U.S. patent application number 12/504336 was filed with the patent office on 2009-11-05 for combined regulation of neural cell production.
This patent application is currently assigned to Stem Cell Therapeutics Inc. Invention is credited to Tetsuro Shingo, Bradley G. Thompson, Samuel Weiss.
Application Number | 20090274668 12/504336 |
Document ID | / |
Family ID | 27502115 |
Filed Date | 2009-11-05 |
United States Patent
Application |
20090274668 |
Kind Code |
A1 |
Thompson; Bradley G. ; et
al. |
November 5, 2009 |
Combined Regulation of Neural Cell Production
Abstract
This invention relates to a method of selectively producing
neural cells, including neurons or glial cells, in vitro or in
vivo. Also provided are methods of treating or ameliorating
neurodegenerative disease or medical conditions by producing neural
cells. Thus, a combination of factors is used to achieve two steps:
increasing the number of neural stem cells and instructing the
neural stem cells to selectively become neurons or glial cells.
Inventors: |
Thompson; Bradley G.;
(Calgary, CA) ; Weiss; Samuel; (Calgary, CA)
; Shingo; Tetsuro; (Okayama, JP) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
P.O BOX 1022
Minneapolis
MN
55440-1022
US
|
Assignee: |
Stem Cell Therapeutics Inc
Calgary
CA
|
Family ID: |
27502115 |
Appl. No.: |
12/504336 |
Filed: |
July 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11343419 |
Jan 30, 2006 |
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12504336 |
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10231493 |
Aug 30, 2002 |
7048934 |
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11343419 |
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60316365 |
Aug 30, 2001 |
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60316579 |
Aug 31, 2001 |
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60322514 |
Sep 14, 2001 |
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60386404 |
Jun 7, 2002 |
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Current U.S.
Class: |
424/93.7 ;
435/377 |
Current CPC
Class: |
A61P 25/14 20180101;
A61P 21/02 20180101; A61P 25/08 20180101; A61K 35/12 20130101; A61K
38/2257 20130101; A61K 38/27 20130101; A61P 25/16 20180101; A61P
25/18 20180101; C12N 5/0623 20130101; A61P 25/28 20180101; A61P
25/00 20180101; A61K 38/30 20130101; A61P 21/00 20180101; A61K
38/2257 20130101; A61K 2300/00 20130101; A61K 38/27 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/93.7 ;
435/377 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 5/00 20060101 C12N005/00; A61P 25/00 20060101
A61P025/00 |
Claims
1. A method for producing neuronal precursor cells or glial
precursor cells, comprising: (a) providing at least one neural stem
cell; (b) contacting the neural stem cell with a factor selected
from the group consisting of prolactin, growth hormone, estrogen,
ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF),
transforming growth factor alpha (TGF.alpha.) and epidermal growth
factor (EGF) in an amount sufficient to increase the number of
neural stem cells; and (c) contacting the neural stem cells from
step (b) to a factor selected from the group consisting of
erythropoietin (EPO), pituitary adenylate cyclase activating
polypeptide (PACAP), prolactin, serotonin, bone morphogenetic
protein (BMP) and cAMP in an amount sufficient to enhance the
production of neuronal precursor cells or glial precursor cells
from the neural stem cells; with the proviso that when the factor
in step (b) is EGF or FGF, the factor in step (c) is PACAP or
prolactin.
2. The method of claim 1 wherein step (b) is performed prior to
step (c).
3. The method of claim 1 wherein steps (b) and (c) are performed
concurrently.
4. The method of claim 1 wherein the neural stem cell is not an
embryonic cell.
5. The method of claim 1 wherein the neural stem cell is an adult
neural stem cell.
6. The method of claim 1 wherein the neural stem cell is located in
a mammal.
7. The method of claim 6 wherein the neural stem cell is provided
by transplanting neural stem cells into the mammal.
8. The method of claim 7 wherein the transplanted neural stem cells
have been expanded in culture prior to being transplanted into the
mammal.
9. The method of claim 7 wherein the transplanted neural stem cells
are syngeneic with the mammal.
10. The method of claim 6 wherein the neural stem cell is located
in the subventricular zone of the forebrain of the mammal.
11. The method of claim 6 wherein the mammal is suffering from or
suspected of having a neurodegenerative disease or condition.
12. The method of claim 11 wherein the disease or condition is
brain injury.
13. The method of claim 12 wherein the brain injury is a
stroke.
14. The method of claim 11 wherein the disease or condition is
selected from the group consisting of Alzheimer's disease, multiple
sclerosis (MS), Huntington's disease, amyotrophic lateral
sclerosis, and Parkinson's disease.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/316,365, filed Aug. 30, 2001; Ser. No.
60/316,579, filed Aug. 31, 2001; Ser. No. 60/322,514, filed Sep.
14, 2001; and Ser. No. 60/386,404, filed Jun. 7, 2002. The entire
disclosure of each of these priority applications is hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to a method of selectively producing
neural cells, such as neurons or glial cells, in vitro or in vivo.
Also provided are methods of treating or ameliorating
neurodegenerative disease or medical conditions by producing neural
cells.
REFERENCES
[0003] U.S. Patent Application Publication No. 2002 0098178 A1.
[0004] U.S. Pat. No. 5,023,252. [0005] U.S. Pat. No. 5,128,242.
[0006] U.S. Pat. No. 5,198,542. [0007] U.S. Pat. No. 5,208,320.
[0008] U.S. Pat. No. 5,268,164. [0009] U.S. Pat. No. 5,326,860.
[0010] U.S. Pat. No. 5,506,107. [0011] U.S. Pat. No. 5,506,206.
[0012] U.S. Pat. No. 5,527,527. [0013] U.S. Pat. No. 5,547,935.
[0014] U.S. Pat. No. 5,614,184. [0015] U.S. Pat. No. 5,623,050.
[0016] U.S. Pat. No. 5,686,416. [0017] U.S. Pat. No. 5,723,115.
[0018] U.S. Pat. No. 5,750,376. [0019] U.S. Pat. No. 5,773,569.
[0020] U.S. Pat. No. 5,801,147. [0021] U.S. Pat. No. 5,833,988.
[0022] U.S. Pat. No. 5,837,460. [0023] U.S. Pat. No. 5,851,832.
[0024] U.S. Pat. No. 5,885,574. [0025] U.S. Pat. No. 5,955,346.
[0026] U.S. Pat. No. 5,977,307. [0027] U.S. Pat. No. 5,980,885.
[0028] U.S. Pat. No. 6,015,555. [0029] U.S. Pat. No. 6,048,971.
[0030] U.S. Pat. No. 6,191,106. [0031] U.S. Pat. No. 6,242,563.
[0032] U.S. Pat. No. 6,329,508. [0033] U.S. Pat. No. 6,333,031.
[0034] U.S. Pat. No. 6,413,952. [0035] U.S. Pat. No. 6,429,186.
[0036] WO 96 40231. [0037] WO 97 48729. [0038] Bernichtein, S., et
al. S179D-human PRL, a pseudophosphorylated human PRL analog, is an
agonist and not an antagonist. Endocrinology 142(9):3950-3963
(2001). [0039] Fernandez-Pol, J. A. Epidermal growth factor
receptor of A431 cells, Characterization of a monoclonal
anti-receptor antibody noncompetitive agonist of epidermal growth
factor action. J. Biol. Chem. 260(8):5003-5011 (1985). [0040]
Johnson, D. L., et al. Erythropoietin mimetic peptides and the
future. Nephrol. Dial. Transplant. 15(9):1274-1277 (2000). [0041]
Kaushansky, K. Hematopoietic growth factor mimetics. Ann. N.Y.
