Methods and compositions for treating neural degeneration

Abstract

To provide adult neural stem cells and various adult pluripotent cells for the treatment of neurodegenerative diseases, new methods and compositions for treatment of neurodegenerative diseases.SOLUTION: Disclosed herein are methods and compositions for the use of marrow adherent stem cells and their descendants e.g. bone marrow-derived neural regenerating cells in the treatment of various neurodegenerative disorders. In certain embodiments, bone marrow-derived neural regenerating cells transplanted to sites of neural degeneration stimulate growth and / or survival of host neurons.SELECTED DRAWING: None

Description

[Technical field]

[0001] Field The methods and compositions disclosed herein are in the field of neurodegeneration (eg, neurodegenerative diseases) and treatment thereof. [Background technology]

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 60 / 964,763, filed August 15, 2007, U.S. Provisional Application No. 61 / 000,972, filed October 30, 2007, U.S. Provisional Application No. 61 / 005,181, filed December 3, 2007, and U.S. Provisional Application No. 61 / 125,991, filed April 30, 2008; the disclosures of which are incorporated by reference in their entireties for all purposes.

[0003] Statement of Federal Support Not applicable.

[0004] background Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis degeneration (ALS) are characterized by the death and / or degeneration of neurons and neural tissue. Since terminally differentiated neurons are not limited in their ability to proliferate, a possible treatment for conditions characterized by neurodegeneration is to provide new neural cells to replace the death and / or loss of nerves and neural tissue. So far, neural cell transplantation has been attempted in animal experiments using embryonic or adult neural stem cells, ES cells and embryonic neural cells. However, such use faces a major obstacle to its application in humans. The use of embryonic stem cells or neural cells is accompanied by ethical issues, and the problem of ensuring a stable supply is also a concern. The proven differentiation ability of ES cells currently attracts great attention, but the cost and labor required to induce differentiation into specific cell types, as well as the risk of teratoma formation after transplantation, are factors that delay the stable application of this technology. On the other hand, patients undergoing transplantation of adult neural stem cells are exposed to tremendous risks and burdens. These cells are found in a very limited core of the central nervous system (i.e., deep in the brain) and therefore must be removed by cranial incision. Finally, attempts to treat neurodegeneration and neuronal injury by transplantation of other types of pluripotent cells, such as hematopoietic stem cells, have had limited success due to the difficulty of controlling the differentiation of the transplanted cells.

[0005] As a result of the above-mentioned difficulties associated with the use of embryonic cells, there is a need for adult neural stem cells and various adult pluripotent cells, new methods and compositions for the treatment of neurodegenerative diseases. Summary of the Invention

[0006] overview Novel methods of treating neurodegenerative diseases disclosed herein utilize cells that, when transplanted into the site of neurodegeneration, act on surrounding non-neuronal cells by inducing differentiation of the surrounding non-neuronal cells into neural or neural tissue, by promoting the recovery of damaged neural cells or dying neurons, and / or by promoting the survival of host neural cells. Additional novel therapeutic methods include transplantation of cells that inhibit the action of host cells that mediate or participate in the process of neural cell death and / or degeneration.

[0007] Thus, disclosed herein are methods for nerve regeneration that can be used, for example, in the treatment of neurodegenerative diseases and neurodegenerative disorders. Such methods include transplantation of bone marrow derived cells that have been induced to acquire certain properties that promote nerve regeneration at or near the site of neurodegeneration. Thus, in one embodiment, the disclosed methods for nerve regeneration and treatment of neurodegeneration include transplantation of bone marrow adherent stromal cells or bone marrow derived nerve regenerating cells at or near the site of neurodegeneration.

[0008] The disclosed methods and compositions are useful for the treatment of degeneration in either the central or peripheral nervous system. For example, the disclosed methods and compositions can be used to treat central nervous system diseases such as Parkinson's disease, amyotrophic lateral sclerosis (ALS), stroke, and Alzheimer's disease. Neurodegeneration or physical damage to nerves in the peripheral nervous system can also be treated using the disclosed methods and compositions.

[0009] In one embodiment, the bone marrow derived neural regenerating cells are derived from bone marrow stromal cells (also known as bone marrow adherent stromal cells) that have been modified to express the amino acid sequence of the Notch intracellular domain (NICD). In one embodiment, the bone marrow stromal cells are transfected with a nucleic acid or polynucleotide comprising a sequence encoding the Notch intracellular domain, and the bone marrow derived neural regenerating cells derived from the transfected cells are used for transplantation. In one embodiment, the Notch intracellular domain consists of amino acids 1703-2504 of the human Notch protein. Ellison et al. (1991) Cell 66: 649-661.

[0010] Without being bound to any particular theory, in certain embodiments, the transplanted bone marrow adherent stromal cells and bone marrow derived neural regenerative cells exert their regenerative effect by supporting the growth of host nerve fibers (e.g., axons) at or near the site of neurodegeneration. In other embodiments, the transplanted bone marrow adherent stromal cells and bone marrow derived neural regenerative cells exert their regenerative effect by promoting the survival of host neurons at or near the site of neurodegeneration. Their regenerative effect is obtained from the secretion of one or more trophic factors by the transplanted cells. Alternatively, regeneration is obtained from the production of one or more extracellular matrix molecules (s) by the transplanted cells that promote the growth and / or survival of host neural cells. The regenerative effect is also obtained from the combined effect of secreted diffusible factors (e.g., growth factors) and non-diffusible factors H (e.g., extracellular matrix).

[0011] Accordingly, the present disclosure includes, but is not limited to, the following number of aspects: 1. A method for providing trophic support to damaged neural tissue in a host, comprising: A method comprising transplanting bone marrow adherent stromal cells or bone marrow derived neural regenerating cells at or near the site of neurodegeneration in a host. 2. The method of embodiment 1, wherein the site of neurodegeneration is in the central nervous system. 3. The method of embodiment 1, wherein the site of neurodegeneration is in the peripheral nervous system. 4. The method of embodiment 2, wherein said neurodegeneration is associated with Parkinson's disease. 5. The method of embodiment 1, wherein said bone marrow-derived neural regenerating cells are progeny of bone marrow stromal cells that have been transfected with a polynucleotide comprising a sequence encoding a Notch intracellular domain (NICD). 6. The method of embodiment 5, wherein the NICD consists of amino acids 1703 to 2504 of Notch-1 protein. 7. The method of embodiment 1, wherein said bone marrow adherent stromal cells or said bone marrow derived neural regenerating cells support the growth of host neural fibers at or near a site of neurodegeneration. 8. The method of embodiment 7, wherein the nerve fiber is an axon. 9. The method of embodiment 1, wherein said bone marrow adherent stromal cells or said bone marrow derived neural regenerating cells promote survival of host neurons at or near a site of neurodegeneration. 10. The method of embodiment 1, wherein said bone marrow adherent stromal cells or said bone marrow derived neural regenerating cells secrete one or more trophic factors. 11. A method for nerve regeneration, comprising transplanting bone marrow adherent stromal cells or bone marrow derived nerve regenerating cells at or near the site of nerve degeneration. 12. The method of embodiment 11, wherein the site of neurodegeneration is in the central nervous system. 13. The method of embodiment 11, wherein the site of neurodegeneration is in the peripheral nervous system. 14. The method of embodiment 12, wherein the neurodegeneration is associated with Parkinson's disease. 15. The method of embodiment 11, wherein the bone marrow-derived neural regenerating cells are progeny of bone marrow stromal cells that have been transfected with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD). 16. The method of embodiment 15, wherein the NICD consists of amino acids 1703 to 2504 of Notch-1 protein. 17. The method of embodiment 11, wherein the bone marrow adherent stromal cells or the bone marrow derived neural regenerating cells support the growth of host neural fibers at or near a site of neurodegeneration. 18. The method of embodiment 17, wherein the nerve fiber is an axon. 19. The method of embodiment 11, wherein the bone marrow adherent stromal cells or the bone marrow derived neural regenerating cells promote survival of host neurons at or near a site of neurodegeneration. 20. The method of embodiment 11, wherein the bone marrow adherent stromal cells or the bone marrow derived neural regenerating cells secrete one or more trophic factors. 21. A method for providing a nutrient carrier to damaged neural tissue, comprising transplanting bone marrow adherent stromal cells or bone marrow derived neural regenerating cells at or near the site of neurodegeneration in a host. 22. The method of embodiment 21, wherein the nutrient carrier promotes survival of host neural cells. 23. The method of embodiment 21, wherein the nutrient carrier inhibits death of host neural cells. 24. The method of embodiment 23, wherein the cell death is by apoptosis. 25. The method of embodiment 21, wherein the nutrient carrier enhances function of a host neuronal cell. 26. The method of embodiment 21, wherein the nutrient carrier stimulates growth of host neural cells. 27. The method of embodiment 26, wherein nerve fiber growth is stimulated. 28. The method of embodiment 27, wherein the nerve fiber is an axon. 29. The method of embodiment 27, wherein the nerve fiber is a dendrite. 30. The method of embodiment 26, wherein new synapses are formed. 31. The method of embodiment 21, wherein the nutrient carrier promotes the rescue of host stem cells to damaged tissue. 32. The method of embodiment 21, wherein the damaged nervous tissue is in the central nervous system. 33. The method of embodiment 21, wherein the damaged nerve tissue is in the peripheral nervous system. 34. The method of embodiment 32, wherein the damaged neural tissue is associated with Parkinson's disease. 35. The method of embodiment 21, wherein said bone marrow-derived neural regenerating cells are progeny of bone marrow adherent stromal cells that have been transfected with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD). 36. The method of embodiment 35, wherein the NICD consists of amino acids 1703 to 2504 of Notch-1 protein. 37. The method of embodiment 21, wherein the bone marrow derived neural regenerating cells or the bone marrow derived neural regenerating cells secrete one or more trophic factors. 38. The method of embodiment 17, wherein the bone marrow adherent stromal cells or the bone marrow derived neural regenerating cells secrete one or more of vascular endothelial growth factor, hepatocyte growth factor, bone morphogenetic protein 4, Dkk-1, fibroblast growth factor-7, heparin-binding epidermal growth factor-like growth factor, interleukin-6, interleukin-8, monocyte chemotactic protein-1, matrix metalloproteinase-1, platelet-derived growth factor AA, and alpha-transforming growth factor. [Brief description of the drawings]

