Therapeutic agent for spinal cord injury subject from acute phase to subacute phase

Abstract

Provided is a therapeutic agent for spinal cord injury from acute to subacute phases, particularly a therapeutic agent for severe spinal cord injury, which is difficult to treat in the past. The invention discloses a therapeutic agent for spinal cord injury from an acute stage to a subacute stage. The present invention is characterized by comprising: (1) (a) a hepatocyte growth factor protein or a substance having a c-Met phosphorylation effect equivalent to that of a hepatocyte growth factor protein, or (b) a gene encoding a hepatocyte growth factor protein or a gene encoding a substance having a c-Met phosphorylation effect equivalent to that of a hepatocyte growth factor protein; and (2) pluripotent stem cells.

Description

Technical Field

[0001] The present invention relates to a therapeutic agent for spinal cord injury from the acute phase to the subacute phase, and particularly to a therapeutic agent for severe spinal cord injury. In addition, the present invention relates to an agent for improving motor dysfunction for spinal cord injury from the acute phase to the subacute phase (particularly an agent for improving lower limb motor function, an agent for improving walking ability, a preparation for obtaining walking ability), an inhibitor of syringomyelia formation at the spinal cord injury site, an inhibitor of spinal cord atrophy of the injured spinal cord, an inhibitor of demyelination after spinal cord injury, a promoter of remyelination after spinal cord injury, and a promoter of migration / axon elongation at the spinal cord injury site, and a method for increasing the survival rate of pluripotent stem cells for transplantation. It should be noted that the respective agents of the present invention are sometimes collectively referred to as "the preparations of the present invention". This application claims the priority of Japanese Application No. 2022-143261, which is incorporated herein by reference. Background Art

[0002] (Spinal Cord Injury) Spinal cord injury refers to a disease state in which the spinal cord parenchyma is damaged by trauma or the like, and sensory, motor, and autonomic nervous system paralysis below the injury site is manifested. The total number of patients in Japan reaches more than 150,000, and about 5,000 new patients are added every year. However, it is considered that the central nervous system of mammals does not regenerate once damaged, and effective treatment methods have not been established yet.

[0003] (Hepatocyte Growth Factor) Hepatocyte Growth Factor (hereinafter sometimes simply referred to as "HGF" in this specification) protein was discovered as a bioactive protein having growth-promoting activity on mature hepatocytes. Through subsequent studies, it has been found that HGF protein acts not only on hepatocytes but also on many epithelial cells, vascular endothelial cells, etc. through the c-Met receptor, and is involved in the repair and regeneration of tissue and organ damage. HGF protein can be mass-produced as a recombinant protein by biotechnological techniques, and recombinant HGF protein is expected to be applied as a therapeutic drug for hepatitis, liver cirrhosis, kidney diseases, trauma, etc. On the other hand, through many recent studies in gene expression and functional analysis including methods such as gene knockout / gene knock-in mice, it has been shown that HGF protein also has the effect of promoting the survival of nerve cells and the elongation of neurites, and is also an important factor as a neurotrophic factor. The HGF protein exhibits neurotrophic factor activity on nerve cells such as hippocampal neurons, dopaminergic neurons, cerebellar granule cells, sensory neurons, and motor neurons. In particular, the HGF protein exhibits a strong survival-promoting effect on motor neurons, and its activity can rival that of the neurotrophic factor (GDNF) known to have the strongest survival-promoting effect on motor neurons and derived from a glial cell line. Based on such neurotrophic activity, it has been reported that the HGF protein can be used as a therapeutic agent for various nervous system diseases represented by amyotrophic lateral sclerosis (ALS) and spinal cord injury (see: Patent Document 1).

[0004] (Prior non-patent literature) Non-patent Document 1 discloses that "it is expected that hepatocyte growth factor and iPS cell-derived NSCs are effective for regeneration after spinal cord injury when administered to the spinal cord injury." However, the composition of the preparation of the present invention is not disclosed or suggested. Non-patent Document 2 discloses "a treatment method for applying a gelatin-furfurylamine (FA) hydrogel in combination with CBD-HGF or HGF to animals with spinal cord compression injury." However, the composition of the preparation of the present invention is not disclosed or suggested.

[0005] Patent Document 2 discloses "a cylindrical sustained-release preparation containing HGF protein obtained by mixing an aqueous solution of HGF protein and a phosphate buffer solution of telopeptide-free collagen, followed by lyophilization and compression molding." However, the composition of the preparation of the present invention is not disclosed or suggested. Patent Document 3 discloses "a therapeutic agent for nerve injury containing pluripotent stem cells derived from differentiated cells." However, the composition of the preparation of the present invention is not disclosed or suggested. Prior art documents Patent documents

[0006] Patent Document 1: Japanese Unexamined Patent Application Publication No. 2018-44000 Patent Document 2: Japanese Patent No. 5419045 Patent Document 3: Japanese Unexamined Patent Application Publication No. 2009-215191 Non-patent documents

[0007] Non-patent Document 1: Int. J. Mol. Sci. 2019, 20(15), 3838 Non-patent Document 2: Sci. Rep. 2018, 8: 917. Summary of the invention Technical problem to be solved by the invention

[0008] To provide a therapeutic agent for spinal cord injury from the acute phase to the subacute phase, which has been difficult to treat in the past, especially for severe spinal cord injury. Furthermore, a therapeutic agent that can also be applied to severe spinal cord injury where there is insufficient effect, such as inability to walk while bearing weight, when using HGF alone or iPS alone can be provided.

[0009] The present invention has been completed by confirming that a preparation containing the following (1) and (2) has a therapeutic effect and an effect of improving motor dysfunction on severe spinal cord injury from the acute phase to the subacute phase, especially on severe spinal cord injury that has no effect when using HGF alone or iPS alone. (1) HGF protein; (2) Neural stem cells and / or neural stem cell - progenitor cells derived from iPS cells.

