U.S. patent application number 11/368919 was filed with the patent office on 2006-09-28 for use of materials for treatment of central nervous system lesions.
Invention is credited to Mari Dezawa, Keita Mori.
Application Number | 20060216276 11/368919 |
Document ID | / |
Family ID | 36953931 |
Filed Date | 2006-09-28 |
United States Patent
Application |
20060216276 |
Kind Code |
A1 |
Dezawa; Mari ; et
al. |
September 28, 2006 |
Use of materials for treatment of central nervous system
lesions
Abstract
Disclosed are methods and materials for treatment of central
nervous system lesions. Preferred methods and materials comprise
neuronal precursor cells and/or marrow adherent stem cell-derived
neuronal cells.
Inventors: |
Dezawa; Mari; (Kyoto,
JP) ; Mori; Keita; (Mountain View, CA) |
Correspondence
Address: |
Innovation Legal Group
1165 Rosefield Way
Menlo Park
CA
94025
US
|
Family ID: |
36953931 |
Appl. No.: |
11/368919 |
Filed: |
March 6, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60659335 |
Mar 7, 2005 |
|
|
|
Current U.S.
Class: |
424/93.7 ;
435/368 |
Current CPC
Class: |
A61P 25/00 20180101;
A61K 35/30 20130101; A61P 9/10 20180101 |
Class at
Publication: |
424/093.7 ;
435/368 |
International
Class: |
A61K 35/30 20060101
A61K035/30; C12N 5/08 20060101 C12N005/08 |
Claims
1. A method comprising: providing neuronal precursor cells; and
administering the neuronal precursor cells to a patient suffering
from a central nervous system lesion in an amount sufficient to
facilitate functional recovery of the patient.
2. The method of claim 1, wherein the neuronal precursor cells are
administered locally.
3. The method of claim 2, wherein the neuronal precursor cells are
administered to a central nervous system lesion of the patient.
4. The method of claim 1, wherein the neuronal precursor cells are
administered intraparenchymally.
5. The method of claim 1, wherein the neuronal precursor cells
comprise human neuronal precursor cells.
6. The method of claim 1, wherein providing neuronal precursor
cells comprises: providing marrow adherent stem cells;
transdifferentiating the marrow adherent stem cells into neuronal
precursor cells.
7. The method of claim 1, wherein the central nervous system lesion
is caused by ischemic stroke or hemorrhagic stroke.
8. The method of claim 1, further comprising allowing the neuronal
precursor cells to migrate from a site of administration to other
locations in the patient.
9. The method of claim 8, wherein another location in the patient
comprises a central nervous system lesion.
10. The method of claim 1, further comprising administering an
immunosuppressive agent to the patient.
11. The method of claim 1, wherein the functional recovery is a
partial functional recovery.
12. The method of claim 1, wherein the neuronal precursor cells are
allogeneic with respect to the patient.
13. A graft forming unit comprising: neuronal precursor cells
present in an amount sufficient to facilitate functional recovery
of a patient suffering from a central nervous system lesion
following administration of the neuronal precursor cells to the
patient; and a pharmaceutically acceptable carrier.
14. The graft forming unit of claim 13, wherein the
pharmaceutically acceptable carrier comprises a solvent, a
dispersion media, an antibacterial agents, or an antifungal
agent.
15. The graft forming unit of claim 13, wherein the graft comprises
neuronal precursor cells in an amount ranging from about 10,000 to
about 100 million neuronal precursor cells.
16. The graft forming unit of claim 13, wherein the neuronal
precursor cells comprise a label.
17. The graft forming unit of claim 13, wherein the neuronal
precursor cells comprise human neuronal precursor cells.
18. A method comprising: providing marrow-adherent stem
cell-derived neuronal cells; and administering the marrow-adherent
stem cell-derived neuronal cells to a patient suffering from a
central nervous system lesion in an amount sufficient to facilitate
functional recovery of the patient.
19. The method of claim 18, wherein the marrow-adherent stem
cell-derived neuronal cells are administered locally.
20. The method of claim 19, wherein the marrow-adherent stem
cell-derived neuronal cells are administered to a central nervous
system lesion of the patient.
21. The method of claim 18, wherein the marrow-adherent stem
cell-derived neuronal cells are administered
intraparenchymally.
22. The method of claim 18, wherein the marrow-adherent stem
cell-derived neuronal cells comprise human marrow-adherent stem
cell-derived neuronal cells.
23. The method of claim 18, wherein providing marrow-adherent stem
cell-derived neuronal cells comprises: providing marrow adherent
stem cells; and inducing the marrow adherent stem cells to form
marrow-adherent stem cell-derived neuronal cells.
24. The method of claim 23, further comprising:
transdifferentiating the marrow adherent stem cells into neuronal
precursor cells; and inducing the neuronal precursor cells to form
neuronal cells.
25. The method of claim 18, wherein the central nervous system
lesion is caused by ischemic stroke or hemorrhagic stroke.
26. The method of claim 18, further comprising administering an
immunosuppressive agent to the patient.
27. The method of claim 18, wherein the functional recovery is a
partial functional recovery.
28. The method of claim 18, wherein the marrow-adherent stem
cell-derived neuronal cells are allogeneic with respect to the
patient.
29. A graft forming unit comprising: marrow-adherent stem
cell-derived neuronal cells present in an amount sufficient to
facilitate functional recovery of a patient suffering from a
central nervous system lesion following administration of the
marrow-adherent stem cell-derived neuronal cells to the patient;
and a pharmaceutically acceptable carrier.
30. The graft forming unit of claim 29, wherein the
pharmaceutically acceptable carrier comprises a solvent, a
dispersion media, an antibacterial agents, or an antifungal
agent.
31. The graft forming unit of claim 29, wherein the graft comprises
neuronal precursor cells in an amount ranging from about 10,000 to
about 100 million neuronal precursor cells.
32. The graft forming unit of claim 29, wherein the marrow-adherent
stem cell-derived neuronal cells comprise a label.
33. The graft forming unit of claim 29, wherein the marrow-adherent
stem cell-derived neuronal cells comprise human marrow-adherent
stem cell-derived neuronal cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/659,335, filed Mar. 7, 2005.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to treatment of central nervous system
lesions, particularly to treatment of stroke.
[0004] 2. Description of the Related Art
[0005] Lesions can form in central nervous system ("CNS") tissue
for a number of reasons. One of the leading causes of CNS lesions
is stroke. Stroke is characterized by the sudden loss of
circulation to an area of the brain, resulting in a corresponding
loss of neurologic function. Also called cerebrovascular accident
or stroke syndrome, stroke is a nonspecific term encompassing a
heterogeneous group of pathophysiologic causes, including
thrombosis, embolism, and hemorrhage. Recent reports indicate an
incidence exceeding 500,000 new strokes of all types per year.
Stroke is a leading killer and disabler. Combining all types of
stroke, it is the third leading cause of death and the first
leading cause of disability. At current trends, this number is
projected to jump to one million per year by the year 2050. When
the direct costs (care and treatment) and the indirect costs (lost
productivity) of strokes are considered together, strokes cost US
society $43.3 billion per year. Strokes currently are classified as
either hemorrhagic or ischemic. Acute ischemic stroke refers to
strokes caused by thrombosis or embolism and accounts for 80% of
all strokes.
[0006] The four major neuroanatomic ischemic stroke syndromes are
caused by disruption of their respective cerebrovascular
distributions.
[0007] Anterior cerebral artery occlusions primarily affect frontal
lobe function, producing altered mental status, impaired judgment,
contralateral lower extremity weakness and hypesthesia, and gait
apraxia.
[0008] Middle cerebral artery (MCA) occlusions commonly produce
contralateral hemiparesis, contralateral hypesthesia, ipsilateral
hemianopsia (blindness in one half of the visual field), and gaze
preference toward the side of the lesion. Agnosia is common, and
receptive or expressive aphasia may result if the lesion occurs in
the dominant hemisphere. Since the MCA supplies the upper extremity
motor strip, weakness of the arm and face is usually worse than
that of the lower limb.
[0009] Posterior cerebral artery occlusions affect vision and
thought, producing homonymous hemianopsia, cortical blindness,
visual agnosia, altered mental status, and impaired memory.
[0010] Vertebrobasilar artery occlusions are notoriously difficult
to detect because they cause a wide variety of cranial nerve,
cerebellar, and brainstem deficits. These include vertigo,
nystagmus, diplopia, visual field deficits, dysphagia, dysarthria,
facial hypesthesia, syncope, and ataxia. Loss of pain and
temperature sensation occurs on the ipsilateral face and
contralateral body. In contrast, anterior strokes produce findings
on one side of the body only.
[0011] These occlusions may occur for a variety of reasons. Emboli
may arise from the heart, the extracranial arteries or, rarely, the
right-sided circulation (paradoxical emboli). The sources of
cardiogenic emboli include valvular thrombi (e.g., in mitral
stenosis, endocarditis, prosthetic valves); mural thrombi (e.g., in
myocardial infarction [MI], atrial fibrillation, dilated
cardiomyopathy); and atrial myxomas. MI is associated with a 2-3%
incidence of embolic stroke, of which 85% occur in the first month
after MI.
[0012] Lacunar infarcts account for 13-20% of all cerebral
infarctions and usually involve the small terminal vasculature of
the subcortical cerebrum and brainstem. Lacunar infarcts commonly
occur in patients with small vessel disease, such as diabetes and
hypertension. Small emboli or an in situ process called
lipohyalinosis is thought to cause lacunar infarcts. The most
common lacunar syndromes include pure motor, pure sensory, and
ataxic hemiparetic strokes. By virtue of their small size and
well-defined subcortical location, lacunar infarcts do not lead to
impairments in cognition, memory, speech, or level of
consciousness.
[0013] The most common sites of thrombotic occlusion are cerebral
artery branch points, especially in the distribution of the
internal carotid artery. Arterial stenosis (i.e., turbulent blood
flow), atherosclerosis (i.e., ulcerated plaques), and platelet
adherence cause the formation of blood clots that either embolize
or occlude the artery. Less common causes of thrombosis include
polycythemia, sickle cell anemia, protein C deficiency,
fibromuscular dysplasia of the cerebral arteries, and prolonged
vasoconstriction from migraine headache disorders. Any process that
causes dissection of the cerebral arteries also can cause
thrombotic stroke (e.g., trauma, thoracic aortic dissection,
arteritis). Occasionally, hypoperfusion distal to a stenotic or
occluded artery or hypoperfusion of a vulnerable watershed region
between two cerebral arterial territories can cause ischemic
stroke.
[0014] Turning to hemorrhagic stroke, the terms intracerebral
hemorrhage (ICH) and hemorrhagic stroke are used interchangeably in
this discussion and are regarded as a separate entity from
hemorrhagic transformation of ischemic stroke. ICH accounts for
approximately 20% of all strokes and is associated with higher
mortality rates than cerebral infarctions. Patients with
hemorrhagic stroke present with similar focal neurologic deficits
but tend to be more ill than patients with ischemic stroke.
Patients with intracerebral bleeds are more likely to have
headache, altered mental status, seizures, nausea and vomiting,
and/or marked hypertension; however, none of these findings
distinguish reliably between hemorrhagic and ischemic strokes.
[0015] In ICH, bleeding occurs directly into the brain parenchyma.
The usual mechanism is thought to be leakage from small
intracerebral arteries damaged by chronic hypertension. Other
mechanisms include bleeding diatheses, iatrogenic anticoagulation,
cerebral amyloidosis, and cocaine abuse. ICH tends to be found in
certain sites in the brain, including the thalamus, putamen,
cerebellum, and brain stem. In addition to the area of the brain
injured by the hemorrhage, the surrounding brain can be damaged by
pressure produced by the mass effect of the hematoma. A general
increase in intracranial pressure may occur. The 30-day mortality
rate for hemorrhagic stroke is 40-80%. Approximately 50% of all
deaths occur within the first 48 hours.
[0016] Other causes for CNS lesions are conventionally known,
including trauma and various diseases of the CNS.
[0017] Treating CNS lesions implicates neurogenesis, i.e. the
(re)generation of neurons in a region of a patient's tissue that is
of interest, including but not limited to replacement of damaged
neurons in a central nervous system lesion. Unfortunately, neuronal
(CNS) tissue is well-known for its limited reparative/regenerative
capacity. The generation of new neurons in the adult is largely
restricted to two regions, the SVZ lining the lateral ventricles,
and the subgranular zone of the dentate gyrus. Limited neuronal
replacement has been demonstrated resulting from endogenous
precursor stem cells that had migrated from the SVZ.
[0018] Some initial success has been reported with certain
neurogenesis methods but these methods have not been clinically
successful. Accordingly, what is needed are methods and
compositions that overcome problems noted in the art for treatment
of central nervous system lesions.
SUMMARY OF THE INVENTION
[0019] In an aspect, the invention relates to a method comprising:
providing neuronal precursor cells; and administering the neuronal
precursor cells to a patient suffering from a central nervous
system lesion in an amount sufficient to facilitate functional
recovery of the patient.
[0020] In another aspect, the invention relates to a graft forming
unit comprising: neuronal precursor cells present in an amount
sufficient to facilitate functional recovery of a patient suffering
from a central nervous system lesion following administration of
the neuronal precursor cells to the patient; and a pharmaceutically
acceptable carrier.
[0021] In still another aspect, the invention relates to a method
comprising: providing marrow-adherent stem cell-derived neuronal
cells; and administering the marrow-adherent stem cell-derived
neuronal cells to a patient suffering from a central nervous system
lesion in an amount sufficient to facilitate functional recovery of
the patient.
[0022] In yet another aspect, the invention relates to a graft
forming unit comprising: marrow-adherent stem cell-derived neuronal
cells present in an amount sufficient to facilitate functional
recovery of a patient suffering from a central nervous system
lesion following administration of the marrow-adherent stem
cell-derived neuronal cells to the patient; and a pharmaceutically
acceptable carrier.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1 shows results from the MCAo procedure.
[0024] FIG. 2 shows results from the MCAo procedure.
[0025] FIG. 3 shows results from the MCAo procedure.
[0026] FIG. 4 shows results from the MCAo procedure.
[0027] FIG. 5 shows results from the MCAo procedure.
[0028] FIG. 6 shows results from the MCAo procedure.
[0029] FIG. 7 shows results from the MCAo procedure.
[0030] FIG. 8 shows results from the MCAo procedure.
[0031] FIG. 9 shows results from the MCAo procedure.
[0032] FIG. 10 shows results from the MCAo procedure.
[0033] FIG. 11 shows results from the MCAI procedure.
[0034] FIG. 12 shows results from the MCAI procedure.
[0035] FIG. 13 shows results from the MCAI procedure.
