NZ611902B2 - Culture method to obtain and maintain a pure or enriched population of mammalian neural stem cells and/or neural progenitor cells that are prone to differentiate into oligodendrocyte-lineage cells in vitro - Google Patents
Culture method to obtain and maintain a pure or enriched population of mammalian neural stem cells and/or neural progenitor cells that are prone to differentiate into oligodendrocyte-lineage cells in vitro Download PDFInfo
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Classifications
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/44—Thiols, e.g. mercaptoethanol
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/10—Growth factors
- C12N2501/105—Insulin-like growth factors [IGF]
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- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/0618—Cells of the nervous system
- C12N5/0623—Stem cells
Abstract
Disclosed is an enriched population of expanded human neural cells wherein the cells are progenitor cells or stem cells, wherein the cells have been cultured under conditions effective to enrich for the expanded human neural cells, wherein the conditions effective to enrich for expanded neural cells comprise a cell culture medium comprising at least one growth supplement in an effective amount, at least two growth factors, and at least one survival factor; wherein the at least one growth supplement is 1-thioglycerol and the effective amount of the growth supplement in the cell culture medium is at least 10 ?M of 1-thioglycerol; wherein the cells maintain their capability to differentiate into neurons, astrocytes, and oligodendrocytes; wherein the cells maintain their ability to differentiate into oligodendrocyte lineage cells efficiently throughout subsequent passages of the culture; and wherein the population of cells express at least the cell surface antigens CD133, CD140a, A2B5 and PSA-NCAM. Also disclosed is the use of said enriched population of cells in the preparation of a medicament for the treatment of demyelinating disease or neurodegenerative disease. comprise a cell culture medium comprising at least one growth supplement in an effective amount, at least two growth factors, and at least one survival factor; wherein the at least one growth supplement is 1-thioglycerol and the effective amount of the growth supplement in the cell culture medium is at least 10 ?M of 1-thioglycerol; wherein the cells maintain their capability to differentiate into neurons, astrocytes, and oligodendrocytes; wherein the cells maintain their ability to differentiate into oligodendrocyte lineage cells efficiently throughout subsequent passages of the culture; and wherein the population of cells express at least the cell surface antigens CD133, CD140a, A2B5 and PSA-NCAM. Also disclosed is the use of said enriched population of cells in the preparation of a medicament for the treatment of demyelinating disease or neurodegenerative disease.
Description
PCT/132012/000030
CULTURE METHOD TO OBTAIN AND MAINTAIN A PURE OR ENRICHED
TION OF IAN NEURAL STEM CELLS AND/OR NEURAL
PROGENITOR CELLS THAT ARE PRONE TO DIFFERENTIATE INTO-
ENDROCYTE-LINEAGE CELLS IN VITRO
FIELD OF THE INVENTION
This invention relates generally to the field of cell biology of neural
stem cells and neural progenitor cells. More specifically, this invention provides
a pure or enriched population of mammalian neural stem cells and/or neural
progenitor cells that are prone to differentiate into oligodendrocyte-Iineage cells
in vitro, suitable for use in biological research, drug screening and human
therapy.
CROSS-REFERENCE TO RELATED APPLICATlONS
This application is related to US. provisional patent application
number 61/431,944 filed on y 12, 2011 and 61/558,527 filed on
November 11, 2011.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR
DEVELOPMENT
Not Applicable.
BACKGROUND OF THE lNVENTlON
During development of the central nervous , primitive,
multipotent neural stem cells (NSC) proliferate, giving rise to transiently dividing
progenitor cells that eventually differentiate into the various cell types that
central nervous system mainly consists of
e the adult brain. The adult
and oligodendrocytes. The
neurons and inal cells, which e astrocytes
progenitor cells for neurons, astrocytes and oligodendrocytes originate
sequentially from neural stem cells in the developing brain (see Figure 1).
PCT/1B2012/000030
Neuronai progenitor cells form first and differentiate into many types of
neurons. Astrocytes develop second and on to support neuron survival.
Finally, oligodendrocyte progenitor cells start to appear and migrate throughout
the central nervous system. They then differentiate into mature
oligodendrocytes, which produce myelin necessary for proper neuronal
function.
Since endrocytes play an important role in supporting the
central nervous system, a pure or enriched population of oligodendrocytes or
their predecessor cells (i.e., oligodendrocyte pre—progenitor cells and/or
endrocyte progenitor cells) would be useful for cell ies and
regenerative. medicine such as in the treatment of neurological disorders
including congenital demyeiinating diseases (for example, Krabbe disease or
Pelizaeus—Merzbacher e), spinal cord injury and other ions that
result from defects in the myelin sheath that insulates nerve cells. These cells
also can be used for research and for identifying new drugs for the treatment of
sclerosis and schizophrenia.
many neurological disorders such as multiple
Mature oligodendrocytes do not proliferate and do not survive well
in culture, and the ability to obtain oligodendrocytes directly from tissue
s in ties sufficient for use in research or human therapy is
extremely difficult. As a result, the use of oligodendrocytes for these purposes
is hindered by the lack of availability of these cells.
One solution to this problem involves obtaining neural stem cells
and/or neural progenitor cells from tissue, expanding the cells in culture to
obtain a sufficiently large quantity .of cells which can subsequently differentiate
into oligodendrocytes. Differentiation can take place either in vitro or in vivo,
such as in the case of transplantation. This would result in a large population of
oligodendrocytes or their progenitors or pre—progenitors for use in research and
human therapy.
However, scientists have struggled to identify culture conditions that
permit long term culture and mass expansion of oligodendrocyte progenitors
and/or pre—progenitors — particularly from vor non-human primates —
PCT/132012/000030
wherein the ing expanded cell population is primarily comprised of cells
that retain the ability to differentiate into oligodendrocytes.
Several scientists have reported obtaining oligodendrocyte
Raff et al,
progenitor cells from rats ((Raff et al, J. Neurosci, 311289, 1983;
Proc. Natl. Acad. Sci.
Nature, 303:390, 1983; Espinosa de los Monteros et al,
U. S. A., 90:50, 1993). These proliferative oligodendrocyte itors are
differentiate in vitro into
known as O-2A progenitors because of their ability to
either oligodendrocytes or type 2 ytes. Other istshave identified rat
culture ((Gallo, Armstrong
or mouse oligodendrocyte pre-progenitors in primary
Neurosci. Res,
RC, J. Neurosci., 151394, 1995; Grinspan, schini B, J.
412540, 1995; Decker et al, Mol. Cell. Neurosci, 161422, 2000). These cells
and are expected
are thought to be precursors of oligodendrocyte progenitors
to be more beneficial in cell therapy because of their superior migration
capacity as compared to oligodendrocyte progenitors. Unfortunately, scientists
have been unable to effectively expand these cells for long s of time in
vitro. In st, scientists have ed culturing 02A itors from rat
factor
optic nerve or spinal cord using B104 conditioned medium or growth
ations such as (i) platelet derived growth factor—AA (PDGF-AA) with
basic fibroblast growth factor (bFGF or basic FGF) and neurotrophin—3 (NT—3),
NT—3.
or (ii) PDGF—AA with ciliary neurotrphic factor (CNTF) and However, no
cell types from primate tissue
one has succeeded in mass expansion of these
using these growth factors.
Thus, it remains very difficult to obtain and expand a pure or
enriched population of oligodendrocytes and/or their predecessor cells from
s other than rat or mouse. It is particularly difficult to obtain and
expand these cells from humans and non—human primates. Therefore, a great
need exists for methods for generating pure or enriched populations of
mammalian neural stem cells or progenitor cells which are prone to differentiate
into oligodendrocyte—lineage cells invitro.
BRIEF Y OF THE lNVENTlON
human
The present invention relates to an isolated expandable
cell
neural cell wherein the cell is a progenitor cells or stem cell, wherein the
maintains its capability to differentiate into neurons, astrocytes,
.oligodendrocytes, wherein the cell maintains its ability to differentiate into
oligodendrocyte lineage cells efficiently throughout subsequent passages,
CD133 and CD140cr.
wherein the cell expresses at least cell surface antigens
of in vitro culturing
The present invention also relates to a method
the cell is a itor cell or stem cell
an expandable neural cell wherein
wherein said cell maintains
ed from a mammalian central nervous system
and oligodendrocytes and
its capability to differentiate into neurons, astrocytes,
cells efficiently, wherein
its ability to differentiate into oligodendrocyte—lineage
least one cell from a human
the method comprises ing and dissociating at
of 37°C, in an atmosphere
fetal neural tissue; culturing the cell at a ature
defined serum—free
comprising 120% d 5% 002, and in a chemically
at least 5 ng/ml PDGF-AA, at
culture medium, wherein the medium comprises
and ing the cell
least 0.5 ng/ml bFGF, and at least 10 [JM 1—thioglycerol;
to obtain the expandable human neural cell.
] ' of ng a
The present invention r relates to a method
condition caused by a loss of myelin or a loss of oligodendrocytes comprising
of a composition
administering to a subject a therapeutically effective amount
is able to maintain
comprising an isolated expandable human neural cell which
and oligodendrocytes,
its capability to differentiate into neurons, astrocytes,
wherein the cell maintains its ability to differentiate into oligodendrocyte lineage
and wherein the cell
cells ently throughout subsequent passages,
CD133 and CD140or.
expresses at least cell surface antigens
The present invention also relates to an in vitro culture comprising
from a mammalian central nervous
at least one isolated neural cell obtained
system wherein the cell is submerged in chemically defined serum—free culture
PCT/1B2012/000030
medium which has at least 5 ng/ml PDGF—AA, - at least 5 ng/ml bFGF, and at
least 10 prl 1—thioglycerol.
neural
The present invention moreover relates to a ceutical
human neural cell.
stem cell composition comprising an isolated expandable
to the use of a
] The present invention additionally relates
to treat a
pharmaceutical neural stem cell composition in a medicament
condition.
of in vitro culturing
The present invention also relates to a method
cells isolated from a
and expanding neural stem cells and/or neural progenitor
cultured and expanded cells
ian central nervous system wherein said
cells. The
maintain their ability to differentiate into oligodendrocyte~lineage
culture of cells in the present invention is an adhesion e.
enriched
The present ion further relates to an ed pure or
and/or neural progenitor
population of expanded mammalian neural stem cells
cells (ie. 04—
cells that are prone to differentiate into oligodendrocyte—lineage
in Figure 7 and Figure 15)
positive cells with spider web morphology as shown
in vitro.
mammalian
The t invention moreover relates to
differentiation from
oligodendrocyte-lineage cells via in vitro expansion and
isolated from mammalian
neural stem cells and/or neural progenitor cells
l nervous system.
BRIEF DESCRIPTION OF THE DRAWlNGS
and neural
Figure 1 depicts the development of neural stem cells
the brain- neurons, astrocytes
progenitor cells into the three main cell types in
and oligodendrocytes; '
of various
Figure 2 shows the ison of marker expression
CNS cells;
Fetal Stem Cells (clone
. Figure 3 depicts contrast images of Human
#2b) in slides A—F;
WO 95730
HFSC cells (clone #2b) in
] Figure 4 illustrates the expansion rate of
the presence of different combinations of growth s;
of PDGF~AA (100 ng/ml)
Figure 5 illustrates the effects of high dose
cells (clone #Zb);
and 1—thioglycerol on the proliferation of HFSC
Figure 6 illustrates a growth curve of HFSC cells (clone #2b);
HFSC cells in
] Figure 7 depicts spontaneous differentiation of
serum—free medium in slides A—F;
inverted
Figure 8 is a phase contrast images taken with an
at various
microscope showing the morphology of HFSC cells (clone #3)
passages;
Figure 9 illustrates a growth curve of HFSC cells (clone #3);
inverted
Figure 10 is a phase contrast images taken with an
4A and 48) at
microscope showing the morphology of HFSC cells (clone
various passages in slides A—F;
Figure 11 illustrates a growth curve of HFSC cells (clone #4A and
#48);
] Figure 12 illustrates the immuno-phenotype.of undifferentiated
HFSC cells in slides A—S;
Figure 13, in slides A-H, shows flow cytometry data rating
proportion of undifferentiated HFSC cells (clone #2b, passage 13);
HFSC
Figure 14 illustrates the immuno-phenotype of differentiated
cells (clone #3, passage 15); and
cells (clone
Figure 15 illustrates the differentiation potential of HFSC
#2b, passage 15) in slides A—E.