[0010] The present invention includes the following. 1. A therapeutic agent for spinal cord injury from the acute phase to the subacute phase, comprising: (1) (a) Hepatocyte growth factor protein or a substance having the same c-Met phosphorylation effect as hepatocyte growth factor protein, or (b) a gene encoding hepatocyte growth factor protein or a gene encoding a substance having the same c-Met phosphorylation effect as hepatocyte growth factor protein; and (2) Pluripotent stem cells. 2. The therapeutic agent according to 1 above, wherein the pluripotent stem cells are neural stem cells and / or neural stem cell - progenitor cells derived from iPS cells. 3. The therapeutic agent according to 1 above, wherein the spinal cord injury is severe spinal cord injury. 4. The therapeutic agent according to 1 above, wherein the substance having the c-Met phosphorylation effect is hepatocyte growth factor protein, the pluripotent stem cells are neural stem cells and / or neural stem cell - progenitor cells derived from iPS cells, and the spinal cord injury from the acute phase to the subacute phase is severe spinal cord injury. 5. The therapeutic agent according to 4 above, wherein the hepatocyte growth factor protein is administered intrathecally. 6. The therapeutic agent according to 5 above, wherein the hepatocyte growth factor protein is administered into the subarachnoid space. 7. The therapeutic agent according to any one of 1 to 6 above, wherein the treatment is to promote the regeneration of the injured spinal cord and / or improve the complications associated with the injured spinal cord. 8. The therapeutic agent according to 7 above, wherein promoting the regeneration of the injured spinal cord is to promote axon elongation, inhibit spinal cord atrophy, improve the survival rate of the pluripotent stem cells, inhibit demyelination, improve remyelination and / or inhibit syringomyelia. 9. The therapeutic agent according to item 7 above, wherein the promotion of the regeneration of the injured spinal cord is the activation of endogenous nerve cells, the promotion and assistance of the reconstruction of nerve circuits, and / or the increase in functional nerve fibers. 10. The therapeutic agent according to item 7 above, wherein the complication accompanying the injured spinal cord is motor dysfunction. 11. The therapeutic agent according to item 10 above, wherein the motor dysfunction is lower limb motor dysfunction. 12. The therapeutic agent according to item 11 above, wherein the motor dysfunction is the loss of the ability to walk. 13. A therapeutic agent for spinal cord injury from the acute phase to the subacute phase, containing hepatocyte growth factor protein; wherein, the hepatocyte growth factor protein is administered intrathecally to the spinal cord injury site, and further, iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site. 14. A therapeutic agent for spinal cord injury from the acute phase to the subacute phase, containing iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells, wherein, during or after the supply of the hepatocyte growth factor protein to the spinal cord injury site by intrathecal administration, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site. 15. An agent for improving motor dysfunction in spinal cord injury from the acute phase to the subacute phase, comprising: (1) hepatocyte growth factor protein; and (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is administered intrathecally to the spinal cord injury site, and further, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site. 16. The agent for improving motor dysfunction according to item 15 above, wherein the motor function is lower limb motor function or walking function. 17. An inhibitor of demyelination after spinal cord injury for spinal cord injury from the acute phase to the subacute phase, comprising: (1) hepatocyte growth factor protein; and (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is administered intrathecally to the spinal cord injury site, and further, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site. 18. A remyelination enhancer after spinal cord injury for spinal cord injury from the acute phase to the subacute phase, comprising: (1) Hepatocyte growth factor protein; and (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is supplied to the spinal cord injury site by intrathecal administration, and further, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site. 19. A syringomyelia inhibitor for spinal cord injury from the acute phase to the subacute phase, comprising: (1) Hepatocyte growth factor protein; and (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is supplied to the spinal cord injury site by intrathecal administration, and further, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site. 20. A spinal cord atrophy inhibitor for spinal cord injury from the acute phase to the subacute phase, comprising: (1) Hepatocyte growth factor protein; and (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is supplied to the spinal cord injury site by intrathecal administration, and further, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site. 21. An axon elongation promoter for the injured site in spinal cord injury from the acute phase to the subacute phase, comprising: (1) Hepatocyte growth factor protein; and (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is supplied to the spinal cord injury site by intrathecal administration, and further, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site. 22. A promoter for neural circuit reconstruction and / or increase of functional nerve fibers in the injured site in spinal cord injury from the acute phase to the subacute phase, comprising: (1) Hepatocyte growth factor protein; and (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is administered intrathecally to the spinal cord injury site, and further, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site. 23. A method for treating spinal cord injury from the acute stage to the subacute stage, comprising: (1) A step of supplying to the spinal cord injury site by intrathecal administration or intramedullary spinal cord administration (a) a hepatocyte growth factor protein or a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein, or (b) a gene encoding the hepatocyte growth factor protein or a gene encoding a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein; and (2) A step of administering pluripotent stem cells to the spinal cord injury site. 24. A kit for treating spinal cord injury from the acute stage to the subacute stage, comprising: (1) (a) A hepatocyte growth factor protein or a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein, or (b) a gene encoding the hepatocyte growth factor protein or a gene encoding a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein; and (2) Pluripotent stem cells; The hepatocyte growth factor protein or a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein, or the gene encoding the hepatocyte growth factor protein or the gene encoding a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein is administered to the spinal cord injury site by intrathecal administration or intramedullary spinal cord administration, and further, the pluripotent stem cells are administered to the spinal cord injury site. 25. A method for improving the survival and viability of pluripotent stem cells for transplantation, which improves the survival and viability of pluripotent stem cells for transplantation by bringing hepatocyte growth factor into contact with the pluripotent stem cells for transplantation. Advantages of the Invention

[0011] The preparation of the present invention has one or more of the following effects. (1) The effect of improving the motor dysfunction caused by spinal cord injury (the effect of restoring lower limb motor function, the effect of obtaining walking ability); (2) The effect of inhibiting syringomyelia in the spinal cord injury site; (3) The effect of inhibiting the atrophy of the injured spinal cord; (4) The effect of promoting migration and axon elongation in the spinal cord injury site; (5) The effect of improving the survival and viability of transplanted cells; (6) The effect of inhibiting demyelination after spinal cord injury; (7) Improve the effect of remyelination after spinal cord injury; (8) Enhance the effect of neurotransmission in the spinal cord injury site. Brief Description of the Drawings

[0012] Figure 1 Is a summary of the production of the spinal cord injury model. Figure 2 Is the motor function evaluation based on the BBB score. Figure 3 Is the motor function evaluation based on the lower limb stride length. Figure 4 Is the motor function evaluation based on the confirmed walking ability. Figure 5 Is the HE staining result and HNA fluorescence staining result of the collected spinal cord tissue (the HNA fluorescence staining in the bottommost figure is the result of the combined group). Figure 6 Is the measurement result of the spinal cord region area. Figure 7 Is the STEM121 fluorescence staining result of the collected spinal cord tissue. Figure 8 Is the detection result of HNA-positive cells, APC-positive cells, GFAP-positive cells, HuC / D-positive cells, and Ki-67 positive cells. Figure 9 Is the measurement result of the Ki-67 positive cell rate (Percentage of Ki positive cell (%)) and Nestin positive cell rate (Percentage of Nestin positive cell (%)). Figure 10 Is the evaluation of serotonergic neurons in the injured peripheral area. Figure 11 Is the Myelin goldblack staining result of the collected spinal cord tissue. Figure 12 Is the measurement result of the myelin area in the injured peripheral area. Figure 13 Is a summary of the measurement method of the survival rate of the transplanted cells. Figure 14 Is the evaluation of the fluorescence of the transplanted cells based on IVIS. Figure 15 Is the measurement result of the survival rate of the transplanted cells after transplantation. Figure 16 Is a summary of the therapeutic effect mechanism of the therapeutic agent of the present invention. Figure 17It is based on the results of kinematic gait analysis and electrophysiological analysis using MEP. Figure 18 It is the evaluation of regenerated nerve fibers (NF-H) in the periphery of the injury. Figure 19 It is the evaluation of motor function according to the BBB score after increasing the number of cases. Figure 20 It is the evaluation of motor function according to the lower limb stride length after increasing the number of cases. Detailed implementation mode

[0013] (Object of the present invention) The object of the present invention (the preparation of the present invention) relates to a therapeutic agent for spinal cord injury from the acute phase to the subacute phase, particularly to a therapeutic agent for severe spinal cord injury. In particular, it can also be used as a therapeutic agent for severe spinal cord injury that has no effect when using HGF alone or iPS alone. In addition, the present invention relates to an agent for improving motor dysfunction (particularly an agent for improving walking ability, a preparation for obtaining walking ability), an inhibitor of syringomyelia in the spinal cord injury site, an inhibitor of spinal cord atrophy, and a promoter of migration and axonal elongation in the spinal cord injury site for spinal cord injury from the acute phase to the subacute phase (particularly a therapeutic agent for severe spinal cord injury, especially severe spinal cord injury that has no effect when using HGF alone or iPS alone). The preparation of the present invention contains the following: (1) (a) HGF protein or a substance having the same c-Met phosphorylation effect as HGF, or (b) a gene encoding HGF or a gene encoding a substance having the same c-Met phosphorylation effect as HGF; (2) Pluripotent stem cells. The above (a) and (b) are preferably used for intrathecal administration or intramedullary spinal cord administration. More specifically, more preferably, the above (a) is used for intrathecal administration, and the above (b) is used for intramedullary spinal cord administration. The present invention also takes the use of the above (1) and / or the above (2) in the preparation of a therapeutic agent for spinal cord injury, an agent for improving motor dysfunction, an inhibitor of demyelination after spinal cord injury, a preparation for enhancing remyelination after spinal cord injury, a preparation for enhancing remyelination after spinal cord injury, an inhibitor of syringomyelia of the spinal cord, an inhibitor of spinal cord atrophy, a promoter of axonal elongation at the injury site, and a promoter of neural circuit reconstruction and / or increase in functional nerve fibers as an object.

[0014] (Spinal cord injury) The preparation of the present invention can be used to treat spinal cord injuries from the acute phase to the subacute phase. Preferably, it can be used to treat severe spinal cord injuries that are difficult to treat with conventional treatment methods (especially difficult to obtain walking ability). It should be noted that severe refers to patients who are unable to bear weight on their lower limbs. It should be noted that treatment includes improvement, alleviation, prevention of recurrence, and cure.

[0015] (Treatment target) The treatment target of the preparation of the present invention may be any subject with a spinal cord, without particular limitation. For example, humans, monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, rabbits, hamsters, guinea pigs, chimpanzees, etc. can be exemplified.

[0016] (HGF protein and gene encoding HGF) As the HGF protein of the present invention, the type is not particularly limited, and HGF proteins derived from various animals (natural HGF proteins or recombinant proteins prepared by genetic engineering techniques) can be appropriately used. In the present invention, for example, it is preferred to use an HGF protein derived from an animal to which the preparation of the present invention is applied. For example, when the preparation of the present invention is applied to humans, an HGF protein derived from humans (hereinafter sometimes also referred to as human HGF protein) is appropriately used. More preferably, it is a recombinant human HGF protein. In addition, the HGF protein used in the present invention may also be a deletion type (dHGF) lacking 5 amino acid residues.

