U.S. patent application number 13/082067 was filed with the patent office on 2011-07-28 for platelet derived growth factor (pdgf)-derived neurospheres define a novel class of progenitor cells.
This patent application is currently assigned to STEM CELL THERAPEUTICS INC.. Invention is credited to Andrew K. Chojnacki, Samuel Weiss.
Application Number | 20110182853 13/082067 |
Document ID | / |
Family ID | 26964336 |
Filed Date | 2011-07-28 |
United States Patent
Application |
20110182853 |
Kind Code |
A1 |
Weiss; Samuel ; et
al. |
July 28, 2011 |
PLATELET DERIVED GROWTH FACTOR (PDGF)-DERIVED NEUROSPHERES DEFINE A
NOVEL CLASS OF PROGENITOR CELLS
Abstract
The present invention is related to the discovery of a novel
class of neural progenitor cells, which proliferate in response to
platelet derived growth factor (PDGF) and differentiate into
neurons and oligodendrocytes but not astrocytes. Progeny of the
progenitor cells can be obtained by culturing brain tissue in PDGF
without serum, epidermal growth factor (EGF), fibroblast growth
factor 2, or transforming growth factor alpha. Upon subculturing
into EGF-containing media, these progeny cells can proliferate and
form neurospheres, whereas PDGF has no such effect.
Inventors: |
Weiss; Samuel; (Calgary,
CA) ; Chojnacki; Andrew K.; (Calgary, CA) |
Assignee: |
STEM CELL THERAPEUTICS INC.
Calgary
CA
|
Family ID: |
26964336 |
Appl. No.: |
13/082067 |
Filed: |
April 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10131230 |
Apr 25, 2002 |
7943376 |
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13082067 |
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60287214 |
Apr 27, 2001 |
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60307070 |
Jul 20, 2001 |
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Current U.S.
Class: |
424/85.2 ;
514/6.5; 514/8.2 |
Current CPC
Class: |
C12N 2503/02 20130101;
A61K 38/1858 20130101; A61P 25/16 20180101; C12N 2501/135 20130101;
C12N 5/0623 20130101; C12N 2501/11 20130101; A61P 25/00 20180101;
A61K 35/12 20130101 |
Class at
Publication: |
424/85.2 ;
514/8.2; 514/6.5 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61K 38/18 20060101 A61K038/18; A61K 38/28 20060101
A61K038/28; A61P 25/00 20060101 A61P025/00; A61P 25/16 20060101
A61P025/16 |
Claims
1-18. (canceled)
19. A method of inducing proliferation of a neural progenitor cell
in mammal, the method comprising administering an effective amount
of a platelet derived growth factor (PDGF) to the mammal.
20. The method of claim 19, wherein the mammal has a
neurodegenerative condition or disease.
21. The method of claim 20, wherein the neurodegenerative condition
or disease is Parkinson's disease.
22. The method of claim 19, wherein the PDGF is administered into
the brain of the mammal.
23. The method of claim 19, further comprising administering one or
more additional growth factors or biological agents.
24. The method of claim 23, wherein the one or more additional
growth factors or biological agents are selected from the group
consisting of BGF, PDGF, FGF-1, FGF-2, TGF-.alpha., TGF-.beta.,
nerve growth factor (NGF), ciliary neurotrophic factor (CNTF),
brain derived neurotrophic factor (BDNF), neurotrophin 3,
neurotrophin 4, leukemia inhibitory factor (LIF), bone morphogenic
protein 2 (BMP-2), insulin-like growth factor, insulin, growth
factor, prolactin, interleukin, forskolin, cAMP and cAMP analog,
pituitary adenylate cyclase activating polypeptide (PACAP), phorbal
ester, estrogen, and ovarian hormone.
25. A method of treating or ameliorating a neurodegenerative
condition or disease in a mammal, the method comprising
administering an effective amount of a platelet derived growth
factor (PDGF) to the mammal.
26. The method of claim 25, wherein the neurodegenerative condition
or disease is Parkinson's disease.
27. The method of claim 25, wherein the PDGF is administered into
the brain of the mammal.
28. The method of claim 25, further comprising administering one or
more additional growth factors or biological agents.
29. The method of claim 28, wherein the one or more additional
growth factors or biological agents are selected from the group
consisting of BGF, PDGF, FGF-1, FGF-2, TGF-.alpha., TGF-.beta.,
nerve growth factor (NGF), ciliary neurotrophic factor (CNTF),
brain derived neurotrophic factor (BDNF), neurotrophin 3,
neurotrophin 4, leukemia inhibitory factor (LIF), bone morphogenic
protein 2 (BMP-2), insulin-like growth factor, insulin, growth
factor, prolactin, interleukin, forskolin, cAMP and cAMP analog,
pituitary adenylate cyclase activating polypeptide (PACAP), phorbal
ester, estrogen, and ovarian hormone.