Acad. Sci. 938:131-138 (2001). [0042] Kolb, B., et al. Nerve growth
factor treatment prevents dendritic atrophy and promotes recovery
of function after cortical injury. Neuroscience 76(4):1139-1151
(1997). [0043] Livnah, O., et al. Functional mimicry of a protein
hormone by a peptide agonist: the EPO receptor complex at 2.8 A.
Science 273(5274):464-471 (1996). [0044] Mode, A., et al. The human
growth hormone (hGH) antagonist G120RhGH does not antagonize GH in
the rat, but has paradoxical agonist activity, probably via the
prolactin receptor. Endocrinology 137(2):447-454 (1996). [0045]
Moro, O., et al. Maxadilan, the vasodilator from sand flies, is a
specific pituitary adenylate cyclase activating peptide type I
receptor agonist. J. Biol. Chem. 272(2):966-70 (1997). [0046]
Rochefort, C., et al. Enriched odor exposure increases the number
of newborn neurons in the adult olfactory bulb and improves odor
memory. J. Neurosci. 22(7):2679-2689 (2002). [0047] Shirnazaki, T.,
et al. The ciliary neurotrophic factor/leukemia inhibitory
factor/gp130 receptor complex operates in the maintenance of
mammalian forebrain neural stem cells. J. Neurosci.
21(19):7642-7653 (2001). [0048] Shingo, T.; et al. Erythropoietin
regulates the in vitro and in vivo production of neuronal
progenitors by mammalian forebrain neural stem cells. J. Neurosci.
21(24):9733-9743 (2001). [0049] Wrighton, N. C., et al. Small
peptides as potent mimetics of the protein hormone erythropoietin.
Science 273(5274):458-464 (1996).
[0050] All of the publications, patents and patent applications
cited above or elsewhere in this application 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
[0051] In recent years, neurodegenerative disease has become an
important concern due to the expanding elderly population which is
at greatest risk for these disorders. Neurodegenerative diseases
include the diseases which have been linked to the degeneration of
neural cells in particular locations of the central nervous system
(CNS), leading to the inability of these cells to carry out their
intended function. These diseases include Alzheimer's Disease,
Multiple Sclerosis (MS), Huntington's Disease, Amyotrophic Lateral
Sclerosis, and Parkinson's Disease. In addition, probably the
largest area of CNS dysfunction (with respect to the number of
affected people) is not characterized by a loss of neural cells but
rather by abnormal functioning of existing neural cells. This may
be due to inappropriate firing of neurons, or the abnormal
synthesis, release, and processing of neurotransmitters. These
dysfunctions may be the result of well studied and characterized
disorders such as depression and epilepsy, or less understood
disorders such as neurosis and psychosis. Moreover, brain injuries
often result in the loss of neural cells, the inappropriate
functioning of the affected brain region, and subsequent behavior
abnormalities.
[0052] Consequently, it is desirable to supply neural cells to the
brain to compensate for degenerate or lost neurons/glial cells in
order to treat neurodegenerative diseases or conditions. One
approach to this end is to transplant neural cells into the brain
of the patient. This approach requires a source of large amounts of
neural cells, preferably from the same individual or a closely
related individual such that host-versus-graft or graft-versus-host
rejections can be minimized. As it is not practical to remove a
large amount of neurons or glial cells from one person to
transplant to another, a method to culture large quantity of neural
cells is necessary for the success of this approach.
[0053] Another approach is to induce the production of neural cells
in situ to compensate for the lost or degenerate cells. This
approach requires extensive knowledge about whether it is possible
to produce neural cells in brains, particularly adult brains, and
how.
[0054] The development of techniques for the isolation and in vitro
culture of multipotent neural stem cells (for example, see U.S.
Pat. Nos. 5,750,376; 5,980,885; 5,851,832) significantly increased
the outlook for both approaches. It was discovered that fetal
brains can be used to isolate and culture multipotent neural stem
cells in vitro. Moreover, in contrast to the long time belief that
adult brain cells are not capable of replicating or regenerating
brain cells, it was found that neural stem cells may also be
isolated from brains of adult mammals. These stem cells, either
from fetal or adult brains, are capable of self-replicating. The
progeny cells can again proliferate or differentiate into any cell
in the neural cell lineage, including neurons, astrocytes and
oligodendrocytes. Therefore, these findings not only provide a
source of neural cells which can be used in transplantations, but
also demonstrate the presence of multipotent neural stem cells in
adult brain and the possibility of producing neurons or glial cells
from these stem cells in situ.
[0055] It is therefore desirable to develop methods of efficiently
proliferating neural stem cells for two purposes: to obtain more
stem cells and hence neural cells which can be used in
transplantation therapies, and to identify methods which can be
used to produce more stem cells in situ.
SUMMARY OF THE INVENTION
[0056] This invention relates to a two-step method of producing
neural cells in vitro or in vivo. We discovered that neurogenesis
and gliogenesis by multipotent neural stem cells (NSCs) involve
proliferation and directed differentiation. As shown in FIG. 1, EGF
(or its adult homolog TGF.alpha.) induces the
self-renewal/expansion of the NSC population. The NSCs will
undergo-spontaneous differentiation in a default pathway to become
glial precursor cells (GPCs). This spontaneous differentiation can
be attenuated by ciliary neurotrophic factor (CNTF). GPCs will
differentiate into the glial cells, which differentiation is
promoted by EGF. Alternatively, NSCs can be instructed by EPO
and/or PACAP/cAMP to differentiate to neuronal precursor cells
(NPCs), which make neurons only.
[0057] Therefore, a two-step process can be used to produce neurons
or glial cells: (1) increasing the number of NSCs; and (2)
promoting differentiation of the NSCs to either neurons or glial
cells by subjecting the NSCs to appropriate conditions which
selectively promotes production of neurons or glial cells.
[0058] Accordingly, one aspect of the present invention provides a
method for producing neuronal precursor cells or glial precursor
cells, comprising: [0059] (a) providing at least one neural stem
cell; [0060] (b) contacting the neural stem cell with a factor
selected from the group consisting of prolactin, growth hormone,
estrogen, ciliary neurotrophic factor (CNTF), pituitary adenylate
cyclase activating polypeptide (PACAP), fibroblast growth factor
(FGF), transforming growth factor alpha (TGF.alpha.) and epidermal
growth factor (EGF) in an amount sufficient to increase the number
of neural stem cells; and [0061] (c) contacting the neural stem
cells from step (b) to a factor selected from the group consisting
of erythropoietin (EPO), PACAP, prolactin, serotonin, bone
morphogenetic protein (BMP) and cAMP in an amount sufficient to
enhance the production of neuronal precursor cells or glial
precursor cells from the neural stem cells; [0062] with the proviso
that when the factor in step (b) is EGF or FGF, the factor in step
(c) is PACAP or prolactin.
[0063] Thus, step (b) is performed to increase the number of neural
stem cells, which can be achieved by at least one of the following:
[0064] (i) increasing proliferation of the neural stem cell, such
as by providing EGF; [0065] (ii) inhibiting spontaneous
differentiation of the neural stem cell, such as by providing CNTF;
or [0066] (iii) promoting survival of the neural stem cell, such as
by providing an estrogen.
[0067] These two steps, increasing NSCs numbers and enhancing
neuron or glia production, may be performed sequentially or
concurrently. It is preferable that step (b) is performed prior to
step (c).