ED DESCRIPTION OF THE DRAWINGS
Figure 1 depicts thedevelopment of neural stem cells and neural
progenitor cells into the three main cell types in the brain- neurons, astrocytes
and oligodendrocytes. Solid arrowed lines indicated the progression of one cell
HFSC cell to neuronal—restricted
type to another. The dashed line from
PCT/182012/000030
the HFSC cell have a lower tendency to
precursors (NRP) indicates that
line from the HFSC cell to the 02A
become neuronal fate. The heavy bolded
to produce
cells indicates that the HFSC cell has a r tendency
oligodendrocyte—lineage cells. The multi—potentiality of HFSC cell to
shown in Example
differentiate into neuron, astrocyte and oligodendrocyte was I
cell to entiate into
7 (see Figure 14). The tendency of HFSC
oligodendrocyte—tineage cells was shown in Example 2 (see Figure 7) and
Example 8 (see Figure 15).
of s CNS
Figure 2 shows a ison of marker expression
in Example 7 (see
cells. This invention disclosed the phenotype of HFSC cell
in this figure. As
Figure 12 and Figure 13) and summarizes the same
and has
discussed later, a HFSC cell is not the same as any other cell type
both features of neural stem cell and oligodendrocyte Type—2 Astrocyte
progenitor (02A).
Cells (clone
Figure 3 depicts contrast images of Human Fetal Stem
taken with an
#2b) in slides A—F. Figure 3, slide A is a phase contrast image
in DMEM/F12
inverted microscope showing HFSC cells (clone #2b) cultured
827 supplement
ning glutamine and HEPES and supplemented with
(lnvitrogenTM), non-essential amino acids (NEAA) (lnvitrogenTM), 1.5 mM
1 mM N—
pyruvate rogenTM), 55 pM B-mercaptoethanol (lnvitrogenTM),tand
medium"
acetyl-L—cysteine ), (in combination referred to as 1
incubator
hereinafter) with 20 ng/ml PDGF—AA and 10 ng/ml bFGF in an
from
maintained at 37°C, 5% Oz, and 5% C02 incubator. The cells shown are
formed spheres and these spheres were plated
passage 0, day 7- The cells
without dissociating Spheres
onto a poly—ornithine coated culture plate directly
at passage 1.
taken with an
Figure 3, slide B & slide C, are phase contrast images
cultured as described in
inverted microscope showing HFSC cells (clone #2b)
The cells shown are from
the description of Figure 3, slide A hereinabove.
passage 1, day ‘l and passage 2, day 14, respectively. The cells plated directly
and spread out from spheres
onto a poly-ornithine coated culture plate attached ‘
PCT/18201 2/000030
(Figure 3, slide 8). The passaged cells could expand successfully in this
slide C).
e condition in subsequent e (Figure 3,
with an
Figure 3, slides D—F are phase contrast images taken
inverted microscope showing HFSC cells (clone #2b) after le passages
and cultured in HFSCM1 medium with 100 nglml PDGF-AA, 1O nglml bFGF,
maintained at 37°C, 5%
ng/ml lGF—1 and 50 pM 1—thioglycerol in an incubator
Most HFSC cells were phase dark cells clustering
- 02, and 5% 002 incubator.
from the clusters
with surrounding cells. The scattered cells that separated
cells (so-
tended to differentiate spontaneously into process-bearing multipolar
called "spider’s web—like" morphology) that is characteristic to pro-
arrowheads
oligodendroblasts or immature oligodendrocyte (indicated by white
less
in Figure 3, slides D & E) but their frequency of ance was usually
F are from passage 8,
than 1%. The cells shown in Figure 3, slides D, E, and
day 8, passage 11, day 11 and passage 19, day 11, respectively.
' in
Figure 4 illustrates the expansion rate of HFSC cells (clone #2b)
nglml PDGF~
the presence of ent combinations of growth factors: (1):
AA +10 nglml bFGF; (2): 20 nglml PDGF—AA + 10 nglml bFGF + 5 nglml NT—B;
nglml
(3): 20 nglml PDGF—AA + 10 nglml bFGF + 10 nglml lGF-1; (4):
PDGF—AA + 10 nglml bFGF + 5 nglml NT—3 + 10 nglml lGF-1. The cells were
in each
harvested at day 11 of passage 3 (P3D11) and number of live cells
condition that
condition was d. Then, they were passaged in the same
The cells were harvested at
was used at passage 3 at the same cell density.
each ion was
day 8 of passage 4 (P4D8) and number of live cells in
counted (these cells were harvested before they became sub—confluent
because they started forming spheres). Condition (4) was most effective at
Condition (3) was ive at both passages.
passage 3 but not at passage 4.
Figure 5 illustrates the effects of high dose of PDGF-AA (100 nglml)
and 1-thioglycerol on the proliferation of HFSC cells (clone #Zb); cultured in
of growth
HFSCM1 medium in the presence of the following combinations
factors: (1): 20 ng/mi PDGF—AA + 10 ng/ml bFGF + 10 nglml [GP—1; (2): 20
50 MA 1—thioglycerol; (3):
ng/ml PDGF-AA +10 nglml bFGF + 10 ng/ml lGF-1 +
PCT/IBZOIZ/00003O
100 nglml PDGF-AA +10 ng/ml bFGF +10 nglml lGF—l; (4): 100 nglml PDGF-
AA +10 nglml bFGF + 10 ng/ml lGF—1 + 50 pM l-thioglycerol; (5): 100 nglml
cells were harvested
A +10 nglml bFGF + 50 prl l—thioglycerol. The
before they became sub—
at day 7 of passage 5 (these cells were ted
confluent because they started forming s as they did at passage 4)
of 20
number of live cells in each condition was counted. The combination
cell
nglml A + 10 nglml bFGF was also tested, but the recoVered
number was too low to evaluate (less than 1 x 10‘1 cells which was under the
countable range) and its .data was ated from this figure. The condition (1)
. The
could expand cells at passage 3 but couldn’t expand cells at passage
addition of 50 MA 1—thioglycerol [condition (2)] or the increase of PDGF-AA
concentration to 100 nglml [condition (3)] had a very little positive effect on
expansion rate. When the addition of 50 uM 1—thioglycerol and the increase of
PDGF—AA tration to 100 nglml were combined [condition (4)], the cell
expansion rate improved dramatically and the cells could be expanded
successfully. When lGF-‘l was eliminated from this condition [condition (5)], the
ion rate decreased to <1, indicating that lGF—1 also promoted HFSC cell
proliferation and/or survival.
Figure 6 shows a growth curve for a human HFSC cells (Clone #Zb)
in the
(open circle with black line). HFSC cells (Clone #2b) were initially cultured
bFGF. 10 nglml lGF—l was added from
presence of 10 nglml PDGF-AA and 1D nglml
increased from 20 nglml to 100 nglml
passage 3. The concentration of PDGF-AA was
from passage 6. However, they have very little or no effects on the expansion rate of
the cells. 50 [1M 1—thioglycerol was added from passage 7. HFSC ceils (Clone #Zb)
started to grow rapidly in the presence of 100 ng/ml PDGF-AA, 1O nglml bFGF,
nglml IGF—1 and 50 pM 1-thioglycerol. 1'0 nglml NT—3 was added at passage 17. NT—3
enhanced the cell growth a little bit but its effects disappeared after a passage, This
panel also es growth curves for human HFSC cells (Clone #2b) frozen at day
of passage 10 (P10 Day 6: open circle with dashed line) and day 11 of passage
(P11 Day 11: open circle with dot line). The frozen cells could be expanded at the
similar speed after thawing,
of HFSC cells in
Figure 7 depicts spontaneous differentiation
contrast images
free medium in slides A-F. Slides A and B are phase
of HFSC
taken with an inverted microscope Showing the morphological change
slides A & B) after being
cells (clone #Zb) at passage 12, day 9 (Figure 7,
ng/mi
cultured in HFSClVH medium supplemented with 20 ng/ml PDGF—AA,
slide A) or without 1-
bFGF, 10 ng/ml lGF—1 and 50 pM 1-thioglycerol (Figure 7.
thioglycerol (Figure 7, slide 8). HFSC'cells were entiated spontaneously
Even in the same
t replenishing bFGF between changing medium.
condition, HFSC cells didn't differentiate if bFGF was replenished everyday and
seemed to grow slowly. In addition, HFSC cells were blocked to differentiate
used.
and formed clusters when 40 ng/ml or higher PDGF~AA was By
and without
decreasing the A concentration from 100 ng/ml to 20 ng/ml
into
replenishing bFGF, the cells could be differentiated spontaneously
process—bearing multipolar cells with spider3 weblike logy, which
expressed the O4 n (Figure 7, slides C & D) and/or GalC antigen e ‘
cells [i.e.,
7, slides E & F), a defining characteristic of oligodendrocyte—lineage .
immature
pro—oligodendroblast (O4—positive and GalC—negative),
oligodendrocyte sitive and GalC- positive)].
Figure 8 is a phase contrast images taken with an inverted
at various
microscope showing the morphology of HFSC cells (clone #3)
stem cells were initially expanded in the
passages. The conventional neural
8, slide A at Day 15 of
ce of bFGF and EGF for 15 days (see Figure
with 20
Passage 0). After that, the cells were cultured in the HFSClVl‘l medium
ng/ml PDGF-AA and 10 ng/ml bFGF in an incubator maintained at 37°C,
increased to 100
Oz, and 5% C302 incubator. PDGF—AA concentration was
50 pM
ng/ml and 10 ng/ml of lGF—1 was added from passage 4 (see Figure 9).
14thioglycerol was added from passage 8 (see Figure 9). Their morphology
became almost identical with clone #Zb after several passages.
Figure 9 shows a growth curve for a human HFSC cells (Clone #3) (open
cultured in the presence of
circle with black line). HFSC cells (Clone #3) were lly
neural stem cells. Then,
ng/ml EGF and 10 ng/ml bFGF to expand conventional
PCT/IBZOIZ/000030
of PDGF-AA and 10 ng/ml
the growth factor ation was changed to 20 nglml
bFGF from e 1. 1O nglml IGF-1 and 10 nglml NT~3 were added from passage
2. Based on the data shown in Figure 4, NT-3 was removed form this culture and the
concentration of PDGF‘AA was increased from 20 nglml to 100 nglml from passage
of the cells as shown
'However, they have very little or no effects on the expansion rate
in clone #2b. 50 pM 1-thioglycerol was added from passage 5, and then HFSC cells
nglml
(Ctone #3) started to grow rapidly in the presence of 100 ng/mt PDGF—AA,
This
bFGF, 10 nglml lGF-1 and 50 pM 1~thioglycerol as HFSC cells (Clone #2b).
7 of
panel‘also es growth curves for human HFSC cells (Clone #3) frozen at day
dashed line). The frozen cells could be
passage 10 (P10 Day 7: open circle with
expanded at the simiiar speed after thawing.
