U.S. patent application number 13/190811 was filed with the patent office on 2012-08-02 for transplantation of bone marrow stromal cells for treatment of neurodegenerative diseases.
This patent application is currently assigned to HENRY FORD HEALTH SYSTEM. Invention is credited to Michael Chopp, Yi Li.
Application Number | 20120195865 13/190811 |
Document ID | / |
Family ID | 46324439 |
Filed Date | 2012-08-02 |
United States Patent
Application |
20120195865 |
Kind Code |
A1 |
Li; Yi ; et al. |
August 2, 2012 |
TRANSPLANTATION OF BONE MARROW STROMAL CELLS FOR TREATMENT OF
NEURODEGENERATIVE DISEASES
Abstract
The present invention relates to a treatment of an autoimmune
demyelinating disease/disorder. Also included in the present
invention is the use of bone marrow stromal cells for the treatment
of multiple sclerosis (MS).
Inventors: |
Li; Yi; (Canton, MI)
; Chopp; Michael; (Southfield, MI) |
Assignee: |
HENRY FORD HEALTH SYSTEM
Detroit
MI
|
Family ID: |
46324439 |
Appl. No.: |
13/190811 |
Filed: |
July 26, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11431290 |
May 9, 2006 |
8017112 |
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13190811 |
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11027881 |
Dec 30, 2004 |
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11431290 |
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09980614 |
Apr 17, 2002 |
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PCT/US00/12875 |
May 11, 2000 |
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11027881 |
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60134344 |
May 14, 1999 |
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Current U.S.
Class: |
424/93.21 ;
424/93.7 |
Current CPC
Class: |
A61P 25/28 20180101;
A61K 38/185 20130101; A61K 35/28 20130101; A61K 38/18 20130101;
A61K 35/30 20130101 |
Class at
Publication: |
424/93.21 ;
424/93.7 |
International
Class: |
A61K 35/28 20060101
A61K035/28; A61P 25/28 20060101 A61P025/28; A61K 48/00 20060101
A61K048/00 |
Claims
1. A method of treating a mammal having an autoimmune demyelinating
disease/disorder, the method comprising isolating a stromal cell
from a bone marrow sample, and administering said stromal cell to
said mammal, wherein the presence of said stromal cell in the CNS
of the mammal effects treatment of said disease/disorder, further
wherein the presence of said stromal cell in the CNS of the mammal
reduces axonal loss.
2. The method of claim 1, wherein said stromal cell is selected
from the group consisting of an autologous stromal cell, an
allogenic stromal cell, a syngeneic stromal cell, and a xenogeneic
stromal cell, with respect to said mammal.
3. The method of claim 1, wherein said mammal is a human.
4. The method of claim 1, wherein said stromal cell is derived from
a human donor.
5. The method of claim 1, wherein said autoimmune demyelinating
disease/disorder is selected from the group consisting of a genetic
disease, multiple sclerosis (MS), and a neurodegenerative
disease.
6.-8. (canceled)
9. The method of claim 1, wherein said stromal cell administered to
said CNS remains present or replicates in said CNS.
10. The method of claim 1, wherein said stromal cell administered
to said CNS does not result in a cell replacement therapy.
11. The method of claim 1, wherein said stromal cell administered
to said CNS induces expression of a growth factor within
neighboring cells.
12.-13. (canceled)
14. The method of claim 1, wherein prior to administering said
stromal cell to said mammal, said cell is cultured in vitro for a
period of time.
15. The method of claim 1, wherein prior to administering said
stromal cell to said mammal, said stromal cell is transfected with
an isolated nucleic acid encoding a therapeutic protein, wherein
when such protein is secreted by said stromal cell, said protein
serves to effect treatment of said disease/disorder.
16. The method of claim 1, wherein said stromal cell is
administered to said mammal by a route selected from the group
consisting of intreavascular, intracerebral, parenteral,
intraperitoneal, intravenous, epidural, intraspinal, intrastemal,
intra-articular, intra-synovial, intrathecal, intra-arterial,
intracardiac, and intramuscular.
17. The method of claim 1, wherein said stromal cell is
administered to said mammal at the site of injury.
18. The method of claim 1, wherein said stromal cell is
administered to said mammal at an adjacent site to the site of
injury.
19. (canceled)
20. The method of claim 1, wherein said stromal cell present in the
CNS activates the proliferation of neighboring cells.
21. The method of claim 1, wherein said stromal cell is
administered concomitantly with a growth factor.
22. The method of claim 1, wherein said stromal cell administered
to said mammal prevents axonal fiber loss in the cells of the
mammal.
23. The method of claim 1, wherein said stromal cell administered
to said mammal prevents demyelination in the cells of the
mammal.
24. (canceled)
25. The method of claim 1, wherein said stromal cell in the CNS of
the mammal induces angiogenesis.
26. The method of claim 1, wherein said stromal cell in the CNS of
the mammal induces neurogenesis.
27. The method of claim 1, wherein said stromal cell in the CNS of
the mammal induces synaptogenesis.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
patent application Ser. No. 11/027,881, filed Dec. 30, 2004, which
is a continuation-in-part of U.S. patent application Ser. No.
09/980,614, filed on Apr. 17, 2002, which is a national phase
application filed under 35 U.S.C. .sctn.371, claiming the benefit
of priority of International Application No. PCT/US00/12875, filed
May 11, 2000, which claims the benefit under 35 U.S.C. .sctn.119(e)
of U.S. Provisional Application No. 60/134,344, filed May 14, 1999,
all of which are herein incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] Most central nervous system (CNS) injuries include stroke,
trauma, hypoxia-ischemia, multiple sclerosis, seizure, infection,
and poisoning directly or indirectly involve a disruption of blood
supply to the CNS. These injuries share the same common pathologic
process of rapid cerebral edema leading to irreversible brain
damage and eventually to brain cell death.
[0003] One common injury to the CNS is stroke which is the
destruction of brain tissue as a result of intracerebral hemorrhage
or ischemia. Stroke may be caused by reduced blood flow or ischemia
that results in deficient blood supply and death of tissues in one
area of the brain (infarction). The causes of ischemic stroke
include blood clots that form in the blood vessels in the brain
(thrombus) and blood clots or pieces of atherosclerotic plaque or
other material that travel to the brain from another location
(emboli). Bleeding (hemorrhage) within the brain may also cause
symptoms that mimic stroke.
[0004] The CNS tissue is highly dependent on blood supply and is
very vulnerable to interruption of blood supply. Without
neuroprotection, even a brief interruption of the blood flow to the
CNS can cause neurological deficit. The brain is believed to
tolerate complete interruption of blood flow for a maximum of about
5 to 10 minutes. It has been observed that after blood flow is
restored to areas of the brain that have suffered an ischemic
injury, secondary hemodynamic disturbances have long lasting
effects that interfere with the ability of the blood to supply
oxygen to CNS tissues. Similarly, interruption of the blood flow to
the spinal cord, for even short periods of time, can result in
paralysis.
[0005] Recognition of the "ischemic penumbra," a region of reduced
cerebral blood flow in which cell death might be prevented, has
focused attention on treatments that might minimize or reverse
brain damage when the treatments are administered soon after stroke
onset. To date, several classes of neuroprotective compounds have
been investigated for acute stroke. They have included calcium
channel antagonists, N-methyl-D-aspartate (NMDA) receptor
antagonists, free radical scavengers, anti-intercellular adhesion
molecule 1 antibody, GM-1 ganglioside, .gamma.-aminobutyric acid
agonists, and sodium channel antagonists, among others. Results
from various trials have yielded disappointing efficacy results and
some evidence of safety problems, including increased mortality or
psychotic effects which resulted in their early termination.
[0006] Multiple sclerosis (MS) is another disease of the CNS. MS is
an inflammatory demyelinating disease, which typically displays a
relapsing-remitting course characterized by episodes of
neurological disability followed by periods of partial or complete
clinical remission (Lucchinetti et al., 2000, Ann. Neurol.
47:707-717; Hemmer et al., 2002, Nat. Rev. Neurosci. 3:291-301).
Most patients later enter a progressive phase of steady decline of
neurological function. Severe axonal loss and neuronal death are
frequent in MS (Ferguson et al., 1997, Brain 120 (Pt. 3):393-399;
Trapp et al., 1998, N. Engl. J. Med 338:278-285; Peterson et al.,
2001, Ann. Neurol. 50:389-400; Bjartmar et al., 2003, Neurotox.
Res. 5:157-164). Axonal loss is a major cause of permanent
neurological deficit in MS (Wujek et al., 2002, J. Neuropathol.
Exp. Neurol. 61:23-32; Bjartmar et al., 2003, J. Neurol. Sci.
206:165-171; Medana et al., 2003, Brain 126:515-530). Chronically
demyelinated axons may degenerate due to a lack of myelin-derived
trophic support (Bjartmar et al., 2003, J. Neurol. Sci.
206:165-171); however, no current therapies for MS are known
provide at axonal protection (Bectold, et al., 2004, Ann. Neurol.
55:607-616).
[0007] Cellular therapy serves as an alternative to drug therapy.
It has been demonstrated that intracerebral transplantation of
donor cells from embryonic tissue may promote neurogenesis (Snyder
et al., 1997 Adv Neurol. 72:121-32). Intrastriatal fetal graft has
been used to reconstruct damaged basal ganglia circuits and to
ameliorate behavioral deficits in a mammalian model of ischemia
(Goto et al., 1997 Exp Neurol. 147:503-9). Fetal hematopoietic stem
cells (HSCs) transplanted into the adult organism or adult HSCs
transplanted into an embryo results in a chimera that reflects the
endogenous cells within the microenvironment into which the cells
were seeded (Geiger et al., 1998, Immunol Today 19:236-41).
Pluripotent stem cells are harbored in the adult CNS and the adult
brain can form new neurons (Gage, 1998 Curr. Opin. Neurobiol.
8:671-6; Kempermann and Gage, 1998 Nat Med. 4:555-7).
[0008] Bone marrow contains at least two types of stem cells,
hematopoietic stem cells and stem cells of non-hematopoietic
tissues variously referred to as mesenchymal stem cells or marrow
stromal cells (MSCs) or bone marrow stromal cells (BMSCs). These
terms are used synonymously throughout herein. MSCs are of interest
because they are easily isolated from a small aspirate of bone
marrow and they readily generate single-cell derived colonies. The
single-cell derived colonies can be expanded through as many as 50
population doublings in about 10 weeks, and can differentiate into
osteoblasts, adipocytes, chondrocytes (Friedenstein et al., 1970
Cell Tissue Kinet. 3:393-403; Castro-Malaspina et al., 1980 Blood
56:289-301; Beresford et al., 1992 J. Cell Sci. 102:341-351;
Prockop, 1997 Science 276:71-74), myocytes (Wakitani et al., 1995
Muscle Nerve 18:1417-1426), astrocytes, oligodendrocytes, and
neurons (Azizi et al., 1998 Proc. Natl. Acad. Sci. USA
95:3908-3913); Kopen et al., 1999 Proc. Natl. Acad. Sci. USA
96:10711-10716; Chopp et al., 2000 Neuroreport II 3001-3005;
Woodbury et al., 2000 Neuroscience Res. 61:364-370). For these
reasons, MSCs are currently being tested for their potential use in
cell and gene therapy of a number of human diseases (Horwitz et
al., 1999 Nat. Med. 5:309-313; Caplan, et al. 2000 Clin. Orthoped.
379:567-570).
[0009] MSCs constitute an alternative source of pluripotent stem
cells. Under physiological conditions they maintain the
architecture of bone marrow and regulate hematopoiesis with the
help of different cell adhesion molecules and the secretion of
cytokines, respectively (Clark and Keating, 1995 Ann NY Acad Sci
770:70-78). MSCs grown out of bone marrow by their selective
attachment to tissue culture plastic can be efficiently expanded
(Azizi et al., 1998 Proc Natl Acad Sci USA 95:3908-3913; Colter et
al., 2000 Proc Natl Acad Sci USA 97:3213-218) and genetically
manipulated (Schwarz et al. 1999 Hum Gene Ther 10:2539-2549).
[0010] MSC are also referred to as mesenchymal stem cells because
they are capable of differentiating into multiple mesodermal
tissues, including bone (Beresford et al., 1992 J Cell Sci
102:341-351), cartilage (Lennon et al., 1995 Exp Cell Res
219:211-222), fat (Beresford et al., 1992 J. Cell. Sci.
102:341-351) and muscle (Wakitani et al., 1995 Muscle Nerve
18:1417-1426). In addition, differentiation into neuron-like cells
expressing neuronal markers has been reported (Woodbury et al.,
2000 J Neurosci Res 61:364-370; Sanchez-Ramos et al., 2000 Exp
Neurol 164:247-256; Deng et al., 2001 Biochem Biophys Res Commun
282:148-152), suggesting that MSC may be capable of overcoming germ
layer commitment.
[0011] The concept of transplantation of bone marrow has been
studied by others. For example, in the Azizi et al. reference, the
investigators transplanted human bone marrow stromal cells (hBMSCs)
into the brains of albino rats (Azizi et al., 1998 Proc Natl Acad
Sci USA 95:3908-3913). Their primary observations were that hBMSCs
can engraft, migrate and survive in a manner similar to rat
astrocytes. Further, it has been demonstrated that the bone marrow
cells when implanted into the brain of adult mice can differentiate
into microglia and macroglia (Eglitis et al., Proc Natl Acad Sci
USA 1997 94:4080-5). Again, this occurred when the bone marrow
cells were transplanted into the brain of normal mice. There have
been many attempts made to use bone marrow stromal cells in cell
therapy in an animal model. However, there has been little evidence
of using bone marrow stromal cells in a diseased animal model or
otherwise an animal that is suffering from a disease. Thus, there
is a long felt need in the art for efficient and directed means of
treating a neurodegenerative disease such as MS in a mammal. The
present invention satisfies this need.
BRIEF SUMMARY OF THE INVENTION
[0012] According to the present invention, there is provided a
method of treating a mammal suffering from a central nervous system
(CNS) injury and/or a neurodegenerative disease. The method
includes the steps of culturing bone marrow stromal cells and
transplanting or otherwise administering the bone marrow stromal
cells into the brain of a mammal in need thereof. In addition, the
present invention encompasses a composition comprising bone marrow
cells and embryonic brain tissue for the use in the treatment of
CNS injury and/or neurodegeneration.
[0013] Also provided is a method of activating the differentiation
of neural cells in an injured brain comprising the steps of
transplanting bone marrow stromal cells adjacent to the injured
brain cells by way of intravascular (intraarterial, intravenous)
administration of the bone marrow stromal cells to the mammal and
having the bone marrow stromal cells activate the endogenous
central nervous system stem cells to differentiate into
neurons.
[0014] In another aspect, the invention includes a method of
stimulating brain parenchymal cells to express an array of trophic
factors including but not limited to NGF, BDNF, VEGF, and bFGF. The
method comprises the steps of transplanting bone marrow stromal
cells adjacent to the injured brain cells by way of intravascular
(intraarterial or intravenous) administration of the bone marrow
stromal cells to the mammal. The expression of neurothrophic
factors by parenchymal cells stimulated by MSCs provides a
therapeutic benefit.
[0015] A method of treating injured and degenerative brain using
the cells of the present invention is also provided. The method
comprises the steps of preparing bone marrow stromal cells and
transplanting bone marrow stromal cells near the injured brain
cells by way of intravascular administration of the cells.
[0016] In addition to using bone marrow stromal cells, whole bone
marrow and cellular components of bone marrow have been employed
(i.e. mesenchymal stem cells (MSCs); hematopoietic stem cells
(HSCs) to treat stroke and traumatic brain injury. Cellular
components of bone marrow were cultured in a special medium and in
medium comprising neurotrophins (i.e. Nerve Growth Factor (NGF),
Brain-derived neurotrophic factor (BDNF)). Cells were injected
either directly into the brain, into the internal carotid artery or
into a femoral vein. The outcome of having the cells administered
into the brain were measured using double staining
immunohistochemistry techniques to morphologically identify
phenotypic transformation of bone marrow cells, and behavioral and
functional tests to identify neurological deficits of the mammal.
The data presented herein demonstrate that treatment of among
others, stroke, spinal cord injury, or traumatic brain injury with
whole bone marrow or cellular components significantly reduces
functional deficits. Bone marrow cells also express phenotypes of
parenchymal cells.
[0017] In addition, mice treated with the neurotoxin
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce
symptoms of Parkinson's disease, were treated with bone marrow
cells or bone marrow stromal cells by delivering the cells by the
route, including but not limited to, intracerebral and
intravascular delivery of the cells to the mammal. Parkinson's
symptoms were significantly reduced in mice treated with either
bone marrow cells or bone marrow stromal cells. These data
demonstrate that these cells can be employed to treat neural injury
and neurodegenerative disease.
[0018] Furthermore, a mouse experimental autoimmune
encephalomyelitis (EAE) model which mimics multiple sclerosis (MS)
was employed to demonstrate that administration of bone marrow
stromal cell to a mammal in need thereof can treat a
neurodegenerative disease associated with demyelination.
