson, West Grove, PN) were used as secondary
antibodies, where appropriate.
[0126] For in-vitro characterization of the cells that were
generated for transplantation, small aggregates of cells were
plated on poly-D-lysine (10 .mu.g/ml) and fibronectin (5 .mu.g/ml;
both from Sigma) pre-coated cover slips in a central well plates in
NPM without growth factors. Half of the cultures were fixated in 4%
paraformaldehyde After 4 hours and stained for A2B5, nestin,
Musashi and PSA-.psi.NCAM. The rest of the cultures were fixed
after 7 days of differentiation, and stained for .beta. tubulin III
and GFAP. The cell surface markers NG2, O4, and GalC were stained
in living cells followed by fixation in 4% paraformaldehyde. The
cells were incubated with primary antibody for 45 min followed by
30 min incubation with a secondary antibody. Mounting medium
containing 4V, 6-diamidino-2-phenylindole (DAPI; Vector,
Burlingame, Calif.) was used for nuclei counter staining.
[0127] For characterization of the in-vivo location and
differentiation of the transplanted cells, double immunofluorescent
staining was performed on 6-8 .mu.m axial frozen brain sections.
The sections were incubated with primary antibody overnight at
4.degree. C. followed by 50 min incubation with a secondary
antibody at room temperature.
[0128] Images were taken by a fluorescent (Nikon E600, Kanagawa,
Japan) or confocal microscope (Zeiss, Feldbach, Switzerland). Three
hundred cells were scored within random fields at .times.1000
magnification using the Nikon E600 fluorescent microscope. The
percentage of each cell phenotype was determined by dividing the
number of positively stained cells by the total number of DAPI
stained nuclei.
[0129] Co-Cultures of hESC-Derived Uncommitted Neural Precursors
and LNCs:
[0130] Lymph nodes were excised from naive mice. LNCs were cultured
as single-cell suspensions, as described previously 20 with 2.5
.mu.g/ml ConA or in control medium. Neural precursors were
irradiated with 3,000 Rad for 1 minute and then added directly to
the LNC culture medium with nonstimulated or stimulated LNCs
(10.sup.5 NPs/2.times.10.sup.5 LNCs)
[0131] In-Vitro Proliferation Assay:
[0132] The proliferation of LNCs after 72-hour incubation in-vitro
was evaluated by means of a standard .sup.3H-thymidine
incorporation assay, as described previously (20), In all
fluorescent activated cell sorter (FACS) experiments, cells were
pre-coated with anti--mouse CD16/CD32 (BD Pharmingen,) to block
unspecific binding, and T cells were identified by cell-surface
labeling with APC-labeled anti-Thy1.2 (BD Pharmingen). All samples
were analyzed in a FACSCalibur apparatus using the Cell Quest
software (BD Biosciences, San Jose, Calif.). The proliferation of T
cells obtained from naive mice was evaluated by FACS analysis for
the incorporation of the cell division tracking dye
5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), as
described previously (21), For CFSE FACS analysis, LNCs were pulsed
with 3 .mu.M CFSE (Molecular Probes, Eugene, Oreg.) for 10 minutes,
washed, and further cultured with or without ConA for 72 hours.
CFSE-labeled, non-activated cells were used as control samples. The
fraction of T cells that entered cell cycle was calculated by the
formula:
total events in cycle n 2 n ( for n .gtoreq. 1 ) Total events in
cycle n 2 n ( for n .gtoreq. 0 ) ##EQU00001##
[0133] T-cell activation was analyzed by staining with PE-labeled
anti-CD25 (Serotec, Bicestar, United Kingdom) for interleukin-2
receptor .alpha. (IL-2R.alpha.),
[0134] Statistical Analysis:
[0135] Clinical evaluations of CEAE mice and quantification of the
pathological features were performed by examiner, blinded to the
experimental group. The results are presented as mean.+-.SD. For
comparison of clinical and pathological parameters between
experimental and control groups, student's t-test was used. For
analysis of the relationship between the inflammatory process and
the tissue damage, regression analysis was used.