[0068] The factors can be provided by any method established in the
art. For example, they can be administered intravascularly,
intrathecally, intravenously, intramuscularly, subcutaneously,
intraperitoneally, topically, orally, rectally, vaginally, nasally,
by inhalation or into the brain. The administration is preferably
performed systemically, particularly by subcutaneous
administration. The factors can also be provided by administering
to the mammal an effective amount of an agent that can increase the
amount of endogenous factors in the mammal. For example, the level
of prolactin in an animal can be increased by using prolactin
releasing peptide.
[0069] When the factors are not directly delivered into the brain,
a blood brain barrier permeabilizer can be optionally included to
facilitate entry into the brain. Blood brain barrier permeabilizers
are known in the art and include, by way of example, bradykinin and
the bradykinin agonists described in U.S. Pat. Nos. 5,686,416;
5,506,206 and 5,268,164 (such as
NH.sub.2-arginine-proline-hydroxyproxyproline-glycine-thienylala-
nine-serine-proline-4-Me-tyrosine.PSI.(CH.sub.2NH)-arginine-COOH).
Alternatively, the factors can be conjugated to the transferrin
receptor antibodies as described in U.S. Pat. No. 6,329,508;
6,015,555; 5,833,988 or 5,527,527. The factors can also be
delivered as a fusion protein comprising the factor and a ligand
that is reactive with a brain capillary endothelial cell receptor,
such as the transferrin receptor (see, e.g., U.S. Pat. No.
5,977,307).
[0070] Although mammals of all ages can be subjected to this
method, it is preferable that the mammal is not an embryo. More
preferably, the mammal is an adult.
[0071] The mammal may suffer from or be suspected of having a
neurodegenerative disease or condition. The disease or condition
may be a brain injury, such as stroke or an injury caused by a
brain surgery. The disease or condition may be aging, which is
associated with a significant reduction in the number of neural
stem cells. The disease or condition can also be a
neurodegenerative disease, particularly Alzheimer's disease,
multiple sclerosis, Huntington's disease, amyotrophic lateral
sclerosis, or Parkinson's disease.
[0072] Alternatively, the neural stem cell may be in a culture in
vitro. The cell may be from an animal of any age. Preferably, the
animal is not an embryo, and most preferably the animal is an
adult.
[0073] Another aspect of the present invention provides a method of
treating or ameliorating a neurodegenerative disease or medical
condition, comprising (a) administering to a mammal a factor which
is capable of increasing the number of neural stem cells; and (b)
subjecting the mammal to a condition which enhances the production
of a lineage restricted cell; whereby production of the lineage
restricted cell is enhanced. For example, neurons can be produced
to compensate for lost or malfunctioning neurons by administering
EGF and EPO. Other factors which are capable of increasing the
number of NSCs, such as CNTF, FGF, prolactin, growth hormone,
IGF-1, PACAP or estrogen, can also be used instead of EGF or in
addition to EGF. Likewise, other factors which can enhance neuron
production, such as PACAP or factors which increases cAMP level,
can be used in the place of EPO or in addition to EPO.
[0074] To produce glial cells to compensate for lost or
malfunctioning glial cells, EGF can be administered, which
stimulates NSC proliferation, and the resulting NSC will
differentiate to glial cells by default. Optionally, inhibitors of
the neuronal pathway, such as antibodies of EPO and cAMP signaling
inhibitors, can be used to promote glial production. Preferably, a
factor that promotes glial formation, such as BMP, is also used to
further produce glial cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] FIG. 1 diagrams a model for neurogenesis and gliogenesis by
neural stem cells (NSCs). EGF (or its adult homolog TGF.alpha.)
induces the self-renewal/expansion of the NSC population. The NSCs
will undergo spontaneous differentiation as a default pathway to
become glial precursor cells (GPCs). This spontaneous
differentiation can be attenuated by CNTF. GPCs differentiate into
astrocytes and/or oligodentrocytes, which differentiation is
promoted by EGF. Alternatively, NSCs can be instructed by EPO
and/or PACAP/cAMP to differentiate to neuronal precursor cells
(NPCs), which make neurons only.
DETAILED DESCRIPTION OF THE INVENTION
[0076] This invention relates to a method of selectively producing
neural cells, including neurons or glial cells, in vitro or in
vivo. Also provided are methods of treating or ameliorating
neurodegenerative disease or medical conditions by producing neural
cells. Thus, a combination of factors is used to achieve two steps:
increasing the number of neural stem cells and instructing the
neural stem cells to selectively become neurons or glial cells.
[0077] Prior to describing the invention in further detail, the
terms used in this application are defined as follows unless
otherwise indicated.
DEFINITIONS
[0078] A "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, daughter cells which result from stem cell
divisions include stem cells. The neural stem cells are capable of
ultimately 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). Thus, the neural stem cells referred to herein are
multipotent neural stem cells.
[0079] A "neurosphere" is a group of cells derived from a single
neural stem cell as the result of clonal expansion. A "primary
neurosphere" refers to the neurospheres generated by plating as
primary cultures brain tissue which contains neural stem cells. The
method for culturing neural stem cells to form neurospheres has
been described in, for example, U.S. Pat. No. 5,750,376. A
"secondary neurosphere" refers to the neurospheres generated by
dissociating primary neurospheres and allowing the individual
dissociated cells to form neurospheres again.
[0080] A "neural cell" is any cell in the neural lineage.
Preferably a neural cell is a neuron or glial cell.
[0081] A polypeptide which shares "substantial sequence similarity"
with a native factor is at least about 30% identical with the
native factor at the amino acid level. The polypeptide 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 the native factor at the amino acid
level.
[0082] The phrase "percent identity" or "% identity" of an analog
or variant with a native factor refers to the percentage of amino
acid sequence in the native factor which are also found in the
analog or variant when the two sequences are aligned. Percent
identity can be determined by any methods or algorithms established
in the art, such as LALIGN or BLAST.
[0083] A polypeptide possesses a "biological activity" of a native
factor if it is capable of binding to the receptor for the native
factor or being recognized by a polyclonal antibody raised against
the native factor. Preferably, the polypeptide is capable of
specifically binding to the receptor for the native factor in a
receptor binding assay.
[0084] A "functional agonist" of a native factor is a compound that
binds to and activates the receptor of the native factor, although
it does not necessarily share a substantial sequence similarity
with the native factor.
[0085] A "prolactin" is a polypeptide which (1) shares substantial
sequence similarity with a native mammalian prolactin, preferably
the native human prolactin, a 199-amino acid polypeptide
synthesized mainly in the pituitary gland; and (2) possesses a
biological activity of the native mammalian prolactin. Thus, the
term "prolactin" encompasses prolactin analogs which are the
deletional, insertional, or substitutional mutants of the native
prolactin. Furthermore, the term "prolactin" encompasses the
prolactins from other species and the naturally occurring variants
thereof.
[0086] In addition, a "prolactin" may also be a functional agonist
of a native mammalian prolactin receptor. For example, the
functional agonist may be an activating amino acid sequence
disclosed in U.S. Pat. No. 6,333,031 for the prolactin receptor; a
metal complexed receptor ligand with agonist activities for the
prolactin receptor (U.S. Pat. No. 6,413,952); G120RhGH, which is an
analog of human growth hormone but acts as a proactin agonist (Mode
et al., 1996); or a ligand for the prolactin receptor as described
in U.S. Pat. Nos. 5,506,107 and 5,837,460.