[0036] FIG. 14 shows results from the MCAI procedure.
[0037] FIG. 15 shows results from the MCAI procedure.
[0038] FIG. 16 shows results from the MCAI procedure.
[0039] FIG. 17 shows results from the MCAI procedure.
[0040] FIG. 18 shows results from the MCAI procedure.
[0041] FIG. 19 shows results from the MCAI procedure.
[0042] FIG. 20 shows results from the MCAI procedure.
[0043] FIG. 21 shows results from the TGI procedure.
[0044] FIG. 22 shows results from the TGI procedure.
[0045] FIG. 23 shows results from the TGI procedure.
[0046] FIG. 24 shows results from the TGI procedure.
[0047] FIG. 25 shows results from the TGI procedure.
[0048] FIG. 26 shows results from the TGI procedure.
[0049] FIG. 27 shows results from the TGI procedure.
[0050] FIG. 28 shows results from the TGI procedure.
[0051] FIG. 29 shows histological results from the Examples.
[0052] FIG. 30 shows histological results from the Examples.
[0053] FIG. 31 illustrates functional recovery and graft
survival.
[0054] FIG. 32 shows results from the MCAo procedure.
[0055] FIG. 33 shows results from the MCAo procedure.
[0056] FIG. 34 shows results from the MCAI procedure.
[0057] FIG. 35 shows results from the MCAI procedure.
[0058] FIG. 36 shows results from the TGI procedure.
[0059] FIG. 37 shows results from the TGI procedure.
[0060] FIG. 38 illustrates graft survival.
[0061] FIG. 39 illustrates graft survival.
[0062] FIG. 40 shows the results of a beam balance test. On day 28
after transplantation, the mean score for the MNC group showed a
significant improvement, compared with the MASC and control groups.
*: p<0.05, **: p<0.01
[0063] FIG. 41 shows the results of a limb placing test. The mean
scores for the NMC group and the MASC group were significantly
different to that of the control group on day 21 and day 28. There
was no significant difference between the MNC and MASC groups. *:
p<0.05, **: p<0.01
[0064] FIG. 42 shows the results of a Morris water maze test. For
the final set, the mean latency time for the MNC group was
significantly different to those for the MASC and control
groups.
[0065] FIG. 43 shows the results of a water maze "spatial probe
trial". Among the three groups, the best results were obtained for
the MNC group, and statistical differences were obtained between
the MNC group and the other groups. *: p<0.05, **: p<0.01
[0066] FIG. 44 shows results from Bederson testing performed in
Example 9.
[0067] FIG. 45 shows results from EBST performed in Example 9.
[0068] FIG. 46 illustrates graft survival according to Example
9.
[0069] FIG. 47 shows graft survival according to Example 9.
[0070] FIG. 48 shows the results of Nissl staining according to
Example 9.
DETAILED DESCRIPTION OF THE INVENTION
A. INTRODUCTION
[0071] The inventors have unexpectedly and surprisingly discovered
that the problems and limitations noted above can be overcome by
practicing the invention disclosed herein. In particular, the
inventors have unexpectedly discovered that it is possible to
provide NPCs and/or MNCs and administer those NPCs and/or MNCs to a
patient suffering from a central nervous system lesion in an amount
sufficient to facilitate functional recovery of the patient.
[0072] The approach disclosed herein has several advantages over
the prior art. First, it provides for a dose-response relationship
that can allow a physician to tailor the surgical procedure to
repair the central nervous system lesion on a patient by patient
basis. Second, it provides for an allogeneic approach to
engraftment. This is useful for characterizing the NPCs and/or MNCs
and/or graft forming units and providing GFU to GFU (or NPC to NPC,
or MNC to MNC) consistency, both in terms of the cell batches and
transplantation procedure. Further, use of NPCs allows for more
precise reconstruction of the central nervous system, as compared
to use of other multi-potent cells. This is because a significant
majority of neuronal precursor cells, more than other types of
multi-potent cells, will adopt a cell fate of neuronal cells when
differentiating, rather than differentiating into other cell types.
This can be important when trying to provide control over
transplantation outcome and limits the possibilities of undesirable
(or undifferentiated) growth of transplanted cells. Additionally,
use of MNCs is desirable because the cells are more differentiated
multi-potent cells which may provide for improved functional
recovery.
[0073] The present invention will now be described in more
detail.
DEFINITIONS
[0074] All publications cited in this specification are hereby
incorporated by reference for all purposes and in their entirety as
if each individual publication were specifically and individually
indicated to be incorporated by reference.
[0075] "Administering" means providing NPCs and/or inventive grafts
to a patient.
[0076] "Area" means a region or defined volume. For instance, an
area of the central nervous system would be a region or defined
volume located in the central nervous system.
[0077] "Central nervous system ischemic event" or "CNS ischemic
event" means any occurrence that results in a lack or
physiologically significant reduction of blood flow to an area of
the central nervous system of a patient. In a preferred embodiment,
a CNS ischemic event comprises an ischemic stroke.
[0078] "Central nervous system lesion" or "CNS lesion" means an
area of damaged, malfunctioning, or diseased neuronal central
nervous system tissue, or a penumbra surrounding such damaged,
malfunctioning, or diseased neuronal central nervous system tissue,
damaged by a CNS ischemic event or by a hemorrhage (e.g., in a
preferred embodiment, hemorrhagic stroke).
[0079] "Central nervous system tissue" means a tissue
conventionally associated with the central nervous system. Brain
tissue and spinal cord tissue are non-limiting examples of central
nervous system tissue. Certain embodiments of the present invention
concern central nervous system tissue, wherein the central nervous
system tissue has been damaged by an ischemic event. Such damage
may occur as conventionally understood, through oxygen deprivation,
and other associated cascades and by-products of such deprivation
and associated cascades.
[0080] "Functional recovery" means the recovery of CNS function
with respect to a CNS lesion as determined either by measurement of
neurobiological parameters characteristic of that function (i.e.
CBF, EEG, cortical expansion, etc.), or by measurement of
behavioral function (e.g. rearing or auditory startle in murine
models, or other models disclosed herein or known in the art).
Recovery is determined by the tendency of the measured variable to
approximate the values observed in a normal or control population.
Functional recovery can be complete, i.e. the recovery returns the
value of the measured parameter to the value observed in the normal
or control population, as determined by appropriate statistical
methodology. Functional recovery can also be incomplete or partial.
For instance, a patient can experience complete functional recovery
of a measured parameter, or 75% recovery, or 50% recovery, etc.
[0081] "Functionally recovered area of the central nervous system"
means to CNS tissue formerly involved in a lesion and subsequently
functionally recovered through the practice of the present
invention.
[0082] "Graft Forming Unit" or "GFU" means a composition that (1)
comprises NPCs and/or MNCs together with a pharmaceutically
acceptable carrier, (2) that is intended for administration to a
patient. In a preferred embodiment, mixtures of NPCs and MNCs are
expressed contemplated. In other preferred embodiments NPCs are
present substantially without MNCs. In still other preferred
embodiments MNCs are present substantially without NPCs.
[0083] "Marrow adherent stem cells" means a type of mitotic
multi-potent cell that gives rise to a variety of cell types: bone
cells (osteocytes), cartilage cells (chondrocytes), fat cells
(adipocytes), and other kinds of connective tissue cells such as
those in tendons.
[0084] "MASC-derived Neuronal Cells (MNCs)" means post-mitotic
neurons that (1) are derived from marrow adherent stem cells, and
(2) that express neuron markers immunohistochemically and exhibit
neuron properties in electrophysiological analysis. Suitable
methods of generating MNCs in vitro may be found in PCT/JP03/01260.
MNCs produced using other techniques known in the art may also be
used in the practice of this invention, so long as they meet the
definition of MNCs set forth herein. In an embodiment, human MNCs
are MAP-2+, neurofilament-M+, and beta tubulin III+ (i.e. TuJ-1+).
These markers may be used to isolate MNCs using FACS following
production of MNCs using the techniques disclosed in
PCT/JP03/01260. Suitable methods of handling MNCs are known
conventionally, including those methods disclosed, for example, in
U.S. Pat. No. 6,833,269 to Carpenter.
[0085] "MCAo" means middle cerebral artery occlusion.
[0086] "MCAI" means middle cerebral artery ligation.
[0087] "Neurogenesis" means the (re)generation of neurons and
neuronal tissue in a region of a patient's tissue that is of
interest, including but not limited to replacement of damaged
neurons in a central nervous system lesion.
[0088] "Neuronal Precursor Cells (NPCs)" means cells that are
mitotic, express nestin and other cell markers specific for neural
precursor/neural progenitor cells, and are derived from MASCs. NPCs
can differentiate into neurons, glia, and oligodendrocytes, and
precursors of any of the foregoing. In an embodiment, NPCs can be
produced from marrow-adherent stem cells (MASCs) according to
methods disclosed in PCT/JP03/01260. NPCs produced using other
techniques known in the art may also be used in the practice of
this invention, so long as they meet the definition of NPCs set
forth herein. Preferably, NPCs comprise human NPCs, although NPCs
of other mammalian species are also encompassed within the scope of
this invention. In an embodiment, NPCs, preferably human NPCs are
CD29+, CD90+, CD105+, CD31-, CD34- and CD45-. These markers may be
used to isolate NPCs, preferably human NPCs, using FACS following
production of NPCs using the techniques disclosed in
PCT/JP03/01260. Suitable methods of handling NPCs are known
conventionally, including those methods disclosed, for example, in
published United States patent application 20020012903 to Goldman
et al.
[0089] "Neuron(s)" means any of the impulse-conducting cells that
constitute the brain, spinal column, and nerves, consisting of a
nucleated cell body with one or more dendrites and a single axon.
Biochemically, neurons are characterized by reaction with
antibodies for Map, neurofilament-M, and beta-tubulin III (i.e.
TuJ-1). Neural cells are also characterized by the presence of
neurotransmitter synthetases or neurotransmitter-related proteins
and by the secretion of neurotransmitters, for example neuropeptide
Y and substance P.
[0090] "Neuronal" means neurons, glia, and oligodendrocytes, and
precursors of any of the foregoing.
[0091] "Patient" means an animal, typically a mammal, and more
typically, a human, in need of treatment for a disease or
disorder.
[0092] "Pharmaceutically acceptable carrier" means any and all
solvents, dispersion media, coatings, antibacterial agents,
antifungal agents, cryoprotectants isotonic and absorption delaying
agents, and the like, that are compatible with pharmaceutical
administration of NPCs or of MNCs. The use of such media and agents
is well known in the art. Except insofar as any conventional media
or agent is incompatible with NPCs or with MNCs, use thereof in the
inventive GFUs is contemplated.
[0093] "Systemically" means throughout, or throughout substantial
portions of, the patient.
[0094] "Tissue" means a part of an organism consisting of an
aggregate of cells having a similar structure and function. A
preferred tissue, according to the invention, is nerve tissue.
[0095] "TGI" means transient global ischemia.
[0096] "Transplantation", which is used synonymously with
"engraftment," means the placement of non-endogenous cells in an
area of a patient. Transplantation may be allogeneic, or non-self
cells being transplanted. Transplantation may also be autologous,
or self cells being transplanted, e.g. from one tissue to another
in the same patient.
[0097] "Transdifferentiated" means development of a cell along a
lineage different from that classically associated with that cell
type.
B. NPCs, AND PHARMACEUTICAL COMPOSITIONS THEREOF
[0098] In an embodiment, NPCs are used in the practice of this
invention as part of GFUs that are transplanted into patients. The
intent is that the NPCs grow and differentiate into neuronal cells
that play a role in the functional recovery of a central nervous
system lesion in the patient. For example, NPCs could differentiate
into neurons that replace damaged endogenous neurons.
Alternatively, NPCs could differentiate into glial cells or neurons
that secrete growth factors. These growth factors may have a
trophic activity on damaged neurons and aid their functional
recovery. In that manner, treatment of central nervous system
lesions is possible.
[0099] Preferred NPCs and preferred methods of providing such NPCs
are disclosed in PCT/JP03/01260, to Dezawa et al., entitled Method
of Differentiating/Inducing Bone Marrow Interstitial Cells Into
Nerve Cells and Skeleton Muscle Cells by Transferring Notch Gene
("Dezawa"). In particular, the "neural precursor cells" of Dezawa,
as described throughout Dezawa and in particular in Example 7, may
be used as the NPCs of the present invention. Dezawa discloses that
MASCs may be transdifferentiated into neural precursor cells that
are then useful as the NPCs of the present invention.
[0100] In embodiments, GFUs may be useful in the practice of this
invention. Pharmaceutically acceptable carriers useful in GFUs of
the present invention can include: sterile isotonic buffers, FRS,
isolyte, sterile diluents such as water, normal saline, fixed oils,
polyethylene glycols, glycerine, propylene glycol, or other
synthetic solvents; antibacterial or antifungal agents such as
ascorbic acid, thimerosal, trimethoprim-sulfamethoxazole, nalidixic
acid, methenamine hippurate or nitrofurantoin macrocrystals and the
like; antioxidants such as ascorbic acid or sodium bisulfite;
chelating agents such as EDTA; buffers such as acetates, citrates,
or phosphates; and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide.
[0101] In an embodiment, graft forming units suitable for use in
the present invention comprise sterile compositions that comprise
the NPCs. For intravenous administration, suitable pharmaceutically
acceptable carriers may include physiological saline, normasol,
isolyte, plasma-lyte, or phosphate buffered saline (PBS). In all
cases, the GFU must be sterile (other than any NPCs or MNCs that
are present) and should be fluid to the extent that easy
syringability exists (proper fluidity can be maintained, for
example, by using materials such as lecithin, by maintaining a
certain particle size in the case of dispersion, and by including
surfactants). The GFU must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi,
as described above. In certain cases, it will be preferable to
include, for example, sugars, polyalcohols such as mannitol or
sorbitol, sodium chloride, LiCl, Na butyrate, and sodium
orthovanadate in the GFU. Generally, the inventive GFUs may be
prepared by incorporating the NPCs into a sterile vehicle which
contains a basic dispersion medium and optionally other ingredients
from those enumerated above.
[0102] It is especially advantageous to formulate the GFUs of the
invention in graft forming unit dosage forms for ease of
administration and uniformity of dosage. Graft forming unit dosage
form as used herein refers to physically discrete units suited as
unitary dosages for the subject to be treated. In an embodiment
each GFU dosage form contains a predetermined quantity of NPCs
calculated to produce the desired therapeutic effect in association
with the required pharmaceutical carrier. The specification for the
graft forming unit dosage forms of the invention are dictated by
and directly dependent on the unique characteristics of the NPCs,
the particular therapeutic effect to be achieved, and the
limitations inherent in the art of compounding NPCs for the
treatment of individuals. The number of NPCs in each graft unit
dosage form preferably can vary from about 1000 cells to about 1
billion cells, preferably from about 10,000 cells to about 100
million cells, more preferably from about 50,000 cells to about 50
million cells. The concentration of NPCs in each graft unit dosage
form preferably can vary from about 100 cells/.mu.L to about
100,000 cells/.mu.L, and more preferably from about 1,000
cells/.mu.L to about 50,000 cells/.mu.L.
