JPWO2020012991A1 - Mesenchymal stem cells and neuropathy therapeutic agents - Google Patents

Abstract

An object of the present invention is to provide a novel therapeutic agent for neuropathy. The present invention for solving the above problems is HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1. , ASCL1, NRP2, one or more of which are highly expressed, are mesenchymal stem cells.

Description

The present invention relates to mesenchymal stem cells and therapeutic agents for neuropathy.

With the progress of an aging society, neurodegenerative diseases, which are disorders of nerve cells in the brain, continue to increase year by year. Neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and spinocerebellar degeneration. In Alzheimer's disease, nerve cells in places such as the cerebral cortex and hippocampus are shed, and memory capacity declines. In Parkinson's disease, nerve cells in a place called the substantia nigra are shed, resulting in impaired motor function. Furthermore, cerebral infarction and cerebral hemorrhage due to stroke are known as diseases caused by damage to nerve cells in the brain, in addition to neurodegenerative diseases.

In addition, perinatal neonatal cerebral disorders occur at a frequency of 1 to 2 in 1,000 live births and cause cerebral palsy for the rest of their lives. Perinatal cerebral disorders that cause such cerebral palsy include hypoxic-ischemic encephalopathy, cerebral hemorrhage, and periventricular leukomalacia, and these main pathological conditions are elevated active oxygen caused by mitochondrial dysfunction. , Macrophage activation and associated hypercytokineemia, an inflammatory condition. The above-mentioned neurodegenerative diseases, strokes, and cerebral palsy can be said to be common in that the causes and symptoms of the diseases are different, but the number of nerve cells is reduced.

Therapeutic agents such as donepezil for Alzheimer's disease and levodopa for Parkinson's disease are used, and these therapeutic agents restore the information processing function of the neural network due to the decrease in nerve cells by supplementing the chemical transmitter signal. The purpose is to make it, and the decrease of nerve cells itself cannot be suppressed. In addition, none of the existing therapeutic agents for neurodegenerative diseases and strokes have a protective effect on nerve cells. Therefore, the development of a new therapeutic agent having an action of protecting nerve cells is desired.

Mesenchymal stem cells are pluripotent progenitor cells first isolated from bone marrow by Friedenstein in 1982 (see Non-Patent Document 1). It has been clarified that mesenchymal stem cells are present in various tissues such as bone marrow, umbilical cord, and adipose tissue, and mesenchymal stem cell transplantation is expected as a new therapeutic method for various intractable diseases ( See Patent Documents 1 and 2). Recently, it is known that there are cells having the same function as stromal cells such as adipose tissue, placenta, umbilical cord, and fetal membrane. Therefore, mesenchymal stem cells are sometimes referred to as stromal cells (Mesenchymal Stromal Cells).

Japanese Unexamined Patent Publication No. 2012-157263 Japanese Patent Application Laid-Open No. 2012-508733

Pittenger F.M.et al.Science, (1999), 284, pp.143-147

An object of the present invention is to provide a novel therapeutic agent for neuropathy under the above-mentioned circumstances.

As a result of diligent research to solve the above problems, the present inventors have HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, Mesenchymal stem (stromal) cells (MSCs) in which one or more of DLL1, HEYL, BMP2, NTN1, ASCL1, and NRP2 are highly expressed are effective in treating neuropathy. And completed the present invention. According to the present invention, it is possible to provide an effective therapeutic agent for the treatment of neuropathy. That is, the gist of the present invention is as follows.

That is, the present invention relates to:
[1] One of HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, NRP2 Mesenchymal stem cells characterized by high expression of one or more.
[2] The mesenchymal stem cell according to [1], which is derived from an allogeneic family.
[3] The mesenchymal stem cell according to [1] or [2], which is derived from umbilical cord tissue or adipose tissue.
[4] The mesenchymal stem cell according to any one of [1] to [3] prepared by the suspension culture method.
[5] The therapeutic agent for neuropathy containing mesenchymal stem cells according to any one of [1] to [4].

According to the present invention, it is possible to provide a novel therapeutic agent for neuropathy.

