US20060216276A1 - Use of materials for treatment of central nervous system lesions - Google Patents

Use of materials for treatment of central nervous system lesions Download PDF

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US20060216276A1
US20060216276A1 US11/368,919 US36891906A US2006216276A1 US 20060216276 A1 US20060216276 A1 US 20060216276A1 US 36891906 A US36891906 A US 36891906A US 2006216276 A1 US2006216276 A1 US 2006216276A1
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cells
neuronal
transplantation
marrow
patient
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Mari Dezawa
Keita Mori
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Sanbio Inc
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Sanbio Inc
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Priority to US12/462,143 priority patent/US8092792B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention relates to treatment of central nervous system lesions, particularly to treatment of stroke.
  • CNS central nervous system
  • Stroke is characterized by the sudden loss of circulation to an area of the brain, resulting in a corresponding loss of neurologic function.
  • 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.
  • 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.
  • the four major neuroanatomic ischemic stroke syndromes are caused by disruption of their respective cerebrovascular distributions.
  • Anterior cerebral artery occlusions primarily affect frontal lobe function, producing altered mental status, impaired judgment, contralateral lower extremity weakness and hypesthesia, and gait apraxia.
  • MCA Middle cerebral artery 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.
  • Posterior cerebral artery occlusions affect vision and thought, producing homonymous hemianopsia, cortical blindness, visual agnosia, altered mental status, and impaired memory.
  • 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.
  • 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 myocardial infarction
  • MI myocardial infarction
  • atrial fibrillation dilated cardiomyopathy
  • atrial myxomas atrial myxomas.
  • MI is associated with a 2-3% incidence of embolic stroke, of which 85% occur in the first month after MI.
  • 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.
  • thrombotic occlusion 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
  • 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).
  • thrombotic stroke e.g., trauma, thoracic aortic dissection, arteritis.
  • hypoperfusion distal to a stenotic or occluded artery or hypoperfusion of a vulnerable watershed region between two cerebral arterial territories can cause ischemic stroke.
  • ICH intracerebral hemorrhage
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention in another aspect, 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.
  • the invention in still another aspect, 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.
  • 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.
  • FIG. 1 shows results from the MCAo procedure.
  • FIG. 2 shows results from the MCAo procedure.
  • FIG. 3 shows results from the MCAo procedure.
  • FIG. 4 shows results from the MCAo procedure.
  • FIG. 5 shows results from the MCAo procedure.
  • FIG. 6 shows results from the MCAo procedure.
  • FIG. 7 shows results from the MCAo procedure.
  • FIG. 8 shows results from the MCAo procedure.
  • FIG. 9 shows results from the MCAo procedure.
  • FIG. 10 shows results from the MCAo procedure.
  • FIG. 11 shows results from the MCAI procedure.
  • FIG. 12 shows results from the MCAI procedure.
  • FIG. 13 shows results from the MCAI procedure.
  • FIG. 14 shows results from the MCAI procedure.
  • FIG. 15 shows results from the MCAI procedure.
  • FIG. 16 shows results from the MCAI procedure.
  • FIG. 17 shows results from the MCAI procedure.
  • FIG. 18 shows results from the MCAI procedure.
  • FIG. 19 shows results from the MCAI procedure.
  • FIG. 20 shows results from the MCAI procedure.
  • FIG. 21 shows results from the TGI procedure.
  • FIG. 22 shows results from the TGI procedure.
  • FIG. 23 shows results from the TGI procedure.
  • FIG. 24 shows results from the TGI procedure.
  • FIG. 25 shows results from the TGI procedure.
  • FIG. 26 shows results from the TGI procedure.
  • FIG. 27 shows results from the TGI procedure.
  • FIG. 28 shows results from the TGI procedure.
  • FIG. 29 shows histological results from the Examples.
  • FIG. 30 shows histological results from the Examples.
  • FIG. 31 illustrates functional recovery and graft survival.
  • FIG. 32 shows results from the MCAo procedure.
  • FIG. 33 shows results from the MCAo procedure.
  • FIG. 34 shows results from the MCAI procedure.