30. A pharmaceutical composition comprising platelet derived growth
factor (PDGF) for administration to a mammal for treating or
ameliorating a neurodegenerative condition or disease.
31. The pharmaceutical composition of claim 30, wherein the
neurodegenerative condition or disease is Parkinson's disease.
32. The pharmaceutical composition of claim 30, wherein the
pharmaceutical composition is for administration to the brain of
the mammal.
Description
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/287,214, filed Apr. 27, 2001, and U.S.
Provisional Application Ser. No. 60/307,070 filed Jul. 20, 2001,
both of which are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention is related to the discovery of a novel
class of neural progenitor cells which can differentiate into
neurons and oligodendrocytes but not astrocytes, and methods of
proliferating these progenitor cells by using platelet derived
growth factor (PDGF).
REFERENCES
[0003] U.S. Pat. No. 5,750,376. [0004] U.S. Pat. No. 5,980,885.
[0005] U.S. Pat. No. 5,851,832. [0006] Dirks R and Bloomers H.
1996. Signals controlling the expression of PDGF. Mol. Biology
Reports 22: 1-24, [0007] Ek B. Westermark B, Wasteson A. and Heldin
C H. 1982. Stimulation of tyrosine-specific phosphorylation by
platelet-derived growth factor. Nature 295(5848):419-420. [0008]
Hannink M and Donoghue D J, 1989. Structure and function of
platelet-derived growth factor (PDGF) and related proteins. Biochim
Biophys Acta. 989(1):1-10. [0009] Nishimura J, Huang J S, and Deuel
T F, 1982. Platelet-derived growth factor stimulates
tyrosine-specific protein kinase activity in Swiss mouse 3T3 cell
membranes Proc Natl Acad Sci USA. 79(14):4303-4307
[0010] All of the above publications, patents and patent
applications are herein incorporated by reference in their entirety
to the same extent as if the disclosure of each individual
publication, patent application or patent was specifically and
individually indicated to be incorporated by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0011] The development of the neural system has been an intensively
studied area. For example, neurodegenerative disease has become an
important concern due to the expanding elderly population which is
at greatest risk for these disorders. These diseases, which include
Alzheimer's Disease, Multiple Sclerosis (MS), Huntington's Disease,
Amyotrophic Lateral Sclerosis, and Parkinson's Disease, have been
linked to the degeneration of neural vi cells in particular
locations of the central nervous system (CNS), leading to inability
of these cells or the brain region to carry out their intended
function. Therefore, it is desirable to find out how neural cells,
including neurons, oligodendrocytes and astrocytes, are generated.
With such findings, neural cells can then be produced in vivo or in
vitro to compensate for the degenerate or injured neural cells.
[0012] A major progress in this study was the discovery of
multipotent neural stem cells (for example see U.S. Pat. Nos.
5,750,376; 5,980,885; 5,851,832). Briefly, these stem cells may be
isolated from both fetal and adult brains, and cultured in vitro
indefinitely. These cells retain the ability to proliferate in
response to growth factors, or differentiate into all lineages of
neural cells (neurons and glia cells, including astrocytes and
oligodendrocytes) in response to differentiation stimuli. To date,
epidermal growth factor (EGF), transforming growth factor alpha
(TGF-.alpha.) and fibroblast growth factor-2 (FGF-2) are the only
factors known to induce the proliferation of single precursor cells
that can give rise to neurons, oligodendrocytes, and astrocytes.
However, the role of other regulatory factors or cells in the
development of the neural system remains to be uncovered.
SUMMARY OF THE INVENTION
[0013] The present invention relates to the discovery of a novel
class of progenitor cells which can proliferate from brain tissue
in the presence of platelet derived growth factor (PDGF). Epidermal
growth factor (EGF), transforming growth factor alpha (TGF-.alpha.)
or fibroblast growth factor-2 (FGF-2) are not required. The progeny
of these progenitor cells are capable of differentiating into
neurons and oligodendrocytes, but not astrocytes. In addition,
while these progeny cells can self-renew and expand when
subcultured into media containing EGF, they do not proliferate in
response to PDGF. Therefore, these cells are a novel class of
progenitor cells.
[0014] Accordingly, one aspect of the present invention provides a
method of producing progeny of a neural progenitor cell wherein the
progenitor cell is capable of differentiating into neurons and
oligodendrocytes but not astrocytes, comprising culturing brain
tissue in the presence of platelet derived growth factor (PDGF)
under conditions that allow proliferation of said progenitor
cell.
[0015] In a preferred embodiment of the present invention, the
progeny cells are capable of proliferating in response to epidermal
growth factor (EGF) but not PDGF.
[0016] Preferably, the brain tissue is cultured in the absence of
serum, EGF, fibroblast growth factor 2 (FGF-2), transforming growth
factor alpha (TGF-.alpha.), or any combination thereof. The brain
tissue may be obtained from embryonic or adult brains. When the
tissue is obtained from embryonic brains, it is preferably from the
ganglionic eminence and more preferably from medial ganglionic
eminence.