[0017] The human HGF protein is preferably, for example, a protein encoded by DNA composed of the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. More specifically, a protein composed of the amino acid sequence represented by SEQ ID NO: 3, a protein composed of the amino acid sequence represented by SEQ ID NO: 4, a protein composed of the amino acid sequence represented by SEQ ID NO: 5, a protein composed of the amino acid sequence represented by SEQ ID NO: 6, etc. are preferred. Among them, as the human HGF protein, a protein having the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 is preferred, and a protein consisting of the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 is more preferred. For example, the HGF protein consisting of the amino acid sequence represented by SEQ ID NO: 6 is a 5-amino acid deletion type HGF protein (dHGF) in which 5 amino acid residues at positions 131 to 135 of the amino acid sequence represented by SEQ ID NO: 5 are deleted. Both of the proteins having the amino acid sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 are HGF proteins naturally present in the human body (natural type HGF proteins), and have mitogen activity, motogen activity, etc. as HGF.

[0018] The HGF protein used in the present invention also includes a protein having at least about 80% or more, preferably about 90% or more, more preferably about 95% or more sequence identity with the amino acid sequence of HGF proteins (natural type HGF proteins) derived from various animals, and having biological activity as HGF (mitogen activity and motogen activity). Regarding the amino acid sequence, "sequence identity" means the identity of the amino acid residues constituting each sequence when comparing the primary structures of proteins, and "% or more" means the degree of such identity. The HGF protein has the above-mentioned mitogen activity and motogen activity, and can be confirmed, for example, by the method described in J. Biol. Chem. 273, 22913 - 22920, 1998. A protein having mitogen activity and motogen activity measured according to J. Biol. Chem. 273, 22913 - 22920, 1998 and compared with the natural type HGF protein is usually about 50% or more, preferably about 70% or more, more preferably about 80% or more, and further preferably about 90% or more is preferred.

[0019] Examples of the protein having the above sequence identity with the HGF protein include an amino acid sequence obtained by substituting, deleting, and / or inserting one to several amino acid residues in the amino acid sequence represented by SEQ ID NO: 5 or 6, or an amino acid sequence obtained by modifying one to several amino acid residues, etc., and having biological activity as HGF. "Several" generally refers to 1 to 8 (1, 2, 3, 4, 5, 6, 7, 8), usually 8, preferably 6, more preferably 5, further preferably 3, and particularly preferably 2. The inserted or substituted amino acids are preferably natural amino acids, but may also be unnatural amino acids other than the 20 amino acids encoded by genes. As long as the unnatural amino acids have an amino group and a carboxyl group, they can be any compound, and examples include γ-aminobutyric acid and the like.

[0020] Replacing an amino acid residue means replacing an amino acid residue in a polypeptide with another amino acid residue, preferably a conservative replacement. "Conservative replacement" means replacing one to several amino acid residues with other amino acid residues having similar chemical properties in a manner that substantially does not change the activity of the polypeptide. Examples include replacing a hydrophobic amino acid residue with another hydrophobic amino acid residue, or replacing a polar amino acid residue with another polar amino acid residue having the same charge. Functionally similar amino acids capable of such replacement are well-known in the art for each amino acid. Examples of amino acids with non-polar (hydrophobic) side chains include glycine, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. Examples of neutral amino acids among amino acids with polar side chains include serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. Examples of amino acids with a positive charge (basic) include arginine, histidine, lysine, etc. In addition, examples of amino acids with a negative charge (acidic) include aspartic acid, glutamic acid, etc.

[0021] The HGF protein contained in the preparation of the present invention may be only one kind, or two or more of the above-mentioned HGF proteins.

[0022] As long as the HGF protein used in the preparation of the present invention is a HGF protein purified to a degree that can be used as a drug, HGF proteins prepared by various methods can be used. As methods for preparing HGF proteins, various methods are known. For example, it can be obtained by extraction and purification from organs such as the liver, spleen, lung, bone marrow, brain, kidney, placenta of mammals such as rats, cows, horses, sheep, blood cells such as platelets and white blood cells, or plasma, serum, etc. As a method for extracting and purifying HGF protein from the above biological tissues, etc., for example, carbon tetrachloride can be intraperitoneally administered to rats, the livers of rats in a hepatitis state can be removed and crushed, and purified by common protein purification methods such as column chromatography using S-agarose gel, heparin agarose gel, etc., and HPLC.

[0023] Alternatively, primary cultured cells or cell lines that produce HGF protein can also be cultured, and the HGF protein can be isolated and purified from the culture (culture supernatant, cultured cells, etc.). Or, the gene encoding the HGF protein (preferably DNA composed of the base sequence represented by SEQ ID NO: 1 or 2) can be inserted into an appropriate vector by genetic engineering methods, inserted into a suitable host for transformation, and the target recombinant HGF protein can be obtained from the culture of the transformant (for example, refer to Biochem. Biophys. Res. Commun. 180: 1151 - 1158, 1991; J. Clin. Invest. 87: 1853 - 1857, 1991; Protein Expr. Purif. 70: 231 - 235, 2010, etc.). The above host cells are not particularly limited, and various host cells used in conventional genetic engineering methods can be used, such as Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant or animal cells, etc. For example, when using animal cells as host cells, animal cells, such as Chinese hamster ovary (CHO) cells, mouse C127 cells, monkey COS cells, etc., can be transformed with an expression vector containing cDNA encoding the amino acid sequence of human HGF protein, the culture supernatant thereof can be isolated, and purified by the above column chromatography, etc., to obtain the HGF protein.

[0024] As long as the HGF protein obtained in this way has the biological activity as HGF, it can also be an amino acid sequence in which one or more [for example, one to several (the meaning of "several" is the same as above, for example, 1 - 8, preferably 1 - 6, more preferably 1 - 5, further preferably 1 - 3, particularly preferably 1 - 2; the same below)] amino acids in the amino acid sequence of the natural type HGF protein are replaced, deleted, and / or inserted. The replacement is preferably a conservative replacement. In addition, similarly, sugar chain replacement, deletion, or insertion can be performed on the HGF protein. Here, regarding the amino acid sequence, the so-called "one or more amino acid deletions, replacements, and / or insertions" means that one or several (usually one to several) amino acids have undergone deletions, replacements, and / or insertions by known technical methods such as genetic engineering methods and site-directed mutagenesis methods, or to the extent that can occur naturally. The HGF protein with sugar chain replacement, deletion, or insertion refers to, for example, the HGF protein in which the sugar chain added to the natural type HGF protein is removed by treatment with an enzyme, etc., and in addition, the HGF protein in which a mutation is introduced into the amino acid sequence at the sugar chain addition site so that no sugar chain is added, or the HGF protein in which a mutation is introduced into the amino acid sequence so that a sugar chain is added at a site different from the natural sugar chain addition site, etc.

[0025] It should be noted that for the HGF protein used in the present invention, when applied to humans, it is preferably the above-mentioned human-derived protein, but it can also be an HGF protein derived from mammals other than humans (such as monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, rabbits, hamsters, guinea pigs, chimpanzees, etc.). Examples of such HGF proteins include those registered in the NCBI database, etc.: for example, the HGF protein derived from mice (such as Accession No. AAB31855, NP_034557, BAA01065, BAA01064, etc.), the HGF protein derived from rats (such as Accession No. NP_58713, etc.), the HGF protein derived from cows (such as Accession No. NP_001026921, XP874086, BAD02475, etc.), the HGF protein derived from cats (such as Accession No. NP_001009830, BAC10545, BAB21499, etc.), the HGF protein derived from dogs (such as Accession No. NP_001002964, BAC57560, etc.) or the HGF protein derived from chimpanzees (such as Accession No. XP519174, etc.), but is not limited to these. In the HGF protein used in the present invention, the C-terminus can be any one of a carboxyl group (-COOH), a carboxylate [-COOM (M represents a metal)], an amide group (-CONH2), or an ester group (-COOR). Among them, as R in the ester, in addition to C1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl, or n-butyl, C3-8 cycloalkyl groups such as cyclopentyl or cyclohexyl, C6-12 aryl groups such as phenyl or α-naphthyl, phenyl-C1-2 alkyl groups such as benzyl and ethyl, or α-naphthyl-C1-2 alkyl groups such as α-naphthylmethyl, C2-6 alkanoylmethyl groups such as acetoxymethyl or pivaloyloxymethyl are used. When the HGF protein used in the present invention has a carboxyl group or a carboxylate at a position other than the C-terminus, the HGF protein in the present invention also includes an HGF protein in which the carboxyl group or the carboxylate is amidated or esterified. As the ester in this case, for example, the ester of the above C-terminus can be used. In addition, in the HGF protein used in the present invention, there are also included proteins in which the amino group of the methionine residue at the N-terminus in the above protein is protected by a protecting group (such as a C1-6 acyl group such as a formyl group or an acetyl group, which is a C2-6 alkanoyl group), proteins in which the glutamyl group formed by cleavage on the N-terminal side in vivo is pyroglutamylated, proteins in which active groups (such as -OH, -SH, amino group, imidazole group, indole group, guanidine group, etc.) on the side chains of amino acids in the molecule are protected by appropriate protecting groups (such as a C1-6 acyl group such as a formyl group or an acetyl group, which is a C2-6 alkanoyl group), or complex proteins such as so-called glycoproteins obtained by sugar chain binding.