[0087] An "EGF" means a native EGF or any EGF analog or variant
that shares a substantial amino acid sequence similarity with a
native EGF, as well as at least one biological activity with the
native EGF, such as binding to the EGF receptor. Particularly
included as an EGF is the native EGF of any species, TGFA, or
recombinant modified EGF. Specific examples include, but are not
limited to, the recombinant modified EGF having a deletion of the
two C-terminal amino acids and a neutral amino acid substitution at
position 51 (particularly EGF51gln51; U.S. Patent Application
Publication No. 20020098178A1), the EGF mutein (EGF-X.sub.16) in
which the His residue at position 16 is replaced with a neutral or
acidic amino acid (U.S. Pat. No. 6,191,106), the 52-amino acid
deletion mutant of EGF which lacks the amino terminal residue of
the native EGF (EGF-D), the EGF deletion mutant in which the
N-terminal residue as well as the two C-terminal residues (Arg-Leu)
are deleted (EGF-B), the EGF-D in which the Met residue at position
21 is oxidized (EGF-C), the EGF-B in which the Met residue at
position 21 is oxidized (EGF-A), heparin-binding EGF-like growth
factor (HB-EGF), betacellulin, amphiregulin, neuregulin, or a
fusion protein comprising any of the above. Other useful EGF
analogs or variants are described in U.S. Patent Application
Publication No. 20020098178A1, and U.S. Pat. Nos. 6,191,106 and
5,547,935.
[0088] In addition, an "EGF" may also be a functional agonist of a
native mnammalian EGF receptor. For example, the functional agonist
may be an activating amino acid sequence disclosed in U.S. Pat. No.
6,333,031 for the EGF receptor, or an antibody that has agonist
activities for the EGF receptor (Fernandez-Pol, 1985 and U.S. Pat.
No. 5,723,115).
[0089] A "PACAP" means a native PACAP or any PACAP analog or
variant that shares a substantial amino acid sequence similarity
with a native PACAP, as well as at least one biological activity
with the native PACAP, such as binding to the PACAP receptor.
Useful PACAP analogs and variants include, without being limited
to, the 38 amino acid and the 27 amino acid variants of PACAP
(PACAP38 and PACAP27, respectively), and the analogs and variants
disclosed in, e.g., U.S. Pat. Nos. 5,128,242; 5,198,542; 5,208,320;
5,326,860; 5,623,050; 5,801,147 and 6,242,563.
[0090] In addition, a "PACAP" may also be a functional agonist of a
native mammalian PACAP receptor. For example, the functional
agonist may be maxadilan, a polypeptide that acts as a specific
agonist of the PACAP type-1 receptor (Moro et al., 1997).
[0091] An "erythropoietin (EPO)" means a native EPO or any EPO
analog or variant that shares a substantial amino acid sequence
similarity with a native EPO, as well as at least one biological
activity with the native EPO, such as binding to the EPO receptor.
Erythropoietin analogs and variants are disclosed, for example, in
U.S. Pat. Nos. 6,048,971 and 5,614,184.
[0092] In addition, an "EPO" may also be a functional agonist of a
native mammalian EPO receptor. For example, the functional agonist
may be EMP1 (EPO mimetic peptide 1, Johnson et al., 2000); one of
the short peptide mimetics of EPO as described in Wrighton et al.,
1996 and U.S. Pat. No. 5,773,569; any small molecular EPO mimetic
as disclosed in Kaushansky, 2001; an antibody that activates the
EPO receptor as described in U.S. Pat. No. 5,885,574, WO 96/40231,
WO 97/48729, Fernandez-Pol, 1985 or U.S. Pat. No. 5,723,115; an
activating amino acid sequence as disclosed in U.S. Pat. No.
6,333,031 for the EPO receptor; a metal complexed receptor ligand
with agonist activities for the EPO receptor (U.S. Pat. No.
6,413,952), or a ligand for the EPO receptor as described in U.S.
Pat. Nos. 5,506,107 and 5,837,460.
[0093] "Enhancing" or "promoting" the formation of a cell type
means increasing the number of the cell type. Thus, a factor can be
used to enhance neuron formation if the number of neurons in the
presence of the factor is larger than the number of neurons absent
the factor. The number of neurons in the absence of the factor may
be zero or more.
[0094] A "neurodegenerative disease or condition" is a disease or
medical condition associated with neuron loss or dysfunction.
Examples of neurodegenerative 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.
[0095] "Treating or ameliorating" means the reduction or complete
removal of the symptoms of a disease or medical condition.
[0096] A mammal "suspected of having a neurodegenerative disease or
condition" is a mammal which is not officially diagnosed with the
neurodegenerative disease or condition but shows a symptom of the
neurodegenerative disease or condition, is susceptible to the
neurodegenerative disease or condition due to family history or
genetic predisposition, or has previously had the neurodegenerative
disease or condition and is subject to the risk of recurrence.
[0097] "Transplanting" a composition into a mammal refers to
introducing the composition into the body of the mammal by any
method established in the art. The composition being introduced is
the "transplant", and the mammal is the "recipient". The transplant
and the recipient may be syngeneic, allogeneic or xenogeneic.
Preferably, the transplantation is an autologous
transplantation.
[0098] An "effective amount" is an amount of a therapeutic agent
sufficient to achieve the intended purpose. For example, an
effective amount of a factor to increase the number of neural stem
cells is an amount sufficient, in vivo or in vitro, as the case may
be, to result in an increase in neural stem cell number. An
effective amount of a composition to treat or ameliorate a
neurodegenerative disease or condition is an amount of the
composition sufficient to reduce or remove the symptoms of the
neurodegenerative disease or condition. 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
[0099] Neural stem cells (NSCs), such as the ones found in the
adult forebrain, are the likely source of restricted neuronal and
glial progenitors, which repopulate structures such as the
olfactory bulb and corpus callosum, respectively. The mechanisms by
which NSCs give rise to restricted progenitors have been unclear
prior to this invention.
[0100] We found that EGF-responsive NSCs gradually become
restricted to a glial lineage. This process is blocked by CNTF,
acting through notch1, to maintain NSCs in a multipotent stage. We
also found that erythropoietin (EPO) directs the production of
restricted neuronal precursors through a mechanism that utilizes
Mash1.
[0101] Thus, we infused either CNTF or EPO into the lateral
ventricles of adult mice for six days, after which we either
removed the entire adult ependyma/subependyma to examine the total
number of EGF-responsive NSCs or examined the in vivo production of
neuronal precursors. CNTF infusion resulted in a 20-25% increase in
the number of NSCs, most likely by preventing NSC differentiation
into glial precursors. EPO infusion resulted in a 50% reduction in
the number of NSCs and a concomitant doubling in neuronal
precursors. Infusion of anti-EPO antibodies resulted in a 20%
increase in NSCs. Therefore, EGF-responsive NSCs turn over
continuously in vivo, a sub-population of which spontaneously
differentiates into restricted glial precursors, while another
sub-population is directed to the neuronal restricted linage by
EPO.
[0102] This mechanism is illustrated in FIG. 1. Thus, EGF (or its
adult homolog TGF.alpha.) induces the self-renewal/expansion of the
NSC population. The NSCs undergo spontaneous differentiation as a
default pathway to become glial precursor cells (GPCs), which
differentiate into glial cells. This spontaneous differentiation
can be attenuated by CNTF. Alternatively, NSCs can be instructed by
EPO and/or PACAP/cAMP to differentiate to neuronal precursor cells
(NPCs), which make neurons only.
[0103] Based on this mechanism, we developed a two-step method to
produce neural cells. The first step is to increase the number of
neural stem cells, which can be achieved by, for example,
proliferating neural stem cells (e.g., by EGF, FGF-1, FGF-2,
TGF.alpha., estrogen, prolactin, PACAP, growth hormone, and/or
IGF-1), inhibiting spontaneous differentiation of neural stem cells
(e.g., by CNTF), and/or promoting survival of neural stem cells
(e.g., by estrogen). The second step is to enhance neuronal or
glial formation from neural stem cells. For example,
erythropoietin, prolactin, serotonin, PACAP and/or cyclic AMP can
be used to enhance neuron formation, while bone morphogenetic
protein (BMP) can be used to enhance glial formation.
