U.S. patent application number 14/202121 was filed with the patent office on 2014-07-03 for pure populations of astrocyte restricted precursor cells and methods for isolation and use thereof.
This patent application is currently assigned to Governmement of the U.S.A., as represented by the Secretary, Department of Health and Human Services. The applicant listed for this patent is Government of the U.S.A, as represented by the Secretary, Department of Health and Human Services, Government of the U.S.A, as represented by the Secretary, Department of Health and Human Services, University of Utah. Invention is credited to Ying Liu, Tahmina Mujtaba, Mahendra S. Rao, Yuan Yuan Wu.
Application Number | 20140186314 14/202121 |
Document ID | / |
Family ID | 27613451 |
Filed Date | 2014-07-03 |
United States Patent
Application |
20140186314 |
Kind Code |
A1 |
Rao; Mahendra S. ; et
al. |
July 3, 2014 |
PURE POPULATIONS OF ASTROCYTE RESTRICTED PRECURSOR CELLS AND
METHODS FOR ISOLATION AND USE THEREOF
Abstract
An isolated, pure homogeneous population of mammalian astrocyte
restricted precursor cells which is CD44 immunoreactive and which
generate astrocytes but not oligodendrocytes is provided. Methods
for isolating and using these mammalian astrocyte restricted
precursor cells are also provided.
Inventors: |
Rao; Mahendra S.; (Timonium,
MD) ; Mujtaba; Tahmina; (Blaine, MN) ; Wu;
Yuan Yuan; (Salt Lake City, UT) ; Liu; Ying;
(Parkville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Government of the U.S.A, as represented by the Secretary,
Department of Health and Human Services
University of Utah |
Rockville
Salt Lake City |
MD
UT |
US
US |
|
|
Assignee: |
Governmement of the U.S.A., as
represented by the Secretary, Department of Health and Human
Services
Rockville
MD
University of Utah
Salt Lake City
UT
|
Family ID: |
27613451 |
Appl. No.: |
14/202121 |
Filed: |
March 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12433060 |
Apr 30, 2009 |
8673292 |
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14202121 |
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10502224 |
May 17, 2005 |
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PCT/US2003/002356 |
Jan 23, 2003 |
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12433060 |
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60351036 |
Jan 23, 2002 |
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Current U.S.
Class: |
424/93.7 ;
435/325 |
Current CPC
Class: |
A61P 25/28 20180101;
A61K 35/12 20130101; G01N 33/56966 20130101; A61P 43/00 20180101;
A61P 25/14 20180101; A61P 25/02 20180101; A61P 25/00 20180101; A61P
17/02 20180101; A61P 21/04 20180101; A61P 25/08 20180101; C12N
5/0622 20130101; A61K 35/30 20130101; A61P 25/16 20180101; C12N
5/0623 20130101 |
Class at
Publication: |
424/93.7 ;
435/325 |
International
Class: |
A61K 35/30 20060101
A61K035/30 |
Goverment Interests
[0002] This invention was supported in part by funds provided by
the National Institutes of Health (Grant No. 5R29NS35087-05). The
U.S. Government has certain rights in this invention.
Claims
1. An isolated population of mammalian precursor cells which
generate astrocytes but not oligodendrocytes, said cells being
CD44+.
2. A method for treating damaged neural cells in a mammal, said
method comprising administering to a mammal with damaged neural
cells the mammalian precursor cells of claim 1.
3. The method of claim 2 wherein the mammalian precursor cells are
administered to the mammal by direct injection into or near a site
of nerve damage.
4. The method of claim 2 wherein the mammalian precursor cells are
administered to the mammal as an implant seeded or coated with the
mammalian precursor cells.
5. The method of claim 4 wherein the implant is implanted at or
near a site of damaged neural cells in the mammal.
6. The method of claim 2 wherein administering the mammalian
precursor cells to the mammal enhances survival of the damaged
neural cells.
7. The method of claim 2 wherein administering the mammalian
precursor cells to the mammal reduces scar formation.
8. An astrocyte generated or differentiated from a population of
mammalian precursor cells which are CD44+ and which generate
astrocytes but not oligodendrocytes.
9. An astrocyte generated or differentiated from a mammalian
precursor cell which generate astrocytes but not oligodendrocytes,
said precursor cell being nestin+, A2B5-, and E-NCAM- or Pax-6-.