[0026] In the present invention, the "gene encoding the HGF protein" refers to a gene capable of expressing the above HGF protein. As the DNA retaining the gene encoding the HGF protein (hereinafter sometimes also referred to as the DNA encoding the HGF protein), preferably listed are those described in, for example, Nature, 342, 440 (1989); Japanese Patent No. 2777678; Biochem. Biophys. Res. Commun., 1989, Vol. 163, p. 967-973; Proc. Natl. Acad. Sci. U.S.A., 1991, Vol. 88 (No. 16), p. 7001-7005, etc., and DNA encoding the HGF protein derived from humans registered as Accession No. M69718, M73240, AC004960, AY246560, M29145, or M73240, etc. in, for example, GenBank / EMBL / DDBJ. In addition, when the DNA encoding the HGF protein used in the present invention is applied to humans, it is preferably the above-mentioned DNA encoding the HGF protein derived from humans. However, it may also be DNA encoding the HGF protein derived from mammals other than humans (such as monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, rabbits, hamsters, guinea pigs, chimpanzees, etc.). Examples of such DNA encoding the HGF protein include, for example, DNA encoding the HGF protein derived from mice (such as Accession No. S71816, NM_010427, D10213, D10212, etc.) registered in the NCBI database and the like, DNA encoding the HGF protein derived from rats (such as Accession No. NM_017017, etc.), DNA encoding the HGF protein derived from cows (such as Accession No. NM_001031751, AB110822, etc.), DNA encoding the HGF protein derived from cats (such as Accession No. NM_001009830, AB080187, AB046610, etc.), DNA encoding the HGF protein derived from dogs (such as Accession No. NM_001002964, AB090353, etc.), or DNA encoding the HGF protein derived from chimpanzees (such as Accession No. XM_519174, etc.), and are not limited to these. In addition, as a specific example of the DNA encoding the HGF protein, preferably, for example, DNA having the base sequence represented by SEQ ID NO: 1 or 2 is listed. Here, the base sequence represented by SEQ ID NO: 1 corresponds to the base sequence at positions 73 to 2259 of the base sequence of Accession No. M60718, and the DNA composed of this base sequence also corresponds to the DNA encoding the HGF protein composed of the amino acid sequence represented by SEQ ID NO: 3. In addition, in DNA recombination technology, when the HGF protein (SEQ ID NO: 3) expressed and produced in cells is secreted outside the cells, the signal sequence is cleaved, and it becomes the HGF protein composed of the amino acid sequence represented by SEQ ID NO: 5. Therefore, the DNA composed of the base sequence represented by SEQ ID NO: 1 also corresponds to the DNA encoding (producing) the HGF protein composed of the amino acid sequence represented by SEQ ID NO: 5. The base sequence represented by SEQ ID NO: 2 corresponds to the base sequence at positions 66 to 2237 of the base sequence of Accession No. M73240, and the DNA composed of this base sequence also corresponds to the DNA encoding the HGF protein composed of the amino acid sequence represented by SEQ ID NO: 4. This HGF protein (SEQ ID NO: 4) also has its signal sequence cleaved when secreted outside the cells in DNA recombination technology, and it becomes the HGF protein composed of the amino acid sequence represented by SEQ ID NO: 6. Therefore, the DNA composed of the base sequence represented by SEQ ID NO: 2 also corresponds to the DNA encoding (producing) the HGF protein composed of the amino acid sequence represented by SEQ ID NO: 6.

[0027] (Substance having the same c-Met phosphorylation effect as the HGF protein and gene encoding the same) It is known that the HGF protein, which is a substance having c-Met phosphorylation effect, induces phosphorylation of the c-Met receptor and has anti-apoptotic effect, angiogenesis effect, morphogenetic effect, cell division promoting effect, axon elongation effect, etc. (Reference: Proc Jpn Acad Ser B Phys Biol Sci. 2010; 86(6): 588-610). That is, a substance having the same c-Met phosphorylation effect as the HGF protein can be used as an active ingredient of the preparation of the present invention, just like the HGF protein. Examples of substances having c-Met phosphorylation equivalent to that of HGF protein are as follows, but are not particularly limited. Examples include: low molecular weight compounds such as ANG-3777 (Angion, a hepatocyte growth factor (HGF) mimetic), ATH-1017 (Athirapharma); cyclic peptide dimers such as aML5, aMD4, and aMD5; HGF replacement peptide (product code: PG-001; c-Met agonist; PeptiGrowth Ltd.); anti-Met antibody AGMB-101 (AGOMAB, AGMB-101 is a full MET agonist); HGF molecular fragment mutants such as NK1 dimer or K1K1; DNA aptamer, RNA aptamer; Internalin (Listeria protein); and peptides having activity substantially the same as that of HGF protein, etc. A peptide having activity substantially the same as that of HGF protein (hereinafter sometimes abbreviated as HGF partial peptide) refers to any peptide that is a partial peptide of the above HGF protein and has activity substantially the same as that of HGF. In the present invention, the HGF partial peptide preferably contains at least about 20 or more, preferably about 50 or more, and more preferably about 100 or more amino acid sequences in the amino acid sequence constituting the above HGF protein. Specifically, peptides represented by sequences such as those from the N-terminal hairpin loop of human HGF to the first Kringle domain in the HGF amino acid sequence are preferably selected. In the HGF partial peptide of the present invention, the C-terminal can be any of a carboxyl group (-COOH), carboxylate (-COOM), amide group (-CONH2), or ester group (-COOR). In addition, similar to the above HGF protein, the HGF partial peptide includes a partial peptide in which the amino group on the N-terminal methionine residue is protected by a protecting group, a partial peptide in which Gln formed by cleavage on the N-terminal side in vivo is pyroglutamylated, a partial peptide in which substituents on the side chains of amino acids in the molecule are protected by appropriate protecting groups, or a complex peptide such as a so-called glycopeptide obtained by sugar chain binding. Examples of the gene encoding a substance having c-Met phosphorylation used in the present invention include genes encoding the above peptides having c-Met phosphorylation.

[0028] (Method for supplying HGF protein or a substance having c-Met phosphorylation equivalent to that of HGF, gene encoding HGF, or gene encoding a substance having c-Met phosphorylation equivalent to that of HGF) There is no particular limitation if the HGF protein, a substance having c-Met phosphorylation equivalent to that of HGF, a gene encoding the HGF, or a gene encoding a substance having c-Met phosphorylation equivalent to that of HGF (hereinafter sometimes collectively referred to as "HGF protein, etc.") is supplied to the spinal cord injury site by intrathecal administration (particularly intrathecal administration into the subarachnoid space) or intramedullary administration to the spinal cord. Specifically, in the case of administering HGF protein, etc. to a patient, various dosage forms can be adopted, such as liquid preparations, solid preparations, etc. However, generally, only the HGF protein or together with its commonly used carrier is used as an injection, a spray, etc. The above injection can be either an aqueous injection or an oily injection. In the case of an aqueous injection, according to a known method, for example, after dissolving HGF protein, etc. in a solution appropriately adding the following pharmaceutically acceptable additives in an aqueous solvent (water for injection, purified water, etc.), it is filtered and sterilized with a filter, etc., and then filled into a sterile container for preparation. Pharmaceutically acceptable additives include, for example, isotonic agents (sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, borax, glucose, propylene glycol, etc.), buffers (phosphate buffer, acetate buffer, boric acid buffer, carbonate buffer, citrate buffer, Tris (tris(hydroxymethyl)aminomethane) buffer, glutamate buffer, ε-aminocaproic acid buffer, etc.), preservatives (methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium ethylenediaminetetraacetate, boric acid, borax, etc.), thickeners (hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, polyethylene glycol, etc.), stabilizers (sodium bisulfite, sodium thiosulfate, sodium ethylenediaminetetraacetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene, etc.) or pH regulators (hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc.). Appropriate solubilizing aids can also be used, such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.) or nonionic surfactants (such as polysorbate 80, polyoxyethylene hydrogenated castor oil 50, etc.). In the case of an oily injection, for example, sesame oil or soybean oil can be used as the oily solvent, and benzyl benzoate or benzyl alcohol can be used as the solubilizing aid. The prepared injection is usually filled into a suitable ampoule or vial. The content of HGF protein in the injection is usually adjusted to about 0.0002 to about 2.0 w / v%, preferably about 0.001 to about 1.0 w / v%, more preferably about 0.01 to about 0.5 w / v%. It should be noted that for liquid preparations such as injections, it is preferably stored frozen or after removing moisture by freeze-drying, etc. When using the freeze-dried preparation, add distilled water for injection, etc., and redissolve it for use.