[0012] [Figure 1] Figure 1 shows the effect of factors secreted by MASCs on neurite outgrowth in cultures of primary rat neurons, as assessed by MAP2 staining. Neurons were cultured in complete neurobasal medium (positive control; top left panel) or αMEM (negative control; top center panel), or co-cultured for 2 days in αMEM with different numbers of MASCs (as shown) separated from rat neurons by a semipermeable membrane (top right and three bottom panels). [Diagram 2]FIG. 2 shows the relative number of surviving neurons based on an assay of intracellular LDH in primary rat cortical neurons co-cultured with MASC and NRC. The five left bars show the titration results of MASC from Donor 42 (D42) co-cultured with cortical neurons in serum-free αMEM. The number of D42 MASC (x 10-3) subjected to co-culture with the neurons is shown on the abscissa. The sixth bar from the left shows intracellular LDH from positive control neurons grown in complete neuronal medium ("Compl. NB") without co-culture. On the right side of the figure, the effect of co-culture with MASC and NRC is evaluated. The fourth bar from the right shows the relative number of neurons in positive cultures grown in complete neuronal medium ("Compl. NB") without co-culture. The three right bars show the results obtained with neurons cultured in serum-free αMEM. The third bar from the right shows results for neurons that were not co-cultured; the second bar from the right shows results for neurons that were co-cultured with 5,000 D31 MASCs; and the right-most bar shows results for neurons that were co-cultured with 5,000 D31 NRCs. [Diagram 3] Figure 3 shows the relative number of surviving neurons, based on an assay of intracellular LDH, in primary rat cortical neurons co-cultured with MASC and NRC. On the right side of the figure, the effect of co-culture with MASC and NRC in serum-free αMEM is evaluated. The left five bars show the results without co-culture; the third and fourth bars from the right show the results for neurons co-cultured with 5,000 or 10,000 D39 MASC, respectively; and the rightmost bar and the second bar from the right show the results for neurons co-cultured with 5,000 or 10,000 D39 NRC, respectively. [Figure 4] Figure 4 shows cell damage (expressed as fold change in LDH release compared to control) in cultures of primary neurons subjected to oxygen / glucose deprivation (OGD) with or without subsequent co-culture with bone marrow adherent stromal cells (MASC) or neural regenerative cells (NRC). The graph pools results obtained with cells from six donors. For all samples, p<0.05 vs. OGD only. [Diagram 5] FIG. 5 shows cell damage (expressed as fold change in LDH release compared to control) in cultures of primary neurons subjected to oxygen / glucose deprivation (OGD) with or without co-culture with different types of conditioned medium (CM) from bone marrow adherent stromal cells (MASC) or neural regenerative cells (NRC). "CM from NB" refers to conditioned medium from donor cells (MASC or NRC) cultured in neuronal medium; "CM from OGD" refers to conditioned medium from donor cells cultured in neuronal medium conditioned by OGD-injured neurons per se; "CM from CNTRL" refers to conditioned medium from donor cells cultured in neuronal medium conditioned by non-injured neurons per se. For all samples, p<0.05 vs. OGD only. [Figure 6] FIG. 6 shows cell damage (expressed as fold change in LDH release compared to control) in cultures of primary neurons subjected to oxygen / glucose deprivation (OGD) and then co-cultured with different types of conditioned medium (CM) from bone marrow adherent stromal cells (MASC) or neural regenerative cells (NRC). Conditioned medium was diluted 1:1, 1:10, 1:25, 1:50, 1:100 or 1:200 as indicated. Control neurons (NB / B27 Med) were co-cultured with neuronal medium (see Example 7 for composition). The ability of donor cell-conditioned medium to rescue damaged neurons is dose-dependent. *p<0.05 vs. OGD only; #p<0.05 vs. CM 1:1; §p<0.05 vs. CM 1:10; mean±SD. [Figure 7] Figure 7 shows cell damage (expressed as fold change in LDH release compared to control) in rat hippocampal slices subjected to oxygen / glucose deprivation (OGD) with or without subsequent co-culture with bone marrow adherent stromal cells (OGD+MASC) or neural regenerative cells (OGD+NRC). The graph shows the results of co-culture of hippocampal slices with MASC or NRC from six donors. For all samples, p<0.05 vs. OGD only. [Figure 8]Figure 8 shows cell damage (expressed as fold change in LDH release compared to control) in rat hippocampal slices subjected to oxygen / glucose deprivation (OGD) and then co-cultured (24 hours) or not with conditioned medium (CM) from bone marrow adherent stromal cells (MASC) or neural regenerative cells (NRC). For all samples, p<0.05 vs. OGD only. [Figure 9] FIG. 9 shows cell damage (expressed as fold change in LDH release compared to control) in cultures of primary neural neurons subjected to oxygen / glucose deprivation (OGD) and further cultured for 24 hours in neural medium (OGD only; left bar), diluted conditioned medium from NRCs cultured for 48 hours in the presence of hippocampal slices (OGD+NRC CM from CNTRL slices (1:25); middle bar), or diluted conditioned medium from NRCs cultured for 48 hours in the presence of OGD-injured hippocampal slices (OGD+NRC CM from OGD slices (1:25); right bar). *p<0.05 vs. OGD only group; #p<0.05 OGD+NRC CM from CNTRL slices. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Detailed Description The terms "transplant," "graft," and "transplant" refer to the placement of exogenous cells into a particular location in a subject or tissue. "Autologous" transplantation refers to the transplantation of a subject's own cells into a subject (e.g., cells are removed from one site in a subject and transferred to another site in the same subject, with or without additional processing of the cells between removal and transfer). "Allogeneic" transplantation refers to the transfer of cells into a subject, where the cells are obtained from a different individual.

[0014] The terms "bone marrow stromal cells", "bone marrow adherent stromal cells", "bone marrow adherent stem cells", "marrow stem cells", "bone marrow stem cells", "mesenchymal cells" and "MASC" refer to dividing, multifunctional cells obtained from bone marrow that can give rise to a number of differentiated cell types during normal development, such as bone cells, and cells normally found in connective tissues, including, but not limited to, chondrocytes and adipocytes. MASCs may be human cells, or cells from other mammals or vertebrates.

[0015] The terms "neural regenerative cells", "neuronal regenerative cells", "NRCs" and "bone marrow-derived regenerative cells" are used interchangeably to refer to dividing cells derived from bone marrow adherent stromal cells that have the ability to promote growth and survival and / or inhibit apoptosis and death of damaged neural cells such as neurons, glia and their precursors. They are therefore distinct from primary neural precursor cells, such as those obtained from fetal and adult tissues such as the hippocampus and periventricular subependymal sites. NRCs can be human cells or cells derived from other mammals or vertebrates.

[0016] A "trophic factor" or "growth factor" is any molecule that promotes the regeneration, growth and / or survival of damaged cells (e.g., neurons, astrocytes, or oligodendrocytes). Such factors may be secreted by cells other than the damaged cells; for example, by bone marrow adherent stromal cells or neural regenerating cells transplanted at the site of neural injury, or by the progeny of such transplanted cells.

[0017] Degenerative diseases of the nervous system, the remodeling of which is not selective, include a variety of conditions with high incidence in the population, including trauma-induced spinal cord injury, cerebrovascular accidents, glaucoma (which can lead to blindness), and neurodegenerative conditions such as Parkinson's disease, ALS, and Alzheimer's disease. Thus, the neuroregenerative methods and compositions disclosed herein fulfill an urgent human health need. ALS (amyotrophic lateral sclerosis) is a condition in which cell death of spinal motor neurons leads to loss of function of nerves that regulate muscle atrophy, thereby inhibiting the movement of muscles throughout the body (including those associated with breathing), leading to the death of the patient within 2-3 years after onset. There is currently no effective treatment for ALS. The availability of cells from one's own bone marrow that support the regeneration of acetylcholinergic neurons would provide a major benefit, allowing autologous transplantation, which may even serve as a treatment for ALS.

[0018] Alzheimer's disease is a condition characterized by dementia in which central nervous system cell death is accompanied by intracellular neurofibrillary tangles and extracellular plaques containing deposits of β-amyloid protein. There is no cure.

[0019] Parkinson's disease is characterized by the death of a group of neurons that synthesize the neurotransmitter dopamine (DA). These neurons are located in the substantia nigra pars compacta of the midbrain and send axons to the forebrain, such as the caudate and nuclei, collectively referred to as the striatum. DA in the striatum is crucial for regulating movement, and loss of DA in this brain region leads to resting tremor, bradykinesia, difficulty initiating movement, and postural defects. This renders patients unable to initiate and control movement in a normal way. The disease is chronic and progressive, and there is currently no cure for Parkinson's disease, nor is there any treatment that can prevent or reverse the loss of DA neurons. The above diseases and other diseases or conditions characterized by neurodegeneration can be treated by providing new nerve cells at or near the site of neurodegeneration, stimulating the growth and / or regeneration of damaged or dying nerve cells, and / or promoting the survival of damaged or dying nerve cells. Thus, the methods and compositions disclosed herein may be used for the treatment of any type of neurodegeneration or neurotrauma, including, but not limited to, Parkinson's disease, Alzheimer's disease, and ALS.

[0020] The site of neurodegeneration may be anatomically identified or may be known by the etiology of the disease to be treated. Furthermore, the application of this technology is not limited to the field of clinical treatment, but is also useful in the field of artificial organ creation, which is expected to be an important development field in the future. For example, the production of nerve cells and / or neural regenerating cells at the cell culture level will help the creation of hybrid artificial organs, etc.

[0021] The present disclosure provides, inter alia, methods and compositions for treating neurodegenerative diseases by transplanting either bone marrow adherent stromal cells or bone marrow derived neural regenerating cells (i.e., cells from bone marrow adherent stromal cells induced to acquire an enhanced ability to induce growth, survival and / or regeneration of damaged neural cells) at or near the site of neurodegeneration or neurotrauma. Bone marrow stromal cells are easily extracted by bone marrow aspiration in outpatients and due to their high proliferation potential they can be cultured in large quantities within a relatively short period of time. Moreover, a further advantage of their use as starting material for the methods and compositions disclosed herein is that the starting material allows for autologous transplantation (i.e., new neural and nervous tissue is formed as a result of transplanting cells obtained from the patient's own bone marrow stem cells). Losses resulting from immune rejection eliminate the need to administer immunosuppressants, thus allowing for a safer treatment. Moreover, since bone marrow stem cells are obtained from a bone marrow bank, this method is also advantageous from the standpoint of supply.

[0022] Bone marrow adherent stromal cells are obtained, for example, from bone marrow aspirates by culturing in α-MEM + 10% FBS + L-glutamine for 3 days, followed by removal of non-adherent cells. For details, see Example 1. In one embodiment, bone marrow derived neural regenerating cells are obtained by a method comprising transfection of bone marrow adherent stromal cells with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD), as described, for example, in U.S. Patent Application Publication No. 2006-0166362 (July 27, 2006), the disclosures of which are incorporated by reference, and Dezawa et al. (2004) J. Clin. Invest. 113: 1701-1710, the disclosures of which are incorporated by reference. In further embodiments, such cells are obtained as described in U.S. Patent Application Publication No. 2006-0251624 (November 9, 2006), the disclosures of which are incorporated by reference. Alternatively, such cells may be obtained as described in U.S. Patent Application Publication No. 2003-0003090 (January 2, 2003), the disclosure of which is incorporated by reference. A specific method for obtaining neural regenerative cells is described in Example 2 below.