Description
[0001] This patent application is a continuation of U.S.
application Ser. No. 12/433,060, filed Apr. 30, 2009, which is a
continuation of U.S. application Ser. No. 10/502,224, filed May 17,
2005, now abandoned, which is the U.S. National Stage of
PCT/US03/02356, filed Jan. 23, 2003, which claims the benefit of
priority from U.S. Provisional Application Ser. No. 60/351,036,
filed Jan. 23, 2002, which are herein incorporated by reference in
their entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to a homogeneous, pure
population of mammalian astrocyte restricted precursor cells which
are CD44 immunoreactive and generate astrocytes but not
oligodendrocytes. The present invention is also related to methods
for isolating a homogeneous, pure population of these mammalian
astrocyte restricted precursor cells. In addition, the present
invention relates to use of mammalian astrocyte restricted
precursor cells in the development of new transplantation
techniques and to enhance myelination and/or reduce necrosis and
glial scar formation upon administration to animals. The astrocyte
restricted precursor cells and pharmaceutical compositions
comprising the same, may thus be used to treat disorders of the
nervous system resulting from trauma or disease which have in some
way damaged the nerve tissue. These cells are also useful in
identifying mammalian genes specific to selected stages of
development.
BACKGROUND OF THE INVENTION
[0004] Neural development has been well characterized in rodents.
Multipotent cells which are nestin immunoreactive and capable of
differentiating into astrocytes, neurons, and oligodendrocytes have
been identified by multiple investigators at various stages of
development. In addition to multipotent precursors, other more
restricted precursors have also been identified. Different
populations of cells can be distinguished by differences in culture
conditions, self-renewal capability, as well as in their ability to
integrate and to differentiate following transplantation.
[0005] Similar studies using human tissue are indicative of the
existence of multiple types of neural precursors as well.
Multipotent human neural stem cells (hNSCs) have been isolated from
fetal and adult tissue (Chalmers-Redman et al. Neuroscience 1997
76:1121-1128; Svendsen et al. J. Neuroscience Methods 1998
85:141-152; Vescovi et al. Exp. Neurology 1999 156:71-83; Carpenter
et al. Exp. Neurology 1999 158:265-278; Quinn et al. J.
Neuroscience Res. 1999 57:590-602; Piper et al. J. Neurophysiology
2000 84:534-548). These cells give rise to glia and neurons, can be
grown under different culture conditions, and show different growth
factor requirements.
[0006] Human neuron restricted precursors have also been described
(Piper et al. J. Neurophysiol. 2000 84:534-548). Piper et al. used
E-NCAM immunoreactivity to isolate neuronal precursor cells while
Goldman and colleagues used neuron specific promoters to isolate
neuronal precursors (Roy et al. Nat. Med. 2000 6(3):271-7; Roy et
al. J. Neurosci. Res. 2000 59(3):321-31; Wang et al. Dev. Neurosci.
2000 22(1-2):167-76). Human neuronal restricted precursor cells
have been isolated from the adult ventricular zone and hippocampus
as well as from fetal tissue at multiple stages of development.
These cells differ from human neuroepithelial cells by their
expression of early neuronal markers such as NCAM, alpha-1 tubulin
and beta-III tubulin.
[0007] Proliferative adult human oligodendrocyte precursors have
been isolated from adult human white matter (Prabhakar et al. Brain
Res. 1995 672(1-2):159-69, Raine et al. Lab. Invest. 1981
45(6):534-46; Scolding et al. Neuroreport 1995 6(3):441-5; Scolding
et al. Neuroscience 1999 89(1):1-4) using cell surface markers.
Others have used promoter-reporter constructs to isolate
oligodendrocytes and their precursors from fetal and adult tissue.
A2B5 immunoreactivity has been utilized to isolate glial precursors
that are capable of differentiating into astrocytes and
oligodendrocytes (U.S. Pat. No. 6,235,527).
[0008] Quinn and colleagues (J. Neurosci. Res. 1999 57:590-602)
describe a mixed population of multipotent stem cells that can
become altered in their properties after prolonged culture. These
cells have been suggested to be astrocyte restricted precursor
cells. However, oligodendrocyte differentiation has not been
tested. Further, no information on antigenic expression, cytokine
dependence, response to growth factors, expression of GFAP/S100, or
A2B5 is available. The cells of Quinn et al. were obtained by
sequentially passaging multipotent stem cells from cultured human
spinal cord tissue.
[0009] A putative astrocyte precursor cell has also been described
by Barres et al. (J. Neurosci. 1999 19(3):1049-61). This cell was
isolated from the optic nerve and its existence in any other part
of the brain is unknown. This cell is A2B5 immunoreactive and thus
resembles the oligodendrocyte precursor O2A. The cells can be
distinguished from the O2A cells mainly by their failure to develop
into oligodendrocytes under conditions in which the O2A cells
readily generate oligodendrocytes. This cell is Pax-6-positive and
dies when exposed to serum. Immunoreactivity with CD44 is
unknown.
[0010] Siedman et al. (Brain Res. 1997 753(1):18-26) have also
described an astrocyte cell line derived by immortalization of a
glial precursor cell. Little information on this immortalized
precursor cell is available and its antigenic characteristics and
ability to differentiate into neurons have not been disclosed.