[0029] The injection can also be prepared by conventional methods for the preparation of pharmaceutical formulations. When prepared as an injection, as additives, for example, in addition to the injection agent, the above solvents, preservatives, stabilizers, isotonic agents, pH regulators, etc. can be used. As the injection agent, liquefied gas injection agents or compressed gases, etc. can be used. As the liquefied gas injection agent, for example, hydrofluorocarbons (chlorofluorocarbon alternatives such as HCFC22, HCFC-123, HCFC-134a, HCFC-142, etc.), liquefied petroleum gas, dimethyl ether, etc. can be cited. As the compressed gas, for example, soluble gases (carbon dioxide, nitrous oxide, etc.) and insoluble gases (nitrogen, etc.) can be cited.

[0030] In addition, the HGF protein used in the present invention can also be used together with a biodegradable polymer in vivo as a sustained-release formulation (for example, a long-acting formulation). In particular, by using the HGF protein as a long-acting formulation, effects such as a reduction in the number of administrations, persistence of action, and reduction of side effects can be expected. This sustained-release formulation can be prepared according to known methods. The biodegradable polymer used in this sustained-release formulation can be appropriately selected from the following known biodegradable polymers in vivo, for example, including: polysaccharides such as starch, dextran, hyaluronan (hyaluronic acid) or its salts, proteins such as telopeptide-free collagen, collagen or gelatin, polyamino acids such as polyglutamic acid, polylysine, polyleucine, polyalanine or polymethionine, polyesters such as polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, polycaprolactone, poly-β-hydroxybutyric acid, polymalic acid, polyanhydride or fumaric acid-polyethylene glycol-vinylpyrrolidone copolymer, polyalkyl cyanoacrylates such as polyorthoester or polymethyl-α-cyanoacrylate, polycarbonates such as polyvinyl carbonate or polypropylene carbonate, etc. Polyesters are preferred, and lactic acid-glycolic acid copolymer is particularly preferred. When using lactic acid-glycolic acid copolymer, its composition ratio (lactic acid / glycolic acid) (mol%) changes according to the expected sustained-release time. For example, for a sustained-release time of about 2 weeks to about 3 months, preferably about 2 weeks to about 1 month, the composition ratio is about 100 / 0 to about 50 / 50. The weight-average molecular weight of this lactic acid-glycolic acid copolymer is usually about 5,000 to about 20,000. The lactic acid-glycolic acid copolymer can be prepared by known preparation methods, for example, the preparation method described in Japanese Patent Laid-Open No. 61-28521. The ratio of the biodegradable polymer to the HGF protein is not particularly limited. For example, the ratio of the HGF protein to the biodegradable polymer is usually about 0.01 to about 30 w / w%.

[0031] As a method of administration, intrathecal administration (preferably within the subarachnoid space), intramedullary spinal cord administration, continuous intrathecal administration using a sustained-release pump, or direct injection, infusion, or supply of an injection or spray into the tissue with spinal cord injury is preferred. In addition, the dosage can be appropriately selected according to the dosage form, the degree of the disease, or age, etc., but is usually 1 μg to 500 mg per administration, preferably 10 μg to 50 mg. In addition, the administration method can also be appropriately selected according to the dosage form, the degree of the disease, or age, etc., and can be administered as a single-dose once, continuously administered for about 30 minutes to about several weeks (preferably about 24 hours to about 2 weeks) once, or the single-dose administration or continuous administration can be administered continuously at intervals. When administered continuously, the administration interval can be once a day to once every few months. For example, when continuously administered locally (e.g., intrathecally) using a sustained-release pump (e.g., osmotic pump), it can be once every few weeks to once every few months. This continuous administration has the advantages that since the HGF protein is gradually released into the spinal cord injury site for a long time, the effect of HGF can be exerted for a long time, and a better therapeutic effect can be obtained. In addition, it has the advantage of reducing the burden on the patient due to fewer administration times. In addition, if necessary, the HGF protein can be additionally administered to a subcutaneous osmotic pump set once, which can also be regarded as an advantage. In addition, the administration time can be appropriately selected according to the dosage form, the degree of the disease, or age, etc., and is preferably within 14 days after injury, more preferably within 7 days after injury, and particularly preferably within 4 days after injury. In particular, in patients with spinal cord injury, considering that the condition is difficult to stabilize about 72 hours after injury, it is particularly preferred to administer within about 72 hours to 4 days after injury. The above administration time also includes the start time or the first administration time during continuous administration or continuous administration. For example, when administered to humans, the following can be exemplified, but there is no particular limitation. Administration site: Intrathecal administration from the lumbar spine. Start time of administration: Immediately after injury, within 72 hours after injury, or within 48 - 72 hours after injury. Number of administrations: Multiple single-dose administrations (1 - 10 times, 2 - 8 times, 3 - 7 times, 4 - 6 times, or 5 times) every 1 day to 10 days (especially 7 days). End time of administration: Varies according to the degree of injury and the degree of cure, and is preferably about 3 - 10 weeks, 4 - 8 weeks, 5 weeks after injury after the above number of administrations. Dosage of HGF protein: 100 - 2000 μg / per, 200 - 1000 μg / per, 300 - 700 μg / per, or about 400 μg / per.

[0032] (Pluripotent stem cells) The pluripotent stem cells of the present invention are not particularly limited as long as they can achieve the effects of the preparation of the present invention. Examples of pluripotent stem cells include pluripotent stem cells derived from differentiated cells obtained by forcibly expressing the Oct3 / 4 gene, Sox2 gene, and Klf4 gene in differentiated cells (see: Japanese Patent Laid-Open No. 2009-215191), pluripotent stem cells (see: Japanese Patent No. 5603282), embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and the like. Neural stem cells refer to cells that have the ability of self-renewal and the ability to differentiate into neural progenitor cells (neural stem cells / progenitor cells). In addition, neural progenitor cells refer to cells that are undifferentiated but have differentiated one stage from neural stem cells, and can self-proliferate and ultimately differentiate into nerve cells. Neural stem cells or neural progenitor cells are preferably cells obtained by differentiating and inducing from pluripotent stem cells. The method of differentiating and inducing pluripotent stem cells into neural stem cells or neural progenitor cells is not particularly limited and can be a commonly used method. For example, neural spheres can be obtained by culturing pluripotent stem cells in suspension in a medium containing at least one of bFGF and EGF. In addition, the obtained neural spheres can be dissociated into single cells, and then cultured in suspension again in a medium containing bFGF, and the formation of neural spheres can be repeated multiple times again. For example, human induced pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, 1231A3 cells can be obtained (see: WO2021 / 045217). Neural stem cells and / or neural stem cell / progenitor cells derived from iPS cells can preferably be exemplified by neural stem cells and / or neural stem cell / progenitor cells derived from human iPS cells (YZWJs513 hiPSc) derived from human umbilical cord blood, etc.

[0033] (Transplantation method of pluripotent stem cells) The transplantation and administration methods of pluripotent stem cells include transplantation, injection, etc., but are not particularly limited. For example, methods of transplanting and administering pluripotent stem cells to one or more sites of spinal cord injury can be exemplified. In addition, it can also be administered by intravenous injection, etc. to reach the spinal cord injury site. In addition, it is preferable to add a γ-secretase inhibitor (GSI) to clinical-grade iPS cell-derived neural stem cell / progenitor cells (YZWJs513 hiPSC-NS / PCs) one day before cell transplantation. For example, in the case of transplantation into humans, the following can be exemplified. Transplantation start time: about 2 to 10 weeks or 4 to 6 weeks after injury. Number of transplantations: 1 to 5 times, 1 to 3 times, 1 to 2 times, or 1 time. Cell transplantation amount: 100,000 to 50 million / per, 300,000 to 10 million / per, 500,000 to 5 million / per, 1 million to 3 million / per, or approximately 2 million / per.