[0104] The present method can be used in vivo or in vitro. In
vitro, the present invention will result in large quantities of
neural cells, which can be used in research or therapeutical
purposes. In particular, the neural cells can be used in
transplantation treatment for neurodegenerative diseases or
conditions. In vivo, the present method can increase the number of
neural stem cells in situ, and enhance neuronal or glial formation
from the enlarged pool of neural stem cells. The resulting neural
cells can migrate to appropriate places in the nervous system to
enhance neurological functions, or compensate for lost or
dysfunctional neural cells. In addition, the in vivo and in vitro
applications can be combined. Thus, neural cells, particularly
neural stem cells produced by the present method in vitro, can be
transplanted into an animal, and factors of the second step can be
provided to the animal to enhance differentiation of neural cells
in vivo. Optionally, factors of the first step may be provided to
the animal as well to further increase the number of neural stem
cells that can be subsequently turned to neurons or glial
cells.
[0105] One particularly interesting neurodegenerative condition is
aging. We have found that the number of neural stem cells in the
subventricular zone is significantly reduced in aged mice.
Accordingly, it will be of particular interest to ameliorate
problems associated with aging by the present invention.
[0106] In addition, the neural stem cell in the subventricular zone
is the source of olfactory neurons, and olfactory dysfunction is a
hallmark of forebrain neurodegenerative diseases, such as
Alzheimer's, Parkinson's and Huntington's diseases. Disruption of
neuronal migration to the olfactory bulb leads to deficits in
olfactory discrimination, and doubling the new olfactory
interneuons enhances new odor memory (Rochefort et al., 2002).
Therefore, the present invention can be used to enhance olfactory
discrimination or olfactory memory, as well as physiological
functions that are associated with olfaction and olfactory
discrimination, such as mating, offspring recognition and
rearing.
[0107] Another particularly important application of the present
invention is the treatment and/or amelioration of brain injuries,
such as stroke (Example 2). A brain injury mimicking a stroke was
introduced into the motor cortex of rats, and the injured rats
showed abnormal behavioral conducts that correlated with the
location of the injury. The rats then received prolactin or growth
hormone for 7 days, both of which can increase neural stem cell
proliferation. Subsequently, the rats received a vehicle control or
erythropoietin for 7 days to enhance neuron formation. The rats
were then observed for a period of time for behavioral testing, and
sacrificed for anatomical analysis.
[0108] The results indicate that both prolactin and growth hormone
treatments led to an improvement of motor functions in the injured
rats. The addition of erythropoietin further enhanced the effect,
particularly when combined with prolactin. The anatomical analysis
also shows that the number of migrating neurons and/or neural stem
cells was increased by every treatment comprising prolactin or
growth hormone. In fact, the combination of prolactin and
erythropoietin even resulted in complete or partial filling of the
cavities created by the brain injury in a majority of the rats.
Therefore, these factors, particular combinations of which, can be
used to produce neural cells and restore neurological functions in
animals with brain injuries.
[0109] An intriguing observation is that prolactin and growth
hormone led to the restoration of different behavioral functions.
Thus, the rats recovered from asymmetrical forelimb usage in
balancing after receiving growth hormone, while prolactin acted to
correct abnormal positioning of the forelimb during swimming.
Therefore, different factors may lead to different cellular
migration patterns or the production of different cells, which
participate in different neural functions. Accordingly, it is
preferable that multiple factors are combined in the treatment of
diseases or conditions that have complicated symptoms. Preferred
combinations include: [0110] (a) prolactin and at least one factor
that enhances neuronal or glial differentiation, such as EPO,
PACAP, cyclic AMP and/or BMP; [0111] (b) EGF and at least one
factor that enhances neuronal or glial differentiation, such as
prolactin, EPO, PACAP, cyclic AMP and/or BMP, particularly
prolactin and/or PACAP; [0112] (c) at least one factor that
increases neural stem cell number in conjunction with prolactin;
[0113] (d) at least one factor that increases neural stem cell
number in conjunction with PACAP; [0114] (e) at least one factor
that increases neural stem cell number in conjunction with EPO; and
[0115] (f) combinations of the above.
[0116] Particularly preferred combinations include EGF and EPO, EGF
and prolactin, EGF and PACAP, EGF and growth hormone (and/or
IGF-1), EGF and prolactin and growth hormone (and/or IGF-1), EGF
and prolactin and PACAP, prolactin and growth hormone (and/or
IGF-1), prolactin and growth hormone (and/or IGF-1) and EPO,
prolactin and PACAP and growth hormone (and/or IGF-1). Most
preferred combinations include EGF and PACAP, EGF and prolactin,
and prolactin and PACAP. Preferably, FGF is not used.
Compositions
[0117] The present invention provides compositions comprising at
least one factor that is capable of increasing neural stem cell
numbers and at least one factor that is capable of enhancing
differentiation of neural stem cells. It should be noted that some
factors are capable of both functions, such prolactin. PACAP, in
addition to enhancing neuronal differentiation, also enhances
proliferation of neural stem cells in the presence of another
mitogen.
[0118] The factors that are useful in the present invention include
their analogs and variants that share a substantial similarity and
at least one biological activity with the native factors. For
example, although the major form of prolactin found in the
pituitary gland has a molecular weight of 23 kDa, variants of
prolactin have been characterized in many mammals, including
humans. Prolactin variants can result from alternative splicing of
the primary transcript, proteolytic cleavage and other
post-translational modifications. A prolactin variant of 137 amino
acids has been described in the anterior pituitary, which is likely
to be a product of alternative splicing. A variety of proteolytic
products of prolactin have been characterized, particularly the
14-, 16- and 22-kDa prolactin variants, all of which appear to be
prolactin fragments truncated at the C-terminus. Other
post-translational modification reported for prolactin include
dimerization, polymerization, phosphorylation, glycosylation,
sulfation and deamidation.
[0119] The prolactin useful in the present invention includes any
prolactin analog, variant or prolactin-related protein which is
capable of increasing neural stem cell number. A prolactin analog
or variant is a polypeptide which contains at least about 30% of
the amino acid sequence of the native human prolactin, and which
possesses a biological activity of prolactin. Preferably, the
biological activity of prolactin is the ability to bind prolactin
receptors. Although several isoforms of the prolactin receptor have
been isolated, for example the long, intermediate and short forms
in rat, the isoforms share the same extracellular domain which
binds prolactin. Therefore, any receptor isoform can be used to
assay for prolactin binding activity. Specifically included as
prolactins are the naturally occurring prolactin variants,
prolactin-related protein, placental lactogens, S179D-human
prolactin (Bernichtein et al., 2001), prolactins from various
mammalian species, including but not limited to, human, other
primates, rat, mouse, sheep, pig, and cattle, and the prolactin
mutants described in U.S. Pat. Nos. 6,429,186 and 5,955,346.
[0120] Similarly, in addition to native EGF, an EGF analog or
variant can also be used, which should share a substantial amino
acid sequence similarity with the native EGF, as well as at least
one biological activity with the native EGF, such as binding to the
EGF receptor. Particularly included as an EGF is the native EGF of
any species, TGF.alpha., or recombinant modified EGF. Specific
examples include, but are not limited to, the recombinant modified
EGF having a deletion of the two C-terminal amino acids and a
neutral amino acid substitution at position 51 (particularly
EGF51gln51; U.S. Patent Application Publication No. 20020098178A1),
the EGF mutein (EGF-X.sub.16) in which the His residue at position
16 is replaced with a neutral or acidic amino acid (U.S. Pat. No.