  • FIG. 35 shows results from the MCAI procedure.
  • FIG. 36 shows results from the TGI procedure.
  • FIG. 37 shows results from the TGI procedure.
  • FIG. 38 illustrates graft survival
  • FIG. 39 illustrates graft survival.
  • 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
  • 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.
  • FIG. 42 shows the results of a Morris water maze test.
  • the mean latency time for the MNC group was significantly different to those for the MASC and control groups.
  • 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
  • FIG. 44 shows results from Bederson testing performed in Example 9.
  • FIG. 45 shows results from EBST performed in Example 9.
  • FIG. 46 illustrates graft survival according to Example 9.
  • FIG. 47 shows graft survival according to Example 9.
  • FIG. 48 shows the results of Nissl staining according to Example 9.
  • the inventors have unexpectedly and surprisingly discovered that the problems and limitations noted above can be overcome by practicing the invention disclosed herein.
  • 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.
  • 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.
  • administering means providing NPCs and/or inventive grafts to a patient.
  • 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.
  • 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.
  • a CNS ischemic event comprises an ischemic stroke.
  • 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).
  • 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.
  • “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.
  • “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.
  • “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.
  • GFU Global Forming Unit
  • mixtures of NPCs and MNCs are expressed contemplated.
  • NPCs are present substantially without MNCs.
  • MNCs are present substantially without NPCs.
  • 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.
  • MNCs “MASC-derived Neuronal Cells” 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+).
  • 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.
  • MCAo middle cerebral artery occlusion
  • MCAI middle cerebral artery ligation
  • 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.
  • Neuronal Precursor Cells 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.
  • MSCs marrow-adherent stem cells
  • NPCs comprise human NPCs, although NPCs of other mammalian species are also encompassed within the scope of this invention.
  • 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.
  • 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.
  • Neuronal means neurons, glia, and oligodendrocytes, and precursors of any of the foregoing.
  • Patient means an animal, typically a mammal, and more typically, a human, in need of treatment for a disease or disorder.
  • “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.
  • 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.
  • TGI transient global ischemia
  • 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.
  • Transdifferentiated means development of a cell along a lineage different from that classically associated with that cell type.
  • 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.
  • NPCs could differentiate into neurons that replace damaged endogenous neurons.
  • 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.
  • 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”).
  • 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.
  • 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.
  • graft forming units suitable for use in the present invention comprise sterile compositions that comprise the NPCs.
  • suitable pharmaceutically acceptable carriers may include physiological saline, normasol, isolyte, plasma-lyte, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • 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.
  • 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.
  • Graft forming unit dosage form refers to physically discrete units suited as unitary dosages for the subject to be treated.
  • 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/ ⁇ L to about 100,000 cells/ ⁇ L, and more preferably from about 1,000 cells/ ⁇ L to about 50,000 cells/ ⁇ L.
  • the GFUs can be included in a container, pack, or dispenser together with instructions for administration.
  • the grafts are preferably stored at approximately 37° C.
  • 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.
  • pre-clinical i.e. non-human
  • 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.
  • brain sections may be doubly-immunostained for green fluorescent protein (GFP) or other cell labels, ⁇ -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.
  • GFP green fluorescent protein
  • ⁇ -tubulin III ⁇ -tubulin III
  • NeuN a neuron-specific protein
  • GFAP glial fibrillary acidic protein
  • O4 an oligodendrocyte-specific protein
  • 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.
  • 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.).
  • 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.
  • labeling may be performed according to the transfection protocols available from the manufacturer, such as the Invitrogen system referred to above.
  • 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° C.
  • 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.
  • 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”).
  • 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.
  • 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.
  • 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.
  • graft forming units suitable for use in the present invention comprise sterile compositions that comprise MNCs.
  • suitable pharmaceutically acceptable carriers may include physiological saline, Cremophor EL.TM. (BASF; Parsippany, N.J.), normasol, isolyte, plasma-lyte, or phosphate buffered saline (PBS).
  • 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.
  • 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.