[0136] Derivation of Enriched Population of Committed
Oligodendrocyte Progenitor Cells (OPCs):
[0137] Initial neural differentiation of hESC was induced as
described above for the derivation of enriched population of
uncommitted neural precursors. Following two weeks of culturing of
hESC clusters in NPM supplemented with noggin (700 ng/ml), and the
oligodendroglial mitogens bFGF (20 ng/ml) and EGF (20 ng/ml), the
cultures were treated with retinoic acid (RA) (sigma) (10 .mu.M) to
induce caudal specification of the neuralized cells. Following 7
days of RA treatment, cultures were propagated as floating spheres
in the oligodendroglial specific, modified Sato medium [22]
supplemented with the oligodendroglial mitogens bFGF (20 ng/ml),
EGF (20 ng/ml) and PDGF-AA (50 ng/ml), and the oligodendroglial
survival and maturation factors neurotrophine 3 (NT3) (5 ng/ml) and
triiodothyronine (T3) (40 ng/ml).
[0138] To derive an expandable population of neural precursors
enriched for oligodendroglial progenitors (committed), initial
neuralization of hESCs was induced as above with or without noggin
supplementation. After 2 weeks of culturing in NPM supplemented
with mitogens+/-noggin the clusters were treated with RA (1-10
.mu.M; preferably 1 .mu.M) and the hedgehog agonist, Purmurphamine
(Merck) (0.5-2 .mu.M; preferably 0.5 .mu.M) to induce
ventral-caudal specification of the neuralized cells. Following
7-21 days, preferably 21 days of treatment, the cultures were
further propagated for three to 20 more weeks as floating spheres
in modified Sato medium supplemented with low concentrations of
purmorphamine (0.2-0.5 .mu.M; preferably 0.5 .mu.M), bFGF (20
ng/ml), EGF (20 ng/ml), (NT3; 5 ng/ml) and ascorbic acid (200 nM).
For final differentiation, the clusters were plated on
poly-D-Lysine (10 .mu.g/ml) and fibronectin (5 .mu.g/ml) coated
coverslips in modified Sato medium supplemented with NT3 (5 ng/ml),
ascorbic acid (200 nM) T3 (40 ng/ml) IGF1 (long/ml;) with or
without Rock inhibitor (10 .mu.M; Sigma) for 2-21 days.
Results:
[0139] Characterization of hESC-Derived Neural Progeny
[0140] To evaluate the therapeutic potential of transplanted
hESC-derived neural progeny in the animal model of MS, uncommitted
neural precursors were derived from hESC by culturing clusters of
undifferentiated hESC in suspension in chemically defined medium
supplemented with noggin, bFGF and EGF and after 3 weeks further
propagated in the same medium without noggin supplementation. It
was demonstrated that these neural precursors were multipotent and
can give rise both in vivo and in vitro to progeny representing the
three major neural lineages including neurons, astrocytes and
oligodendrocytes.
[0141] Prior to the transplantation of the hESC-derived neural
precursors into the brain ventricles of chronic EAE rats, the
characterization of their differentiation in vitro was once again
established, which was consistent with previous published results
[17]. Enriched populations of neural precursors in floating spheres
were generated by culturing hESC clusters in serum free medium
supplemented with noggin for 3 weeks. The spheres were further
expanded 5 weeks in the same medium supplemented with mitogens
(e.g. bFGF and EGF) before transplantation. The human neurospheres
that were prepared for transplantation were highly enriched with
uncommitted neural precursors, as indicated by the expression of
A2B5, Musashi, nestin and PSA-NCAM (FIGS. 1A-1D, respectively). At
this point differentiation of the neural precursors was induced by
plating the spheres on fibronectin-coated coverslips and by mitogen
withdrawal. Immunofluorescent staining, performed 7 days later,
demonstrated that the neural precursors differentiated mainly into
neurons and astrocytes, as was indicated by the expression of the
neuronal marker .beta.III tubulin by 67% of the differentiating
cells and the astrocyte marker GFAP by 12% of the cells (FIGS.
1E-1F). The expression of the oligodendroglial marker, O4 by
differentiating cells, was marginal (<0.01%, not shown).
The Effect of Transplanted hESC-Derived Uncommitted Neural
Precursors on Clinical Course of EAE:
[0142] GFP expressing hESC-derived neural precursors were
transplanted into the brain ventricles of chronic MOG EAE mice. As
described in the Materials and Methods sections, the transplanted
(n=15) and control (n=21) groups of MOG EAE mice were scored daily
during a 38 days period for clinical signs of EAE (FIG. 2).