6,191,106), the 52-amino acid deletion mutant of EGF which lacks
the amino terminal residue of the native EGF (EGF-D), the EGF
deletion mutant in which the N-terminal residue as well as the two
C-terminal residues (Arg-Leu) are deleted (EGF-B), the EGF-D in
which the Met residue at position 21 is oxidized (EGF-C), the EGF-B
in which the Met residue at position 21 is oxidized (EGF-A),
heparin-binding EGF-like growth factor (HB-EGF), betacellulin,
amphiregulin, neuregulin, or a fusion protein comprising any of the
above. Other useful EGF analogs or variants are described in U.S.
Patent Application Publication No. 20020098178A1, and U.S. Pat.
Nos. 6,191,106 and 5,547,935.
[0121] As another example, useful PACAP analogs and variants
include, without being limited to, the 38 amino acid and the 27
amino acid variants of PACAP (PACAP38 and PACAP27, respectively),
and the analogs and variants disclosed in, e.g., U.S. Pat. Nos.
5,128,242; 5,198,542; 5,208,320; 5,326,860; 5,623,050; 5,801,147
and 6,242,563.
[0122] Erythropoietin analogs and variants are disclosed, for
example, in U.S. Pat. Nos. 6,048,971 and 5,614,184.
[0123] Further contemplated in the present invention are functional
agonists of prolactin or additional factors useful in the present
invention. These functional agonists bind to and activate the
receptor of the native factor, although they do not necessarily
share a substantial sequence similarity with the native factor. For
example, maxadilan is a polypeptide that acts as a specific agonist
of the PACAP type-1 receptor (Moro et al., 1997).
[0124] Functional agonists of EPO have been extensively studied.
EMP1 (EPO mimetic peptide 1) is one of the EPO mimetics described
in Johnson et al., 2000. Short peptide mimetics of EPO are
described in, e.g., Wrighton et al., 1996 and U.S. Pat. No.
5,773,569. Small molecular EPO mimetics are disclosed in, e.g.,
Kaushansky, 2001. Antibodies that activate the EPO receptor are
described in, e.g., U.S. Pat. No. 5,885,574; WO 96/40231 and WO
97/48729).
[0125] Antibodies that have agonist activities for the EGF receptor
are described, e.g., in Fernandez-Pol, 1985 and U.S. Pat. No.
5,723,115. In addition, activating amino acid sequences are also
disclosed in U.S. Pat. No. 6,333,031 for the EPO receptor, EGF
receptor, prolactin receptor and many other cell surface receptors;
metal complexed receptor ligands with agonist activities for the
prolactin and EPO receptors can be found in U.S. Pat. No.
6,413,952. Other methods of identifying and preparing ligands for
receptors, e.g., EPO and prolactin receptors, are described, for
example, in U.S. Pat. Nos. 5,506,107 and 5,837,460.
[0126] It should be noted that the effective amount of each analog,
variant or functional agonist may be different from that for the
native factor or compound, and the effective amount in each case
can be determined by a person of ordinary skill in the art
according to the disclosure herein. Preferably, the native factors,
or analogs and variants that share substantial sequence similarity
with the native factors, are used in the present invention.
[0127] Pharmaceutical compositions are also provided, comprising
the factors as described above, and a pharmaceutically acceptable
excipient and/or carrier.
[0128] The pharmaceutical compositions can be delivered via any
route known in the art, such as parenterally, intrathecally,
intravascularly, intravenously, intramuscularly, transdermally,
intradermally, subcutaneously, intranasally, topically, orally,
rectally, vaginally, pulmonarily or intraperitoneally. Preferably,
the composition is delivered into the central nervous system by
injection or infusion. More preferably it is delivered into a
ventricle of the brain, particularly the lateral ventricle.
Alternatively, the composition is preferably delivered by systemic
routes, such as subcutaneous administration. For example, we have
discovered that prolactin, growth hormone, IGF-1, PACAP and EPO can
be effectively delivered by subcutaneous administration to modulate
the number of neural stem cells in the subventricular zone.
[0129] When the composition is not directly delivered into the
brain, and factors in the composition do not readily cross the
blood brain barrier, a blood brain barrier permeabilizer can be
optionally included to facilitate entry into the brain. Blood brain
barrier permeabilizers are known in the art and include, by way of
example, bradykinin and the bradykinin agonists described in U.S.
Pat. Nos. 5,686,416; 5,506,206 and 5,268,164 (such as
NH.sub.2-arginine-proline-hydroxyproxyproline-glycine-thienylalanine-seri-
ne-proline-4-Me-tyrosine.PSI.(CH.sub.2NH)-arginine-COOH).
Alternatively, the factors can be conjugated to the transferrin
receptor antibodies as described in U.S. Pat. No. 6,329,508;
6,015,555; 5,833,988 or 5,527,527. The factors can also be
delivered as a fusion protein comprising the factor and a ligand
that is reactive with a brain capillary endothelial cell receptor,
such as the transferrin receptor (see, e.g., U.S. Pat. No.
5,977,307).
[0130] The pharmaceutical compositions can be prepared by mixing
the desired therapeutic agents with an appropriate vehicle suitable
for the intended route of administration. In making the
pharmaceutical compositions of this invention, the therapeutic
agents are usually mixed with an excipient, diluted by an excipient
or enclosed within such a carrier which can be in the form of a
capsule, sachet, paper or other container. When the
pharmaceutically acceptable excipient serves as a diluent, it can
be a solid, semi-solid, or liquid material, which acts as a
vehicle, carrier or medium for the therapeutic agent. Thus, the
compositions can be in the form of tablets, pills, powders,
lozenges, sachets, cachets, elixirs, suspensions, emulsions,
solutions, syrups, aerosols (as a solid or in a liquid medium),
ointments containing, for example, up to 10% by weight of the
therapeutic agents, soft and hard gelatin capsules, suppositories,
sterile injectable solutions, and sterile packaged powders.
[0131] Some examples of suitable excipients include artificial
cerebral spinal fluid, lactose, dextrose, sucrose, sorbitol,
mannitol, starches, gum acacia, calcium phosphate, alginates,
tragacanth, gelatin, calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl
cellulose. The formulations can additionally include: lubricating
agents such as talc, magnesium stearate, and mineral oil; wetting
agents; emulsifying and suspending agents; preserving agents such
as methyl- and propylhydroxy-benzoates; sweetening agents; and
flavoring agents. The compositions of the invention can be
formulated so as to provide quick, sustained or delayed release of
the therapeutic agents after administration to the patient by
employing procedures known in the art.
[0132] For preparing solid compositions such as tablets, the
therapeutic agent is mixed with a pharmaceutical excipient to form
a solid preformulation composition containing a homogeneous mixture
of a compound of the present invention. When referring to these
preformulation compositions as homogeneous, it is meant that the
therapeutic agents are dispersed evenly throughout the composition
so that the composition may be readily subdivided into equally
effective unit dosage forms such as tablets, pills and
capsules.
[0133] The tablets or pills of the present invention may be coated
or otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permit the inner component
to pass intact into the duodenum or to be delayed in release. A
variety of materials can be used for such enteric layers or
coatings, such materials including a number of polymeric acids and
mixtures of polymeric acids with such materials as shellac, cetyl
alcohol, and cellulose acetate.