FIG. 1 is a diagram showing the results of comparing the mRNA expression levels of various factors in mesenchymal stem cells obtained by culturing in various media. FIG. 2 is a diagram showing the results of comparing the mRNA expression levels of various factors in planar cultured cells (ADH) and suspension cultured cells (SUS) for mesenchymal stem cells. It is a figure which shows the administration effect to the rat transient ischemic attack model of the mesenchymal stem cell obtained by culture by various culture methods (body weight). It is a figure which shows the administration effect to the rat transient ischemic attack model of the mesenchymal stem cell obtained by culture by various culture methods (neurosymptom score). It is a figure which shows the administration effect to the rat transient ischemic attack model of the mesenchymal stem cell obtained by culture by various culture methods (the number of steps). It is a figure which shows the administration effect to the rat transient ischemic attack model of the mesenchymal stem cell obtained by culture by various culture methods (tape peeling test). It is a time-lapse image showing the state of cell-cell interaction between nerve cells and mesenchymal stem cells. It is a figure which shows the nerve cell death inhibitory effect of the mesenchymal stem cell obtained by culturing in the serum-free medium (Rohto). It is a figure which shows the dose-dependent nerve cell activation effect of the mesenchymal stem cell obtained by culturing in the serum-free medium (Rohto). It is a figure which shows the nerve cell activation effect of the mesenchymal stem cell obtained by the plane (ADH) or suspension (SUS) culture using serum-free medium (Rohto) (comparison with fibroblast). It is a figure which shows the nerve cell activation effect of the mesenchymal stem cell obtained by culturing in a serum-free medium (Rohto) (comparison with the cell cultured in another medium). It is a cell photograph which shows the nerve cell death inhibitory effect of the mesenchymal stem cell obtained by culturing in a serum-free medium (Rohto) (comparison with the cell cultured in another medium). It is a figure which shows the nerve cell death inhibitory effect of the mesenchymal stem cell obtained by culturing in a serum-free medium (Rohto) (comparison with the cell cultivated in another medium).

Hereinafter, the mesenchymal stem cells and the therapeutic agent for neuropathy of the present invention will be described in detail.

[Mesenchymal stem cells]
The mesenchymal stem cells of the present invention are HGF (hepatocyte growth factor), SHH (sonic hedgehog), OLIG2 (oligodendrocyte transcription factor 2), VEGFA (vascular endothelial growth factor A), NEUROG1 (neurogenin 1), GRPR (gastrin releasing peptide). receptor), IL1R1 (interleukin-1 receptor 1), CRHR2 (corticotropin releasing hormone receptor 2), CCKAR (cholecystokinin A receptor), APOE (apolipoprotein E), PAX3 (paired box 3), PAX5 (paired box 5), EGF ( epidermal growth factor), CXCL1 (CXC motif chemokine ligand 1), GDNF (glial cell derived neurotrophic factor), NRCAM (neuronal cell adhesion molecule), DLL1 (delta like canonical Notch ligand 1), HEYL (hes related family bHLH transcription factor with High expression of any one or more of YRPW motif-like), BMP2 (bone morphogenetic protein 2), NTN1 (netrin 1), ASCL1 (achaete-scute family bHLH transcription factor 1), NRP2 (neuropilin 2) It is characterized by being.

HGF is a type of growth factor and is known to have a neuroprotective effect. SHH is a gene belonging to the hedgehog family and is known to be involved in the protection of nerve cells against oxidative stress. VEGFA is a type of growth factor and is known to be involved in the protection of nerve cells by inducing angiogenesis. IL1R1 is a receptor for IL1, which is a type of inflammatory cytokine, and is known to be involved in the inflammatory response. DLL1 belongs to the DSL family, which is a Notch ligand, and is known to be involved in the regulation of Notch signaling.

The above HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, NRP2 are highly expressed. Being includes that each mRNA expression is highly expressed, each protein is highly expressed, or both are highly expressed.

Further, the mesenchymal stem cell of the present invention may express the above-mentioned factors higher than other cells, but specifically, the mesenchymal stem cell of the present invention may be subjected to conventional culture conditions (for example,). It suffices if the above factors are highly expressed as compared with the mesenchymal stem cells obtained by culturing in MEM-α medium containing 10% FBS). It is preferably expressed twice or more, more preferably five times or more, still more preferably ten times or more, and particularly preferably 100 times or more, as compared with the mesenchymal stem cells obtained under conventional culture conditions.

Compared with fibroblasts, the mesenchymal stem cells of the present invention have HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, Any one or more of HEYL, BMP2, NTN1, ASCL1 and NRP2 may be highly expressed. It is preferably expressed twice or more, more preferably five times or more, still more preferably ten times or more, and particularly preferably 100 times or more with respect to skin fibroblasts.

In the present invention, the mesenchymal stem cell has an ability to differentiate into one or more cells belonging to the mesenchymal system (osteocytes, myocardial cells, chondrocytes, tendon cells, adipocytes, etc.), and maintains the ability. It means a cell that can proliferate. The term mesenchymal stem cell used in the present invention means the same cell as a stromal cell, and does not particularly distinguish between the two. It may also be simply referred to as mesenchymal cells. Tissues containing mesenchymal stem cells include, for example, adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrial membrane, placenta, amniotic membrane, chorion, decidua, dermatitis, skeletal muscle, bone membrane, dental follicle, periodontal ligament, etc. Examples include dental pulp and tooth germ. For example, adipose tissue-derived mesenchymal stem cells mean mesenchymal stem cells contained in adipose tissue, and may be referred to as adipose tissue-derived stromal cells. Of these, adipose tissue-derived mesenchymal stem cells, umbilical band-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and placenta-derived mesenchymal stem cells are used from the viewpoints of effectiveness in treating neuropathy diseases and availability. Stem cells and mesenchymal stem cells derived from dental pulp are preferable, mesenchymal stem cells derived from adipose tissue and mesenchymal stem cells derived from umbilical cord are more preferable, and mesenchymal stem cells derived from umbilical cord are most preferable.