[0034] (Therapeutic agent for spinal cord injury from acute phase to subacute phase) The therapeutic agent for spinal cord injury from acute phase to subacute phase of the present invention comprises the following: (1) (a) HGF protein or a substance having the same c-Met phosphorylation effect as the HGF protein, or (b) a gene encoding the HGF or a gene encoding a substance having the same c-Met phosphorylation effect as the HGF protein; (2) Pluripotent stem cells. In addition, as another mode of the therapeutic agent for spinal cord injury from acute phase to subacute phase of the present invention, it comprises the following: (1) HGF protein; It should be noted that the HGF protein is supplied to the spinal cord injury site by intrathecal administration, and further, iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site. In addition, as another mode of the therapeutic agent for spinal cord injury from acute phase to subacute phase of the present invention, it comprises the following: (1) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells. It should be noted that during the supply of the HGF protein to the spinal cord injury site by intrathecal administration, or simultaneously with the supply, or after the supply, iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

[0035] (Therapeutic method for spinal cord injury from acute phase to subacute phase) The therapeutic method for spinal cord injury from acute phase to subacute phase of the present invention comprises the following steps. (1) A step of supplying (a) hepatocyte growth factor protein or a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein, or (b) a gene encoding the hepatocyte growth factor protein or a gene encoding a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein to the spinal cord injury site by intrathecal administration or intramedullary spinal cord administration; (2) A step of administering pluripotent stem cells to the spinal cord injury site.

[0036] (Kit for treating spinal cord injury from acute phase to subacute phase) The kit for treating spinal cord injury from the acute phase to the subacute phase of the present invention comprises the following. (1) (a) Hepatocyte growth factor protein or a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein, or (b) a gene encoding the hepatocyte growth factor protein or a gene encoding a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein; (2) Pluripotent stem cells. The hepatocyte growth factor protein or a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein, or the gene encoding the hepatocyte growth factor protein or the gene encoding a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein is supplied to the spinal cord injury site by intrathecal administration or intramedullary spinal cord administration, and then the pluripotent stem cells are administered to the spinal cord injury site.

[0037] As a preferred embodiment of the therapeutic agent for spinal cord injury from the acute phase to the subacute phase, the treatment method for spinal cord injury from the acute phase to the subacute phase, and the kit for treating spinal cord injury from the acute phase to the subacute phase of the present invention, the substance having c-Met phosphorylation effect is HGF protein, the pluripotent stem cells are iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells, and the spinal cord injury is severe spinal cord injury. In addition, as a preferred embodiment of the therapeutic agent for spinal cord injury from the acute phase to the subacute phase, the treatment method for spinal cord injury from the acute phase to the subacute phase, and the kit for treating spinal cord injury from the acute phase to the subacute phase of the present invention, the HGF protein is supplied to the spinal cord injury site by intrathecal administration, and then the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

[0038] Examples of treating using the therapeutic agent for spinal cord injury from the acute phase to the subacute phase, the treatment method for spinal cord injury from the acute phase to the subacute phase, and the kit for treating spinal cord injury from the acute phase to the subacute phase of the present invention include: promoting the regeneration of the injured spinal cord and / or improving the complications associated with the injured spinal cord. Examples of promoting the regeneration of the injured spinal cord include: promoting axon elongation, inhibiting the atrophy of the injured spinal cord, increasing the survival rate of pluripotent stem cells, inhibiting demyelination after spinal cord injury, increasing remyelination after spinal cord injury, and / or inhibiting the formation of spinal cord cavities. Promoting the regeneration of the injured spinal cord in the present invention includes: activating endogenous nerve cells, promoting and assisting the reconstruction of neural circuits and / or increasing functional nerve fibers. In addition, promoting the regeneration of the injured spinal cord in the present invention includes enhancing neurotransmission, and can restore the reduction and attenuation of neurotransmission caused by abnormal neurotransmission, disorders, etc. Furthermore, promoting the regeneration of the injured spinal cord in the present invention further includes: regulating the immune response, inhibiting inflammation, and / or promoting and assisting angiogenesis. Examples of complications accompanying the injured spinal cord include motor dysfunction. Examples of motor dysfunction include motor dysfunction caused by lower limb motor dysfunction and loss of walking ability. In the present invention, the walking includes gait.

[0039] (Agent for improving motor dysfunction in acute to subacute spinal cord injury) The agent for improving motor dysfunction in acute to subacute spinal cord injury of the present invention comprises the following. (1) HGF protein; (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells. The HGF protein is supplied to the spinal cord injury site by intrathecal administration, and further, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site. Examples of motor function are lower limb motor function and walking ability.

[0040] (Agent for inhibiting cavitation of the spinal cord in acute to subacute spinal cord injury) The agent for inhibiting cavitation of the spinal cord in acute to subacute spinal cord injury of the present invention comprises the following. (1) HGF protein, a carrier loaded with the HGF protein and capable of sustained release; (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells. The HGF protein is supplied to the spinal cord injury site by intrathecal administration, and the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

[0041] (Agent for inhibiting atrophy of the spinal cord in acute to subacute spinal cord injury) The agent for inhibiting atrophy of the spinal cord in acute to subacute spinal cord injury of the present invention comprises the following. (1) HGF protein, a carrier loaded with the HGF protein and capable of sustained release; (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells. The HGF protein is supplied to the spinal cord injury site by intrathecal administration, and the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

[0042] (Agent for promoting axon elongation at the injury site in acute to subacute spinal cord injury) The axon elongation promoter for the injured site in acute to subacute spinal cord injury of the present invention comprises the following. (1) HGF protein, and a carrier loaded with the HGF protein and capable of sustained release; (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells. The HGF protein is supplied to the spinal cord injury site by intrathecal administration, and the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

[0043] (Method for improving the survival rate of pluripotent stem cells for transplantation) The method for improving the survival rate of pluripotent stem cells for transplantation of the present invention includes at least the following steps. (1) A step of bringing hepatocyte growth factor into contact with pluripotent stem cells for transplantation. Through Example 7 below, it was confirmed that by bringing hepatocyte growth factor into contact with pluripotent stem cells for transplantation, the survival rate of the pluripotent stem cells for transplantation increased. The contact includes binding hepatocyte growth factor in the same physical space (preferably the transplantation site) so as to interact with at least a part of the pluripotent stem cells for transplantation.

[0044] In the preparation of the preparation, therapeutic agent, and therapeutic agent of the present invention, the following combinations can be preferably exemplified, but there is no particular limitation. (1) Hepatocyte growth factor protein or a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein, pluripotent stem cells; (2) A gene encoding hepatocyte growth factor protein or a gene encoding a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein, pluripotent stem cells; (3) Hepatocyte growth factor protein or a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein, iPS cell-derived neural stem cells; (4) A gene encoding hepatocyte growth factor protein or a gene encoding a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein, iPS cell-derived neural stem cells; (5) Hepatocyte growth factor protein, iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells. In addition, the pluripotent stem cells in (1) can also be combined with the hepatocyte growth factor protein in (5). As a combination of hepatocyte growth factor protein and pluripotent stem cells, the combinations of (1) to (5) are not particularly limited. Specific examples are given below to illustrate the present invention in detail, but the present invention is not limited to these examples. All animal experiments were conducted in accordance with the regulations of the Animal Experiment Committee of Keio University (Approval No. 7207 of Keio University). Example 1

