Composition expressed at time of biobridge formation after traumatic brain injury

Abstract

To provide a composition that allows biobridging between the subventricular zone and the damaged brain site by implanting cells after a traumatic brain injury of a subject.SOLUTION: The composition is expressed when a biobridge is formed between the subventricular zone and the damaged brain site by implanting cells after a traumatic brain injury of a subject and comprises matrix metalloproteinase 9 at a therapeutically effective amount. The cells are obtained in a process comprising: (a) a step of providing the culture product of bone marrow adherent stem cells; (b) a step of bringing the cell culture product of the step (a) into contact with a polynucleotide that includes a sequence encoding the Notch intracellular domain, the polynucleotide not encoding the full length of the Notch protein; (c) a step of selecting cells comprising the polynucleotide of the step (b); and (d) a step of additionally culturing without selecting the cell selected in the step (c).SELECTED DRAWING: None

Description

Technical Field

[0001] Citation of Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 61 / 647,893, filed May 16, 2012. The specification and drawings of U.S. Provisional Patent Application No. 61 / 647,893 are hereby incorporated by reference in their entirety for all purposes.

[0002] Statement Regarding Federal Support Part of the research described herein was supported by a grant from the National Institute of Neurological Disorders and Stroke. The United States Government may have certain rights in the invention disclosed herein.

[0003] Field The present disclosure is in the field of cell therapy for neurological disorders.

Background Art

[0004] Background Stem cells were first adopted for the thorough examination of cell development1 and form the basis of regenerative medicine, including cell-based therapies for the treatment of neurological disorders2,3. Stem cells also exist in adulthood8, self-renew, differentiate into multiple lineages9, contribute to normal homeostasis10, and have the ability to be therapeutically beneficial endogenously11-14, or after transplantation15-21, in damaged organs, i.e., the brain. The subventricular zone (SVZ) of the lateral ventricle and the granular cell layer of the hippocampal dentate gyrus are the two major stem cell niches in the adult brain22,23, although quiescent neural stem cells (NSCs) have been detected in other brain regions24. Induction of endogenous stem cells after injury provides new opportunities in regenerative medicine2,3,11-21.

[0005] Cells other than pluripotent stem cells have also been used in the treatment of disorders of the central nervous system. For the treatment of stroke, SB623 cells (expressing exogenous Notch intracellular domain) were The cells derived from the marrow adherent stem cells that are present in the bone marrow are called endothelial cells. It is used by implantation at or near the site of the attack. See, e.g., U.S. Patent No. 8,092, , No. 792 and Yasuhara et al. (2009) Stem Cells Devel. 18:1501-1 See page 513. U.S. Patent No. 7,682,825 is a patent for the treatment of several disorders of the central and peripheral nervous system. Further uses of SB623 cells in the treatment of cancer are described.

[0006] Despite these scientific advances and some early clinical studies,25-27 cell therapy remains a A fundamental gap in our understanding of the law is the ability of transplanted cells to induce cell proliferation in injured neural tissue. To date, knowledge of the mechanisms that promote graft survival and persistence has been limited. Increased efficacy of cell transplants in providing therapeutic benefit in hematological and non-hematological disorders Therefore, the survival and persistence of transplanted cells is considered to be the key to successful therapy. Much effort has been directed at prolonging the life of large numbers of transplanted cells. An effective method of cell therapy that does not require sex would be advantageous.

[0007] Traumatic brain injury (TBI) refers to injury to the brain resulting from an external mechanical force. TBI can be caused by: Arising from falls, firearm wounds, sports accidents, construction accidents and vehicle accidents, among other causes Victims of TBI suffer from several physical, cognitive, social, emotional and / or or may suffer behavioral disorders.

[0008] There is little that can be done to reverse the initial physical damage of TBI. Therefore , treatment options mainly consist of stabilization to prevent further injury during the acute phase, and subsequent rehabilitation . Due to these limited options, additional methods and compositions for the treatment of TBI are needed.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0010]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0011] Summary The inventors have discovered that transplantation of SB623 cells (i.e., cells derived from myeloid adherent stem cells in which the exogenous Notch intracellular domain is expressed) can be used in the treatment of traumatic brain injury (TBI). Animals that received transplantation of SB623 cells after TBI showed significantly improved motor and nerve functions in addition to a significant reduction in injury to the cortical core and the cortical area surrounding the injury, compared to injured animals that received only vehicle injections.

[0012] Contrary to expectations, the inventors found that the survival and persistence of a large number of transplanted cells was SB623​ It was also found that it is not required for the therapeutic benefit of cell transplantation. Surprisingly, the therapeutic benefit is sufficient to initiate a robust and stable functional recovery and can be obtained by minimal acute graft survival. This addresses two major problems: the necessity of a sufficient supply of transplantable cells and the necessity of long-term graft survival.

[0013] The inventors have also discovered that the beneficial effect of SB623 cell transplantation in the treatment of TBI results from the formation of a biological bridge (the "biobridge") between the neurogenic niche in the subventricular zone (SVZ) and the damaged brain site. This biobridge, visualized immunohistochemically and laser-captured, initially expressed high levels of extracellular matrix metalloproteinases and was characterized by a stream of transplanted cells. Over time after transplantation, the transplanted cells were replaced by newly formed host cells, and few transplanted cells remained in the biobridge. Thus, the transplanted SB623 cells initially formed a pathway that promoted the subsequent migration of host neurogenic cells from the neurogenic niche to the brain injury site between the neurogenic SVZ and the damaged cortex.

[0014] This series of events reveals a novel method for the treatment of TBI, namely, the transplantation of SB623 cells that form a temporary pathway to direct the migration of host neurogenic cells. That is, the transplanted SB623 cells initially form a biobridge between the neurogenic niche and the damaged site, but once this biobridge is formed, the transplanted cells are replaced by host neurogenic cells that migrate to the damaged site. These findings demonstrate that from the neurogenic niche to the damaged ​​​​​​​​​​​​​Long-distance migration of host cells to the damaged brain region is achieved through transplanted SB623 cells that act as a biobridge for the initiation of the endogenous repair mechanism It has been shown that it can be achieved through transplanted SB623 cells that act as .