The mesenchymal stem cells in the present invention may be derived from the same species as the subject (subject) to be treated, or may be derived from a different species. Examples of the species of mesenchymal stem cells in the present invention include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, and rats, and preferably cells derived from the same species as the subject (subject) to be treated. .. The mesenchymal stem cells in the present invention may be derived from a subject (subject) to be treated, that is, an autologous cell, or may be derived from another subject of the same species, that is, an allogeneic cell. It is preferably an allogeneic cell.

Since mesenchymal stem cells are less likely to cause rejection of allogeneic subjects, pre-prepared donor cells are expanded and cryopreserved in the mesenchymal system in the disease therapeutic agent of the present invention. It can be used as a stem cell. Therefore, the mesenchymal stem cells in the present invention can be easily commercialized and can obtain a stable and constant effect as compared with the case where self-mesenchymal stem cells are prepared and used. It is more preferable that they are allogeneic.

In the present invention, the mesenchymal stem cell means an arbitrary cell population including the mesenchymal stem cell. The cell population is at least 20% or more, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98. % Or 99% or more are mesenchymal stem cells.

In the present invention, the umbilical cord is a white tubular tissue that connects the foetation and the placenta, and is composed of umbilical veins, umbilical arteries, glue-like tissues (Wharton's Jelly), umbilical cord matrix itself, and many other mesenchymal stem cells. Including. The umbilical cord is preferably obtained from an animal of the same species as the subject (administration target) using the therapeutic agent for the disease of the present invention, and more preferably for humans in consideration of administering the therapeutic agent for the disease of the present invention to humans. Umbilical cord.

In the present invention, the adipose tissue means a tissue containing stromal cells including adipocytes and microvascular cells, and is, for example, a tissue obtained by surgically excising or aspirating subcutaneous fat of a mammal. Adipose tissue can be obtained from subcutaneous fat. It is preferably obtained from an animal of the same species as the adipose tissue-derived mesenchymal stem cell to be administered, which will be described later, and more preferably human subcutaneous fat in consideration of administration to humans. The subcutaneous fat supply individual may be alive or dead, but the adipose tissue used in the present invention is preferably a tissue collected from a surviving individual. When collected from an individual, liposuction includes, for example, PAL (power assist) liposuction, erconia laser liposuction, body jet liposuction, etc., and ultrasonic waves from the viewpoint of maintaining the state of cells. It is preferable not to use.

In the present invention, the bone marrow refers to parenchyma that fills the lumen of bone and is a hematopoietic organ. Bone marrow fluid exists in the bone marrow, and the cells existing in it are called bone marrow cells. Bone marrow cells include erythrocytes, granulocytes, megakaryocytes, lymphocytes, adipocytes and the like, as well as mesenchymal stem cells, hematopoietic stem cells, vascular endothelial progenitor cells and the like. Bone marrow cells can be harvested from, for example, human ilium, long bone, or other bone.

In the present invention, each tissue-derived mesenchymal stem cell such as adipose tissue-derived mesenchymal stem cell, umbilical band-derived mesenchymal stem cell, and bone marrow-derived mesenchymal stem cell refers to adipose tissue-derived mesenchymal stem cell and umbilical band-derived mesenchymal stem cell, respectively. It means an arbitrary cell population including mesenchymal stem cells derived from each tissue such as stem cells and mesenchymal stem cells derived from bone marrow. The cell population is at least 20% or more, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98. % Or 99% or more are adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells.

The mesenchymal stem cells in the present invention are HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1. , In addition to high expression of any one or more of NRP2, for example, growth characteristics (eg, population doubling ability from passage to aging, doubling time), nuclear type analysis (eg, normal) Nuclear type, maternal or neonatal lineage, surface marker expression by flow cytometry (eg, FACS analysis), immunohistochemistry and / or immunocytokine (eg, epitope detection), gene expression profiling (eg, gene chip array; Reverse transcription PCR, real-time PCR, polymerase chain reaction such as conventional PCR), miRNA expression profiling, protein array, protein secretion such as cytokine (eg, plasma coagulation analysis, ELISA, cytokine array), metabolite (metabolome analysis), book It may be characterized by other methods known in the art and the like.