Accordingly, the invention includes a method of treating a
neurodegenerative disease associated with inflammatory
demyelination using bone marrow stromal cells. Preferably, the
neurodegenerative disease is MS.
[0019] Also encompassed in the present invention is a composition
comprising an aggregate, composed of neural stem cells from the
fetal neurosphere, MSCs from adult bone marrow and cerebro-spinal
fluid from adult Wistar rats (called NMCspheres). These NMCspheres
have been successfully used to treat stroke and brain trauma, and
can be employed to treat neurodegenerative disease.
[0020] Accordingly, the present invention encompasses methods and
compositions for the culturing of bone marrow stromal cells in
neurotrophins, and the intraparenchymal and intravascular
administration of these cells (cultured in the presence or absence
of a growth factor), for therapy and the treatment of stroke,
trauma and Parkinson's disease using bone marrow. In addition, the
cells can be used to treat a neurodegenerative disease associated
with demyelination.
[0021] The invention relates to a method of treating a mammal
having a disease, disorder or condition of the CNS. The method
comprises obtaining a bone marrow sample from donor, isolating a
stromal cell from the bone marrow sample, and administering the
isolated stromal cell to the CNS of the mammal, wherein the
presence of the isolated stromal cell in the CNS effects treatment
of the disease, disorder or condition.
[0022] In one aspect, the presence of the isolated stromal cell in
the CNS of the mammal induces angiogenesis. In another aspect, the
presence of the isolated stromal cell in the CNS of the mammal
induces neurogenesis. In yet another aspect, the presence of the
isolated stromal cell in the CNS of the mammal induces
synaptogenesis.
[0023] In one aspect, the presence of the isolated stromal cell in
the CNS of the mammal does not induce an immune response against
the stromal cell.
[0024] In another aspect, the mammal is a human.
[0025] In another aspect, the donor is a human who is not suffering
from a disease, disorder or condition of the central nervous
system.
[0026] In yet another aspect, the human donor is allogeneic,
syngeneic or xenogeneic with the human patient.
[0027] In a further aspect, the human donor is the human
patient.
[0028] In one aspect, the disease, disorder or condition of the CNS
is selected from the group consisting of a genetic disease, an
ischemic induced injury, a spinal cord injury, stroke and
Parkinson's disease.
[0029] In yet another aspect, the disease, disorder or condition of
the CNS is an inflammatory demyelinating disease. More preferably,
the disease, disorder or condition of the CNS is MS.
[0030] In another aspect, the disease, disorder or condition is
injury to the tissues or cells of the CNS. In yet another aspect,
the disease, disorder or condition is within the brain of the
patient.
[0031] In a further aspect, the isolated stromal cell administered
to the CNS remains present in the CNS. In another aspect, the
isolated stromal cell administered to the CNS replicates in the
CNS.
[0032] In yet another aspect, the stromal cell administered to the
CNS does not result in a cell replacement therapy. Preferably, the
stromal cell induces endogenous neighboring cells to express a
growth factor. More preferably, the endogenous neighboring cell is
a parenchymal or vascular cell.
[0033] In yet another aspect, prior to administering the isolated
stromal cell, the cell is cultured in vitro.
[0034] In one aspect, the isolated stromal cell is administered to
the mammal by a route selected from the group consisting of
intravascular, intracerebral, parenteral, intraperitoneal,
intravenous, epidural, intraspinal, intrastemal, intra-articular,
intra-synovial, intrathecal, intra-arterial, intracardiac, and
intramuscular.
[0035] In another aspect, the isolated stromal cell in the CNS of
the mammal secretes a factor selected from the group consisting of
a growth factor, a trophic factor and a cytokine. In a further
aspect, the secreted factor is selected from the group consisting
of leukemia inhibitory factor (LIF), brain-derived neurotrophic
factor (BDNF), epidermal growth factor receptor (EGF), basic
fibroblast growth factor (bFGF), FGF-6, glial-derived neurotrophic
factor (GDNF), granulocyte colony-stimulating factor (GCSF),
hepatocyte growth factor (HGF), IFN-.gamma., insulin-like growth
factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8,
monocyte chemotactic protein (MCP-1), mononuclear phagocyte
colony-stimulating factor (M-CSF), neurotrophic factors (NT3),
tissue inhibitor of metalloproteinases (TIMP-1), TIMP-2, tumor
necrosis factor (TNF-.beta.), vascular endothelial growth factor
(VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR),
bone morphogenetic protein 4 (BMP4), IL1-a, IL-3, leptin, stem cell
factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet
derived growth factor-BB (PDGFBB), transforming growth factors beta
(TGF.beta.-1) and TGF.beta.-3.
[0036] In yet a further aspect, prior to administering the isolated
stromal cell, the isolated stromal cell is transfected with an
isolated nucleic acid encoding a therapeutic protein, wherein when
the protein is secreted by the stromal cell and the secreted
protein serves to effect treatment of said disease, disorder or
condition.
[0037] The invention includes administering an isolated stromal
cell to the mammal at the site of injury.
[0038] In another aspect, the stromal cell is administered to the
mammal at an adjacent site to the site of injury. In one aspect,
following administering the stromal cell into the mammal, the
stromal cell migrates to the site of injury.
[0039] In a further aspect, the stromal cell present in the CNS
activates the proliferation of an endogenous neighboring cell. In a
further aspect, the endogenous neighboring cell is an
astrocyte.
[0040] In one aspect, the stromal cell activates the MEK/Akt
pathway in neighboring cells. In another aspect, the stromal cell
activates the PI3K/Erk pathway in neighboring cells. In a further
aspect, the stromal cell activates the differentiation of
neighboring cells.
[0041] In yet another aspect, the stromal cell exhibits at least
one marker characteristic of a cell of the CNS. In further aspect,
the marker is selected from the group consisting of class III
.beta.-tubulin, the M subunit of neurofiliments, tyrosine
hydroxylase, gluatmate receptor subunits of the GluR1-4 and GluR6
classes, glial fibrillary acidic protein, myelin basic protein,
brain factor 1, NeuN, NF-M, NSE, nestin, and trkA.
[0042] The invention includes administering an isolated stromal
cell concomitantly with a growth factor to the mammal.
[0043] In one aspect, the stromal cell administered to the patient
prevents axonal fiber loss in the cells of the mammal.
[0044] In another aspect, the stromal cell administered to the
patient prevents or reduces demyelination in the cells from the
mammal.
[0045] The invention includes administering an isolated stromal
cell to the mammal in the absence of immunosupppressive agents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0047] FIG. 1, comprising FIG. 1A through Figure B, is a diagram of
the three regions of the rat brain after two hours of middle
cerebral artery occlusion (MCAo) with bone marrow
transplantation.
[0048] FIG. 2, comprising FIG. 2A through FIG. 2L is an image
depicting bone marrow cells in an H&E prepared section in the
immunoreactivity of representative proteins in the (ischemic
boundary zone) IBZ of a series of adjacent sections from rats
sacrificed four days after bone marrow transplantation (FIG. 2A
through FIG. 2H). FIG. 2I depicts the neuronal specific nuclear
protein, NeuN. FIG. 2J demonstrates that the bone marrow
transplantation of the cells adjacent to the ependymal cells
exhibited reactivity for the neuronal marker, MAP-2; and FIG.
2K-FIG. 2L depict that the cells of the (subventricular zone) SVZ
express Neuro D and glial fibrillary acidic protein (GFAP) protein
markers.
[0049] FIG. 3, comprising FIG. 3A through FIG. 3H, is a series of
images depicting H&E prepared sections of cerebral tissue after
MCAo and having bone marrow cells transplanted into the mammal
after MCAo.
[0050] FIG. 3, comprising FIG. 3I through FIG. 3J, is an image
depicting the TUNEL staining exhibiting apoptotic-like cells within
the bone marrow grafting at four days following
transplantation.
[0051] FIG. 4, comprising FIG. 4A through FIG. 4C, depicts data
from the adhesive-removal test, the rotarod-motor test and the
Neurological Severity Score (NSS), respectively.
[0052] FIG. 5, comprising FIG. 5A through FIG. 5B, depicts grafts
demonstrating that mice treated with transplanted MSCs exhibited a
significant improvement in the duration on the rotarod and an
improved neurological function compared to vehicle treated
mammals.
[0053] FIG. 6, comprising FIG. 6A through FIG. 6B, depicts that
rats that received MSC intraarterial transplantation exhibited
significant improvement on the adhesive-removal test and the
modified Neurological Severity Scores (mNSS) at 14 days following
transplantation compared with control mammals.
[0054] FIG. 7, comprising FIG. 7A through FIG. 7B depicts
functional data from rats receiving administration of MSCs
intravenously compared with control-ischemia rats not receiving
MSCs.
[0055] FIG. 8 depicts rotarod data from mice subjected to
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
neurotoxicity;
[0056] FIG. 9, comprising FIG. 9A through FIG. 9D depicts the
morphological changes, i.e. most shrunk pigmented neurons
disappeared and only few of them were observed in the substantia
nigra at 45 days after MSC transplantation in MPTP induced
Parkinson's diseased (MPTP-PD) mice; viable 5-bromo-2-deoxyuridine
(BrdU) immunoreactive cells identified in the injected area and
migrated to variable distances into the host striatum at 45 days
after transplantation of the MSCs; double staining shows that
scattered BrdU reactive cells express tyrosine hydroxylase (TH)
immunoreactivity within the grafts;
[0057] FIG. 10 depicts data from the Basso-Beaftie-Bresnahan (BBB)
test from mammals subjected to spinal cord injury.
[0058] FIG. 11 is an image depicting the composite MSC neurosphere
nine days after cell-neurosphere integration.
[0059] FIG. 12, comprising FIGS. 12A and 12B, is a series of images
demonstrating that treatment with hMSCs improves survival rate and
neurological functional recovery in experimental autoimmune
encephalomyelitis (EAE) mice. FIG. 12A demonstrates that the
survival rates for hMSCs treated mice at weeks 10, 20, 35, and 45
were significantly higher than those in the PBS group (p<0.01).
FIG. 12B demonstrates that functional scores were significantly
lower among mice treated with hMSCs compared with PBS treated mice
as early as 1 week up to 45 weeks (p<0.05).
[0060] FIG. 13, comprising FIGS. 13A through 13D, is a series of
images demonstrating that hMSC treatment increases axonal density
in the white matter of EAE brain. FIGS. 13A-13B and FIGS. 13C-13D
depict reduced area of axonal loss in the striatum and corpus
callosum, respectively, between the hMSC treatment group compared
with the PBS treatment group.
[0061] FIG. 14, comprising FIGS. 14A through 14C, is a series of
images demonstrating that administration of hMSCs increases NGF
expression in the CNS of EAE mice. FIG. 14A is an image depicting
NGF cell expression in the EAE brain treated with hMSCs or PBS.
FIG. 14B is a graph depicting increased numbers of NGF reactive
cells in the brain at 1, 10, 20, 35 and 45 weeks compared with the
PBS treatment. FIG. 14C is an image depicting that about 50-70% of
NGF.sup.+ cells co-localizes with NeuN.sup.+ cells.
[0062] FIG. 15 is a graph demonstrating that hMSCs are present in
EAE brain following transplantation as measured by the presence of
MAB1281 cells.
[0063] FIG. 16, comprising FIGS. 16A-16I, is a series of images
depicting the phenotype of the transplanted hMSC. FIGS. 16D-16F
demonstrates that less than about 5% of MAB1281.sup.+ cells
co-localized with NG2.sup.+ cells. FIGS. 16A-16C and 16C-16I
demonstrate that about 10% of MAB1281.sup.+ cells co-localized with
GFAP.sup.+ cells and MAP-2.sup.+ cells, respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0064] The present invention provides a method of treating neural
injury and neurodegeneration using transplantation of bone marrow
stromal cells. It has been determined that bone marrow stromal
cells present within injured brain and/or spinal cord produce an
array of factors including, but not limited to, cytokines and
growth factors. The bone marrow stromal cells activate among
others, endogenous stem cells and ependymal cells in the brain, to
proliferate and differentiate into parenchymal cells including, but
not limited to, neurons. These new neurons can be present at sites
adjacent to the sites of injury. Thus, the bone marrow stromal
cells activate endogenous CNS stem cells to differentiate into
among others, neurons. The bone marrow stromal cells also produce
factors including, but not limited to cytokines and growth factors,
that promote repair and plasticity of the brain. In addition, the
bone marrow stromal cells can induce angiogenesis.
[0065] In addition, the invention includes a method of
transplanting bone marrow stromal cells to a mammal in need
thereof, such as a mammal having MS, where the bone marrow stromal
cells induce expression of growth factors within CNS cells. In one
aspect, the bone marrow stromal cells stimulate brain parenchymal
cells to express NGF. The expression of NGF by the parenchymal
cells provides an elevated level of NGF present in the CNS that
otherwise would be at a lower level in the absence of bone marrow
stromal cells. The elevated level of NGF present in the CNS
provides a therapeutic benefit, including but not limited to
stimulating axonal repair, prevent demyelination, reducing axonal
loss, stimulating oligodendrocyte growth, stimulated
oligodendrocyte differentiation, enhancing survival of
differentiated oligodendrocytes, and exhibiting immunomodulatory
effects. Therefore, in some instances, the therapeutic effect from
transplantation of bone marrow stromal cell is not due to cell
replacement therapy where the transplanted bone marrow stromal
cells differentiate into neuronal cells for replacement of damaged
endogenous neuronal cells, but rather the interaction with
endogenous cells to induce endogenous cells to secrete growth
factors such as NGF.
[0066] The present invention encompasses methods of culturing bone
marrow stromal cells, and methods for administering the cells of
the present invention to a mammal. The cells can be transplanted
into the penumbral tissue, which is a tissue adjacent to a lesion.
The tissue adjacent to the lesion provides a receptive environment,
similar to that of a developmental brain, for the survival and
differentiation of the bone marrow stromal cells. It is based on
this activity that the bone marrow stromal cells are useful in
treating neural injury and neurodegeneration wherein brain and/or
spinal cord damage has occurred.
[0067] In addition, bone marrow stromal cells are effective in
treating neural injury and degeneration when these cells are
administered intravascularly, i.e. intraarterially or
intravenously. Therefore, after such brain injury, when the brain
tissue is damaged, in an effort to compensate for the lost tissue,
the administration of bone marrow stromal cells can provide a
sufficient source of cells to promote compensatory responses of the
brain to such damage.
[0068] The cells of the present invention can be administered into
including, but not limited to ischemic brain, injured brain,
injured spinal cord and into brain that exhibits symptoms of
Parkinson's disease. In some instances, the cells are administered
to a brain of a mammal that exhibits symptoms of MS. In any event,
transplantation of the cells into the brain can also be performed
with co-transplantation of growth factors including, but not
limited to brain derived neurotrophic factor (BDNF) and nerve
growth factor (NGF). The cells of the invention are cultured with
NGF prior to transplantation into a recipient.
[0069] Transplantation can be performed at various time points
(i.e., from four hours to two days after stroke, from one to seven
days after trauma, seven days after spinal cord injury and fourteen
days after initiation of Parkinson's disease) after experimental
stroke in both the rat and the mouse. The data presented herein
indicate that the transplantation of bone marrow or components into
ischemic brain results in differentiation of the bone marrow cells
into the brain parenchymal cells, including but not limited to
neurons. In addition, endogenous brain stem cells are activated to
proliferate and differentiate into parenchymal cells. The cells of
the invention migrate to different regions within the brain
including, but not limited to, the hippocampus, the olfactory bulb
and the cortex. There is also improved functional outcome in rats
treated with bone marrow transplantation cultured with or in
combination with growth factors. The disclosure herein demonstrates
that the cells of the invention can also be used to provide
improved functional outcomes in higher mammals, including but not
limited to humans.
[0070] The disclosure presented herein also indicates that the
transplantation of bone marrow stromal cells into a brain of a
mammal that exhibits characteristics of MS or otherwise a mammal
suffering from immune-mediated demyelination, reduces axonal loss
in the brain. In addition, the transplantation of bone marrow
stromal cells contributes to expression of NGF from endogenous
parenchymal cells. The secretion of NGF by endogenous parenchymal
provides both neurotrophic and immunomodulatory effects.
Definitions
[0071] As used herein, each of the following terms has the meaning
associated with it in this section.
[0072] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0073] The term "about" will be understood by persons of ordinary
skill in the art and will vary to some extent on the context in
which it is used.
[0074] As used herein, the term "autologous" is meant to refer to
any material derived from the same individual to which it is later
to be re-introduced into the individual.
[0075] As used herein, the term "allogeneic" is meant to refer to
any material derived from a different mammal of the same
species.
[0076] "Xenogeneic" refers to any material derived from a mammal of
a different species.
[0077] As used herein, the term "bone marrow stromal cells
(BMSCs)," "stromal cells," "mesenchymal stem cells" or "MSCs" are
used interchangeably and refer to the small fraction of cells in
bone marrow which can serve as stem cell-like precursors to
osteocytes, chondrocytes, and adipocytes. Bone marrow stromal cells
have been studied extensively (Castro-Malaspina et al., 1980, Blood
56:289-30125; Piersma et al., 1985, Exp. Hematol 13:237-243;
Simmons et al., 1991, Blood 78:55-62; Beresford et al., 1992, J.