[0015] Accordingly, the present disclosure provides, among other things, the following embodiments: 1. A method for treating a traumatic brain injury in a subject, the method comprising administering to the subject's brain a therapeutically effective amount of SB623 cells, wherein the SB623 cells are obtained by: (a) providing a culture of bone marrow adherent stem cells (MSCs); (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD), wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection cells (MSC); (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD), wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection cells (MSC); (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD), wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection cells (MSC); (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD), wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection cells (MSC); (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD), wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection cells (MSC); (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD), wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection obtained by the method. 2. The method according to embodiment 1, wherein the subject is a human. 3. The method according to any one of embodiments 1 or 2, wherein the MSC is obtained from a human. 4. Cells for transplantation into a subject for the treatment of traumatic brain injury, the cells being obtained by a process comprising: (a) providing a culture of MSCs; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding NICD, wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection cells for transplantation into a subject for the treatment of traumatic brain injury, the cells being obtained by a process comprising: (a) providing a culture of MSCs; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding NICD, wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection cells for transplantation into a subject for the treatment of traumatic brain injury, the cells being obtained by a process comprising: (a) providing a culture of MSCs; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding NICD, wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection cells for transplantation into a subject for the treatment of traumatic brain injury, the cells being obtained by a process comprising: (a) providing a culture of MSCs; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding NICD, wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection cells for transplantation into a subject for the treatment of traumatic brain injury, the cells being obtained by a process comprising: (a) providing a culture of MSCs; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding NICD, wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection cells for transplantation into a subject for the treatment of traumatic brain injury, the cells being obtained by a process comprising: (a) providing a culture of MSCs; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding NICD, wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection 5. The cells according to embodiment 4, wherein the subject is a human. 6. The cell according to any one of embodiments 4 or 5, wherein the MSC is obtained from a human. 7. A method for inducing the migration of endogenous neurogenic cells from a neurogenic niche to a brain injury site, the method comprising administering a therapeutically effective amount of SB623 cells to the brain of a subject, wherein the SB623 cells are obtained by: (a) providing a culture of MSC; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD), wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting the cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection. 8. The method according to embodiment 7, wherein the neurogenic niche is the subventricular zone. 9. The method according to any one of embodiments 7 or 8, wherein the brain injury is a traumatic brain injury. 10. The method according to any one of embodiments 7 to 9, wherein the subject is a human. 11. The method according to any one of embodiments 7 to 10, wherein the MSC is obtained from a human. 12. A method for stimulating the proliferation of neurogenic cells of a subject, the method comprising administering a therapeutically effective amount of SB623 cells to the brain of a subject, wherein the SB623 cells are obtained by: (a) providing a culture of MSC; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD), wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting the cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection. 13. The method according to any one of embodiments 7 to 12, wherein the polynucleotide is a short hairpin RNA (shRNA). 14. The method according to any one of embodiments 7 to 13, wherein the polynucleotide is introduced into the cell culture by transfection. 15. The method according to any one of embodiments 7 to 14, wherein the selection step (c) is performed by fluorescence-activated cell sorting (FACS). 16. The method according to any one of embodiments 7 to 15, wherein the culturing step (d) is performed for about 1 to about 14 days. 17. A pharmaceutical composition comprising SB623 cells obtained by: (a) providing a culture of MSC; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD), wherein the polynucleotide does not encode a full-length Notch protein; (c) selecting the cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection. 18. The pharmaceutical composition according to embodiment 17, wherein the polynucleotide is a short hairpin RNA (shRNA). 19. The pharmaceutical composition according to embodiment 17 or 18, further comprising a pharmaceutically acceptable carrier. 20. Use of the pharmaceutical composition according to any one of embodiments 17 to 19 for the manufacture of a medicament for treating a brain injury. 21. Use of the pharmaceutical composition according to any one of embodiments 17 to 19 for the manufacture of a medicament for stimulating the proliferation of neurogenic cells of a subject. 22. A method for treating a brain injury, the method comprising administering a therapeutically effective amount of the pharmaceutical composition according to any one of embodiments 17 to 19 to a subject in need thereof. 23. A method for stimulating the proliferation of neurogenic cells of a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition according to any one of embodiments 17 to 19 to a subject in need thereof. 24. The method according to embodiment 23, wherein the subject has a brain injury. A method obtained by the step of further culturing the selected cells of step (c) without selection. The method thus obtained. 13. The method according to embodiment 12, wherein the brain injury is a traumatic brain injury. 14. The method according to any one of embodiments 12 or 13, wherein the subject is a human. 15. The method according to any one of embodiments 12 to 14, wherein the MSC is obtained from a human. 16. A method for inducing the proliferation of neurogenic cells and their migration to the brain injury site of a subject, the method comprising administering a therapeutically effective amount of SB623 cells to the brain of the subject, wherein the SB623 cells are obtained by: (a) providing a culture of MSCs; and (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD), wherein the polynucleotide does not encode a full-length Notch protein; and (c) selecting the cells comprising the polynucleotide of step (b); and (d) further culturing the selected cells of step (c) without selection. The method thus obtained. 17. The method according to embodiment 16, wherein the brain injury is a traumatic brain injury. 18. The method according to any one of embodiments 16 or 17, wherein the subject is a human. 19. The method according to any one of embodiments 16 to 18, wherein the MSC is obtained from a human. 18. The method according to any one of embodiments 16 or 17, wherein the subject is a human. 19. The method according to any one of embodiments 16 to 18, wherein the MSC is obtained from a human. In certain embodiments, for example, the following are provided: (Item 1) Cells for transplantation into a subject for the treatment of traumatic brain injury, (a) providing a culture of MSCs; (b) contacting the cell culture of step (a) with a polynucleotide comprising a sequence encoding NICD, wherein the polynucleotide does not encode a full-length Notch protein; A step that does not encode a protein, and (c) Selecting a cell comprising the polynucleotide of step (b); (d) Further culturing the selected cell of step (c) without selection. A cell obtained by a process comprising: (Item 2) The cell according to item 1, wherein the subject is human. (Item 3) The cell according to item 1 or item 2, wherein the MSC is obtained from human. (Item 4) A method for treating a traumatic brain injury of a subject, comprising administering a therapeutically effective amount of the cell according to any one of items 1 to 3 to the brain of the subject. (Item 5) A method for inducing the migration of endogenous neurogenic cells from a neurogenic niche to a brain injury site, comprising administering a therapeutically effective amount of the cell according to any one of items 1 to 3 to the brain of the subject. (Item 6) The method according to item 5, wherein the neurogenic niche is the subventricular zone. (Item 7) The method according to item 5, wherein the brain injury is a traumatic brain injury. (Item 8) A method for stimulating the proliferation of neurogenic cells of a subject, comprising administering a therapeutically effective amount of the SB623 cell according to any one of items 1 to 3 to the brain of the subject. (Item 9) A method for inducing the proliferation of neurogenic cells and their migration to the brain injury site of a subject, comprising administering a therapeutically effective amount of the SB623 cell according to any one of items 1 to 3 to the brain of the subject. (Item 10) The method according to item 9, wherein the brain injury is a traumatic brain injury. BRIEF DESCRIPTION OF THE DRAWINGS

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[0024] Detailed Description Methods and compositions for the treatment of traumatic brain injury (TBI) are disclosed herein. Methods and compositions for the modulation of the migration of stem cells (e.g., neural stem cells, neuronal stem cells) in the brain are also disclosed herein. Methods and compositions for the modulation of the migration of stem cells (e.g., neural stem cells, neuronal stem cells) in the brain are also disclosed herein.

[0025] The inventors have made the unexpected discovery that the behavioral and histological improvements resulting from cell transplantation after TBI do not require large-scale graft survival or long-term graft persistence. In fact, only modest acute graft survival is required to provide these therapeutic benefits. Thus, the inventors have discovered a novel method for nerve repair with a threshold amount of transplanted cells that can induce the SVZ to generate and drive new cells into the impacted cortical area without the need to persist in the brain. Thus, a minimal effective dose of transplantation and acute survival of the transplanted cells are sufficient to initiate complex endogenous restoration mechanisms to suppress substantial brain injury.

[0026] ​​​​In the practice of the present disclosure, unless otherwise specified, cell biology, toxicology, molecular biology, biochemistry, cell culture, immunology, neurology, surgery, recombinant DNA, and related fields within the scope of the art are employed using standard methods and conventional techniques. Such techniques are described in the literature and are thus available to those of ordinary skill in the art. For example, Alberts, B. et al., "Molecular Biology of the Cell", 5th Edition, Garland Science, New York, NY, 2008; Voet, D. . et al. "Fundamentals of Biochemistry: Life at the Molecular Level", 3rd Edition , John Wiley & Sons, Hoboken, NJ, 2008; Sambrook, J. et al., "Molecular C loning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press , 2001; Ausubel, F. et al., "Current Protocols in Molecular Biology", J ohn Wiley & Sons, New York, 1987 and periodic updates; Freshney, R.I., "C ulture of Animal Cells: A Manual of Basic Technique", 4th Edition, John Wil ey & Sons, Somerset, NJ, 2000; as well as the series "Methods in Enzymolo gy", Academic Press, San Diego, CA.

[0027] Mesenchymal stem cells (MSCs) The present disclosure treats TBI by transplanting SB623 cells into the brain injury site of a subject , provided is a method for modulating stem cell migration. SB623 cells are obtained from mesenchymal stem cells (MSCs) by expressing the intracellular domain of the Notch protein in myeloid adherent stem cells (MSCs), which are also known as myeloid adherent stromal cells and mesenchymal stem cells. MSCs are obtained by selecting adherent cells (i.e., cells that adhere to tissue culture plastic) from bone marrow. Exemplary disclosures of MSCs are provided in U.S. Patent Application Publication No. 2003 / 0003090; Prockop (1997) Science 276:71-74 and Jiang (2002) Nature 418 vol. 41-49. Methods for the isolation and purification of MSCs can be found, for example, in U.S. Patent No. 5,486,359; Pittenger et al. (1999) Science 284:143-1

[0028] 47 and Dezawa et al. (2001) Eur. J. Neurosci. 14:1771-1776 pages. Human MSCs are commercially available (e.g., BioWhittak er, Walkersville, MD) or can be obtained from donors, for example, by bone marrow aspiration and subsequent selection of adherent bone marrow cells. See, for example, WO2005 / 10055 2. MSCs can also be isolated from umbilical cord blood. See, for example, Campagnoli et al. (2001 Blood 98:2396-2402; Erices et al. (2000) Br. J. Haematol. 10 9:235-242 and Hou et al. (2003) Int. J. Hematol. 78:256 -261. Additional sources of MSCs include, for example, menstrual blood and placenta.