(Method of preparing mesenchymal stem cells)
HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, NRP2, or The method for preparing mesenchymal stem cells in which two or more are highly expressed is not particularly limited, but can be prepared, for example, as follows. That is, mesenchymal stem cells are separated and cultured from tissues such as fat, umbilical cord, and bone marrow according to a method known to those skilled in the art, and HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, using an antibody that specifically binds to PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or NRP2. It can be obtained by separating CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or NRP2 high-expressing cells with a cell sorter, magnetic beads, or the like. In addition, HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, in mesenchymal stem cells by culturing using a specific medium. By inducing the expression of HEYL, BMP2, NTN1, ASCL1 or NRP2, HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1 , HEYL, BMP2, NTN1, ASCL1 or NRP2 high expression mesenchymal stem cells can also be obtained. In the cell population obtained by this induction, more than 50% of the cell population is HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, High expression of HEYL, BMP2, NTN1, ASCL1 or NRP2 is preferred, with 70% or more HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, High expression of GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or NRP2 is more preferable, and 80% or more are HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3. , PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or NRP2 are more preferably highly expressed, with 90% or more being HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, High expression of CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or NRP2 is particularly preferred, substantially HGF, SHH, OLIG2, VEGFA, Most preferably, it is a homogeneous cell population with high expression of NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or NRP2. Below, between HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or NRP2 high expression The method for preparing leaf stem cells will be specifically described.

Mesenchymal stem cells can be prepared by methods well known to those skilled in the art. Below, as an example, a method for preparing mesenchymal stem cells derived from umbilical cord tissue and mesenchymal stem cells derived from adipose tissue will be described.

The umbilical cord can be recovered by appropriately removing the placenta from the placenta delivered by vaginal delivery and cesarean section and the puerperal tissue including the umbilical cord. After removing the umbilical cord blood from the collected umbilical cord, sterile or bacteriostatic treatment may be performed. Cord blood removal is performed by rinsing with an anticoagulant solution such as a heparin-containing solution. Aseptic or bacteriostatic treatment is not particularly limited, but is a medium supplemented with povidone iodine or one or more antibiotics and / or antifungal agents such as penicillin, streptomycin, amphotericin B, gentamicin, and nystatin. Alternatively, it may be immersed in a buffer. Further, if necessary, a step of selectively lysing red blood cells may be included. As a method for selectively lysing erythrocytes, a method well known in the art can be used, for example, incubation in a hypertonic medium or a hypotonic medium by lysis with ammonium chloride.

The umbilical cord-derived cell of the present invention means a cell population prepared using the umbilical cord as a raw material, and may be obtained by a known production method. For example, the cell is produced by a method including the following steps (i) to (iii). can do:
(I) Step of cutting the umbilical cord;
(Ii) The step of culturing the umbilical cord obtained in step (i); and (iii) the step of subculturing.

Further, as another method for preparing the cells, instead of (i) the step of cutting the umbilical cord, (i') a step of dissociating the tissue by enzymatically treating the umbilical cord may be included. Further, in addition to the step of (i) cutting the umbilical cord, a step of (i') dissociating the tissue by enzymatically treating the umbilical cord may be included.

In the step (i) of the present invention for cutting the umbilical cord, the umbilical cord obtained by the above method is cut by a mechanical force (shred force or shear force) including the amniotic membrane, blood vessels, perivascular tissue and Walton jelly. Can be done by Although not particularly limited, the umbilical cord section obtained by cutting is exemplified in the size of ^{1 to 10 mm 3} , 1 to 5 mm ³ , 1 to 4 mm ³ , 1 to 3 mm ³ or 1 to 2 mm ^3. In the step of dissociating the tissue by enzymatically treating the (i') umbilical cord of the present invention, the umbilical cord obtained by the above method is enzymatically treated with the amniotic membrane, blood vessels, perivascular tissue and Walton jelly. This can be done in the step of dissociating the tissue. The enzyme treatment is not particularly limited, and examples thereof include enzyme treatment using one or more kinds of enzymes such as collagenase, dispase and hyaluronidase.

In the step of culturing the umbilical cord obtained in the steps (ii) and (i) of the present invention, the umbilical cord obtained in the step (i) is used as an appropriate cell on a solid surface using an appropriate cell medium. Incubate under density and culture conditions.

The medium used in this step is not particularly limited as long as it can culture mesenchymal stem cells, but such a medium is obtained by adding serum to the basal medium and / or albumin, transferrin, fatty acid, insulin. , Sodium selenate, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol and the like may be added with one or more serum substitutes. If necessary, substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts are added to these media. You may.

Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, MEM-α medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and MCDB201. Examples thereof include a medium and a mixed medium thereof.