[0023] The cells described herein may be suspended in a physiologically compatible implant carrier. As used herein, the term "physiologically compatible carrier" refers to a carrier that is compatible with the other components of the preparation and is not harmful to the recipient thereof. Those of skill in the art are familiar with physiologically compatible carriers. Suitable examples include cell culture media (e.g., Eagle's Minimum Essential Medium), phosphate buffered saline, Hank's Balanced Salt Solution ± glucose (HBSS), and multiple electrolyte solutions such as Plasma-Lyte® A (Baxter).

[0024] Transplantation of either bone marrow adherent stromal cells or bone marrow derived neural regenerative cells involves any method known to those of skill in the art, including, for example, injection or surgical implantation of the cells. The cells may be transplanted by themselves, in a buffer or suspension as a "graft forming unit (GFU)", or in association with a matrix or support material. See, for example, U.S. Patent No. 6,989,271 and U.S. Patent Application Publication No. 2006-0216276, the disclosures of both of which are incorporated by reference.

[0025] The volume of cell suspension administered to a patient will vary depending on the site of implantation, the goal of treatment, and the amount of cells in the solution. Typically, the amount of cells administered to a patient is a "therapeutically effective amount". As used herein, a therapeutically effective amount refers to the number of transplanted cells required to treat a particular disease. For example, if the treatment is Parkinson's disease, transplantation of a therapeutically effective amount of cells will typically result in a reduction in the amount and / or severity of symptoms associated with the disease, such as stiffness, akinesia, and gait disorders, and possibly an increase in tyrosine hydroxylase-expressing nerve fibers.

[0026] The cells may be implanted into spaces containing cerebrospinal fluid, such as the subarachnoid space or ventricles, or extraneurally, within the parenchyma of the brain. As used herein, the term "extraneural" refers to a site in the patient that is not within the central or peripheral nervous system, such as the celiac ganglion or sciatic nerve. "Central nervous system" includes all structures within the dura. Typically, the cells are administered by injection into the subject's brain. The exact needle size will depend on the species being treated, but should preferably be 1 mm in diameter or less in any given subject.

[0027] The disclosed methods and compositions for nerve regeneration can be used for the treatment of neurodegeneration in the central and peripheral nervous system.Central nervous system diseases suitable for treatment include, for example, stroke, Parkinson's disease, Alzheimer's disease, ALS, and various types of spinal cord or brain injury.Peripheral nervous system diseases suitable for treatment disclosed herein include, for example, physical injury of peripheral nerves; drug-induced, diabetic or spontaneous neuropathies; neuropathic pain; sensory or motor deficits; dyskinesia; hereditary motor and sensory neuropathies; infectious polyneuropathy and inflammatory neuropathies.

[0028] In some embodiments, transplanted cells, including bone marrow adherent stromal cells and / or their progeny, undergo further differentiation after transplantation to become neurons (e.g., cholinergic neurons or dopaminergic neurons) and / or glial cells, which, in some embodiments, nevertheless retain certain immunological markers characteristic of bone marrow stromal cells.

[0029] In further embodiments, the transplanted cells induce the restoration of damaged neurons and / or glial cells. Such restoration may be mediated by cell-cell contact (between the transplanted cells and the damaged neurons) or by secretion of one or more factors (diffusible or non-diffusible) by the transplanted cells that rescue the damaged neurons. Rescue of astrocytes and / or microglia is important in the treatment of many neurological diseases (e.g., ALS) because all of these cell types secrete factors that in turn support the survival and growth of motor neurons.

[0030] In further embodiments, the transplanted cells induce endogenous cells (e.g., fibroblasts, mesenchymal cells, stromal cells, endothelial cells, and / or neural stem cells) to differentiate into new neurons and / or glial cells. Such differentiation can be mediated by cell-cell contact (between the transplanted cells and one or more endothelial cells) or by secretion of factors that induce endothelial cell differentiation.

[0031] In yet a further embodiment, the transplanted cells secrete trophic factors that induce growth of existing neural processes into the site of neurodegeneration, thereby reversing neurodegeneration and / or restoring neural tissue lost due to degeneration.

[0032] Certain examples below demonstrate the use of the disclosed methods and compositions in 6-hydroxydopamine (6-OHDA)-treated rat brain, a well-known and well-accepted model of Parkinson's disease. In this model, 6-OHDA causes a progressive loss of dopaminergic neurons that minimizes the pathogenesis of Parkinson's disease. These particular results show that bone marrow-derived neural regenerative cells can restore damaged or diseased dopaminergic neurons and thus can be used as a cell therapy for Parkinson's disease and other types of neurodegeneration. Further examples show that either bone marrow adherent stromal cells or neural regenerative cells can restore damage caused by oxygen / glucose deprivation in cultured neurons and the hippocampus. Further examples describe the identification and quantification of multiple trophic factors secreted by bone marrow adherent stromal cells or neural regenerative cells. EXAMPLES

[0033] Example 1: Preparation of bone marrow adherent stromal cells (MASC) Bone marrow aspirates obtained from human donors were divided into 12.5 ml aliquots in 50 ml tubes, and 12.5 ml of growth medium (containing 10% FBS in αMEM supplemented with penicillin / streptomycin and 2 mM L-glutamine) was added to each tube. The contents of the tubes were mixed by inversion and centrifuged for 8 minutes at 200 x g. The upper clear phase was discarded, the volume of the lower phase was adjusted to 25 ml with fresh growth medium, and the tubes were mixed and centrifuged again. The upper layer was again removed. The volume of the lower phase in each tube was again adjusted to 25 ml, and the contents of the tubes were pooled in a 250 ml tube. After determination of cell concentration by trypan blue exclusion and determination of the number of nucleated cells, 100 x 10 6 Cells were seeded into T225 flasks at a density of total nucleated cells / flask in 40 ml growth medium per flask. Flasks were incubated at 37° C. in a CO2 incubator for 3 days, during which time MASCs attached to the flasks.

[0034] After 3 days, non-adherent cells were removed by shaking the flasks and discarding the culture medium. Each flask was washed three times with 40 ml of αMEM supplemented with penicillin / streptomycin; then, 40 ml of pre-warmed (37°C) growth medium was added to each flask and the cells were incubated in a CO2 incubator. During this time, the medium was replaced with 40 ml of fresh growth medium every 3-4 days and the cells were monitored for colony growth and cell density.

[0035] When the cultures reached 25-30% confluence (usually 10,000-20,000 cells / colony, 10-14 days), MASCs (passage M0) were harvested from up to 10 T-225 flasks at a time. The medium was removed from the flasks and the adherent cells were rinsed twice with 20 ml of DPBS w / o Ca / Mg (DPBS - / -, HyClone). 10 ml of 0.25% trypsin / EDTA (Invitrogen, Carlsbad, CA) was added to each flask and the flasks were incubated at room temperature for approximately 5 minutes. When the cells had detached and the colonies had dispersed into single cells, the trypsin was incubated by adding 10 ml of growth medium followed by gentle mixing. The cell suspension was removed from the flasks and pooled in a 250 ml tube. The tubes were subjected to centrifugation at 200 x g for 8 minutes. The supernatant was carefully removed and the wet cell pellet was approximately 1x10 6 The cells were resuspended in growth medium to an estimated cell concentration of 10 cells / ml. Viable cell counts were determined and the cells were diluted to 2 x 10 6 Cells were seeded into T225 flasks at a concentration of 10 cells / flask (passage M1). Cells were grown for 3–5 days or until 85–90% confluent, with medium changes every 2–3 days. At 85–90% confluence, passage M1 cells were harvested by trypsinization and cultured at 2 x 10 6Cells were replated at 1000 x 1000 cells / T225 flask to generate passaged M2 cultures. If necessary, M2 cultures were fed with fresh medium every 3 days. When passaged M2 cultures approached 85-90% confluence (usually within 3-5 days), they were either harvested for transfection to generate NRCs (Example 2 below) or frozen for future use (Example 3 below).

[0036] Example 2: Preparation of neural regenerative cells (NRCs) NRCs were generated either directly from MASC harvested from passaged M2 cultures or from passaged M2 MASC that had been frozen as described in Example 3 and thawed and recovered as described in Example 4.

[0037] A. Preparation of the transfection mixture Neural regenerating cells were generated by transfection of passaged M2 MASC with a plasmid encoding the Notch intracellular domain. The plasmid (pCI-Notch) contained a pCI-neo backbone (Promega, Madison, WI) in which a sequence encoding amino acids 1703-2504 of the human Notch-1 protein encoding the intracellular domain was inserted into the multiple cloning site. For each flask of MASC, 5 ml of transfection mixture was used containing 40 μg of plasmid and 0.2 ml of Fugene 6® solution. To generate the transfection mixture, an appropriate amount of Fugene 6® solution (depending on the number of flasks of cells to be transfected) was added to αMEM in a sterile 250 ml tube using a glass pipette. The solution was mixed gently and incubated at room temperature for 5 minutes. An appropriate amount of plasmid DNA was then added in portions to the Fugene 6® / αMEM mixture, slowly added, and incubated at room temperature for 30 minutes.

[0038] Prior to the addition of pCI-Notch DNA to the Fugene 6® / αMEM mixture, 5 ml was taken and placed in a 15 ml tube and 40 μg of pEGFP plasmid was added. As a control for transfection efficiency, this solution was used to transfect one flask of cells.

[0039] B. Transfection For transfection, passage M2 MASCs were harvested by trypsinization (as described in Example 1) and plated at 2.5 x 10 cells in 40 ml of growth medium per T225 flask. 6 Cells were seeded at a density of 1000 x 1000 μg / flask. When cells reached 50-70% confluence (usually within 18-24 hours), they were prepared for transfection by replacing their growth medium with 35 ml / flask of transfection medium (αMEM + 10% FBS without penicillin / streptomycin). Three hours after introduction of the transfection medium, 5 ml of the transfection mixture (section A above) was added to each T-225 flask by pipetting directly into the medium so as not to touch the growth surface, then gently mixing. For determination of transfection efficiency, a control T-225 flask was transfected with 40 μg of pEGFP plasmid. After incubating the medium at 37°C for 24 hours in transfection medium, the transfection medium was replaced with αMEM + 10% FBS + penicillin / streptomycin.