[0103] The GFUs can be included in a container, pack, or dispenser
together with instructions for administration. The grafts are
preferably stored at approximately 37.degree. C.
[0104] In certain embodiments it may be desirable to label the NPCs
prior to transplantation. This may be desirable in pre-clinical
(i.e. non-human) models in order to track the migration of
transplanted NPCs, differentiation of transplanted NPCs, survival
of transplanted NPCs, and so on. Various cell labeling methods may
be employed depending on the pre-clinical circumstances under which
the labels are to be read. For instance, fluorescent proteins
(green fluorescent protein, red fluorescent protein, etc.) may be
used as in circumstances in which a detector can be suitably placed
near the transplant site.
[0105] When analyzing engrafted brains in non-clinical situations,
immunohistochemical analysis may be useful. In an embodiment, brain
sections may be doubly-immunostained for green fluorescent protein
(GFP) or other cell labels, .beta.-tubulin III, NeuN (a
neuron-specific protein), glial fibrillary acidic protein (GFAP),
or O4 (an oligodendrocyte-specific protein) to identify neuronal,
astrocytic, glial, or oligodendrocytic profiles. The number of
positive profiles for a given antibody and the number of cells
expressing GFP may be estimated according to the Abercrombie
correction formula. The volume of distribution and total GFP (or
other label) positive profiles may be calculated by determining the
area of the brain containing at least 10% GFP-positive (or other
label-positive) profiles in every fifth section and multiplying by
the distance from the anterior aspects of the brain that contain
GFP-positive (or other label-positive) profiles.
[0106] In an embodiment, when labeling NPCs using GFP, the
following materials may be useful:
[0107] Materials: Cryo-preserved NPCs, PBS (Invitrogen 14190-136),
HTS-FRS (BioLife Solutions 99-609-DV), GFP-Lentivirus stock
suspension with a titer of approximately 10.sup.7/ml, Hexamidine
Bromide (polybrene) (Sigma (H-9268)-1 or 2 frozen aliquots @ 10
mg/ml), Sterile Water, USP, Opti-MEM (Invitrogen), and Fetal Bovine
Serum (Hyclone).
[0108] A GFP-Lentivirus stock suspension may be obtained
commercially, or made using a commercially available kits such as
the ViraPower Lentiviral Expression System (available from
Invitrogen, Carlsbad Calif.). In particular, the pLenti6/V5 Gateway
Vector may be combined with a GFP cassette, according to the
manufacturer's directions, to eventually produce suitable
GFP-lentivirus suspensions.
[0109] In an embodiment, labeling may be performed according to the
transfection protocols available from the manufacturer, such as the
Invitrogen system referred to above.
[0110] In another embodiment, when labeling NPCs using GFP, the
following methods may be useful: Cell handling procedures, except
the centrifugations steps, preferably are performed in a Biohazard
Safety Cabinet Level-2. A polybrene stock solution may be prepared
by dissolving 10 mg of polybrene in 1 ml of Sterile Water, USP, and
filtering through a 0.25 micron filter. The resultant, filtered
stock solution can be divided into aliquots and stored protected
from light at -20.degree. C.
[0111] The day before viral infection, plate NPCs in a T225 flask
containing 30 ml of the cell culture medium at a density of 2
million cells per flask; and culture cells in a 37.degree. C./5%
CO.sub.2 incubator overnight
[0112] On the day of viral infection, thaw the lentivirus stock at
RT and the polybrene stock solution in a 37 Deg C. water bath. In a
50 ml falcon tube, add 45 ml of pre-warmed (37.degree. C.) 10% FBS
in alpha MEM and 5 ml of the thawed viral stock to obtain a medium
with a MOI around 10. Add the thawed polybrene at a final
concentration of 10 ug/ml (1,000.times. dilution). Remove the old
medium from the T225 flask, and add the viral mixture into the
flask and rock gently back and forth 3-4 times. Return the flask
into the 37.degree. C./5% CO.sub.2 incubator.
[0113] On the following day, remove completely the viral medium
from the flask. Wash 6.times.30 ml with 10% FBS in alpha MEM.
Collect 5 ml from each wash for infectivity testing. Replace with
fresh culture medium, and put the flask back into the
incubator.
[0114] Next day, harvest viral infected cells, count and re-suspend
them to a total volume of 360 ul in HTS-FRS and transferred to a
1.5 ml sterile, DNAse-free, RNAse-free, pyrogen-free microfuge
tube. The infected cell concentration may be set to match an
appropriate transplantation volume. The cells may then be held on
wet ice until use for graft administration.
MNCs, and Pharmaceutical Compositions Thereof
[0115] In an embodiment, MNCs are used in the practice of this
invention as part of grafts that are transplanted into patients.
The intent is that the MNCs play a role in the functional recovery
of a region of a patient's tissue that is of interest. In that
manner, treatment of central nervous system lesions is
possible.
[0116] Preferred MNCs and preferred methods of providing such MNCs
are disclosed in PCT/JP03/01260, to Dezawa et al., entitled Method
of Differentiating/Inducing Bone Marrow Interstitial Cells Into
Nerve Cells and Skeleton Muscle Cells by Transferring Notch Gene
("Dezawa"). In particular, the "neural cells" of Dezawa, as
described throughout Dezawa and in particular in Example 1, may be
used as the MNCs of the present invention. Dezawa discloses that
marrow-adherent stem cells may be transdifferentiated into neuronal
cells that are then useful as the MNCs of the present
invention.
[0117] In a preferred embodiment, MNCs may be produced from NPCs
using neurotrophic agents. Useful neurotrophic agents include but
are not limited to basic-fibroblast growth factor (bFGF), and
ciliary neurotrophic factor (CNTF). Suitable methods of using
neurotrophic agents with NPCs in vitro may be found in
PCT/JP03/01260.
[0118] In embodiments, GFUs may be useful in the practice of this
invention. Pharmaceutically acceptable carriers useful in GFUs of
the present invention can include: sterile isotonic buffers, FRS,
isolyte, sterile diluents such as water, normal saline, fixed oils,
polyethylene glycols, glycerine, propylene glycol, or other
synthetic solvents; antibacterial or antifungal agents such as
ascorbic acid, thimerosal, trimethoprim-sulfamethoxazole, nalidixic
acid, methenamine hippurate or nitrofurantoin macrocrystals and the
like; antioxidants such as ascorbic acid or sodium bisulfite;
chelating agents such as EDTA; buffers such as acetates, citrates,
or phosphates; and agents for the adjustment of tonicity such as
sodium chloride or dextrose. PH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide.
[0119] In an embodiment, graft forming units suitable for use in
the present invention comprise sterile compositions that comprise
MNCs. For intravenous administration, suitable pharmaceutically
acceptable carriers may include physiological saline, Cremophor
EL..TM.. (BASF; Parsippany, N.J.), normasol, isolyte, plasma-lyte,
or phosphate buffered saline (PBS). In all cases, the GFU must be
sterile (other than any NPCs or MNCs that are present) and should
be fluid to the extent that easy syringability exists (proper
fluidity can be maintained, for example, by using materials such as
lecithin, by maintaining a certain particle size in the case of
dispersion, and by including surfactants). The GFU must be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi, as described above. In certain cases, it
will be preferable to include, for example, sugars, polyalcohols
such as mannitol or sorbitol, sodium chloride, LiCl, Na butyrate,
and sodium orthovanadate in the GFU. Generally, the inventive GFUs
may be prepared by incorporating the NPCs into a sterile vehicle
which contains a basic dispersion medium and optionally other
ingredients from those enumerated above.
[0120] It is especially advantageous to formulate the GFUs of the
invention in graft forming unit dosage forms for ease of
administration and uniformity of dosage. Graft forming unit dosage
form as used herein refers to physically discrete units suited as
unitary dosages for the subject to be treated. In an embodiment
each GFU dosage form contains a predetermined quantity of MNCs
calculated to produce the desired therapeutic effect in association
with the required pharmaceutical carrier. The specification for the
graft forming unit dosage forms of the invention are dictated by
and directly dependent on the unique characteristics of the MNCs,
the particular therapeutic effect to be achieved, and the
limitations inherent in the art of compounding MNCs for the
treatment of individuals. The number of MNPs in each graft unit
dosage form preferably can vary from about 1000 cells to about 1
billion cells, preferably from about 10,000 cells to about 100
million cells, more preferably from about 50,000 cells to about 50
million cells. The concentration of MNCs in each graft unit dosage
form preferably can vary from about 100 cells/.mu.L to about
100,000 cells/.mu.L, and more preferably from about 1,000
cells/.mu.L to about 50,000 cells/.mu.L.
[0121] The GFUs can be included in a container, pack, or dispenser
together with instructions for administration. The grafts are
preferably stored at approximately 4.degree. C.
[0122] In certain non-clinical embodiments it may be desirable to
label the MNCs prior to transplantation. This may be desirable in
order to track the migration of transplanted MNCs, further changes
to transplanted MNCs, survival of transplanted MNCs, and so on.
Various cell labeling methods may be employed depending on the
circumstances under which the labels are to be read. For instance,
fluorescent proteins (green fluorescent protein, red fluorescent
protein, etc.) may be used as in circumstances in which a detector
can be suitably placed near the transplant site. Labeling may be
performed using conventional methods, such as the Invitrogen
GFP-lentiviral system noted above.
[0123] When analyzing engrafted brains in non-clinical situations,
immunohistochemical analysis may be useful. In an embodiment, brain
sections may be doubly-immunostained for green fluorescent protein
(GFP) or other cell labels, .beta.-tubulin III, NeuN (a
neuron-specific protein), glial fibrillary acidic protein (GFAP),
or O4 (an oligodendrocyte-specific protein) to identify neuronal,
astrocytic, glial, or oligodendrocytic profiles. The number of
positive profiles for a given antibody and the number of cells
expressing GFP may be estimated according to the Abercrombie
correction formula. The volume of distribution and total GFP (or
other label) positive profiles may be calculated by determining the
area of the brain containing at least 10% GFP-positive (or other
label-positive) profiles in every fifth section and multiplying by
the distance from the anterior aspects of the brain that contain
GFP-positive (or other label-positive) profiles. In an embodiment,
MNCs may be fluourescenty labeled using retroviral infection via
the pBabe neo-GFP vector. M. Dezawa et al., "Sciatic nerve
regeneration in rats induced by transplantation of in vitro
differentiated bone-marrow stromal cells." Eur J Neurosci.
2001;14:1771-6. The procedure may be modified such that other
fluourescent proteins may be incorporated into the vector.
C. NPC TRANSPLANTATION
[0124] In an embodiment, NPCs and/or GFUs according to the
invention may be administered using conventional protocols and
routes of administration, and amounts of NPCs and/or GFUs to be
administered to patients can be optimized using conventional dose
ranging techniques. NPCs and/or GFUs according to the present
invention may be administered alone or in combination with other
substances or compositions. Routes of administration may be chosen
from conventional routes of administration known to one of skill in
the art.
[0125] It is contemplated that transplantation will be carried out
by a variety of methods, including but not limited to infusion
through an injection cannula, needle or shunt, or by implantation
within a carrier, e.g., a biodegradable capsule, but other routes
of administration, are also within the scope of the invention.
[0126] NPCs and/or GFUs according to the invention may be
administered systemically to a patient, in which instance
parenteral routes such as intravenous (i.v.), or intra-arterial
(such as through internal or external carotid arteries)
administration are preferred routes of systemic administration.
Systemic administration techniques can be adapted from techniques
used to administer precursor cells generally, such as those
disclosed in D Lu et al., "Intraarterial administration of marrow
stromal cells in a rat model of traumatic brain injury." J
Neurotrauma. August 2001;18(8):813-9.
[0127] In embodiments, NPCs and/or GFUs according to the invention
may be administered locally to a patient's central nervous system
lesion. In a preferred embodiment, the NPCs and/or GFUs of the
present invention may be administered through an intraparenchymal
route. An advantage of administering the NPCs and/or GFUs locally
to a patient's central nervous system lesion is that the patient's
immune system may be less active inside the blood-brain barrier.
Therefore, the chances of immunorejection of the NPCs by the host
may be reduced, and the chances of graft survival may be increased
even though immunosuppressants still may be required. Another
advantage of local administration is more precise targeting of NPCs
to the CNS lesion.
[0128] When transplanting into a central nervous system lesion,
transplantation may be carried out using stereotactic surgical
procedures. In such procedures, the patient is anesthetized. The
patient's head is placed in an MRI compatible stereotactic frame
and the micropositioner with micro-injector placed over the skull.
Burr holes may be made in the patient's skull using a dental drill
or other suitable instrument to expose areas of the dura just above
the target sites.
[0129] In an embodiment, a needle pass using a 26-gauge needle and
Hamilton micro-syringe (or other suitable size syringe) may be
made, in which the needle is manually guided to the graft sites
using MRI images to insure proper placement of the NPCs and/or GFU.
Injections, preferably as bolus injections, may be made to the
graft site(s). Infusions rates can vary, preferably infusion
volumes are from about 0.1 to about 10 .mu.L/min, more preferably
from about 0.5 to about 5 .mu.L/min, and still more preferably from
about 1.0 to about 3.0 .mu.L/min. In an embodiment, the needle may
be left in place for a period of time, preferably ranging from
about 1 to about 10 minutes, more preferably about 5 minutes,
following infusion. Following the period wherein the needle is left
in place, the needle may be raised a short distance, preferably
about 1 mm to about 10 mm, more preferably about 2 mm and then held
in place for an additional period of time, preferably ranging from
about 5 minutes to about 30 minutes, more preferably about 15
minutes. The syringe may then be removed from the patient, the
wound site can be closed in anatomical layers, and the patient
monitored for recovery from anesthesia.
[0130] Analgesics, (e.g., buprenorphine) and antibiotics (e.g.,
Cephazolin, 50 mg/kg, IM, b.i.d..times.5 days) may be administered,
as needed, as part of the surgical/post-surgical procedures.
Antibiotic treatment may be continued post-surgically for an
extended period, preferably up to 30 days following surgery, to
suppress opportunistic infection.