SUMMARY OF THE INVENTION
[0011] The present invention is directed to astrocyte restricted
precursor cells, pharmaceutical compositions comprising the same,
and methods of utilizing the astrocyte restricted precursor cells
to treat mammals with damage to the nervous systems. The astrocyte
restricted precursor cells of the present invention are not
immortalized. These cells do not express A2B5. Further, these cells
differ from stem and progenitor cell populations in their
expression of CD44 and their ability to differentiate into
astrocytes under conditions in which other populations
differentiate into neurons or oligodendrocytes.
[0012] Thus, one aspect of the present invention relates to an
isolated, pure homogeneous population of mammalian astrocyte
restricted precursor cells which is CD44 immunoreactive and can
generate astrocytes but not oligodendrocytes.
[0013] Another aspect of the present invention relates to a method
for isolating a pure homogeneous population of mammalian astrocyte
restricted precursor cells. In the method of the present invention,
the pure homogeneous population of astrocyte restricted precursor
cells is isolated from a heterogeneous or mixed population of
mammalian cells via CD44 immunoreactivity.
[0014] Another aspect of the present invention relates to methods
for development of new transplantation techniques using these
mammalian astrocyte restricted precursor cells.
[0015] Another aspect of the present invention relates to
pharmaceutical compositions comprising the mammalian astrocyte
restricted precursor cells and methods of using these compositions
to treat patients with damage of the nervous system. In one
embodiment, the compositions and methods are used to enhance
myelination of mammalian neuronal cells. In another embodiment, the
compositions and methods are used to reduce glial scar formation
and necrosis.
[0016] Another aspect of the present invention relates to methods
for identifying mammalian genes specific to selected stages of
development using these astrocyte restricted precursor cells.
DETAILED DESCRIPTION OF THE INVENTION
[0017] For cell replacement in the nervous system, differentiated
cells are ultimately required. However, extensive studies have
shown that differentiated cells do not survive well following
transplantation. Therefore, some researchers have focused their
efforts on use of precursor cells which have been shown to survive
and integrate into the intact or damaged brain.
[0018] The present invention relates to a pure, homogeneous
population of mammalian astrocyte restricted precursor cells which
can be isolated from various sources of mammalian neural tissue
and/or cells including, but not limited to, mammalian embryonic or
fetal tissue, mammalian embryonic stem (ES) cell cultures, and
glial restricted precursor cells. The present invention also
relates to methods for isolating a pure, homogeneous population of
astrocyte restricted precursor cells from such tissues and
cells.
[0019] For purposes of the present invention, by "pure" it is meant
a population of cells in which greater than 95%, more preferably
99%, exhibit the same characteristics.
[0020] In a preferred embodiment, the mammalian tissue or cells
from which the astrocyte restricted precursor cells are isolated is
either rodent or human. However, as will be understood by those of
skill in the art upon reading this disclosure, the methods for
isolation taught herein are also routinely adaptable to cells or
tissues from other mammals including, but not limited to, non-human
primates, equines, canines, felines, bovines, porcines, ovines,
lagomorphs, and the like
[0021] As demonstrated herein, the astrocyte restricted precursor
cells of the present invention express CD44. Prior to
differentiations these cells also express nestin. Unlike the
putative astrocyte restricted cells of Barres et al. (J. Neurosci
1999 19(3):1049-61), the astrocyte restricted precursor cells of
the present invention do not express A2B5. Nor do the cells of the
present invention express PSANCAM. The cells of the present
invention grow well in FGF and EGF. The CD44-positive cells of the
present invention do not express GFAP, vimentin or S-100 initially,
but have the capacity to differentiate into GFAP, vimentin and/or
S-100 positive cells. Upon differentiation, the cells of the
present invention maintain their CD44 immunoreactivity but lose
expression of nestin. Thus, the CD44 positive cells of the present
invention can be readily distinguished from glial-restricted
precursor cells, multipotent stem cells, neuronal precursors and
the putative astrocyte precursor described by Barres et al. (J.
Neurosci. 1999 19(3):1049-61) based on antigen expression, cytokine
dependence and differentiation ability. See Table 1 which provides
a comparison of characteristics of the cells of Barres et al with
the astrocyte restricted precursor (ARP) cells of the present
invention.
TABLE-US-00001 TABLE 1 ARP Cells of Characteristic Barres et al.
Present Invention A2B5 Expression ++ -- CD44 Expression n.d. ++
Pax-6 Expression ++ -- Clonal analysis n.d. ++ Transplant n.d. ++
experiments Serum Exposure death differentiation n.d. = not
determined
The astrocyte restricted precursor cells are present in the
developing mammalian brain prior to acquisition of GFAP
immunoreactivity. In addition, the CD44+ astrocyte restricted
precursor cells can be generated from glial-restricted precursors
(GRP) and can be distinguished from GRP cells by antigen
expression, cytokine dependency and differentiation ability.