Statistical analysis of the clinical scores revealed that
transplanted hESC-derived NPs significantly attenuated the clinical
signs of EAE, as was indicated by reduced maximal clinical scores
and reduced cumulative scores in transplanted (.box-solid.) versus
control (.tangle-solidup.) animals (FIG. 2 and Table 1). The
severity of the disease was measured by calculating maximal
clinical score, and cumulative clinical score (defined in the notes
below(.sup.1,2)), for transplanted and control groups. Both
clinical parameters of transplanted animals were significantly
improved in comparison to controls. It is noted that reduced
clinical signs in transplanted animals were evident as early as the
acute phase of the disease.
TABLE-US-00001 TABLE 1 Parameters of EAE severity in transplanted
and control animals cells transplanted (N = 15) Control (N = 21) P
value Max clinical score.sup.1 2.65 .+-. 1.32 3.95 .+-. 1.25 0.0044
Cumulative clinical 58.06 .+-. 53.57 93.89 .+-. 51.58 0.0475
score.sup.2 .sup.1Maximal clinical score = Mean of the maximal
clinical scores during the experimental period. .sup.2Cumulative
clinical score = Mean of the sum of the daily clinical scores
during the experimental period.
In-Vivo Localization and Differentiation Fate of Transplanted
hESC-Derived Neural Precursors:
[0143] After the 38 days period of the behavioral follow up
(described above), the transplanted and control animals were
sacrificed for histopathological analysis. Immunofluorescent
staining of brain sections demonstrated that the transplanted
neural precursors, identified by the expression of human-specific
mitochondria (FIGS. 3A, 3C-3G), /human nuclei antigens (FIG. 3B) or
by expression of GFP (FIGS. 3A (insert), 3H) survived in the brain
tissue.
[0144] The neural precursors migrated extensively from the brain
lateral ventricles exclusively into white matter areas such as the
corpus callosum and the periventricular white matter (CC in FIG.
3A) and were not observed in grey matter areas such as subcortical
grey matter (SGM in FIG. 3A). Costaining with anti-O4 which is an
oligodendroglial marker, was used to identify the white matter
(FIG. 3A). Nuclei were counterstained with DAPI. The high migratory
properties of hESCs-derived neural precursors in response to
inflammation is believed to be central to any beneficial effect of
transplanted cells whether by their own regenerative potential or
by reducing the disease process in their local surrounding.
[0145] Immunofluorescent stainings demonstrated that most of the
transplanted cells either remained as uncommitted precursors,
identified by the expression of the RNA binding protein, Musashi
(FIG. 3B) or committed into common bipotential
neuronal/oligodendroglial precursors, expressing the bHLH
transcription factors Olig1 (FIG. 3C) and Olig2 (FIG. 3D). Some
transplanted cells further differentiated into neuronal precursors
expressing NGN2 (FIG. 3E), oligodendroglial progenitors, expressing
NG2 (FIG. 3F), astrocytes expressing GFAP (FIG. 3G) and mature
oligodendrocytes expressing markers such as GalC (FIG. 3H). The
incidence of differentiation into these cell types was .about.1%
per cell type.
[0146] Serial H&E-stained sections covering the entire brain
did not reveal teratomas or any other tumor formation in
transplanted mice.
[0147] These data showed that the transplanted hESC-derived neural
precursors had the potential to undergo differentiation in vivo
towards several cell types including oligodendrocytes and therefore
they may be used for regeneration and remyelination in MS. However,
the use in these studies, of an experimental animal model which
allows only very limited remyelination, and consequently the
relatively small amount of differentiation into mature
oligodendrocytes highlighted that mechanisms other than
remyelination by the transplanted cells may underline the observed
therapeutic effect in this specific model.