  • Graft forming unit dosage form refers to physically discrete units suited as unitary dosages for the subject to be treated.
  • 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/ ⁇ L to about 100,000 cells/ ⁇ L, and more preferably from about 1,000 cells/ ⁇ L to about 50,000 cells/ ⁇ L.
  • the GFUs can be included in a container, pack, or dispenser together with instructions for administration.
  • the grafts are preferably stored at approximately 4° C.
  • 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.
  • brain sections may be doubly-immunostained for green fluorescent protein (GFP) or other cell labels, ⁇ -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.
  • GFP green fluorescent protein
  • ⁇ -tubulin III ⁇ -tubulin III
  • NeuN a neuron-specific protein
  • GFAP glial fibrillary acidic protein
  • O4 an oligodendrocyte-specific protein
  • 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.
  • 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.
  • 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.
  • 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.
  • a carrier e.g., a biodegradable capsule
  • 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.
  • 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.
  • NPCs and/or GFUs according to the invention may be administered locally to a patient's central nervous system lesion.
  • 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.
  • transplantation may be carried out using stereotactic surgical 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.
  • a needle pass using a 26-gauge needle and Hamilton micro-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 ⁇ L/min, more preferably from about 0.5 to about 5 ⁇ L/min, and still more preferably from about 1.0 to about 3.0 ⁇ L/min.
  • 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.
  • Analgesics e.g., buprenorphine
  • antibiotics e.g., Cephazolin, 50 mg/kg, IM, b.i.d. ⁇ 5 days
  • Antibiotic treatment may be continued post-surgically for an extended period, preferably up to 30 days following surgery, to suppress opportunistic infection.
  • 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.
  • 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®, Injection; manufactured by Novartis Pharma AG, Basel, Switzerland for Novartis Pharmaceuticals Corporation (Novartis), East Hanover, N.J.
  • 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.
  • 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.
  • 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.
  • a carrier e.g., a biodegradable capsule
  • NPCs and/or GFUs according to the invention may be administered locally to a patient's central nervous system lesion.
  • 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.
  • 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.
  • 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.
  • a carrier e.g., a biodegradable capsule
  • MNCs and/or GFUs according to the invention may be administered locally to a patient's central nervous system lesion.
  • 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.
  • transplantation may be carried out using stereotactic surgical 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.
  • a needle pass using a 26-gauge needle and Hamilton micro-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 ⁇ L/min, more preferably from about 0.5 to about 5 ⁇ L/min, and still more preferably from about 1.0 to about 3.0 ⁇ L/min.
  • 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.
  • Analgesics e.g., buprenorphine
  • antibiotics e.g., Cephazolin, 50 mg/kg, IM, b.i.d. ⁇ 5 days
  • Antibiotic treatment may be continued post-surgically for an extended period, preferably up to 30 days following surgery, to suppress opportunistic infection.
  • 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.
  • 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®, Injection; manufactured by Novartis Pharma AG, Basel, Switzerland for Novartis Pharmaceuticals Corporation (Novartis), East Hanover, N.J.
  • 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.
  • allogeneic transplantation (same species graft forming units) is preferable.
  • allogeneic transplantation mimics the clinical setting in which allogeneic transplantation of NPCs in patients suffering from central nervous system lesion may take place.
  • MCAo middle cerebral artery occlusion
  • MCAI middle cerebral artery ligation
  • TGI transient global ischemia
  • 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.
  • 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).
  • allogeneic transplantation (same species grafts forming units) is preferable.
  • 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.
  • the MASC group demonstrated slight improvements in behavioral assessment tests compared with control group, but not as much as the NMC group.
  • MNC transplantation 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.
  • MNCs in the cortex, striatum and hippocampus demonstrated extension of neuritis in the host brain, which could not be observed in the MASC-group.
  • 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.
  • mice 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.).
  • 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.
  • 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.
  • the elevated body swing test 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.
  • 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 ⁇ 40 ⁇ 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • NPCs cryopreserved human embryonal carcinoma-derived neurons
  • 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.
  • test animals underwent the MCAo procedure as described above, and were evaluated weekly for four weeks port-transplantation with the following results.