Examples of the serum include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, pig serum, sheep serum, rabbit serum, rat serum and the like. .. When serum is used, 5 v / v% to 15 v / v%, preferably 10 v / v% may be added to the basal medium.

Examples of the fatty acid include, but are not limited to, linoleic acid, oleic acid, linoleic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, stearic acid and the like. Examples of the lipid include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Amino acids include, but are not limited to, for example, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine and the like. .. Examples of the protein include, but are not limited to, ecotin, reduced glutathione, fibronectin, β2-microglobulin and the like. Examples of the polysaccharide include glycosaminoglycans, and among glycosaminoglycans, hyaluronic acid, heparan sulfate and the like are exemplified, but the polysaccharides are not limited thereto. Growth factors include, for example, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocellular growth factor (HGF), epithelial growth factor (EGF). , Binding tissue growth factor (CTGF), vascular endothelial cell growth factor (VEGF) and the like, but are not limited thereto. From the viewpoint of using the umbilical cord-derived mesenchymal stem cells obtained in the present invention for cell transplantation, it is preferable to use a medium containing no heterologous components such as serum (Zenofree). Such a medium is provided as a medium prepared in advance for mesenchymal stem cells (stromal cells) from, for example, PromoCell, Lonza, Biological Industries, Veritas, R & D Systems, Corning, Rohto and the like. Has been done.

In the present invention, the "solid surface" means any material that enables the binding and adhesion of adipose tissue-derived mesenchymal stem cells in the present invention. In certain embodiments, such a material is a plastic material that has been treated to facilitate the binding and adhesion of mammalian cells to its surface. The shape of the culture vessel having a solid surface is not particularly limited, but a petri dish, a flask, or the like is preferably used. The cells are washed after incubation to remove unbound cells and cell debris.

In the present invention, cells that finally remain bound and adhered to the solid surface can be selected as a cell population of umbilical cord tissue-derived mesenchymal stem cells.

The mesenchymal stem cells derived from the umbilical cord tissue of the present invention can also be produced by using the suspension culture production method. As a suspension culture production method, there are a method of aggregating cells and stirring and culturing them as a cell mass on a sphere, a method of adhering cells on microcarriers and culturing by stirring the microcarriers, and the like. For stirring, there are a method of rotating a stirring blade in a container with a stirrer, a method of placing a bag containing a culture solution and cells on a shaker, and shaking the bag together to suspend the culture solution. The medium used in the suspension culture production method is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells, and the above-mentioned medium is exemplified. The microcarrier is not particularly limited as long as it can be used in suspension culture, and examples thereof include polyester, polystyrene, glass, and dextran.

The adipose tissue-derived mesenchymal stem cells may be obtained, for example, by the production method described in US Pat. No. 6,777,231, and can be produced, for example, by a method including the following steps (i) to (iii). it can:
(I) Step of obtaining cell suspension by enzymatic digestion of adipose tissue;
(Ii) The steps of precipitating cells and resuspending the cells in a suitable medium; and (iii) culturing the cells on a solid surface and removing cells that do not show binding to the solid surface.

As the adipose tissue used in the step (i), it is preferable to use a washed adipose tissue. Washing can be performed by vigorous stirring and precipitation with a physiologically compatible aqueous saline solution (eg, phosphate buffered saline (PBS)). This is to remove impurities (also called debris, for example, damaged tissue, blood, red blood cells, etc.) contained in adipose tissue from the tissue. Therefore, washing and sedimentation are generally repeated until debris is totally removed from the supernatant. Since the remaining cells exist as clumps of various sizes, enzymes that weaken or destroy intercellular connections in the washed cell clumps (eg, for example) in order to dissociate the cells while minimizing damage to the cells themselves. Treatment with collagenase, dispase, trypsin, etc.) is preferred. The amount and duration of such enzymes will vary depending on the conditions used and are known in the art. The cell mass can be degraded by other treatment methods such as mechanical agitation, ultrasonic energy, thermal energy, etc., in place of or in combination with such enzymatic treatment, but with minimal cell damage. In order to suppress it, it is preferable to carry out only the enzyme treatment. When an enzyme is used, it is desirable to inactivate the enzyme using a medium or the like after an appropriate period in order to minimize harmful effects on cells.

The cell suspension obtained in step (i) includes a slurry or suspension of aggregated cells and various contaminant cells such as erythrocytes, smooth muscle cells, endothelial cells, and fibroblasts. Therefore, the aggregated cells and these contaminating cells may be subsequently separated and removed, but the separation and removal are omitted because they can be removed by adhesion and washing in the step (iii) described later. You may. Separation and removal of contaminating cells can be achieved by centrifugation, which forces the cells into a supernatant and a precipitate. The resulting precipitate containing contaminating cells is suspended in a physiologically compatible solvent. Suspended cells may contain erythrocytes, but the lysis step is not always necessary because erythrocytes are excluded by selection by adhesion to the solid surface, which will be described later. As a method for selectively lysing erythrocytes, a method well known in the art can be used, for example, incubation in a hypertonic medium or a hypotonic medium by lysis with ammonium chloride. After lysis, the lysate may be separated from the desired cells, for example by filtration, centrifugation, or density fractionation.