[0040] C. Secretion of transplanted cells Cells that had integrated plasmid DNA were selected 48 hours after transfection by replacing the medium with 40 ml / flask of selection medium (growth medium containing 100 μg / ml G-418). Fresh selection medium was provided for 3 days and again 5 days after selection initiation. After 7 days, the selection medium was removed and the cells were cultured in 40 ml growth medium. Cultures were grown for approximately 3 weeks (18-21 days) and recultured every 2-3 days with fresh growth medium. Cells were harvested approximately 3 weeks after selection initiation, when surviving cells began to form colonies. The medium was removed from the flask using an aspirator pipette and replaced with 20 ml Ca 2+ / Mg 2+ DPBS without any added phosphate-buffered saline was added to each flask at room temperature. The growth surface was gently rinsed, the washing solution was removed by aspiration, and the rinsing step was repeated. Then, 10 ml of pre-warmed (37°C) 0.25% trypsin / EDTA was added to each flask, the growth surface was rinsed, and the flasks were incubated at room temperature for 5-10 minutes. The cultures were monitored under a microscope to ensure complete detachment of the cells. When detachment was complete, trypsin was inactivated by the addition of 10 ml of growth medium / flask. The mixture was rinsed over the growth surface. Mixing was performed by pipetting 4-5 times with a 10 ml pipette, and the suspension was transferred to a 50 ml conical centrifuge tube. Cells from several flasks were pooled in a single tube. If clumps were present, the cells were allowed to settle and the suspension was removed to a separate tube. The cell suspension was centrifuged at 200 x g for 8 minutes at room temperature. The supernatant was removed by aspiration. The cell pellet was loosened by gently tapping the tube and approximately 10 ml of CaCl2+ was added. 2+ / Mg 2+ Free DPBS was added to each tube, and the cells were resuspended by pipetting up and down 4-5 times with a 10 ml pipette to obtain a homogenous suspension.

[0041] D. Expansion of Transfected Cells Measure the cell count of the suspension of transfected and selected cells and culture at 2 x 10 cells. 610 cells / flask were seeded into a T-225 flask (giving a seeding of approximately 30% viable cells). This culture is called M2P1 (passage number 1). The M2P1 culture was grown in fresh medium for 2-3 days. When the cells reached 90-95% confluence (usually 4-7 days after passaging), they were harvested and cultured at 2 x 10 6 The cells / flask were replated to generate passaged M2P2. When the M2P2 culture reached 90-95% confluence, it was harvested for cryopreservation (Example 3) or further assays.

[0042] Example 3: Cryopreservation MASCs and NRCs were frozen for storage according to the following procedure. MASCs were typically frozen after passage M2, and NRCs were typically frozen after passage M2P2. Processing 4-5 flasks simultaneously, medium was aspirated from the culture flask, 10 ml of 0.25% trypsin / EDTA (room temperature) was added to each flask, the culture surface was gently rinsed for ≤30 seconds, and aspirated off. 10 ml of warmed (37°C) 0.25% trypsin / EDTA was then added to each flask, the growth surface was rinsed, and the flasks were incubated at room temperature for 5-10 minutes. Cultures were monitored by microscopy to ensure complete detachment of the cells.

[0043] When detachment was complete, 10 ml of αMEM growth medium was added to each flask, the culture surface was rinsed, and the detached cells were mixed by pipetting 4-5 times with a 10 ml pipette. The cell suspension was transferred to a sterile 250 ml conical centrifuge tube and large clumps of cells were removed. Cells from 15-20 flasks were pooled into one 250 ml tube.

[0044] The tubes were subjected to centrifugation at 200 x g for 8 minutes at room temperature. The supernatant was removed by aspiration. The pellet was loosened by gently tapping the tubes, and approximately 25 ml of DPBS (- / -) was added to each tube. The cells were resuspended by gently pipetting 4-5 times with a 10 ml pipette to obtain a homogenous suspension. Clumps in the suspension were removed by pipetting each sample through a sterile 70 μm sieve placed in the mouth of a 50 ml tube.

[0045] The cell suspension was collected in a 250 ml centrifuge tube and any remaining clumps were removed. The final volume was adjusted to 200 ml with DPBS (- / -) and the sample was subjected to centrifugation at 200 x g for 8 min at room temperature. The supernatant was removed by aspiration. The cell pellet was loosened by tapping and 20 ml of DPBS (- / -) was added to the tube and the cells were resuspended by mixing well and pipetting with a 10 ml pipette. The final volume was adjusted with DPBS (- / -) and approximately 0.5-1.0 x 10 6 The estimated concentration of cells / ml was usually about 4-5 ml / harvest T225 flask, or about 200 ml for a 40-flask harvest.

[0046] The viable cell number was counted for the above suspension and then subjected to centrifugation at 200 × g for 8 min. The supernatant was aspirated and the cell pellet was resuspended in cold CryoStor solution (BioLife Solutions, Bothell, WA) and diluted to 12 × 10 6 The concentration was adjusted to 100 cells / ml. 1 ml aliquots were dispensed into vials, sealed and placed in a Cryo Cooler at 4° C. The vials were transferred to CryoMed (Thermo Forma) freezer racks and frozen.

[0047] Example 4: Thawing and Recovery Frozen cells (MASC or NRC) were stored in liquid nitrogen. When required for an experiment, they were rapidly thawed and cultured as follows: A tube of frozen cells was placed at 37°C until thawed. The thawed cell suspension (1 ml) was immediately placed in 10 ml of growth medium and gently resuspended. The suspension was centrifuged at 200 x g, the supernatant removed, and the cells were resuspended in growth medium and cultured for 10 min. 6 The estimated concentration of cells / ml was calculated. Viable cells were counted by trypan blue exclusion assay and the cells were diluted to 2 x 10 6 Cells were seeded at a density of 100 cells / T225 flask. Cells were cultured at 37°C in a CO2 incubator for 3-4 days until cell growth resumed.

[0048] Example 5: Survival of transplanted bone marrow neural regenerating cells into the rat brain Adult male athymic rats were stereotaxically embedded in neurobasal tissue with 38,000 bone marrow derived neural regenerating cells obtained essentially as described in Example 2. Rats were sacrificed at 2 hours, 48 ​​hours and 14 days after transplantation. Cell survival was measured using immunocytochemistry for human cell nuclei (HuNu) antibodies by localizing transplanted human cells in perfused sections. Sections were counterstained with Nisel or hematoxylin-ecogine to assess potential toxicity resulting from transplantation. Survival of transplanted cells was 14% at 5 hours, 12% at 48 hours and 9% at 14 days after transplantation. This represents a high level of cell survival compared to many studies of embryonic cell and neural progenitor cell transplantation, where survival of transplanted cells is in the range of <5%.

[0049] Example 6: Induction of dopaminergic neurons following transplantation of bone marrow-derived neural regenerative cells in a rat model of Parkinson's disease This example shows that transplantation of bone marrow-derived neural regenerative cells induces the growth and regeneration of dopaminergic neurons in a rat model of Parkinson's disease. The physiological principles governing neuronal growth (e.g., neurite outgrowth), development, regeneration, and aging have been extensively studied in the rat and have been found to be similar to those governing humans. In particular, partial lesions of the nigrostriatal projection, which approach the cause of Parkinson's disease in humans, can be performed in rats by injecting 6-hydroxydopamine (6-OHDA) into the striatum.

[0050] In two independent experiments, rat brains were treated with 6-hydroxydopamine (6-OHDA); subsequently, bone marrow-derived neural regenerative cells were transplanted into the injury site of specific 6-OHDA-injured brains. The transplanted cells survived and remained tyrosine hydroxylase (TH)-negative, while simultaneously inducing TH-positive (i.e., dopaminergic) nerve fibers into the transplantation site. Amphetamine-induced nuclear translocation of c-fos protein, an indicator of dopamine release, was also observed in some cases. The results implicate bone marrow-derived neural regenerative cells in the recovery, regeneration, and rescue of dopaminergic neurons in a rat model of Parkinson's disease.

[0051] A. Processing of bone marrow-derived neural regenerative cells for transplantation Bone marrow derived neural regenerative cells prepared as described in Example 2 above were frozen in liquid nitrogen until use. To thaw, the frozen vial containing the cells was immediately placed in a 37°C water bath and left there until the contents were completely thawed. The vial was quickly removed and the cells transferred to a 15 ml conical centrifuge tube containing 10 ml of cold growth medium (α-MEM + 10% FBS). The preparation was centrifuged at 200xg for 8 minutes at room temperature in a swinging bucket rotor to form a pellet of cells. The supernatant was carefully removed and the cells were resuspended in DPBS w / o Ca. 2+ -Mg 2+ Add about 0.5 x 10 6A final cell concentration of 1.0 cells / ml was obtained. A cell count was performed at this stage to obtain an accurate cell number and to check viability. The cells were then centrifuged at 200xg for 8 min at room temperature. 1.4 ml of DPBS w / o CaCl2+ were added. 2+ -Mg 2+ The pellet was resuspended by adding 100 ml of 1% CO and the cells were transferred to a 1.5 ml tube. The sample was then centrifuged at 200xg for 8 minutes. Most of the buffer was removed and the cells were then immediately centrifuged again at 200xg for 1 minute and the remaining buffer was removed using a P-200 pipettor.

[0052] The pellet volume was then estimated and the final target volume was calculated using the following formula:

[0053]

number

[0054] Cell counting was performed to check the final concentration and viability before transplantation. After cell transplantation, the remaining cells were also checked for cell number and viability to ensure the quality of the transplantation method.

[0055] B. Creation of 6-OHDA Lesions In two independent experiments, 38 adult male Fischer 344 rats were unilaterally lesioned by injection of 6-OHDA into the right striatum. Rats were anesthetized with isoflurane and then head-immobilized in a stereotaxic apparatus equipped with a continuous isoflurane anesthesia cone. Respiration and pain reflexes were continuously monitored. A mid-skull incision was made, the skin was removed, and the injection needle was zeroed with a bregma suture. The injection site was marked and a burr hole made for each set of coordinates. Injection coordinates were A / P: +0.2, M / L: -3.0, D / V: -5.5.

[0056] The injection needle (10 μl Hamilton syringe, 26G needle) was lowered at a rate of 1 mm / min. After approaching the injection site, the needle was kept in place for 4 minutes before the injection started. The injection was then started (injection solution: 0.2 mg / ml ascorbic acid and 16 μg 6-OHDA, injection volume: 2.8 μl, rate: 0.5 μl / min). After the injection, the needle was kept in place for another 4 minutes to allow the injected solution to diffuse from the injection site. The needle was then slowly removed (1 mm / min). The skin was sutured with discontinuous stitches (at least 5) and the wound was treated with Fougera 2 antibacterial ointment and LMX4 local anesthesia to avoid infection and discomfort of the animal.

[0057] C. Cyclosporine Treatment Daily subcutaneous administration of 10 mg / kg cyclosporine A was performed to suppress immune responses to the transplanted cells. Prior to injection, Sandimmune (50 mg / ml) was diluted 1:5 with sterile saline (to give a final concentration of 10 mg / ml). Infusions were initiated 24 hours prior to cell implantation and continued daily for the remainder of the experiment.