[0017] Also provided are the progeny cells produced by culturing
brain tissue in the presence of PDGF as described above.
[0018] Another aspect of the present invention provides a method of
screening drugs, comprising:
(a) providing a population of the progeny cells of the present
invention; (b) contacting the progeny cells with a candidate drug;
and (c) determining the effect of the candidate drug on the progeny
cells.
[0019] If the candidate drug leads to a desired effect, the drug
can be further tested and developed. The desired effect may be, for
example, proliferation of the progeny cells, activation or
inhibition of an enzyme that is associated with a disease or
medical condition, or binding of a receptor in the cell.
[0020] Another aspect of the present invention provides a method of
identifying genes that are involved in proliferation or
differentiation of the progenitor cells, comprising providing a
cDNA library prepared from a population of proliferated cells,
providing a cDNA library prepared from a population of
differentiated cells, and comparing the two cDNA libraries. cDNAs
that are present selectively in the proliferated cell library are
likely involved in proliferation, while cDNAs that are present
selectively in the differentiated cell library are likely involved
in differentiation. These cDNAs can then be further characterized
according to established methods in the art.
[0021] In addition, the present invention also provides a method of
identifying genes that participate in astrocyte differentiation,
comprising comparing a cDNA library prepared from differentiated
multipotent neural stem cells to a cDNA library prepared from
differentiated progeny cells of the progenitor cells described
herein. Since multipotent neural stem cells differentiate to
neurons, oligodendrocytes and astrocytes, while the progeny cells
of the present invention differentiate to neurons and
oligodendrocytes only, cDNAs present in the neural stem cell
library but not the library of the progeny cells will likely
participate in astrocyte differentiation. These cDNAs can then be
further characterized according to established methods in the
art.
[0022] Accordingly, also provided are cDNA libraries prepared from
the progenitor cells or progeny, as well as nucleic acid or protein
microarrays prepared using the nucleic acids or proteins of the
progenitor cells and progeny.
[0023] Still another aspect of the present invention provides a
method of modifying the progeny cells described herein, comprising
introducing a nucleic acid into the progeny cells to result in
alteration in the genetic material in the cells. The resultant
modified cells are also provided.
[0024] Yet another aspect of the present invention provides a
method of treating or ameliorating a disease or medical condition
associated with neuron or oligodendrocyte loss or dysfunction,
comprising transplanting an effective amount of the progeny cells
to a mammal harboring the disease or medical condition. Optionally,
other biological agents can be administered to the mammal as well,
including, e.g., EGF, PDGF, FGF-1, FGF-2, TGF-.alpha., TGF-.beta.,
nerve growth factor (NGF), ciliary neurotrophic factor (CNTF),
brain derived neurotrophic factor (BDNF), neurotrophin 3,
neurotrophin 4, leukemia inhibitory factor (LIF), hone morphogenic
protein 2 (BMP-2), insulin-like growth factors, insulin, growth
factor, prolactin, interleukins, forskolin, cAMP or cAMP analogs,
pituitary adenylate cyclase activating polypeptide (PACAP) phorbol
esters, estrogen and ovarian hormones. These biological agents may
be administered prior to, concurrently or after transplantation of
the progeny cells. The transplanted cells may be syngeneic,
allogeneic or xenogeneic to the transplantation recipient.
Preferably, the transplant is syngeneic or allogeneic, and most
preferably syngeneic.
[0025] Another aspect of the present invention provides a method of
inducing proliferation of a neural progenitor cell that
differentiate into neurons and oligodendrocytes but not astrocytes,
comprising administering an effective amount of platelet derived
growth factor (PDGF) to a mammal. The PDGF, or an agent known to
induce or activate PDGF, can be administered via any route known in
the art. PDGF is preferably administered into the brain of the
mammal. This method can be combined with the transplantation
described above.
[0026] Also provided are pharmaceutical compositions comprising
progeny cells of the present invention. The pharmaceutical
compositions preferably further comprise a pharmaceutically
acceptable excipient and, or a pharmaceutically acceptable
carrier.
DETAILED DESCRIPTION OF THF INVENTION
[0027] The present invention relates to the discovery of a novel
class of progenitor cells which are derived from brain tissue and
proliferate in the presence of platelet derived growth factor
(PDGF) to form neurospheres. These PDGF-generated neurospheres
contain progeny cells which are capable of differentiating
primarily into neurons and oligodendrocytes. In addition, while
these progeny cells can self-renew and expand when subcultured into
EGF-containing media, they do not proliferate in response to
PDGF.
[0028] Prior to describing the invention in further detail, the
terms used in this application are defined as follows unless
otherwise indicated.