Figure 10 is a phase contrast images taken with an inverted
microscope showing the morphology of HFSC cells (clone 4A and 48) at
various passages in slides A—F.. The cells were cultured in the HFSCM1
medium and 50 uM 1—thioglycerol with 20 nglml A, 20 nglml bFGF and
ng/ml of lGF—1 (clone #4A) or 100 nglml PDGF-AA, 20 nglml bFGF and
nglml of lGF-1 (clone #48) in an incubator maintained at 37°C, 5% 02, and 5%
C02 incubator. Clone #4A grew slower than clone #48 initially but started to
clone #48 from passage 2. They became
grow at around the same speed as
clone
almost homogeneous and their morphology became almost cal with
#2b or #3 after 3 passages,
Figure 11 shows a growth curve for a human HFSC cells (Clone #4A:
#48: open circle with black line). HFSC cells (Clone
open square with dashed line and
#4A) were cultured in the presence of 20 nglml of PDGF-AA, 20 nglml bFGF,
cultured in the
nglml, lGF—1 and 50 pM 1—thioglycerol. HFSC cells (Clone #48) were
ng/ml bFGF and 20 nglml lGF—1 and 50 [1M 1—
presence of 100 nglml of PDGF—AA,
thioglycerol. The cell number of both clones was decreased dramatically at first
this decline was more prominent in clone #4A. After this l decline in cell number,
they started to expand rapidly This panel also includes a growth curve for human
with
HFSC cells (Clone #48) frozen at day 601‘ passage 5 (P5 Day 6: open circle
dashed line).
Figure 12 iliustrates the immuno~phenotype of undifferentiated
HFSC .cells in slides A—S; (clone #2b, passage 12—16) cultured in the presence
PCT/IBZOIZ/OOOOISO
ng/ml lGF—1, and 50 ulVl 1-
of 100 ng/ml PDGF—AA, 10 ng/ml bFGF,
80x2, Nestin, Olig2, PDGF~
thioglycerol, and staining positive for either CD133,
"Ra, NGZ, A285, PSA—NCAM, GFAP or Vimentin. DAPl was used to
90% of the cells were
rstain cell nuclei. These figures showed at least
NGZ, A285 and vimentin
positive for CD133, 80x2, Nestin,.Olig2, d,
The staining of PSA—NCAM was a little
but there were no GFAP—positive cells.
looked ve for PSA-NCAM.
bit weak but still more than 90% of cells
Figure 13, in slides A—H, shows flow cytometry data illustrating
13). Black line
proportion of undifferentiated HFSC cells (clone #2b, passage
and the gray—filled rams
histograms represented the iso—type control
most of HFSC cells
represented each tested antigen.‘ This data showed that
13 slide 8),
were CD133—positive e 13, slide A), (DDS-positive (Figure
slide D), A285
CD140a-positive (Figure 13, slide C), NG2-positive (Figure 13,
slide F), and PSA—NCAM—
positive (Figure 13, slide E), O4—positive (Figure 13,
(data not
positive (Figure 13, slide G). As tested by an immunocytochemistry
shown), CD44 was negative e 13, slide H).
differentiated HFSC
Figure 14 illustrates the immuno—phenotype of
medium for 31 days
cells (clone #3, passage 15) cultured in serum—containing
Neurofilament—L,
and stained with antibodies that recognize neuron (Blll Tublin,
followed by a
and MAP2), oligodendrocyte.(04, MBP) and yte (GFAP)
counterstain cell nuclei.
fluorescent secondary antibody. DAPl was used to
marker. To evaluate
HFSC cell could differentiate into cells ve for each
ined with anti-Blll
co—localization of neuronal axon and myelin, cells were
slides A—C), anti-
Tublin antibody and anti—MBP antibody e 14,
slides D-F), anti-
Neurofilament-L antibody and anti—MBP antibody (Figure 14,
slides G-l).
IVIAP2 antibody and anti—MBP antibody e 14, Only a few
To evaluate co-
neuronal axon and myelin seemed to be co—localized.
co-stained with 04 antibody and
localization of O4 antigen and MBP, cells were
anti-MBP antibody (Figure 14, slides J—L). Most signals for MBP were co—
could also differentiate into
localized with signal for O4 antigen. HFSC cells
14. slides MO). in
astrocyte that was positive for GFAP antigen (Figure
2012/000030
in many
addition, many cells were positive for vimentin which was expressed
epithelial cells including astrocyte and mesenchymal cells. Furthermore,
undifferentiated cells were also positive for vimentin (data not shown).
cells (clone
Figure 15 illustrates the differentiation potential of HFSC
#Zb, passage 15) in slides A-E. The cells were differentiated in DMEM/F12
containing gl'utamine and HEPES and mented with B27 supplement,
100 ng/ml IGF—
supplement and 50 pM 1—thioglycerol with 10 ng/ml PDGF—AA,
1, 10 ng/ml BDNF, and 100 ull/l pCPT—cAMP. The cells were stained with anti-
after
GD3 antibody and 04 antibody following fluorescent secondary antibody
4-day differentiation. Undifferentiated cells expressed GD3 stronger than
oligodendrocyte progenitor cells and O4 vice versa (arrowhead of Figure 15,
cell
slide A, B and C shows undifferentiated cells). The most of differentiated
showed a multipolar morphology with weak GD3 signal and strong 04 signal,
indicating they were oligodendrocyte progenitor cells or pro-oligodendroblast.
Other cell types (e.g. astrocyte and neuron) were defined as a tion
GDS—nesgative and ative cells. Figure 15, slide D shows the ratio of
More
each cell population from 10 different of the differentiated cells.
than 70% of total cells differentiated into oligodendrocyte progenitor cells
(75.8% i 2.09%). Morethan 20% of total cells were still undifferentiated cells
(23.5% :1: 2.03%). The other cell type were less than 1 % of total cells (0.7% i:
0.41%). The ratio of oligodendrocyte progenitor cells and other cell types to
differentiated cells (oligodendrocyte progenitor cells plus other cell types) were
calculated and shown in Figure 15, slide E. Oligodendrocyte progenitor cell
other cell types were 0.9% i-
was 99.1% i‘0.56% of differentiated cells whereas
0.56% of differentiated cells.
DETAILED DESCRlPTION OF THE RED
EMBODIMENTS OF THE ION
The present invention provides a method for culturing and
ing neural stem cells or neural itor cells isolated from a
- PCT/IBZOIZ/000030
mammalian l nervous system to produce a pure or enriched population
neural stem cells or neural progenitor cells that have the ability to differentiate
Neural stem
into oligodendrocytes or oligodendrocyte—lineage cells in vitro.
neuronal
cells and neural progenitor cells both generate progeny that are either
cells (such as neuronal progenitors or mature neurons) or glial cells including
enewable
astrooytes and oligodendrocytes. While neural stem cells are
but are not
(i.e., able to proliferate nitely), neural itor cells may be,
necessarily, capable of self renewal. The culture methods of the present
invention can produce an expanded cell population that can be differentiated
into at least 70%, 80%, 90% or 95% oligodendrocyte—lineage cells, which are
oligodendrocyte progenitor cells and oligodendrocytes, of differentiated cells.
The ing abbreviations and definitions are used throughout this
application:
The term “HFSC cell” or human fetal spinal cord-derived cell, refers
neural stem cells
to the pure or enriched tion of expanded mammalian
and/or neural progenitor cells that are described in this ion.
' [00056] The term “HFSCM1 ” refers to DMEM/F12 containing
glutamine and HEPES and supplemented with 827 supplement (lnvitrogenTM),
non-essential amino acids (NEAA) (InvitrogenTM), 1.5 mM pyruvate
(lnvitrogenTM), 55 pM B-mercaptoethanol (lnvitrogenm), and 1 mM N-acetyl—L-
cysteine (Sigma) in combination.
The term “glial cells” refers to non~neuronal cells of the central
nervous system and encompasses mature oligodendrocytes, astrocytes
committed progenitor cells for either or both of these cell types.
The term potent progenitor cells” refers to neural progenitor
limited
cells that have the potential to give rise to cells from multiple, but a
number of, es.
“Pluripotency” (derived from the Latin ”plurimus" or "very many“ and
“potentia” or "powered") refers to a stem cell that has the potential to
differentiate into any of the three germ layers: endoderm (interior stomach
lining, gastrointestinal tract, the lungs), rm (muscle, bone, blood,
PCT/IBZOIZ/000030
urogenital), or ectoderm (epidermal tissues and nervous ). Neural stem
cells are otent and not pluripotent. Embryonic stem cells are pluripotent
and not multipotent.
A “committed progenitor cell” is a progenitor cell that is committed,
This is in contrast to a
or destined, to become a specific type of mature cell.
multipotent or a pluripotent progenitor cell, which has the potential to become
cells which
one of two or more types of mature cells (such as O—ZA progenitor
on timing
can become either oligodendrocytes or type-2 astrocytes, depending
and environmental factors).
] An “oligodendrocyte" is a type of gliatcell whose main function is to
insulate nerve cell axons in the central s system of some vertebrates.
The term “oligodendrocyte—lineage cells” refers to oligodendrocytes,
pro-oligodendroblast and oligodendrocyte progenitors (e.g.02A progenitor).
This term does not include restricted sors or neural stem cells:
The terms ”oligodendrocyte progenitor cells" and “oligodendrocyte
progenitors" are used interchangeably throughout this application and refer to
cells that are committed to forming more progenitor cells and/or'progeny that
are oligodendrocytes in preference to neurons or non-neurological tissue.
Unless otherwise specified, they may, but do not necessarily. have the
capability of making other types of glial cells (such as type—2 astrocytes). This
term as used herein does not encompass oligodendrocyte pre-progenitors or
glial-restricted precursors (see Figure 1).
dendrocyte pre—progenitors" are predecessor cells of
oligodendrocyte progenitors.
"Pro—oligodendroblasts” are predecessor cells to post-mitotic
oligodendrocytes.
“Expanding" cells in culture means to se cell number in the
which stimulate cell
presence of culture medium containing supplements
proliferation.
The cell “expansion rate" refers to the cell number on a particular
date divided by the initial cell number at the start of culture.
“Expanded" neural progenitor cells or neural stem cells as used
neural stem cells that are derived
herein refers to neural progenitor cells or
neural stem cells that have proliferated
from ed neural progenitor cells or
in vitro, producing the expanded cell population.
"Passaging" cells (also known as “subculturing” or “splitting" cells)
alive and growing under
refers to a technique that enables cells to be kept
of time by dissociating cells
laboratory culture conditions for extended periods
and then
from one another (with enzymes like trypsin or collagenase
vessel. Cells can be
transferring a small number of cells into a new e
cultured for a longer time if they are passaged at regular als, as
it avoids
cell density.
the premature senescence associated with prolonged high
] A “growth environment” is an environment in which the cells
vitro. Features of the
interest will proliferate, differentiate and/or mature in
environment include the medium in which the cells are Cultured, any growth
and a supporting
factors or differentiation—inducing factors that may be present,
ure (such as a substrate on a solid surface) if present
] General Techniques
General s in cell biology, protein chemistry, and antibody
techniques can be found in t Protocols in Protein Science (J.E. Colligan
Bonifacino et
et al., eds, Wiley & Sons); Current Protocols in Cell Biology (J.S.
et al.
al., Wiley 8: Sons) and t Protocols in lmmunology (J.E. Colligan
eds., Wiley & Sons). Cell culture methods are described generally in the
Basic Technique (R.l.
current edition of Culture of Animal Cells: A Manual 'of
Harrison
Freshney etl, Wily & Sons); General Techniques of Cell Culture (MA.
& l.F. Rae, Cambridge Univ. Press). Tissue Culture supplies and reagents are
ble from commercial vendors such as Chemicon®. Millipore®, R&D
Systems®, lnvitrogenTM, Nalgene—NuncTM ational, Sigma—AldrichTM, and
ScienCell.