Cell. Sci. 102:341-3 51; Liesveld et al., 1989, Blood 73:1794-1800;
Liesveld et al., Exp. Hematol 19:63-70; Bennett et al., 1991, J.
Cell. Sci. 99:131-139). Bone marrow stromal cells may be derived
from any animal. In some embodiments, stromal cells are derived
from humans.
[0078] "Differentiation medium" is used herein to refer to a cell
growth medium comprising an additive or a lack of an additive such
that a stem cell, embryonic stem cell, ES-like cell, MSCs,
neurosphere, NSC or other such progenitor cell, that is not fully
differentiated when incubated in the medium, develops into a cell
with some or all of the characteristics of a differentiated
cell.
[0079] As used herein, the term "disease, disorder or condition of
the central nervous system" is meant to refer to a disease,
disorder or a condition which is caused by a genetic mutation in a
gene that is expressed by cells of the central nervous system such
that one of the effects of such a mutation is manifested by
abnormal structure and/or function of the central nervous system,
such as, for example, neurodegenerative disease or primary tumor
formation. Such genetic defects may be the result of a mutated,
non-functional or under-expressed gene in a cell of the central
nervous system. The term should also be construed to encompass
other pathologies in the central nervous system which are not the
result of a genetic defect per se in cells of the central nervous
system, but rather are the result of infiltration of the central
nervous system by cells which do not originate in the central
nervous system, for example, metastatic tumor formation in the
central nervous system. The term should also be construed to
include trauma to the central nervous system induced by direct
injury to the tissues of the central nervous system. The term
should also include a neurodegenerative disease associated with
demyelination of cells of the CNS. An example of such a disease is
multiple sclerosis (MS).
[0080] "Neural stem cell" or "NSC" is used herein to refer to
undifferentiated, multipotent, self-renewing neural cell. A neural
stem cell is a clonogenic multipotent stem cell which is able to
divide and, under appropriate conditions, has self-renewal
capability and can terminally differentiate into among others,
neurons, astrocytes, and oligodendrocytes. Hence, the neural stem
cell is "multipotent" because stem cell progeny have multiple
differentiation pathways. A neural stem cell is capable of self
maintenance, meaning that with each cell division, one daughter
cell will also be, on average, a stem cell.
[0081] "Neurosphere" is used herein to refer to a neural stem
cell/progenitor cell wherein Nestin expression can be detected,
including, inter alia, by immunostaining to detect Nestin protein
in the cell. Neurospheres are aggregates of proliferating neural
stem and progenitor cells and the formation of neurosphere is a
characteristic feature of neural stem cells in in vitro
culture.
[0082] As used herein, "central nervous system" should be construed
to include brain and/or the spinal cord of a mammal. The term may
also include the eye and optic nerve in some instances.
[0083] As used herein "endogenous" refers to any material from or
produced inside an organism, cell or system.
[0084] "Exogenous" refers to any material introduced from or
produced outside an organism, cell, or system.
[0085] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and the
non-coding strand, used as the template for transcription of a gene
or cDNA, can be referred to as encoding the protein or other
product of that gene or cDNA.
[0086] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. Nucleotide sequences that encode proteins and RNA
may include introns.
[0087] An "isolated nucleic acid" refers to a nucleic acid segment
or fragment which has been separated from sequences which flank it
in a naturally occurring state, e.g., a DNA fragment which has been
removed from the sequences which are normally adjacent to the
fragment, e.g., the sequences adjacent to the fragment in a genome
in which it naturally occurs. The term also applies to nucleic
acids which have been substantially purified from other components
which naturally accompany the nucleic acid, e.g., RNA or DNA or
proteins, which naturally accompany it in the cell. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (e.g., as a cDNA
or a genomic or cDNA fragment produced by PCR or restriction enzyme
digestion) independent of other sequences. It also includes a
recombinant DNA which is part of a hybrid gene encoding additional
polypeptide sequence.
[0088] In the context of the present invention, the following
abbreviations for the commonly occurring nucleic acid bases are
used. "A" refers to adenosine, "C" refers to cytosine, "G" refers
to guanosine, "T" refers to thymidine, and "U" refers to
uridine.
[0089] A "vector" is a composition of matter which comprises an
isolated nucleic acid and which can be used to deliver the isolated
nucleic acid to the interior of a cell. Numerous vectors are known
in the art including, but not limited to, linear polynucleotides,
polynucleotides associated with ionic or amphiphilic compounds,
plasmids, and viruses. Thus, the term "vector" includes an
autonomously replicating plasmid or a virus. The term should also
be construed to include non-plasmid and non-viral compounds which
facilitate transfer of nucleic acid into cells, such as, for
example, polylysine compounds, liposomes, and the like. Examples of
viral vectors include, but are not limited to, adenoviral vectors,
adeno-associated virus vectors, retroviral vectors, and the
like.
[0090] "Expression vector" refers to a vector comprising a
recombinant polynucleotide comprising expression control sequences
operatively linked to a nucleotide sequence to be expressed. An
expression vector comprises sufficient cis-acting elements for
expression; other elements for expression can be supplied by the
host cell or in an in vitro expression system. Expression vectors
include all those known in the art, such as cosmids, plasmids
(e.g., naked or contained in liposomes) and viruses that
incorporate the recombinant polynucleotide.
[0091] The phrase "substantially homogeneous population of cells"
as used herein should be construed to mean a population of cells
wherein at least 75% of the cells exhibit the same phenotype.
[0092] A "therapeutic" treatment is a treatment administered to a
subject who exhibits signs of pathology for the purpose of
diminishing or eliminating those signs.
[0093] A "therapeutically effective amount" of a compound is that
amount of compound which is sufficient to provide a beneficial
effect to the subject to which the compound is administered. Also,
as used herein, a "therapeutically effective amount" is the amount
of cells which is sufficient to provide a beneficial effect to the
subject to which the cells are administered.
Description
[0094] The present invention is based on the discovery that MSCs
can differentiate into neurons and other parenchymal cells. In
addition, the present invention is based on the discovery that MSCs
can secrete a factor including, but not limited to NGF, BDNF, VEGF,
and bFGF, that is beneficial to neighboring and/or distal cells.
The disclosure herein demonstrates that when MSCs are introduced
into a mammal, the MSCs activate endogenous cells to proliferate
and differentiate. In some instances, the MSCs induce endogenous
cells to express NGF. Preferably, the endogenous cells express and
secrete NGF.
[0095] The present disclosure also demonstrates that MSCs when
introduced to a site, or near a site of brain injury and/or spinal
cord injury, produce and secrete an array of factors including, but
not limited to trophic factors, cytokines and growth factors. The
factors secreted by the MSCs serve to activate, among others,
endogenous stem cells and subepedymal/epedymal cells in the brain
and/or spinal cord to proliferate and differentiate into
parenchymal cells, including, but not limited to neurons. Thus, the
present invention includes a method of using MSCs to promote repair
and plasticity of a CNS tissue in a mammal including, but not
limited to brain and spinal cord that has undergone disease,
disorder or condition associated with a defect in the CNS.
Preferably, the disease is MS.
[0096] The cells of the present invention can also be used to
secrete an angiogenic factor including, but not limited to vascular
growth factor, endothelial cell growth factor, and the like. MSCs
can be used to induce angiogenesis within the tissue in which the
MSCs are present. Thus, the invention provides a method of
promoting neovascularization within a tissue using such cells. In
accordance with this method, the cells are introduced to the
desired tissue under conditions sufficient for the cell to produce
the angiogenic factor. The presence of the factor within the tissue
promotes neovascularization within the tissue.
[0097] The mode of administration of the cells of the invention to
the CNS of the mammal may vary depending on several factors
including the type of disease being treated, the age of the mammal,
whether the cells are differentiated or not, whether the cells have
heterologous DNA introduced therein, and the like. An example of
administration of the cells into a brain tissue is provided herein
in the experimental Examples section. In that example, cells are
introduced into the brain of a mammal by intracerebral or
intravascular transplantation. Cells may be introduced to the
desired site by direct injection, or by any other means used in the
art for the introduction of compounds into the CNS.
[0098] The cells can be administered into a host in a wide variety
of ways. Preferred modes of administration are intravascular,
intracerebral, parenteral, intraperitoneal, intravenous, epidural,
intraspinal, intrastemal, intra-articular, intra-synovial,
intrathecal, intra-arterial, intracardiac, or intramuscular. In
some embodiments, MSCs are administered to the brain by direct
transplantation as described herein in the experimental Examples
section. In other embodiments, MSCs are administered to the central
nervous system, i.e., the spinal cord, by simple injection.
[0099] Transplantation of the cells of the present invention can be
accomplished using techniques well known in the art as well as
those described herein or as developed in the future. The present
invention comprises a method for transplanting, grafting, infusing,
or otherwise introducing the cells into a mammal, preferably, a
human. Exemplified herein are methods for transplanting the cells
into brains of various mammals, but the present invention is not
limited to such anatomical sites or to those mammals. Also, methods
for bone transplants are well known in the art and are described
in, for example, U.S. Pat. No. 4,678,470, pancreas cell transplants
are described in U.S. Pat. No. 6,342,479, and U.S. Pat. No.
5,571,083, teaches methods for transplanting cells to any
anatomical location in the body.
[0100] In order to transplant the cells of the present invention
into a mammal, for example a rat, the rat is anesthetized,
preferably with approximately 3.5% halothane, and anesthesia is
maintained with 1.0% halothane in 70% N.sub.2O and 30% O.sub.2 or
any cocktail well known in the art. The rat is then positioned on a
stereotaxic instrument. A midline incision is made in the scalp and
a is hole drilled for the injection of the cells. Rats receive
implants of the cells into the right striatum using a glass
capillary attached to a 10 .mu.l Hamilton syringe. Each rat
receives a total of about 1.times.10.sup.5 cells. Following
implantation, the skin was sutured closed with either thread or
staples. After recovery, the rats are behaviorally tested and
sacrificed for histological and immunological analysis to determine
the differentiation of both the implanted cell and the endogenous
cells of the CNS in vivo.
[0101] The cells of the present invention can be transplanted into
a human. Preferably, the cells are from the patient for which the
cells are being transplanted into (autologous transplantation). One
preferable mode of administration is as follows. In the case where
cells are not from the patient (allogeneic transplantation), at a
minimum, blood type or haplotype compatibility should be determined
between the donor cell and the patient. Surgery is performed using
a Brown-Roberts-Wells computed tomographic (CT) stereotaxic guide.
The patient is given local anesthesia in the scalp area and
intravenously administered midazolam. The patient undergoes CT
scanning to establish the coordinates of the region to receive the
transplant. The injection cannula usually consists of a 17-gauge
stainless steel outer cannula with a 19-gauge inner stylet. This is
inserted into the brain to the correct coordinates, then removed
and replaced with a 19-gauge infusion cannula that has been
preloaded with about 30 .mu.l of tissue suspension. The cells are
slowly infused at a rate of about 3 .mu.l/min as the cannula is
withdrawn. Multiple stereotactic needle passes are made throughout
the area of interest, approximately 4 mm apart. The patient is
examined by CT scan postoperatively for hemorrhage or edema.
Neurological evaluations are performed at various post-operative
intervals, as well as PET scans to determine metabolic activity of
the implanted cells.
[0102] Between about 10.sup.5 and about 10.sup.13 cells per 100 kg
person are administered to a human per infusion. In some
embodiments, between about 1.5.times.10.sup.6 and about
1.5.times.10.sup.12 cells are infused per 100 kg person. In some
embodiments, between about 1.times.10.sup.9 and about
5.times.10.sup.11 cells are infused per 100 kg person. In some
embodiments, between about 4.times.10.sup.9 and about
2.times.10.sup.11 cells are infused per 100 kg person. In some
embodiments, between about 5.times.10.sup.8 cells and about
1.times.10.sup.10 cells are infused per 100 kg person.
[0103] In some embodiments, a single administration of cells is
provided. In some embodiments, multiple administrations are
provided. In some embodiments, multiple administrations are
provided over the course of 3-7 consecutive days. In some
embodiments, 3-7 administrations are provided over the course of
3-7 consecutive days. In some embodiments, 5 administrations are
provided over the course of 5 consecutive days.
[0104] In some embodiments, a single administration of between
about 10.sup.5 and about 10.sup.13 cells per 100 kg person is
provided. In some embodiments, a single administration of between
about 1.5.times.10.sup.8 and about 1.5.times.10.sup.12 cells per
100 kg person is provided. In some embodiments, a single
administration of between about 1.times.10.sup.9 and about
5.times.10.sup.11 cells per 100 kg person is provided. In some
embodiments, a single administration of about 5.times.10.sup.10
cells per 100 kg person is provided. In some embodiments, a single
administration of 1.times.10.sup.10 cells per 100 kg person is
provided.
[0105] In some embodiments, multiple administrations of between
about 10.sup.5 and about 10.sup.13 cells per 100 kg person are
provided. In some embodiments, multiple administrations of between
about 1.5.times.10.sup.8 and about 1.5.times.10.sup.12 cells per
100 kg person are provided. In some embodiments, multiple
administrations of between about 1.times.10.sup.9 and about
5.times.10.sup.11 cells per 100 kg person are provided over the
course of 3-7 consecutive days. In some embodiments, multiple
administrations of about 4.times.10.sup.9 cells per 100 kg person
are provided over the course of 3-7 consecutive days. In some
embodiments, multiple administrations of about 2.times.10.sup.11
cells per 100 kg person are provided over the course of 3-7
consecutive days. In some embodiments, 5 administrations of about
3.5.times.10.sup.9 cells are provided over the course of 5
consecutive days. In some embodiments, 5 administrations of about
4.times.10.sup.9 cells are provided over the course of 5
consecutive days. In some embodiments, 5 administrations of about
1.3.times.10.sup.11 cells are provided over the course of 5
consecutive days. In some embodiments, 5 administrations of about
2.times.10.sup.11 cells are provided over the course of 5
consecutive days.
[0106] In a one embodiment of the invention, the cells of the
present invention are administered to a mammal suffering from a
disease, disorder or condition involving the CNS, in order to
augment or replace the diseased and damaged cells of the CNS. MSCs
are preferably administered to a human suffering from a disease,
disorder or condition involving the CNS. The MSCs are further
preferably administered to the brain or spinal cord of the human.
In some instances, the cells are administered to the adjacent site
of injury in the human brain. The precise site of administration of
the cells depend on any number of factors, including but not
limited to, the site of the lesion to be treated, the type of
disease being treated, the age of the human and the severity of the
disease, and the like. Determination of the site of administration
is well within the skill of the artisan versed in the
administration of such cells. Based on the present disclosure, the
cells can be administered to the patient via intracarotid or
intravenous routes.
[0107] In another embodiment, the therapeutic benefit of
administering bone marrow stromal cells to a mammal in need thereof
is not the result of a cell replacement therapy. That is, the
administered bone marrow stromal cells provide a therapeutic
benefit by inducing endogenous CNS cells to express and secrete a
growth factor. In one aspect, the bone marrow stromal cells
stimulate brain parenchymal cells to express and secrete a factor
including, but not limited to NGF, BDNF, VEGF, and bFGF. By way of
example, the expression and secretion of NGF by the parenchymal
cells provides an elevated level of NGF compared to the level of
NGF present in an otherwise identical CNS not treated with bone
marrow stromal cells. In any event, the elevated level of NGF
present in the CNS provides a therapeutic benefit, including but
not limited to stimulating axonal repair, prevent demyelination,
reducing axonal loss, stimulating oligodendrocyte growth,
stimulated oligodendrocyte differentiation, enhancing survival of
differentiated oligodendrocytes, and exhibiting immunomodulatory
effects.
[0108] There are several ways in which MSCs can be used in a
mammal, preferably, a human, to treat diseases of the central
nervous system. For example, the cells can be used as precursor
cells that differentiate following introduction into the CNS or as
cells which have been differentiated into neural cells prior to
introduction into the CNS. In either situation, the cells can be
differentiated to express at least one characteristic of a cell of
the CNS including, but not limited to class III .beta.-tubulin, the
M subunit of neurofiliments, tyrosine hydroxylase, glutamate
receptor subunits of the GluR1-4 and GluR6 classes, glial
fibrillary acidic protein, myelin basic protein, brain factor 1,
NeuN, NF-M, NSE, nestin, and trkA.
[0109] The data presented herein establish that MSCs, when
transplanted into a mammal, express proteins characteristic of
astrocytes (positive for glial fibrillary acidic protein, GFAP, a
marker for early astrocytes) and neurons (positive for microtubule
associate protein-2, MAP-2, a marker for neurons). It is
anticipated that MSCs which are introduced into the CNS can
differentiate into other cell types including, but not limited to
oligodendrocytes, Schwann cells and parenchymal cells.