[0148] To determine whether transplantation had an effect on
endogenous remyelination we calculated the G ratios of axons from
toluidine blue stained spinal cord semi-thin sections. Axons with a
G ratio >0.8 were considered remyelinated. Analysis of the G
ratios values of axons from the transplanted and control groups
revealed more demyelinated axons in the non-transplanted group
(FIGS. 31-3J) and a number of remyelinated axons in both groups
(FIGS. 31-3J). Albeit low remyelination, it is believed that
subject to specific conditions selected, local transplantation of
hESC-derived neural precursors (committed and/or uncommitted) will
result in a meaningful remyelination preferably to an extent at
least as equal to the protective anti-inflammatory effect obtained
thereby or above (see below).
Transplanted hESC-Derived Neural Precursors Attenuate the
Inflammatory Process and the Progression of Host Tissue Damage
[0149] A time course experiment was performed in which the
evolution of the inflammatory process and the tissue damage was
compared in neural precursor-transplanted and control EAE mice.
Following the induction of MOG EAE and transplantation as described
above, mice from transplanted and control groups were sacrificed at
4 time points which represented 4 critical stages in the course of
EAE: Day 10 in which immune cells begin to infiltrate the CNS
although the disease does not yet manifest clinically; Day 13 in
which there are early clinical signs and inflammation is more
robust; Day 20 which represents the peak of the acute phase of MOG
EAE; and Day 50 which represents the chronic phase. In each time
point (n=5 per time point) histochemical and pathological analysis
of spinal cord sections was performed to quantify the severity of
inflammation, demyelination and axonal damage.
[0150] To measure the extent of inflammation, the numbers of immune
cell infiltrations, numbers of CD3+ T cells and numbers of Mac3+
macrophages/activated microglia per mm.sup.2 of the sections were
examined. Demyelination and axonal injury were measured by luxol
fast blue loss and Bielschowsky staining, respectively.
[0151] Regression analysis of T cells and macrophages the areas of
axonal loss at days 13 and 20 post EAE induction demonstrated that
in both transplanted and control groups the extent of the
inflammatory process was strongly correlated to the severity of
tissue damage at these time points (r.sup.2=0.86, P=0.00002, FIG.
4). An evidence for initial infiltration of immune cells into the
CNS was detected in both groups as early as 10 days post EAE
induction. However, the numbers of CD3+ cells and Mac3+ cells were
significantly decreased in the transplanted animals, starting from
Day 13 and Day 20 post EAE induction, respectively (FIGS. 5A, 5D,
5G, and Table 2). The difference between the groups in the numbers
of immune cells in the CNS even increased at the later time points
examined (Table 2). In addition, axonal damage and demyeliantion
were first detected in both groups at day 13 post induction (FIGS.
5J, 5M and Table 2). Both parameters became significantly reduced
in the transplanted group as compared to the controls at day 20
post induction and the difference between the groups in the amount
of axonal damage even increased at the last time point we examined
(FIGS. 5J, and 5M, and Table 2). To summarize, FIGS. 5A-50 show
that in neural precursors-transplanted mice an attenuation of the
inflammatory process, indicated by less immune-cell infiltrates
(FIG. 5A), less T cells (FIG. 5D) and less macrophages/activated
microglia (FIG. 5G) was evident from Day 13 post EAE induction and
became significant from Day 20 and on. Demyelination, indicated by
loss of Kluver Barrera staining (FIG. 5J) and axonal damage,
indicated by Bielschowsky staining (FIG. 5M) were both
significantly reduced from Day 20 post EAE induction and on.
Representative images of H&E staining FIGS. 5B, 5C), CD3 (FIGS.
5E, 5F) and Mac3 immunostaining (FIGS. 5H-5I), Kluver Barrera
staining (FIGS. 5K-5L) and Bielschowsky silver staining (FIGS.
5N-50) taken from Day 20 post EAE induction, demonstrate the
reduction in transplanted versus control mice in the numbers of
immune cell infiltrates, T cells, macrophages, areas of
demyelination and areas of axonal damage, respectively.
[0152] Quantification of apoptotic CD3+ T cells in the
histopathological sections showed 3.2.+-.2.4% pyknotic T cell
nuclei in control EAE CNS and 2.7.+-.2.5% in transplanted CNS.
[0153] Thus, the effect of transplantation was not mediated by
induction of T-cell apoptosis in the CNS of EAE mice. This
determination supports the understanding that the neural
progenitors provide a protective effect (by preventing immune cells
from penetrating the CNS) and that there is no induction of
apoptosis of immune cells in the CNS.