[0045] (Materials and Methods) (1) Recombinant human HGF protein A recombinant human HGF protein composed of the amino acid sequence represented by SEQ ID NO: 6 was used. This protein was prepared using CHO cells according to the method described in Biochem. Biophys. Res. Commun. 180: 1151-1158, 1991. (2) Preparation of spinal cord injury model An osmotic pump was prepared aseptically. HGF protein (concentration: 1 mg / mL, dissolved in PBS) or PBS was injected into an Alzet mini osmotic pump (manufactured by ALZA Corporation, Model 2002). A silicone tube (manufactured by IMAMURA Co., Ltd., catheter) with an inner diameter of 0.3 mm and an outer diameter of 0.7 mm filled with HGF protein or PBS in the lumen was connected to the pump discharge part, and the connection part was covered with another silicone tube (manufactured by IMAMURA Co., Ltd.) with an inner diameter of 1.0 mm and an outer diameter of 2.0 mm. After culturing at 37°C for 12 hours, it was used for the experiment. Adult female SD rats (about 8 weeks old) were anesthetized by intraperitoneal administration of 14 w / v% chloral hydrate. After removing the vertebral arches of the tenth and twelfth thoracic vertebrae, the osmotic pump (pre-filled with HGF protein solution by the above method) was implanted subcutaneously on the right dorsal side of the spine, and the catheter passed through the muscle layer from the subcutaneous tissue and was guided to the vertebral arch of the twelfth thoracic vertebra. Next, a severe crush injury of 240 kDyne was made on the tenth thoracic spinal cord using an IH impactor (manufactured by Precision Systems). Then, the dura mater and arachnoid mater of the twelfth thoracic spinal cord were incised together in the cephalocaudal direction, the catheter was inserted into the subarachnoid space, and the tip of the catheter was advanced directly above the injured spinal cord. Then, the muscle layer and skin were sutured to complete the surgery. (3) Administration of HGF protein After the above surgery (corresponding to the acute phase after crush injury), the HGF protein solution was administered intrathecally through the osmotic pump for 2 weeks (the dosage of recombinant human HGF protein, 200 μg / 2 weeks). It should be noted that the control group was only administered PBS. (4) Transplantation of iPS cell-derived neural stem cells and progenitor cells One day before cell transplantation, γ-secretase inhibitor (DAPT) was administered to clinical-grade iPS cell-derived neural stem cell-progenitor cells (YZWJs513hiPSC-NS / PCs). On the 9th day after injury (equivalent to the subacute phase after crush injury), clinical-grade iPS cell-derived neural stem cell-progenitor cells (YZWJs513 hiPSC-NS / PCs) were transplanted into 1 site at the center of the injury (1×10 6 cells). After that, the hindlimb motor function of the rats was evaluated by the Basso, Beattie and Bresnahan (BBB) score every 1 week, and the observation period was up to the 84th day after injury, and then the spinal cord was collected. (5) Each group According to the above (1) to (4), each group was prepared (reference: Figure 1 ). Combination group: Administration of recombinant human HGF protein + transplantation of iPS cell-derived neural stem cell-progenitor cells; HGF alone group: Recombinant human HGF protein + administration of PBS instead of transplantation of iPS cell-derived neural stem cell-progenitor cells; TP alone group: Administration of PBS instead of recombinant human HGF protein + transplantation of iPS cell-derived neural stem cell-progenitor cells; Control group: Administration of PBS instead of recombinant human HGF protein + administration of PBS instead of transplantation of iPS cell-derived neural stem cell-progenitor cells.

[0046] (6) Evaluation method Immunostaining, myelin gold black staining, and evaluation method using IVIS photon count of fluorescent protein were performed on the collected spinal cord tissues. The references for each evaluation method are "Grafted human-induced pluripotent stem-cell-derived neurospheres promote motor functional recovery after spinal cord injury in mice, 2011", "In vivo imaging of engrafted neural stem cells: its application in evaluating the optimal timing of transplantation for spinal cord injury, 2005", "High resolution neurochemical gold staining method for myelin in peripheral and central nervous system at the light-and electron-microscopic level, 2009". Example 2

[0047] (Motor function evaluation) As a motor function evaluation (especially for lower limb motor function evaluation), the Basso-Beattie-Bresnahan (BBB) score (Basso et al. J. Neurotrauma vol. 12, pp. 1-21, 1995) and the stride length of the Digigait small animal walking analysis system were measured. The results are shown in Figures 2 to 4 . Furthermore, the walking analysis based on kinematics and the motor evoked potentials (MEP) were measured. The results are shown in Figure 17 . Regarding the BBB score, the combination group (the group administered with the preparation of the present invention) showed a slow increase until day 84, but the TP alone group and the HGF alone group did not increase until day 84 ( Figure 2 ). Regarding the length of the lower limb stride in the Digigait small animal walking analysis system, the combination group was significantly increased compared with the TP alone group and the HGF alone group ( Figure 3 ). In addition, as shown in Figure 4As described, in a severe spinal cord injury model in the subacute phase, the combination group achieved walking ability. It should be noted that, according to previous views, in a severe spinal cord injury model in the subacute phase, the HGF alone group (administration of HGF protein) did not achieve walking ability. Furthermore, in the walking analysis based on kinematic analysis, the bending of the hind limb joints in the combination group increased, and an improvement in walking function was observed. In the electrophysiological analysis based on MEP, a significant increase in the amplitude of the motor evoked potential waveform in the combination group was observed, confirming the enhancement of nerve transmission ( Figure 17 ). From these results, the preparation of the present invention not only has the effect of improving the motor function of patients with spinal cord injury from the acute phase to the subacute phase, but also has the effect of enabling patients with severe spinal cord injury to achieve walking ability. Example 3

[0048] (Evaluation by histological analysis) The HE staining results and HNA (Human neutrophil alloantigens) fluorescence staining results of the collected spinal cord tissues are shown in Figure 5 . It was confirmed that in the combination group (the group administered with the preparation of the present invention), the cavitation caused by the crush injury disappeared and was replaced by nerve cells compared with other groups. The measurement results of the spinal cord area are shown in Figure 6 . It was confirmed that the spinal cord area was larger in the combination group compared with other groups. The STEM121 (mouse monoclonal antibody that can specifically react with the cytoplasmic protein of human cells) fluorescence staining of the collected spinal cord tissues is shown in Figure 7 . In the combination group (the group administered with the preparation of the present invention), extensive migration and axonal elongation towards the cranial-caudal side of the transplanted cells (nerve stem cells and progenitor cells derived from human iPS cells) were confirmed. As described above, the preparation of the present invention has the effects of inhibiting spinal cord cavitation, inhibiting atrophy of the injured spinal cord, and promoting migration and axonal elongation. Example 4

[0049] (Evaluation of differentiation of transplanted cells) The survival of iPS-derived nerve stem cells and progenitor cells is marked by HNA-positive cells. Positive cells of APC (Adenomatous polyposis Coli, a marker for mature oligodendrocytes), GFAP (glial fibrillary acidic protein), HuC / D (neuronal-specific RNA-binding protein), and Ki-67 positive cells were detected ( Figure 8 ). According to the detection results, it was observed that human iPS cell-derived neural stem cells and progenitor cells, which were transplanted cells, differentiated into three neural lineages (neurons, astrocytes, and oligodendrocytes). By measuring the Ki-67 positive cell rate (Percentage of Ki positive cell (%)) and the Nestin positive cell rate (Percentage of Nestin positive cell (%)), no proliferation of proliferative cells and undifferentiated cells was observed in both the combined group and the TP alone group, and the neoplastic change was negative ( Figure 9 ). Thus, it was confirmed that the preparation of the present invention does not have adverse reactions (especially carcinogenesis of transplanted cells) of transplanted cells (pluripotent stem cells). Example 5

[0050] (Evaluation of regenerated nerve fibers and serotonergic neurons in the peripheral area of injury) Serotonergic neurons (5-HT), which play an important role in the recovery of lower limb motor function after spinal cord injury, were measured. The results are shown in Figure 10 . In addition, regenerated nerve fibers (NF-H), which play an important role in the recovery of lower limb motor function after spinal cord injury, were measured. The results are shown in Figure 18 . In the combined group (the group administered with the preparation of the present invention), cells that were positive for 5-HT were confirmed from the rostral side to the center of the injury. In addition, compared with the TP alone group, many 5-TH positive fibers were confirmed at 4 mm caudal to the injury site in the combined group. Compared with the TP alone group, an increase in serotonergic neurons was confirmed in the combined group (the group administered with the preparation of the present invention) from the center of the injury to the caudal side (4 mm). In addition, compared with the TP alone group, a significant increase in NF-H positive nerve fibers was confirmed in the combined group (the group administered with the preparation of the present invention) at the injury site and 1 mm rostral and caudal to it. Thus, the preparation of the present invention has the effect of restoring lower limb motor function. Example 6

[0051] (Quantitative evaluation of myelin sheath area around injury) Regarding remyelination after spinal cord injury, which is greatly related to the recovery of lower limb motor function and improvement of gait after spinal cord injury, quantitative evaluation of myelin sheath area was performed using Myelin Gold Black staining. The results are shown in Figure 11 , Figure 12 . In the control group, cavitation and demyelination were observed around the lesion center. In the HGF alone group, inhibition of demyelination was observed around the lesion center. In the TP alone group, remyelination was observed in the lesion center. In the combination group, myelin area was significantly increased due to inhibition of demyelination and remyelination compared to the other groups. Therefore, the preparation of the present invention has the effect of enhancing the demyelination inhibition and remyelination inhibition after spinal cord injury. Example 7

[0052] (Evaluation of the survival rate of transplanted cells) EF1-fffluc (fluorescent protein fused to luciferase: M0I4) was added to the cells (YZWJs513) and subcultured (9 th The subcultured cells (YZWJs513) were transplanted into spinal cord injury model rats (Example 1). After transplantation, fluorescein (800 μg / per), a fluorescent protein, was intraperitoneally administered every week, and the survival rate of the transplanted cells was measured by IVIS imaging ( Figure 13 The results are shown in Figure 14 , Figure 15 . In the combination group (group administered with the preparation of the present invention), it was confirmed that the survival rate of transplanted cells was increased compared with the TP alone group. Therefore, the preparation of the present invention has the effect of improving the survival rate of transplanted cells (pluripotent stem cells). Example 8

[0053] The results of the BBB score and the stride length of the Digigait small animal walking analysis system in the number of cases (combination group: 12, HGF alone group: 11, TP alone group: 10, control group: 11) increased from the number of cases in Example 2 (combination group: 16, HGF alone group: 14, TP alone group: 15, control group: 15) are shown in the following table. Figure 19 and Figure 20 .