Specialized reference books relevant to this disclosure include
McGraw—Hill 2000;
Principles of Neuroscience, 4"1 n, Kandel et al. eds,
M. H.
CNS Regeneration: Basic Science and Clinical Advances, Tuszynski &
I’CT/132012/000030
Neuron: Cell and Molecular
J.H Kordower, eds, Academic Press, 1999; The
Biology, 3 rd edition, l. B. Levitan and L.K. rek, Oxford U. Press, 2001;
et al. eds, Cambridge U. Press
Glial Cells: Their'Role in Behavior, PR. Laming
Matsas &
1998; The onal Roles of Glial Cells in Health and Disease,
Jessen &
Tsacopoulos eds, Plenum Pub. Corp, 1999: Glial Cell Development,
Adrian
Richardson eds, Oxford U. Press, 2001; and Man of Steel, Hill, 1996.
in the context of cell ontogeny, the adjective “differentiated" is a
r along the
ve term. A differentiated cell is a cell that has progressed
pmental pathway than the cell to which it is being compared. Thus,
neural stem cells can differentiate to e-restricted itors. These, _
to age
turn, can differentiate into cells further along the pathway or
differentiated cells, such as mature neurons or oligodendrocytes.
Differentiated cells of this invention can be characterized according
of oligodendrocytes.
to whether they s phenotypic markers characteristic
Classic immunocytochemical markers for these cells that may be present
depending on the maturity of the cell population are the following:
80x2: a marker for pluripotent stem cells and neural stem cells.
Nestin: a marker for neural stem cells.
CD133: a cell surface marker for neural stem cells.
PDGF—Receptor alpha (PDGF~RG)I the a chain of the platelet—
derived growth factor receptor. A marker for oligodendrocytes and their
progenitors.
CD140a: the same as PDGF—Ror. CD140a antibody recognizes an
extracellular domain of PDGF—Rd. A cell surface marker for oligodendrocytes
and their progenitors.
CD9: a cell surface glycoprotein that is know to complex with
cell surface
integrins and other transmembrane 4 superfamily proteins. A
marker for germline stem cell, neural stem cell, oligodendrocyte,
mesenchymal stem cell.
PSA—NCAM: polysiaiylated-neural cell adhesion molecules. A cell
neuronal progenitor,
surface marker for neuronal-restricted precursor (NRP),
— ~PCT/1132012/000030 u u v u
This marker is negative in
neurobiast and oligodendrocyte pre—progenitor.
giial——restricted precursor (GRP)
icted precursor (GRP)
A2851a cell surface marker for glial—
cell (OPC) and type 2
glial progenitor cells and oligodendrocyte progenitor
neuronal—restricted precursor
astrocytes. This cell surface marker is ve in
(NRP). ‘
A cell surface marker for
N62: a chondroitin sulfate proteoglycan.
cells.
macrophages and oligodendrocyte progenitor
GD3: Ganglioside GD3. A marker for oligodendrocyte pre-
progenitor and oligodendrocyte progenitors
04: a marker for oligodendrocytes and their progenitors.
] Galactocerebroside C (GaIC): a marker for immature
oligodendrocytes.
Myelin basic protein (MBP): a marker for mature oiigodendrocyte.
in cell—cell interactions,
CD44: a cell-surface glycoprotein involved
for onic acid. A cell surface
cell adhesion and migration and a receptor
cells.
marker for some epithelial cells and astrocyte lineage
for astrocytes.
Glial fibrillary acidic protein (GFAP): a marker
and neurons.
[3]“ Tublin: a marker for neuronal progenitors
] Neurofilament—L: a marker for mature neurons.
Microtubule—associated protein 2 (MAPZ): a marker for mature
neurons.
suitable
Tissue specific markers can be detected using any
for cell surface
immunological que, such as flow immunocytochemistry
of fixed cells or tissue
markers, or immunohistochemistry (for example,
detailed method for flow
sections) for intracellular or cell—surface s. A
Blood, 9611740, 2000.
cytometry analysis is ed in Galiacher et ai,
if a icantly
Expression of a cell—surface antigen is defined as positive
in a standard
detectable amount of antibody will bind to the antigen
after on of the
lmmunocytochemistry or flow cytometry assay, optionally
or other conjugate to
cells, and optionally using a labeled secondary antibody
PCT/132012/000030
it is often cial to
amplify labeling. To facilitate use in research or therapy,
the characteristics
maximize the proportion of cells in the tion that have
of oligodendrocytes or their progenitors. It is possible to obtain populations of
cells that are at least 50%, 60%, 70%, 90% or 95% specific lineage cells,
identified as being positive for one or more of the phenotypic markers
characteristic of such cells.
For eutic applications relating to reconstitution of neural
form
function, it is often desirable to minimize the ability of the cell population to
and cells of non—
other cell types, particularly erentiated stem cells,
ectodermal lineage. Depending on the application, it may also be
neuronal lineage and
advantageous to ze the proportion of cells of the
and their committed
their committed progenitors or cells of the astrocyte lineage
invention
progenitors. The contamination of the populations according to this
these other types of
have less than 30%, 20%, 10% or 5% contamination with
cells.
The methods of the present invention cannot result in the
pment of an entire human sm.
The method'of the present invention involves culturing isolated
central
neural stem cells and/or neural progenitor cells from a mammalian
the expansion of the cells
nervous system in a defined medium that permits
method of the present
through multiple es. The cells cultured using the
invention retain their ability, throughout expansion, to differentiate into
oligodendrocyte-lineage cells. The cells cultured using the method of the
present invention can be passaged more than 6 times and ed over
into
1,000 times while retaining their, ability to subsequently differentiate
oligodendrocyte—lineage cells. In some embodiments, the expanded cell
population resulting from the culture method of the present ion comprises
of cells having at least 30%, 50%, 70% or
or can differentiate into a population
serum-free culture
80% oligodendrocyte—lineage cells of differentiated cells in
condition. ln a red embodiment, the expanded cell culture population
invention comprises at least
resulting from the culture method of the present
PCT/IBZOIZ/OOOO30
cells. In preferred
90% oligodendrocyte—lineage cells of differentiated
In some ments the
embodiments, the expanded cells are otent.
into oligodendrocyte—
ty of expanded cells are capable of differentiating
lineage cells upon culturing in decreased PDGF—AA medium (i. e., 20 ng/ml
50 uM 1-thioglycerol
PDGF~AA, preferably with 10 ng/ml bFGF with or without
bFGF between
and optionally with at least 10 ng/ml lGF—t) without replenishing
changing medium. ln some embodiments the majority of expanded cells are
in10
capable of differentiating into oligodendrocyte—lineage cells upon culturing
and 10 ng/ml BDNF
ng/ml of PDGF-AA, 100 ng/ml lGF—1, 100 MA pCPT-CAMP
and supplemented with B27
in 12 containing ine and HEPES
supplement, N2 supplement and 50 pM glycerol.
The isolated mammalian neural stem cells and/0r neural progenitor
the central nervous
cells for use in the present invention may be obtained from
but not limited to, a
system of a mammalian, ably a primate such as,
human. endrocyte progenitors and pre~progenitors are known to exist in
from
white matter of the central nervous system As such suitable sources
not limited
which to isolate cells for use in the present invention include, but are
cord. In addition, isolated stem
to, the optic nerve, corpus callosum and spinal
such
cells may be derived from a mammalian fetus, preferably a primate fetus,
methods known in the art. In some
as but not limited to a human fetus, using
fetal spinal
embodiments, the isolated stem cells are prepared from human
cord tissue obtained from a human fetal spinal column. In a preferred
obtained from 8»
embcdiment, isolated cells for use in the t invention are
human fetal
24 weeks gestational age, preferably 12—18 weeks gestational age
for example,
spinal cord. Human fetal spinal columns can be ed,
commercially through companies such as Advanced Bioscience Resources,
informed consent
lnc. (Alameda, CA, USA) with the lRB permission and an
with
from a donor. Spinal cord tissue can be dissected from the spinal column,
The tissue then can be
the meninges and peripheral nerves d.
dissociated, washed and placed in a culture vessel containing a growth medium
that permits cell proliferation.
limited to, culture
Suitable culture vessels may include, but are not
combination of poly-amino acids
vessels with a culture surface having one or a
tissue culture plastic and es
(e.g., poly—lysine and/or rnithine),
Cells generally may be plated at
treated with laminin, vitronectin or fibronectin.
to 105 cells/cmz, ably at a density of
a density ranging from 10“
approximately 3 x 104 to 5 x 104 cells/cm? Poly~ornithine or poly—lysine may be
et al, J. ci.,
used to coat e vessels as reported previously (Raff
3:1289, 1983; Raff et al, Nature, 303:390, 1983; Protocols for Neural Cell
Culture, 3rd edition Humana Press, Inc. ).
Culture vessels may be coated with 1
2 to 20 pglml, more preferably 5 to 15
to 40 pg/ml of poly——ornithine preferably
pg/ml. The th of cell ment can vary depending on vendor, surface
vessels. The optimal
modification, format and specific lot of culture
for each source of culture
concentration of coating materials can be determined
s using methods known in the art. In some embodiments a two—hour
is performed to coat
incubation with 10 pg/ml of poly—ornithine or poly-lysine
In some
vessels, for example, from BD Faicon (Sparks, MD, USA).
embodiments a 30—minute incubation with 5 pg/ml of poly-ornithine or poly—
from Nalgen Nunc
lysine can be performed to coat vessels, for example,
International ster, NY USA)
cells
The isolated neural stem cells and/or neural itor
in a serum-
obtained from a mammalian central nervous system are cultured
cell expansion without
, free chemically defined culture medium that permits
ing differentiation of the cells (for example, into neurons, astrocytes
such as, but
oligodendrocytes). The culture medium comprises a base medium
: Nutrient Mixture F-12, 1:1
not limited to, Dulbecco’s Modified Eagle
Medium, RPMI-
(DMEM/F12) (Invitrogen®) (e.g., lscove’s Modified Dulbecco’s
be supplemented with various
1640 and Neurobasal). The base medium may
Such components may
components to t cell‘health and survival.
non-esSential amino acids [NEAA
include, but are not limited to, at least 0.25%
(Invitrogen®)— a 1% solution contains 100 pM of L—Alanine, L—Asparagine H20,
and L—Serine], at least 1.0
L-Aspartic Acid, L-Glutamic Acid, Glycine, L~Proline,
PCT/IBZOIZ/00003O
W0 2012/095730
1% 827 ment
mM glutamine, at least 0.5 mM pyruvate, at least
(lnvitrogen®),‘at least 0.1 mM N-acetyl—cysteine and/or at least 10 uiVl B-
mercaptoethanol. In some embodiments the base medium may comprise
N821 (as disclosed in Y. Chen et al., J. Neurosci. Methods, 171:239, 2008) in
place of 827 supplement. 827 supplement contains bovine serum albumin,
retinol
transferrin, insulin, terone, osterone, triiodo-l—thyronine,
ic acid, Linolenic
e, DL tocopherol, DL tocopherol acetate, Biotin,
reduced, catalase,
acid, ethanolamine, Na Selenite, L—carnitine, glutathione
superoxide dismutase, Degalactose and cine. lnvitrogen has disclosed
its ingredients but hasn’t disclosed their concentration. However,
concentration of each ingredients of their original formulation, B18 supplement,
based on this information and the
was disclosed. N821 was developed
concentration of each gradient was disclosed. lt worked in neuronal culture as
good as 827 supplement. In addition, N821 could be used to culture neural
from human embryonic
stem cells and oligodendrocyte progenitors derived
stem cells. Therefore, this supplement is thought to be a good ate to
replace B27 supplement. In a preferred embodiment, the e medium
comprises DMEM/F12 supplemented With 1-4 mM, more preferably 2.5
glutamine; 10—25 mM, more preferably 15 mM HEPES; 0.5—2.0 mM, more
preferably 1 mM pyruvate; 1 to 4%, more preferably 2% 827 supplement; 0.25—
3%, more preferably 1% NEAA; 1-200 pM, more preferably 50 0M
-100
thioglycerol; 0.1-3 mM, more preferably 1 mM N~acetyl~cysteine; and/or
pM, more preferably 55 0M aptoethanol.