[0022] Clonal analysis indicates that a subset of nestin+ cells
that are GFAP- when grown in culture differentiate solely into
astrocytes. This subset is quite large and constitutes
approximately 11% of the cells analyzed.
[0023] A variety of markers were examined to identify a cell
surface marker that would label this nestin+/GFAP- population of
cells. It was found that CD44 is specific for this glial
population. CD44 expression co-localized with astrocytic markers
such as GFAP and S-100. CD44+ cells were RC1 negative and did not
co-express A2B5. A small subset of the CD44+ cells were nestin
immunoreactive but GFAP negative, thus indicating that these cells
represented an astrocyte precursor cell population.
[0024] While the number of CD44 positive cells is small, generally
in the range of 1-10% of the total number of cells present at any
stage of development from E15 to adult, it increases after culture
in conditions that promote astrocyte differentiation. CD44 positive
cells divide in culture and express low levels of GFAP. Expression
of GFAP increases after differentiation while the expression of
CD44 is down regulated. CD44 positive cells do not express A2B5 or
PLP and, thus, can be distinguished from the bipotential
glial-restricted precursor cell. While CD44 expression has been
described in other cell types such as macrophages and astrocytes
following injury, under the differentiation conditions used herein,
CD44 expression was limited to astrocytes and, thus, can be used in
accordance with procedures taught herein to identify astrocyte
restricted precursor cells.
[0025] Thus, as demonstrated herein, CD44 expression can be used to
identify and isolate astrocyte restricted precursor cells from
various sources of neural tissue including, but not limited to,
mammalian ES cell cultures and mammalian fetal or embryonic tissue
as well as glial-restricted precursor cells or GRPS methods for
isolating glial restricted precursor cells are described in U.S.
Pat. No. 6,235,527 the teachings of which are herein incorporated
by reference in their entirety. This population of astrocyte
restricted precursor cells is not immortalized. Further, population
of cells does not express A2B5 and differs from stem and progenitor
populations in its expression of CD44 and its ability to
differentiate into astrocytes under conditions in which other
populations differentiate into neurons or oligodendrocytes.
[0026] Various methods for isolating the CD44 positive astrocyte
restricted precursor cells from mixed populations of cells can be
used.
[0027] In one embodiment, mammalian neural tubes are dissociated at
a stage after astrocyte development, for example week 10 onward in
humans or after E16 in rodents, and dissociated cells are
triturated to a single cell suspension and labeled with an
anti-CD44 antibody. Labeled cells are visualized using a
fluorescently labeled secondary antibody targeted to the first
antibody and labeled cells are isolated using a selection
process.
[0028] Examples of selection processes useful in the present
invention include, but are not limited to immunopanning, magnetic
bead sorting and/or FACS sorting. Detailed magnetic bead and FACS
sorting protocols are well known in the art and can be routinely
adapted to use of CD44 as the selection marker. Further, as will be
understood by those of skill in the art upon reading this
disclosure, negative as well as positive selection methods can be
used. Thus enrichment of the astrocyte restricted precursor cells
of the present invention can be achieved by reselecting from a
mixed population cells that express CD44 but do not express A2B5 or
E-NCAM and vice versa. Positive and negative selection processes
can be used in any sequence and antibodies with a binding profile
similar to A2B5 or E-NCAM can be used.
[0029] In another embodiment, neural tubes are dissociated at any
stage after neural tube closure, for example E8.5 in mouse, E10.5
in rat, and week 5 gestation in human, and cells are maintained in
adherent culture for 5 to 40 days. Cells are then removed from
culture and CD44 positive cells are isolated via a selection
process as described in the preceding paragraph.
[0030] In another embodiment, A2B5+ cells are isolated. Cells are
then induced to differentiate in culture by growth in astrocyte
promoting conditions. By astrocyte promoting conditions it is meant
to include, but is not limited to, addition of bone morphogenetic
proteins (BMPs), oncostatin M, serum, Leukemia Inhibitory
Factor/Ciliary Neurotrophic Factor (LIF/CTNF) and other members of
the cytokine family such as interleukin-6 either singly or in
combination for a minimum period of three days. In a preferred
embodiment these agents are added at concentrations in the range of
1-5 ng/mL. CD44+ cells are then isolated via a selection process as
described above.
[0031] Recently, human fetal tissue derived neural cells have
become available through commercial sources such as Cambrex (East
Rutherford, N.J.), Clonexpress (Gaithersberg, Md.), ScienCell
Research Laboratories (San Diego, Calif.) and Clonetics (San Diego,
Calif.). These cells serve as a source of neural tissue and/or
cells for isolation of the mammalian astrocyte restricted precursor
cells of the present invention in accordance with the methods
taught herein.