[0134] The liquid forms in which the novel compositions of the
present invention may be incorporated for administration orally or
by injection include aqueous solutions, suitably flavored syrups,
aqueous or oil suspensions, and flavored emulsions with edible oils
such as corn oil, cottonseed oil, sesame oil, coconut oil, or
peanut oil, as well as elixirs and similar pharmaceutical
vehicles.
[0135] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as described herein. The compositions are
administered by the oral or nasal respiratory route for local or
systemic effect. Compositions in preferably pharmaceutically
acceptable solvents may be nebulized by use of inert gases.
Nebulized solutions may be inhaled directly from the nebulizing
device or the nebulizing device may be attached to a face mask
tent, or intermittent positive pressure breathing machine.
Solution, suspension, or powder compositions may be administered,
preferably orally or nasally, from devices which deliver the
formulation in an appropriate manner.
[0136] Another formulation employed in the methods of the present
invention employs transdermal delivery devices ("patches"). Such
transdermal patches may be used to provide continuous or
discontinuous infusion of the therapeutic agent of the present
invention in controlled amounts. The construction and use of
transdermal patches for the delivery of pharmaceutical agents is
well known in the art. See, for example, U.S. Pat. No. 5,023,252,
herein incorporated by reference. Such patches may be constructed
for continuous, pulsatile, or on demand delivery of pharmaceutical
agents.
[0137] Other suitable formulations for use in the present invention
can be found in Remington's Pharmaceutical Sciences.
[0138] The following abbreviations have the following meanings.
Abbreviations not defined have their generally accepted
meanings.
[0139] EGF=epidermal growth factor
[0140] PDGF=platelet derived growth factor
[0141] DMSO=dimethylsulfoxide
[0142] CNTF=ciliary neurotrophic factor
[0143] EPO=erythropoietin
[0144] NSC=neural stem cell
[0145] GPC=glial precursor cell
[0146] NPC=neuronal precursor cell
[0147] PACAP=pituitary adenylate cyclase activating polypeptide
[0148] cAMP=cyclic AMP
Materials and Methods
Neural Stem Cell Culture
[0149] The protocols for neural stem cell culture are described in
detail in U.S. Pat. No. 5,750,376 or Shingo et al., 2001. Briefly,
embryonic neural stem cells were prepared from E14 medial and
lateral ganglionic eminences. Adult neural stem cells were prepared
from the subventricular zone of adult mice. The tissue was cultured
in basal medium containing 20 ng/ml EGF, or other growth factors as
indicated in each case, to form neurospheres. The composition of
the basal medium is as follows: DMEM/F12 (1:1); glucose (0.6%);
glutamine (2 mM); sodium bicarbonate (3 mM); HEPES (5 mM); insulin
(25 .mu.g/ml); transferrin (100 .mu.g/ml); progesterone (20 mM);
putrescine (60 .mu.M); and selenium chloride (30 nM).
[0150] Seven days later, the neurospheres (primary neurospheres)
were passaged by mechanical dissociation and reseeded as single
cells (passage 1). For secondary neurospheres, the single cells
were then cultured for seven days to form secondary
neurospheres.
Infusion of Growth Factors
[0151] Two-month-old CD-1 mice (Charles-River, Laval, Quebec,
Canada) were anesthetized with sodium pentobarbital (120 mg/kg,
i.p.) and implanted with an osmotic pump (Alzet 1007D; Alza
Corporation, Palo Alto, Calif.). The cannula was located in the
right lateral ventricle (antero-posterior+0.2 mm, lateral+0.8 mm to
bregma, and dorsoventral -2.5 mm below dura with the skull leveled
between lambda and bregma). Human recombinant EPO (1000 I U/ml),
rabbit anti-EPO neutralizing antibody (100 .mu.g/ml), rabbit IgG
(100 .mu.g/ml), rat recombinant CNTF (33 .mu.g/ml), or human
recombinant EGF (33 .mu.g/ml) was dissolved in 0.9% saline
containing 1 mg/ml mouse serum albumin (Sigma). Each animal was
infused for 6 consecutive days at a flow rate of 0.5 .mu.l/hr,
resulting in a delivery of about 25 IU of EPO, 3 .mu.g of
antibodies, or 400 ng of CNTF or EGF per day.
Test Animals for the Stroke Study
[0152] Adult male Long-Evans rats (250-350 g) were obtained from
Charles River Breeding Farms and were adapted to the colony for two
weeks prior to any treatment. A week before surgery the rats were
given a baseline testing on the behavioral tests.
Focal Ischemic Injury and Infusion
[0153] The animals for the stroke study received unilateral
devascularization of the sensorimotor cortex. Using Isoflurane
anesthesia, the skin was incised and retracted and the overlying
fascia were removed from the skull. A skull opening was made at the
following coordinates, taking care not to damage the dura: AP +4.0
to -2.0; L 1.5 to 4 (the parasagittal ridge; Kolb et al., 1997).
The dura was cut and retracted from the skull opening. A cotton
swab soaked in sterile saline was gently rubbed across the exposed
pia and the vessels were removed. A hole was then drilled in the
contralateral hemisphere to provide an opening for the cannulae
attached to the osmotic minipump at AP-0.5; L 1.5. An osmotic
minipump was placed under the skin between the shoulder blades and
a tube connected under the skin to the cannulae, which was attached
to the skull with fast-drying cement. Once hemostasis had been
achieved the scalp was sutured closed with 5-O sterile suture. The
animals were given a single injection of Banamine (an analgesic)
and returned to their home cage. Sham animals received only
anesthesia, the bone opening, and the skin was incised and
sutured.
[0154] Six days later the animals were assessed using the
behavioral test. On the following day the animals were
re-anesthetized and the minipump was replaced with a second one
containing the appropriate solutions. Sham animals were only
anesthetized. The animals were retested 7, 14, and 28 days later to
yield behavioral measures on weeks 1, 2, 3, 4, and 6.
Forelimb Inhibition Test
[0155] This test has been shown to constitute a sensitive measure
of functional integrity of regions of anterior neocortex. In normal
rats, swimming is accomplished by propulsion from the hind limbs.
The forelimbs are normally inhibited from any stroking and are held
immobile and together under the animal's neck. Inhibition of the
forelimbs was assessed by filming animals while swimming. Animals
were introduced into one end of an aquarium (30 w.times.90
l.times.43 h cm) filled to a depth of 25 cm with room temperature
water (.about.25.degree. C.) and filmed as they swim to a 9.5 cm
square visible platform. This platform projects 2 cm above the
surface of the water and is positioned at the opposite end of the
aquarium. Scoring of inhibition was done by counting the number of
strokes, if any, made by each forelimb in three swims along the
length of the aquarium. A swim was deemed scorable only if the
animal did not touch the sides of the aquarium during the swimming
trial.
Forelimb Asymmetry Test
[0156] Forepaw asymmetry of the animals was determined by filming
them from below while in an acrylic cylinder 25 cm in diameter, on
an acrylic platform. Preference was determined by separately
counting the number of times in 5 minutes that an animal reared and
placed the left or right forepaw on the surface of the cylinder in
a gesture of postural stabilization. This test allows a measure of
asymmetry in forelimb use, a measure that shows a reliable bias to
using the limb ipsilateral to the injury.
Brain Anatomical Analysis
[0157] At the conclusion of week 6 the animals were given an
overdose of Euthanol and intracardially perfused with 0.9% saline
and 4% paraformaldehyde in picric acid. The brains were
cryoprotected and cut on a Vibratome at 40 microns. Five sets of
sections were kept every 400 microns. Two sets were stained, one
with Cresyl Violet and one with Doublecortin. The remaining sets
were saved. The Cresyl Violet staining was performed on the slides
whereas the Doublecortin was performed as an immunohistochemical
procedure on free-floating sections. The Cresyl Violet staining
allows assessment of lesion extent whereas the Doublecortin stains
for a microtubule associated protein that is present in migrating
neuronal progenitor cells.