[0131] Additional techniques for implantation may be found in K S
Bankiewicz et al., "Technique for bilateral intracranial
implantation of cells in monkeys using an automated delivery
system." Cell Transplantation, 9(5):595-607 (2000).
[0132] In certain embodiments, immunosuppressive agents may be
administered together with the inventive grafts and/or NPCs. These
agents may help to suppress rejection of the NPCs by the patient's
immune system, particularly when the graft and/or NPCs are
administered systemically. Examples of immunosuppressants useful in
the practice of this invention include, but are not limited to
antimetabolites such as azathioprine, alkylating agents such as
cyclophosphamide, folic-acid antagonists such as methotrexate or
mercaptopurine (6-MP), mycophenolate (CellCept), Cyclosporine-A and
Tacrolimus (FK-506). A preferred immunosuppressive agent is CsA.
CsA may obtained from a variety of sources, including as
Sandimmune.RTM., Injection; manufactured by Novartis Pharma AG,
Basel, Switzerland for Novartis Pharmaceuticals Corporation
(Novartis), East Hanover, N.J.
[0133] Immunosuppressants may be administered by a variety of
routes, including oral, i.p., and i.v. Dosing of immunosuppressants
may vary according to the nature of the immunosuppressant and the
patient. In an embodiment, the immunosuppressant may be dosed two
days prior to transplantation and continuing at suitable intervals
thereafter. In an embodiment, the immunosuppressant may be dosed
beginning on the day of grafting (approximately four hours
post-procedure) and continuing at 24-hour intervals thereafter.
Dosage ranges preferably may vary from about 0.5 mg/kg/day to about
100 mg/kg/day, more preferably from about 5 mg/kg/day to about 75
mg/kg/day, still more preferably from about 5 mg/kg/day to about 50
mg/kg/day. Intravenous injections may be administered as a bolus,
at a rate ranging preferably from about 0.005 to about 0.100
mL/minute, more preferably at about 0.050 mL/minute.
[0134] NPCs and/or GFUs according to the invention may be
administered using conventional protocols and routes of
administration, and amounts of NPCs and/or GFUs to be administered
to patients can be optimized using conventional dose ranging
techniques. NPCs and/or GFUs according to the present invention may
be administered alone or in combination with other substances or
compositions. Routes of administration may be chosen from
conventional routes of administration known to one of skill in the
art.
[0135] It is contemplated that transplantation will be carried out
by a variety of methods, including but not limited to infusion
through an injection cannula, needle or shunt, or by implantation
within a carrier, e.g., a biodegradable capsule, but other routes
of administration, are also within the scope of the invention.
[0136] In embodiments, NPCs and/or GFUs according to the invention
may be administered locally to a patient's central nervous system
lesion. In a preferred embodiment, the NPCs and/or GFUs of the
present invention may be administered through an intraparenchymal
route. An advantage of administering the NPCs and/or GFUs locally
to a patient's central nervous system lesion is that the patient's
immune system may be less active inside the blood-brain barrier.
Therefore, the chances of immunorejection of the NPCs by the host
may be reduced, and the chances of graft survival may be increased
even though immunosuppressants still may be required. Another
advantage of local administration is more precise targeting of NPCs
to the CNS lesion.
D. MASC-DERIVED NEURONAL CELL TRANSPLANTATION
[0137] In an embodment, MNCs and/or GFUs according to the invention
may be administered using conventional protocols and routes of
administration, and amounts of MNCs and/or GFUs to be administered
to patients can be optimized using conventional dose ranging
techniques. MNCs and/or GFUs according to the present invention may
be administered alone or in combination with other substances or
compositions. Routes of administration may be chosen from
conventional routes of administration known to one of skill in the
art.
[0138] It is contemplated that transplantation will be carried out
by a variety of methods, including but not limited to infusion
through an injection cannula, needle or shunt, or by implantation
within a carrier, e.g., a biodegradable capsule, but other routes
of administration, are also within the scope of the invention.
[0139] In embodiments, MNCs and/or GFUs according to the invention
may be administered locally to a patient's central nervous system
lesion. In a preferred embodiment, the MNCs and/or GFUs of the
present invention may be administered through an intraparenchymal
route. An advantage of administering the MNCs and/or GFUs locally
to a patient's central nervous system lesion is that the patient's
immune system may be less active inside the blood-brain barrier.
Therefore, the chances of immunorejection of the MNCs by the host
may be reduced, and the chances of graft survival may be increased
even though immunosuppressants still may be required. Another
advantage of local administration is more precise targeting of MNCs
to the CNS lesion.
[0140] When transplanting into a central nervous system lesion,
transplantation may be carried out using stereotactic surgical
procedures. In such procedures, the patient is anesthetized. The
patient's head is placed in an MRI compatible stereotactic frame
and the micropositioner with micro-injector placed over the skull.
Burr holes may be made in the patient's skull using a dental drill
or other suitable instrument to expose areas of the dura just above
the target sites.
[0141] In an embodiment, a needle pass using a 26-gauge needle and
Hamilton micro-syringe (or other suitable size syringe) may be
made, in which the needle is manually guided to the graft sites
using MRI images to insure proper placement of the MNCs and/or GFU.
Injections, preferably as bolus injections, may be made to the
graft site(s). Infusions rates can vary, preferably infusion
volumes are from about 0.1 to about 10 .mu.L/min, more preferably
from about 0.5 to about 5 .mu.L/min, and still more preferably from
about 1.0 to about 3.0 .mu.L/min. In an embodiment, the needle may
be left in place for a period of time, preferably ranging from
about 1 to about 10 minutes, more preferably about 5 minutes,
following infusion. Following the period wherein the needle is left
in place, the needle may be raised a short distance, preferably
about 1 mm to about 10 mm, more preferably about 2 mm and then held
in place for an additional period of time, preferably ranging from
about 5 minutes to about 30 minutes, more preferably about 15
minutes. The syringe may then be removed from the patient, the
wound site can be closed in anatomical layers, and the patient
monitored for recovery from anesthesia.
[0142] Analgesics, (e.g., buprenorphine) and antibiotics (e.g.,
Cephazolin, 50 mg/kg, IM, b.i.d..times.5 days) may be administered,
as needed, as part of the surgical/post-surgical procedures.
Antibiotic treatment may be continued post-surgically for an
extended period, preferably up to 30 days following surgery, to
suppress opportunistic infection.
[0143] Additional techniques for implantation may be found in K S
Bankiewicz et al., Technique for bilateral intracranial
implantation of cells in monkeys using an automated delivery
system. Cell Transplantation, 9(5):595-607 (2000).
[0144] In certain embodiments, immunosuppressive agents may be
administered together with the inventive grafts and/or MNCs. These
agents may help to suppress rejection of the MNCs by the patient's
immune system. Examples of immunosuppressants useful in the
practice of this invention include, but are not limited to
antimetabolites such as azathioprine, alkylating agents such as
cyclophosphamide, folic-acid antagonists such as methotrexate or
mercaptopurine (6-MP), mycophenolate (CellCept), Cyclosporine-A and
Tacrolimus (FK-506). A preferred immunosuppressive agent is CsA.
CsA may obtained from a variety of sources, including as
Sandimmune.RTM., Injection; manufactured by Novartis Pharma AG,
Basel, Switzerland for Novartis Pharmaceuticals Corporation
(Novartis), East Hanover, N.J.
[0145] Immunosuppressants may be administered by a variety of
routes, including oral, i.p., and i.v. Dosing of immunosuppressants
may vary according to the nature of the immunosuppressant and the
patient. In an embodiment, the immunosuppressant may be dosed two
days prior to transplantation and continuing at suitable intervals
thereafter. In an embodiment, the immunosuppressant may be dosed
beginning on the day of grafting (approximately four hours
post-procedure) and continuing at 24-hour intervals thereafter.
Dosage ranges preferably may vary from about 0.5 mg/kg/day to about
100 mg/kg/day, more preferably from about 5 mg/kg/day to about 75
mg/kg/day, still more preferably from about 5 mg/kg/day to about 50
mg/kg/day. Intravenous injections may be administered as a bolus,
at a rate ranging preferably from about 0.005 to about 0.100
mL/minute, more preferably at about 0.050 mL/minute.
E. EXPERIMENTAL OBSERVATIONS AND ADVANTAGES OF NPCs
[0146] Although both autologous and allogeneic transplantation of
NPCs, including pharmaceutical compositions that comprise NPCs, are
contemplated by this invention, allogeneic transplantation (same
species graft forming units) is preferable. In an embodiment,
allogeneic transplantation mimics the clinical setting in which
allogeneic transplantation of NPCs in patients suffering from
central nervous system lesion may take place. Disclosed herein are
the results from stereotaxically transplantation of NPCs, according
to the invention, into the brains of adult male Sprague-Dawley rats
that have been subjected to middle cerebral artery occlusion
(MCAo), middle cerebral artery ligation (MCAI) or transient global
ischemia (TGI). These models are useful in understanding the
efficacy of the present invention in the treatment of central
nervous system lesions. Further discussion of these models can be
found in the literature, particularly C. Borlongan et al.,
"Transplantation of cryopreserved human embryonal carcinoma-derived
neurons (NT2N cells) promotes functional recovery in ischemic
rats." Exp Neurol. 1998;149:310-21; and C. Borlongan et al., "Glial
cell survival is enhanced during melatonin-induced neuroprotection
against cerebral ischemia." FASEB J. 2000;14:1307-17. Each stroke
animal received a graft comprising one of three cell doses: about
40,000, 100,000 and 200,000 viable NPCs (these numbers are
understood to be approximate as used hereinafter). Transplantation
was carried out at about 6 weeks post-stroke, and animals were
immunosuppressed daily with Cyclosporine-A (10 mg/kg, i.p.)
throughout the post-transplantation survival time. Locomotor and
cognitive performance of transplanted rats was characterized weekly
over a period of 4 weeks post-transplantation, and again once at 12
weeks post-transplantation. Histological examination of the extent
of cerebral ischemia and graft survival was examined in randomly
selected animals at 5 weeks and 12 weeks post-transplantation.
[0147] The following tests were used in the outcome determination
of both the stroke operation and transplantation procedures as set
forth in more detail below. The manner of performing these tests is
set forth elsewhere herein, and would also be understood by one of
skill in the art. TABLE-US-00001 TABLE 1 PARAMETERS OF NPC
TRANSPLANT EFFICACY Test Objective EBST Reveals locomotor deficits
after stroke and recovery after transplantation Neurological exam
Reveals sensory-motor abnormalities after stroke and recovery after
transplantation Morris water maze Reveals cognitive deficits after
stroke and recovery after transplantation TTC histology Reveals
extent of cerebral infarction GFAP Reveals extent of cerebral
infarction and host immune response to transplant GFP viral vector
Reveals survival and migration of grafted NPCs Neu-N Reveals
neuronal phenotypic expression of grafted NPCs Legend: EBST,
elevated body swing test; TTC, triphenyltetrazolium chloride; GFAP,
glial fibrially acidic protein
[0148] The data obtained in the Examples below revealed that, in
the models studied, animals transplanted with NPCs displayed
significant improvements in both locomotor and cognitive
performance compared to their pre-transplantation baseline
performance. The two higher doses of about 100,000 and about
200,000 cells promoted better behavioral effects compared to the
lower cell dose of about 40,000 cells, thus suggesting a
dose-response relationship. Robust recovery from stroke-induced
behavioral deficits was seen as early as one week
post-transplantation and sustained over the four weeks
post-transplantation period. Significant improvements in motor
performance (using the elevated body swing test and Bederson test)
were observed in all three stroke types. In contrast, significant
improvements in cognitive performance (using the Morris Water Maze)
were more robust and stable in MCAo and TGI transplanted animals
compared to MCAI transplanted animals. All stroke transplanted
animals looked healthy and there were no observable overt adverse
effects during the study period.
[0149] The type of stroke appears to be a factor in functional
recovery, in that while all stroke animals displayed significant
improvements in motor performance, MCAo and TGI transplanted
animals showed better recovery in cognitive performance compared to
MCAI transplanted animals. The demonstration of significant
recovery of both motor and cognitive functions in MCAo and TGI
transplanted animals suggests that these two stroke models which
produced basal ganglia and hippocampal damage, respectively, are
responsive to NPC transplantation. Extrapolating these observations
to clinical application would indicate that patients with fixed
basal ganglia and hippocampal stroke may benefit from NPC
transplantation.
[0150] Histological results at 5 and 12 weeks post-transplantation
indicate that NPC graft survival mediated the observed functional
effects. The data suggest that transplanting 100,000 and 200,000
NPCs produced better behavioral recovery than the lower dose of
40,000 cells. The correlational analyses between graft survival and
behavioral effects further support that surviving NPCs promoted the
motor and cognitive recovery in stroke animals. Of note, graft
survival was determined using the lentivirus labeling approach, and
this strategy was shown to be reliable for marking grafted NPCs.
Furthermore, with this method, NPC migration was easily
tracked.
[0151] Depending on the stroke type, it appears that the more
severe the brain damage, as seen in both MCAo and MCAI, the better
the migration of NPCs. In contrast, the mild brain damage caused by
TGI appears to have resulted in less migration of the cells. The
observed ability of NPCs to travel long-distance to the site of
injury indicates its potential to migrate to and exert reparative
effects on specific stroke target sites. The results provided in
the Examples below support the view that NPCs that migrate are more
likely to differentiate into neuronal phenotypes. There are many
factors that might have contributed to this preferential
differentiation of migrated cells, including but not limited to
host microenvironment and type of stroke (location and degree/type
of cell death).
Experimental Observations and Advantages of MNCs
[0152] Although both autologous and allogeneic transplantation of
MNCs, including pharmaceutical compositions that comprise MNCs, are
contemplated by this invention, allogeneic transplantation (same
species grafts forming units) is preferable. In an embodiment,
allogeneic transplantation mimics the clinical setting in which
allogeneic transplantation of MNCs in patients suffering from
central nervous system lesions may take place. The results from
animals trials of allogenic transplantation of MNCs in animal
models of stroke are provide in Section J below. These models are
useful in understanding the efficacy of the present invention in
the treatment of central nervous system lesions.
[0153] The results in Section H suggest that the MNC group showed
significant improvements in the behavioral assessment tests
compared with the control and MASC group. In histological analysis,
the infarct volume measured at 41 days after MCAo did not show
significant difference among three groups. Compared with MASCs,
MNCs demonstrated higher survival ratio and larger proportion of
MNCs showed neuronal marker positivity and neurite extetion in the
host brain.
[0154] The MASC group demonstrated slight improvements in
behavioral assessment tests compared with control group, but not as
much as the NMC group.