[0032] Use of human ES cell lines as a source of the astrocyte
restricted precursor cells was also demonstrated in three human
cell lines (H1, H7, H9). Human ES cells have been previously shown
to differentiate into neuronal progenitors that subsequently
generate differentiated neurons (Carpenter et al. Exp. Neurol. 2001
172(2):383-97). In the present invention, dividing precursor cells
that expressed neuronal or glial markers were first identified in
ES cells. Differentiation conditions were similar to those
described herein and used for generating neurons. Specifically, the
first stage of differentiation of the ES cells was induced by the
formation of embryonic bodies (EBs) in FBS media with or without 10
.mu.M all trans-RA. After 4 days in suspension, EBs were plated
onto fibronectin coated plates in defined proliferation media
supplemented with 10 ng/mL hEGF, 10 ng/mL hbFGF, 1 ng/mL hPDGF-AA,
and 1 ng/mL hIGF-1. In these conditions, the EBs adhered to the
plates and cells began to migrate and proliferate on the plastic,
forming a monolayer. After 3 days in these conditions many cells
with neuronal morphology were present. Similar results were found
with each human ES cell line.
[0033] Multiple types of dividing cell populations can be
identified in cultures of differentiating ES cells based on
antibodies that recognize cell surface epitopes. These include
A2B5+ cells, PSANCAM+ cells and CD44+ cells. Double labeling
experiments following differentiation showed that the CD44+ cells
of the ES cells were a unique population of cells that were similar
morphologically, antigenically and in their ability to
differentiate into astrocytes to the astrocyte restricted cells of
the present invention isolated from other sources of neural tissue
and cells.
[0034] The astrocyte restricted precursor cells of the present
invention have a variety of uses.
[0035] For example, these cells can be used in nonhuman mammalian
models to develop new transplantation techniques.
[0036] In addition, these cells can be used therapeutically in
mammals, more preferably humans, in diseases characterized by
neural damage and more particularly astrocyte degeneration. In
particular, administration of the astrocyte restricted precursor
cells of the present invention is expected to be useful in
enhancing myelination of neurons. These cells are also useful in
identifying new drugs which enhance survival and proliferation of
these cells upon administration.
[0037] The cells can also be used in the reduction of scars. It is
well known that fetal astrocytes can incorporate into the brain
when transplanted. Fetal cells, as opposed to adult cells, reduce
adult glial cell proliferation and scar formation, thereby
promoting repair. Astrocyte restricted precursor cells of the
present invention can be administered at or near a lesioned site or
area of damage one week to several weeks after injury to reduce
endogenous adult glial cell proliferation and reduce scar
formation.
[0038] Accordingly, the present invention also relates to
pharmaceutical compositions comprising these astrocyte restricted
precursor cells for use in treatment of mammals with neural damage.
In a preferred embodiment, the cells are provided in injectable
form or on implants to promote directed axon regeneration and
reduce glial scar formation in the forebrain, and/or in damaged
spinal axons of the central nervous system. Such compositions are
useful in promoting CNS nerve regeneration and/or enhancing
myelination and/or reducing glial scar formation. Compositions
comprising astrocyte restricted precursor cells can be applied, in
various different formulations as described infra, to regions or
areas of nerve damage. Such compositions can be administered to
mammals having nervous system damage resulting from various causes
including, but not limited to, trauma, surgery, ischemia,
infection, metabolic disease, nutritional deficiency, malignancies
and paraneoplastic syndromes, toxic agents, and degenerative
disorders of the nervous system. Examples of neurodegenerative
disorders which can be treated using compositions of the present
invention include, but are not limited to, Alzheimer's disease,
Parkinson's disease, Huntington's chorea, amyotrophic lateral
sclerosis, progressive supranuclear palsy and peripheral
neuropathies. Compositions comprising the astrocyte restricted
precursor cells of the present invention can also be applied to a
wound to reduce scar formation. For example, following surgery, a
composition comprising these cells can be applied in accordance
with the presence invention to reduce scar formation from a lesion
due to, for example, arteriovenous malformation, necrosis,
bleeding, and craniotomy, which can secondarily lead to epilepsy.
The compositions of the present invention can also be used in the
treatment of epilepsy by stabilizing the epileptic focus and
reducing scar formation.