[0110] Further, the disclosure herein demonstrates that following
introduction of MSCs into a mammal, the cells can secrete various
factors. Such factors include, but are not limited to, growth
factors, trophic factors and cytokines. In some instances, the
secreted factors can have a therapeutic effect in the mammal. The
secreted factors can activate the cell from which the factor was
secreted from. In addition, the secreted factor can activate
neighboring and/or distal endogenous cells to proliferate and/or
differentiate. Preferably an MSC secretes a cytokine or growth
factor such as human growth factor, fibroblast growth factor, nerve
growth factor, insulin-like growth factors, hemopoietic stem cell
growth factors, members of the fibroblast growth factor family,
members of the platelet-derived growth factor family, vascular and
endothelial cell growth factors, members of the TGF.beta. family
(including bone morphogenic factor), or enzymes specific for
congenital disorders.
[0111] MSCs can also secrete factors, trophic factors, and
cytokines including, but not limited to, leukemia inhibitory factor
(LIF), brain-derived neurotrophic factor (BDNF), epidermal growth
factor receptor (EGF), basic fibroblast growth factor (bFGF),
FGF-6, glial-derived neurotrophic factor (GDNF), granulocyte
colony-stimulating factor (GCSF), hepatocyte growth factor (HGF),
IFN-.gamma., insulin-like growth factor binding protein (IGFBP-2),
IGFBP-6, IL-1ra, IL-6, IL-8, monocyte chemotactic protein (MCP-1),
mononuclear phagocyte colony-stimulating factor (M-CSF),
neurotrophic factors (NT3), tissue inhibitor of metalloproteinases
(TIMP-1), TIMP-2, tumor necrosis factor (TNF-.beta.), vascular
endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen
activator receptor (uPAR), bone morphogenetic protein 4 (BMP4),
IL1-a, IL-3, leptin, stem cell factor (SCF), stromal cell-derived
factor-1 (SDF-1), platelet derived growth factor-BB (PDGFBB),
transforming growth factors beta TGF.beta.-1 and TGF.beta.-3.
[0112] The data presented herein establishes that the cells can
successfully graft to the CNS tissue. Further, the cells can
migrate to different regions within the brain including, but not
limited to hippocampus, olfactory bulb and cortex. These cells may
therefore replace cells in the CNS which have been lost as a result
of a genetic disease, trauma, or other injury. Further, these cells
can activate endogenous cells to proliferate and/or
differentiate.
[0113] In addition, prior to the introduction of the cells into the
CNS, the cells may be genetically engineered to produce molecules
such as trophic factors, growth factors, cytokines, neurotrophins,
and the likes, which are beneficial to cells which are already
present in the CNS. For example, MSCs can be cultured and
genetically engineered cells prior to their introduction into a
recipient, and following the introduction of the engineered cell
into the recipient, the cells are able to repair the defected CNS
tissue.
[0114] Based on these considerations, the types of diseases which
are treatable using the cells of the present are limitless. For
example, among neonates and children, the cells may be used for
treatment of a number of genetic diseases of the CNS, including,
but not limited to, Tay-Sachs disease and the related Sandhoff's
disease, Hurler's syndrome and related mucopolysaccharidoses and
Krabbe's disease. To varying extents, these diseases also produce
lesions in the spinal cord and peripheral nerves. In addition, in
neonates and children, treatment of head trauma during birth or
following birth is treatable by introducing the cells into the CNS
of the individual. CNS tumor formation in children is also
treatable using the methods of the present invention.
[0115] With respect to adult diseases of the CNS, the cells of the
present invention are useful for treatment of Parkinson's disease,
Alzheimer's disease, spinal cord injury, stroke, trauma, tumors,
degenerative diseases of the spinal cord such as amyotropic lateral
sclerosis, Huntington's disease, epilepsy and the like. Treatment
of multiple sclerosis may also be possible.
[0116] Other neurodegenerative diseases include but are not limited
to, AIDS dementia complex; demyelinating diseases, such as multiple
sclerosis and acute transferase myelitis; extrapyramidal and
cerebellar disorders, such as lesions of the ecorticospinal system;
disorders of the basal ganglia or cerebellar disorders;
hyperkinetic movement disorders, such as Huntington's Chorea and
senile chorea; drug-induced movement disorders, such as those
induced by drugs that block CNS dopamine receptors; hypokinetic
movement disorders, such as Parkinson's disease; progressive
supra-nucleopalsy; structural lesions of the cerebellum;
spinocerebellar degenerations, such as spinal ataxia, Friedreich's
ataxia, cerebellar cortical degenerations, multiple systems
degenerations (Mencel, Dejerine Thomas, Shi-Drager, and
Machado-Joseph), systermioc disorders, such as Rufsum's disease,
abetalipoprotemia, ataxia, telangiectasia; and mitochondrial
multi-system disorder; demyelinating core disorders, such as
multiple sclerosis, acute transverse myelitis; and disorders of the
motor unit, such as neurogenic muscular atrophies (anterior horn
cell degeneration, such as amyotrophic lateral sclerosis, infantile
spinal muscular atrophy and juvenile spinal muscular atrophy);
Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy
body disease; Senile Demetia of Lewy body type; Wernicke-Korsakoff
syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; Subacute
sclerosing panencephalitis hallerrorden-Spatz disease; and Dementia
pugilistica. See, e.g., Berkow et. al., (eds.) (1987), The Merck
Manual, (15.sup.th edition), Merck and Co., Rahway, N.J., which
reference, and references cited therein, are entirely incorporated
herein by reference.
[0117] In some aspects of the invention, an individual suffering
from a disease, disorder, or a condition that affects the CNS that
is characterized by a genetic defect may be treated by
supplementing, augmenting and/or replacing defective or deficient
neurological cells with cells that correctly express a normal
neurological cell gene. The cells which are to be introduced into
the individual may be derived from a different donor (allogeneic)
or they may be cells obtained from the individual to be treated
(autologous). In addition, the cells to be introduced into the
individual can by obtained from an entirely different species
(xenogeneic). The cells may also be genetically modified to correct
the defect. But this is not the only instance where the cells can
be genetically modified.
[0118] In another aspect of the invention, an individual suffering
from a disease, disorder or a condition of the central nervous
system can be treated as follows. Isolated MSCs are obtained and
expanded in culture. The cells are then administered to the
individual in need thereof. It is envisioned that some of the
isolated/expanded cells that are administered to the individual
develops into normal cells of the central nervous system. Thus,
repopulation of the central nervous system tissue with an expanded
population of MSCs serves to provide a population of normal central
nervous system cells which facilitate correction of the defect in
the central nervous system tissue. In addition, the cells that are
introduced into the individual can secrete agents including, but
not limited to growth factors, trophic factors, cytokines and the
like to activate endogenous cells of the individual to proliferate
and differentiate.
[0119] Based upon the disclosure herein, it is envisioned that the
MSCs of the present invention can be administered to the individual
in need thereof without the requirement of using immunosuppressive
drug therapy. It is recognized that cells from disparate
individuals invariably results in the risk of graft rejection.
However, it was observed that MSCs did not induce an immune
response when the cells were administered to an allogeneic
recipient. Further, it was observed that the presence of an
immunosuppressive drug, for example cyclosporine A (CsA) during
transplantation of MSCs to an allogeneic mammal, did not contribute
anymore significant effects on neurological functional recovery
compared to when MSCs were administered to an otherwise identical
mammal without receiving an immunosuppressive drug. Therefore, as
more fully discussed elsewhere herein, an aspect of the invention
includes using allogeneic MSCs for transplantation.
[0120] The invention also includes methods of using MSCs of the
present invention in conjunction with current mode, for example the
use of immunosuppressive drug therapy, for the treatment of host
rejection to the donor tissue or graft versus host disease. An
advantage of using MSCs in conjunction with immunosuppressive drugs
in transplantation is that by using the methods of the present
invention to ameliorate the severity of the immune response
following transplantation, the amount of immunosuppressive drug
therapy used and/or the frequency of administration of
immunosuppressive drug therapy can be reduced. A benefit of
reducing the use of immunosuppressive drug therapy is the
alleviation of general immune suppression and unwanted side effects
associated with immunosuppressive drug therapy.
[0121] In another aspect of the invention, the cells are
pre-differentiated into, for example, neurons prior to
administration of the cells into the individual in need thereof.
MSCs can be differentiated in vitro by treating the cells with
differentiation factors including, but are not limited to
antioxidants, epidermal growth factor (EGF), and brain derived
neurotrophic factor (BDNF). It has been demonstrated that treatment
of the cells with these factors induced the cells to undergo
morphologic changes consistent with neuronal differentiation, i.e.,
the extension of long cell processes terminating in growth cones
and filopodia. In addition, it was observed that these agents
induced the expression of neuronal specific proteins including, but
are not limited to nestin, neuron-specific enolase (NSE),
neurofilament M (NF-M), neuron-specific nuclear protein (NeuN), and
the nerve growth factor receptor trkA.
Treating CNS Disorders
[0122] Treating a human patient having a disease, disorder, or a
condition that affects the CNS, encompasses among others,
intracerebral grafting of MSCs or MSC-differentiated cells to the
CNS, including the region of the CNS having the injury or a region
adjacent to the site of injury. MSC-differentiated cells include,
for example, oligodendrocyte precursors that have been
differentiated by culturing MSCs in a differentiation medium. The
cells of the invention can be injected into a number of sites,
including the intraventricular region, the parenchyma (either as a
blind injection or to a specific site by stereotaxic injections),
and the subarachnoid or subpial spaces. Specific sites of injection
can be portions of the cortical gray matter, white matter, basal
ganglia, and spinal cord. Without wishing to be bound to any
particular theory, any mammal affected by a CNS disorder, as
described elsewhere herein, can be so treated by one or more of the
methodologies described herein.
[0123] Conventional techniques for grafting are described, for
example, in Bjorklund and Stenevi (1985, Neural Grafting in the
Mammalian CNS, eds. Elsevier, pp 169-178), the contents of which
are incorporated by reference. Procedures include intraparenchymal
transplantation, achieved by injecting the cells of the invention
into the host brain tissue. However, transplantation of the cells
of the invention can be effected in a number of CNS regions.
[0124] According to the present invention, administration of cells
into selected regions of a patient's brain may be made by drilling
a hole and piercing the dura to permit the needle of a microsyringe
to be inserted. Alternatively, the cells can be injected
intrathecally into a spinal cord region. The cell preparation of
the invention permits grafting of the cells to any predetermined
site in the brain or spinal cord. It also is possible to effect
multiple grafting concurrently, at several sites, using the same
cell suspension, as well as mixtures of cells.
[0125] The data disclosed herein demonstrate the effects of
administering the cells of the present invention to a mammal that
has undergone a disease, disorder, or a condition that affects the
CNS, otherwise a CNS injury, for example stroke using intraarterial
(IA) or intravenous (IV) delivery systems. The effects of MSCs
injected via IA or IV in an injured mammal was assessed by
analyzing neurological function, neurogenesis, and angiogenesis in
mammals that were subjected to ischemic conditions. Quantitative
analysis using immunohistochemistery techniques indicated that
angiogenesis was significantly enhanced by the administration of
the cells of the present invention. The data disclosed herein
demonstrated that no significant differences were observed with
respect to neurological function, neurogenesis, and angiogenesis in
mammals that received either IA or IV administration of the cells.
Based on the present disclosure, MSCs delivered to the ischemic
brain through both intracarotid and intravenous routes provide
therapeutic benefits to a mammal that has undergone stroke.
However, the invention should in no way be construed to be limited
to any one method of administering MSCs. Rather, any method of
administration of the cells should be construed to be included in
the present invention. Further, the invention should in no way be
limited to stroke, rather, any disease, disorder or condition of
the CNS can be treated using compositions and methods of the
present invention.
[0126] Treatment of a patient, according to the invention, can take
advantage of the migratory ability of MSCs, and using them to
provide a peptide, protein or other substance to a region of the
CNS affected by a dysfunction or deficiency relating to that
substance. As such, the cells of the invention may contain
exogenous DNA encoding a product that is missing in an individual
suffering from a CNS disorder. For example, the DNA can code for a
transmitter, such as acetylcholine or GABA, or a receptor for such
a transmitter. If an individual is suffering from a
glutamate-induced injury, it may be desirable to introduce into the
patient a gene coding for a glutamate transporting protein, which
can reduce glutamate-induced cytotoxicity.
[0127] In a further approach, DNA that encodes a growth factor or a
cytokine can be transfected to MSCs, which then are administered to
a patient suffering from a CNS disorder, the etiology or
elaboration of which is associated with a deficit or dysfunction in
the gene expression product. To this end, the invention includes,
for example, the use of a gene that, upon expression, produces
factors including, but are not limited to NGF, brain-derived
neurotrophic factor (BDNF), glial cell line-derived neurotrophic
factor (GDNF), insulin-like growth factor (IGF-1) and ciliary
neurotrophic factor (CNTF). In addition, the selected gene can
encode leukemia inhibitory factor (LIF) or any other of the other
cytokines, disclosed, for example, by Reichardt et al. (1997,
Molecular and Cellular Approaches to Neural Development, Oxford
University Press: 220-263), supra, that promotes cell survival or
differentiation.
[0128] A therapeutic procedure according to the present invention
can be effected by injecting cells, preferably stereotaxically,
into the cortex or the basal ganglia. Thereafter, the diffusion and
uptake of a ligand secreted by an MSC is beneficial in alleviating
the symptoms of a disorder where the subject's neural cells are
defective in the production of such a gene product. Thus, an MSC
genetically modified to secrete a neurotrophic factor, such as
nerve growth factor (NGF), is used to prevent degeneration of
cholinergic neurons that might otherwise die without treatment.
Alternatively, MSCs to be grafted into a subject with a disorder
characterized by a loss of dopamine neurons, such as Parkinson's
disease, can be modified to contain exogenous DNA encoding L-DOPA,
the precursor to dopamine.
[0129] According to the present invention, other CNS disorders
likewise can be treated, including Alzheimer's disease, ganglioside
storage diseases, CNS damage due to stroke, and damage in the
spinal cord. For example, Alzheimer's disease is characterized by
degeneration of the cholinergic neurons of the basal forebrain. The
neurotransmitter for these neurons is acetylcholine, which is
necessary for their survival. Engraftment of an MSC containing an
exogenous gene encoding for a factor that would promote survival of
these neurons, can be accomplished by the method of the invention
described herein.
[0130] The use of MSCs for the treatment of a disease, disorder, or
a condition that affects the CNS provides an additional advantage
in that the MSCs can be introduced into a recipient without the
requirement of an immunosuppressive agent. Successful
transplantation of a cell is believed to require the permanent
engraftment of the donor cell without inducing a graft rejection
immune response generated by the recipient. Typically, in order to
prevent a host rejection response, nonspecific immunosuppressive
agents such as cyclosporine, methotrexate, steroids and FK506 are
used. These agents are administered on a daily basis and if
administration is stopped, graft rejection usually results.
However, an undesirable consequence in using nonspecific
immunosuppressive agents is that they function by suppressing all
aspects of the immune response (general immune suppression),
thereby greatly increasing a recipient's susceptibility to
infection and other diseases.
[0131] The present invention provides a method of treating a
disease, disorder, or a condition that affects the CNS by
introducing MSCs into the recipient without the requirement of
immunosuppressive agents. The present invention relates to the
discovery that administration of an allogeneic or a xenogeneic MSC,
or otherwise an MSC that is genetically disparate from the
recipient, into a recipient provides a benefit to the recipient.
The present disclosure demonstrates that administration of such an
MSC into a mammal that was subjected to MCAo (to induce stroke
conditions) did not exhibit host rejection to the MSC. The
disclosure presented herein demonstrates that administration of
MSCs into the diseased mammal exhibit significant neurological
recovery as measured by Adhesive-Removal and mNSS tests without the
observation of the MSCs inducing a cytotoxic T lymphocyte response.
Further, there was no significant difference in the neurological
recovery between groups that received transplantation of MSCs in
the presence or absence of an immunosuppressive agent such as
cyclosporine. Thus, the present invention provides a method of
administering MSCs to a recipient having a disease, disorder, or a
condition that affects the CNS without inducing an immune response
by the recipient against the MSCs. Therefore, the present invention
provides a method of using MSCs to treat a disease, disorder or
condition without the requirement of using immunosuppressive agents
when administering MSCs to a recipient. There is, therefore, a
reduced susceptibility for the recipient of the transplanted MSCs
to incur infection and other diseases, including cancer relating
conditions that is associated with immunosuppression therapy.
Genetic Modification
[0132] The cells of the present invention can also be used to
express a foreign protein or molecule for a therapeutic purpose or
for a method of tracking their integration and differentiation in a
patient's tissue. Thus, the invention encompasses expression
vectors and methods for the introduction of exogenous DNA into the
cells with concomitant expression of the exogenous DNA in the cells
such as those described, for example, in Sambrook et al. (1989,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York), and in Ausubel et al. (1997, Current
Protocols in Molecular Biology, John Wiley & Sons, New
York).
[0133] The isolated nucleic acid can encode a molecule used to
track the migration, integration, and survival of the cells once
they are placed in the patient, or they can be used to express a
protein that is mutated, deficient, or otherwise dysfunctional in
the patient. Proteins for tracking can include, but are not limited
to green fluorescent protein (GFP), any of the other fluorescent
proteins (e.g., enhanced green, cyan, yellow, blue and red
fluorescent proteins; Clontech, Palo Alto, Calif.), or other tag
proteins (e.g., LacZ, FLAF-tag, Myc, His.sub.6, and the like)
disclosed elsewhere herein. Alternatively, the isolated nucleic
acid introduced into the cells can include, but are not limited to
CFTR, hexosaminidase, and other gene-therapy strategies well known
in the art or to be developed in the future.