In step (ii), the suspended cells may be washed once or multiple times in succession, centrifuged, and resuspended in the medium in order to increase the purity of the mesenchymal stem cells. Alternatively, cells may be separated based on cell surface marker profiles or based on cell size and granularity.

The medium used for resuspension is not particularly limited as long as it can culture mesenchymal stem cells, but such a medium is a basal medium to which serum is added and / or albumin, transferrin, fatty acid, etc. It may be prepared by adding one or more serum substitutes such as insulin, sodium selenate, cholesterol, collagen precursor, trace element, 2-mercaptoethanol, 3'-thiolglycerol and the like. If necessary, substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts are added to these media. You may.

Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, MEM-α medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and MCDB201. Examples thereof include a medium and a mixed medium thereof.

Examples of the serum include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, pig serum, sheep serum, rabbit serum, rat serum and the like. .. When serum is used, 5 v / v% to 15 v / v%, preferably 10 v / v% may be added to the basal medium.

Examples of the fatty acid include, but are not limited to, linoleic acid, oleic acid, linoleic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, stearic acid and the like. Examples of the lipid include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Amino acids include, but are not limited to, for example, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine and the like. Examples of the protein include, but are not limited to, ecotin, reduced glutathione, fibronectin, β2-microglobulin and the like. Examples of the polysaccharide include glycosaminoglycans, and among glycosaminoglycans, hyaluronic acid, heparan sulfate and the like are exemplified, but the polysaccharides are not limited thereto. Growth factors include, for example, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocellular growth factor (HGF), epithelial growth factor (EGF). , Binding tissue growth factor (CTGF), vascular endothelial cell growth factor (VEGF) and the like, but are not limited thereto. From the viewpoint of using the adipose-derived mesenchymal stem cells obtained in the present invention for cell transplantation, it is preferable to use a medium containing no heterologous components such as serum (Zenofree). Such a medium is provided as a medium prepared in advance for mesenchymal stem cells (stromal cells) from, for example, PromoCell, Lonza, Biological Industries, Veritas, R & D Systems, Corning, Rohto and the like. Has been done.

Subsequently, in step (iii), on a solid surface without differentiating the cells in the cell suspension obtained in step (ii), using the appropriate cell medium described above, the appropriate cell density and Incubate under culture conditions. In the present invention, the "solid surface" means any material that enables the binding and adhesion of adipose tissue-derived mesenchymal stem cells in the present invention. In certain embodiments, such a material is a plastic material that has been treated to facilitate the binding and adhesion of mammalian cells to its surface. The shape of the culture vessel having a solid surface is not particularly limited, but a petri dish, a flask, or the like is preferably used. The cells are washed after incubation to remove unbound cells and cell debris.

In the present invention, cells that finally remain bound and adhered to the solid surface can be selected as a cell population of adipose tissue-derived mesenchymal stem cells.

In order to confirm that the selected cells are mesenchymal stem cells in the present invention, the surface antigen may be analyzed by a conventional method using flow cytometry or the like. In addition, the ability to differentiate into each cell lineage may be tested, and such differentiation can be performed by conventional methods.

The mesenchymal stem cells in the present invention can be prepared as described above, but may be defined as cells having the following characteristics;
(1) Shows adhesiveness to plastic under culture conditions in standard medium.
(2) Surface antigens CD73 and CD90 are positive, CD45 is negative, and (3) can be differentiated into osteoocytes, adipocytes, and chondrocytes under culture conditions.

From the mesenchymal stem cells obtained by the above steps, HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, By selectively separating cells highly expressing NTN1, ASCL1 or NRP2 protein by immunological methods using cell sorters, magnetic beads, etc., HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1 , CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or mesenchymal stem cells highly expressing NRP2 protein can be obtained. Identification that can induce the expression of HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or NRP2 HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, Induces BMP2, NTN1, ASCL1 or NRP2 expression and efficiently HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL , BMP2, NTN1, ASCL1 or NRP2 high expression mesenchymal stem cells can also be obtained. As an example, a specific method of selective separation by an immunological method using a cell sorter will be described below.