Definition
[0029] A "multipotent neural stem cell", or "neural stem cell", is
a stem cell in the neural cell lineage. A stem cell is a cell which
is capable of reproducing itself. In other words, when a stem cell
divides, at least some of the resulting daughter cells are also
stem cells. The neural stem cells, and their progeny, are capable
of differentiating into all the cell types in the neural cell
lineage, including neurons, astrocytes and oligodendrocytes
(astrocytes and oligodendrocytes are collectively called glia or
glial cells). Therefore, the neural stem cells are multipotent
neural stem cells. Multipotent neural stem cells are described, for
example, in U.S. Pat. Nos. 5,750,376; 5,980,885; and 5,851,832.
[0030] The adult neural stem cells preferably refer to the neural
stem cells located in or derived from the subventricular zone (SVZ)
of the forebrain of adult mammals, which are different from the
proliferating cells in the adult hippocampus.
[0031] The "progeny" of the novel progenitor cells described herein
refers to any and all cells derived from the progenitor cells as a
result of proliferation or differentiation. In particular, the
progeny cells include the cells in the primary neurospheres which
are prepared by culturing brain tissue in the presence of PDGF but
not EGF, FGF-2, or TGF-.alpha..
[0032] A "neurosphere" or "sphere", as used herein, is a cluster of
cells derived from a single neural cell.
[0033] A "platelet derived growth factor", or "PDGF" is a protein
factor which (1) shares substantial sequence identity with the
native human PDGF; and (2) possesses a biological activity of the
native human PDGF. Native PDGF consists of two polypeptide chains
selected from Chain A and Chain B. Chain A and Chain B are similar.
For example, the human Chain A and Chain B share a 56% sequence
identity in the mature PDGF molecule. A PDGF molecule may consist
of A-A, A-B or B-B. A discussion of the structural and functional
relationship of PDGF can be found, for example, in Hannink et al.,
1989.
[0034] A protein which shares "substantial sequence identity" with
the native human PDGF consists of at least one polypeptide that is
at least about 30% identical with Chain A or Chain B of the native
human PDGF at the amino acid level. The PDGF is preferably at least
about 40%, more preferably at least about 60%, yet more preferably
at least about 70%, and most preferably at least about 80%
identical with Chain A or Chain B of the native human PDGF at the
amino acid level. Thus, the term "PDGF" encompasses PDGF analogs
which are the deletional, insertional, or substitutional mutants of
the native PDGF. Furthermore, the term "PDGF" encompasses the PDGFs
from other species, the naturally occurring variants, and different
post-translationally modified forms (such as the glycosylated and
phosphorylated forms) thereof.
[0035] The phrase "percent identity" or "% identity" with the
native PDGF refers to the percentage of amino acid sequence in
Chain A or Chain B of the native human PDGF which are also found in
the PDGF analog when the two sequences are best aligned (including
gaps). Percent identity can be determined by any methods or
algorithms established in the art, such as LALIGN or BLAST.
[0036] A factor possesses a "biological activity of PDGF" if it is
capable of binding to any known PDGF receptor and stimulates the
tyrosine kinase activity of the receptor (Ek et al., 1982;
Nishimura et al., 1982).
[0037] A "PDGF-derived neurosphere" or "PDGF-generated neurosphere"
is a neurosphere produced from brain tissue in the presence of
PDGF. These neurospheres are primary neurospheres since they are
generated from brain tissue without cell passaging.
[0038] An "EGF-derived neurosphere" or "EGF-generated neurosphere"
is a neurosphere produced from brain tissue in the presence of EGF.
These neurospheres are primary neurospheres since they are
generated from brain tissue without cell passaging.
[0039] A "secondary neurosphere" is a neurosphere generated by
dissociating (passaging) a primary neurosphere and culturing the
dissociated cells under conditions which result in the formation of
neurospheres from single cells.
[0040] A "neural disease or condition associated with neuron or
oligodendrocyte loss or dysfunction" is a disease or medical
condition that is caused by or otherwise associated with
neuron/oligodendrocyte loss or dysfunction. Examples of these
diseases or conditions include neurodegenerative diseases, brain
injuries or CNS dysfunctions. Neurodegenerative diseases include,
for example, Alzheimer's Disease, multiple sclerosis (MS), macular
degeneration, glaucoma, diabetic retinopathy, peripheral
neuropathy, Huntington's Disease, amyotrophic lateral sclerosis,
and Parkinson's Disease. Brain injuries include, for example,
stroke (e.g., hemorrhagic stroke, focal ischemic stroke or global
ischemic stroke), and traumatic brain injuries (e.g. injuries
caused by a brain surgery or physical accidents). CNS dysfunctions
include, for example, depression, epilepsy, neurosis and
psychosis.
[0041] "Treating or ameliorating" means the reduction or complete
removal of the symptoms of a disease or medical condition.
[0042] An "effective amount" is an amount of a therapeutic agent
sufficient to achieve the intended purpose. The effective amount of
a given therapeutic agent will vary with factors such as the nature
of the agent, the route of administration, the size and species of
the animal to receive the therapeutic agent, and the purpose of the
administration. The effective amount in each individual case may be
determined empirically by a skilled artisan according to
established methods in the art.