TABLE-US-00002 TABLE 2 Histopathological analyses of inflammatory
parameters, demyelination, and axonal damage in the spinal cord of
C57BL/6 mice at 10, 13, 20, and 30 days after MOG35-55 EAE
induction Pathological parameter Control EAE (n = 5) Transplanted
(n = 5) P Value Inflammation No. Immune cell Day 10 p.i.* 0.18 .+-.
0.2 0.1 .+-. 0.13 0.5 infiltrations mm.sup.2 Day 13 p.i.* 1.43 .+-.
0.5 1.15 .+-. 0.4 0.32 (H&E) Day 20 p.i.* 7.1 .+-. 1.4 5.22
.+-. 0.6 0.044 Day 50 p.i.* 5.1 .+-. 0.9 2.41 .+-. 0.7 0.006 No.
CD3 + T Cells/ Day 10 p.i.* 2.5 .+-. 2.9 1.66 .+-. 2.3 0.65
mm.sup.2 Day 13 p.i.* 19.16 .+-. 4.2 14.66 .+-. 4.3 0.047 Day 20
p.i.*0 152.9 .+-. 22.7 120.2 .+-. 11.1 0.016 Day 50 p.i.* 108.05
.+-. 20.9 57.26 .+-. 13 0.029 No. Day 10 p.i.* 4.79 .+-. 6 3.5 .+-.
4.8 0.73 MAC3 + Macrophages/ Day 13 p.i.* 40.2 .+-. 8.5 34.16 .+-.
5.8 0.24 mm.sup.2 Day 20 p.i.* 240 .+-. 32.9 190 .+-. 19.8 0.027
Day 50 p.i.* 179.44 .+-. 15.9 106.04 .+-. 22.5 0.005 Axonal
Pathology % Axonal Day 10 p.i.* 0 0 -- injury/Section Day 13 p.i.*
1.3 .+-. 0.57 1.23 .+-. 0.53 0.758 (Bielschowsky) Day 20 p.i.* 4.65
.+-. 1 3.41 .+-. 0.4 0.038 Day 50 p.i.* 5.22 .+-. 0.83 3.58 .+-.
0.25 0.024 Demyelination % Demyelination/ Day 10 p.i.* 0 0 --
Section (Klyver Day 13 p.i.* 0.68 .+-. 0.15 0.6 .+-. 0.14 0.409
Barrera - PAS) Day 20 p.i.* 4.2 .+-. 0.36 2.82 .+-. 0.5 0.002 Day
50 p.i.* 4.36 .+-. 0.24 2.85 .+-. 1.4 0.002 *post injection
[0154] The above data demonstrated the potential of the
hESC-derived neural precursors to differentiate in vivo towards the
oligodendroglial fate and the potential of the transplanted
hESC-derived neural precursors to attenuate the inflammatory
process and consequently the host neural parenchymal pathology of
EAE mice.
hESC-Derived Neural Precursors Inhibit Activation and Proliferation
of Lymph-Node Cells, in Response to Concavalin A (ConA).
[0155] It was previously demonstrated that neural precursors
derived from brains of newborn mice exhibited a bystander
inhibitory effect on T-cell activation and proliferation in vitro
(13). In order to determine whether the hESC-derived neural
precursors have similar immunosuppressive properties, the neural
precursors were co-cultured with lymph-node cells (LNCs). First,
the .sup.3H-thymidine incorporation assay was employed to test
whether hESC-derived NPs exert a direct suppressor effect on the
in-vitro proliferation of LNCs obtained from naive C57BL mice. The
human neural precursors inhibited LNC proliferation in response to
ConA, in a dose dependent manner (FIG. 6A). A maximal effect of 91%
inhibition in 3H-thymidine incorporation was obtained when NPs/LNC
ratio of 1:2 was used. This ratio was therefore used for the
following in-vitro experiments.
[0156] The effect of hESC-derived neural precursors on T-cell
activation and proliferation was then investigated. To this end,
the induction of IL-2R.alpha., a marker for T-cell activation, in
Thy1.2+ T cells was measured. In LNCs co-cultured with the human
neural precursors the fraction of IL-2R.alpha. T cells was reduced
by 32%, and a similar decrease was observed in the mean
fluorescence intensity of IL-2R.alpha. (FIGS. 6B-6C). In addition,
naive LNCs were labeled with CFSE and stimulated with ConA in the
presence or absence of neural precursors. FACS analysis showed that
the human neural precursors reduced the fraction of cycling T cells
from 49% to 21% (FIG. 6D-6E).