[0134] Tracking the migration, differentiation and integration of
the cells of the present invention is not limited to using
detectable molecules expressed from a vector or virus. The
migration, integration, and differentiation of a cell can be
determined using a series of probes that would allow localization
of transplanted MSCs. Such probes include those for human-specific
Alu, which is an abundant transposable element present in about 1
in every 5000 base pairs, thus enabling the skilled artisan to
track the progress of the transplanted cell. Tracking transplanted
cell may further be accomplished by using antibodies or nucleic
acid probes for cell-specific markers detailed elsewhere herein,
such as, but mot limited to, NeuN, MAP2, neurofilament proteins,
and the like.
[0135] The invention also includes an MSC which, when an isolated
nucleic acid is introduced therein, and the protein encoded by the
desired nucleic acid is expressed therefrom, where it was not
previously present or expressed in the cell or where it is now
expressed at a level or under circumstances different than that
before the transgene was introduced, a benefit is obtained. Such a
benefit may include the fact that there has been provided a system
wherein the expression of the desired nucleic acid can be studied
in vitro in the laboratory or in a mammal in which the cell
resides, a system wherein cells comprising the introduced nucleic
acid can be used as research, diagnostic and therapeutic tools, and
a system wherein mammal models are generated which are useful for
the development of new diagnostic and therapeutic tools for
selected disease states in a mammal.
[0136] A cell expressing a desired isolated nucleic acid can be
used to provide the product of the isolated nucleic acid to another
cell, tissue, or whole mammal where a higher level of the gene
product can be useful to treat or alleviate a disease, disorder or
condition associated with abnormal expression, and/or activity.
Therefore, the invention includes an MSC expressing a desired
isolated nucleic acid where increasing expression, protein level,
and/or activity of the desired protein can be useful to treat or
alleviate a disease, disorder or condition involving the CNS.
[0137] The MSC can be genetically engineered to express a growth
factor, for example NGF, prior to the administration of the
engineered MSC into the recipient. The engineered MSC expresses and
secretes NGF at a larger amount compared with an MSC that has not
been genetically modified to express such a factor. A benefit of
using a genetically modified MSC in the treatment of a disease,
disorder, or a condition that affects the CNS is to increase the
therapeutic effects of having MSCs present in the recipient. The
increased therapeutic effect is attributed to the increase
secretion of NGF from the engineered MSC. With the increased
secretion of NGF from the engineered MSC, a larger amount of NGF is
present for neighboring cells or distal cells to benefit from the
NGF. In addition, the increase amount of NGF present in the
recipient allows a decrease in the time frame from which a patient
can be treated.
Methods of Affecting/Modulating Cell Survival
[0138] The skilled artisan, when armed with the disclosure herein,
can readily appreciate that the present invention encompasses novel
methods and compositions for modulating/affecting cell survival,
for example, increasing cellular survival through increased Akt and
Erk1 at both the protein level and the RNA level. The present
invention is based on the discovery that when MSCs were co-cultured
with astrocytes that have been subjected to ischemic conditions, it
has been observed that the presence of MSCs with the post-ischemic
astrocyte in culture reduced the amount of cell death of the
astrocytes. The present disclosure demonstrates that when
astrocytes were incubated in ischemic conditions such as incubation
of the astrocytes in an anaerobic chamber, and then co-cultured
with MSCs, there was a significant reduction in the cell death and
apoptotic phenotypes exhibited by the astrocytes compared with
post-ischemic astrocytes cultured in the absence of MSCs.
[0139] The data herein demonstrates that co-culturing astrocytes
with MSCs upregulated the phosphorylation of Erk1 and Akt in
astrocytes. While not wishing to be bound to any particular theory,
it is believed that MSCs contribute to the survival of neighboring
astrocytes by activating cellular proliferation and survival
signaling pathways post-translationally. It was demonstrated that
astrocytes that were treated with MEK inhibitor (U0126), which
inhibits Erk1 phoshporylation, or PI3K inhibitor (LY29004), which
inhibits Akt phosphorylation, underwent significant apoptosis and
cell death similar to the post-ischemic control group. As such, the
inhibition of molecular pathways leading to the activation of Erk1
and/or Akt inhibits the ability of a cell to survive conditions
that cause cell death and/or apoptosis that otherwise activation of
such pathways would overcome the cell death and/or apoptosis
conditions. Based on the present disclosure, the duration for which
Erk1 and/or Akt are activated increases the ability of a cell that
has been subjected to cell death/apoptotic conditions to survival
from such conditions. In addition, the intensity for which Erk1
and/or Akt is activated in a cell increases the survival potential
of a cell from cell death/apoptotic conditions. As such, the
present invention comprises a method of using MSCs to activate
survival signals, such as activation of Erk1 and/or Akt in a cell
in order to confer protection to the cell from cell death/apoptotic
conditions.
[0140] In addition to the ability of MSCs to activate cellular
pathways in neighboring cells, the present disclosure also
demonstrates that MSCs can induce neighboring post-ischemic
astrocytes to increase the transcription of various growth factors
including, but are not limited to bFGF, BDNF, and VEGF. Based upon
the present disclosure, one skilled in the art would appreciate
that MSCs can enhance the recovery of post-ischemia astrocytes by
stimulating the activation of MEK/Akt and PI3K/Erk pathways in
astrocytes, and increasing growth factor production by
astrocytes.
Axonal and Myelination Remodeling
[0141] Axonal loss and demyelination are frequently observed to be
associated with a disease, disorder, or a condition that affects
the CNS. Axonal loss and demeylination is believed to contribute to
neurological functional impairment in CNS conditions, for example,
in ischemic cerebrovascular diseases and inflammatory demyelinating
diseases, such as MS.
[0142] The disclosure presented herein demonstrates that
administration of MSCs to a diseased mammal having a condition
including, but not limited to an ischemic condition, an axonal
degeneration, or demyelination, improves neurological functional
recovery in the diseased mammal. The present disclosure also
demonstrates that the administered cells play a role in the
formation and/or maintenance of axonal fibers in an injured or
otherwise diseased brain. In some instances, the administered cells
prevent axonal fiber loss in the brain of the diseased mammal.
Mammals that were subjected to ischemic conditions or conditions of
inflammatory demyelination and subsequently treated with the
administration of MSCs demonstrated significant reduced areas of
demyelination and reduced areas of axon loss compared with an
otherwise identical mammal not treated with MSCs. While not wishing
to be bound to any particular theory, the improved neurological
functions exhibited by the diseased mammal following treatment with
MSCs is attributed to the reduced demyelination and axon loss.
[0143] In some instances, the therapeutic effects of administering
MSCs to the mammal suffering from a neurodegenerative disease, such
as MS, is not the result of cell replacement therapy. That is, the
disclosure presented herein demonstrates that a therapeutic effect
from the administered MSCs can be attributed to the fact that the
MSCs stimulate endogenous brain parenchymal cells to express NGF.
The expression of NGF by endogenous brain parenchymal cells
stimulates axonal repair in both acute and chronic diseases. Thus,
in some instances, a therapeutic outcome from transplantation of
MSCs to a mammal in need thereof does not require differentiation
of MSCs into cells of the CNS, such as neurons, to replace the
damaged or injured cells.
[0144] Based on the present disclosure, one skilled in the art
would appreciate that the loss of axonal fibers and demyelination
of the axons contribute to neurological impairment. Axons play a
major role in the neurological functions of a mammal. Axons are
insulated by a myelin sheath, which greatly increases the rate at
which an axon can transport a signal. Any loss in axonal fibers or
conditions of demeylination retards neurons from properly
functioning, for example, significant impairment in sensory, motor
and other types of functioning when nerve signals reach their
targets either too slowly, asynchronously (when some axons in a
nerve conduct faster than others), intermittently (when conduction
is impaired only at high frequencies), or not at all. As such, the
present invention provides compositions and methods for treating a
neurological impairment by preventing degradation of axonal fibers
and preventing demyelination. In addition, the present invention
encompasses compositions and methods for using MSCs to remodel
axonal fibers and myelination.
[0145] The above discussion provides a factual basis for the use of
bone marrow stromal cell transplantation for the treatment of
neural injury and neurodegeneration. The methods used with and the
utility of the present invention can be shown by the following
non-limiting examples and accompanying figures.
[0146] Standard molecular biology techniques known in the art and
not specifically described were generally followed as in Sambrook
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, New York (1989), and in Ausubel et al., Current
Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.
(1989) and in Perbal, A Practical Guide to Molecular Cloning, John
Wiley & Sons, New York (1988), and in Watson et al.,
Recombinant DNA, Scientific American Books, New York and in Birren
et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4
Cold Spring Harbor Laboratory Press, New York (1998) and
methodology as set forth in U.S. Pat. Nos. 4,66,828; 4,683,202;
4,801,531; 5,192,659 and 5,272,057 and incorporated herein by
reference. Polymerase chain reaction (PCR) was carried out
generally as in PCR Protocols: A Guide To Methods And Applications,
Academic Press, San Diego, Calif. (1990). In-situ (in-cell) PCR in
combination with Flow Cytometry can be used for detection of cells
containing specific DNA and mRNA sequences (Testoni et al, 1996,
Blood 87:3822.).
[0147] Standard methods in immunology known in the art and not
specifically described are generally followed as in Stites et al.
(eds), Basic and Clinical Immunology (8th Edition), Appleton &
Lange, Norwalk, Conn. (1994) and Mishell and Shiigi (eds), Selected
Methods in Cellular Immunology, W.H. Freeman and Co., New York
(1980).
EXAMPLES
Example 1
Treatment of Stroke (Rat) with Intracerebral Transplantation of
MSC
Description of Intracerebral Transplantation of Bone Marrow Derived
MSCs After Cerebral Ischemia in the Rat
[0148] Adult male Wistar rats were used in this study (n=28). Rats
were subjected to middle cerebral artery occlusion (MCAo) for two
hours using the intraluminal occlusion model. Following MCAo, the
control group (rats subjected to MCAo without receiving
transplantation of MSCs (n=8)), was compared with the experimental
groups, which included injection into the ischemic boundary zone
(IBZ) at 24 hours after MCAo: phosphate buffered saline (n=4); non
NGF cultured bone marrow MSCs (n=8); and NGF cultured MSCs (n=8).
Approximately 4.times.10.sup.4 cells in 10 .mu.l total fluid volume
were injected into the rat following MCAo. The rats were sacrificed
14 days after MCAo.
Behavioral Outcome Measurements
[0149] Behavioral data from the battery of functional tests
(rotarod, adhesive-removal and Neurological Severity Score tests
(NSS)) demonstrated that motor and somatosensory functions were
impaired by the ischemic insult by way of subjecting the rats to
MCAo. It was observed that no significant differences of the
rotarod, adhesive-removal and NSS tests were detected among the
groups prior to surgery and before transplantation. Significant
recovery of somatosensory behavior (p<0.05) and NSS (p<0.05)
were detected in mammals transplanted with MSCs following MCAo
compared with mammals not receiving transplantation of MSCs
following MCAo mammal (FIGS. 1A, 1C). Mammals that received
transplantation of MSCs that were cultured with NGF displayed
significant recovery in motor (p<0.05), somatosensory
(p<0.05) and NSS (p<0.05) behavioral tests at two weeks
post-transplantation with NGF, compared with transplantation of
MSCs alone. FIGS. 1A, 1B, 1C show data from the adhesive-removal
test, the rotarod-motor test and the NSS, respectively. These data
clearly demonstrate that treatment of stroke with intracranial
transplantation of MSCs provides significant therapeutic benefit
and that MSCs when cultured in NGF provides superior therapeutic
benefit compared with MSCs cultured without NGF, as indicated in
the motor test data (FIG. 1B).
Example 2
Treatment of Stroke (Mouse) with Intracerebral Transplantation of
MSC
Intrastriatal Transplantation of MSCs into Mice After Stroke:
Embolic MCAo and Transplantation
[0150] Experimental adult mice (C57BL/6, weighing about 27-35 g)
were subjected to MCAo and following MCAo, the mice received
transplantation of MSCs (n=5). Control mice were subjected to MCAo
alone (n=8). Experimental groups received either injection of PBS
into the ischemic striatum (n=5); or transplantation of MSCs into
the normal striatum (n=5). MCAo was induced using an embolic model
developed in our laboratory (Zhang et al., 1997). Briefly, using a
facemask, mice were anesthetized with 3.5% halothane and anesthesia
was maintained with 1.0% halothane in 70% N.sub.2O and 30% O.sub.2.
A single intact fibrin-rich in 24 hour old homologous clot (8
mm.times.0.000625 mm.sup.2, 0.18:1) was placed at the origin of the
MCAo via a modified PE-50 catheter. Surgical and physiological
monitoring procedures were identical to those previously published
(Zhang et al., 1997). Four days after MCAo (n=18), the mice were
mounted on a stereotaxic frame (Stoelting Co. Wood Dale, Ill.).
Using aseptic technique, a burr hole (1 mm) was made on the right
side of the skull to expose the dura overlying the right cortex.
Semisuspended MSCs (1.times.10.sup.5 in 3:1 PBS) were slowly
injected over a 10-minute period into the right striatum (AP=0 mm,
ML=2.0 mm, and DV=3.5 mm from the bregma). Without wishing to be
bound to any particular theory, this position approximates the
ischemic boundary zone in the striatum. The needle was retained in
the striatum for an additional 5 minutes interval to avoid donor
reflux. Mice were sacrificed at 28 days after stroke.
Behavioral Testing
[0151] Each mouse was subjected to a series of behavioral tests
(rotarod-motor test, Neurological Severity Score) to evaluate
various aspects of neurological function by an investigator who was
blinded to the experimental groups. Measurements were performed
prior to stroke and at 28 days after stroke.
Results
[0152] BrdU reactive MSCs survived and migrated a distance of
approximately 2.2 mm from the grafting areas toward the ischemic
areas. BrdU reactive cells expressed neuronal (.about.1% NeuN) and
astrocytic proteins (.about.8% glial fibrillary acidic protein,
GFAP). Functional recovery from a rotarod test (p<0.05) and
modified Neurological Severity Score tests (NSS, including motor,
sensory and reflex, p<0.05) were significantly improved in the
mice receiving MSCs following MCAo treatment compared with mice not
receiving MSCs following MCAo treatment (FIG. 2). FIG. 2 shows that
mice treated with transplanted MSCs exhibited a significant
improvement in the duration on the rotarod (FIG. 2) and an improved
neurological function (FIG. 2) compared to vehicle treated mammals.
The findings suggest that the intrastriatal transplanted MSCs
survive in the ischemic brain and improve functional recovery of
adult mice.
Example 3
Treatment of Stroke (Mouse) with Intravascular Administration of
MSC
Description of Experiments
[0153] Experiments were performed on adult male Wistar rats (n=30)
weighing about 270 to 290 g. In all surgical procedures, anesthesia
was induced in rats with 3.5% halothane, and maintained with 1.0%
halothane in 70% N.sub.2O and 30% O.sub.2 using a face mask. The
rectal temperature was controlled at 37.degree. C. with a feedback
regulated water heating system. Transient MCAo was induced using a
method of intraluminal vascular occlusion, as described above. Two
hours after MCAo, reperfusion was performed by withdrawal of the
suture until the tip cleared the internal carotid artery.
Intracarotid Administration of MSCs
[0154] Intra-carotid transplantation of MSCs was carried out at 24
hours after MCAo (n=23). A modified PE-50 catheter was advanced
from the same site of this external carotid artery into the lumen
of the internal carotid artery until it rested 2 mm proximal to the
origin of the MCA (FIG. 1). Approximately 2.times.10.sup.6 MSCs in
200 .mu.l PBS (n=6) or control fluid (200 .mu.l PBS, n=8) were
injected over a 10-minute period into each experimental rat.
Immunosuppressants were not used in any mammal. All rats were
sacrificed at 14 days after MCAo.
Intravenous Administration of MSCs
[0155] For intravenous administration of MSCs, a femoral vein was
cannulated and either 1.5.times.10.sup.6 MSCs or 3.times.10.sup.6
MSCs were injected.
Behavioral Tests and Immunohistochemistry
[0156] Each rat was subjected to a series of behavioral tests (NSS
and adhesive removal test) to evaluate neurological function before
MCAo, and at 1, 4, 7 and 14 days after MCAo. Single and double
immunohistochemistry were employed to identify cell specific
proteins of BrdU reactive MSCs.
Results
[0157] For intra-arterial administration, BrdU reactive cells
(.about.21% of 2.times.10.sup.6 transplanted MSCs) distributed
throughout the territory of the MCAo by 14 days after ischemia.