[0054] (Summary of Embodiments) According to the results of Examples 1 to 7, based on Figure 16The mechanism described in [reference] believes that the preparation of the present invention has the following effects. (1) Effect of improving motor dysfunction caused by spinal cord injury (effect of restoring lower limb motor function, effect of obtaining walking ability); (2) Effect of inhibiting syringomyelia in the spinal cord injury site; (3) Effect of inhibiting atrophy of the injured spinal cord; (4) Effect of promoting migration and axon elongation in the spinal cord injury site; (5) Effect of improving the survival rate of transplanted cells; (6) Effect of inhibiting demyelination after spinal cord injury; (7) Effect of improving remyelination after spinal cord injury; (8) Effect of enhancing neurotransmission in the spinal cord injury site. In addition, it is considered that a substance having the same c-Met phosphorylation effect as the HGF protein has the same effect as the HGF protein. Industrial applicability

[0055] A therapeutic agent for spinal cord injury from the acute phase to the subacute phase can be provided.

Claims

1. A therapeutic agent for spinal cord injury from the acute phase to the subacute phase, characterized in that, Comprising: (1) (a) Hepatocyte growth factor protein or a substance having c-Met phosphorylation equivalent to that of hepatocyte growth factor protein, or (b) a gene encoding hepatocyte growth factor protein or a gene encoding a substance having c-Met phosphorylation equivalent to that of hepatocyte growth factor protein; and (2) Pluripotent stem cells.

2. The therapeutic agent for treating spinal cord injury from the acute stage to the subacute stage according to claim 1, wherein, The pluripotent stem cells are iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells.

3. The spinal cord injury therapeutic agent from the acute stage to the subacute stage according to claim 1, wherein, The spinal cord injury is a severe spinal cord injury.

4. The spinal cord injury therapeutic agent from the acute stage to the subacute stage according to claim 1, wherein, The substance having the c-Met phosphorylation is hepatocyte growth factor protein, the pluripotent stem cells are iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells, and the spinal cord injury from the acute phase to the subacute phase is a severe spinal cord injury.

5. The therapeutic agent for spinal cord injury from the acute stage to the subacute stage according to claim 4, wherein, The hepatocyte growth factor protein is administered intrathecally.

6. The spinal cord injury therapeutic agent from the acute stage to the subacute stage according to claim 5, wherein, The hepatocyte growth factor protein is administered into the subarachnoid space.

7. A therapeutic agent for spinal cord injury from the acute phase to the subacute phase according to any one of claims 1 to 6, wherein, The treatment is to promote the regeneration of the injured spinal cord and / or improve the complications associated with the injured spinal cord.

8. The therapeutic agent for spinal cord injury from the acute phase to the subacute phase according to claim 7, wherein, The promotion of the regeneration of the injured spinal cord is to promote axon elongation, inhibit spinal cord atrophy, improve the survival rate of the pluripotent stem cells, inhibit demyelination, improve remyelination and / or inhibit syringomyelia.

9. The therapeutic agent for spinal cord injury from the acute stage to the subacute stage according to claim 7, wherein, The promotion of the regeneration of the injured spinal cord is the activation of endogenous nerve cells, the promotion and assistance of the reconstruction of nerve circuits and / or the increase of functional nerve fibers.

10. The therapeutic agent for spinal cord injury from the acute stage to the subacute stage according to claim 7, wherein, The complication associated with the injured spinal cord is motor dysfunction.

11. The spinal cord injury therapeutic agent from the acute stage to the subacute stage according to claim 10, wherein, The motor dysfunction is lower limb motor dysfunction.

12. The therapeutic agent for spinal cord injury from the acute phase to the subacute phase according to claim 11, wherein, The motor dysfunction is the loss of walking ability.

13. A therapeutic agent for spinal cord injury from the acute stage to the subacute stage, characterized in that, Containing hepatocyte growth factor protein; Wherein, the hepatocyte growth factor protein is supplied to the spinal cord injury site by intrathecal administration, and further, iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

14. A therapeutic agent for spinal cord injury from the acute phase to the subacute phase, characterized in that, Containing iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells, Wherein, during or after the supply of the hepatocyte growth factor protein to the spinal cord injury site by intrathecal administration, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

15. A motor dysfunction improver for spinal cord injury from the acute stage to the subacute stage, characterized in that, Comprising: (1) Hepatocyte growth factor protein; and (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is supplied to the spinal cord injury site by intrathecal administration, and further, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

16. The motor function impairment improver for spinal cord injury from the acute stage to the subacute stage according to claim 15, wherein, The motor function is lower limb motor function or walking function.

17. A demyelination inhibitor after spinal cord injury for spinal cord injury from the acute phase to the subacute phase, characterized in that, Comprising: (1) Hepatocyte growth factor protein; and (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is supplied to the spinal cord injury site by intrathecal administration, and further, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

18. A myelin re - sheathing enhancer after spinal cord injury for spinal cord injury from the acute phase to the sub - acute phase, characterized in that, Comprising: (1) Hepatocyte growth factor protein; and (2)iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is supplied to the spinal cord injury site by intrathecal administration, and then, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

19. A syringomyelia inhibitor for spinal cord injury from the acute stage to the subacute stage, characterized in that, Comprising: (1) Hepatocyte growth factor protein; and (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is supplied to the spinal cord injury site by intrathecal administration, and then, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

20. A spinal cord atrophy inhibitor for spinal cord injury from the acute phase to the subacute phase, characterized in that, Comprising: (1) Hepatocyte growth factor protein; and (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is supplied to the spinal cord injury site by intrathecal administration, and then, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

21. An axon elongation promoter for an injured site in spinal cord injury from the acute phase to the subacute phase, characterized in that, Comprising: (1) Hepatocyte growth factor protein; and (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is supplied to the spinal cord injury site by intrathecal administration, and then, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

22. A promoter for reconstructing nerve circuits and / or increasing functional nerve fibers at the injury site in spinal cord injury from the acute phase to the subacute phase, characterized in that, Comprising: (1) Hepatocyte growth factor protein; and (2) iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells; The hepatocyte growth factor protein is supplied to the spinal cord injury site by intrathecal administration, and then, the iPS cell-derived neural stem cells and / or neural stem cell-progenitor cells are administered to the spinal cord injury site.

23. A treatment method for spinal cord injury from the acute stage to the subacute stage, characterized in that, Comprising: (1) A step of supplying (a) a hepatocyte growth factor protein or a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein, or (b) a gene encoding the hepatocyte growth factor protein or a gene encoding a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein to the spinal cord injury site by intrathecal administration or intramedullary spinal cord administration; And (2) A step of administering pluripotent stem cells to the spinal cord injury site.

24. A kit for the treatment of spinal cord injury from the acute phase to the subacute phase, characterized in that, Comprising: (1) (a) A hepatocyte growth factor protein or a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein, or (b) a gene encoding the hepatocyte growth factor protein or a gene encoding a substance having the same c-Met phosphorylation effect as the hepatocyte growth factor protein; And (2) Pluripotent stem cells; The hepatocyte growth factor protein, a substance having c-Met phosphorylation equivalent to that of the hepatocyte growth factor protein, or a gene encoding the hepatocyte growth factor protein or a gene encoding a substance having c-Met phosphorylation equivalent to that of the hepatocyte growth factor protein is administered intrathecally or intramedullary to the spinal cord, and then the pluripotent stem cells are administered to the spinal cord injury site.

25. A method for improving the survival and survival rate of pluripotent stem cells for transplantation, characterized in that, The survival rate of the pluripotent stem cells for transplantation is increased by contacting the hepatocyte growth factor with the pluripotent stem cells for transplantation.

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