[0155] Another advantage of MNC transplantation according to the
invention is the greater survival rate of MNC as compared, for
instance, with the multipotent MASCs. One month following
transplantation, approximately 30-45% of transplanted MNCs were
detected while only 10-20% of transplanted MASCs were detected. The
greater survival rate of MNCs may provide an advantage in
functional recovery.
[0156] In the current study, some MNCs in the cortex, striatum and
hippocampus demonstrated extension of neuritis in the host brain,
which could not be observed in the MASC-group. Hence the
significant behavioral improvements in the MNC group suggested that
the transplanted MNCs maintained neuronal characteristics in the
host brain, and contributed to the functional recovery in the MCAo
rat model.
G. NPC EXAMPLES
[0157] The Examples set forth herein are meant to be illustrative,
and in no way limiting, of the scope of the present invention.
[0158] Experimental procedures. All animals initially received
MCAo, MCAI or TGI stroke surgery. At about six weeks after the
surgery, animals were tested on elevated body swing test, Bederson
test, and Morris water maze task. Only animals that displayed
significant motor deficits were subsequently used for
transplantation surgery and randomly assigned to one of the
following treatments. Sample size for each arm of the study is
given in Table 2. TABLE-US-00002 TABLE 2 TREATMENT CONDITIONS Total
rats used in this study Graft type (Approx.) Cell dose Stroke Type
Sample size NPC 40,000 MCAo 8 MCAl 10 TGI 8 100,000 MCAo 10 MCAl 10
TGI 8 200,000 MCAo 10 MCAl 10 TGI 8
[0159] All animals underwent stroke surgery, received transplants
of 3 needle passes (MCAo and MCAI) or 2 needle passes (TGI), and
were treated with daily cyclosporine-A (10 mg/kg, i.p.).
[0160] The animals underwent weekly testing for the first 4 weeks
post-transplant. Half of animals were euthanized at 5 weeks
post-transplant for histological analyses of the cerebral
infarction and graft survival, phenotypic expression, and
migration. The rest of the animals were again tested behaviorally
and thereafter euthanized at 12 weeks post-stroke in order to
assess long-term behavioral and histological effects of NPCs. For
clarity, a schematic diagram is provided below. ##STR1##
[0161] Cell Labelling Using GFP-Lentiviral vector system: The
GFP-lentivirus system was supplied by Dr. Didier Trono of the
University of Geneva (Geneva, Switzerland). NPCs were labeled using
the following general scheme. Minor variations in method were
tolerated.
Materials Needed
[0162] 10 mg/mL polybrene stock solution/sterile filtered (Sigma)
[0163] Opti-MEM media (Gibco/Invitrogen) [0164] 1% Fetal Bovine
serum with antibiotics (Penicillin/Streptomycin) [0165] 6 well
plate approximately 1.times.10.sup.6 San-Bio cells [0166] Viral
suspension Detailed Procedure [0167] 1. Warm cell media in
37.degree. C. incubator. [0168] 2. Add polybrene to 10 .mu.g/mL
[0169] (Add 10 .mu.L of a 10 mg/mL polybrene stock solution to 10
mL media. Mix well.) [0170] 3. In a separate tube, Add 1 mL viral
suspension to 1 mL media containing polybrene. [0171] 4. Rapidly
thaw a SanBio cell aliquot in a 37.degree. C. water bath. Rinse
vial with 70% Ethanol; wipe dry. [0172] 5. Add entire contents to a
15 mL centrifuge tube containing 10 mL pre-warmed PBS; mix gently
and spin at 1000 RPMs on a low speed clinical centrifuge, room
temperature, 5 minutes. [0173] 6. Gently pipet out the supernate
and resuspend cell pellet in 2 mL prewarmed media containing virus.
(from step 3) [0174] 7. Transfer contents to one well of a 6-well
plate; place in the 37.degree. C. incubator; Incubate for 3-hours
[0175] 8. Wash cells in 10 mL prewarmed PBS, twice. [0176] 9.
Resuspend cells in 20 .mu.L PBS or media of choice. Transfer to a
1.5 mL [0177] Eppendorf tube. Keep on ice. Cells are ready for
transplantation.
[0178] Behavioral tests: Animals were subjected to the following
sensorimotor and cognitive behavioral measures in the pharmacology
studies of NPCs according to the invention:
[0179] Elevated Body Swing Test (EBST)
[0180] Morris Water Maze (MWM)
[0181] Bederson Neurological Scale
[0182] Elevated Body Swing Test (EBST)
[0183] The elevated body swing test (EBST) measures basic postural
reflexes and asymmetrical trunk function. The EBST test has been
demonstrated to show a long lasting deficit following MCAo and MCAI
ischemia in the rodent. C. Borlongan et al., "Locomotor and passive
avoidance deficits following occlusion of the middle cerebral
artery." Physiol Behav. 1995, 58:909-17. See also C. Borlongan et
al., "Early assessment of motor dysfunctions aids in successful
occlusion of the middle cerebral artery." Neuroreport. 1998b;
9:3615-21. It has also been evaluated in neural transplantation
paradigms for chronic stroke. C. Borlongan et al., "Early
assessment of motor dysfunctions aids in successful occlusion of
the middle cerebral artery." Neuroreport. 1998; 9:3615-21.
[0184] EBST involves handling the animal by its tail and recording
the direction of the swings. The test apparatus consisted of a
clear Plexiglas box (40.times.40.times.35.5 cm). The animal was
gently picked up at the base of the tail, and elevated by the tail
until the animal's nose is at a height of 2 inches (5 cm) above the
surface. The direction of the swing, either left or right, was
counted once the animals head moved sideways approximately 10
degrees from the midline position of the body. After a single
swing, the animal is placed back in the Plexiglas box and allowed
to move freely for 30 seconds prior to retesting. These steps are
repeated 20 times for each animal. Normally, intact rats display a
50% swing bias, that is, the same number of swings to the left and
to the right. A 75% swing bias would indicate 15 swings in one
direction and 5 in the other during 20 trials. Previous work with
the EBST has noted that lesioned animals display >75% biased
swing activity at one month after a nigrostriatal lesion; asymmetry
is stable for up to six months
[0185] Bederson Neurological Exam
[0186] The Bederson Neurological scale measures sensorimotor tasks.
J. Bederson et al., "Rat middle cerebral artery occlusion:
evaluation of the model and development of a neurologic
examination." Stroke. 1986;17:472-6; M. Altumbabic, "Intracerebral
hemorrhage in the rat: effects of hematoma aspiration." Stroke.
1998;29:1917-22. Previous work has shown measurable deficit over
time as measured by the Bederson model in both the MCAo and the
MCAI stroke models in rat.
[0187] About one hour after the EBST, the Bederson Neurological
exam is conducted following the procedures previously described. A
neurologic score for each rat is obtained using 4 tests which
include: [0188] (a) observation of spontaneous ipsilateral
circling, graded from 0 (no circling) to 3 (continuous circling);
[0189] (b) contralateral hindlimb retraction, which measures the
ability of the animal to replace the hindlimb after it is displaced
laterally by 2 to 3 cm, graded from 0 (immediate replacement) to 3
(replacement after minutes or no replacement); [0190] (c) beam
walking ability, graded 0 for a rat that readily traverses a
2.4-cm-wide, 80-cm-long beam to 3 for a rat unable to stay on the
beam for 10 seconds; and [0191] (d) bilateral forepaw grasp, which
measures the ability to hold onto a 2-mm-diameter steel rod, graded
0 for a rat with normal forepaw grasping behavior to 3 for a rat
unable to grasp with the forepaws.
[0192] The scores from all 4 tests, which are done over a period of
about 15 minutes on each assessment day, are added to give a
neurologic deficit score (maximum possible score, 12).
[0193] Morris Water Maze (MWM)
[0194] The Morris Water Maze assesses several aspects of cognitive
functioning, including task acquisition and retention, search
strategies, and perseveration. The water maze task is presumed to
be sensitive to damage in several brain areas affected by MCAo
including striatum and frontal cortex.
[0195] About one hour after the Bederson Neurological exam, animals
are introduced to Morris water maze in order to assess spatial
memory. The Morris water maze consists of an inflatable tank, 6
feet in diameter and 3 feet deep. The tank was filled with 12 cm of
water and made opaque by adding 300 ml of milk. An 11-cm-tall
platform made of clear Plexiglass with a circular surface 10 cm in
diameter was placed into 1 of 4 positions in the pool. The platform
is 1 cm below the surface of the water and thus hidden from the
view of an animal in the water. The pool is divided into four
quadrants of equal surface area. The test rat was placed in the
pool facing the side of the tank and released at 1 of 4 starting
positions (north, south, east, or west), which was randomly
determined, and were located arbitrarily at equal distances on the
pool rim. The platform was located in the middle of the south-west
quadrant 25 cm from the pool rim. The start point was changed after
each trial. The animal was given approximately 60 seconds to find
the platform and allowed to rest on the platform for approximately
30 seconds and placed back in starting position, for a total of 3
tests from starting positions determined at random. If the rat
failed to find the hidden platform within approximately 60 seconds,
it was placed on the platform and allowed to rest on the platform
for approximately 30 seconds. After the rest period, the rat was
placed back in the tank and was tested again for 2 more trials. The
training day consisted of 3 trials. The testing day was conducted
on day 2 (for probe trial, see below). After each training trial
and testing, the rat was then placed in a cage on top of a heating
pad. The swim paths were monitored by a video camera connected to a
computer through an image analyzer. Escape latency time to reach
the platform and path length the animal swam to find the platform
were used to assess acquisition of the water-maze task. Swimming
speed=path length/escape latency was used to assess the motoric
activity of rats in this task. To assess recall of the platform
position, a 60-second probe trial with no platform in the pool was
undertaken on day 2; the percentage of time spent in the former
platform position was monitored.
[0196] MCAo stroke surgery: All surgical procedures were conducted
under aseptic conditions. MCAo stroke procedures were taken from
the literature, in particular from C. Borlongan et al., "Chronic
cyclosporine-A injection in rats with damaged blood-brain barrier
does not impair retention of passive avoidance." Neurosci Res.
1998, 32:195-200. Determination in each animal of successful
occlusion was attained using a Laser Doppler that revealed
significant (>75%) reduction in cerebral blood flow during the
1-hour occlusion. MCAo produced consistent striatal damage.
[0197] MCAI stroke surgery: The MCAI surgical procedure is
described generally in Y. Wang et la., "Glial Cell-Derived
Neurotrophic Factor Protects Against Ischemia-Induced Injury in the
Cerebral Cortex." 1997, J. Neuroscience; 17 (11):4341-4348. The
Laser Doppler was also used to verify arterial ligation. MCAI
produces consistent cortical damage.
[0198] TGI stroke surgery: A 4-vessel occlusion technique was used.
Under deep anesthesia, animals received a ventral midline cervical
incision. The vertebral arteries were isolated through the alar
foramina of the first cervical vertebra and microclips were used to
ligate both common carotid arteries for 15 minutes. This technique
has been shown to produce global cerebral ischemia, with consistent
hippocampal damage.
[0199] Neuronal Precursor Cells: Neuronal Precursor Cells were
provided by SanBio, Inc. (Mountain View Calif.). These cells were
produced generally according to the teachings of
PCT/JP03/01260.
[0200] Grafting Procedures: All surgical procedures were conducted
under aseptic conditions. Under equithesin (3 ml/kg i.p.)
anesthesia (animals checked for pain reflexes), the animals were
implanted with NPCs directly into the striatum for MCAo, the cortex
for MCAI or the hippocampus for TGI, using a 26-gauge implantation
cannula. See generally Y. Wang et al., "Glial Cell-Derived
Neurotrophic Factor Protects Against Ischemia-Induced Injury in the
Cerebral Cortex" 1997, J. Neuroscience; 17 (11):4341-4348 and C.
Borlongan et al., "Transplantation of cryopreserved human embryonal
carcinoma-derived neurons (NT2N cells) promotes functional recovery
in ischemic rats." Exp Neurol. 1998;149:310-21. Cryopreserved NPCs
were obtained using the methods disclosed herein. Viability cell
counts, using Tryphan Blue exclusion method, were conducted prior
to transplantation and immediately after the transplantation on the
last animal recipient. The pre-determined cell doses (40,000,
100,000 and 200,000) referred to number of viable cells.
Transplantation surgery were carried out within 2 hours after
thawing the cells. Infusion rate was 1 ul of cell solution per
minute. Following infusion, a 3-minute absorption period was
allowed before the needle was retracted. A heating pad and a rectal
thermometer maintained body temperature at about 37 Deg C.
throughout surgery and until recovery from anesthesia.
[0201] Statistical analysis: Behavioral data were analyzed using
repeated measures of ANOVA with statistical significance set at
p<0.05. Posthoc tests included compromised t-tests to reveal
significant differences (p's<0.05) between treatment
conditions.
EXAMPLE 1
MCAo Results--Weekly for Four Weeks Post-Transplantation
[0202] The test animals underwent the MCAo procedure as described
above, and were evaluated weekly for four weeks
port-transplantation with the following results.
[0203] EBST: For the weekly testing over the four weeks
post-transplantation, overall ANOVA revealed significant main
treatment effects (F2,21=57.06, p<0.0001) (FIG. 1).
Dose-dependent significant effects (200,000>100,000>40,000)
were also observed (p's<0.01). The motor asymmetry was
significantly reduced in each of the four weeks
post-transplantation compared to baseline (p's<0.0001), with the
most robust recovery seen at one week post-transplantation
(p's<0.0001), and with stable recovery displayed for the
subsequent three weeks post-transplantation. Posthoc tests revealed
that the significant reduction in motor asymmetry at 1 week
post-transplantation did not differ across the three cell doses,
but dose-dependent effects were seen at 2, 3 and 4 weeks
post-transplantation (p's<0.05) (FIG. 2).
[0204] Bederson test: For the weekly testing over the four weeks
post-transplantation, overall ANOVA revealed significant main
treatment effects (F2,21=9.65, p<0.001) (FIG. 3). The higher
doses of 200,000 and 100,000 cells promoted better improvement in
neurologic deficit scores than the low dose of 40,000 cells
(p's<0.01). Improvements in neurologic deficit scores were
significantly reduced in each of the four weeks
post-transplantation compared to baseline (p's<0.0001), with a
trend towards better improvement over time in that transplanted
animals performed better at 2, 3 and 4 weeks post-transplantation
compared to 1 week post-transplantation (p's<0.0001). Posthoc
tests revealed that the significant reduction in neurologic deficit
scores at 1 week post-transplantation did not differ across the
three cells doses, but the high doses 100,000 and 200,000 produced
better recovery than the low dose 40,000 cells at 2 weeks
(p's<0.05), and dose-dependent effects
(200,000>100,000>40,000) were seen at 3 and 4 weeks
post-transplantation (p's<0.05) (FIG. 4).