Furthermore, oxygen may facilitate differentiation of neural
progenitor cells. Therefore, to reduce cell entiation, the cells may be
cultured in a 1—20% 02 growth environment. In a red embodiment, the
cells are cultured in a culture flask in an incubator providing a 37°C, 1—10%,
environment. After establishing HFSC
more preferably 5% Oz, 5% CO; growth
there was no
cells, the effect of oxygen concentration was assessed but
to 5% Oz
increase of differentiated celis in 20% Oz condition compared
of HFSC
condition in the culture condition to expand HFSC cells. The growth
PCT/11320 12/000030
cells in 5% 0; condition was a little bit faster than that in 20% 0; condition.
of p53 in
Oxygen is a cause of oxidative stress and known to induce mutation
rodent cells. To reduce a risk of mutation, we kept culturing HFSC cells in 5%
02 condition.
2] The neural stem cells and/or neural progenitor cells are cultured in
culture medium further comprising growth factors to stimulate proliferation for
isolating the cells. The culture medium may contain at least 5, 10, 20 or 40
nglml platelet—derived growth factor-AA (PDGF—AA), at least 2.5, 5 or 10 nglml
basic FGF , and/or at least 10, 25 or 50 mm 1—thioglycerol to isolate the
cells. In some embodiments the culture medium further comprises at least 1, 5
PDGF—
or 10 nglml insulin—like growth factor—1 (lGF—1). in some embodiments
AA may be replaced with PDGF-BB, PDGF—AB, PDGF—CC, or D. ln
some embodiments bFGF may be replaced with other member of fibroblast
growth s (e.g. FGF-4 or FGF—9). In some ments lGF—1 may be
ed with lGF—Z. In a preferred embodiment, the e medium comprises
40-60 uM 1—thioglycerol, 40—200 nglml PDGF—AA, 5—100 nglml bFGF, and 5-
100 nglml lGF—1 to isolate the cells.
The isolated neural stem cells and/or neural progenitor cells are
cultured in culture medium r comprising growth factors to stimulate
proliferation after isolating the cells. The culture medium may contain at least
1, 2, or 5 nglml platelet—derived growth factor-AA (PDGE—AA), at least 0.5, 1 or
ng/ml basic FGF (bFGF), and/or at least 10, 25 or 50 uM glycer0l to
expand the cells. In some embodiments the culture medium further comprises
at least 1, 2 or 5 nglml insulin-Iikegrowth factor—1 (lGF—1). In a preferred
embodiment, the culture medium comprises 40—60 uM 1-thioglycerol, 5—100
nglml PDGF—AA, 1—50 nglml bFGF, and 5-100 nglml lGF—‘l to expand the cells
after the isolation of HFSC cells.
The isolated neural stem cells and/or neural progenitor cells grown
under culture conditions of the present invention exhibit a doubling time of 50-
120 hours. In preferred embodiments, the ng time is about 60 to 100
hours. The cells can continue this proliferation rate through at least 8, 11, 14 or
' PCT/IBZOIZ/OOOOSO
17 passages. The cells may be expanded at least 100, 250. or preferably at
least 500 times per month. ln preferred embodiments, the cells cultured using
the method of the present invention exhibits an expansion rate >1 for more than
18 passages.
Examples
Example 1 base
-— HFSC cell culture and expansion; Identification of
medium and growth ‘factcrs
Several medium and supplements were tested in a preliminary
experiment using HFSC cells d from human 12—week fetal spinal cord,
which was obtained from Advanced Bioscience Resources, Inc. (Alamada, CA,
USA) with an ed consent of a donor. The cells were cultured in
DMEMzF—12 (1:1) (DMEM/F12) (lnvitrogenTM, Carlsbad, CA, USA)
supplemented with 2 mM ine llnvitrogenTM), 1 mM pyruvate
(lnvitrogenTM) and 2% 827 supplement (lnvitrogenTM) lly. Various growth
factors were examined whether they stimulate growth of HFSC cells. Most
effective growth factors were the ation of PDGF—AA and bFGF. The
cells could grow in the presence of PDGF-AA and bFGF but showed vacuoles
and looked unhealthy. Several supplements were tested and it was observed
that DMEM/F12 supplemented with 1% NEAA in addition to 2 mM glutamine, 1
mM pyruvate and 2% 827 supplement could decrease vacuoles and increase
cell number ly (data not shown). Other supplements including 1 mM N-
acetyl—cysteine (Sigma—AldrichTM, St. Louis, MO, USA) and 55 pM [3-
mercaptoethanol (lnvitrogenTM) seemed to improve the cells’ status but the
improvements were not as prominent as with NEAA (data not shown). Thus,
DMEM/F12 supplemented with 2 mM glutamine, 1 mM pyruvate, 2% 827
supplement, 1% NEAA, 1mM yl—cysteine and 55 pM B—mercaptoethanol
to culture
was identified as optimal base medium and was used thereafter
HFSC cells.
Human 15-week fetal spinal cord was dissected from the spinal
column, with the es and peripheral nerves removed. The tissue then
PCT/IBZOIZ/OOOOSO
and washed and cells obtained from human
was dissociated with Accutase
vessel ning the following growth
fetal spinal cord were placed in a culture
and ’l5 mM HEPES, 2%
medium: DMEM/F12 containing 2.5 mM glutamine
1.5 mM pyruvate rogenTM), 55
827 supplement (lnvitrogenTM), 1% NEAA,
i lel N-acetyl—L~cysteine (Sigma-
uM B-mercaptoethanol (lnvitrogenTM).
AldrichTM TM), 20 ng/ml PDGF-AA (R&D Systems, Inc., Minneapolis, MN, USA)
then placed in an incubator
and 10 ng/ml bFGF (R&D Systems). Cells were
added daily to
maintained at 37°C, 5% Oz, and 5% (302. bFGF (10 ng/ml) was
2-3 days during passage.
the e medium. Medium was Changed every
cells were not so many in the
Based on the preliminary experiments, g
and bFGF and many cells stopped proliferating or died
presence of PDGF—AA
PDGF—Ro
within a few weeks. This result seemed to be reasonable because
To remove cells that are not
expressing cells are usually less than 5% of cells.
responsive to PDGF-AA and bFGF, cells were cultured in an low adhesion
The cells that are responsive to
culture plate (Corning lnc., Corning, NY, USA).
while cells that are
PDGF-AA and bFGF made spheres (see Figure 3, slide A)
not responsive to them did not form spheres. Spheres were collected at lower
1,000 rpm for
speed of centrifugation (300 rpm for collecting s vs.
After 9 days, cells
ting single cells) for medium change and passaging.
onto poly—ornithine—coated culture vessels. After
were harvested and passaged
The cells exhibited a
this period, cells were passaged every 7-14 days.
Figure 3, slides B and C)
heterogeneous morphology at these early stages (see
within a month. The proliferation
and many of the cells stopped proliferating
cells eventually did not expand. ln
rate became slower over time and the
es in their
on, the cells began to appear unhealthy, forming
cytoplasm.
condition for HSCF cell
Example 2 —— Identification of optimal growth
expansion
of PDGF—AA and bFGF in the
As mentioned in Example 1, inclusion
of the HFSC cells initially, but the
culture medium supported adequate growth
NT—3 (R&D Systems) and lGF-1
eration rate slowed after 2 passages.
PCT/IBZOIZ/000030
(Sigma-AldrichTM) were tested to ine ifthey could enhance the
eration of HFSC cells. The presence of 20 nglml PDGF—AA with 10 nglml
bFGF was insufficient to stimulate the proliferation rate of the cells (the
expansion rate was <1) (see Figure 4). Subsequent addition of 5 nglml NT-3
and/or10 nglml lGF-1 resulted in an enhanced proliferation rate (the expansion
rate became >1). Combination of NT—3 and lGF—1 was most effective at
further ed to
passage 3. The cells obtained from each condition were
confirm their effects. The cells were harvested earlier because the cells started
g s. At passage 4, the ry of proliferation rate by
combination of NT—3 and IGF—1 was decreased and single addition of lGF-1
became most effective at passage 4. Combination of NT—3 and lGF—1 might
cause differentiation of cells or form more spheres and lost while changing
medium. Thus, the addition of lGF—‘l was identified as a most effective survival
factor and was used thereafter to culture HFSC cells. However, the
proliferation rate of the HFSC cells began to slow again at the end of passage 4
and the cell number was decreased comparing to the seeded cell number.
Therefore, another supplement, 1-thioglycerol, was examined whether it could
enhance the proliferation of HFSC cells at passage 5.
Figure 5 shows the effect of 1-thioglycerol on the proliferation of
HFSC cells. The l growth factor combination (20 nglml PDGF-AA +. 10
nglml bFGF) couldn‘t expand cells at all (data was not shown in this figure
because it was under the countable range). The growth factor combination
used from passage 3 (20 nglml PDGF~AA + 10 nglml bFGF + 10 nglml lGF—1)
+ 10 nglml
was more effective than this combination (20 nglml PDGF-AA
bFGF) but unable to stimulate HFSC cell proliferation well after e 4
e 4). Many cofactors d to the biosynthesis and degradation of fatty
acids and related long-chain hydrocarbons feature thiols. 1—thioglycerol was
next tested on the HFSC cells since it is one of thiol—based antioxidants and
has been reported to stimulate the proliferation of some cells (i.e. mouse
embryonic cortical and hippocampal neurons, mouse bone marrow mast cells,
and human B cell lines) in culture. In addition, higher dose of PDGF—AA (100
PCT/IBZOIZ/000030
ng/ml) was also tested whether it could complement the decrease of vity
to growth factors.
The addition of 50 pM 1—thioglycerol in the presence of 20 nglml
PDGF~AA, 10 ng/ml bFGF and 10 ng/ml tGF-1 stimulated proliferation ly
but the cell number was still decreased (expansion rate <1). This result was
r to that obtained in the absence of 1-thiogiyceroi when the A
concentration was increased to 100 ng/ml in the presence of 10 nglml bFGF +
ng/ml'lGF—1. However, when both 50 pM 1—thioglycerol and increased
PDGF-AA (100 nglml) were included in the culture medium (along with 10
nglml bFGF and 10 nglml , the two components appeared to work
synergisticaliy, significantly increasing the cell number (expansion rate >1).
When lGF-1 was eliminated from this ment cocktail (i.e. total
supplementation was 100 nglml PDGF—AA + 10 ng/mi bFGF + 50 11M
thioglycerol), the expansion rate decreased to <1, ting that lGF-1 also
promoted HFSC cell proliferation and/or survival. However, if HFSC cells were
cultured in this condition longer, HFSC cells might be expanded even in this
condition. This data indicated that the addition of 50 pM 1—thioglycerol and the
increase of PDGF—AA concentration to 100 ng/ml in addition to 10 nglml bFGF
+ 10 nglml lGF—1 were important to expand the cells. Furthermore, addition of
lGF-1 might not be mandatory but was still effective even in the presence of 50
uM 1~thioglycerol and 100 nglml PDGF—AA to increase the expansion rate. The
combination of 50 pM 1—thiogiycerol and 100 nglml PDGF—AA in addition to 10
nglml bFGF + 10 nglml lGF-1 was used fter to culture HFSC cells.