The mesenchymal stem cells prepared above are treated with a trypsin / EDTA solution or the like, and the cell suspension obtained is centrifuged (room temperature, 400 G, 5 minutes) to remove the supernatant. Staining Buffer (1% BSA-PBS) was added to the ^{cells to prepare 1 × 10 6} cells / 500 μL, and the cell suspension concentration was made uniform by pipetting, and then 50 μL was placed in a new 1.5 mL microtube. Dispense one by one. A primary antibody (Mouse anti-human TFPI, manufactured by Sekisui diagnostics, ADG4903) was added to the dispensed cell suspension at a concentration of 5 to 20 μg / mL, suspended, and then kept in the dark and refrigerated for 30 minutes to 1 hour. React. After washing 3 times with 1 mL of Staining Buffer, add Staining Buffer to make 50 μL, add a secondary antibody (Anti Mouse IgG alexar488, Thermofisher scientific, A21202) at a concentration of 1 to 10 μg / mL and suspend. React for 30 minutes to 1 hour under shading and refrigeration. After washing 3 times with 1 mL of Staining Buffer, add 300 μL of PI Buffer (prepared by adding 28.8 μL of Propidium iodide solution (SIGMA, P4864) to 14.4 mL of Staining buffer), suspend well, and suspend well. Separation can be performed by fluorescence activated cell sorting (FACS) through a tube.

(Cyropreservation of mesenchymal stem cells)
The mesenchymal stem cells in the present invention may be cells that have been cryopreserved and thawed as appropriate as long as they have a disease therapeutic effect. In the present invention, cryopreservation can be performed by suspending mesenchymal stem cells in a cryopreservation solution well known to those skilled in the art and cooling the cells. Suspension can be carried out by, if necessary, exfoliating the cells with a release agent such as trypsin, transferring to a cryopreservation container, appropriately treating the cells, and then adding a cryopreservation solution.

The cryopreservation solution may contain DMSO (Dimethyl sulfoxide) as a frost damage protective agent, but DMSO has a property of inducing differentiation of mesenchymal stem cells in addition to cytotoxicity, and therefore has a DMSO content. It is preferable to reduce. Alternatives to DMSO are exemplified by glycerol, propylene glycol or polysaccharides. When DMSO is used, it contains a concentration of 5% to 20%, preferably a concentration of 5% to 10%, more preferably a concentration of 10%. In addition to this, the additives described in WO2007 / 058308 may be included. As such cryopreservation liquids, for example, cryopreservation provided by Bioverde, Japan Genetics Co., Ltd., Reprocell, Xenoac, Cosmo Bio, Kojin Bio Co., Ltd., Thermo Fisher Scientific Co., Ltd., etc. A liquid may be used.

When the above-mentioned suspended cells are cryopreserved, they may be frozen at a temperature between −80 ° C. and −100 ° C. (for example, −80 ° C.), using an arbitrary freezer capable of achieving the temperature. obtain. Although not particularly limited, the cooling rate may be appropriately controlled by using a program freezer in order to avoid a sudden temperature change. The cooling rate may be appropriately selected depending on the components of the cryopreservation solution, and may be performed according to the manufacturer's instructions of the cryopreservation solution.

The storage period is not particularly limited as long as the cells cryopreserved under the above conditions retain the same properties as before freezing after thawing, but for example, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 1 year or more, or more. Since cell damage can be suppressed by storing at a lower temperature, the cells may be transferred to a gas phase (from about −150 ° C. or lower to −180 ° C. or higher) on liquid nitrogen for storage. When storing in the gas phase on liquid nitrogen, it can be carried out using a storage container well known to those skilled in the art. Although not particularly limited, for example, when storing for 2 weeks or more, it is preferable to store in a gas phase on liquid nitrogen.

The thawed mesenchymal stem cells may be appropriately cultured before the next cryopreservation. The culture of the mesenchymal stem cells is carried out using the medium capable of culturing the above-mentioned mesenchymal stem cells, and is not particularly limited, but at a culture temperature of about 30 to 40 ° C., preferably about 37 ° C., in CO _2- containing air. It may be done in an atmosphere. The CO ₂ concentration is about 2-10%, preferably about 5-10%. In culturing, cells are detached with a release agent such as trypsin after reaching the appropriate confluency for the culture vessel (eg, 50% to 80% of the culture vessel is occupied by cells). Then, the cells may be seeded in a separately prepared culture vessel at an appropriate cell density to continue the culture. When seeding cells, typical cell densities are 100 cells / cm ^{2 to} 100,000 cells / cm ² , 500 cells / cm ^{2 to} 50,000 cells / cm ² , 1,000 to 10,000 cells. Examples include / cm ² , 2,000 to 10,000 cells / cm ² . In certain embodiments, the cell density is 2,000 to 10,000 cells / cm ² . It is preferable to adjust the time to reach the appropriate confluency from 3 days to 7 days. During culturing, the medium may be changed as needed.