Example 1
The Effect of CNTF and EPO In Vivo
[0158] To determine the effect of CNTF and EPO in vivo, CNTF or EPO
was infused into adult mice for six days as described in Materials
and Methods. The brain tissue was then harvested and used to grow
neural stem cells as an indication of the number of neural stem
cells in the brain after infusion. Alternatively, the brain tissue
was stained for tyrosine hydroxylase or Mash1 to determine the
extent of neurogenesis.
[0159] As described in detail in Shimazaki et al., 2001, CNTF
infusion led to a significant increase of the number of primary
neurospheres that can be obtained from the brain (about 25%).
Moreover, confusion of EGF and CNTF increased the number of neural
stem cells by about 40%. Therefore, CNTF is capable of increasing
neural stem cell numbers, particularly in combination with EGF.
CNTF does not stimulate proliferation of neural stem cells,
however, as CNTF did not increase the number of BrdU positive cells
when BrdU was also given to the animals.
[0160] Since CNTF does not promote proliferation or survival of
neural stem cells, we hypothesized that CNTF inhibits spontaneous
differentiation of neural stem cells. By spontaneously
differentiating into a lineage-restricted cell, neural stem cell
will not be able to self-renew, and the number of neural stem cells
will decrease while the number of differentiated cells increase.
Therefore, if CNTF inhibits this spontaneous differentiation, a
neurosphere produced in the presence of CNTF should be more
expandable and multipotent than a neurosphere produced in its
absence.
[0161] Accordingly, we compared the expandability and multipotency
of neurospheres that were produced in EGF alone or EGF plus CNTF.
For expandability, pass 1 neurospheres were dissociated and
replated at clonal density to generate pass 2 neurospheres, and the
number of pass 2 neurospheres that were derived from a single pass
1 sphere was counted. The results show that the pass 1 neurospheres
generated in EGF plus CNTF led to significantly more pass 2
spheres, indicating that these pass 1 spheres contained more
expandable cells than spheres generated in EGF alone. For
multipotency, the percentages of neuron, oligodendrocyte and
astrocyte that could be derived from each neurosphere were
determined, and the results show that neurospheres produced in EGF
alone generated 4 times as many glial cells than those produced in
EGF plus CNTF. Therefore, neural stem cells differentiate to glial
cells by default, which can be inhibited by CNTF.
[0162] EPO, on the other hand, reduced the number of neural stem
cells by about 50% and increased neurogenesis. Therefore, even
though neural stem cells spontaneously differentiate to the glial
lineage, a portion of neural stem cells can be induced to form
neuronal progenitor cells by EPO. Furthermore, infusion of anti-EPO
antibodies, but not non-specific IgG, led to an increase of neural
stem cells, indicating that there is an on-going neurogenesis
process in vivo that involves EPO.
Example 2
The Effect of Factor Combinations in a Stroke Model
[0163] In order to determine the effect of various combinations of
factors in animals that suffer a brain injury, focal ischemic
injuries were introduced into the brains of rats as a model of
stroke. As a result of the brain injury, the animals had lesions in
the motor cortex and behaved abnormally in two behavioral tests.
One is the forelimb inhibition test, a sensitive measure of
functional integrity of regions of the anterior neocortex. Normal
rats inhibit the use of forelimbs when they swim, but when one side
of the motor cortex was injured in this experiment, the rats failed
to inhibit the use of the contralateral forelimb as the motor
cortex controls the contralateral side of the body. In the other
test, the forelimb asymmetry test, normal rats use both forelimbs
equally when they try to balance themselves. The injured animals,
however, preferred to use the ipsilateral forelimb, presumably
because they could not control their contralateral forelimbs.
[0164] The animals then received various test factors, and the
effects of these factors on the forelimb inhibition test and brain
anatomy were assessed. As controls, a sham control group received a
sham brain injury and no test factors, and a vehicle control group
received the brain injury as well as infusions of artificial
cerebral spinal fluid (CSF). The treatments each test group
received are summarized below:
TABLE-US-00001 Second Infusion Group Brain Injury First Infusion
(days 1-7) (days 8-14) 1 sham none none 2 yes CSF CSF 3 yes
prolactin CSF 4 yes prolactin erythropoietin (EPO) 5 yes growth
hormone CSF 6 yes growth hormone erythropoietin (EPO)
[0165] The schedule and procedure of the brain injury, infusion,
behavioral test and anatomical analysis are described in Materials
and Methods.
[0166] A. The Effect of Prolactin and Prolactin Plus EPO
[0167] Before the brain injury, all rats inhibited both forepaws in
the forelimb inhibition test, which is expected from normal rats.
After the injury, all ischemic groups (Groups 2-6) failed to
inhibit the contralateral forepaw, but they continued to inhibit
the ipsilateral forepaw. Upon infusion of the test factors, the two
prolactin groups (Groups 3 and 4) showed greater forepaw
inhibition. In fact, by the end of the last week (4 weeks after
completion of the infusions), the prolactin plus EPO group (Group
4) was indistinguishable from the controls. Therefore, prolactin,
and particularly the combination of prolactin and EPO, resulted in
a recovery from a representative symptom of stroke.
[0168] Anatomically, the prolactin group showed a high degree of
doublecortin staining in the brain, indicating that prolactin
induced extensive neurogenesis. The rats in the prolactin plus EPO
group had an expanded subventricular zone, indicating a significant
cell increase in this area. In addition, many doublecortin positive
cells appeared in the legioned area, white matter and the lateral
ventricle. A stream of doublecortin positive cells could be
observed between the subventricular zone and the lesioned area.
Since doublecortin is a marker of migrating neuronal progenitor
cells, these results indicate that neural stem cells gave rise to
neuronal progenitor cells upon treatment, and the progenitor cells
migrated to the lesioned area. The new growth in the lesioned area
was so extensive that the cavities created by the ischemic injury
were completely or partially filled up in a majority of the rats in
this group. These anatomical results thus strongly support the
behavioral study that prolactin, or the combination of prolactin
and EPO, can be used to treat brain injuries such as stroke.
[0169] B. The Effect of Growth Hormone and Growth Hormone Plus
EPO
[0170] The results of the forelimb asymmetry test indicate that
although the extent of asymmetry decreased at the end of week six
in all the test groups, the groups receiving growth hormone (Groups
3 and 4) showed a faster and more extensive recovery in the first
four weeks. These results are consistent with those from the
anatomical analysis, which show that growth hormone alone (Group 3)
resulted in increased doublecortin positive cells, and the
combination of growth hormone and EPO (Growth 4) led to migration
of doublecortin positive cells around the lateral ventricle.
[0171] Accordingly, growth hormone, either alone or in conjunction
with EPO, improved a motor neuron-related function in a stroke
model as well as neuron formation/migration in the brain,
indicating that growth hormone can be used to treat or ameliorate
brain injuries.
[0172] Thus, prolactin and the combination of prolactin and EPO
improved the motor function of injured rats in the forelimb
inhibition test but not the forelimb asymmetry test, while growth
hormone and its combination with EPO had the reversed effect. These
results demonstrate that different factors can stimulate different
neural pathways and enhance the recovery of different neuronal
circuits, indicating that it is important to combine various
factors for a more complete and effective therapeutic result.
* * * * *