Methods
[0043] Fibroblast growth factor-2, transforming growth factor
.alpha., and epidermal growth factor can induce the in vitro
proliferation of multipotent neural stem cells, derived from the
E14 mouse basal forebrain or adult brain tissue, into neurospheres
of undifferentiated cells. To date, these are the only factors
which have been reported to induce the proliferation of single
precursor cells that can give rise to neurons, oligodendrocytes,
and astrocytes.
[0044] In the present invention, we investigated if PDGF alone
could stimulate the formation of neurospheres in defined media. As
shown in Example 1, PDGF induces the formation of neurospheres from
dissociated cells of the E14 mouse basal forebrain in the absence
of EGF, TGF-.alpha., and FGF-2. The formation of these neurospheres
was inhibited by Tyrphostin AG 1296, an inhibitor of PDGF receptor
phosphorylation, indicating that PDGF-derived neurosphere formation
is mediated by PDGF receptor kinase action (Example 2).
[0045] The PDGF-generated neurospheres consist of cells that are
different from EGF-generated neurospheres. When primary brain
tissue culture is exposed to EGF, multipotent neural stem cells
proliferate and form neurospheres. As shown in Example 4, these
primary neurospheres can be dissociated into single cells, cultured
under clonal conditions in the presence of EGF or PDGF, and expand
to form secondary neurospheres. In contrast, when PDGF-generated
neurospheres were dissociated, the constituent cells could not
self-renew or produce secondary neurospheres when subcultured back
into PDGF. However, PDGF-generated neurospheres did
self-renew/expand when subcultured into EGF (Example 3).
[0046] The differentiation patterns of PDGF- and EGF-derived
neurospheres are also different. Primary PDGF-generated
neurospheres differentiate primarily into neurons and
oligodendrocytes (Example 5), as opposed to the neurospheres
derived from multipotent neural stem cells that differentiate into
neurons, oligodendrocytes and astrocytes, wherein the percentage of
astrocyte is typically 60-70%.
[0047] Consistent with the results described above, further
evidence indicates that the EGF-generated neurospheres and
PDGF-generated neurospheres are produced from cells with different
spatial distribution patterns. As shown in Example 6, ganglionic
eminence from E14 embryos was dissected into two portions, medial
ganglionic eminence (MGE) and lateral ganglionic eminence (LGE).
MGE and LGE were then dissociated, cultured in either EGF or PDGF,
and allowed to form neurospheres. In the presence of PDGF,
neurospheres were produced primarily from MGE-derived cells, with
LGE producing very few neurospheres. However, both MGE and LGE were
capable of efficiently producing neurospheres in the presence of
EGF. These results thus indicate that the EGF-generated spheres and
PDGF-generated spheres do not come from the same cells.
[0048] Furthermore, we also discovered that when both EGF and PDGF
are present in the culture media, more neurospheres are produced
than with either EGF or PDGF alone. Although there are several
possible explanations for this observation, the result is again
consistent with the notion that PDGF induces the formation of
neurospheres from a novel progenitor cell, which is not the
multipotent neural stem cell.
[0049] The present invention thus provides a method of producing
progeny of a novel class of progenitor cells, which, in response to
PDGF, proliferate to neurospheres with unique proliferation and
differentiation properties. These neurospheres can be obtained by
culturing brain tissue in defined media in the absence of EGF,
TGF-.alpha.. FGF-2, serum or any combination thereof. The brain
tissue can be derived from any mammalian brain, including adult and
embryonic brains. Preferably, the brain tissue is harvested from
the forebrain, particularly the striatum. The brain tissue is more
preferably ganglionic eminence, and most preferably medial
ganglionic eminence. The brain tissue is preferably from a primate,
rodent, feline, canine, domestic livestock (such as cattle),
particularly human.
[0050] These progenitor cells, as well as their progeny, can be
used to produce neurons and oligodendrocytes. Since multipotent
neural stem cells typically produce about 60-70% astrocytes, the
progenitor cells of the present invention provide a more enriched
source for neurons and oligodendrocytes. As such, the progenitor
cells and their progeny can be used to treat or ameliorate neural
diseases or conditions associated with neuron or oligodendrocyte
loss or dysfunction, such as Alzheimer's Disease, multiple
sclerosis (MS), macular degeneration, glaucoma, diabetic
retinopathy, peripheral neuropathy, Huntington's Disease,
amyotrophic lateral sclerosis, Parkinson's Disease, stroke (e.g.,
hemorrhagic stroke, focal ischemic stroke or global ischemic
stroke), traumatic brain injuries (e.g. injuries caused by a brain
surgery or physical accidents), depression, epilepsy, neurosis and
psychosis.