[0029] See, for example, Campagnoli et al. (2001 Blood 98:2396-2402; Erices et al. (2000) Br. J. Haematol. 10 9:235-242 and Hou et al. (2003) Int. J. Hematol. 78:256 -261. Additional sources of MSCs include, for example, menstrual blood and placenta. .

[0030] Notch intracellular domain The Notch protein is a transmembrane receptor found in all metazoans that affects cell differentiation through intracellular signaling. Contact between the Notch extracellular domain and Notch ligands (e.g., Delta, Serrate, Jagged) results in two proteolytic cleavages of the Notch protein, the second of which is catalyzed by γ-secretase and releases the Notch intracellular domain (NICD) into the cytoplasm. In the mouse Notch protein, this cleavage occurs between amino acids gly1743 and val1744. The NICD translocates to the nucleus where it acts as a transcription factor and recruits additional transcriptional regulatory proteins (e.g., MAM, histone acetylase) to relieve the transcriptional repression of various target genes (e.g., Hes1). For further details and information regarding Notch signaling, see, for example, Artavanis-Tsakonas et al. (1995) Science 268:225-232; Mumm and Kopan (2000) Develop. Biol. 228:151-165 and Ehebauer et al. (2006) Sci. STKE 2006(364), cm7. [DOI: 10.1126 / stke.3642006cm7].

[0031]

[0032] Cell culture and transfection Standard methods of cell culture are known in the art. For example, R. I. Freshney "Culture of Animal Cells: A Manual of Basic Technique", 5th Edition, Wiley, New See York, 2005.

[0033] Methods for introducing exogenous DNA into cells (i.e., transfection), as well as methods for selecting cells containing exogenous DNA, are also well known in the art. For example, Sa mbrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Ha rbor Laboratory Press, 2001; Ausubel et al., "Current Protocols in Molecu lar Biology", John Wiley & Sons, New York, 1987 and periodic updates are referred to.

[0034] SB623 cells In one embodiment for the preparation of SB623 cells, a culture of MSCs is contacted with a polynucleotide comprising a sequence encoding the Notch intracellular domain (NICD), for example by transfection, followed by enrichment of the transfected cells by drug selection and further culture. See, for example, U.S. Patent No. 7,682,825 (March 2 23, 2010); U.S. Patent Application Publication No. 2010 / 0266554 (October 21, 2010) and WO2009 / 023251 (February 19, 2009); the entire disclosures of which are incorporated by reference in their entirety for the purpose of describing the isolation of bone marrow adherent stem cells and the conversion of bone marrow adherent stem cells into SB623 cells (referred to in those documents as "neural progenitor cells" and "neural regenerative cells"). In these methods, any polynucleotide encoding the Notch intracellular domain ( ).

[0035] ). For example, a vector) can be used, and any method for the selection and enrichment of transfected cells can be used. For example, in certain embodiments, the MSC contains a sequence encoding the Notch intracellular domain and is transfected with a vector that also contains a sequence encoding a drug resistance marker (e.g., resistance to G418). In further embodiments, two vectors are used for the transfection of MSC, one containing a sequence encoding the Notch intracellular domain and the other containing a sequence encoding a drug resistance marker. In these embodiments, after transfection of the cell culture with the vector, selection is achieved by adding to the cell culture a selection agent (e.g., G418) in an amount sufficient to kill cells that do not contain the vector but spare cells that contain the vector. Non-selection involves the removal of the selection agent or a reduction in its concentration to a level that does not kill cells that do not contain the vector. After selection (e.g., for 7 days), the selection agent is removed and the cells are cultured further (e.g., for 2 passages). Thus, the preparation of SB623 cells involves transient expression of an exogenous Notch intracellular domain in MSC. For this purpose, MSC can be transfected with a vector containing a sequence that encodes the Notch intracellular domain and does not encode the full-length Notch protein. All such sequences are well known to those skilled in the art and are immediately available. For example, Del Amo et al. (1993) Genomics 15:259-264 presents the complete amino acid sequence of the mouse Notch protein, and Mumm and Kopan (2000) Devel. Bio For example, in certain embodiments, the MSC contains a sequence encoding the Notch intracellular domain and is transfected with a vector that also contains a sequence encoding a drug resistance marker (e.g., resistance to G418). In further embodiments, two vectors are used for the transfection of MSC, one containing a sequence encoding the Notch intracellular domain and the other containing a sequence encoding a drug resistance marker. In these embodiments, after transfection of the cell culture with the vector, selection is achieved by adding to the cell culture a selection agent (e.g., G418) in an amount sufficient to kill cells that do not contain the vector but spare cells that contain the vector. Non-selection involves the removal of the selection agent or a reduction in its concentration to a level that does not kill cells that do not contain the vector. After selection (e.g., for 7 days), the selection agent is removed and the cells are cultured further (e.g., for 2 passages). In further embodiments, two vectors are used for the transfection of MSC, one containing a sequence encoding the Notch intracellular domain and the other containing a sequence encoding a drug resistance marker. In these embodiments, after transfection of the cell culture with the vector, selection is achieved by adding to the cell culture a selection agent (e.g., G418) in an amount sufficient to kill cells that do not contain the vector but spare cells that contain the vector. Non-selection involves the removal of the selection agent or a reduction in its concentration to a level that does not kill cells that do not contain the vector. After selection (e.g., for 7 days), the selection agent is removed and the cells are cultured further (e.g., for 2 passages). In these embodiments, after transfection of the cell culture with the vector, selection is achieved by adding to the cell culture a selection agent (e.g., G418) in an amount sufficient to kill cells that do not contain the vector but spare cells that contain the vector. Non-selection involves the removal of the selection agent or a reduction in its concentration to a level that does not kill cells that do not contain the vector. After selection (e.g., for 7 days), the selection agent is removed and the cells are cultured further (e.g., for 2 passages). In these embodiments, after transfection of the cell culture with the vector, selection is achieved by adding to the cell culture a selection agent (e.g., G418) in an amount sufficient to kill cells that do not contain the vector but spare cells that contain the vector. Non-selection involves the removal of the selection agent or a reduction in its concentration to a level that does not kill cells that do not contain the vector. After selection (e.g., for 7 days), the selection agent is removed and the cells are cultured further (e.g., for 2 passages). In these embodiments, after transfection of the cell culture with the vector, selection is achieved by adding to the cell culture a selection agent (e.g., G418) in an amount sufficient to kill cells that do not contain the vector but spare cells that contain the vector. Non-selection involves the removal of the selection agent or a reduction in its concentration to a level that does not kill cells that do not contain the vector. After selection (e.g., for 7 days), the selection agent is removed and the cells are cultured further (e.g., for 2 passages). In these embodiments, after transfection of the cell culture with the vector, selection is achieved by adding to the cell culture a selection agent (e.g., G418) in an amount sufficient to kill cells that do not contain the vector but spare cells that contain the vector. Non-selection involves the removal of the selection agent or a reduction in its concentration to a level that does not kill cells that do not contain the vector. After selection (e.g., for 7 days), the selection agent is removed and the cells are cultured further (e.g., for 2 passages). In these embodiments, after transfection of the cell culture with the vector, selection is achieved by adding to the cell culture a selection agent (e.g., G418) in an amount sufficient to kill cells that do not contain the vector but spare cells that contain the vector. Non-selection involves the removal of the selection agent or a reduction in its concentration to a level that does not kill cells that do not contain the vector. After selection (e.g., for 7 days), the selection agent is removed and the cells are cultured further (e.g., for 2 passages). In these embodiments, after transfection of the cell culture with the vector, selection is achieved by adding to the cell culture a selection agent (e.g., G418) in an amount sufficient to kill cells that do not contain the vector but spare cells that contain the vector. Non-selection involves the removal of the selection agent or a reduction in its concentration to a level that does not kill cells that do not contain the vector. After selection (e.g., for 7 days), the selection agent is removed and the cells are cultured further (e.g., for 2 passages).