The HFSC cells were further expanded in the presence of 100
nglml PDGF-AA, 10 nglml bFGF, 10 nglml iGF—1 and 50 pM 1—thioglycerol after
passage 6. The cells began to proliferate more rapidly under these conditions
after passage and were expanded 3-4 times within a week. The doubling time
The HFSC cells were able to
was imately 60—100 hours in this condition.
maintain this proliferative state even after passage 8 (see Figure 3, slide D),
and e 19 (see Figure 3, slide F).
passage 11 (see Figure 3, slide E)
Thus, defined medium comprising 100 ng/ml PDGF—AA, 10 nglml bFGF, 10
the optimal culture
ng/ml lGF-1 and 50 ulVl 1—thioglycerol was identified as
cells and/or neural
condition for long term expansion of the neural stem
progenitor cells.
of HSCF cell
e 3 — Spontaneous differentiation technique
While testing various culture conditions, it was observed that when
HFSC cells were cultured with the removal of bFGF from the medium, many
cells (indicative of
the cells seemed to differentiate into bipolar or multipolar
oligodendrocyte) and died soon. To avoid these cell death, a neous
differentiation technique was developed.
Culture medium was usually changed every 2 days for expanding
HFSC cells and it was t to be very important to keep HFSC cells in
proliferative state. To enhance cell differentiation, medium was changed every
of PDGF-AA
3 or4 days for this experiment. Basic FGF and high concentration
cells but they were also important
were thought to block differentiation of HFSC
for HFSC cells to survive. Therefore, PDGF—AA concentration was decreased
lGF—1 with
from 100 to 20 ng/ml in the presence of 10 ng/ml bFGF, 10 ng/ml
cells couldn’t survive well
50pM glycerol or without 1—thioglycerol. HFSC
if bFGF was removed. Usually, bFGF were replenished everyday to keep
HFSC cells in proliferative state. When replenishing bFGF was stopped, many
HFSC cells ted from their clusters and formed complex web—like
and/or immature
processes (indicative of pro—oligodendroblasts I
oligbdendrocytes) and survived well as shown in Figure 7, slidesA and B.
These process—bearing cells With spidefs web—like morphology
as shown in Figure 7, slides
were positive for O4 antigen and/or GalC antigen
C—F, orerthey were thought to be pro~oligodendroblast sitive
GalC~negative) or immature oligodendrocyte (O4—positive and GalC— positive).
be entiated but
Even in the presence of 1—thioglycerol, HFSC cells could
in the
complexity of process looked simpler when 1-thioglycerol was t
In addition, other cell types
culture medium as shown in Figure 7, slides A & B.
HFSC cells were t
(eg, astrocyte 'and neuron) were rarely observed and
PCT/IBZOIZ/000030
cells. This
to be prone to differentiate into only oligodendrocyte-lineage
cells in a
technique enabled to observe differentiated oligodendrocyte-lineage
y state.
These data further indicate that the culture conditions of the t
stem cells and/or
invention are particularly useful for ing isolated neural
neural progenitor cells that are prone to differentiate into oligodendrocytes,
characteristics
since most of the differentiated cells exhibited oligodendr’ocyte
and resemble oligodendrocytes or pro—oligodendrocytes.’
cell
Example 4- Induction of HFSC cell from conventional neural stem
As shown in Figure 1, tional neural stem cell is thought to be
' this relationship, it was examined
a predecessor of HFSC cell. To confirm
neural stem cell that
whether HFSC cell could be induced from conventional
from a second tissue sample of human fetal
was reported previously. The cells
of 10
spinal cord (11 weeks gestation) were lly cultured in the presence
mM glutamine, 15
nglml EGF and 10 nglml bFGF in DMEM/F12 containing 2.5
mM-HEPES, 2% 827 supplement, 1% NEAA, 1.5 mM pyruvate, 55 uM [3—
mercaptoethanol, and ’l lel N—acetyl—bcysteine for 15 days to expand
conventional neural stem cells. Many different cell types were present initially
the end of the primary
but some expandable cell tion was obtained at
A at Day 15 of Passage
culture. These s are illustrated in Figure 8, slide
condition as clone
0. After the first passage the cells were ed in the same
nglml lGF-1, 100 nglml
#Zb (i.e. HFSCM1 medium with 10 nglml bFGF,
PDGF-AA and 50 11M 1—thioglycerol) through 15 passages.
The morphology of the cells from the expandable cell population
at around
obtained at the end of the primary culture became homogenous
to form clusters and spheres, similar to those
passage 5 and the cells tended
In addition, this clone
that formed with clone #213 (see Figure 8, slides B~F).
cells spontaneously (data not
could differentiate into oligodendrocyte—lineage
#2b (data not shown)
shown), showed the same immunevphenotype as clone
flow cytometry as described in
and the same marker sion pattern by
PCT/lBZOlZ/(lOQOLEO
for future testing (e.g.
Example 7. The cells from this clone were frozen down
Figure 12). This data ts that HFSC cell could be induced from
is thought
tional neural stem cell and that conventional neural stem cell
to be a predecessor of HFSC cell.
{000117} PDGF-AA was thoUghtto work through PDGF receptor a. This
members (PDGF-AA,
receptor is known to be stimulated by all PDGF family
used to
PDGF-BB, PDGF—AB. PDGF—CC and PDGF—DD). B was
HFSC cell clone
examine whether PDGF-BB could replace PDGF—AA using
condition
#2b and clone #3. PDGF—BB could expand (both clones in the same
except for PDGF—AA and it was proved that PDGF—AA could be successfully
replaced with other PDGF family members.
Example 5— Confirmation of optimal cell cuiture components
While the inventor tested various culture conditions after HFSC cell
for each
(clone #2b) was established, the inventor noticed that dose se
growth factor has been changed. To isolate HFSC cell (clone #2b), higher
concentration of PDGF—AA (100 ng/ml) was ary in addition to 50 pM
thioglycerol. After this clone became proliferate constantly, higher
this
concentration of PDGF—AA (100 ng/ml) was no more ed to expand
clone. Expansion rate was saturated at around 10-20 ng/ml of PDGF-AA and
had no onal effects on their
higher tration of PDGF—AA (100 ng/ml)
growth. This may be because of a long-term culture or a continuous usage
1—thioglycerol. To establish clone #2b and clone #3 of HFSC cells, 1—
of higher
thioglycerol was used after several passages. To examine the effect
of 1—
concentration of PDGF—AA, new ciones were established in the presence
thioglycerol from-an initial culture. In addition, the response to bFGF and lGF—1
When higher
seemed to be saturated at 20 ng/ml and 40 ng/ml, respectively.
cells could
concentration of lGF—1 (50600 ng/ml) was used, more differentiated
be seen (it looked like around 1%). Therefore. 20 ng/ml lGF—1 was considered
The usage of 20 ng/mi bFGF and 20
to be preferable to expand HFSC cells.
540%. compared to the usage of 10
ng/ml lGF—1 improved the expansion rate
ng/ml bFGF and 10 ng/ml lGF-1. Therefore, 20 ng/ml bFGF and 20 ng/ml lGF-
1 were used for this experiment.
HFSC cells from a third sample (12 weeks gestation) were cultured
in the identified optimal
to see it higher tration of A is required
culture medium components and if they would provide similar growth
in the same
characteristics in another batch of cells. The cells were cultured
medium and 50 pM 1—
culture medium as clone #2b and #3 (Le. HFSCM1
PDGF—AA in
thioglycerol) with 20 ng/ml (clone #4A) or 100 ng/ml (clone #48)
of the first passage,
addition to 20 ng/ml bFGF and 20 ng/ml lGF—1.; At the end
that of clone #48 (see
cell number of clone.#4A was less than one third of
Figure 11, slide A and slide B). However, clone #4A started to proliferate at the
similar speed as clone #48 after passage 1 even with lower PDGF-AA
concentration than clone #48. The morphology of the cells became
form rs and spheres,
homogenous at passage 3 and the cells tended to
slides C—F). In
similar to those. that formed with clone #2b or #3 (see Figure 10,
cells
addition, these cells could differentiate into oligodendrocyte—lineage
neously as clone #2b and #3 did. This result ted that higher
isolate HFSC
concentration of PDGF-AA was not mandatory but preferable to
not necessary once HFSC
cells and that higher concentration of PDGF-AA was
cells were established. Furthermore, this e method could provide similar
growth characteristics in another batch of cells and it was confirmed that this
The cells from this clone were frozen down for
process could be repeatable.
future testing.
and grow after
Example 6— Ability of expanded HFSC cells to recover
freeze-thaw cycle
of 8%
0] The cells of clone #2b were cryopreserved in the presence
frozen at
DMSO at passage 10 11 and 12 The HFSC cells (clone #2b)
passage 11 have been deposited at ATCC (accession number PTA—12291)
the presence of 8% DMSO at
The cells of clone #3 were also cryopreservedin
of clone #4A and #48 grew faster than clone #2b
passage 9 and 10. The cells
VVO 2012/095730
or #3, therefore they were cryopreserved in the presence of 8% DMSO at
4 and 5 (clone #48) in the same
passage 4 and 5 (clone #4A) or passage 3,
condition. The same medium to culture the HFSC cell 1 medium and
50 pM 1—thiogiycerol) was used for ng the HFSC cells. The cells were
later thawed and cultured in the above described free HFSCM1 medium
and 50 uM 1—thioglycerol with 10 ng/ml bFGF, 10 ng/ml lGF—1, 100 ng/ml
PDGF-AA or 20 ng/mi bFGF, 20 ng/mi lGF—1, 20 ng/ml PDGF-AA. These cells
were ed to proliferate at a similar rate as before freezing (see Figure 6,
Figure 9 and Figure 11). The frozen cells were used for later experiments
.shown in s 12—15.
Example 7 —— Characterization of expanded and undifferentiated HFSC cells
When the HFSC cells of Example 2 were cultured in the presence
of 100 ng/ml PDGF-AA, 10 ng/ml bFGF, 10 ng/ml lGF-1 and 50 pM 1-
thiogiycerol, they grew in clusters and/or spheres as shown in Figure 3, slides
D-F. The scattered cells that separated from the clusters tended to differentiate
into oligodendrocytes as shown in Figures 3, slides D and E. Even when the .
cells were passaged in a single cell state, they eventually began to gather and
form clusters again. Their shape in this proliferative state resembled
oligodendrocyte. pre-progenitor cells (see Ben—Hur et al, J. Neurosci., 1825777,
1998; Gago et al, Mol. Cell. Neurosci, 22:162. 2003), but not like O-2A
progenitors which grow in a bipolar morphology without forming any clusters.
Even rat oligodendrocyte pre-progenitor celis were reported a long time ago,
the human counterpart has not been reported. However, it was unnecessary to
identify the precise stage of endrocyte predecessor cell in the ed
cell culture since they retained their ability to differentiate into oligodendrocytes
(as shown below) regardless.