[0051] The progenitor cells and their progeny can be cultured in
vitro and transplanted into a mammal to compensate for lost neurons
or oligodendrocytes. In this treatment, the progeny may be neurons
and oligodendrocytes that have been induced to differentiate in
vitro, or precursor cells from PDGF-derived neurospheres. Growth
factors or other biological agents can be co-administered into the
mammal to facilitate proliferation and/or differentiation of neural
cells. These growth factors and biological agents include, but are
not limited to, EGF, PDGF, FGF-1, FGF-2, TGF-.alpha., TGF-.beta.,
nerve growth factor (NGF), ciliary neurotrophic factor (CNTF),
brain derived neurotrophic factor (BDNF), neurotrophin 3,
neurotrophin 4, leukemia inhibitory factor (LIF), bone morphogenic
protein 2 (BMP-2), insulin-like growth factors, insulin, growth
factor, prolactin, interleukins, forskolin, cAMP or cAMP analogs,
pituitary adenylate cyclase activating polypeptide (PACAP) phorbol
esters, estrogen and ovarian hormones. It is also contemplated that
other cells, such as multipotent neural stem cells, can be
transplanted into the same mammal to provide additional source of
neural cell. These other cells, growth factors or biological agents
can be given to the mammal prior to, concurrently with, or after
transplantation of the progenitor cells and progeny of the present
invention.
[0052] Alternatively, PDGF can be administered in vivo to induce
proliferation of the progenitor cells and ultimately compensate for
lost neurons and/or oligodendrocytes. PDGF, or agents known to
induce or activate PDGF, can be administered by any route. PDGF is
preferably administered into the brain, more preferably a ventricle
in the brain and midst preferably the lateral ventricle. Any agents
known to induce or activate PDGF can also be used (e.g., see Dirks
et al., 1996). Growth factors and/or other biological agents, as
described above, can optionally be administered prior to,
concurrently with, or after administration of PDGF.
[0053] The novel progenitor cells and their progeny can also be
used to identify genes that are involved in proliferation or
differentiation of these cells. For example, a cDNA library can be
prepared using the neurospheres produced by culturing brain tissue
in PDGF as disclosed herein. The neurospheres are then exposed to
biological agents that induce the spheres to proliferate or
differentiate, and another cDNA library is prepared using the
proliferated or differentiated cells. By comparing the two cDNA
libraries (e.g., by subtraction cloning), genes that participate in
proliferation or differentiation can be identified. Those genes
that are up-regulated in the process of proliferation may include,
without being limited to, genes encoding transcription factors,
enzymes and growth factor receptors that stimulate proliferation or
inhibit differentiation. The genes that are down-regulated during
proliferation may include, without being limited to, gene encoding
transcription factors, enzymes and growth factor receptors that
inhibit proliferation or induce differentiation to neurons and/or
oligodendrocytes. Similarly, the genes that are up-regulated in the
process of differentiation may include, without being limited to,
genes encoding transcription factors, enzymes and growth factor
receptors that inhibit proliferation or stimulate differentiation
to neurons and/or oligodendrocytes. The genes that are
down-regulated during differentiation may include, without being
limited to, gene encoding transcription factors, enzymes and growth
factor receptors that induce proliferation or inhibit
differentiation to neurons and/or oligodendrocytes.
[0054] Since the progenitor cells of the present invention do not
differentiate to astrocytes and multipotent neural stem cells do,
the present invention also provides a method of identifying factors
or genes that control astrocyte formation. For example, the cDNA
library of differentiating neural stem cells may be subtracted with
the cDNA library of proliferating neural stem cells to remove
proliferation-related genes and house-keeping genes. Thereafter,
the subtracted library can be further subtracted with the cDNA
library prepared front cells of the present invention that have
been induced to differentiate. Differentiating factors that are
selective for astrocytes should remain, while other differentiating
factors are likely to be removed by this second subtraction.
[0055] The progenitor cells and progeny can also be used to
identify potential therapeutic agents for diseases. For example,
the cells can be exposed to various candidate drugs and the effect
of the candidates determined. Depending on the purpose of the drug
screening, the practitioner may look for, for instance, the
expression of certain neural marker, the alteration of activity
level of an enzyme, the formation of a specialized cell type, or
increased cell numbers. Candidate drugs that result in the desired
effect can then be further tested and developed.
[0056] It should be noted that the progeny cells of the present
invention can be modified by genetic engineering. The modified
cells can then be transplanted into a mammal or used to study
neurobiology. The methods of modification and nucleic acids to be
used in such modification will vary depending on the purpose of the
modification. For example, the cells may be modified to produce a
biological agent, to knock out a gene, or to express a reporter
gene that can be used to detect the effect of candidate drugs in a
drug screening system. The methods and nucleic acids to be used can
be determined by people of ordinary skill according to the
disclosure herein and knowledge in the art,
Compositions
[0057] The present invention provides a progenitor cell that
responds to PDGF to form neurospheres in the absence of EGF, FGF-2,
TGF-.alpha., serum, and the combination thereof. Also provided are
neurospheres obtained as described above, which comprise progeny
cells of the progenitor. The progeny can differentiate to neurons
and oligodendrocytes but not astrocytes. cDNA libraries and
microarrays containing the nucleic acids or proteins of the progeny
cells are also provided, as well as progeny cells that have been
modified by genetic engineering techniques.