[0039] Pharmaceutical compositions of the present invention
comprise an effective amount of the isolated astrocyte restricted
precursor cells of the present invention and a pharmaceutically
acceptable carrier. By "effective amount" it is meant a composition
comprising approximately 100,000 to about one million cells. As
will be understood by one of skill upon reading this disclosure
however, cell number may vary depending upon the selected site of
administration. Examples of pharmaceutically acceptable carriers
include, but are not limited to liquid vehicles such as sterile
saline, buffered saline, dextrose and water and semi-liquid or
gel-like vehicles which may further comprise a media which impedes,
at least in part, the mobility of the cells so as to localize the
cells at the site of damage. Alternatively, pharmaceutically
acceptable carriers may comprise a solid vehicle such as an implant
seeded or coated with the cells.
[0040] The pharmaceutical compositions can be delivered by a wide
range of methods to promote CNS nerve regeneration, enhance
myelination and/or reduce scar formation. Exemplary methods
adaptable for use with the compositions of the present invention
are set forth in U.S. Pat. No. 5,202,120, the teachings of which
are herein incorporated by reference in their entirety.
[0041] In one embodiment, the cells are delivered by direct
application, for example, by direct injection of the cells in a
vehicle into or near the site of nerve damage. In this embodiment,
it may be preferred to deliver the cells in a vehicle comprising a
media which impedes, at least in part, the mobility of the cells so
as to localize the cells at the site of damage. Examples of media
which can impede cell mobility include, but are not limited, pastes
or gels, such as biodegradable gel-like polymers of fibrin or
hydrogels. These semi-solid media also provide the advantage of
impeding migration of scar producing mesenchymal components such as
fibroblasts into the site.
[0042] In another embodiment, the cells can be delivered via a
pharmaceutical compositions comprising a polymer implant or using
surgical bypass techniques. For example, the astrocyte restricted
precursor cells can be seeded or coated onto a polymer implant.
Various polymer implants with differing composition, geometries and
pore size which can be used in this embodiment have been described.
Examples include, but are in no way limited to, implants comprising
nitrocellulose, polyanhydrides and acrylic polymers. In a preferred
embodiment, an implant with a pore size of at least 0.45 .mu.m is
used.
[0043] The geometry of the implant is selected based upon its
intended use at the damage site. For example, an elongated
triangular implant may be selected to promote nerve regeneration
into the spinal cord dorsal root entry zone while a
pentagonal-shaped implant may be used to promote nerve regeneration
in the corpus callosum.
[0044] In another embodiment, the polymers may serve as synthetic
bridges over which nerve regeneration is promoted and scar
formation reduced by application of the astrocyte restricted
precursor cells at the ends or in the vicinity of the ends of the
synthetic bridge. For example, an acrylic polymer tube with
astrocyte restricted precursor cells of the present invention at
one or more ends, or throughout the tube, can be used to bridge
lesions rostrally or bypass lesions, for example, of the spinal
cord, over which nerve regeneration can be induced.
[0045] Examples of such tubes are set forth in European Patent
Publication 286284, and in references by Aebischer et al. (Brain
Res. 1988 454:179-187 and Prog. Brain Res. 1988 78:599-603) and
Winn et al. (Exp. Neurol. 1989 105:244-250).
[0046] The cells of the present invention can also be used in
combination with surgical bypass techniques to promote nerve
regeneration and/or to reduce scar formation in a selected region.
Examples of such techniques which can be routinely adapted to use
with the compositions of the present invention are set forth in
U.S. Pat. No. 5,202,120, which is herein incorporated by reference
in its entirety.
[0047] The astrocyte restricted precursor cells of the present
invention are also useful in the identification of genes specific
to selected stages of development. In one embodiment, the cells can
serve as a source of mRNA for generation of cDNA libraries that are
specific to the stage development of the cells.
[0048] The cells can also be used in the generation of cell lines
and cell-specific antibodies for use therapeutically and
diagnostically as well.
[0049] The following nonlimiting examples are provided to further
illustrate the present invention.
EXAMPLES
Example 1
Culture of Human Neural Stem Cells
[0050] Human neural progenitor cells derived from fetal tissue were
acquired from Cambrex. Frozen aliquots of cells were thawed and
plated on fibronectin/laminin-coated multiwell dishes in Neural
Progenitor Cell Basal Medium (NPBM, Cambrex) supplemented with
human recombinant basic fibroblast growth factor, human recombinant
epidermal growth factor, "neural survival factors", 5 mg/mL
gentamicin, and 5 mg/mL amphotericin-B (Singlequots, Cambrex).
Cultures were incubated at 37.degree. C., 5% CO.sub.2 and fixed 24
hours later. These wells were subsequently processed for
immunocytochemistry to assess the starting population of Cambrex
cells. In parallel, Cambrex cells were thawed and immediately
plated on fibronectin/laminin-coated flasks (Greiner) and cultured
in Neuroepithelial Precursor (NEP) medium that consisted of
DMEM-F12 (Life Technologies) supplemented with additives as
described by Bottenstein and Sato, basic fibroblast growth factor
(bFGF, 10 ng/ml, Peprotech, Rocky Hill, N.J.), and chick embryo
extract (CEE, 10%). Unattached cells typically formed floating
spheres. After 24 hours in culture, spheres were removed, gently
triturated, and re-combined with the attached cells. NEP media was
exchanged every other day.