[0036] Thus, the preparation of SB623 cells involves transient expression of an exogenous Notch intracellular domain in MSC. For this purpose, MSC can be transfected with a vector containing a sequence that encodes the Notch intracellular domain and does not encode the full-length Notch protein. All such sequences are well known to those skilled in the art and are immediately available. For example, Del Amo et al. (1993) Genomics 15:259-264 presents the complete amino acid sequence of the mouse Notch protein, and Mumm and Kopan (2000) Devel. Bio Thus, the preparation of SB623 cells involves transient expression of an exogenous Notch intracellular domain in MSC. For this purpose, MSC can be transfected with a vector containing a sequence that encodes the Notch intracellular domain and does not encode the full-length Notch protein. All such sequences are well known to those skilled in the art and are immediately available. For example, Del Amo et al. (1993) Genomics 15:259-264 presents the complete amino acid sequence of the mouse Notch protein, and Mumm and Kopan (2000) Devel. Bio Thus, the preparation of SB623 cells involves transient expression of an exogenous Notch intracellular domain in MSC. For this purpose, MSC can be transfected with a vector containing a sequence that encodes the Notch intracellular domain and does not encode the full-length Notch protein. All such sequences are well known to those skilled in the art and are immediately available. For example, Del Amo et al. (1993) Genomics 15:259-264 presents the complete amino acid sequence of the mouse Notch protein, and Mumm and Kopan (2000) Devel. Bio Thus, the preparation of SB623 cells involves transient expression of an exogenous Notch intracellular domain in MSC. For this purpose, MSC can be transfected with a vector containing a sequence that encodes the Notch intracellular domain and does not encode the full-length Notch protein. All such sequences are well known to those skilled in the art and are immediately available. For example, Del Amo et al. (1993) Genomics 15:259-264 presents the complete amino acid sequence of the mouse Notch protein, and Mumm and Kopan (2000) Devel. Bio For example, Del Amo et al. (1993) Genomics 15:259-264 presents the complete amino acid sequence of the mouse Notch protein, and Mumm and Kopan (2000) Devel. Bio For example, Del Amo et al. (1993) Genomics 15:259-264 presents the complete amino acid sequence of the mouse Notch protein, and Mumm and Kopan (2000) Devel. Bio Pages 151-165 of Volume 1,228 surround the so-called S3 cleavage site that releases the intracellular domain, and provide the amino acid sequence from the mouse Notch protein. In summary, these references provide those skilled in the art with any peptide containing a Notch intracellular domain that is not a full-length Notch protein, thereby providing those skilled in the art with any polynucleotide containing a sequence encoding a Notch intracellular domain that does not encode a full-length Notch protein. The aforementioned documents (Del Amo and Mumm) are hereby incorporated by reference in their entirety for disclosing the amino acid sequence of the full-length Notch protein and the amino acid sequence of the Notch intracellular domain respectively. Similar information is available for Notch proteins and nucleic acids from additional species including rats, Xenopus laevis, Drosophila melanogaster, and humans. For example, see Weinmaster et al. (1991) Development 113:199-205; Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No. NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are hereby incorporated by reference in their entirety for disclosing the amino acid sequences of the full-length Notch proteins and the amino acid sequences of the Notch intracellular domains of several different species. In a further embodiment, the SB623 cells are such that the MSCs are exogenous to the Notch extracellular domain containing any peptide, thereby providing those skilled in the art with any polynucleotide containing a sequence encoding a Notch intracellular domain that does not encode a full-length Notch protein. The aforementioned documents (Del Amo and Mumm) are hereby incorporated by reference in their entirety for disclosing the amino acid sequence of the full-length Notch protein and the amino acid sequence of the Notch intracellular domain respectively. Similar information is available for Notch proteins and nucleic acids from additional species including rats, Xenopus laevis, Drosophila melanogaster, and humans. For example, see Weinmaster et al. (1991) Development 113:199-205; Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No. NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are hereby incorporated by reference in their entirety for disclosing the amino acid sequences of the full-length Notch proteins and the amino acid sequences of the Notch intracellular domains of several different species. In a further embodiment, the SB623 cells are such that the MSCs are exogenous to the Notch extracellular domain containing any peptide, thereby providing those skilled in the art with any polynucleotide containing a sequence encoding a Notch intracellular domain that does not encode a full-length Notch protein. The aforementioned documents (Del Amo and Mumm) are hereby incorporated by reference in their entirety for disclosing the amino acid sequence of the full-length Notch protein and the amino acid sequence of the Notch intracellular domain respectively. Similar information is available for Notch proteins and nucleic acids from additional species including rats, Xenopus laevis, Drosophila melanogaster, and humans. For example, see Weinmaster et al. (1991) Development 113:199-205; Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No. NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are hereby incorporated by reference in their entirety for disclosing the amino acid sequences of the full-length Notch proteins and the amino acid sequences of the Notch intracellular domains of several different species.

[0037] Similar information is available for Notch proteins and nucleic acids from additional species including rats, Xenopus laevis, Drosophila melanogaster, and humans. For example, see Weinmaster et al. (1991) Development 113:199-205; Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No. NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are hereby incorporated by reference in their entirety for disclosing the amino acid sequences of the full-length Notch proteins and the amino acid sequences of the Notch intracellular domains of several different species. Similar information is available for Notch proteins and nucleic acids from additional species including rats, Xenopus laevis, Drosophila melanogaster, and humans. For example, see Weinmaster et al. (1991) Development 113:199-205; Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No. NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are hereby incorporated by reference in their entirety for disclosing the amino acid sequences of the full-length Notch proteins and the amino acid sequences of the Notch intracellular domains of several different species. Similar information is available for Notch proteins and nucleic acids from additional species including rats, Xenopus laevis, Drosophila melanogaster, and humans. For example, see Weinmaster et al. (1991) Development 113:199-205; Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No. NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are hereby incorporated by reference in their entirety for disclosing the amino acid sequences of the full-length Notch proteins and the amino acid sequences of the Notch intracellular domains of several different species. Similar information is available for Notch proteins and nucleic acids from additional species including rats, Xenopus laevis, Drosophila melanogaster, and humans. For example, see Weinmaster et al. (1991) Development 113:199-205; Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No. NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are hereby incorporated by reference in their entirety for disclosing the amino acid sequences of the full-length Notch proteins and the amino acid sequences of the Notch intracellular domains of several different species. Similar information is available for Notch proteins and nucleic acids from additional species including rats, Xenopus laevis, Drosophila melanogaster, and humans. For example, see Weinmaster et al. (1991) Development 113:199-205; Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No. NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are hereby incorporated by reference in their entirety for disclosing the amino acid sequences of the full-length Notch proteins and the amino acid sequences of the Notch intracellular domains of several different species. Similar information is available for Notch proteins and nucleic acids from additional species including rats, Xenopus laevis, Drosophila melanogaster, and humans. For example, see Weinmaster et al. (1991) Development 113:199-205; Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No. NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are hereby incorporated by reference in their entirety for disclosing the amino acid sequences of the full-length Notch proteins and the amino acid sequences of the Notch intracellular domains of several different species. Similar information is available for Notch proteins and nucleic acids from additional species including rats, Xenopus laevis, Drosophila melanogaster, and humans. For example, see Weinmaster et al. (1991) Development 113:199-205; Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No. NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are hereby incorporated by reference in their entirety for disclosing the amino acid sequences of the full-length Notch proteins and the amino acid sequences of the Notch intracellular domains of several different species. Similar information is available for Notch proteins and nucleic acids from additional species including rats, Xenopus laevis, Drosophila melanogaster, and humans. For example, see Weinmaster et al. (1991) Development 113:199-205; Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No. NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are hereby incorporated by reference in their entirety for disclosing the amino acid sequences of the full-length Notch proteins and the amino acid sequences of the Notch intracellular domains of several different species. Similar information is available for Notch proteins and nucleic acids from additional species including rats, Xenopus laevis, Drosophila melanogaster, and humans. For example, see Weinmaster et al. (1991) Development 113:199-205; Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No. NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are hereby incorporated by reference in their entirety for disclosing the amino acid sequences of the full-length Notch proteins and the amino acid sequences of the Notch intracellular domains of several different species.

[0038] Similar information is available for Notch proteins and nucleic acids from additional species including rats, Xenopus laevis, Drosophila melanogaster, and humans. For example, see Weinmaster et al. (1991) Development 113:199-205; Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No. NM_017167 (and the references cited therein); SwissProt P46531 (and the references cited therein); SwissProt Q01705 (and the references cited therein); and GenBank CAB40733 (and the references cited therein). The aforementioned references are hereby incorporated by reference in their entirety for disclosing the amino acid sequences of the full-length Notch proteins and the amino acid sequences of the Notch intracellular domains of several different species. By introducing a nucleic acid containing a sequence encoding the Notch intracellular domain into MSC so as not to express it It is prepared by. This can be achieved, for example, by transfecting MSC with a vector containing a sequence encoding the Notch intracellular domain that does not encode a full-length Notch protein. This can be achieved by transfection.