[0058] The present invention further provides pharmaceutical
compositions comprising the progenitor cells, or particularly the
progeny cells, of the present invention. These pharmaceutical
compositions are useful, for example, in transplantation treatment
for subjects with a disease or condition associated with neuron or
oligodendrocyte loss or dysfunction. The pharmaceutical
compositions preferably further comprise a pharmaceutically
acceptable excipient and/or a pharmaceutically acceptable
carrier.
[0059] The following examples are offered to illustrate this
invention and are not to be construed in any way as limiting the
scope of the present invention.
EXAMPLES
[0060] In the examples below, the following abbreviations have the
following meanings. Abbreviations not defined have their generally
accepted meanings.
[0061] .degree. C.=degree Celsius
[0062] hr=hour
[0063] min=minute
[0064] .mu.M=micromolar
[0065] mM=millimolar
[0066] M=molar
[0067] ml=milliliter
[0068] .mu.l=microliter
[0069] mg=milligram
[0070] .mu.g=microgram
[0071] FBS=fetal bovine serum
[0072] DTT=dithiothrietol
[0073] SDS=sodium dodecyl sulfate
[0074] PBS=phosphate buffered saline
[0075] DMEM=Dulbecco's modified Eagle's medium
[0076] .alpha.-MEM=.alpha.-modified Eagle's medium
[0077] .beta.-ME=.beta.-mercaptoethanol
[0078] EGF=epidermal growth factor
[0079] FGF=fibroblast growth factor
[0080] PDGF=platelet derived growth factor
[0081] TGF-.alpha.=transforming growth factor alpha
[0082] DMSO=dimethylsulfoxide
[0083] MGE=medial ganglionic eminence
[0084] LGE=lateral ganglionic eminence
Example 1
PDGF Induces Production of Primary Neurospheres
[0085] E14 striatum was mechanically dissociated as previously
described (for example see U.S. Pat. No. 5,750,376; 5,980,885; or
5,851,832) and plated at 10,000 cells/ml in 6 well plates in
defined culture media plus 100 ng/ml of PDGF-AA (Peprotech). The
composition of defined culture media is as follows: [0086] DMEM/F12
(1:1) [0087] glucose (0.6%) [0088] glutamine (2 mM) [0089] sodium
bicarbonate (3 mM) [0090] HEPES (5 mM) [0091] insulin (25 .mu.g/ml)
[0092] transferrin (100 .mu.g/ml) [0093] progesterone (20 nM)
[0094] putrescine (60 .mu.M) [0095] selenium chloride (30 nM)
[0096] Neurospheres, which are clusters of neural cells derived
from single cells, formed after 7 days of culture in vitro (DIV).
Six wells per experiment were counted for neurosphere production
and the results are shown below:
TABLE-US-00001 Average number of Experiment# neurospheres
produced/well 1 14.5 2 9.2 3 34.3* 4 8.6 5 7.7 6 31.5* average =
17.7 .+-. 5.0 *PDGF appears to be approximately 3 fold more potent
when used immediately after reconstitution in defined culture
media.
[0097] In control experiments, wherein PDGF was omitted from the
culture media, no neurospheres formed. Therefore, PDGF is capable
of inducing neurosphere production from single precursor cells in
the absence of serum, EGF, TGF-.alpha. and FGF-2.
Example 2
Tyrphostin AG 1296 Inhibits the Production of Primary Neurospheres
by PDGF but not EGF
[0098] In order to determine if the PDGF-induced primary
neurosphere formation is mediated by the PDGF receptor kinase, a
selective PDGF receptor kinase inhibitor, Tyrphostin AG 1296, was
added to neurosphere culture. Primary cells were cultured at 10,000
cells/ml in the presence of either EGF or PDGF, plus 5 .mu.M of
Tyrphostin AG 1296 (Sigma) or DMSO in the same volume as Tyrphostin
(DMSO being the solvent for Tyrphostin). Results are expressed
below as the percentage of neurospheres formed, with the number of
PDGF- or EGF-derived neurospheres arbitrarily set at 100%,
respectively'.
TABLE-US-00002 TABLE 1 The Effect of Tyrphostin on neurosphere
formation in response to EGF or PDGF PDGF or EGF alone with DMSO
with Tyrphostin PDGF 100% 97% 10% EGF 100% 101% 55% n = 3 to 9
[0099] Therefore, Tyrphostin AG 1296 almost abolished primary
neurosphere formation induced by PDGF, indicating that formation of
the PDGF-derived neurospheres is mediated via PDGF receptor kinase
action. EGF-derived neurosphere formation was also inhibited by
Tyrphostin AG 1296 to some extent. Although the reasons for the
inhibition of EGF action are not clear, it is possible that
Tyrphostin AG 1296 is also a partial inhibitor for the EGF
receptor.