In order to characterize the immuno-phenotype of the
undifferentiated HFSC cells, the cells at passage 11-15 were iated into a
single cell state with se (lnnovative Cell Technologies, San Diego, CA,
. USA) and grown in poiy-ornithine—coated 24—well culture piates and cultured
PCT/IBZOIZ/OOOOZSO
ng/ml bFGF, 10 ng/ml
3-7 days in the ce of 100 ng/ml PDGF-AA,
with 4%
lGF— 1 and 50 uM 1 -thioglycerol The cells were then fixed
paraformaldehyde and then washed with PBS. For staining surface antigens
GalC and PSA-NCAM, cells were
like CD133 oi, NG2 A285, 04, O1
and stained with
d with PBS ning 3% normal gout serum (NGS)
antibodies. For staining intracellular antigens like 80x2, nestin, Olig2, myelin
Blll Tublin, Neurofilament-L and MAP2,
basic protein (MBP), Vimentin, GFAP,
X—100 in ice-cold PBS for 10 min
cells were permeabilized with 0.1% Triton
before blocking. CD133/1 mouse lgG1 monoclonal antibody (Clone A0133,
(Upstate), anti—N62
Miltenyi Biotec), anti—PDGF—‘Ro rabbit polyclonal antibody
mouse lgM monoclonal
rabbit polyclonal antibody (Millipore), anti—PSA—NCAM
antibody (Millipore), A285 mouse lgM monoclonal antibody (Millipore),
01 mouse lgM monoclonal
mouse lgM monoclonal antibody (R&D Systems),
anti-Nestin
antibody (Millipore), anti—80x2 rabbit polyclonal antibody (Millipore),
mouse monoclonal
mouse lgG1 monoclonal antibody (Millipore), anti-OligZ
BP rat
lgG2a dy, anti-GalC monoclonal lgGB antibody (Millipore),
monoclonal
monoclonal lgG2a antibody (Millipore), anti-Vimentin mouse lgG1
antibody (Millipore), anti-
antibody (Santa Cruz), anti-GFAP rabbit polyclonal
anti—Neurofilament-L
lSlll Tublin monoclonal mouse lgG1 antibody; (Millipore),
anti-MAP2 rabbit polyclonal
mouse monoclonal lgG1 dy (Cell Signaling),
dy (Miilipore) and anti—MAPZ mouse lgG1 onal antibody
1:600 (N62),
(Millipore) were used at 1:300'(CD133/1), 1:300 (PDGF—Ro),
1:1000 (01), 121000 (30x2),
121000 (PSA—NCAM), 121000 , 121000 (04),
1:50 (MBP), 1.2500 (Vimentin),
1:200 (Nestin), 1:200 (Olig2), 1:100 (GalC),
1:1000 (GFAP), 1:100 (l3lll Tublin), 1:200 filament-L), 121000 (MAPZ,
PBS containing 3% NGS. After
polyclonal) or 1:200 (MAPZ, onal) in
with 3 changes of PBS
overnight incubation at 4°C, wells were washed
cell staining for some cell surface
containing 3% N68. ln some case, a live
used to reduce non—specific
antigen (N62, A285, 04, O1 and GD3) was
each primary antibody before
signals. In such case, cells were stained with
fixation without blocking. Anti-N62 rabbit poiyclonal antibody, A285 mouse
IglVl monoclonal antibody, 04 mouse lglVl monoclonal antibody, 01 mouse lglVl
monoclonal antibody, and isialoganglioside GD3 mouse monoclonal lgGE
antibody (Millipore) were used at 1:150 (N62), 1:100 (A285), 1:200 (04), 1:100‘
(01) and 1:200 ((303) in PBS containing 0.5% BSA. After 30 minutes,
incubation at room ature, wells were washed with 3 s of PBS
containing 0.5% BSA. The secondary antibodies, DyLight 488—Conjugated
AffiniPure Goat anti—rabbit lgG (Fcy Fragment c), DyLight 488—conjugated
AffiniPure Goat anti—mouse lgG (Fcy Fragment specific), DyLight 488—
conjugated AffiniPure Goat anti—rabbit lgG (H+L), DyLight 488—conjugated
AffiniPure Goat anti—rat lgG (H+L), DyLight 594-conjugated AffiniPure Goat
anthmouse lgG (H+L), and/or t 594-conjugated AffiniPure Goat anti—
mouse lgM (u chain specific) (all secondary antibodies were purchased from
Jackson lmmunoResearch tories, lnc.) were used at dilution of 1:500 for
1 hour at room temperature. The cells were then washed with 2 changes
PBS. DAPl was used to counterstain cell nuclei. The cells were then observed
using an Olympus 1X81 ed for epifluorescence.
Most of the HFSC cells were CD133—positive (see Figure 12, slide A
& B),.Sox2—positive (see Figure 12, slides C & D) and nestin-positive (see
Figure 12, slide E & F), indicative of neural stem cells. Most of the HFSC cells
cells for motor neuron and
were OligZ—positive (often tive of progenitor
oligodendrocyte) (see Figure 12, slides G & H), PDGF-Ra-positive (often
tive of oligodendrocyte~lineage cells) (see Figure 12, slides I & J), A285-
positive (A285 is y absent from neural stem cells and found on glial
progenitor cells, oligodendrocyte progenitor cells and type—2 astrocytes) (see
cells. Most of
Figure 12, slides M & N), indicative of oligodendrocyte progenitor
slides O 8: P) but
the HFSC cells were PSA~NCAM—positive (see Figure 12,
absent from
there were variety in their expression levels (PSA—NCAM is usually
neural stem cells and found on neuronal progenitor cells and oligodendrocyte
pre—progenitor cells). No GFAP~positive cells could be seen while most of them
At least 90% of HFSC cell
were vimentin—positive (see Figure 12, slide 0-8).
seemed to be positive for above markers except for GFAP but the precise
counting was very difficult because HFSC cell tended to make rs and to
be detached from culture vessels very easily during fixation and staining. To
quantify their purity, a flow cytometry is was done and shown in Figure
4] ln addition, undifferentiated HFSC cells were weakly stained with
04 antibody that is a marker for pro—oligodendroblast (O4-positive, GalC—
negative) and immature oligodendrocyte (Oil—positive, GalC—positive) by
immunocytochemistry but it was difficult to distinguish with weak ng and
ecific staining. Some pro—oligodendroblast Which shows strong 04—
positive cells with multipolar morphology could be seen in this culture but their
frequency of appearance was less than 1% of total cells.
in addition, undifferentiated HFSC cells were weakly stained with
anti—MAP2 antibody but their morphology was not like . When the
antibody was used with the cells differentiated in the presence of serum,
neuron was identified with strong signals and neuronal morphology (see Figure
14, slide H). Such strong signal of MAP2 with neuronal logy was not
identified in undifferentiated HFSC cells. This weak signal disappeared when
the cells were differentiated, so that this staining seemed to be specific and
undifferentiated HFSC cells might not be non—specific staining.
Overall, HFSC cell expressed specific markers for neural stem cell
, 80x2, and Nestin) and specific markers for oligodendrocyte-lineage
cells (Olig2, NGZ, A285, and 04). These data suggested that HFSC cell might
be an intermediate cell between neural stem cell and oligodendrocyte
progenitor cell. Furthermore, HFSC cell sed PSA—NCAM in addition
above antigens, indicative to be the human counterpart of rat oligodendrocyte
pre-progenitor cell.
aiic acid (PSA) of PSA—NCAM is a long, negatively charged,
cell—surface glycan with an enourmous hydrated volume that serves to
modulate the distance between cells. PSA is involved in a number of plasticity—
related responses in the adult CNS, including changes in circadian and
al patterns, adaptations to pain and stress, and aspects of learning and
WQ 2012/095730 PCT/IBZOIZ/000030
One of the roles of PSA
memory (RutishauserNat. Rev. Neurosci., 9:26, 2008).
in the neonatal nervous System is in the migration of oligodendrocyte
progenitors. When PSA is removed from migrating 02A progenitors, migration
of 02A itor was inhibited in wound model (Barrel-Moran et al, J.
Neurosci. Res, 72:679, 2003). Another role is controlling differentiation timing
of cells. PSA is expressed on both developing axons and oligodendrocyte
cells correlates with the onset of
precursors, and its down regulation on these
myelination. PSA is also related plasticity-associated responses of the adult
CNS. Given the ability of PSA to regulate developmental and adult plasticity, it
follows that PSA—expressing cell could have the therapeutic value in situation in
which tissues have been damaged by injury or disease. Axonal regrowth in the
PSA—expressing region (engineered PSA sion in the scar or on grafted
Schwann cells) were observed through the scar in trauma model. HFSC cell is
the same effect on treating
an endogenous ressing cell and will have
the trauma like brain injury or spinal cord injury.
To further characterize the HFSC cells (clone #2b), cells that were
frozen at passage 11 'were thawed, cultured in the growing condition bed
above and passaged every 7—9 days. The cells ed for 9 days at
passage13 were then ted to flow cytometry using the ing
antibodies: PE-conjugated anti—CD133/1 mouse lgG1 monoclonal antibody
(Clone A0133, yi Biotec); PE-conjugated CD140a mouse lgGZa
monoclonal dy (Clone 0R1, BD Pharmingen); PE—conjugated CD9 mouse
lgG1 monoclonal antibody (Clone M—L13, BD Pharmingen); PE—conjugated
CD44 mouse lgGZb monoclonal antibody (Clone 644-25, BD Pharmingen);
PE—conjugated anti—PSA—NCAM mouse lgM monoclonal antibody (2—28,
Miltenyi-Biotec); PE—conjugated A285 mouse lglVl monoclonal antibody (Clone
105H829, Miltenyi Biotec); jugated O4 mouse lgM monoclonal antibody
(Clone O4, Miltenyi Biotec); and PE—conjugated 62 mouse lgGi
monoclonal antibody (R&D Systems). Briefly, after dissociation with Accutase,
with 2 mM EDTA and
the cells were washed and resuspended in ice—cold PBS
number was
0.5% BSA and kept on ice. After cell number was counted and cell
2012/000030
adjusted to 1 x 107ceils/ml using ld PBS with 2 mM EDTA and 0.5% BSA.
pl of cell suspension 00 cells) was transferred into each 1.5—ml tube.
Primary antibodies then were added into each tube following the
manufacturer's recommendation. PE—conjugated isotype controls for each
antibody were used to set appropriate gates. After 20—min incubation on ice,
cells were washed with ice-cold PBS with 2 mM EDTA and 0.5% BSA and
resuspended in fixation buffer (BD Bioscience). After 20 minutes fixation on ice,
cells were washed and resuspended in ice—cold PBS with 2 mM EDTA and
0.5% BSA. Fluorescence of celis was measured using FACS Canto ll (BD
Bioscience) and each data was analyzed using Gatelogic software (lnivai
Technologies Pty Ltd.)
As show in Figure 13, all or most of the HFSC ceils (clone #2b)
were CD133-positive (100% of , CD9-positive (100% of , CD140a—
positive (98.8% of cells), of cells). AZBS-positive (99.9%
, NGZ—positive (89.8%
of cells), O4-positive (94.6% of cells), PSA—NCAM—positive (68.9% of cells), and
CUM—negative (0.4% of cells were positive). By immunocytochemistry, the 04
signal could be hardly distinguished from non-specific staining and was much
weaker than the 04 signal of pro-oligodendroblasts or oiigodendrocytes. From
the result of flow try, the 04 signal of HFSC cells (clone #Zb) could be
separated from isotype control and HFSC cells (clone #2b) appeared weakly
positive for O4 antigen. HFSC cells (clone #3) showed almOst the same
phenotype by flow try. They were CD133—positive (98.4% of cells),
ODS-positive (99.4% of cells), CD140o—positive (91.5% of celis), NGZ—positive
(63.4% of cells), A285~positive (99.8% of cells), O4—positive (71.8% of cells),
PSA-NCAM-positive (74.7% of ceils), and CD44-negative (0.3% of cells were
positive).
Example 8 - Differentiation potential of expanded HFSC cells in serum—
containing medium
To test the entiation ial of the expanded cells, the HFSC
cells (clone #3) were passaged to separate/single cell stage and cultured in
PCT/lBZOlZ/00003O
serum-containing medium [Oligodendrocyte Precursor Cell Differentiation
Medium (OPCDM)] (ScienCellTM Research Laboratories) to stimulate
differentiation. The cells were then stained with antibodies that recognize
and anti—MAP2
neurons (anti—Blll Tublin antibody, anti-Neurofiiament-L antibody
antibody), oligodendrocyte progenitor cells and endrocytes [04 antibody,
01 antibody, anti—GalC antibody and anti—myelin basic n (MBP) antibody],
and astrocytes (anti—GFAP antibody) followed by a fluorescent dye—conjugated
secondary dy ht 488 or DyLight 594, Jackson lmmunoResearch).
DAPI was used to counterstain cell nuclei.