[0039] Further details regarding the preparation of SB623 cells and methods for making cells having properties similar to those of SB62 3 cells that can be used in the methods disclosed herein are found in U.S. Patent No. 7,682,825; U.S. Patent No. 8,133,725 and U.S. Patent Application Publication Nos. 2010 / 0266554 and 2011 / 0229442, the disclosures of which provide further details regarding SB623 cells and alternative methods for their preparation, as well as methods for making cells having properties similar to those of SB623 cells and are hereby incorporated herein by reference. See also Dezawa et al. (2004) J. Clin. Invest. 113:1701-1710. For the purpose of providing, and for the purpose of providing a method for making cells having properties similar to those of SB623 cells are hereby incorporated herein by reference. See also Dezawa et al. (2004) J. Clin. Invest. 113:1701-1710. 113:1701-1710.

[0040] Reversal of symptoms of TBI by transplantation of SB623 cells As a treatment for TBI, the efficacy of SB623 cell transplantation was tested in a rat model system. To confirm that all animals exhibited normal behavior at baseline (i.e., before cerebral seizure), adult male Sprague-Dawley rats (8 weeks old) were evaluated in motor and nervous system tests (all performed by two researchers blinded to the treatment conditions throughout the study). Next, the animals were subjected to experimental traumatic brain injury (TBI) and 7 days (all performed by two researchers blinded to the treatment conditions throughout the study). Next, the animals were subjected to experimental traumatic brain injury (TBI) and 7 days after TBI, the animals were transplanted with SB623 cells. Subsequently, to confirm typical motor and nervous system dysfunctions induced by TBI , the same behavioral tests were performed. Following these tests (7 days after TBI), animals were randomly assigned to one of two groups to receive either stereotactic transplantation of Notch-induced bone marrow-derived stem cells (SB623 cells) at 26, 29 or vehicle injection into the cortex (see Example 3).

[0041] The inventors found that animals that received transplantation of SB623 cells both 1 and 3 months after TBI showed significant improvement in motor and nervous system function in addition to a significant reduction in injury to the cortical core and the cortical area surrounding the injury compared to animals that received only vehicle (see Examples). These behavioral and physical improvements were achieved with modest graft survival of 0.60% and 0.16% at 1 and 3 months after TBI, respectively. Other parts of the brain affected by TBI include the striatum and the hippocampus. Therefore, transplantation of SB6 23 cells into the striatum and hippocampus can also be used for the treatment of TBI affecting these areas. In summary, transplantation of SB623 cells into brain-injured animals provided robust functional recovery despite lack of graft persistence.

[0042] Formation of a biobridge by transplantation of SB623 cells Examination of host tissue in brain-injured animals that received transplantation of SB623 cells 1 month after TBI revealed a sharp increase in endogenous cell proliferation (detected by expression of Ki67) and differentiation of neurogenic cells (detected by expression of nestin) in the cortical area surrounding the injury and the subventricular zone (SVZ). Cells migrating along the corpus callosum (CC) of these animals (doublecortin The flow of (expressing) was also detected. In contrast, animals that received only the vehicle and underwent experimental TBI showed only limited cell proliferation, slight neural differentiation, and scattered migration in the cortical area around the injury. Furthermore, newly formed cells were found in extremely small numbers in the subventricular zone of these control animals (see Examples).

[0043] Three months after TBI, the brains from animals that received SB623 cell transplantation showed not only neural cells (expressing both nestin and doublecortin) migrating along and beyond the CC from the SVZ to the impacted cortex, but also a much higher level of cell proliferation and neural differentiation surrounding the cortical area of the injury. Brains from injured animals that received only the vehicle showed a much higher level of cell proliferation three months after TBI than one month after TBI, but the newly formed cells appeared to be "trapped" within the SVZ and corpus callosum, and only a few cells were able to reach the impacted cortex. Quantitative analysis of the immunoreactivity of Ki67, nestin, and doublecortin in the SVZ, CC, and injured cortical area showed statistically significant differences in the expression of these markers between animals that received SB623 cell transplantation and those that received only the vehicle.

[0044] In a separate experiment, the neurospheres formed by endogenous cells migrating from the SVZ to the injury site were isolated by laser capture microdissection (Espina et al. (2006) Nature Prot. 1:586 - 603) and their neurogenicity was analyzed. In this experiment, three groups of animals were analyzed: (1) animals that received TBI and subsequently received SB623 cell transplantation seven days after TBI ​​​​​​​​​, (2) animals that received TBI and then received vehicle injection 7 days after TBI, and (3) sham surgery were performed, and age-matched control adult Sprague-Dawley rats (n = 3 per group). Zymograph assays of laser-captured biobridges from animals that received TBI revealed a 2-fold and 9-fold upregulation of the expression / activity of matrix metalloproteinase 9 (MMP-9), respectively, in animals that received SB623 cell transplantation compared to animals that received vehicle injection or sham surgery 1 month and 3 months after transplantation (Example 11). MMP has been associated with the recovery of chronic brain injury29, and inhibition of MMP activity has been shown to suppress the migration of neurogenic cells from the SVZ to the injured tissue and delay neurovascular remodeling30. Therefore, MMP can play a role in promoting the migration of host cells to the damaged brain area as part of the process by which SB623 cells provide functional recovery from TBI.

[0045] In summary, the inventors have discovered that transplantation of SB623 cells remodels the injured brain by forming a biobridge between the SVZ and the cortex surrounding the injury. Now, this method of cell therapy can be used to form similar biobridges between neurogenic and non-neurogenic sites to promote the injury-specific migration of cells across tissues that would otherwise be a barrier to cell motility.

[0046]

[0047] Formulations, Kits, and Routes of Administration Also provided are therapeutic compositions comprising the SB623 cells disclosed herein. Such compositions generally include SB623 cells and a pharmaceutically acceptable carrier. Auxiliary active compounds​​​​​​​​​ It can also be incorporated into the SB623 cell composition.

[0048] The therapeutic compositions disclosed herein are particularly useful for treating TBI and modulating stem cell migration in the brain. Thus, a "therapeutically effective amount" of a composition comprising SB623 cells is any amount that reduces the symptoms of TBI or stimulates the migration of stem cells in the brain. For example, the dosage may vary with the number of cells being about 100, 500, 1,000, 2,500, 5,000, 10,000, 20,000, 50,000, 100,000, 300,000, 500 ,000, 1,000,000, 5,000,000 to 10,000,000, or more (or any integer therebetween), and the dosing frequency may be, for example, once a day, twice a week, once a week, twice a month, once a month, depending on, for example, body weight, route of administration, severity of the disease, etc. Thus, a therapeutically effective amount can include multiple administrations of the same or different amounts of SB623 cells. In certain embodiments, a single administration of SB623 cells is a therapeutically effective amount.

[0049] Various pharmaceutical compositions and techniques for their preparation and use are known to those skilled in the art in view of the present disclosure. For a detailed list of suitable pharmacological compositions and their administration techniques, see Remington's Pharmaceutical Sciences, 17th Edition, 1985; Brunton et al., "Goodman and Gilman's The Pharmacological Basis of Therapeutics", McGraw -Hill, 2005; University of the Sciences in Philadelphia (ed.), "Rem ington's Pharmaceutical Sciences", Lippincott Williams & Wilkins, 2005; and Katzung (ed.), "Basic & Clinical Pharmacology", McGraw-Hill, 2004. ington: "The Science and Practice of Pharmacy", Lippincott Williams & W ilkins, 2005; and University of the Sciences in Philadelphia (ed.) ,"Remington: The Principles of Pharmacy Practice", Lippincott Williams & Wilkins, 2008, and other texts can be referred to.

[0050] The cells described herein may be suspended in a physiologically compatible carrier for transplantation . As used herein, the term "physiologically compatible carrier" refers to a carrier that is compatible with SB623 cells and any other components of the formulation and is not harmful to its recipient. Those skilled in the art are familiar with physiologically compatible carriers. Examples of suitable carriers 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 (trademark) A (Baxter) is included.