Example 3
PDGF-Derived Primary Neurospheres can be Subcultured into EGF but
not PDGF Containing Media
[0100] Single PDGF primary neurospheres prepared as described in
Example 1 were transferred into 96-well plates and mechanically
dissociated in either the presence of 20 ng/ml EGF (Peprotech) or
100 ng/ml PDGF-AA. The formation of secondary neurospheres was
assayed after 7 or more days in culture in vitro. The results are
shown below.
TABLE-US-00003 Average # of secondary neurospheres formed/well PDGF
0 EGF 3.12 .+-. 1.64 (n = 3)
[0101] Therefore, the cells in PDGF-derived primary neurospheres
cannot proliferate in response to PDGF. In contrast, these cells
can proliferate and form secondary neurospheres in response to
EGF.
Example 4
EGF-Derived Primary Neurospheres can be Subcultured into Either
EGF- or PDGF-Containing Media
[0102] To determine if the neurospheres derived in EGF-containing
media have different proliferation properties as those of
PDGF-generated neurospheres, primary EGF neurospheres were
generated from embryonic day 14 striatum by culturing dissociated
striatum in EGF (20 ng/ml) containing define culture media at a
cell density of 200,000 cells/ml. Subsequently, individual
neurospheres were isolated, placed in individual wells in either
PDGF or EGF containing media, and dissociated mechanically. The
numbers of secondary neurospheres that came from a single primary
EGF-generated neurospheres are given below (eight wells for each
condition; numbers indicate average number of secondary
neurospheres/well):
TABLE-US-00004 EGF-containing media PDGF-containing media Exp. #1
27 21.5 Exp. #2 14.1 26.7
[0103] Accordingly, EGF-derived neurospheres, in contrast to
PDGF-derived neurospheres, contain cells which can proliferate in
response to either EGF or PDGF to form secondary neurospheres.
Clearly, the PDGF-derived neurospheres define a novel class of
progenitor cells which are distinct from multipotent neural stem
cells which give rise to the EGF-derived neurospheres.
Example 5
Primary PDGF-Derived Neurospheres Differentiate into Neurons and
Oligodendrocytes
[0104] Primary PDGF-derived spheres, derived either clonally
(10,000 cells/ml) or in high density culture (200,000 cells/ml),
were plated without dissociation onto poly-1-ornithine coated
coverslips and allowed to differentiate for 2-5 days in vitro in
the absence of serum. These PDGF derived spheres, whether produced
clonally or in high density culture, yielded differentiated neurons
and a smaller number of oligodendrocytes. No astrocytes could be
detected. Again, these results indicate that the primary
neurospheres formed in response to PDGF define a novel class of
progenitor cells.
Example 6
The PDGF-Induced Neurospheres are Derived from Different Cells as
the EGF-Induced Neurospheres
[0105] In order to locate the cells that give rise to the novel
progenitor cells described herein, we dissected ganglionic eminence
to two portions. Thus, the ganglionic eminence was isolated from
E14 embryos of mice, and the medial ganglionic eminence (MGE) was
separated from the lateral ganglionic eminence (LGE). MGE and LGE
were then dissociated and cultured as described in Example 1. The
resulting primary culture was exposed to EGF or PDGF in addition to
the defined culture media, and the number of neurospheres were
counted and summarized below.
TABLE-US-00005 TABLE 2 Neurosphere formation using medial,
ganglionic eminence and lateral ganglionic eminence PDGF EGF Source
of brain MGE LGE MGB LGE tissue Average 10.22 .+-. 1.11 1.27 .+-.
0.45 16.9 .+-. 2.87 9.64 .+-. 1.59 number of neurospheres n = 2 or
3
[0106] These results show that the PDGF-induced neurospheres are
primarily derived from MGE. In contrast, the EGF-induced
neurospheres can be produced efficiently using both MGE and LGE,
and the MGE produces more spheres in response to FOP than LGE.
Accordingly, it is highly unlikely that the same cell type give
rise to both PDGF- and EGF-induced neurospheres. Instead, the cells
that give rise to PDGF-induced neurospheres are located primarily
in the MGE, while the cells that form EGF-induced spheres are
located in both MGE and LGE.
Example 7
Combination of PDGF and EGF
[0107] We also tested the effect of combining PDGF and EGF on the
number of neurospheres formed. Thus, brain tissue was prepared as
described in Example 1 and cultured in the presence of PDGF, EGF,
or the combination of PDGF and EGF. The number of neurospheres from
each experiment was then counted and shown in Table 3.
TABLE-US-00006 TABLE 3 Combined effect of PDGF and EGF PDGF EGF
PDGF + EGF Average number of 73.36 .+-. 7.13 80.5 .+-. 8.57 137.75
.+-. 11.7 neurospheres
[0108] These results indicate that there is an additive effect when
PDGF and EGF are combined. This additive effect is consistent with
the notion that PDGF and EGF stimulate different cells to
proliferate and form neurospheres.
* * * * *