[0058] D. Transplantation Seven days after treatment with 6-OHDA, bone marrow-derived neural regenerating cells were transplanted into the injury site. Transplantation was performed by stereotactic injection of 0.5 μl of a suspension of bone marrow-derived neural regenerating cells at various cell doses (6,000-21,000 cells / μl) in PBS into the injured striatum of 19 rats. Nine control rats received no further treatment (lesion-only group). Ten control rats received PBS at the same coordinates. Ten deposits occurred around the injury site and two deposits per needle trajectory (coordinates: 1-2.: A / P: +0.5, M / L: -2.5, D / V: -4.4 / -5.8; 3-4.: A / P:+0.5, M / L: -3.5, D / V: -4.8 / -0.2; 5-6.: A / P: 0.0, M / L: -3.0, D / V: -4.8 / -6.2; 7-8.: A / P: -0.3, M / L: -2.7, D / V: -4.2 / 5.4; 9-10.: A / P: -0.3, M / L: -3.8, D / V: -5.0 / -6.2). Injections were performed with a 10 μl Hamilton syringe with a 26G needle (straight, 30 angle tip). To accommodate cell injections, the injection protocol was modified. The needle was kept in place for 1 min before and after injection. Injection parameters were as follows: 0.5 μl / deposit, 1 μl / min rate. New burr holes for extra injection sites were created at the beginning of the surgery. Implanted cell numbers were 6,000 cells / implant (n=3), 8,000 cells / implant (n=3), 12,000 cells / implant (n=4), and 21,000 cells / implant (n=3).

[0059] Euthanasia Three weeks after transplantation, rats were sacrificed and their brains were examined for the presence of dopaminergic (i.e., TH-positive) neurons. Two hours before sacrifice, rats were given an IP injection of DL-amphetamine (5 mg / kg) to induce c-fos expression in striatal target neurons that receive projections from dopaminergic axons. Rats were then overdosed with pentobarbital. Corneal and toe pinch reflexes were checked to ensure adequate anesthesia before undergoing intracardiac perfusion with saline (400 ml) followed by 4% paraformaldehyde (400 ml).

[0060] F. Tissue Processing After intracardiac perfusion with paraformaldehyde, brains were removed, blocked, postfixed overnight, and then transferred successively to 10%, 20%, and 30% sucrose solutions after the tissue was submerged in each solution. Frozen coronal sections 40 μm thick were collected from each brain and kept in cryoprotectant solution at -20°C until staining.

[0061] G. Immunohistochemistry / staining Immunoreaction for tyrosine hydroxylase (TH), the enzyme that catalyzes the rate-limiting step in dopamine synthesis, was used as a test for the presence of dopaminergic neurons. Two methods were used. First, frozen sections were washed three times with PBS and then blocked for 1 h at room temperature in PBS containing 0.3% Triton X100 and 3% normal goat serum (blocking solution). Primary antibody (rabbit anti-TH, Chemicon, 1:1000) was then added to the blocking solution and incubation was continued overnight at room temperature on a shaker. Sections were then washed briefly with PBS and incubated with secondary antibody (biotinylated goat anti-rabbit, 1:500) for 2 h at room temperature. DAB staining was performed using the Vectastain ABC kit and DAB substrate kit (Vector Laboratories) according to the manufacturer's protocol.

[0062] In the second method, frozen sections obtained from the striatum were washed three times with Tris-buffered saline (TBS), incubated with 0.3% H2O2 in TBS for 15 min, blocked in 10% normal goat serum (NGS), 0.5% Triton x-100 (TX-100) in TBS for 20 min at room temperature, and washed briefly with TBS / 0.1% TX-100. Sections were then incubated with 1:2000 polyclonal rabbit anti-TH antibody (Chemicon) in 1% NGS and 0.3% TX-100 in TBS overnight at room temperature (RT) on a shaker. Sections were then washed briefly with TBS / 0.1% TX-100 and incubated with biotinylated goat anti-rabbit antibody (Vector Laboratories) diluted 1:500 in TBS with 1% NGS and 0.1% TX-100 for 2.5 h at room temperature, followed by incubation with avidin-peroxidase conjugate (Vectastain ABC Elite, Vector Laboratories) in TBS for 2 h. Visualization was performed with DAB (20 mg / ml), 0.8% nickel sulfate, 0.005% H2O2 in 50 mM sodium acetate, 10 mM imidazole buffer (pH 7.0). Between each step, several washes were performed with TBS with 0.1% TX-100.

[0063] Survival of bone marrow-derived neural regenerating cells in tissue sections was detected by staining for human nuclear mitotic apparatus protein or nuclear matrix protein (hNuMA). Following antigen retrieval by incubation in 10 mM sodium acetate buffer (pH 8.5) at 80°C for 30 min, striatal sections were washed three times with PBS and blocked for 1 h at room temperature in 5% NGS, 5% BSA, 1% TX-100 in PBS. Sections were incubated for 48 h at 4°C in mouse monoclonal anti-hNuMA antibody (Calbiochem) diluted 1:50 in blocking solution. Sections were washed seven times with 0.02% TX-100 in PBS and signals were detected by incubation for 90 min at room temperature with secondary antibody conjugated to Cy3 (Jackson ImmunoResearch) diluted 1:250 in blocking solution without TX-100. Hoechst nuclear staining was performed by treating sections with Hoechst 33,342 dye (5 μg / ml in PBS; Invitrogen) for 15 min.

[0064] For double staining of hNuMA and TH, the same protocol as above was used. Mouse monoclonal anti-hNuMA antibody (Calbiochem) and polyclonal rabbit anti-TH antibody (Chemicon) were applied simultaneously at 1:50 and 1:500 dilutions in blocking solution. Visualization was performed by incubating sections for 90 min at room temperature with secondary antibodies conjugated to Cy3 and Cy2 (Jackson ImmunoResearch) at 1:250 dilution in blocking solution without TX-100. After incubation with secondary antibodies, Hoechst nuclear staining was performed as described above.

[0065] H. Nuclear translocation of c-fos The ability of cells to release dopamine in response to amphetamine was measured by induction of c-fos expression and nuclear translocation of c-fos protein. For analysis of c-fos expression and translocation, rats were injected with dl-amphetamine 2 hours before sacrifice. dl-amphetamine causes increased extracellular levels of DA from intact DA fibers. DA stimulates target striatal neurons causing translocation of the immediate early response gene product c-fos to the nuclei of these neurons. Three weeks after transplantation, rats were fixed and sacrificed by transcardial perfusion, and brain sections were stained for c-fos immunoreactivity.

[0066] Amphetamine-induced expression of c-fos in striatal nuclei was measured by immunocytochemistry. Frozen sections were washed three times with PBS, incubated for 15 min with 0.3% H2O2 in PBS, and blocked for 1 h at room temperature with 3% NGS, 2% fetal bovine serum (BSA), and 0.05% TX-100 in PBS. Sections were then incubated overnight at room temperature in polyclonal anti-c-fos antibody (Santa Cruz) diluted in 1% NGS, 1% BSA, and 0.05% TX-100 in PBS. Sections were washed several times with 0.05% TX-100 in PBS, and then incubated for 2.5 h with biotinylated goat anti-rabbit secondary antibody (Vector Laboratories) diluted 1:500 in 1% NGS and 1% BSA. This was followed by incubation with avidin-peroxidase conjugate (Vectastain ABC Elite, Vector Laboratories) in PBS for 2 h and visualization with DAB (20 mg / ml), 0.8% nickel sulfate, 0.005% H2O2 in 50 mM sodium acetate, 10 mM imidazole buffer (pH 7.0), with several washes in TBS with 0.1% TX-100 between each step.

[0067] After staining the cells for c-fos-immunoreactivity as described above, positive cell nuclei were counted using Neurolucida software (version 7.50.4, MicroBrightField Bioscience, Williston, VT). Sampling methods differed slightly depending on the level examined. At the level of the graft (bregma, 0.48 mm), a grid was set at 2X magnification with its center corresponding to the graft center. Using the "contour" function, three contour plots were drawn; one on either side and one below the grid. The area bounded by the contour on either side was calculated as 0.25 mm. 2 (area was 250 μm x 1000 μm). The area bounded by the lower contour was half that, i.e., 0.125 mm 2 The magnification was then increased to 20X and the NeuroLucida "meander scan" function was activated. The contours were then measured in 180 x 180 μm increments for c-fos positive cells using a computer cursor to mark cells as they were observed. A mirror point was placed on the contralateral side of the graft using the center of the midline of the brain section as a reference. Again, a grid was placed with its center corresponding to this mirror point. Three contour plots were drawn at 2X magnification and c-fos positive cells were marked at 20X magnification.

[0068] I. Additional Tests for Dopaminergic Neuronal Function (DA-Dependent Behaviors) Further tests for recovery and / or regeneration of dopaminergic neurons include amphetamine-induced rotation and forelimb positioning.

[0069] Dopaminergic neurons were labeled by injection of fluorogold (a retrograde tracer) at the injury / transplantation site, and the number of fluorogold-positive neurons was assayed after transplantation.

[0070] The phenotype of the transplanted cells and the surrounding host cells was examined by staining for various markers: neuronal markers: MAP2, βIII tubulin, glial cell marker: glial fibrillary acidic protein (GFAP), bone marrow adherent stromal cell marker: CD105.

[0071] J. Results In all rats transplanted with bone marrow-derived neural regenerating cells, a high density of tyrosine hydroxylase (TH)-immunoreactive fibers (a hallmark of dopaminergic neurons) was observed around the transplant site, whereas in control rats injected with PBS, few or no tyrosine hydroxylase-immunoreactive fibers were observed at the injury site.

[0072] Although a small number of transplanted bone marrow-derived neural regenerating cells survived at the injection site, as detected by expression of a human-specific nuclear matrix protein, tyrosine hydroxylase immunoreactivity was not observed in some of the surviving transplanted cells, suggesting that bone marrow-derived neural regenerating cells did not differentiate into dopaminergic neurons and that the tyrosine hydroxylase-immunoreactive fibers observed around the transplant were of host brain origin.

[0073] In sections co-stained for TH and human nuclear matrix protein, there was no clear overlap between hNuMA and TH staining, providing evidence for a host origin of the TH-positive fibers surrounding the graft. In addition, a positive correlation was observed between the number of viable cells and the density of TH-immunoreactive fibers near the graft. This effect was most pronounced in rats receiving higher doses of cell transplants. In the first experiment, all three rats receiving 21,000 cells / μl had more viable cells and a higher density of TH-IR fibers than rats receiving the lower dose of cells. In the second experiment, five of six rats receiving 20,000 cells / μl had more viable cells and six of six had increased TH-IR fibers near the graft site.

[0074] When c-fos expression and nuclear translocation were assayed, two of the six rats showed very high levels of c-fos-positive nuclei around the transplant sites. In the other rats, multiple areas of c-fos-positive nuclei around the transplanted neural regenerators were also observed. These observations suggest that the increase in TH-immunoreactive fibers is associated with an amphetamine-induced increase in DA near the NRC transplants in some rats (not in the entire striatum, but not in all transplants).

[0075] Taken together, these results indicate that transplantation of bone marrow-derived neural regenerative cells into the site of neural regeneration induces the growth, survival and / or regeneration of host dopaminergic neurons into the transplanted tissue, possibly by secreting one or more trophic factors. Thus, transplantation of bone marrow-derived neural regenerative cells provides a method for the restoration of dopaminergic neurons in Parkinson's disease and other neurodegenerative conditions.