[0051] The volume of the SB623 cell suspension administered to a subject will vary depending on the transplant site, treatment goal, and the number of cells in the solution. Generally, the amount of cells administered is a therapeutically effective amount. As used herein ,"therapeutically effective amount" or "effective amount" refers to the number of transplanted cells required to achieve the treatment of a particular disorder, i.e., to result in a reduction in the amount and / or severity of symptoms associated with that disorder . For example, in the case of TBI, transplantation of a therapeutically effective amount of SB623 cells results in a reduction and / or reversal of TBI symptoms, e.g., motor activity and and It brings about the restoration of the performance of the nervous system and the stimulation of the migration of host neurogenic cells. A therapeutically effective amount varies depending on the type and degree of brain injury and may also vary depending on the overall condition of the subject as well.

[0052] The disclosed therapeutic composition may also include pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, i.e., carriers. These carriers can, for example, stabilize the SB623 cells and / or promote the survival of SB623 cells in the body. Each carrier should be "acceptable" in the sense that it is compatible with the other components of the formulation and does not damage the subject . Some examples of materials that can serve as pharmaceutically acceptable carriers include the following : sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol , mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide ; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffering solution; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifying agents and lubricants ​Wetting agents, such as sodium lauryl sulfate and magnesium stearate, and coloring agents , release agents, coating agents, sweeteners, flavors and fragrances, preservatives and antioxidants may be present in the composition .

[0053] Exemplary formulations include, but are not limited to, those suitable for parenteral administration, such as intrapulmonary, intravenous, intra arterial, intraocular, intracranial, intrathecal, or subcutaneous administration, such as those encapsulated in micelles, liposomes or drug release capsules (the active agent is incorporated into a biocompatible coating designed for slow release); ingestible formulations; formulations for topical use, such as eye drops, creams, ointments and gels; and other formulations such as inhalants, aerosols and sprays . The dosage of the disclosed composition will vary depending on the degree and severity of the need for treatment, the activity of the administered composition, the overall health of the subject and other considerations well known to those of ordinary skill in the art .

[0054] In a further embodiment, the compositions described herein are delivered into the intracranial space at or near the site of traumatic brain injury . Such localized delivery allows for non-systemic delivery of the composition, thereby reducing the burden on the body of the composition as compared to systemic delivery . Local delivery can be achieved, for example, by intracranial injection or through the use of a variety of medical implantable devices including, but not limited to, stents and catheters, or can be achieved by inhalation, venesection or surgery. Methods of coating, implanting, embedding or otherwise attaching the desired agent to medical devices such as stents and catheters are well established in the art and are contemplated herein .

[0055] ​​ Another aspect of the disclosure relates to a kit for effecting administration of SB623 cells to a subject, optionally together with another therapeutic agent. In one embodiment, the kit includes a composition of SB623 cells formulated in a pharmaceutically acceptable carrier suitable for transplantation.

Examples

[0056] In the studies disclosed herein, rats were subjected to experimental traumatic brain injury (TBI), and 7 days later, rats with sufficient motor and nervous system deficits received transplantation of SB623 cells or vehicle into the injury area. Values of motor and nervous system performance were evaluated before TBI (baseline value), again 7 days after TBI (before transplantation), and then monthly for 3 months after TBI.

[0057] After completion of the behavioral tests 1 and 3 months after TBI, animals randomly selected by cardiac perfusion with 4% paraformaldehyde were euthanized (n = 10 per group). For evaluation of the persistence of transplanted cells, histological appearance of brain tissue inside and outside the injury area, expression of various neural markers inside and outside the injury area, and zymogenic activity inside and outside the injury area, their brains were removed and sectioned.

[0058] Transplantation outcomes were evaluated using the following criteria: 1) motor behavior by the elevated body swing test (EBST) and Rotorod; 2) nervous system performance by the Bederson modified neurological examination; 3) lesion volume by histology (H&E stained sections); 4) graft survival by immunohistochemistry using an antibody (HuNu) that specifically detects human cells; and 5) immunohistochemical analysis based on mechanisms of neuroprotection and / or regeneration using antibodies against transplanted human cells and host cells. ​​​​​​​​​​​​​

[0059] (Example 1) Preparation of MSC and SB623 cells Bone marrow aspirate from adult human donors was obtained from Lonza Walkersville, Inc . (Walkersville, MD), and plated in α-MEM (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine (Invitrogen, Carlsba d, CA) and penicillin / streptomycin (Invitrogen). Cells were cultured at 37 °C and 5% CO2 for 3 days to obtain a monolayer of adherent cells. After removal of non-adherent cells, cultivation was continued for 2 weeks under the same conditions. During this period, 0.25% trypsin / EDTA was used to passage the cells twice. A portion of the cells from the second passage was frozen as MSC.

[0060] The remaining cells from the second passage were plated and transfected using Fugene6 (Roche Diagnostics, India apolis, IN) together with a plasmid containing the sequence encoding the Notch intracellular domain operably linked to the cytomegalovirus promoter (pCMV-hNICD1-SV40-Neo (registered trademark)). This plasmid also contained sequences encoding resistance to neomycin and G418 under the transcriptional control of the SV40 promoter. Transfected cells were cultured at 37 °C and 5% CO2 in the growth medium described in the previous paragraph supplemented with 100 μg / ml of G418 (Invitrogen, Carlsbad, CA). After 7 days, G 418-resistant colonies were expanded and the culture was passaged twice. After the second passage, the cells were collected and ​​​​Frozen as SB623 cells.

[0061] For further research, the MSCs and SB623 cells prepared as described herein were thawed and used as needed.

[0062] (Example 2) Induction of TBI in a rat model At baseline (before TBI surgery), a total of 40 animals identified as showing normal behavior (50 - 60% bias swing activity in EBST; 60 - second stay period on Rotorod; and average Anderson score of 0 - 0.5 maximum) were subjected to TBI surgery as follows.

[0063] All surgical procedures were performed under aseptic conditions. Adult male Sprague - Dawley rats were anesthetized with 1.5% isofluorane, and pain reflexes were checked. Under deep anesthesia, the animals were subjected to a moderate TBI model as follows. Each animal was placed in a stereotaxic frame and maintained under anesthesia with 1 - 2% isofluorane administered through a gas mask. After exposing the skull, a 4 - mm bone resection craniotomy was performed on the left frontoparietal cortex, with its center at - 2.0 mm AP and + 2.0 mm ML relative to the bregma. Using a 3 - mm diameter compressed air - actuated metal impactor, the brain was impacted at a speed of 6.0 m / s, reaching a depth of 1.0 mm below the dura mater layer and remaining in the brain for 150 milliseconds. The impactor rod was angled 15° from vertical so as to be perpendicular to the tangent plane of the brain surface at the impact site. To verify consistency, the speed and duration were measured using a linear variable displacement transducer (Macrosensors, Pennsauken, NJ) connected to the impactor.

[0064] Following controlled cortical impact injury, after bleeding had ceased, the incision was sutured. Feedback controlled integrated heating pad and rectal thermometer unit enabled maintenance of body temperature within normal limits. All animals were monitored until recovery from anesthesia. Additionally, animals were weighed and observed daily for three consecutive days following induction of TBI, and then twice weekly, and monitored daily throughout the entire study period for any signs indicating health status or problems or complications.

[0065] (Example 3) Transplantation of SB623 cells Of the animals that had received TBI, only those animals with the following degrees of motor dysfunction on day 7 after TBI were selected for the transplantation study: at least 75% bias swing activity on EBST; a stay period of 30 seconds or less on Rotorod; and an average Bederson score of at least 2.5. The selected animals were randomly assigned to either the group that received SB623 transplantation (n = 20) or the group that received vehicle injection (n = 20). The target area for transplantation was the ipsilateral cortex, which corresponded to the cortical area around the lesion based on the target site previously established for similar stereotactic implants.

[0066] All surgical procedures were performed under aseptic conditions. The animals were anesthetized with 1.5% isoflurane and pain reflexes were checked. When deep anesthesia was achieved (determined by loss of pain reflex), the hair around the area of the surgical incision (skull area) was shaved, leaving sufficient margins to prevent contamination of the surgical site. Thereafter, the surgical site was scrubbed twice with surgical antiseptic and sterile drapes were applied.