Some BrdU reactive cells expressed proteins characteristic of
astrocytes (glial fibrillary acidic protein, GFAP) and neurons
(microtubule associated protein-2, MAP-2). Rats with MSC
intra-arterial transplantation exhibited significant improvement on
the adhesive-removal test (p<0.05) (FIG. 3) and the modified
Neurological Severity Scores (p<0.05) (FIG. 3) at 14 days,
compared with controls. The data for intravenous administration of
MSCs were very similar, in that significant functional improvement
was present with rats treated with MSCs compared to placebo treated
rats. FIG. 4 shows functional data from rats receiving
administration of MSCs intravenously compared to control-ischemia
rats not receiving MSCs. A significant improvement is noted in the
speed in which the rats removed the sticky tabs from their paws at
seven and 14 days after stroke, compared to control mammals (FIG.
4). The overall neurological function of rats receiving MSCs
administered intraarterially was significantly improved compared to
control-ischemia rats at 14 days after stroke. The findings suggest
that MSCs injected intra-arterially are localized and directed to
the territory of MCAo and these cells foster functional improvement
after cerebral ischemia. In addition, intravenous administration of
MSCs also provides a significant improvement in functional outcome.
Thus, the data presented demonstrated that vascular administration
is a feasible and effective route of administration of
therapeutically beneficial MSCs.
Example 4
Treatment of Traumatic Brain Injury (Rat) with Intracerebral
Transplantation of MSC
Description
[0158] Experiments were performed on 66 male Wistar rats weighing
about 250-350 grams. A controlled cortical impact device was used
to induce injury to the rats (Dixon et al. 1991 J. Neuroscience
Methods 39:253-262). Injury was induced by impacting the left
cortex with a pneumatic piston containing a 6 mm diameter moving at
a rate of 4 mm/second and producing 2.5 mm compression. BrdU
labeled MSCs were harvested from donor mammals and implanted into
the ipsilateral hemisphere, as in the stroke experiments. MSCs were
transplanted into brain 24 hours after injury. Rats receiving
transplantation of MSCs were sacrificed at 4 days (n=4), 1 week
(n=15), 2 weeks (n=4) and 4 weeks (n=4) after transplantation.
Control mammals were divided into 3 groups: 1) rats subjected to
injury without transplantation and sacrificed at 8 days (n=4) and
29 days (n=4) after injury; 2) mammals injected with PBS one day
after injury and sacrificed at 4 days (n=4), 7 days (n=4), 14 days
(n=4) and 28 days after PBS injection; 3) sham control rats with
craniotomy but no injury or transplantation were sacrificed 8 days
(n=4) and 29 days (n=4) after craniotomy.
Outcome Measures (Behavior, Histology)
[0159] An accelerating rotarod test was employed to measure motor
function. Measurements were performed at 2, 5, 15, and 29 days
after injury. After sacrifice, brain sections were stained with
hematoxylin and eosin and double-labeled immunohistochemistry was
performed to identify MSC cell type.
Results
[0160] Histological examination revealed that after transplantation
MSCs survived, proliferated and migrated towards the injury site.
BrdU labeled MSCs expressed markers for astrocytes and neurons.
Rats transplanted with MSCs exhibited a significant improvement in
motor function compared with control mammals which did not received
transplantation of MSCs. The data indicate that intracerebral
transplantation of MSCs significantly improves neurological
function after traumatic brain injury. In a complementary set of
experiments, treated rats were also subjected to traumatic brain
injury and received transplantation of MSCs; however, in this
experiment MSCs were delivered to the brain by means of
intraarterial (intracarotid artery) administration. The data were
observed to be similar to intracranial transplantation. MSCs
migrated readily into the injured region of brain and these cells
expressed protein markers of brain cells (astrocytes, neurons).
Thus, the present disclosure indicate that traumatic brain injury
can be treated with MSC administered intracerebrally or via a
vascular route.
Example 5
Treatment of Parkinson's (Mouse) with Intracranial Transplantation
of MSCs
Description of MPTP Method and Results
[0161] Adult male C57BL/6 mice, 8 week-old, weighing about 20-35 g,
were employed in this study. In order to obtain severe and long
lasting lesions, mice were treated with intraperitoneal injections
of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
hydrochloride (30 mg/kg, Sigma) in saline once a day for seven
consecutive days (210 mg/kg total dose). Mice were transplanted
with BrdU labeled MSCs (3.times.10.sup.5/3 .mu.l) directly into the
right striatum, stereotaxically.
Behavioral Tests
[0162] Mice subjected to each MPTP injection, presented and
retained behavioral abnormalities (akinesia, postural instability,
tremor and rigidity) for several hours, as reported in literature
(Heikkila et al., 1989).
[0163] Drug-free evaluation of Parkinson's disease (PD) using
rotarod test was described by Rozas et al. (1997, 1998). MPTP-PD
mice with or without MSC transplantation were tested on a rotarod
at an increasing speed (16 rev/minute and 20 rev/minute) after the
last MPTP injections (five trials per day to obtain stable values)
without any additional enhanced drug injection. A trial was
terminated when the mice fell from the rotarod. Significant
improvement in motor function (p<0.05) was observed at 35 days
after MPTP injection in Parkinson's disease mice treated with MSC
transplantation compared with control MPTP-injected mice alone.
FIG. 5 shows rotarod data from mice subjected to MPTP
neurotoxicity. Two experiments were performed; the mice were placed
on the rotarod rotating at 16 rpm or at 20 rpm. The data
demonstrated that mice treated with MSCs showed a significant
increase in duration on the rotarod at both angular velocities
compared to MPTP mice given PBS intracerebrally. Mice treated with
MSCs cultured with NGF appeared to have incremental benefit
compared to MSC treatment, although the differences were not
observably significant.
Morphological Changes
[0164] Viable BrdU immunoreactive cells were identified in the
injected area and migrated to variable distances into the host
striatum (FIG. 1B) at 35 days. Double staining shows that scattered
BrdU reactive cells (FIG. 1C) express tyrosine hydroxyls (a
dopamine marker) immunoreactivity (FIG. 1D) within the grafts.
Conclusions
[0165] These data demonstrate that intracerebral transplantation of
MSCs reduces Parkinson's disease symptoms in mice.
Example 6
Treatment of Spinal Cork Injury (Rat) with Intralesional
Transplantation of MSCs
Description of Spinal Cord Injury (SPI)
[0166] Impact injury was induced using the weight-drop 10 g from a
height of 25 mm, `NYU impact` model) to produce a spinal cord
injury of moderate severity. Adult male Wistar rats (300.+-.5 g)
were anesthetized with pentobarbital (50 mg/kg, intraperitoneally),
and a laminectomy was performed at the T9 level.
Transplantation and Behavioral Testing
[0167] MSCs 2.5.times.10.sup.5/4:1 were injected into the epicenter
of injury at 7 days after SPI. The Basso-Beatie-Bresnahan (BBB)
Locomotor Rating scores were obtained before and after
transplantation (Basso et al., 1995). FIG. 7 shows data from the
BBB test from mammals subjected to spinal cord injury, and
receiving MSC transplantation or simply given the same volume of
vehicle. All rats had a score of 21 (normal score) before spinal
cord injury and a score of zero at 6 hours after contusion. In the
rats subjected to contusion with PBS injection, scores improved
from 6.7 (1 week) to 11.5 (5 weeks). The control group had an early
improvement in neurologic function, which plateaued by the third
week. The rats subjected to contusion with MSC transplantation had
a significantly improved score of 7.0 (1 week) and 15.3 (5 weeks).
The MSC treated group exhibited a steady recovery that had not
plateaued by the fifth weeks, which was the end point of the
experiment. The MSC treated rats had significant improvement on BBB
scores with the p-value, 0.01 for overall and each individual time
point for treatment effect. In functional terms, the contused rats
in the MSC treated group could walk with consistent weight
supported plantar steps with forelimb and hindlimb coordination. In
contrast, the contused rats in the PBS control group exhibited
obvious motor function deficits.
Histological Analysis
[0168] Cells derived from MSCs, identified by BrdU
immunoreactivity, survived and were distributed throughout the
damaged tissue (T9, FIG. 1A) from 1 week to 4 weeks after MSC
transplantation. BrdU reactive cells migrated 5 mm both caudal and
rostral from the epicenter of transplanted cells (FIG. 1B). FIG. 2A
shows that the antibody against Rip did not react with damaged
oligodendrocytes in contused rats with non treated PBS injection.
In contrast, after spinal cord injury and receiving MSC
transplantation (FIG. 2B), intense Rip immunoreactivity clearly
demarcated myelinated small and large diameter fibers. Double
immunostaining (FIGS. 2C-D) demonstrates that scattered BrdU
reactive cells express the neuronal marker, NeuN.
Conclusions
[0169] Treatment of moderate to severe spinal cord injury in a
mammal by administering MSCs into the site of injury provides
significant improvement of motor function. The MSCs express protein
markers of neurons and oligodendrocytes, indicating that these
cells when placed within the spinal cord acquire characteristics of
parenchymal cells.
Example 6
Neurosphere (NMCsphere)--a New Composite for the Treatment of CNS
Injury and Disease
Description of Neurosphere Experiment
[0170] Aggregates, composed of neural stem cells from fetal
neurosphere, mesenchymal stem cells from adult bone marrow and
cerebro-spinal fluid from adult Wistar rats (called NMCspheres)
were used in the following experiments. Fetal brain cells were
pre-labeled with
1,1'-dioctadecy-6,6'-di(4-su1fophey1)-3,3,3',3'-tetramethylindocarbocyani-
ne (Dil) and bone marrow mesenchymal cells from adult rats were
pre-labeled with 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO)
and/or bromodeoxyuridine (BrdU). Using laser scanning confocal
microscopy (three-dimensional) and immunohistochemical analysis on
paraffin and frozen sections, it was identified that:
1. Cell-cell interaction: Within the NMCsphere, cells derived from
bone marrow mesenchymal stem cells, rapidly form a scaffold (1 day)
and a network (9 days, FIG. 8) overtime, in vitro. FIG. 8 depicts
the composite MSC neurosphere nine days after cell-neurosphere
integration. The MSC, identified by DO and BrdU form an
axonal-dendritic like network (yellow-green). 2. Cell-cell
interaction: Within the NMCsphere, cells derived from neural stem
cells have a longer life span than within neurosphere alone. The
NMCspheres express proteins, i.e., nestin that is normally found in
immature neural cells; glial fibrillary acidic protein (GFAP) that
is a specific marker for differentiated astrocytes; myelin basic
protein (MBP) that is a marker of oligodendrocytes; and
neuron-specific class III .beta.-tubulin (TuJ1) that is a marker
for immature neurons and microtubule associated protein 2 (MAP-2)
that is a marker for neuronal cell bodies and dendrites. 3.
NMCsphere-microenvironment: The size and structure of the
NMCspheres are influenced by the microenvironment of the medium,
i.e., they grow better in the IMDM with stem cell factor than with
standard DMEM. 4. Secretion of NMCspheres: Adding the supernatant
from the cultured NMCsphere into the medium DMEM and IMDM for
neurospheres and MSCs, respectively, stimulated the growth of both
neurospheres and MSCs. Obvious cell-cell connection and
proliferation was induced with this supernatant. This suggests the
NMCspheres secrete supporting substances for stem cells. These
substances can be used to enhance neurogenesis. 5. Cerebro-spinal
fluid (CSF) provides an optimal microenvironment to form NMCspheres
that is superior to conventional medium.
Example 7
Treatment of Stroke and Brain Trauma with NMCsphere
[0171] Protocol for MSC & neurosphere transplantation in rats
after MCAo and traumatic brain injury (TBI).
MCAo
[0172] BrdU prelabeled MSCs and neurospheres were mixed and
cultured in flasks for 7 days. At 24 hours after MCAo, rats were
anesthetized with halothane and the composite NMSsphere was
injected into the brain of MCAo rats (n=4). The mammals were
mounted on a stereotaxic apparatus (Model 51603, Stoelting Co.,
Wood Dale, Ill.). Twenty spheres (diameter less than 0.2 mm) in 5
ml PBS were injected vertically by a Hamilton syringe into the
right striatum at the coordinates LM=2.5 mm, VD=4.5 mm and AP=O to
the bregma, and into the right cortex at LM=2.5 mm, VD=2 mm and
AP=O mm. Without wishing to be bound to any particular theory, this
position approximates the ischemic boundary zone. Three microliters
of spheres were initially injected into the striatum and 2 ml into
the cortex over a 10-minute period in each spot. The needle was
retained in the cortex for an additional 5 minute interval to avoid
bone marrow reflux from the injected areas to the brain surface.
After injection, bone wax (W810, Ethicon) was placed on the skull
to prevent the leakage of the solution. Rats were sacrificed at 14
days after MCAo.
Traumatic Brain Injury (TBI)
[0173] BrdU prelabeled MSCs and neurospheres were mixed and
cultured in flasks for 7 days. At 4 days after TBI rats (n=4) were
anesthetized with chloride hydrate and placed onto the stereotactic
frame, and then exposed to the previous injured area. A pipette
with a glass tip (0.5 mm of diameter) containing 15 prepared mixed
NMCspheres (diameter of 0.25 mm) in 20 UL PBS was fixed onto the
stereotactic frame. The tip of the needle was inserted at the
central site of the injured area, 2.5 mm away from brain surface.
Spheres were injected into the brain over 5 minutes, and then kept
for an additional 5 minute interval to avoid reflux. In both sets
of experiments (stroke and TBI) functional outcome measurements
were measured using the rotarod and adhesive removal tests.
Results
[0174] Functional benefit in both stroke and TBI was evident in
rats treated with NMCspheres. These data indicate that NMCspheres
can be employed for the treatment of stroke and brain injury. This
composite, is a new material with potential for the treatment of
CNS injury and neurodegeneration.
Example 8
Description of Novel Medium (With and Without Growth Factors)
Employed for the Culturing of MSCs for the Treatment of Neural
Injury and Neurodegeneration
[0175] Primary bone marrow cells were obtained at 48 hours after
treating adult Wistar rats with 5-fluorouracil (5-FU, 150 mg/kg)
and cultured in the Iscove's Modified Dulbecco's Medium (IMDM)
supplemented with 10% fetal bovine serum (FBS) and stem cell factor
(100 ng/ml). Adherent MSCs were resuspended in fresh IMDM with
nerve growth factor (NGF, 200 ng/ml), brain derived neurotrophic
factor (BDNF, 100 ng/ml) and epidermal growth factor (EGF, 20
ng/ml) up to one month. Control MSCs were cultured in the IMDM
without neural growth factors. Antibodies against neuronal nuclei
(NeuN), microtubule associated protein-2 (MAP-2) and glial
fibrillary acidic protein (GFAP) were used for immunocytochemical
identification of cultured cells.
[0176] The data indicates that cells derived from adult bone marrow
stem and progenitor cells can grow in large quantities in culture
and express proteins characteristic of neurons and astrocytes.
Neurotrophic growth factors enhance the neural expression of cells
derived from bone marrow cells in vitro. Immunocytochemical
staining shows that control MSCs cultured without neurotrophic
growth factors expressed the neuronal marker, NeuN (.about.1%, FIG.
1A) and the astrocytic marker, GFAP (.about.3%, FIG. 1B) at a
baseline level. However, MSCs treated with neurotrophic growth
factors (i.e., NGF) express NeuN (.about.3%, FIG. 1C) and GFAP
(.about.30%, FIG. 1D) at an elevated level.
[0177] Bromodeoxyuridine, which is incorporated into dividing
cells, and identifies newly formed DNA, was added to the medium at
72 hours before transplantation. Using immunoperoxidase with
3,3'-diaminobenzidine (DAB, brown) and counter staining by
hematoxylin, bone marrow cells are identified by the antibody
against BrdU. The number of MSCs labeled with BrdU was observed to
be .about.90% in vitro.
Discussion
[0178] The data demonstrate that cultured adult bone marrow cells,
particularly marrow stromal cells (MSCs), survive and differentiate
into parenchymal like cells in the adult rodent brains after
ischemia, brain and spinal cord trauma, and Parkinson's disease,
and that bone marrow promotes prominent proliferation,
differentiation and migration of ventricular zone/subventricular
zone (VZ/SVZ) NSCs.
[0179] Pluripotent bone marrow cells become glia in normal rat
brain (Azizi et al., 1998), and facilitate cell proliferation and
cell-specific differentiation after MCAo. The bone marrow
transplantation experiment requires a sensitive means of monitoring
the fate of the bone marrow cells. Help came from the bone marrow
cells carrying tracers and markers, such as BrdU, CD34, nestin,
PCNA. Pluripotent hematopoietic stem cells and mesenchymal stem
cells from the adult bone marrow exposed to the new ischemic
microenvironment after MCAo are triggered to proliferate and
differentiate into neuronal (MAP-2, NeuN) and glial cell (GFAP)
phenotypes. Fresh bone marrow or stroma humoral factors are also a
source of differentiating factors and provides the chemotatic
microenvironment to enhance the proliferation, migration and
differentiation of neural stem cells from VZ/SVZ.