[0076] Example 7: Co-culture with MASCs and NRCs enhances survival of and neurite outgrowth from primary neurons This example shows that primary cortical neurons grow well in complete neuromedium, but grow poorly in serum-free αMEM, forming fewer and shorter neurites. However, when primary cortical neurons are cultured in a medium that does not support neuritogenesis (i.e., serum-free αMEM) and separated from either MASC or NRC by a semipermeable membrane, neurite outgrowth is enhanced. This suggests that MASC and NRC produce soluble factors that support neuronal growth and neuritogenesis. The extent of the rescue effect appears to be similar for a given amount of MASC or NRC.

[0077] A. Preparation of Culture Plates One day before the experiment, Millicell 96-well cell culture insert plates (Millipore MACAC02S5, 0.4 μm pore size) were coated with a 10 μg / ml solution of poly-D-lysine (Sigma-Aldrich, St. Louis, MO). The lysine solution was aspirated, the plates were dried for 10 min, washed with PBS, and filled with complete neuronal medium (Neurobasal® medium supplemented with 2% B-27 and 0.5 mM GlutaMAX®), after which the plates were incubated at room temperature for 1 h. Treated plates filled with neuronal medium were stored at 4°C until use.

[0078] B. Donor Cell Preparation Donor cells (MASC and NRC) were prepared and frozen as described in Examples 1-3. Cells to be tested (MASC and NRC from donors D31 and D39) and MASC from donor D42 (used in this example as an internal control) were thawed and cultured at 2 x 10 in T225 flasks in 10% FBS supplemented with αMEM + penicillin and streptomycin (growth medium). 6 Cells / flask were seeded for 5-7 days at 37°C / 5% CO2. For MASCs, the medium was also supplemented with 2 mM L-glutamine. Cells were removed by replacing the medium with a small volume of 0.25% (w / v) trypsin, 1 mM EDTA, collected in a 50 ml tube, and growth medium was added (5-10 ml) to inactivate trypsin. The tubes were centrifuged at 200xg for 5 min, and the cell pellet was resuspended in 5-10 ml of complete medium. An aliquot of the cell solution was counted in a hemocytometer (Hausser Scientific). For D42 standard donor cells, 4 x 10 ml of growth medium was added. 5 Adjust cell concentration to 2x10 / ml for test cells (D31 and D39 MASC and NRC). 5 The cell concentration was adjusted to 1 / ml.

[0079] Millicell 96-well cell culture insert plates (Millipore MACAC02S5, 0.4 μm pore size) were prepared for cell seeding by filling the inserts with 25 μl / well of growth medium. A portion of the cell suspension described in the previous paragraph sufficient to provide the desired number of donor cells was added to each well; e.g., 20, 6.7, 2.2, and 0.75 x 10 3 For seeding cells / insert, 4 x 10 5 50, 17, 5.5, and 2 μl of the 100 μl / ml cell suspension and 0.33, 44.5, and 48 μl of growth medium were added to the wells, respectively. The inserts were then lowered into a 96-well feeding compartment containing 250 μl / well of the same growth medium. The cells were cultured overnight at 37° C., 5% CO2, and used the next day for co-culture experiments.

[0080] C. Preparation of Rat Cortical Neurons Rat cortex (E 17) was obtained from BrainBits (Springfield, IL) in medium provided by the supplier. For isolation and preparation of cortical neurons, the medium was removed and replaced with 2 ml of 0.25% (w / v) trypsin, 1 mM EDTA (Gibco / Invitrogen, Carlsbad, CA). The mixture was incubated in a 37°C water bath for 5-7 min. The trypsin solution was removed and the tissue was rinsed with 2 ml of αMEM supplemented with 10% fetal bovine serum (FBS). 2 ml of 0.25 mg / ml DNase I in Dulbecco's modified PBS was added to the trypsinized tissue and the suspension was mixed by vortexing for 30 s. The suspension was further mixed by pipetting up and down in a 1 ml micropipette tip. Any large tissue pieces remaining were discarded. The cell suspension was then placed in a 15 ml centrifuge tube and subjected to centrifugation at 200xg for 1 min. The supernatant was removed and the cells were resuspended in 2-3 ml of complete neuronal medium (Neurobasal® supplemented with 2% B-27 and 0.5 mM GlutaMAX®). A portion of this neuronal cell suspension was dispensed into microcentrifuge tubes at 40,000 cells / tube, the cells were pelleted, the medium was aspirated, and the cell pellets were frozen at -20°C. These cells were used as standards in the LDH assay (see section F below).

[0081] D. Co-culture of rat primary cortical neurons with MASCs or NRCs On the day of the experiment, the polylysine-coated lower compartment prepared in Section A above was warmed in a 37° C. incubator for approximately 30 minutes. The neuronal medium was removed from the lower compartment and replaced with αMEM, except for a few wells that retained the neuronal medium, to be used as a positive control. Rat cortical neurons prepared in Section C above were plated into the wells of the lower compartment at a density of 5,000 cells / well (96-well plate) and allowed to adhere to the plate for 1 hour at 37° C.

[0082] Alternatively, donor cells as described in section B above were transferred to serum-free αMEM by removing the medium from the wells above the membrane and replacing it with 75 μl of serum-free αMEM, then inserting the upper compartment into the lower compartment also containing serum-free αMEM, and incubating at 37°C for approximately 30 minutes. The upper compartment containing donor cells (MASC and NRC) in serum-free αMEM was then inserted into the lower compartment containing primary cortical neurons in serum-free αMEM. Co-culture was carried out at 37°C, 5% CO2 for 5-6 days. For positive controls, wells in the lower compartment contained neuronal medium rather than serum-free αMEM, and no donor cells were present in the corresponding insert compartment.

[0083] E. Immunohistochemistry After co-culture, some cultures were tested for neurite outgrowth by assaying the expression of MAP-2 (a neuronal marker). For this assay, cells in the low well were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) for 20 min and then blocked with 0.3% Triton X-100, 5% normal donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA) for 1 h at room temperature. An antibody against MAP-2 (mouse monoclonal, Sigma-Aldrich, St Louis, MO) was added to the blocking solution and incubation continued for 1 h at room temperature. After the 1 h incubation, cells were washed with PBS and incubated with Cy3-conjugated AffiPure F(ab')2 fragment of donkey anti-mouse IgG (minimal cross-reactivity, Jackson Immunoresearch Laboratories, West Grove, PA) diluted 1:1000 for 1 h at room temperature. Cells were washed with PBS, then wells were filled with PBS and staining was assayed on an Axiovert 40CFL (Zeiss, Germany); photographs were taken on an AxioCam MRm.

[0084] Figure 1 shows the effect of co-culture with MASCs on neurite outgrowth in primary rat cortical neurons, as assayed by immunocytochemistry for MAP-2 as described in the previous paragraph. MAP-2 labeling showed extensive neurite outgrowth when neurons were cultured in complete neuronal medium (top left panel of Figure 1), and few short neurite outgrowth when neurons were cultured in serum-free αMEM (top center panel of Figure 1). When neurons were co-cultured with MASCs in αMEM, the number of treatments correlated positively with the number of MASCs co-cultured with the neurons (top right and bottom panels of Figure 1). A similar supportive effect on neurite outgrowth from primary cortical neurons was observed in co-cultures with NRCs.

[0085] F. LDH Assay for Cell Viability The survival of certain samples of neurons co-cultured with MASC and NRC in serum-free αMEM was assessed by measuring the levels of intracellular lactate dehydrogenase (LDH). For these samples, the inserts containing donor cells were removed and the medium was aspirated from the lower compartment, which was subsequently washed with PBS. 0.15 ml of 2% (v / v) Triton X-100 was then added to each well to lyse cells that had adhered to the plate. 0.1 ml of Triton lysate was removed from each well and transferred to a new well for LDH assay using the Roche LDH assay kit. A standard curve was generated using serial dilutions of frozen cells obtained as described in section C above, dissolved in 500 μl of 2% Triton X-100.

[0086] For the LDH assay, 100 μl of a mixture of reagents A and B from the LDH kit, prepared according to the manufacturer's protocol, was added to each well. After approximately 20-30 minutes, color was measured with a SpectraMax Plus (Molecular Devices) at a wavelength of 490 nm and a reference wavelength of 650 nm. Typically, color was allowed to develop until the standard containing the highest cell concentration gave a reading of approximately 1 OD unit. Measurements were analyzed using Soft Max Pro Software, and a standard curve was constructed using a quadratic fit.

[0087] Figures 2 and 3 show the results of two independent experiments in which the intracellular LDH assay was used to measure the relative number of surviving neurons after co-culture in serum-free αMEM with MASC and NRC from two different donors. In two experiments, D42 MASC were also used as an internal control. D42 MASC cells were titrated in each experiment to confirm the ability of the assay to detect differences in the levels of neuronal survival.

[0088] In the experiment shown in Figure 2, donor 31 (5 x 10 3 Equal numbers of MASC and NRC derived from 1000-well cultures (100 / well) produced similar effects on neuronal survival (Figure 2, rightmost two bars). The effects were less than those obtained from the same numbers of D42 MASC, as evidenced by the titration curves of D42 MASC shown in the leftmost five bars of Figure 2.

[0089] In the experiment shown in Figure 3, MASCs and NRCs from donor 39 (D39) were cultured at two different concentrations (10 x 10 3 / well and 5 x 10 3 / well). Again, MASC and NRC showed similar levels of rescue activity (Fig. 3, rightmost four bars). However, in this experiment, D39 MASC and NRC were more active than D42 MASC, which was used as a standard, as evidenced by the titration curve of D42 MASC (Fig. 3, leftmost five bars).

[0090] Example 8: Rescue of damaged neural cells after co-culture with bone marrow stromal cells or neural regenerative cells To provide an in vitro model of neuronal injury, primary cortical neurons were subjected to oxygen / glucose deprivation (OGD), which resulted in significant cell damage within 24 hours. Immediately after OGD, neurons were co-cultured with either human bone marrow adherent stromal cells or human neural regenerating cells (both prepared as described in Examples 1 and 2). These donor cells were separated from the oxygen / glucose deprived neurons by a 0.4 μm pore size membrane that allows the passage of molecules but excludes cell-cell contact. In both cases, the presence of donor cells rescued the neurons following OGD as described below.

[0091] Cortical neurons from E17 Sprague-Dawley rats were cultured at 6,000 cells / cm 2 Neurons were seeded at 100°C on poly-d-lysine-coated 12-well plates and cultured at 37°C / 5% CO2 in Neurobasal® supplemented with 2% B-27 and 0.5 mM GlutaMAX® (Neuronal medium). After 14 days of in vitro maturation, neurons were subjected to OGD (Dulbecco's modified Eagle's medium without 10% oxygen and glucose) for 60 min. Control cultures were maintained in neuronal medium at atmospheric oxygen (normoxia) for the same time. Immediately after, the medium was replaced again with neuronal medium, and inserts containing neural regenerating cells, bone marrow adherent stromal cells, or medium alone were placed in the wells. Neuronal medium was used for all groups (n=6 matched donors / cell type; i.e., MASCs were obtained from 6 different donors and a portion of each donor culture was used for NRC production). Two to four cell wells / group were used for the experiments.