[0205] MWM Acquisition: For the weekly testing over the four weeks
post-transplantation, overall ANOVA revealed no significant main
treatment effects (F2,21=2.87, p=0.08) (FIG. 5). There appears to
be a trend towards longer MWM acquisition time over the 4 weeks
post-transplantation period as revealed by longer acquisition times
at 2, 3 and 4 weeks compared to baseline and 1 week
post-transplantation (p's<0.01) (FIG. 6).
[0206] MWM Probe test: Time to find the platform. For the weekly
testing over the four weeks post-transplantation, overall ANOVA
revealed significant main treatment effects (F2,21=61.33,
p<0.0001) (FIG. 7). The higher doses of 200,000 and 100,000
cells promoted significantly shorter time locating the platform
than the low dose of 40,000 cells (p's<0.0001). Improvements in
the time to find the platform were significantly reduced in each of
the four weeks post-transplantation compared to baseline
(p's<0.0001), with a trend towards better improvement over time
in that transplanted animals performed better at 2, 3 and 4 weeks
post-transplantation compared to 1 week post-transplantation
(p's<0.0001). Posthoc tests revealed that the higher doses of
200,000 and 100,000 produced significantly shorter times to find
the platform than the low dose 40,000 cells across the 4-week
post-transplantation period, with clear dose-dependent response
(200,000>100,000>40,000) seen at 1 and 4 weeks
post-transplantation (p's<0.05) (FIG. 8).
[0207] MWM Probe test: Time spent on the platform area. For the
weekly testing over the four weeks post-transplantation, overall
ANOVA revealed significant main treatment effects (F2,21=15.19,
p<0.0001) (FIG. 9). The higher doses of 200,000 and 100,000
cells promoted significantly longer times spent in the platform
area than the low dose of 40,000 cells (p's<0.01). Times spent
in the platform area were significantly increased in each of the
four weeks post-transplantation compared to baseline
(p's<0.0001), with a trend towards better improvement over time
in that transplanted animals performed better at 3 and 4 weeks
post-transplantation compared to 1 and 2 weeks post-transplantation
(p's<0.0001). Posthoc tests revealed that the higher doses of
200,000 and 100,000 produced significantly longer times spent in
the platform area than the low dose 40,000 cells across the 4-week
post-transplantation period (p's<0.05) (FIG. 10).
EXAMPLE 2
MCAI Results--Weekly for Four Weeks Post-Transplantation
[0208] The test animals underwent the MCAI procedure as described
above, and were evaluated weekly for four weeks
post-transplantation with the following results.
[0209] EBST: For the weekly testing over the four weeks
post-transplantation, overall ANOVA revealed significant main
treatment effects (F2,24=76.30, p<0.0001) (FIG. 11). The higher
doses of 200,000 and 100,000 cells produced better recovery from
motor asymmetry than the low dose of 40,000 cells (p's<0.0001).
The motor asymmetry was significantly reduced in each of the four
weeks post-transplantation compared to baseline (p's<0.0001),
with better recovery displayed at 2, 3 and 4 weeks
post-transplantation. Posthoc tests revealed that the higher cell
doses significantly reduced motor asymmetry better than the low
dose, with dose-dependent effects (200,000>100,000>40,000)
seen at 1 week post-transplantation (p's<0.05) (FIG. 12).
[0210] Bederson test: For the weekly testing over the four weeks
post-transplantation, overall ANOVA revealed significant main
treatment effects (F2,24=3.65, p<0.05) (FIG. 13). The highest
dose of 200,000 cells promoted better improvement in neurologic
deficit scores than the low dose of 40,000 cells (p's<0.05); no
significant differences were found between the medium dose of
100,000 cells and the low dose of 40,000 cells. (FIG. 13)
Improvements in neurologic deficit scores were significantly
reduced in each of the four weeks post-transplantation compared to
baseline (p's<0.0001), and stable over time as there were no
significant differences between cell doses across the weekly
neurologic scores (p's>0.005). Posthoc tests revealed that the
highest dose of 200,000 cells produced better recovery than the low
dose 40,000 cells at 3 and 4 weeks (p's<0.01); no other
significant differences were found between cell doses at earlier
time points post-transplantation (p's>0.05) (FIG. 14).
[0211] MWM Acquisition: For the weekly testing over the four weeks
post-transplantation, overall ANOVA revealed no significant main
treatment effects (F2,24=5.78-16, p>0.05) (FIG. 15). There
appears to be a trend towards longer MWM acquisition time over the
4 weeks transplantation as revealed by longer acquisition times at
1, 2, 3 and 4 weeks post-transplantation compared to baseline
(p's<0.0001) (FIG. 16).
[0212] MWM Probe test: Time to find the platform. For the weekly
testing over the four weeks post-transplantation, overall ANOVA
revealed no significant main treatment effects (F2,24=0.62, p=0.55)
(FIG. 17). Over time post-transplantation, significantly longer
times in locating the platform were noted (p's<0.001) (FIG.
18).
[0213] MWM Probe test: Time spent on the platform area. For the
weekly testing over the four weeks post-transplantation, overall
ANOVA revealed no significant main treatment effects (F2,24=2.01,
p=0.16) (FIG. 19). Although a trend of longer times were spent in
the platform area over the 4-week post-transplantation period
compared to baseline (p's<0.001), transient dose-dependent
effects were only seen at 1 week post-transplantation
(p's<0.05), and not in the other weekly test time periods
(p's>0.05) (FIG. 20).
EXAMPLE 3
TGI Results--Weekly for Four Weeks Post-Transplantation
[0214] The test animals underwent the TGI procedure as described
above, and were evaluated weekly for four weeks
port-transplantation with the following results.
[0215] Bederson test: For the weekly testing over the four weeks
post-transplantation, overall ANOVA revealed significant main
treatment effects (F2,23=47.33, p<0.001) (FIG. 21). The higher
doses of 200,000 and 100,000 cells promoted better improvement in
neurologic deficit scores than the low dose of 40,000 cells
(p's<0.0001). Improvements in neurologic deficit scores were
significantly reduced in each of the four weeks
post-transplantation compared to baseline (p's<0.0001). Posthoc
tests revealed that the significant reduction in neurologic deficit
scores at 1 week post-transplantation did not differ across the
three cell doses, but the high doses 100,000 and 200,000 produced
better recovery than the low dose 40,000 cells at 2, 3 and 4 weeks
(p's<0.005), and dose-dependent effects
(200,000>100,000>40,000) were seen at 4 weeks
post-transplantation (p's<0.05) (FIG. 22).
[0216] MWM Acquisition: For the weekly testing over the four weeks
post-transplantation, overall ANOVA revealed significant main
treatment effects (F2,23=9.88, p<0.001) (FIG. 23). However, this
significant treatment effect was achieved only because the highest
dose of 200,000 cells produced a transient significant improvement
in acquiring the task at 1 week post-transplantation compared to
the two other doses of 100,000 and 40,000 cells (p's<0.005).
Thereafter, longer acquisition times at 2, 3 and 4 weeks
post-transplantation compared to baseline and 1 week
post-transplantation were noted (p's<0.005) (FIG. 24).
[0217] MWM Probe test: Time to find the platform. For the weekly
testing over the four weeks post-transplantation, overall ANOVA
revealed significant main treatment effects (F2,23=30.98,
p<0.0001) (FIG. 25). The higher doses of 200,000 and 100,000
cells promoted significantly shorter time locating the platform
than the low dose of 40,000 cells (p's<0.0001). Improvements in
the time to find the platform were significantly reduced in each of
the four weeks post-transplantation compared to baseline
(p's<0.0001), with a trend towards better improvement over time
in that transplanted animals performed better at 3 and 4 weeks
post-transplantation compared to 1 and 2 weeks post-transplantation
(p's<0.0001). Posthoc tests revealed that the higher dose of
200,000 cells produced significantly shorter times to find the
platform than the other two doses of 100,000 and 40,000 cells at 2
and 3 weeks post-transplantation period (p's<0.05), while the
two higher doses exerted better improvement in finding the platform
compared to the low cell dose at 4 weeks post-transplantation
(p's<0.05) (FIG. 26).
[0218] MWM Probe test: Time spent on the platform area. For the
weekly testing over the four weeks post-transplantation, overall
ANOVA revealed significant main treatment effects (F2,23=54.06,
p<0.0001) (FIG. 27). Significant dose-dependent effects
(200,000>100,000>40,000) were seen over the 4-week
post-transplantation period (p's<0.0001). In addition, over the
4-week post-transplantation period, longer times were spent in the
platform area (p's<0.0001). Posthoc tests revealed that the
higher doses of 200,000 and 100,000 produced significantly longer
times spent in the platform area than the low dose 40,000 cells at
3 and 4 weeks post-transplantation period (p's<0.05). At 2 weeks
post-transplantation, only the highest cell dose produced
significantly longer time spent in the platform area compared to
the other two cell doses (p's<0.05) (FIG. 28).
EXAMPLE 4
Histological Examination at 5 Weeks Post-Transplantation
[0219] Randomly selected animals were euthanized at 5 weeks
post-transplantation (see Table 3). Histological examinations were
limited to 2-3 brain samples for each dose and stroke type.
Accordingly, quantitative analyses could only be performed on graft
survival and migration based on GFP epifluorescence. For the other
histological parameters, specifically on using different antibody
markers to detect phenotypic expression, only a general description
is provided.
[0220] NPC graft survival: GFP epifluorescence revealed
dose-dependent graft survival (200,000>100,000>40,000) across
all three stroke types (F8,32=33.9, p<0.0001) (FIG. 29). Mean
cell counts of GFP-positive cells revealed that the lower dose of
40,000 cells resulted in low numbers of surviving GFP-positive
grafts, while both higher doses of 100,000 and 200,000 resulted in
higher numbers of surviving GFP-positive grafts. These observations
were consistent for MCAo, MCAI and TGI transplanted animals.
However, when percentages for each cell dose were calculated, no
significant differences (F8,32=1.67, p>0.05) (FIG. 30) in the
percent graft survival were obtained across the 3 doses.
TABLE-US-00003 TABLE 3 Five weeks post-transplant histology Graft
type Cell dose Stroke Type Sample size NPC 40,000 MCAo 4 MCAl 5 TGI
4 100,000 MCAo 5 MCAl 5 TGI 4 200,000 MCAo 5 MCAl 5 TGI 4
[0221] Positive correlations between graft survival and functional
recovery: Regression analyses revealed that the higher the number
of surviving NPC grafts (200,000>100,000>40,000), the better
the functional improvement (FIG. 31).
[0222] NPC graft migration: GFP epifluorescence revealed that
majority (approx. 55%-85%) of the transplanted cells remained
within the original transplant site (FIG. 32). In MCAo transplanted
animals, several GFP-positive cells could be easily identified
within the original striatal transplant sites (62%); in MCAI
transplanted animals, GFP-positive cells remained within the
original cortical transplant sites (53%), and; in TGI transplanted
animals, GFP-positive cells remained within the original
hippocampal sites (86%). However, it appears that both MCAo and
MCAI transplanted animals displayed more migration of grafted cells
compared to TGI transplanted animals. Nonetheless, when migration
was observed, the grafted cells remained within the general target
area, in that graft migration in MCAo was observed within the
striatum, in MCAI within the cortex, and in TGI within the
hippocampus. Graft migration in both MCAo and MCAI was
characterized by grafted cells lining the ischemic penumbra in the
striatum and cortex, respectively. Furthermore for MCAo, a medial
to lateral (1.8 mm) and dorsal to ventral (2.3 mm) migration of
cells along the striatal ischemic penumbra was observed. For MCAI,
a medial to lateral (4.4 mm) migration of cells was seen. For TGI
transplanted animals, graft migration was characterized by
GFP-positive SBDPs identified in the CA2 and CA3 regions (1.6 mm
and 0.7 mm away, respectively, from the original CA1 transplant
site).
[0223] NPC phenotypic expression: Grafted NPC cells were positive
for GFAP (about 5%) and a very few cells (2-5 cells per brain) were
also positive for NeuN. Both these markers co-localized with GFP.
These observations were consistent for all doses and all three
types of stroke.
EXAMPLE 5
MCAo Results at Twelve Weeks Post-Transplantation
[0224] At twelve weeks following transplantation, the test animals
that underwent the TGI procedure as described above were evaluated
with the following results.
[0225] EBST: For the 12 weeks post-transplantation testing, overall
ANOVA revealed significant main treatment effects (F2,9=11.84,
p<0.005) (FIG. 32). Posthoc test revealed significantly reduced
motor asymmetry at 12 weeks post-transplantation compared to
baseline (p<0.001).
[0226] Bederson test: For the 12 weeks post-transplantation
testing, overall ANOVA revealed significant main treatment effects
(F2,9=41.83, p<0.001) (FIG. 32). Posthoc testing revealed
significantly reduced neurological deficits at 12 weeks
post-transplantation compared to baseline (p<0.001).
[0227] MWM Acquisition: For the 12 weeks post-transplantation
testing, overall ANOVA revealed no significant main treatment
effects (F2,9=0.36, p=0.71) (FIG. 33). These results indicate no
significant differences in MWM acquisition between baseline and 12
week post-transplantation.
[0228] MWM Probe test: Time to find the platform. For the 12 weeks
post-transplantation testing, overall ANOVA revealed significant
main treatment effects (F2,9=6.18, p<0.05) (FIG. 33). Posthoc
test revealed significantly reduced time to find the platform at 12
weeks post-transplantation compared to baseline (p<0.001).
[0229] MWM Probe test: Time spent on the platform area. For the 12
weeks post-transplantation testing, overall ANOVA revealed
significant main treatment effects (F2,9=6.18, p<0.05) (FIG.
33). Posthoc test revealed significantly increased time in the
platform area at 12 weeks post-transplantation compared to baseline
(p<0.001).
EXAMPLE 6
MCAI Results at Twelve Weeks Post-Transplantation
[0230] At twelve weeks following transplantation, the test animals
that underwent the MCAI procedure as described above were evaluated
with the following results.
[0231] EBST: For the 12 weeks post-transplantation testing, overall
ANOVA revealed significant main treatment effects (F2,9=23.02,
p<0.0005) (FIG. 34). Posthoc test revealed significantly reduced
motor asymmetry at 12 weeks post-transplantation compared to
baseline (p<0.0001).
[0232] Bederson test: For the 12 weeks post-transplantation
testing, overall ANOVA revealed significant main treatment effects
(F2,9=9.29, p<0.01) (FIG. 34). Posthoc test revealed
significantly reduced neurological deficits at 12 weeks
post-transplantation compared to baseline (p<0.0001).