[0180] The VZ/SVZ of the mammalian forebrain is a region of
germinal matrices that develops late in gestation, enlarges, and
then diminishes in size, but persists in a vestigial form
throughout life (Gage 1998). In the normal adult brain, the absence
of forebrain neuronal production reflects not a lack of appropriate
neural stem cells, but rather a tonic inhibition and/or a lack of
postmitotic trophic and migratory support. Although the signals
that trigger the quiescent central nervous system (CNS) stem cells
within the normal VZ/SVZ to enter the cell cycle have yet to be
resolved, the data show that a lesioned CNS is a different
environment than an intact CNS and markedly alters the terminal
differentiated phenotype of the neural stem cells. Importantly, the
VZ/SVZ in the adult forebrain is not a passive ischemia-threatened
zone, located far from the ischemic areas (FIGS. 3F-H), but is an
active tissue providing cells to reconstruct brain. VZ/SVZ cells
proliferate and differentiate into neuronal and glial phenotypes
after MCAo. The survival of neurons arising from adult NSCs is
dictated by both the availability of a permissive pathway for
migration and the environment into which migration occurs. New
neurons depart the VZ/SVZ to enter the brain parenchyma via radial
guide fibers, which emanate from cell bodies in the ventricular
ependyma in adult rat (FIGS. 2K-L), and provide a permissive
pathway for migration as found during development (Rakic 1972).
Mitosis within the graft and VZ/SVZ shows that ischemic injured
brain together with the transplanted cells reverts to an early
stage of development to promote repair. The data are consistent
with the observation that adult brain can form new neurons (Gage
1998).
Example 9
Effects of Bone Marrow Stromal Cells Injected via Different Routes
(Intraarterial Versus Intravenous) After Stroke in Rats
[0181] The data disclosed herein demonstrate the beneficial effects
of administering rat bone marrow stromal cell (rBMSC) to rats that
have undergone stroke using intraarterial (IA) or intravenous (IV)
delivery systems. In the present experiments, comparative effects
of rBMSCs injected via IA or IV on neurological function,
neurogenesis, and angiogenesis in ischemic rats were analyzed.
Young adult rats were subjected into middle cerebral artery
occlusion (MCAo) for two hours. After 24 hours, 2.times.10.sup.6
rBMSCs or phosphate buffered saline (PBS) were infused into the
carotid artery or the tail vein of the rat. The rats were
sacrificed at day one (n=6), day seven (n=6), day fourteen (n=6)
and day twenty eight (n=12) after cell injection, (half mammals for
IA and half for IV at every time point); whereas the control
mammals (n=6 for IA and n=6 for IV) were sacrificed at day twenty
eight after PBS injection. Behavioral tests (an adhesive-removal
test and a modified Neurological Severity Score, mNSS) were
performed at 1, 7, 14, 21 and 28 day after MCAo. BrdU
immunohistochemistry was used to evaluate endogenous neurogenesis
in both the subventricular zone (SVZ) and the subgranular zone
(SGZ). Immunohistochemistry was employed to measure angiogenesis in
the ischemic boundary zone (IBZ). Significant (P<0.05) recovery
of the adhesive-removal test and mNSS were found in both IA and IV
groups as early as day seven and persisted to at least day twenty
eight after cell injections compared with control mammals that did
not receive injection of rBMSCs. Immunohistochemistry results
showed that the number of BrdU positive cells in the SVZ and SGZ
significantly increased (P<0.05) during 7-14 day after stroke
and returned to baseline at day twenty eight. Quantitative analysis
using immunohistochemistery techniques indicated that angiogenesis
was significantly enhanced (P<0.05) by the rBMSC administration
and persisted for at least day twenty eight in the IBZ after the
onset of stroke. It was observed that no significant difference
between IA and IV groups in all the above examinations was detected
at this cell dose. Based on the present disclosure, rBMSCs
delivered to the ischemic brain through both intracarotid and
intravenous routes provide therapeutic benefits after stroke.
Example 10
Correlated Expression of MT1-MMP and IGF-1 Genes with Neurological
Recovery in Ischemic Rats Treated with Human Marrow Stromal
Cells
[0182] The data disclosed herein demonstrate that human bone marrow
stromal cells (hMSCs) can enhance neurogenesis and promote neural
stem cell proliferation and migration following administering into
an ischemic rat. Without wishing to be bound to any particular
theory, it is believed that the neurological recovery in an
ischemic rats using hMSC is determined by enhanced gene expression
in the ischemic area induced by hMSCs. To elucidate the molecular
mechanisms underlying the hMSC effect on stroke, differently
expressed genes in the treated and untreated ischemic brain tissue
were identified. Rats were subjected to permanent occlusion of the
right middle cerebral artery (MCAo) alone (n=18) and were injected
intravenously with 3.times.10.sup.6 hMSCs (n=18) at day one after
MCAo. Functional outcome was measured at 0, 1 and 7 day after MCAo
by a modified Neurological Severity Score, mNSS. A number of genes
including, but not limited to, Membrane-Type 1 matrix
metalloproteinase (MT1-MMP), insulin-like growth factor (IGF-1) and
its receptor IGF-1R in the ischemic boundary zone (IBZ) were tested
using RT-PCR at day 0, 2 and 7 after MCAo in rats treated with
hMSC, compared with control rats not receiving injection of hMSCs.
RT-PCR analysis demonstrated a significant increase of IGF-1 and
MT1-MMP mRNA at day two after MCAo which was observed to persist
for at least day seven. IGF-1 mRNA levels in the IBZ of rats
receiving injection of hMSC at day two and day seven after MCAo
were significantly increased compared with that of non hMSC treated
mammals. MT1-MMP mRNA levels in hMSC treated rats exhibited no
distinct difference from that of the non treated mammal at day two,
but there was a significant increase in MT1-MMP mRNA at day seven
in hMSC treated rates as compared with non treated mammals. It was
also observed that there was no significant difference in IGF1-R
mRNA expression among the normal, ischemic and hMSC treated rats at
any time points tested. These data indicate that hMSC treatment can
achieve a neurorestorative effect through multiple mechanisms in
the IBZ. Elevated IGF-1, in the presence of abundant IGF-1
receptor, can promote neuron survival and regeneration during the
hMSC treatment of these ischemic rats. MT-1 MMP, an important
membrane-bound MMP, has been suggested to play a central role in
mediating cell surface focal proteolysis pathways. IGF-1 has been
demonstrated to up-regulate MT1-MMP expression. The present
disclosure demonstrates that MT1-MMP may be functionally linked to
neuronal survival and regeneration mediated by IGF-1 in the
IBZ.
Example 11
Treatment of Stroke in Rats with Human Bone Marrow Stromal
Cells
[0183] The data disclosed herein addresses whether treatment of
stroke in rats using xenogeneic human bone marrow stromal cells
(hMSCs) necessitates the use of an immunosuppressive agent, for
example, cyclosporin A (CsA), and whether CsA affects the
neurological response to stroke and treatment with hMSCs in rats.
hMSCs were obtained from three healthy human donors. Adult Wistar
rats were subjected to 2 hours of middle cerebral artery occlusion
(MCAo). Four groups of ischemic rats (n=6, per group) were
subjected to: 1) MCAo alone without treatment; 2) 15 mg/kg CsA by
gastric feeding daily beginning at one day after MCAo for 27 days;
3) tail intravenous injection of 3.times.10.sup.6 hMSCs at one day
after MCAo; and 4) co-treatment with hMSCs and CsA after MCAo.
Functional outcome was measured by using an adhesive-removal patch
test and a modified Neurological Severity Score (mNSS) before
stroke and at 1, 7, 14, 21 and 28 day after stroke. A
human-specific antibody (Mab1281) against cellular nuclei was used
to identify hMSCs within the brain tissue. Since unwanted
activation of T-lymphocytes promotes graft rejection, human
graft-versus-rat host cytotoxic T lymphocyte (CTL) response was
measured using a .sup.51Cr assay to determine the lytic effect. It
was observed that no stroke rats died after hMSC injection into the
rats. Significant functional recovery of adhesive-removal
(p<0.05) at 14, 21 and 28 day, and mNSS (p<0.05) at 21 and 28
day was found in rats treated with hMSCs (Group 3 and 4), compared
to control ischemia rats, which did not receive hMSC injection
(Group 1 and 2). Few mAb1281 positive cells (approximately
500-3,000 positive cells per brain) were detected in recipient rats
in Group 3 (1,283.+-.592) and Group 4 (1,431.+-.727); however, no
significant difference was observed between these groups. It was
also observed that CsA did not have an apparent effect on
neurological functional recovery after stroke with or without hMSC
treatment. There was no evidence of hMSC induction of CTL response
both in vitro and in vivo. The data indicate that there is no
apparent complicating immune response that obscures the therapeutic
benefit of hMSC treatment in stroke tats. Thus, CsA
immunosuppression is not needed as an adjunctive therapy when
administering hMSCs to a rat.
Example 12
Allogeneic Rat Marrow Stromal Cells Promote Brain Remodeling
Without Immunologic Sensitization in Stroked Rats
[0184] The present disclosure addresses the effects of allogeneic
(allo-) and syngeneic (syn-) rat bone marrow stromal cell (rBMSC)
for the treatment of stroke with respect to functional outcome
based on immune reaction, glial scar formation and glial-axonal
architecture. Female Wistar rats (n=25) were subjected to middle
cerebral artery occlusion (MCAo) for two hours. At 24 hour after
MCAo, rats were injected intravenously with phosphate buffered
saline (PBS, n=8), syn-Wistar strain rBMSCs (3.times.10.sup.6/rat,
n=8), or allo-ACI strain rBMSCs (3.times.10.sup.6/rat, n=9).
Neurological functional recovery was performed using the
Neurological Severity Score, adhesive-removal patch and Corner
tests. Rats were sacrificed at day twenty eight after treatment,
and were bled to determine antibody titers to rBMSCs. Lymphocytes
collected from mesenteric and cervical lymph nodes were cultured
with irradiated syn- or allo-spleen cells to determine T cell
proliferative responses against donor alloantigens using the Mixed
Lymphocyte Reaction assay. Antibody titers to rBMSCs were
determined by a flow cytometry method. In situ hybridization and
double immunostaining techniques were employed for male
Y-chromosome.sup.+ bearing rBMSC and brain cell type
identification. Significant functional recovery (p<0.05) was
found in both groups treated with rBMSCs (syn- or allo-) compared
to PBS controls, but no difference was detected between syn- and
allo-rBMSC treated rats. Similar numbers of Y-chromosome.sup.+
cells were detected in the syn- and allo-rat brains at twenty eight
days after treatment. Astrocyte proliferation was prominent
(BrdU+-GFAP+, hyperplasia) in the ischemic brain and exceeded cell
proliferations in other cells. It was observed that the thickness
of the scar wall decreased (p<0.05), and the axonal density as
measured by Bielshowsky silver staining increased (p<0.05) in
areas where reactive astrocytes were present (GFAP+ hypertrophy) in
the scar boundary zone (SBZ) and in the subventricular zone (SVZ)
of the rBMSC treated rats, compared with the non-treated rats.
Moreover, axonal projections exhibited an overall orientation
parallel to elongated processes of reactive astrocytes and toward
lesion areas in the SBZ and SVZ of the rBMSC treated rats,
suggesting that rBMSCs may enhance reactive astrocyte-related
axonal repair in adult brain. No evidence of T cell priming or
humoral antibody production rBMSC was observed in recipient mammals
after treatment with allogeneic ACI-rBMSCs. Based on the present
disclosure, both syn- and allo-rBMSC treatment of stroke in rats
improved neurological recovery and enhanced brain remodeling with
no indication of immunologic sensitization.
Example 13
BMSC Confer Post-Ischemic Protection via Increased Akt and Erk and
Growth Factor Production Within Neighboring Astrocytes
[0185] It has been demonstrated that treatment of stroke using bone
marrow stromal cells (BMSCs) significantly improves functional
outcome, and reduces apoptosis in the brain. Astrocytes have been
shown to be the first cells to suffer ischemic insult among all
types of neural cells. The present disclosure provides insight on
the interaction between BMSCs and astrocytes after ischemic insult.
The data disclosed here addressed the effect of rat BMSCs (rBMSCs)
on post-ischemia induced apoptosis and cell death of astrocytes, as
well as the mechanisms of these effects using an in vitro ischemic
model. After a four hour ischemic incubation in an anaerobic
chamber, astrocytes were co-cultured with rBMSCs in non-ischemic
conditions (as post-ischemic incubation) for an additional four
hours. Astrocytes cultured without the presence of rBMSCs (control
group) depicted evident morphological and biochemical apoptotic
features. A large number of condensed nuclei were observed, and
many cells appeared as dark detached spheres or oval-shaped bodies.
A cell viability assay demonstrated that about 35% of the
astrocytes were not viable. However the introduction of rBMSCs
remarkably reduced the apoptosis and cell death (to about 1.5%,
P<0.01) in astrocytes, and most of the astrocytes appeared as a
confluent cobblestone layer. BrdU-immunostaining revealed a higher
proliferation rate in the co-cultured astrocyte group compared to
the control group. Western blot analysis and real-time quantitative
PCR demonstrated that rBMSCs drastically increased Erk1 and Akt at
both protein and RNA level in post-ischemic astrocytes.
Additionally, Western blot analysis also revealed that co-culturing
astrocytes with rBMSCs upregulated the phosphorylation of Erk1 and
Akt in astrocytes. Astrocytes treated with MEK inhibitor (U0126) or
PI3K inhibitor (LY29004) underwent significant apoptosis and cell
death similar to the post-ischemic control group. Co-culturing
astrocytes with rBMSCs significantly (P<0.01) attenuated this
U0126 and LY29004 mediated insult. Furthermore, real-time PCR
demonstrated that rBMSC co-culture increased RNA levels of bFGF,
BDNF, and VEGF in astrocytes that had suffered ischemia. These
results indicate that rBMSCs enhance the recovery of post-ischemia
astrocytes by stimulating the activation of MEK/Akt and PI3K/Erk
pathways in astrocytes, and increasing growth factor production by
astrocytes.
Example 14
Treatment of Stroke in Rats with Human Marrow Stromal Cells
Decreases Axonal Loss and Demyelination
[0186] Axonal loss and demyelination are frequently observed in
ischemic cerebrovascular diseases and contribute to neurological
functional impairment. It has been demonstrated that human marrow
stromal cells (hBMSCs) improved neurological functional recovery in
ischemic rats. The present disclosure, addresses the effect of
hBMSCs on axonal fibers in ischemic brain. Rats were subjected to
permanent middle cerebral artery occlusion (MCAo) and injected
intravenously with 3.times.10.sup.6 hBMSCs or phosphate buffered
saline (PBS) (n=6 per group) at one day after MCAo, and sacrificed
at fourteen days after MCAo. Axon and myelin damage was examined
using Bielshowsky and Luxol fast blue double staining,
respectively, in the MCAo rats receiving hBMSC or PBS treatment.
Nerve fiber damage was found in the white matter (WM) of the
striatum (ST) and corpus callosum (CC) of the ipsilateral
hemisphere after MCAo with PBS treatment, and involved both axonal
and myelinated components. Demyelination was more severe than axon
loss (10.4.+-.2.3% vs 3.7.+-.0.7%), indicating that the myelin is
more susceptible to ischemia than the axon. The surviving WM area
within the ipsilateral CC significantly increased compared to the
corresponding area of the contralateral CC. Enhanced density of
axons was observed in the WM bundles in the ST and WM in the CC of
the ischemic boundary zone, indicating that a self-neurorestorative
mechanism was initiated. It was observed that no significant change
in the contralateral CC area among normal, PBS and hBMSC treatment
groups. hBMSC treated MCAo mammals demonstrated significantly
reduced areas of demyelination (3.7.+-.0.2% vs 10.4.+-.2.3%) and
axon loss (1.8.+-.0.4% vs 3.7.+-.0.7%), in the ipsilateral ST when
compared to PBS treated controls. It was observed that the
morphologically intact areas of the ipsilateral CC were
significantly increased (19.8.+-.4.5% vs 11.+-.6.7%); and the
density of axons were enhanced (27.5.+-.6.3% vs 19.8.+-.5.6%) in
the ST and CC of the ischemic boundary zone in the hBMSC treated
rats compared with the PBS controls. The present disclosure
suggests that axonal and myelination remodeling may contribute to
improved functional recovery after treatment of stroke with
hBMSCs.
[0187] In summary, the data indicate that intracerebral and
intravascular bone marrow transplantation after stroke neural
injury and Parkinson's disease significantly improves functional
recovery. Transplantation also enhances the proliferation and
differentiation of exogenous bone marrow stem cells and endogenous
NSCs. Bone marrow aspirations and biopsies have been employed in
the diagnosis and treatment of clinical diseases. Bone marrow
transplantation provides a new avenue to induce plasticity of the
injured brain and spinal cord and provides a therapeutic strategy
for treatment of neural injury and neurodegeneration.
[0188] In addition, a new substance is identified herein, a
composite of MSCs and neurospheres, which when transplanted into
brain after stroke or trauma, improves functional recovery.
Example 15
Bone Marrow Stromal Cells Reduce Axonal Loss in the Experimental
Autoimmune Encephalomyelitis (EAE) Mice
[0189] The following experiments were designed to investigate the
effects of transplantating human bone marrow stromal cells (hBMSCs)
or otherwise hMSCs in an experimental model of multiple sclerosis
(MS). Such an experimental model involves assessing
remitting-relapsing and axonal loss in experimental autoimmune
encephalomyelitis (EAE) mice.