[0092] The substrate of the inserts is a polyethylene terephthalate (PET) membrane with a pore size of 0.4 μm, allowing molecular exchange while avoiding cell-cell contact. Bone marrow adherent stromal cells or neural regenerative cells were seeded onto the inserts in suitable growth medium, which was rinsed and replaced with neuronal medium before being placed into the wells containing neurons. Donor cells were 80-100% confluent on the inserts (0.9 cm) when added to the wells containing neurons. 2 ). After 24 hours of OGD, cell damage / death was assessed by quantifying lactate dehydrogenase (LDH) release from cultured neurons (i.e., extracellular LDH) and standardizing for appropriate control conditions. Neurons in control conditions (non-OGD) also received inserts containing neural regenerating cells, bone marrow adherent stromal cells, or medium only to account for LDH release from donor cells, since LDH can cross the membrane separating donor cells from the neurons. Thus, results are presented as fold increase in LDH release of neurons subjected to OGD and then co-cultured with inserts of either donor cell type or medium only, compared to neurons under control (non-OGD) conditions co-cultured with inserts containing the same donor cell type or medium. LDH was assessed using the "Cytotoxicity Detection Kit (LDH)" from Roche Applied Science (Indianapolis, IN). See Example 7 for details of the LDH assay. Note that in this example, extracellular LDH was assessed as an indicator of cell death, whereas in Example 7, cells were detached from the medium and lysed for measurement of extracellular LDH.

[0093] The results are shown in Figure 4, which demonstrates that exposure of injured neurons to bone marrow adherent stromal cells or neural regenerating cells significantly reduced cell injury / death (measured as extracellular LDH) following OGD compared to exposure of neurons to medium alone (p<0.05). Furthermore, because the donor cells were not in direct contact with the injured neurons, the observed reduction in cell death is consistent with the secretion of one or more trophic factors by the donor cells.

[0094] Example 9: Rescue of damaged neural cells after co-culture with conditioned medium from bone marrow stromal cells or neural regenerative cells To provide further evidence that trophic factors secreted by bone marrow-adherent stromal cells or neural regenerative cells rescue neurons subjected to ischemic insult, neurons injured by OGD were cultured in conditioned medium from bone marrow-adherent stromal cells or neural regenerative cells. To prepare conditioned medium, donor cells (n=5 matched donors / cell type, 2-4 wells / group) were cultured for 24 hours in one of the following media: 1) fresh neuronal medium (composition as above), 2) neuronal medium in which OGD-injured neurons had been cultured for 24 hours, or 3) neuronal medium in which control neurons had been cultured for 24 hours. The conditioned medium was then withdrawn from the donor cell cultures and added to cultured neurons that had undergone OGD or to control neurons that had not undergone OGD. LDH release was assessed after 24 hours.

[0095] The results are shown in Figure 5. In all cases, injured neurons exposed to conditioned media showed significantly reduced cell damage 24 hours after OGD (measured by LDH release compared to control cells that did not undergo OGD) (p<0.05). No differences were observed among the three donor cell-conditioned media, indicating that any substances released by injured neurons over the 24 hours of co-culture with donor cells did not significantly affect the ability of donor cell-conditioned media to rescue injured neurons. In addition, there was no significant difference in the rescue effect of media conditioned by either bone marrow-adherent stromal cells or neural regenerating cells.

[0096] To determine whether this rescue effect had a dose response, conditioned medium was obtained by culturing donor cells for 24 hours in neuronal medium (n=5 matched donors / cell type, 2-4 wells / group). This conditioned medium was added to either neurons subjected to OGD or control neurons at a dilution of 1:1, 1:10, 1:25, 1:50, 1:100 or 1:200, and neurons were cultured in conditioned medium for 24 hours. The results are shown in Figure 6, which indicates that the rescue of injured neurons by conditioned medium (derived from bone marrow adherent stromal cells and neural regenerating cells) was dose-dependent.

[0097] Example 10: Rescue of injured neurons after co-culture with neural regenerating cells pre-cultured with injured neurons To determine whether the injured neuroenvironment affects the ability of donor cells to rescue injured neurons, bone marrow adherent stromal cells or neural regenerative cells (n=3 donors) were seeded onto PET inserts (see Example 7) and pre-cultured for 24 hours with either neurons previously injured by OGD for 24 hours or with control injured neurons. After this pre-culture, bone marrow adherent stromal cell- or neural regenerative cell-containing inserts were seeded into wells containing neurons that had undergone OGD immediately prior to the addition of the inserts or control uninjured neurons. After a further 24 hours, LDH release was assayed (2-4 wells / group).

[0098] The results of this experiment indicate that bone marrow adherent stromal cells and neural regenerating cells pre-cultured with either injured or non-injured neurons can rescue OGD-injured neurons. MASC and NRC had similar rescue activity, and pre-culture with either control or OGD-injured neurons did not affect the rescue activity of either MASC or NRC. (This may be due to the fact that the conditioned medium was used undiluted in this experiment, and the rescue effect was beyond the discrimination range of the assay.) These results indicate that donor cells may respond to cues from injured neurons that affect the production and / or secretion of one or more diffusible trophic factors.

[0099] Example 11: Preparation of primary hippocampal slices Rat organotypic hippocampal slices (OHS) were obtained from the brains of 9-day-old Sprague-Dawley rat pups according to the following procedure. 1) Female rat pups (P9) were delivered from Charles River Laboratories (Wilmington, MA). Animals were housed in sealed cardboard boxes with adequate food and water provided by Charles River until the time of sacrifice. Animals were left undisturbed for less than 5 hours before treatment. 2) The rat pups were decapitated one at a time. One rat was placed in an airtight box containing dry ice (for CO2 generation). 3) The anesthetized rat pup was removed from the box and immediately submerged in 70% ethanol in a glass beaker, then immediately decapitated with scissors. All skin was removed from the head. 4) The carcass was transferred to a biosafety cabinet. The brain was removed from the head and placed in ice-cold buffer (e.g. PBS). 5) The hippocampal formation was dissected from the brain with curved forceps and placed on a tissue chopper stage (Mcllwain) to be cut into 400 μm slices. 6) Slices were placed on single-well membrane inserts (Millipore, see Examples 7 and 8), which in turn were placed in cell culture plates containing 50% Hank's modified Eagle's medium, 24% Hank's balanced salt solution, 25% horse serum and 1% penicillin / streptomycin; supplemented with 8.5 mM HEPES, 5.5 mM glucose and 0.5 mM GlutaMAX®. 7) The plates containing OHS were then stored in a CO2 incubator at 35°C. 8) After 4 to 6 days of culture, OHS was used for experiments.

[0100] Example 12: Rescue of injured hippocampal slices after co-culture with bone marrow stromal cells or neural regenerative cells Using an in vivo model, we used hippocampal brain slices to test trophic support for injured neurons by bone marrow-adherent stromal cells and neural regenerating cells. The hippocampus was chosen due to the distinct neuronal pathways contained within this structure and the fact that ample literature exists on organotypic hippocampal slice cultures.

[0101] Hippocampal slices (P9 rat, 400 μm cut) were cultured on inserts (Millipore), whose base is a polyethylene terephthalate (PET) membrane with a pore size of 0.4 μm, allowing molecular exchange but inhibiting cell-cell contact. The inserts were placed in wells containing 50% Hank's modified Eagle's medium, 25% Hank's balanced salt solution, 25% horse serum, and 1% penicillin / streptomycin supplemented with 8.5 mM HEPES, 5.5 mM glucose, and 0.5 mM GlutaMAX®. After 1 day, the medium was slowly replaced with Neurobasal® medium supplemented with 2% B-27 and 1.0 mM GlutaMAX® ("slice medium"). After 4-6 days in culture, slices were subjected to OGD (DMEM with 0% oxygen and no glucose) for 90 min. Control cultures were supplemented with slice medium and maintained in atmospheric oxygen (normoxia) for the same time. Immediately after OGD, inserts (n=6 matched donors / cell type, 4-6 inserts / group) were transferred to wells containing (1) bone marrow adherent stromal cells (70-100% confluence) in slice medium, (2) neural regenerating cells (70-100% confluence) in slice medium, or (3) slice medium alone. LDH release was quantified after 24 and 48 hours.

[0102] The results shown in Figure 7 indicate that slices cultured for 24 and 48 hours after OGD in the presence of donor cells (i.e., bone marrow adherent stromal cells or neural regenerating cells) showed significantly less cell damage than those cultured in slice medium alone (p<0.05).

[0103] Cells within the slices are also analyzed by propidium iodide uptake to assess whether cells within the slices are damaged.

[0104] Example 13: Rescue of damaged hippocampal slices following transplantation of bone marrow stromal cells or neural regenerative cells To examine how donor cells affect host tissue when direct cell-cell contact occurs, and to examine the effect of the injury environment on donor cells, donor cells (i.e., bone marrow adherent stromal cells or neural regenerating cells) are injected directly into hippocampal slices.

[0105] To aid in visualization of donor cells, donor cells were infected with a lentivirus containing a sequence encoding green fluorescent protein (GFP) under the transcriptional control of the CMV promoter. After infection at a multiplicity of infection (MOI) of 3 viral particles / cell, approximately 50-60% of donor cells (neuroregenerative or bone marrow adherent stromal cells; n=1 matched donor / cell type) were fluorescent, and stable fluorescence was observed for at least 1 month in culture.

[0106] GFP-positive donor cells were delivered to hippocampal slices by pipetting 0.5 μl of cells (at a concentration of approximately 1,000 cells / μl) onto the slice surface. The behavior of GFP-positive donor cells within thick (200-400 μm) slices was compared with respect to survival, proliferation, migration, and integration into tissues after transplantation into normal or damaged (e.g., ischemic) brain slices and monitored using confocal microscopy. For example, slices were monitored by confocal microscopy at various times (e.g., 2 hours) after delivery of donor cells to determine donor cell number, location (in the dissected tissue and on the z-dimension), and morphology. This analysis was repeated, for example, 2 and 5 days after injection and compared to baseline levels to assess survival / proliferation, migration, and integration. Propidium iodide injection was also used to assess host and donor cell survival. Subsequent analysis included fixation of tissue slices and staining for proliferation, cell death, and phenotypic markers.

[0107] Comparison of the location of transplanted cells with the location of regenerating or recovering host neural cells provides information about the extent of the trophic effect mediated by the transplanted cells. The number and location of transplanted cells are also analyzed to determine the extent of cell division and translocation of transplanted cells. The morphology of the transplanted cells and surrounding host cells is assessed, and the differentiated state of the transplanted cells and surrounding host cells is analyzed by cytochemical and immunological methods.