[0233] MWM Acquisition: For the 12 weeks post-transplantation
testing, overall ANOVA revealed no significant main treatment
effects (F2,9=1.37, p=0.30) (FIG. 35). These results indicate no
significant differences in MWM acquisition between baseline and 12
week post-transplantation.
[0234] MWM Probe test: Time to find the platform. For the 12 weeks
post-transplantation testing, overall ANOVA revealed no significant
main treatment effects (F2,9=0.26, p=0.78) (FIG. 35). These results
indicate no significant differences in MWM probe test, i.e., with
the platform available, between baseline and 12 week
post-transplantation.
[0235] MWM Probe test: Time spent on the platform area. For the 12
weeks post-transplantation testing, overall ANOVA revealed no
significant main treatment effects (F2,9=0.15, p=0.86) (FIG. 35).
These results indicate no significant differences in MWM probe
test, i.e., without the platform, between baseline and 12 week
post-transplantation.
EXAMPLE 7
TGI Results at Twelve Weeks Post-Transplantation
[0236] At twelve weeks following transplantation, the test animals
that underwent the TGI procedure as described above were evaluated
with the following results.
[0237] Bederson test: For the 12 weeks post-transplantation
testing, overall ANOVA revealed significant main treatment effects
(F2,9=184.02, p<0.0001) (FIG. 36). Posthoc test revealed
significantly reduced neurological deficits at 12 weeks
post-transplantation compared to baseline (p<0.0001).
[0238] MWM Acquisition: For the 12 weeks post-transplantation
testing, overall ANOVA revealed no significant main treatment
effects (F2,9=0.31, p=0.74) (FIG. 37). These results indicate no
significant differences in MWM acquisition between baseline and 12
week post-transplantation.
[0239] MWM Probe test: Time to find the platform. For the 12 weeks
post-transplantation testing, overall ANOVA revealed significant
main treatment effects (F2,9=5.14, p<0.05) (FIG. 37). Posthoc
test revealed significantly reduced time to find the platform at 12
weeks post-transplantation compared to baseline (p<0.0001).
[0240] MWM Probe test: Time spent on the platform area. For the 12
weeks post-transplantation testing, overall ANOVA revealed
significant main treatment effects (F2,9=4.39, p<0.05) (FIG.
37). Posthoc test revealed significantly increased time in the
platform area at 12 weeks post-transplantation compared to baseline
(p<0.0001).
EXAMPLE 8
Histological Examination at 12 Weeks Post-Transplantation
[0241] All remaining animals were euthanized at 12 weeks
post-transplantation (see Table 4). Quantitative analyses of graft
survival and migration based on GFP epifluorescence and other
immunohistochemical parameters, specifically on using different
antibody markers to detect phenotypic expression, were conducted.
TABLE-US-00004 TABLE 4 Twelve weeks post-transplant histology Graft
type (Approx.) Cell dose Stroke Type Sample size NPC 40,000 MCAo 4
MCAl 4 TGI 4 100,000 MCAo 4 MCAl 4 TGI 4 200,000 MCAo 4 MCAl 4 TGI
4
[0242] NPC graft survival: GFP epifluorescence revealed partial
dose-dependent graft survival (200,000=100,000>40,000) across
all three stroke types (F8,27=14.88, p<0.0001) (FIG. 38). Mean
cell counts of GFP-positive cells revealed that the lower dose of
40,000 cells resulted in low numbers of surviving GFP-positive
grafts, while both higher doses of 100,000 and 200,000 resulted in
higher numbers of surviving GFP-positive grafts. These observations
were consistent for MCAo, MCAI and TGI transplanted animals.
However, when percentages for each cell dose were calculated, no
significant differences (F8,27=1.37, p>0.05) (FIG. 39) in the
percent graft survival were obtained across the 3 doses. This
suggests that percent graft survival in both low and high doses was
maintained, potentially by CsA immunosuppression.
[0243] NPC graft migration: In agreement with the 5-week histology
results, GFP epifluorescence revealed that majority (approx.
65%-90%) of the transplanted cells remained within the original
transplant site. In MCAo transplanted animals, several GFP-positive
cells could be easily identified within the original striatal
transplant sites (72%); in MCAI transplanted animals, GFP-positive
cells remained within the original cortical transplant sites (64%),
and; in TGI transplanted animals, GFP-positive cells remained
within the original hippocampal sites (91%). It appears that both
MCAo and MCAI transplanted animals displayed more migration of
grafted cells compared to TGI transplanted animals. Moreover, when
migration was observed, the grafted cells remained within the
general target area, in that graft migration in MCAo was observed
within the striatum, in MCAI within the cortex, and in TGI within
the hippocampus. In addition, graft migration in both MCAo and MCAI
was characterized by grafted cells lining the ischemic penumbra in
the striatum and cortex, respectively. Furthermore for MCAo, a
medial to lateral (2.0 mm) and dorsal to ventral (2.5 mm) migration
of cells along the striatal ischemic penumbra was observed. For
MCAI, a medial to lateral (4.5 mm) migration of cells was seen. For
TGI transplanted animals, graft migration was characterized by
GFP-positive SBDPs identified in the CA2 and CA3 regions (1.6 mm
and 0.8 mm away, respectively, from the original CA1 transplant
site).
[0244] NPC phenotypic expression: Across stroke types and doses,
the approximate survival rate is 15%. Within these original
transplant sites, most of the cells retain their beady appearance,
and are not positive for NeuN or GFAP. However, in MCAo
transplanted animals, a few of these cells exhibit NeuN and GFAP.
GFP positive cells were detected that migrated along the striatal
penumbra, cortical penumbra and CA3 of MCAo, MCAI, and TGI
transplanted animals, respectively. Indeed, NeuN immunostaining
reveals that these cells express such marker for mature neurons.
Overall, about 25% of surviving GFP positive cells are NeuN
positive across stroke types and doses; in cells that have migrated
away from the transplant site, about 60% are NeuN positive. These
cells displayed neuronal morphology, characterized by elaborate and
long processes, which are abundant in MCAo transplanted animals.
Further GFP epifluorescence microscopy revealed the distinct
neuronal morphology found in each stroke type, as well as NPC graft
cell-to-cell contact. Some cells (about 5% overall and across
stroke types and doses) exhibit the morphology of glial cell which
was confirmed by GFAP staining. Most, if not all, GFAP positive
cells were found near or within blood vessels.
[0245] Graft-host tissue pathology: There was no evidence of tumor
formation using Nissl staining in striatum; cortex; or
hippocampus.
EXAMPLE 9
Additional NPC Testing in Stroke Models
[0246] The purpose of this study was to examine the therapeutic
benefits of NPCs in stroke animals. Behavioral tests were used to
reveal motor and neurological functions of transplanted stroke
animals. Transplantation was carried out at 1 month post-stroke,
and animals were immunosuppressed daily with Cyclosporin-A (CsA, 10
mg/kg, i.p.) throughout the one-month post-transplantation survival
time. Locomotor and neurological performance of transplanted rats
were characertized at days 7, 14 and 28 post-transplantation.
Successful transplant outcome, as revealed by determination of an
efficacious NPC dose range, was evaluated using locomotor behavior
and neurological performance.
[0247] There were 3 treatment conditions: 0 (medium alone), low
dose 90 k NPC, and high dose 180 k NPC. The number of animals for
each treatment condition corresponded to a required sample size for
statistical analyses (n=10 per group). Animals not reaching the
criteria for behavioral deficits (75% biased swing activity and a
score of 2.5 in neurological exam) were not included in the study.
Thus, animals that reached these criteria and those exceeding these
criteria were included in the study. Typically, most stroke animals
reached such criteria, with at least 8 subjects needed to provide
conclusive statistical analyses. All animals were immunosuppressed
(10 mg/kg CsA, i.p., daily) throughout the study.
[0248] All animals initially received MCAo stroke surgery. At four
weeks after the surgery, animals were tested with the EBST,
followed an hour later by the Bederson neurological exam. Only
animals that displayed significant motor and neurological deficits
were subsequently used for transplantation surgery and randomly
assigned to one of the following treatments: TABLE-US-00005 TABLE 5
TREATMENT CONDITIONS GRAFT TYPE CELL DOSE STROKE TYPE SAMPLE SIZE
NPC 90,000 MCAo 10 NPC 180,000 MCAo 10 Vehicle 0 MCAo 10 Legend:
All animals underwent stroke surgery, received striatal (MCAo)
transplants, and were treated with chronic CsA.
[0249] Animals were again introduced to the same battery of
behavioral tests at days 7, 14 and 28 post-transplantation. For
clarity, a schematic diagram is provided below. TABLE-US-00006
TABLE 6 TIMELINE OF EXPERIMENTAL PROCEDURES ##STR2##
[0250] All surgical procedures were conducted under aseptic
conditions. The animals were anesthetized with equithesin (300
mg/kg, i.p.) and checked for pain reflexes. Under deep anesthesia,
animals underwent the MCA occlusion surgery. The MCA occlusion
technique involves insertion of a suture filament through the
carotid artery to reach the junction of the MCA, thus blocking the
blood flow from the common carotid artery, as well as from the
circle of Willis. The right common carotid artery was identified
and isolated through a ventral midline cervical incision. The
filament size was 4-0, made of sterile, non-absorbable suture
(Ethicon, Inc.), with the diameter of the suture tip tapered to 24
to 26-gauge size using a rubber cement. About 15 to 17 mm of the
suture filament was inserted from the junction of the external and
internal carotid arteries to block the MCA. The right MCA was
occluded for one hour; a one-hour occlusion of the MCA generally
results in maximal infarction. In addition, the length and size of
the tip of the embolus have been found to produce complete MCA
occlusion in animals weighing between 250 to 350 g. A heating pad
and a rectal thermometer promotes maintenance of body temperature
within normal limits. To determine succesful occlusion and
reperfusion, a Laser Doppler was used. The Doppler probe was placed
at the level of the dura directly above the expected infarct
striatal region (AP: +2.0, ML: .+-.2.0, and DV: -4.0 mm) to measure
cerebral blood flow before, during and after occlusion.
[0251] All surgical procedures were conducted under aseptic
conditions. Under equithesin (3 ml/kg i.p.) anesthesia (animals
checked for pain reflexs), the animals were implanted with NPCs or
vehicle directly into the striatum (0.5 mm anterior to bregma, 2.8
mm lateral to midline and 5.0 mm below the dural surface) using a
28-gauge implantation cannula. Cryopreserved human NPCs were
obtained from SanBio, Inc. and thawed just prior to transplantation
surgery. Viability cell counts, using Trypan Blue exclusion method,
were conducted prior to transplantation and immediately after the
transplantation on the last animal recipient. The pre-determined
cell dosages (90,000 and 180,000) were based on pilot studies
demonstrating that these dosages are within therapeutically
effective dosage range.
[0252] Transplantation surgery was carried out within 2 hours after
thawing the cells. Infusion rate was 1 ul of cell solution per
minute. Following infusion, a 3-minute absorption period was
allowed before the needle was retracted. One needle pass was used,
but there were 3 dorsoventral deposits, with each site receiving a
3-ul cell solution. A heating pad and a rectal thermometer allowed
maintenance of body temperature at about 37.degree. C. throughout
surgery and following recovery from anesthesia.
[0253] The one-hour MCAo stroke surgery produced consistent
behavioral impairments at one month post-stroke as revealed by
significant biased swing activity and neurological deficits in EBST
and Bederson exam, respectively, compared to pre-stroke performance
of the animals in both tests. Pair-wise comparisons between
pre-stroke and post-stroke performance of the animals revealed
significant impairments in both tests (p's<0.0001) in all stroke
animals included in this study.
[0254] Following random assignments of the stroke animals to either
vehicle, low dose 90 k NPCs, or high dose 180 k NPCs, ANOVA
revealed significant treatment effects for both tests
(p's<0.0001). Pair-wise comparisons between treatment groups
revealed that as early as day 7 post-transplantation, stroke
animals that were transplanted with NPCs, regardless of the dose,
exhibited significant amelioration of behavioral deficits compared
to vehicle-treated stroke animals (p's<0.05). This behavioral
recovery by NPC transplanted stroke animals was sustained at day 14
and day 28 post-transplantation, in that NPC GFUs again regardless
of the dose promoted significant attenuation of both motor and
neurological impairments compared to vehicle treatment
(p's<0.0001). Closer examination of the two NPC doses revealed
that the high dose 180 k produced significantly better amelioration
of behavioral deficits compared to the low dose 90 k across all
post-transplantation test days for EBST (p's<0.01), and at days
14 and 28 post-transplantation test days for Bederson
(p's<0.0005). Results are shown in FIGS. 44-45.
[0255] The present behavioral data demonstrated the robust
therapeutic benefits of NPCs in that behavioral recovery was
detected as early as day 7 post-transplantation. Results further
revealed that the positive outcome of NPC grafts was stable up to
day 28 post-transplantation (the study cut-off period). Although
both low and high dose of NPCs promoted functional benefits, the
high dose provided significantly better behavioral recovery than
the low dose.
[0256] All stroke animals in this study were immunosuppressed. As
the vehicle-treated stroke animals did not display any observable
behavioral recovery, this eliminated the possible confounding
beneficial effects of the immunosuppressant CsA as seen previously
in studies incorporating delivery of the drug at pre-stroke period,
during or immediately after stroke. The observed behavioral
recovery being limited to NPC transplanted stroke animals indicate
that the source of the therapeutic effects is not likely from the
immunosuppression per se, but from the grafted cells.
[0257] Hematoxylin and eosin (H&E) and Nissl staining was
conducted to measure the maximum infarcted area in each animal
using an NIH imaging system. To calculate infarct volume, the
following formula was used=2 mm (thickness of the slice).times.[sum
of the infarction area in all brain slices (mm.sup.2)].
[0258] At one month post-transplantation, randomly selected animals
were euthanized for immunohistochemistry. Tissues were processed
using standard ABC method using the following procedures. 20 .mu.m
cryostat sectioned tissues were be examined at 4.times.
magnification and digitized using a PC-based Image Tools computer
program. Brain sections were blind-coded and Abercrombie's formula
was used to calculate the total number of immunopositive cells.
[0259] NPC survival following transplantation was assessed using
monoclonal human specific antibody HuNu, human cell surface markers
which do not cross react with rodent cell surface markers or other
rodent proteins. To detect expression of neuronal, glial and
oligodendrocyte phenotype in cell grafts, immunohistochemical
analysis, Neu-N and GFAP, was used, respectively. These cell
surface markers also revealed migration of engrafted NPCs.