[0157] In vitro oligodendroglial differentiation of hESCs was
examined. The experimental protocol included initial induction of
human ESCs to differentiate as free floating clusters, under
defined culture conditions, in the presence of noggin into early
multipotent neural precursors according to the published protocols
[17] as described above. After two weeks, they were treated with
retinoic acid (RA) and propagated as floating spheres in modified
Sato medium and mitogens At sequential week intervals,
differentiation was induced by plating on fibronectin and
withdrawal of the mitogens, and the expression of oligodendroglial
markers were analyzed by immunostaining. After three weeks of
propagation as free floating clusters, followed by plating and
differentiation for 48 hours, the differentiating cells expressed
the oligodendroglial markers NG2 (12%) (FIGS. 7A,6C, 7D, 7F) GD3
(20%) (FIGS. 7B, 7C) and PDGFR.alpha. (15%) (FIGS. 7E, 7F).
Although at this stage of three weeks propagation, when enrichment
for OPCs was obtained, markers of mature oligodendrocytes such as
O4 and GalC were not detected, even after 7-10 days of
differentiation. Only in cultures that were propagated for more
than 5 weeks and induced to differentiate for 7 days, O4 and GalC
were expressed by 3% and 1% of differentiating cells, respectively
(FIGS. 7G, 7I (O4) and FIGS. 7H, 7I (GalC). Nuclei in FIGS. 7C, 7F,
7G-7I were counter stained with DAPI.
[0158] To develop expandable cultures enriched for oligodendroglial
committed progenitors, hESCs clusters were induced to differentiate
as free floating clusters into multipotent uncommitted neural
precursors as above with or without noggin. After two weeks of
initial neural induction the multipotent uncommitted neural
precursors were further cultured for 1-3 weeks, preferably 3 weeks,
as floating spheres in modified Sato medium supplemented with
mitogens, retinoic acid (RA) and the hedgehog (HH) agonist
purmorphamine. During this culture period the early multipotent
neural precursors gradually become enriched with Olig2+
oligodendroglial precursors (30-50% of total cells).
[0159] For further expansion of the oligodendroglial precursors,
the floating spheres were propagated in Sato medium supplemented
with mitogens, and low concentrations (0.5 .mu.M) of purmorphamine.
After 3-20 weeks of propagation as free floating clusters and
plating on fibronectin for 24-48 hours, expression of the OPC
markers Olig2 (FIG. 8A), PDGFR.alpha. (FIG. 8B) and NG2 (FIG. 8C)
were detected in 30%, 20% and 20% of the differentiating cells,
respectively. Following further 7-21 days of differentiation in the
presence of NT3, AA, T3 and IGF1+/-Rock inhibitor, markers of
mature oligodendrocytes such as O4 (FIG. 8D), GalC (FIG. 8E) and
MBP (FIG. 8F) were detected in 20%, 15% and 3% of the plated cells,
respectively.
[0160] In cultures where early neutralization (2 weeks) and caudal
ventral specification with RA and HH agonist (3 weeks) was induced
as above but were further propagated in Sato medium in the absence
of purmorphamine the level of enrichment for Olig2 and O4 following
five weeks of propagation and 1-7 days of differentiation was only
5% and 3%, respectively (FIGS. 8G-8H).
[0161] Taking into consideration all the above results, it was
concluded that transplantation of hESC-derived neural precursors in
any manner, to patients suffering from autoimmune demyelination
disease, e.g. MS, may facilitate both remyelination and attenuation
of the local inflammatory process and thus produce a dual
therapeutic effect. Such oligodendroglial-committed precursors may
also be transplanted in combination with hESC-derived multipotent,
non oligodendroglial-committed precursors, to obtain neural
protection and regeneration by each of the two neural precursor
types respectively.
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Sequence CWU 1
1
1121PRTRattus sp. 1Met Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg
Val Val His Leu 1 5 10 15 Tyr Arg Asn Gly Lys 20
* * * * *