[0190] The present disclosure demonstrates that hBMSCs were able to
reduce axonal loss in an MS model. Briefly, EAE was induced in
SJL/J mice (n=63) by injection with proteolipid protein (PLP). Mice
were injected intravenously with hBMSCs (n=26) or PBS (n=37) on the
day of clinical onset and neurological function was measured daily
(score 0-5) until 45 weeks after onset. Mice were sacrificed at
week 1, 10, 20, 35 and 45. Double staining for Luxol fast blue and
Bielshowsky was used to identify myelin and axons, respectively.
Immunohistochemistry was performed to measure the expression of
nerve growth factor (NGF) and MAB1281, a marker of hBMSCs. hBMSC
treatment significantly reduced the mortality of EAE mice, and
significantly improved functional recovery in EAE mice compared to
PBS treatment. Axonal density in the EAE striatum and corpus
callosum was significantly increased in the hBMSC treatment group
compared with that of the PBS treatment group. NGF.sup.+ cells
significantly increased in the hBMSC treated mice compared to PBS
controls at 1, 10, 20, 35 and 45 weeks. Most of the NGF.sup.+ cells
were identified as brain parenchymal cells. Less than 5% of
MAB1281.sup.+ cells co-localized with NG2.sup.+, a marker of
oligodendrocyte progenitor cells. About 10% of MAB1281.sup.+ cells
co-localized with GFAP and MAP-2, a marker of astrocytes and of
neurons, respectively. It was observed that hBMSCs improved
functional recovery and therefore provides a therapy aimed at
axonal protection in EAE mice, in which NGF plays an important
role.
[0191] The Materials and Methods used in the experiments presented
in this Example are now described.
Cell Culture
[0192] hBMSCs were isolated, grown and tested, using methods
adopted from Zhang et al. (2005, Exp. Neurol. 195:16-26). Briefly,
bone marrow was obtained from adult human donors and the nucleated
cell fraction was cultured with Dulbecco's Modified Eagle
Medium-low glucose media and 10% fetal bovine serum. The adherent
cells were harvested, passaged and cryopreserved in appropriate
dose related aliquots in Plasma-Lyte containing human serum albumin
and dimethyl sulfoxide. The cells are tested for purity at the end
of each passage by flow cytometry. The BMSCs were positive for MHC
class I, CD29, CD90, CD105, CD13, CD44, CD63, CD73 and CD166. The
cells were negative for MHC class II, CD45, CD14 and CD34.
EAE Induction and Animal Groups
[0193] Myelin proteolipid protein (PLP) (p139-151; HSLGKWLGHPDKF,
SEQ ID NO:1; SynPep Corporation, Dublin, Calif.) was used for
immunization. The purity of the peptide was greater than 95% as
measured by High Performance Liquid Chromatography. EAE was induced
in female SJL/J mice (8-10 week old, Jackson Laboratory, Bar
Harbor, Me.) by subcutaneous injection with 25 ug PLP dissolved in
50 ul complete Freund's adjuvant (CFA) (Difco Laboratories,
Livonia, Mich.). On the day of immunization and 48 hours post
immunization, 200 ng pertussis toxin (PT) (List Biological
laboratories, Inc. Campbell, Calif.) in 0.2 ml phosphate buffered
saline (PBS) was injected into the mouse tail vein (Youssef et al.,
2002, Nature 420:78-84; Zhang et al., 2005, Exp. Neurol.
195:16-26). Mice were randomly divided into: 1) hBMSC treatment
group (n=26): hBMSCs (2.times.10.sup.6 per mouse) were administered
intravenously in 1 (one) ml total fluid volume PBS on the day of
clinical symptom onset (score.gtoreq.1); and 2) PBS treatment group
(n=37): PBS (1 ml) was injected into the tail vein of the EAE mice
on the day of clinical symptom onset as EAE controls. An additional
control normal group (n=6) consisted of mice without
immunization.
Neurological Functional Measurement
[0194] Mice in the hBMSC treatment group and PBS treatment group
were scored daily for clinical symptoms of EAE, as follows: 0,
healthy; 1, loss of tail tone; 2, ataxia and/or paresis of
hindlimbs; 3, paralysis of hindlimbs and/or paresis of forelimbs;
4, tetraparalysis; 5, moribund or dead (Pluchino et al., 2003,
Nature 422:688-694). Neurological functions of EAE were tested in
mice treated with hBMSCs or PBS daily until 45 weeks after clinical
symptom onset.
Histopathology and Immunohistochemistry
[0195] EAE mice treated with PBS or hBMSCs were euthanized at 1,
10, 20, 35 and 45 weeks after clinical symptom onset. Brain tissue
(ranging from bregma +1.18 mm to bregma -1.82 mm) were fixed in 4%
of paraformaldehyde and divided into 4 serial sections per mouse.
These tissue blocks were embedded in paraffin and cut into 6 .mu.m
thick coronal slides.
[0196] Double staining for Luxol fast blue and Bielshowsky was used
to demonstrate myelin and axons, respectively (Karnezis et al.,
2004, Nat. Neurosci. 7:736-744; Pluchino et al., 2003, Nature
422:688-694; Furlan et al., 2001, J. Immunol. 167:1821-1829). After
staining, nuclei appeared colorless; myelin turquoise and axons
appeared black on a pale grey/blue background.
[0197] To identify the expression of NGF, a rabbit polyclonal
antibody against NGF (1:300, Santa Cruz Biotechnology Inc., Santa
Cruz, Calif.) was used. To identify the fate of injected hBMSCs in
the CNS of EAE mice, slides were treated with a monoclonal antibody
specific to human nuclei (MAB1281; 1:500, Chemicon, Temecula,
Calif.). Double immunofluorescence labeling was performed to
identify the relationship of NGF with neural cells and hBMSC with
neural cells. Antibodies against MAP-2 or NeuN (markers of
neurons), glial fibrillary acidic protein (GFAP, a marker for
astrocytes) and NG2 (a marker for progenitor oligodendrocyte cell)
were used to identify parenchymal cells. Negative control slides
for each animal received identical preparations for immunostaining,
except for the fact that primary antibodies were omitted.
Quantification and Statistical Analysis
[0198] Neurological functional tests and tissue slides were
evaluated by a blinded examiner to the treatment status of each
animal. Mice were monitored for mortality up to 45 weeks (i.e., if
the mice were not sacrificed earlier than 45 weeks). Mortality
rates were compared between the hBMSC treated and PBS treated
groups using the log-rank survival analysis with Kaplan-Meier
curves plotted for survival rates over time.
[0199] The functional score, ranging from 1 to 5, was measured on
the day before the treatment and daily during and after the
treatment up to 45 weeks after EAE. Subgroups of mice were
sacrificed at week 1 (n=7 in each group), week 10 (n=7 in the PBS
group; n=4 in the hBMSC group), week 20 (n=4 in each group), week
35 (n=6 in the PBS group, n=4 in the hBMSC group) and week 45 (n=4
in each group) for measurement of morphological changes. In the
case where a mouse died prior to an upcoming sacrificed time, the
functional score 5 was given for that sacrifice time.
[0200] Normality of the functional recovery score was evaluated
using Generalized Estimating Equations (GEE) on the ranked data.
Without wishing to be bound by any particular theory, GEE was
chosen because it has fewer restrictions on the data distribution.
Analysis of variance for repeated measures including the
independent factor of the treatment and dependent factor of the
time was also employed. Analyses were performed on scores measured
from week 1 to week 45. The analysis began testing for the
treatment by time interaction, followed by testing the main effect
of hBMSC or time, if no interaction was observed at the 0.05 level.
A subgroup analysis of hBMSC effect at each time point was
conducted, if an interaction or time effect was detected. The hBMSC
by time interaction indicated that the effect of hBMSC on the
functional recovery depended on the time after EAE. Functional
outcome was reported as mean.+-.SD per time point for data
illustration.
[0201] Axonal loss in the white matter of the corpus callosum and
striatum in the EAE brain was assessed. The axonal density was
counted on an average of four brain sections (ranging from bregma
+1.18 mm to bregma -1.82 mm) per mouse (at 40.times.
magnification). Data were obtained using a 3-CCD color video camera
(Sony DXC-970 MD) and interfaced with ImageJ image processing
program (National Institute of Mental Health, Bethesda, Md.). The
axonal density was presented as a proportional area. To measure
immunoreactive cells, numbers of NGF.sup.+ and MAB1281.sup.+ cells
were counted on an average of 4 brain sections (ranging from bregma
+1.18 mm to bregma -1.82 mm) per mouse (at 40.times.
magnification), using a 3-CCD color video camera (Sony DXC-970 MD)
interfaced with the Micro Computer Imaging Device (MCID) analysis
system (Imaging Research Inc. St. Catharines, Ontario, Canada). The
density of immunoreactive cells was calculated by dividing the
number of counted cells by the scan area, presented as numbers per
mm.sup.2. Data was presented as mean.+-.SD. Significance between
the two groups was examined using a t-test. A value of p<0.05
was considered significant.
[0202] The results of the experiments presented in this Example are
now described.
hBMSC Treatment Improves Survival Rate and Neurological Functional
Recovery in EAE Mice
[0203] Without wishing to be bound by any particular theory, since
MS is a chronic disease course, the mortality and function of the
mice were measured up to 45 weeks after clinical onset. It was
observed that mice in the hBMSC treated group had significantly
higher survival rates as compared to the PBS treated group. A total
of 63 EAE mice were employed in the study. Survival rates for hBMSC
treated mice at weeks 10, 20, 35, and 45 were significantly higher
than those in the PBS group (p<0.01) (FIG. 12A).
[0204] Experiments were designed to evaluate whether the
administration of BMSCs on the day of clinical onset was an
effective treatment. It was observed that there were several
remitting-relapsing courses of disease within 45 weeks after
clinical symptom onset (FIG. 12B). The relationship between hBMSC
treatment and time was significant (p<0.05) and therefore,
pair-wise comparisons at each time point were conducted with the
mean and SD of the functional scores. Functional scores were
significantly lower among mice treated with hBMSCs compared with
PBS treated mice as early as 1 week up to 45 weeks. Before week 20,
functional scores were significantly lower in the hBMSC group
compared with the PBS group at 70% time points, after week 20,
there were significant difference between 2 groups at 20% time
points. The significance of hBMSCs effects were sustained to 45
weeks (p<0.05) (FIG. 12B).
hBMSC Treatment Increases Axonal Density in the White Matter of the
EAE Brain
[0205] Due to the effective neurological functional benefit of
hBMSC treatment, experiments were designed to address whether BMSC
treatment affects axonal loss. The proportional area of axonal loss
was significantly reduced in the striatum (FIGS. 13A and 13B) and
corpus callosum (FIGS. 13C and 13D) of the hBMSC treatment group
compared with that of the PBS treatment group at 20, 35 and 45
weeks after clinical onset. These data demonstrate that the long
term functional effect of BMSCs is associated with the reduction of
axonal loss in the EAE brain.
Administration of hBMSCs Increases NGF Expression in the CNS of EAE
Mice
[0206] Without wishing to be bound by any particular theory, it is
believed that BMSC treatment reduces axonal loss and improves
neurological outcome by augmenting expression of NGF in parenchymal
cells. The following experiments were designed to measure NGF cell
expression in the white matter of the striatum and corpus callosum.
It was observed that NGF was present in the normal brain tissue of
mice. After onset of EAE, cellular expression of NGF significantly
decreased in the brain during the acute and chronic phase of EAE.
hBMSC treatment significantly increased the number of NGF reactive
cells in the brain at 1, 10, 20, 35 and 45 weeks compared with the
PBS treatment (FIGS. 14A and 14B). Furthermore, double staining
shows that hBMSCs stimulated the brain parenchymal cells to express
NGF. Approximately, 50-70% of NGF.sup.+ cells co-localized with
NeuN.sup.+ cells (FIG. 14C).
hBMSCs are Present in the EAE Brain
[0207] MAB1281.sup.+ cells were present in the CNS from as early as
1 week up to 45 weeks following hBMSC transplantation. Most of the
cells were located in the striatum and the corpus callosum. The
number of MAB1281.sup.+ cells significantly increased at 10, 20, 35
and 45 weeks compared to the MAB1281.sup.+ cells at 1 week (FIG.
15). Double staining revealed that less than about 5% of
MAB1281.sup.+ cells co-localized with NG2.sup.+ cells and about 10%
of MAB1281.sup.+ cells co-localized with MAP-2.sup.+ cells and
GFAP.sup.+ cells, respectively, (FIG. 16). These data demonstrated
that the therapeutic effect of BMSCs on EAE was not a result of
cell replacement.
BMSCs for the Treatment of Degenerative Diseases of the CNS
[0208] The results presented herein demonstrate that
transplantation of hBMSCs at the day of clinical EAE symptom onset
improved survival rates and reduced disease severity, having a
statistical significance from 1 week up to 45 weeks after disease
compared with PBS treatment.
[0209] Since MS is an immune-mediated demyelinating and
degenerative disease of the CNS, with lesions predominantly
occurring in the CNS white matter, it is believed that the first
step in combating MS is to suppress the immune onslaught. However,
these strategies alone are insufficient for treating the chronic
progressive disability that is the ultimate outcome of the disease
(Chitnis et al., 2005, Curr. Drug Targets Immune Endocr. Metabol.
Disord. 5:11-26).
[0210] The data presented herein revealed that few MAB1281.sup.+
cells co-localized with neural cell markers. Although the injected
hBMSCs express brain cell phenotypic proteins, the data does not
indicate true differentiation and neuronal or glial cell function.
Thus, it is believed that the beneficial outcome from hBMSC
treatment is not a result of a cell replacement therapy. Rather, it
is believed that BMSCs secrete a series of growth factors and
induce expression of growth factors within the parenchymal
cells.
[0211] In the present study, it was observed that NGF expression
increased after hBMSC treatment. NGF stimulates axonal repair
(Walsh et al., 1999, J. Neuroscience 19:4155-4168; Jones et al.,
2003, J. Neurosci. 23:9276-9288) and induces axon growth (Zhou et
al., 2004, Neuron 42:897-912). Moreover, axon repair is present not
only in acute, but also in chronic CNS injury (Grill et al., 1997,
Exp. Neurol. 148:444-452). NGF also enhances the survival of
differentiated oligodendrocytes (Cohen et al., 1996, J. Neurosci.
16:6433-6442), and stimulates oligodendrocyte growth and/or
differentiation (Aloe et al., 1998, Arch. Ital. Biol. 136:247-256).
In addition to its neurotrophic effect, NGF exhibits
immunomodulatory effects (Aloe et al., 1997, Allergy 52:883-894;
Flugel et al., 2001, Eur. J. Immunol. 31:11-22; Fainzilber et al.,
2002, J. Neurosci. 3:1029-1034; Aloe et al., 2004, Ann. Ist Super
Sanita. 40:89-99), such as suppression of MHC II inducibility in
microglia and stimulation of memory B cells and Th2 responses
(Bracci-Laudiero et al., 2002, J. Neuroimmunol. 123:58-65; Bonini
et al., 2003, Int. Arch. Allergy Immunol. 131:80-84;
Bracci-Laudiero et al., 2005, Blood 106:3507-3514;
Stampachiacchiere et al., 2005, J. Neuroimmunol. 169:20-30). It has
also been found that administration of NGF dramatically reduced the
number and size of lesions produced in EAE, upregulated the
anti-inflammatory cytokine IL-10 in glial cells and suppressed
interferon-.gamma. expression by infiltrating T-cells (Villoslada
et al., 2002, J. Exp. Med. 191:1799-1806). Anti-NGF treatment of
rats resulted in more severe EAE pathology (Micera et al., 2000, J.
Neuroimmunol. 104:116-123). Without wishing to be bound by any
particulate theory, it is believed that stimulation of NGF in the
parenchymal cells by hBMSCs contributes to the reduction of axonal
injury and improvement of neurological outcome.
[0212] The results presented herein demonstrate that MAB1281+ cells
significantly increased at 10, 20, 35 and 45 weeks compared to the
MAB1281+ cells at 1 week after hBMSC transplantation. It is
believed that the increased number of hBMSCs present after
transplantation facilitates functional recovery. It was observed
that hBMSCs entered the CNS of EAE mice and contributed to the
decreased mortality and improved neurological functional recovery
from as early as 1 week up to 45 weeks in the mice. The
transplanted cells contributed to the reduced axonal loss in the
EAE brain, and stimulated brain parenchymal cells to express NGF,
which is believed to provide both neurotrophic and immunomodulatory
effects. The results presented herein demonstrate that BMSC
treatment is useful for therapy of autoimmune demyelinating
disorders.
[0213] The invention has been described in an illustrative manner,
and it is to be understood that the terminology which has been used
is intended to be in the nature of words of description rather than
of limitation.
[0214] Obviously, many modifications and variations of the present
invention are possible in light of the above teachings. It is,
therefore, to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described.
[0215] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
[0216] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
* * * * *