[0067] Next, the animals were placed in a stereotaxic fixation device (Kopf Instruments, Tujunga, CA ) was fixed, and a small opening was made in the skull with a bar. The coordinates of the opening were 0.5 mm in front and 1.0 mm lateral to the vertex, and 2.0 mm below the dura surface. These were selected to correspond to the cortical area adjacent to the core lesion site based on the map of Paxinos and W atson (1998). A 26-gauge Hamilton syringe containing the test material was then lowered to the opening. Three deposits of 3 μl each were formed with one pass of the needle. Each deposit was 100, 000 viable cells in 3 μl of Plasmalyte A injected over 3 minutes. After an additional 2-minute absorption time, the needle was withdrawn and the wound was closed with a stainless steel wound clip. A heating pad and rectal thermometer made it possible to maintain the body temperature at approximately 37°C throughout the surgery and after recovery from anesthesia. Control injections contained Plasmalyte A only.

[0068] Treated and control animals were subjected to the elevated body swing test (EBST, Example 4), neurological examination (Example 5), and Rotarod test (Example 6) monthly from baseline (before TBI), 7 days after TBI (just before transplantation ) and then up to 3 months after TBI.

[0069] Furthermore, to determine the degree of injury (Examples 8 and 9); the extent of host cell proliferation, migration, and neural differentiation (Example 10); and the presence of zymogen activity (Example 11), the brains of treated and control animals were histologically characterized 1 and 3 months after TBI.

[0070] (Example 4) Elevated body swing test (EBST) ​​​​​​​​​All researchers who tested the animals were blinded to the treatment conditions. EBST was performed by handling the animals by their tails and recording the direction in which the animals wagged their heads. The test apparatus consisted of a transparent plexiglass box (40×40×35.5 cm). The animals were gently lifted by the base of their tails and raised with the tail until the animals' noses were 2 inches (5 cm) above the surface. When the animals' heads moved approximately 10 degrees laterally from the midline position of the body, the direction (left or right) of the swing was recorded. After one swing, the animals were returned to the plexiglass box and allowed to move freely for 30 seconds before retesting. These steps were repeated for a total of 20 assays for each animal. Intact rats show a 50% swing bias, i.e., the same number of swings to the left and right. A 75% swing bias showed 15 swings in one direction and 5 swings in the other direction during 20 attempts. Previous results using EBST have shown that animals with unilateral lesions show biased swing activity exceeding 75% one month after substantia nigra striatal lesions

[0071] or unilateral hemisphere lesions, and that such asymmetry is stable for up to 6 months3,26. The results of EBST are shown in Figure 1. After TBI, virtually all animals showed biased swing activity. In animals transplanted with SB623 cells, the biased swing activity steadily declined over a 3-month period following TBI and transplantation.

[0072] (Example 5) Modified Bederson neurological examination Approximately 1 hour after the end of EBST, according to the aforementioned procedures 3 and 26 with minor modifications, the modified Bederson neurological examination was performed. The neurological score for each rat was as follows: (1) Forelimb retraction, which measures the ability of the animal to return its forelimb after displacement 2 - 3 cm laterally, was graded from 0 (immediate return) to 3 (return after several seconds or no return); (2) Beam walking ability, graded from 0 for rats that easily crossed a beam 2 .4 cm wide and 80 cm long, to 3 for rats that could not stay on the beam for 10 seconds; and (3) Bilateral forepaw grip, which measured the ability to grip a 2 - mm - diameter steel rod, was graded from 0 for rats with normal forepaw grasping behavior to 3 for rats that could not grasp with their forepaws. The scores were obtained using three tests. The scores from all three tests, which were performed over approximately 15 minutes on each investigation day, were added to give the average neurological deficit score (the maximum possible score, 9 points divided by 3 tests = 3). The results of these neurological examinations are shown in Figure 2. After TBI, the average neurological score was 2.5 (out of 3) in all animals. In animals transplanted with SB623 cells, this score steadily decreased (indicating improved neurological function) over the 3 - month period following TBI and transplantation. The improvement in neurological function in animals transplanted with SB623 cells was statistically significant compared to animals injected with the vehicle (p < 0.05).

[0073] The results of these neurological examinations are shown in Figure 2. After TBI, the average neurological score was 2.5 (out of 3) in all animals. In animals transplanted with SB623 cells, this score steadily decreased (indicating improved neurological function) over the 3 - month period following TBI and transplantation. The improvement in neurological function in animals transplanted with SB623 cells was statistically significant compared to animals injected with the vehicle (p < 0.05).

[0074] (Example 6) Rotorod (registered trademark) test One hour after completion of the neurological examination, the animals were subjected to the Rotorod (registered trademark) test. This test was a rotating treadmill that accelerated from 4 rpm to 40 rpm over 60 seconds. including placing an animal thereon (Rotorod (registered trademark), Accuscan, Inc . Columbus, OH). The total number of seconds the animal was able to remain on the treadmill was recorded and used as an index of coordination. Previous results using the TBI model system showed that lesioned animals were significantly able to remain on the Rotorod for a shorter time compared to sham-operated or normal controls.

[0075] The results of this assay are shown in Figure 3. Non-lesioned animals were able to remain on the treadmill for an average of 60 seconds. The average time on the treadmill decreased to less than 20 seconds 7 days after TBI. In animals transplanted with SB623 cells after TBI, the average time on the treadmill approximately doubled to approximately 40 seconds. These improvements were statistically significant compared to TBI animals injected with vehicle.

[0076] (Example 7) Perfusion and section preparation One and three months after TBI, after completion of the behavioral tests described in Examples 4 - 6, rats randomly selected were euthanized by cardiac perfusion with 4% paraformaldehyde (n = 10 per group). The brains were dissected, post-fixed overnight in 4% paraformaldehyde, and then immersed in 30% sucrose. Starting at -5.2 mm anterior to bregma and moving posteriorly to -8.8 mm from bregma, each forebrain was cut into 40 μm coronal sections. The sections were processed for determination of brain injury and analysis of cell survival in the area surrounding the lesion as described in Examples 8 and 9.

[0077] (Example 8) Measurement of brain injury To determine the degree of brain injury and host cell survival, brain sections were prepared and examined. At least four coronal tissue sections per brain were processed for hematoxylin and eosin (H&E) or Nissl staining. The indirect lesion area, calculated by subtracting the area of the intact hemisphere from the area of the contralateral hemisphere, was used to quantify the brain injury by determining the indirect lesion area calculated by subtracting the area of the intact hemisphere from the area of the contralateral hemisphere. The lesion volume was presented as a percentage of the volume of the lesion compared to the contralateral hemisphere by summing the lesion areas from serial sections. The lesion volume was presented as a percentage of the volume of the lesion compared to the contralateral hemisphere by summing the lesion areas from serial sections. The results quantitatively shown in Figure 4B indicate that animals with TBI that received transplantation of SB623 cells had significantly less injury to the cortical core and the perilesional

[0078] cortical area compared to animals with TBI that received injection of vehicle. cortical area compared to animals with TBI that received injection of vehicle. cortical area compared to animals with TBI that received injection of vehicle.

[0079] (Example 9) Analysis of cell survival in the perilesional lesion area of TBI Randomly selected high-magnification fields corresponding to the perilesional cortical area were examined to count the host cells surviving in this region. The results are shown in Figure 4A.