Example 2
Isolation of Human Neuroepithelial Precursor Cells (hNEPs)
[0051] After 5 days in culture, immunopanning and flow-activated
cell sorting were used to remove ENCAM+, NG2+, and A2B5+ cells.
Briefly, cells were treated with 5 mM EDTA (Life Technologies) and
the suspension plated on an ENCAM antibody (5A5, Developmental
Studies Hybridoma Bank)-coated dish to allow binding of all ENCAM+
cells to the plate. ENCAM antibody-coated dishes were prepared by
sequentially coating tissue culture dishes with an unlabeled
anti-mouse IgM antibody (10 mg/ml) overnight, rinsing dishes with
DPBS, followed by coating with 5A5 hybridoma supernatant for 1-3
hours at 37.degree. C. Plates were washed twice with DPBS prior to
plating neural progenitor cells. After a 30 minute exposure period,
unbound cells (eNCAM- cells) were removed and plated onto a dish
coated with antibodies to NG2 for 30 minutes. NG2 panning dishes
were made by coating dishes with an NG2 antibody (1:100) for 1-3
hours at 37.degree. C. The supernatant was then removed
(ENCAM-/NG2-cells) and immunostained for A2B5. Cells were exposed
to antibodies to A2B5 (1:2, Developmental Studies Hybridoma Bank)
in NEP media for 1 hour at 37.degree. C., 5% CO.sub.2. A secondary
goat anti-mouse IgM-PE labeled antibody was then applied for 1 hour
to stain the membranes of live A2B5+ cells. All cells were then
sent through a flow-activated cell sorter to remove the population
of A2B5+ cells. After sorting, the negative population (human NEPs)
was propagated in NEP media on fibronectin/laminin coated T-75
flasks prior to transplantation studies. NEP media was exchanged
every other day.
Example 3
Generation of Neurons, Oligodendrocytes, and Astrocytes from
hNEPs
[0052] Panned/sorted populations of human NEPs were plated on
fibronectin/laminin-coated 12 mm coverslips in various conditions
to promote differentiation. To induce neuronal differentiation,
cells were exposed to bFGF (10 ng/ml) and NT3 (10 ng/ml,
Peprotech). After 5 days in culture, fixed cultures were stained
using antibodies to beta-III tubulin to assess the capacity of
these cells to differentiate into neurons. For oligodendrocyte
differentiation, cells were plated in a bFGF (10 ng/ml)-containing
medium for 2 days and then were switched to a medium containing
PDGF (10 ng/ml, Upstate Biotech., Waltham, Mass.) and T3 (50 nM)
for 7 days. Antibodies to O4, GalC and MBP were used to identify
oligodendrocytes in culture. For astrocytic differentiation, cells
were cultured for 5 days in the presence of fetal calf serum (10%,
Life Technologies). Astrocytes were identified using antibodies to
CD44, GFAP and S-100.
Example 4
Clonal Cultures and Clonal Propagation
[0053] Mixed cell cultures of human fetal cells (12-22 weeks of
gestation) were obtained from Clonetics and plated in T80 flasks in
the presence of bFGF and CEE (10%). After 3 days in culture cells
were labeled with A2B5 and NG-2. Immunonegative cells were
collected by FACS sorting analysis and replated into flasks in the
presence of bFGF and CEE. After 24 hours cells were labeled with
E-NCAM, sorted by FACS and negative cells were replated at a clonal
density in 10 cm dishes in the presence of bFGF and CEE. Control
dishes were labeled after 24 hours with A2B5, E-NCAM, GFAP and
NG-2. At that time point, 97% of all cells do not express any of
the differentiation markers tested. Single cells were grown at a
clonal density of 50-200 cells/35 mm dish). Cells were maintained
in FGF and CEE for 8-10 days and then CEE was withdrawn to initiate
differentiation. For oligodendrocyte differentiation cultures were
exposed to PDGF and thyroid hormone. After 5-7 days cultures were
labeled with antibodies against GFAP and beta-III tubulin to
determine differentiation into astrocytes and neurons,
respectively. Generation of oligodendrocytes was assessed 7 to 15
days after the initiation of differentiation. Neuronal and glial
differentiation was assessed using antibodies against GFAP, and
beta-III tubulin. For oligodendrocyte differentiation, cultures
were exposed to PDGF and thyroid hormone and differentiation was
assessed using antibodies to O4 and Gal-C.