[0108] Example 14: Rescue of injured hippocampal slices after co-culture with conditioned medium from bone marrow stromal cells or neural regenerative cells The extent to which donor cell-conditioned medium affects injured hippocampal tissue slices was also tested. For this study, conditioned medium was prepared by adding fresh slice medium to plated donor cells (n=5 matched donors / cell type, 5-6 wells / group). After 24 h of culture, donor cell-conditioned medium was collected and added to either wells containing inserts with hippocampal slices that underwent OGD or wells containing control slices that did not undergo OGD. LDH release was assayed after 24 h. Figure 8 shows that donor cell-conditioned medium significantly improved the viability of injured slices (p<0.05), with no significant difference in the rescue effect of medium conditioned by bone marrow-adherent stromal cells or neural regenerating cells.

[0109] Example 15: Rescue of injured neurons after co-culture with conditioned medium from neural regenerating cells pre-cultured with injured hippocampal slices To determine whether the injured neuroenvironment affects the ability of donor cells to rescue injured neurons, neural regenerative cells (n=3 donors) were seeded in 6-well plates and co-cultured with either hippocampal slices injured by OGD or control non-injured slices for 48 hours (Example 12). After this pre-culture, the medium was removed and the two neural regenerative cell-conditioned mediums were diluted 1:25 with neuron medium and added to wells containing neurons that had undergone OGD immediately before the addition of the conditioned medium (see Example 8). After another 24 hours, LDH release was assayed (4 wells / group).

[0110] Figure 9 shows the results of this experiment. Injured neurons cultured in conditioned medium from neural regenerating cells co-cultured with injured brain slices showed significantly less cell damage than those cultured in conditioned medium from neural regenerating cells co-cultured with control non-injured slices (p<0.05). This result provides further evidence that donor cells respond to cues from injured neurons that affect the production and / or secretion of one or more diffusible trophic factors from the donor cells.

[0111] Example 16: Trophic factor secretion by bone marrow stromal cells and neural regenerative cells The data presented above provide evidence that bone marrow stromal cells and stromal cell-derived neural regenerating cells can rescue injured neural cells by the provision of one or more trophic factors. To begin to identify the key trophic factors involved, protein levels of specific candidate trophic factors were quantified in donor cell-conditioned medium. Bone marrow stromal cells and neural regenerating cells (n=5 matched donors / cell type) were cultured at approximately 15,000 cells / cm in αMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. 2 The cells were cultured until they reached a density of 100,000 cells / ml. The medium was then replaced with Opti-MEM® medium (approximately 200,000 cells / ml) and the conditioned medium was harvested after 72 hours. The amounts of the following 10 cytokines were quantified using Quantibody® Custom Array (RayBiotech, Inc.): bone morphogenetic protein-7 (BMP-7), brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), glial cell line-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), nerve growth factor (b-NGF), platelet-derived growth factor (PDGF-BB), insulin-like growth factor-I (IGF-I), and vascular endothelial growth factor (VEG-F). The results are shown in Table 1. Hepatocyte growth factor and vascular endothelial growth factor were found in significant amounts (ie, within the range of the standards tested), with no significant differences between the concentrations present in bone marrow stem cell- or neural regenerative cell-conditioned medium.

[0112] [Table 1]

[0113] Example 17: Further analysis of trophic factor secretion by bone marrow stromal cells and neural regenerative cells Bone marrow stromal cells and neural regenerative cells (n=5 matched donors / cell type) were cultured at approximately 15,000 cells / cm in αMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. 2 The donor cells were cultured to a density of 100,000 cells / ml. The medium was then replaced with Opti-MEM® reduced serum medium (approximately 200,000 cells / ml) and conditioned medium was harvested after 72 hours. To determine which of the 30 selected cytokines were detectable in the donor cell-conditioned medium and to compare the relative amounts of cytokine levels in the different samples, a semi-quantitative custom array (Quantibody™ Array, RayBiotech, Inc., Norcross, GA) was used. The arrays were exposed to conditioned medium from MASCs and NRCs obtained as described previously in this paragraph.

[0114] The results are summarized in Table 2. The first column identifies the factor whose presence was assayed. The second column indicates the number of MASC conditioned medium preparations (out of a total of five) that showed levels of a particular factor that were 1.5-fold higher than the level of the factor in Opti-MEM®. The third and fourth columns indicate the average level of a given factor in the five samples, expressed as the fold change in normalized signal intensity and standard deviation (fourth column) compared to the normalized signal intensity of arrays exposed to Opti-MEM® only (third column). A fold change of 1.5-fold or greater compared to Opti-MEM® only indicates that the cytokine detected in the conditioned medium was due to the presence of donor cells.

[0115] Column 5 indicates the number of NRC conditioned medium preparations (out of a total of five) that had levels of a particular factor that were 1.5-fold greater than the level of that factor in Opti-MEM®. Columns 6 and 7 indicate the average level of a given factor in the five samples, expressed as the fold change in normalized signal intensity and standard deviation (column 7) compared to arrays exposed to Opti-MEM® only (column 6). A fold change of 1.5-fold or greater compared to Opti-MEM® only indicates that the cytokine detected in the conditioned medium was due to the presence of donor cells.

[0116] The eighth column indicates the number of donors (out of a total of five) in which the amount of a particular factor was higher in NRC-conditioned medium than in MASC-conditioned medium. The ninth and tenth columns indicate the ratio of levels in NRC-conditioned medium to levels in MASC-conditioned medium and the standard deviation, respectively, for each factor tested.

[0117] The results show that the following ten cytokines were found in either MASC or NRC donor cell-conditioned medium: bone morphogenetic protein-4 (BMP-4), Dickkopf-1 (IL-1), fibroblast growth factor-7 (FGF-7), heparin-binding epidermal growth factor-like growth factor (HB-EGF), interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), matrix metalloproteinase-1 (MMP-1), platelet-derived growth factor (PDGF-AA), and vascular endothelial growth factor (VEGF). Four of these were consistently found at increased levels in neural regenerative cell-conditioned medium compared to bone marrow stromal cell-conditioned medium: DKK-1, IL-6, IL-8, and MCP-1.

[0118] [Table 2]

[0119] Example 18: Secretion of trophic factors in the presence of heparin Some growth factors are known to contain heparin-binding domains. Therefore, they may not be soluble in the medium because they are secreted by donor cells but instead bind to endogenous heparin on the cells. To test for production of such factors by MASC and / or NRC, heparin (50 μg / ml) was added after transferring the cells to Opti-MEM® and once each day thereafter until conditioned medium was harvested (for a total of 3 days). If such heparin-binding growth factors are produced and secreted by donor cells, endogenous heparin in the medium would complete their binding and increase the chances of them being recovered in the conditioned medium. Conditioned medium was assayed as described in Example 17.

[0120] The results are shown in Table 3. The first column identifies the factor whose presence was assayed. The second column indicates the level of a given factor in conditioned medium from a single sample of MASC. The third column indicates the level of the factor in a MASC sample from the same donor (D46) in which heparin was present during preparation of the conditioned medium. The fourth column indicates the level of a given factor in conditioned medium from a single sample of NRC obtained from the same donor as the MASC characterized in columns 2 and 3. The fifth column indicates the level of a factor in an NRC sample from the same donor (D46) in which heparin was present during preparation of the conditioned medium. The sixth column indicates the level of a given factor in conditioned medium from a single sample of NRC obtained from a different donor (D47). The seventh column indicates the level of a factor in an NRC sample from donor D4) in which heparin was present during preparation of the conditioned medium. The eighth column indicates the fold increase in the level of a factor in heparin-containing cultures, averaged across all three samples (D46 MASC, D46 NRC, and D47 NRC).

[0121] The results show that the addition of heparin to the culture medium increased the levels in the conditioned medium of three factors secreted in the absence of heparin (DKK-I, IL-6, and VEGF). In addition, increased levels of hepatocyte growth factor (HGF), which was not detected in the absence of heparin (Example 16), were observed in the donor cell-conditioned medium. Finally, in one donor, increased levels of transforming growth factor alpha (TGF-α, not detectable in the absence of heparin) were also observed. These results point to the presence of heparin-binding factors secreted by donor MASC and NRC.

[0122] [Table 3]

[0123] Example 19: Quantification of trophic factors secreted by MASC and NRC Conditioned medium was collected from MASCs and NRCs (n=8 matched donors / cell type) as described in Examples 16 and 17, and the concentrations of 10 of the factors identified in Examples 16-18 were measured in the conditioned medium using Quantibody® custom arrays (Ray Biotech, Inc.). The results shown in Table 4 confirm previous findings. With the exception of BMP-4 and HB-EGF, all cytokines tested are present in the conditioned medium by bone marrow adherent stromal cells and / or neural regenerative cells (within the range of standards used in the assay). Also confirming the results obtained in Example 17, four of these proteins (KK-I, IL-6, IL-8, and MCP-I) were found to be consistently at increased levels in neural regenerative cell-conditioned medium compared to bone marrow adherent stromal cell-conditioned medium. The inability to detect BMP-4 in the two cell types and the ability to detect HB-EGF in NRCs may be due to its levels being outside the range of standards for this particular array.

[0124] [Table 4]

Claims

1. 1. A method for providing trophic support to damaged neural tissue in a host, comprising: A method comprising transplanting bone marrow adherent stromal cells or bone marrow derived neural regenerating cells at or near the site of neurodegeneration in a host.

2. The method of claim 1 , wherein the nutrient carrier promotes survival of host neural cells.

3. The method of claim 1 , wherein the nutrient carrier inhibits death of host neural cells.

4. The method of claim 3, wherein the cell death is by apoptosis.

5. The method of claim 1 , wherein the nutrient carrier enhances the function of host neural cells.

6. The method of claim 1 , wherein the nutrient carrier stimulates host neural cells.

7. 7. The method of claim 6, wherein the growth of nerve fibers is stimulated.

8. The method of claim 7 , wherein the nerve fiber is an axon.

9. The method of claim 7 , wherein the nerve fiber is a dendrite.

10. The method of claim 6, wherein new synapses are formed.

11. The method of claim 1 , wherein the nutrient carrier promotes the recruitment of host stem cells to the damaged tissue.

12. The method of claim 1 , wherein the damaged nervous tissue is in the central nervous system.

13. The method of claim 1 , wherein the damaged nerve tissue is in the peripheral nervous system.

14. The method of claim 12, wherein the damaged neural tissue is associated with Parkinson's disease.

15. The method of claim 1, wherein the bone marrow-derived neural regenerating cells are progeny of bone marrow stromal cells that have been transfected with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD).

16. The method of claim 15, wherein the NICD consists of amino acids 1703 to 2504 of the Notch-1 protein.

17. The method of claim 1 , wherein the bone marrow adherent stromal cells or the bone marrow derived neural regenerating cells secrete one or more trophic factors.

18. The method of claim 17, wherein the bone marrow adherent stromal cells or the bone marrow derived neural regenerating cells secrete one or more of vascular endothelial growth factor, hepatocyte growth factor, bone morphogenetic protein 4, Dkk-1, fibroblast growth factor-7, heparin-binding epidermal growth factor-like growth factor, interleukin-6, interleukin-8, monocyte chemotactic protein-1, matrix metalloproteinase-1, platelet-derived growth factor AA, and alpha-transforming growth factor.

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