All three major central nervous system (CNS) phenotypes were
observed following treatments to stimulate differentiation of HFSC cells. When
the HFSC cells were ed in serum—containing , Bill Tublin-positive
cells, Neurofilament-L-positive cells, and MAP—Z—positive cells were detected
(indicative of neurons). There were many Bill -positive cells (Figure 14,
slide B) s small number of cells was positive for Neurofilament—L (Figure
14; slide E) or MAPZ (Figure 14, slide H). This result indicated many of them
into MBP—positive
are immature neurons. HFSC cells could also differentiate
cells. MBP is a major ent of myelin and expressed only in mature
endrocyte. This data indicates that HFSC cells have an ability to
differentiate into mature oligodendrocytes. (Io—localization of MBP and above
neuronal markers was evaluated but only a few co-localization could be seen
(Figure 14, slides C, F, I). This might be due to immaturity of neurons because
myelin will not be wrapped on axon of immature neuron. Co—localization of
MBP and O4 antigen was also evaluated (Figure 14, slides J—L). Most signal of
MBP co-localized with signal of O4 antigen but only half of signal of O4 antigen
co—localized with signal of MBP. This result was reasonable because 04
antigen is expressed in oligodendrocyte progenitors, immature
oligodendrocytes and mature oligodendrocytes while MBP is expressed only
that is
mature oligodendrocyte. These cells were also positive for GalC antigen
not shown).
a marker for immature oligodendrocytes (data GFAP-positive cells
cytes) and vimentin-positive cells were also detected as shown in Figure
PCT/IBZOIZ/OOOOSO
14, slides M-O. These data indicate that the expanded HFSC cells are
multipotent and that, therefore, they are likely neural stem cells, since they are
capable of giving rise to the three major central‘nervous system cell
phenotypes depending on the environment. The HFSC cell (clone #2b) was
also tested in the same condition and showed the same multipotency as the
HFSC cell did (clone #3).
e 9 —‘ Differentiation potential of expanded HFSC cells in serum-free
medium
HFSC cells showed good differentiation potency into
oligodendrocyte by reducing PDGF-AA concentration without replenishing
bFGF as shown in Figure 5. However, the differentiated cells were less than
half of total cells. If HFSC cells were seeded at very low density (< 0.5 x 104
cells/cmz) without bFGF with 10 ng/ml of PDGF-AA and 100 ng/ml lGF-1, most
cells seemed to differentiate but they were lost within a day. To examine their
potency to differentiate into endrocyte further, an induction of
differentiation and a long-term cell survival were thought to be very important
Cyclic AMP (CAMP) is known to induce differentiation of many cell types.
' pCPT-cAMP which is a cell-permeable analog of CAMP was tested whether it
could induce differentiation in HFSC cells. 100 pM pCPT—cAMP could induce
differentiation of HFSC cells at high density (3 x 104 cells/cmz) but cell couldn't
survive well even in the presence of 100 ng/ml lGF-i. BDNF is reported to
e differentiation of oligodendrocyte progenitors and support cell al
of differentiated cells. When HFSC cells were differentiated in the presence of
ng/ml of PDGF—AA, 100 ng/ml IGF—1, 100 pM pCPT—cAMP and 10 ng/ml
BDNF in DMEM/FlZ ning glutamine and HEPES and supplemented with
827 supplement, N2 supplement and 50 uM 1-thioglycerol, at least more than
half of HFSC cells were differentiated into process—bearing cells. Because
HFSC cell ses several oligodendrocyte markers like 04 or N62, the new
method to distinguish HFSC cell and dendrocyte-lineage cell was required.
Based on several trials, the inventor d that undifferentiated cells express
~ - -
GD3 stronger than oligodendrocyte progenitor cells and O4 vice versa
(arrowhead of Figure 11, slide A, slide B and slide C). When the cells were
stained with GD3 and O4, oligodendrocytes could be distinguished using their
staining pattern and their morphology. In addition, other cell types like neuron
or astrocyte also could be identified because they don‘t express GD3 or 04
antigen. Figure 15, slide D shows the ratio of each cell type. Undifferentiated
cells were 23.5% i 2.0% of total cells. Oligodendrocyte progenitor cells were
75.8% i 2.1% of total cells. The other cell types were only 0.9% :t 0.6%. As
mentioned above, the ratio of oligodendrocyte progenitor cells to differentiated
cells dendrocyte progenitor cells plus other cell types) were 99.1% i
0.56% of differentiated cells s other cell types-were 0.9% i 0.56% of
differentiated cells. This data indicates that HFSC cell has the high potential to
differentiate into oligodendrocyte-lineage cells.
Example 10- HFSC cell is enriched or selected by a fluorescent activated
cell sorting (FACS) method or a magnetic sorting method
The present invention disclosed the phenotype of HFSC cell that is
CD133-positive, -positive, CDQ-positive, CD44—negative, AM—
positive, A285-positive', O4—positive, and NG2-positive. This information
enables to select or enrich HFSC cell without culturing. CD133 is. a marker for
neural stem cell and not expressed in progenitor or precursor cells. (309 is also
used as a marker for neural stem cell but some oligodendrocytes are known to
s CD9. PSA-NCAM and A285 are used to detect neuronal-restricted
precursor or glial—restricted precursor. Most neural precursors and progenitors
are thought to express PSA-NCAM and A285 or either PSA—NCAM or A285,
their usage cannot enrich HFSC cell so well, especially in the first and second
trimester. CD140a, NG2, A285 and 04 are used as markers for
endrocyte precursor cell, pro-oligodendroglia and oligodendrocyte. The
expression level of CD140a and NGZ were higher in HFSC cell than
endrocyte precursor cell, pro—oligodendroglia or oligodendrocyte,
s the expression level of A285 and 04 were lower in HFSC cell than
oligodendrocyte precursor cell, pro-oligodendroglia or oligodendrocyte. The
to be more appropriate to enrich HFSC
usage of CD140a and NG2 are thought
cell. Based on above information and the data described in this invention, the
effectiveness of each marker to enrich HFSC cell will be CD140a > NG2 > CD9
> CD133 > A285 > 04, PSA—NCAM but this order will be vary depend on their
gestation week.
However, a single marker will not be enough to select HFSC cells
and combination of 2 markers can select HFSC cell more ically. The
combination of one of neural stem cell markers (CD133 or 009) and one of
oligodendrocyte—lineage markers (CD140a, NG2) will be very effective to select
HFSC cells. Based on above knowledge, the most efficient combinations of
markers to select the HFSC cells will be CD133 and CD140a among these
combinations but other combinations should be also more ive than
selection with a single marker.
] The frequency of appearance of CD133 or CD140a is usually low
(less than 5%) and the appearance of CD140a is later (expression starts from
around 8—week and maximum at around 18-week of gestation week) than that
of CD133. Therefore, the cells expressing both CD133 and CD140a will be
their gestation week. Most
very low (less than 1% of total cells) depending on
of CD133—positive cells may not express CD140a if cells are d from
human fetal tissue at gestation week 15 or earlier. Because CD133-positive
and CD140a—negative cell will express CD140a later, the HFSC cell-can be
ed when the cells are ed in the same condition for HFSC cells after
the initial enrichment of CD133-positive cell.
Claims (17)
1. An enriched population of expanded human neural cells wherein the cells are progenitor cells or stem cells, wherein the cells have been cultured under conditions ive to enrich for the expanded human neural cells, wherein the conditions effective to enrich for expanded neural cells comprise a cell culture medium comprising at least one growth supplement in an effective amount, at least two growth factors, and at least one survival factor, n the at least one growth ment is glycerol and the effective amount of the growth supplement in the cell culture medium is at least 10 μM of 1- thioglycerol, wherein the cells maintain their capability to differentiate into neurons, astrocytes, and oligodendrocytes, wherein the cells in their ability to differentiate into oligodendrocyte lineage cells efficiently throughout subsequent passages of the culture, and wherein the population of cells express at least the cell surface antigens CD133, CD140a, A2B5 and PSA-NCAM.
2. The enriched population of ed human neural cells of claim 1 wherein the at least two growth factors are platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) and are present in a concentration of at least 5 ng/ml of PDGF and at least 2.5 ng/ml of bFGF to isolate the human neural cells in the culture.
3. The ed population of expanded human neural cells of claim 1 wherein the at least two growth factors are platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) and are present in a concentration of about 40 ng/ml to about 200 ng/ml of PDGF and about 5 ng/ml to about 40 ng/ml of bFGF to isolate the human neural cells in the culture.
4. The enriched tion of expanded human neural cells of claim 1 wherein the at least two growth factors are platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) and are present in a concentration of about 100 ng/ml of PDGF and about 20 ng/ml of bFGF to isolate the human neural cells in the culture.
5. The enriched population of expanded human neural cells of claims 2 to 6 wherein said the growth supplement is 1-thioglycerol and the effective amount of growth supplement in the culture medium is at least 50 µM of 1-thioglycerol to isolate the human neural cells in the culture.
6. The enriched population of ed human neural cells of claim 1 n the at least two growth factors are platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) and are present in a concentration of at least 1 ng/ml of PDGF and at least 0.5 ng/ml of bFGF to expand the cells after a cell line is established and in order to maintain the cells.
7. The enriched population of ed human neural cells of claim 1, wherein said the at least two growth s are platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) and are present in a concentration of about 5 ng/ml to about 100 ng/ml of PDGF and about 1 ng/ml to about 50 ng/ml of bFGF to expand human neural cells after an enriched population of human neural cells is established.
8. The enriched population of expanded human neural cells of claim 1, wherein said the at least two growth factors are platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) and are present in a concentration of about 20 ng/ml of PDGF and about 20 ng/ml of bFGF to expand human neural cells after an enriched population of human neural cells is established.
9. The enriched population of expanded human neural cells of claim 1, wherein said the at least one survival factor is Insulin-like growth factor (IGF) and is present in a concentration of at least 1 ng/ml of IGF.
10. The enriched population of expanded human neural cells of claim 1, wherein the at least one survival factor is IGF and is present in a concentration of about 5 ng/ml to about 100 ng/ml of IGF.
11. The ed population of expanded human neural cells of claim 1, n said the at least one survival factor is IGF and is t in a concentration of about 20 ng/ml of IGF.
12. The enriched population of expanded human neural cells of claim 1, wherein the cells can be frozen and thawed without losing its ability to differentiate into neurons, astrocytes, and oligodendrocytes throughout subsequent passages, and their ability to express at least the cell surface antigens CD133, CD140a, A2B5 and PSANCAM.
13. Use of a ition comprising the enriched population of cells of claim 1 in the preparation of a medicament for the treatment of demyelinating disease or neurodegenerative disease, n said demyelinating disease is caused by a loss of myelin or a loss of oligodendrocytes, n said demyelinating disease is selected from the group consisting of spinal cord injury (SCI), multiple sclerosis (MS), hereditary leukodystrophy, transverse athy/myelitis, progressive multiple focal leukoencephalopathy and other ital demyelinating diseases, wherein said neurodegenerative disease is selected from the group consisting of Alzheimer's disease, senile dementia of Alzheimer type (SDAT), Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis (ALS), ischemia, blindness and a egenerative disease caused by loss of neural cells.
14. A pharmaceutical neural stem cell composition sing an enriched population of expanded human neural cells as in claim 1.
15. The ed population of expanded human neural cells of claim 1, wherein said cells have been deposited as ATCC accession number PTA-12291.
16. The enriched population of expanded human neural cells of claim 1, n the cells that were cultured have been derived from a human fetal neural tissue selected from the group ting of spinal cord, cerebral cortex, hippocampus, striatum, basal forebrain, ventral mesencephalon, locus ceruleus, hypothalamus, cerebellum, corpus callosum and optic nerve.
17. The enriched population of expanded human neural cells of claim 1, wherein the cells that were cultured were derived from neural tissue from the human spinal cord at 8-24 weeks of gestation.
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US61/558,527 | 2011-11-11 | ||
PCT/IB2012/000030 WO2012095730A1 (en) | 2011-01-12 | 2012-01-12 | Culture method to obtain and maintain a pure or enriched population of mammalian neural stem cells and/or neural progenitor cells that are prone to differentiate into oligodendrocyte-lineage cells in vitro |
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