Example 5
Generation of Neurons, Oligodendrocytes, and Astrocytes from
hNEPs
[0054] Panned/sorted populations of human NEPs were plated on
fibronectin/laminin-coated 12 mm coverslips in various conditions
to promote differentiation. To induce neuronal differentiation,
cells were exposed to bFGF (10 ng/ml) and NT3 (10 ng/ml,
Peprotech). After 5 days in culture, fixed cultures were stained
using antibodies to beta-III tubulin to assess the capacity of
these cells to differentiate into neurons. For oligodendrocyte
differentiation, cells were plated in a bFGF (10 ng/ml)-containing
medium for 2 days and then were switched to a medium containing
PDGF (10 ng/ml, Upstate Biotech., Waltham, Mass.) and T3 (50 nM)
for 7 days. Antibodies to O4, GalC and MBP were used to identify
oligodendrocytes in culture. For astrocytic differentiation, cells
were cultured for 5 days in the presence of fetal calf serum (10%,
Life Technologies). Astrocytes were identified using antibodies to
CD44, GFAP and S-100 (Morita et al. Dev. Neurosci. 1997 19:210-218;
Gomes et al. Braz. J. Med. Biol. Res. 1999 32:619-631).
Example 6
Human ES Cell Culture
[0055] Male (H1) and female (H7 & H9) huES cell lines were
maintained on MATRIGEL in MEF (primary mouse embryonic fibroblasts)
conditioned medium (CM). CM was generated from huES cell media
(ESM) comprised of 80% Knockout DMEM (Gibco), 20% Knockout Serum
replacement (Gibco), 0.1 mM beta-mercaptoethanol, 1 mM glutamine,
1% non-essential amino acids, supplemented with 4 ng/mL hbFGF
(Gibco). Cultures were passaged by incubation in 200 units/ml
collagenase IV (Gibco) for about 5-10 minutes at 37.degree. C. and
then gently dissociated into small clusters in CM. Cells were
passaged about once every week. Conditioned media was generated
from MEF and collected daily and used immediately for feeding HuES
cultures. Before addition to the HuES cultures this conditioned
media was supplemented with an additional 4 ng/ml of hbFGF (Gibco).
Cells for generating CM were refed with ESM daily and used for 7-10
days.
Example 7
Differentiation of huES Cells
[0056] Embryoid bodies (EBs) were formed from undifferentiated ES
cultures harvested by incubation with 200 u/mL collagenase at
37.degree. C. for 5-10 minutes. The cells were gently scraped from
the dish and resuspended in ultra low attachment polystyrene plates
(Corning) in media composed of KO-DMEM, 20% FBS, 1% non-essential
amino acids, 1 mM glutamate and 0.1 mM beta-mercaptoethanol. In
some experiments, 10 .mu.M all-trans retinoic acid was added to the
EBs in suspension. After 4 days in suspension, EBs were plated onto
poly-1-lysine/FN coated plates in proliferation media comprised of
DMEM/F12 with B27 and N2 supplements (Gibco) and 10 ng/mL hEGF, 10
ng/mL hbFGF (Gibco), 1 ng/mL hPDGF-AA (R&D Systems), 1 ng/mL
hIGF-1 (R&D Systems). After 3 days in these conditions, the
cells were harvested with trypsin and replated in differentiation
media comprised of Neurobasal media supplemented with B27, 10 ng/mL
hNT-3(R&D Systems) and 10 ng/mL hBDNF (R&D Systems). These
cultures were fed 3 times per week and fixed after 14-21 days.
Example 8
Immunocytochemistry
[0057] Cultures were stained using antibodies against A2B5 (1:2,
Developmental Studies Hybridoma Bank), AC133/2 (1:100, Miltenyi
Biotec, Auburn, Calif.), beta-III tubulin (1:1000, Sigma), E-NCAM
(1:2, 5A5, Developmental Studies Hybridoma Bank), GFAP (1:2000,
Dako, Carpinteria, Calif.), NG2 (1:100) and O4 (1:2, Developmental
Studies Hybridoma Bank). Following fixation, cultures were treated
with 0.5% Triton X-100 (Sigma) in PBS for 2 minutes to access
intracellular antigens. Fixed coverslips or plates were then
treated with primary antibodies in a blocking solution containing
Hank's balanced salt solution and 5% calf serum for 1 hour at room
temperature. Following 3 washes with PBS, cultures were incubated
in the appropriate secondary antibodies (1:220) conjugated to
either Texas Red or Alexa 488 (Molecular Probes, Eugene, Oreg.) for
1 hour at room temperature. AC133/2 staining required amplification
with a biotinylated secondary antibody, followed by a
streptavidin-alexa 488 conjugated tertiary antibody. All cultures
were counterstained with DAPI (Molecular Probes) to identify cell
nuclei.
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