  • test animals underwent the MCAI procedure as described above, and were evaluated weekly for four weeks post-transplantation with the following results.
  • test animals underwent the TGI procedure as described above, and were evaluated weekly for four weeks port-transplantation with the following results.
  • NPC graft migration GFP epifluorescence revealed that majority (approx. 55%-85%) of the transplanted cells remained within the original transplant site ( FIG. 32 ).
  • 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%).
  • MCAo and MCAI transplanted animals displayed more migration of grafted cells compared to TGI transplanted animals.
  • 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.
  • 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.
  • MCAI a medial to lateral (4.4 mm) migration of cells was seen.
  • 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).
  • 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.
  • test animals that underwent the TGI procedure as described above were evaluated with the following results.
  • test animals that underwent the MCAI procedure as described above were evaluated with the following results.
  • test animals that underwent the TGI procedure as described above were evaluated with the following results.
  • 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.
  • 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.
  • 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.
  • MCAI a medial to lateral (4.5 mm) migration of cells was seen.
  • 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).
  • 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.
  • 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.
  • CsA Cyclosporin-A
  • 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.
  • a Laser Doppler was used to determine successive occlusion and reperfusion.
  • 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.
  • 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° C. throughout surgery and following recovery from anesthesia.
  • 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.
  • Hematoxylin and eosin (H&E) and Nissl staining was conducted to measure the maximum infarcted area in each animal using an NIH imaging system.
  • H&E Hematoxylin and eosin
  • Nissl staining was conducted to measure the maximum infarcted area in each animal using an NIH imaging system.
  • 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.
  • HuNu monoclonal human specific antibody
  • 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 .
  • 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.
  • GFP green fluorescent protein
  • 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.
  • pCI neo-NICD containing the Notch intracellular domain
  • NICD-transfected MASCs were subcultured once (60-70% confluence) and were incubated in alpha-MEM containing 10% FBS, 5 ⁇ 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.
  • NF-M neurofilament-M
  • ⁇ -tubulin3 beta-tubulin isotype 3
  • rats were anesthetized with intraperitoneal injection of 50 mg/kg sodium pentobarbital and placed onto a sterotaxic frame.
  • the infarct area was produced in the lateral area from approximately 3.5 mm lateral to the midline.
  • the cell suspension composed of 8000-16000 cultured cells in 3 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m-thick cryostat sections were made from each block.
  • Sections were stained with hematoxylin and eosin (H&E) to evaluate the infarct area.
  • H&E hematoxylin and eosin
  • the images of sections were captured using a 1 ⁇ objective lens under a light microscope, and the lesion areas were traced using Scion Image (Scion Corporation, Frederick, Md.).
  • 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.
  • MAP-2 (1:100, Boehringer Mannheim, Germany), ⁇ -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° 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 ⁇ -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).
  • the mean score was not statistically different among the three groups.
  • MASC group showed slight improvement compared with the control, statistically significant difference could not be detected on day 28 and 35 ( FIG. 40 ).
  • 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 NMC group showed a tendency to take shorter latency time to the escape platform than MASC group, a statistically significant difference did not exist.
  • 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.
  • 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.
  • GFP-labeled MNCs were immunopositive for MAP-2 and showed neurite development in the host brain. They were also immunopositive for Tuj-1 and ⁇ -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%).
  • MAP-2 neuronal
  • Tuj1 ⁇ -tubulin3 and NF-M
  • GFAP glial
  • 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.
  • 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.
  • N2N cells cryopreserved human embryonal carcinoma-derived neurons
  • GABA receptor agonist promotes reformation of the striatonigral pathway by transplant derived from fetal striatal promordia in the lesioned striatum. Exp Neurol. 1997;147:503-509.
  • Nishino H and Borlongan C V Restoration of function by neural transplantation in the ischemic brain. Prog Brain Res. 2000;127:461-76.
  • Pakzaban P Isacson O. Neural xenotransplantation: reconstruction of neuronal circuitry across species barriers. Neuroscience. 1994;62:989-1001.

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