[0260] Generally, the NPCs survived well in the striatum with a few
neurons positive for MAP2 expression at 1 month
post-transplantation. The graft survival did not differ
significantly between the two dose levels of NPCs. NPCs reduced the
ischemic cell loss in the stroke penumbra. The two dose levels
showed almost the same extent of neuro-rescue effects. Data from
these analyses is presented in FIGS. 46-48.
H. MNC EXAMPLES
[0261] The Examples set forth below are meant to be illustrative,
and in no way limiting, of the scope of the present invention.
[0262] Experimental Procedures.
[0263] Culturing of MASCs and Neuronal Induction
[0264] MASCs were isolated from Wistar rats generally as described
previously in S. Azizi et al. "Engraftment and migration of human
bone marrow stromal cells implanted in the brains of albino
rats--similarities to astrocyte grafts." Proc Natl Acad Sci USA,
1998;95:3908-13. The MASCs were labeled with green fluorescent
protein (GFP) by retroviral infection using the pBabe neo-GFP
vector generally as described in M. Dezawa et al., "Sciatic nerve
regeneration in rats induced by transplantation of in vitro
differentiated bone-marrow stromal cells." Eur J Neurosci.
2001;14:1771-6.
[0265] Neuronal induction from MASCs is generally as described in
M. Dezawa et al., "Specific induction of neuronal cells from
bone-marrow stromal cells and application for autologous
transplantation J Clin Invest. 2004;113:1701-10. Briefly, a vector
(pCI neo-NICD) containing the Notch intracellular domain (NICD) was
transfected into MASCs using Lipofectamin2000 (Invitrogen Corp.,
Carlsbad, Calif.). Cells were selected by G418 after 11 days. For
induction of MNCs, NICD-transfected MASCs were subcultured once
(60-70% confluence) and were incubated in alpha-MEM containing 10%
FBS, 5 .mu.M FSK (Calbiochem, La Jolla, Calif.), 10 ng/ml bFGF
(Peprotech, London, UK) and 10 ng/ml CNTF (R&D Systems,
Minneapolis, Minn.). Five days later, cells were transplanted into
the MCAo rat model. To characterize the induced MNCs in vitro,
immunocytochemistry was performed. Anti-MAP-2ab (Sigma, St. Louis,
Mo.), neurofilament-M (NF-M) (Chemicon, Temecula, Calif.) and
beta-tubulin isotype 3 (.beta.-tubulin3) (Sigma, St. Louis, Mo.)
were used as neuronal markers, and 90-95% of MNCs were shown to be
immunopositive for these markers.
[0266] MCAo Rat Model:
[0267] Male Wistar rats weighing 200-250 g were kept at room
temperature (24.degree. C.) with a 12-h light-dark cycle, and were
given food and water freely. The MCAo procedure was a modification
of the methods described in J. Koizumi et al., "Experimental
studies of ischemic brain edema. 1. A new experimental model of
cerebral embolism in rats in which recirculation can be introduced
in the ischemic area." Jpn J Stroke. 1986;8:1-8; and E. Longa et
al., "Reversible middle cerebral artery occlusion without
craniectomy in rats." Stroke. 1989;20:84-91. Briefly, under deep
anesthesia induced by a mixture of 1.0-1.5% halothane, 10% O2 and
air, a midline cervical incision was performed, and the left
carotid bifurcation was identified. A probe made of 4-0 Nylon
thread with a silicon rubber-coated head of diameter 0.3 mm was
inserted into the ligated external carotid artery and advanced into
the internal carotid artery to a position 16-18 mm from the
bifurcation. During the surgery, rectal temperature was maintained
between 37.5-38.0.degree. C. using a feedback-heating pad (BWT-100,
Bio Research Center Co. Ltd., Tokyo, Japan). Arterial blood gas
analysis was performed and p02 was maintained at 85-120 mmHg
through control of the anesthetic device. Reperfusion was performed
4 hours after the occlusion through a 10 mm withdrawal of the
probe.
[0268] Transplantation
[0269] On day 7 following the MCAo procedure, rats were
anesthetized with intraperitoneal injection of 50 mg/kg sodium
pentobarbital and placed onto a sterotaxic frame. In a preliminary
experiment, the infarct area was produced in the lateral area from
approximately 3.5 mm lateral to the midline. For transplantation
into the non-necrotic brain parenchyma, the cell suspension,
composed of 8000-16000 cultured cells in 3 .mu.l of phosphate
buffered saline (PBS, pH 7.4), was stereotaxically injected into
the left forebrain from the following 3 locations: +2 mm, 0 mm and
-2 mm anterior to the bregma, and 2 mm lateral to the midline and
at 1.2 mm depth from the cortical surface in each case. Total
numbers of transplanted cells were 24000-48000.
[0270] Three groups of animals were prepared; the control group,
which received only PBS (without cell transplantation) (n=7), the
MASC group, which underwent transplantation of non-treated intact
MASCs (n=10), and the NMC group, into which MNCs were transplanted
(n=10).
[0271] Behavioral Testing
[0272] The severity of neurological damage was evaluated using the
following tests: beam balance test, limb placing test and Morris
water maze test. Beam balance test and limb placing test were
performed on day 7 (just before transplantation), 14, 21, 28 and 35
after MCAo. Morris water maze test was performed from day 36 to 40
following the MCAo procedure.
[0273] Beam Balance Test
[0274] The beam balance test is used to assess gross vestibulomotor
function, and was carried out generally as described previously in
C. Dixon et al., "A fluid percussion model of experimental brain
injury in the rat." J Neurosurg. 1987;67:110-9. Scoring was based
on the following criteria: balancing with a steady posture with
paws on the top of the beam: a score of 0; grasping the sides of
the beam and/or shaky movement: 1; one or more paw(s) slipping off
the beam: 2; attempting to balance on the beam, but falling off: 3;
and falling off the beam with no attempt to balance or hang on:
4.
[0275] Limb Placing Test
[0276] The limb placing test examines sensorimotor integration in
limb placing responses to visual, tactile and proprioceptive
stimuli, and was performed generally as described previously in M.
De Ryck et al. "Photochemical stroke model: flunarizine prevents
sensorimotor deficits after neocortical infarcts in rats." Stroke,
1989;20:1383-1390. A proprioceptive adduction test was also
performed, again generally according to the procedures laid out in
the De Ryck et al. article. For each test, scoring was based on the
following criteria: immediate and complete placing of the limb: a
score of 0; incomplete and/or delayed (>2 seconds) placing,
including interspersed flailing: 1; and no placing: 2. Visual,
forward tactile and lateral tactile, proprioceptive stimuli were
given to right forelimb. Forward tactile and lateral tactile and
proprioceptive stimuli were given to right hindlimb. Proprioceptive
adduction test were performed in both forelimb and hindlimb. Total
score ranges 0-18.
[0277] Morris Water Maze Test
[0278] The Morris water maze test is a useful method to assess
cognitive function. Several modification of this test has been
reported. A version of the test generally as reported in A.
Fukunaga et al., "Differentiation and angiogenesis of central
nervous system stem cells implanted with mesenchyme into ischemic
rat brain." Cell Transplant. 1999;8:435-41 was used. This test was
performed from day 36 to day 40 after MCAo. A pool (diameter 150
cm, depth 35 cm) was prepared. An escape platform (diameter 10 cm)
was located 1 cm beneath the surface of the water rendered opaque
and milky white. Four starting points around the edge of the pool
were designed as N, E, S and W. The platform was kept in the middle
of a quadrant, equidistant from the center and the edge of the
pool. A rat was released into the water from each starting point
and allowed to swim until reaching the platform, and the time taken
to reach the platform was recorded (maximum of 120 seconds). Rats
were trained in the task using two sets of four trials on each of 5
consecutive days. After the first set on the fifth day, instead of
the second set, a spatial probe trial was performed. This test is
to estimate short memory retention. The platform was removed and
the rat was allowed to swim for 60 seconds. The number of times
each animal crossed the platform-located area was measured. The
time spent in the platform-located quadrant was also measured.
[0279] Histological Analysis
[0280] On day 41, rats were sacrificed with administration of a
pentobarbital overdose, and perfused transcardinally with 0.9%
saline followed by periodate-lysine-paraformaldehyde fixative
solution as generally described in I. McLean et al.,
"Periodate-lysine-paraformaldehyde fixative. A new fixation for
immunoelectron microscopy." J Histochem Cytochem. 1974;22:1077-83.
The brain was cut into coronal blocks of 2 mm thickness using Brain
Matrix (BAS Inc. Warwickshire, UK). 10 .mu.m-thick cryostat
sections were made from each block. Sections were stained with
hematoxylin and eosin (H&E) to evaluate the infarct area. The
images of sections were captured using a 1.times. objective lens
under a light microscope, and the lesion areas were traced using
Scion Image (Scion Corporation, Frederick, Md.). The infarct volume
was calculated generally as described previously in R. Swanson et
al., "A semiautomated method for measuring brain infarct volume." J
Cereb Blood Flow Metab. 1990;10:290-29, and expressed as a
percentage of the volume of the contralateral hemisphere.
[0281] For immunostaining, the sections were incubated with primary
antibodies to MAP-2 (1:100, Boehringer Mannheim, Germany),
.beta.-tubulin3 (1:400, Sigma, Missouri), NF-M (1:200, Boehringer
Mannheim, Germany), Tuj-1 (1:100, BAbCO, CA), or GFAP (1:400, Dako,
CA) at 4.degree. C. overnight. Alexa Fluor 546-conjugated
anti-mouse IgG (Molecular Probes, Eugene, Oreg.) (for MAP-2) or
anti-rabbit IgG (Molecular Probes, Eugene, Oreg.) (for
.beta.-tubulin3, NF-M, Tuj-1 and GFAP) was used as the secondary
antibody. TOTO-3 was used for the nuclear staining. Specimens were
inspected using confocal laser scanning microscopy (CLMS) (Radiance
2000, Bio-Rad, Hertfordshire, UK).
[0282] In each rat the total number of GFP-labeled cells in the
whole forebrain was calculated. The number of GFP-labeled cells in
the hippocampus also calculated in the same way.
[0283] Statistical Analysis
[0284] The behavioral evaluation data and infarct volume data were
analyzed using a non-repeated measures ANOVA. When the results were
significant (p<0.05), Student-Newman-Keuls post hoc procedure
was used at a 95% significant level. The values are presented as
mean.+-.standard deviation, unless otherwise stated.
EXAMPLE 10
Behavioral Testing
[0285] One week after MCAo was performed, and just before the
transplantation, severe right side neurological deficits were
apparent, and the mean score for each behavioral test showed no
statistical differences among the three groups.
[0286] Beam Balance Test
[0287] From day 7 to day 21, the mean score was not statistically
different among the three groups. On day 28 and 35, the mean score
of the NMC group showed a significant improvement, compared with
the control (day 28: p=0.0041, day 35: p=0.0001) and MASC groups
(p=0.0471, 0.0007 respectively). Although MASC group showed slight
improvement compared with the control, statistically significant
difference could not be detected on day 28 and 35 (FIG. 40).
[0288] Limb Placing Test
[0289] There were no statistical differences between the mean score
of three groups from day 7 to day 21. On day 28 and 35, the NMC and
MASC groups showed a significant improvement, compared with the
control group (day28: p=0.0022 and 0.085, day35: p=0.0022 and
0.0211 respectively). However, the mean score showed no significant
difference between the NMC and MASC groups throughout the entire
period (FIG. 41).
[0290] Morris Water Maze Test
[0291] The mean latency time recorded in each set of four trials to
locate the submerged escape platform is shown in FIG. 42 for each
of the three groups. The NMC group showed the shortest latency time
compared to the control and MASC group. The mean latency time for
the second set on day 39 and the first set on day 40 demonstrated
significant difference between the NMC and control groups (p=0.0339
and 0.0492 respectively) (FIG. 42). Although the NMC group showed a
tendency to take shorter latency time to the escape platform than
MASC group, a statistically significant difference did not
exist.
[0292] In the spatial probe trial, rats in the NMC group showed a
significant improvement compared with the control (p=0.0419) and
MASC group (p=0.0453) (FIG. 43). The mean time spent in the
platform-located quadrant was the longest in NMC group among three
groups. Significant difference existed between NMC and control
(p=0.0339) (FIG. 43).
EXAMPLE 11
Histological Study
[0293] The infarct area was located in the lateral half of the left
hemisphere including cortex, striatum and hippocampus, and
formation of cysts and scars was observed in most lesioned brains.
The hippocampus of the lesion side was atrophic and showed
partially irregular arrangement or loss of neurons compared with
contralateral side. Infarct volumes were measured in all MCAo
models. The mean infarct volume in the NMC, MASC and control groups
on day 33 were 50.7.+-.10.9%, 51.0.+-.10.2% and 50.9.+-.11.1%
respectively. There was no statistically significant difference
among the three groups.
[0294] Transplanted GFP-labeled MASCs and MNCs were located mainly
at the boundary area between intact tissue and infarct area
including the ipsilateral cortex, corpus callosum, striatum and
hippocampus. The infiltration of inflammatory cells into the
infarct focus was observed. There seemd to be no difference in the
number of inflammatory cells that infiltrated inot the infarct
locus between the MNC and MASC groups.
[0295] A large number of GFP-labeled MNCs were immunopositive for
MAP-2 and showed neurite development in the host brain. They were
also immunopositive for Tuj-1 and .beta.-tubulin3. In the
ipsilateral hippocampus, many cell bodies and neurites of
GFP-labeled MNCs were also shown to be NF-M positive. A large
fraction of GFP-labeled transplanted MNCs were positive for MAP-2
(84.0.+-.8.1%), whereas only a small number of cells were positive
for GFAP (1.0.+-.0.2%).
[0296] In contrast, the large majority of MASCs in the host brain
were negative for both neuronal (MAP-2, Tuj1, .beta.-tubulin3 and
NF-M) and glial (GFAP) markers. The percentages of MAP-2- and
GFAP-positive cells among the GFP-labeled cells were 1.4.+-.0.2%
and 4.8.+-.1.0%, respectively. The formation of neurites in MAP-2
positive MASC could not be found.
[0297] The mean number of MNCs and MASCs in the host forebrain were
13250.+-.1126 and 5850.+-.997. Approximately 30-45% of transplanted
MNCs were detected and, on the other hand, 10-20% of transplanted
MASCs were detected one month after the transplantation. The
survival ratio of MNCs in the ischemic brain was substantially
higher than MASCs. In the hippocampus, the mean number of MNCs was
790.+-.160 and 89% of them were MAP2-positive. The mean number of
MASCs, in contrast, was 470.+-.66 and 0.6% were MAP2-positive,
showing that most of transplanted MASCs were negative for both
neuronal and glial markers.
[0298] In all groups, tumor formation was not observed in the brain
parenchyma up to 41 days after MCAo.
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