[0080] (Example 10) Immunohistochemistry Floating sections were processed for immunofluorescence microscopy. Briefly, 40-μm cryostat sections of tissue were examined at 4× magnification and digitized using a PC-based image tool computer program. Monoclonal human specific antibody HuNu, which does not cross-react with rodent proteins, was used to investigate the engraftment of transplanted SB623 cells. Additional brain sections were processed for mechanism-based immunohistochemical analysis of brain tissue samples, focusing on cell proliferation (Ki67), migration (doublecortin or DCX), and neural differentiation (nestin). cell proliferation (Ki67), migration (doublecortin or DCX), and neural differentiation (nestin). cell proliferation (Ki67), migration (doublecortin or DCX), and neural differentiation (nestin). done. Brain sections were blindly coded and the total number of immunopositive cells was calculated using Abercrombie's formula3, 26. The results of these analyses showed that transplantation of SB623 cells induced the formation of a biobridge between the SVZ and the cortex that had been impacted by highly proliferative,

[0081] neurotically dedicated migratory cells. One month after TBI, immunofluorescence and confocal microscopy revealed endogenous cell proliferation (demonstrated by cells expressing Ki67) and a surge in immature neurodifferentiation (cells expressing nestin) along the corpus callosum (CC) of animals that had received SB623 cell transplantation in the cortical area and subventricular zone (SVZ) surrounding the injury. Brains from animals that received only vehicle showed limited cell proliferation and neurodifferentiation, as well as scattered migration, in the cortical area surrounding the injury, and newly formed cells were rarely present in the SVZ. Three months after TBI, brains from animals that received SB623 transplantation showed substantial cell proliferation and neurodifferentiation surrounding the cortical area surrounding the injury, accompanied by a robust flow of neuro-labeled cells (expressing both nestin and doublecortin) migrating along and beyond the CC from the SVZ to the impacted cortex. In contrast, in brains from vehicle-injected animals, cell proliferation was enhanced, but newly formed cells were "trapped" within the SVZ and CC, and only a few cells were able to reach the impacted cortex. Quantitative analysis of Ki67, nestin, and DCX immunolabeled cells in the SVZ, CC, and CTX revealed statistically significant differences between transplanted and vehicle-injected animals (Figs. 5-7). ​​​​​​​​

[0082] (Example 11) Zymography To test for the presence and / or activity of proteolytic enzymes following transplantation of SB623 cells into the injured brain, its analysis was performed on cohorts of animals separate from those described in Examples 4 - 10 using a cohort of animals. Adult Sprague - Dawley rats were subjected to TBI and then transplanted with SB623 cells or vehicle . A control group of age - matched, sham - operated adult Sprague - Dawley rats was subjected to the same experimental procedure (n = 3 rats per group). One and three months after TBI , tissue corresponding to the biobridge formed by cells migrating from the SVZ to the impact - affected cortex was obtained by laser dissection. After extraction, the tissue was placed in cryotubes and snap - frozen in liquid nitrogen. The tubes were stored at - 80 °C freezer until homogenization . Samples were homogenized in 450 μL of cold - working buffer containing 50 mM Tris - HCl (pH 7.5), 75 mM NaCl, and 1 mM PMS F. The tissue was processed with a homogenizer for 10 minutes and centrifuged at 13000 rpm for 20 minutes at 4 °C. The supernatant was separated , frozen, and kept at - 80 °C until use. The total protein concentration in the supernatant was assayed by the Bradford method

[0083] On the day zymography was performed, a volume equivalent to 50 μg of total protein was loaded onto freshly provided gelatin zymography gels. All gels had a control lane loaded with 0.5 ng of recombinant MMP - 9 used as a standard for both enzyme amount (ng) and gelatinolytic activity (expressed in relative optical density units, see below) . Non - reducing electrophoresis was performed at 100 V . The gels were run at 100 V for non - reducing electrophoresis . The supernatant was separated, frozen, and kept at - 80 °C until use. The total protein concentration in the supernatant was assayed by the Bradford method

[0084] On the day zymography was performed, a volume equivalent to 50 μg of total protein was loaded onto freshly provided gelatin zymography gels. All gels had a control lane loaded with 0.5 ng of recombinant MMP - 9 used as a standard for both enzyme amount (ng) and gelatinolytic activity (expressed in relative optical density units, see below) . All gels had a control lane loaded with 0.5 ng of recombinant MMP - 9 used as a standard for both enzyme amount (ng) and gelatinolytic activity (expressed in relative optical density units, see below) . Non - reducing electrophoresis was performed at 100 V for non - reducing electrophoresis at 100 V Protein was electrophoretically separated on a native gel. After electrophoresis, the gel was washed twice with 125 ml of 2.5% Triton for 20 minutes. Next, it was incubated with the activation buffer (Zymogram Development Buffer, Bio-Rad, Hercules, CA) at 37 °C for 20 hours. The next day, the gel was stained with Coomassie Blue R-250 staining solution (Bio-Rad) for 3 hours and decolorized with decolorizing solution (Bio-Rad) for 25 minutes. The gelatinolytic activity of the sample was investigated by densitometric analysis of the bands (Gel-Pro v3.1, Media Cybernetics, Carlsbad, CA). The molecular weight of the protein in the gel region showing lytic activity was determined by comparison with a prestained standard protein marker (Bio-Rad) run on the same gel. Activity was expressed as the optical density relative to that of 0.5 ng of recombinant MMP-9 run on the gel as a standard.

[0085] Laser capture microdissected (corresponding to brain tissue between the SVZ and the impacted cortex) from animals transplanted with SB623 cells after TBI showed high levels of MMP-9 gelatinolytic activity at 1 month and 3 months after TBI. Levels in SB623-treated animals were significantly higher at both time points than those in microdissected samples from sham-operated animals injected with vehicle (p < 0.05). Microdissected samples from vehicle-injected animals showed a significant increase in MMP-9 activity compared to sham-operated animals at 1 month after TBI, but these levels returned to control levels at 3 months after TBI (i.e., not significantly different from those of sham-operated animals).

[0086] ​​​​​​​​​​​​​​For detection on the blot, wash the membrane with blotting grade nonfat dry milk (Bio-Rad). After washing with 0.1% Tween 20-Tris-buffered saline (TTBS), The membrane was then incubated overnight at 4°C with 1 μg / ml of anti-MMP-9 monoclonal mouse antibody. The membrane was washed again with TTBS and incubated with the secondary antibody (horseradish peroxidase conjugate). Gated goat anti-mouse IgG (1:1,000 dilution, Calbiochem) for 1 h Incubate and finally add horseradish peroxidase developing solution (ECL Advanced Detection The membrane was then developed using an autoradiography film (Hybrid Immunofluorescence Imaging Kit, Amersham). The subjects were exposed to 100 mg of 10 ... The density of the sample bands for the immunogram is compared with the standard band (0.5 ng of recombinant MMP-9). The optical density was expressed as the maximum optical density relative to the mean optical density.

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Behavioral improvement in a primate P arkinson’s model is associated with multiple homeostatic effects of hu man neural stem cells. Proc. Natl. Acad. Sci. U. S. A. 104, 12175-12 180 (2007) 7. Lee, J. P. et al. Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nat. Med. 13, 439-447 (2007) 8. Ma, D. K. et al. Epigenetic choreographers of neurogenesis in th e adult mammalian brain. Nat. Neurosci. 13, 1338-1344 (2010) 9. Hong, S. H. et al. Cell fate potential of human pluripotent ste m cells is encoded by histone modifications. Cell Stem Cell. 9, 24-36 (2011) 10. Kim, Y. et al. Mouse B-type lamins are required for proper org anogenesis but not by embryonic stem cells. Science. 334, 1706-1710 (2 011) 11. Borlongan, C. V. Bone marrow stem cell mobilization in stroke: a ‘bonehead’ may be good after all! Leukemia 25, 1674-1686 (2011) 12. Barha, C. K. et al. 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Claims

1. 1. A cell for transplantation into a subject for the treatment of traumatic brain injury, comprising: (a) providing a culture of MSCs; (b) injecting the cell culture of step (a) into a polynucleotide sequence comprising a sequence encoding NICD; contacting said polynucleotide with a full-length Notch protein; A step that does not encode a protein; (c) selecting cells containing the polynucleotide of step (b); (d) further culturing the selected cells of step (c) without selection; 4. A cell obtained by a process comprising:

2. The cell of claim 1 , wherein the subject is a human.

3. The cell of claim 1 or claim 2, wherein the MSC is obtained from a human.

4. A method for treating traumatic brain injury in a subject, comprising administering to a subject a compound according to any one of claims 1 to 3. administering a therapeutically effective amount of cells to the brain of the subject.

5. A method for inducing migration of endogenous neurogenic cells from the neurogenic niche to sites of brain injury Administering a therapeutically effective amount of the cells according to any one of claims 1 to 3 to the brain of a subject. A method comprising:

6. The method of claim 5 , wherein the neurogenic niche is the subventricular zone.

7. 6. The method of claim 5, wherein the brain injury is a traumatic brain injury.

8. A method for stimulating proliferation of neurogenic cells in a subject, comprising administering to a subject a composition comprising a compound according to any one of claims 1 to 3. A method comprising administering to the brain of a subject a therapeutically effective amount of SB623 cells as described above.

9. A method for inducing neurogenic cells to proliferate and migrate to the site of brain injury in a subject. and administering a therapeutically effective amount of SB623 cells according to any one of claims 1 to 3 to the brain of a subject. The method of claim 1,

10. 10. The method of claim 9, wherein the brain injury is a traumatic brain injury.

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