Thawing of cryopreserved cells can be performed by methods well known to those skilled in the art. For example, a method performed by standing or shaking in a constant temperature bath at 37 ° C. or in a hot water bath is exemplified.

The mesenchymal stem cell of the present invention may be a cell in any state, for example, a cell recovered by exfoliating a cell in culture, or a cell frozen in a cryopreservation solution. Good. It is preferable to use cells obtained by expanding and culturing the same lot of cells in small portions and cryopreserving them, in that the same action and effect can be stably obtained and the handling property is excellent. The mesenchymal stem cells in the cryopreserved state may be thawed immediately before use and may be directly mixed with a solution such as an infusion solution or a medium while being suspended in the cryopreserved solution. Alternatively, the cryopreservation solution may be removed by a method such as centrifugation and then suspended in a solution such as an infusion solution or a medium. Here, the "infusion solution" in the present invention refers to a solution used in the treatment of humans, and is not particularly limited, but for example, a physiological saline solution, a Japanese Pharmacopoeia physiological saline solution, a 5% glucose solution, and a Japanese Pharmacopoeia. Glucose injection, Ringer's solution, Japanese Pharmacopoeia Ringer's solution, Lactobacillus Ringer's solution, Ringer's acetate solution, No. 1 (starting solution), No. 2 (dehydration replacement fluid), No. 3 (maintenance solution), No. 4 (postoperative recovery solution) And so on.

[Treatment for neuropathy]
The therapeutic agent for neurological disorders of the present invention is the above-mentioned HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL. , BMP2, NTN1, ASCL1, NRP2, contains mesenchymal stem cells that highly express any one or more. According to the neuropathy therapeutic agent of the present invention, neuropathy can be effectively protected. The above description of the section on mesenchymal stem cells can be applied to the mesenchymal stem cells containing the therapeutic agent for neuropathy of the present invention.

In addition to the above-mentioned mesenchymal stem cells, the therapeutic agent for neuropathy of the present invention can be added with a pharmaceutically acceptable carrier or addition according to a conventional method according to its use and morphology, as long as the effects of the present invention are not impaired. It may contain an object. Examples of such carriers and additives include tonicity agents, thickeners, sugars, sugar alcohols, preservatives (preservatives), bactericides or antibacterial agents, pH regulators, stabilizers, and chelating agents. , Oil-based bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizers, dispersants, emulsifiers, buffers, solubilizers , Antioxidants, sweeteners, acidity agents, colorants, flavoring agents, fragrances, refreshing agents and the like, but are not limited thereto. Typical components include, for example, the following carriers and additives.

As the carrier, for example, an aqueous carrier such as water or hydrous ethanol; as the tonicity agent (inorganic salt), for example, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc .; as the polyhydric alcohol, for example. , Glycerin, propylene glycol, polyethylene glycol, etc .; Examples of thickeners include carboxyvinyl polymer, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, argic acid, polyvinyl alcohol (completely or partially saponified), polyvinylpyrrolidone, macrogol. Etc.; as sugars, for example, cyclodextrin, glucose, etc .; as sugar alcohols, for example, xylitol, sorbitol, mannitol, etc. (these may be d-form, l-form, or dl-form); preservatives. Examples of the bactericidal agent or antibacterial agent include dibutylhydroxytoluene, butylhydroxyanisole, alkyldiaminoethylglycine hydrochloride, sodium benzoate, ethanol, benzalconium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, sorbic acid, and sorbin. Potassium acid, tromethamole, sodium dehydroacetate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, oxyquinoline sulfate, phenethyl alcohol, benzyl alcohol, biguanide compounds (specifically, polyhexanide hydrochloride (specifically, polyhexanide hydrochloride (specifically) Polyhexamethylene biguanide), etc.), Gloquil (trade name manufactured by Rhodia), etc .; Examples of pH adjusters include hydrochloric acid, boric acid, aminoethylsulfonic acid, epsilon-aminocaproic acid, citric acid, acetic acid, sodium hydroxide. , Potassium hydroxide, Calcium hydroxide, Magnesium hydroxide, Sodium hydrogen carbonate, Sodium carbonate, Boso, Triethanolamine, Monoethanolamine, Diisopropanolamine, Sulfate, Magnesium sulfate, Phosphoric acid, Polyphosphate, Propionic acid, Shu Acids, gluconic acid, fumaric acid, lactic acid, tartaric acid, malic acid, succinic acid, gluconolactone, ammonium acetate, etc .; Stabilizers include, for example, dibutylhydroxytoluene, tromethamole, sodium formaldehyde sulfoxylate (longalit), Tocopherol, sodium pyrosulfate, monoethanolamine, aluminum monostearate, glycerin monostearate, sub Sodium hydrogensulfate, sodium sulfite, etc .; oil-based bases include, for example, olive oil, corn oil, soybean oil, sesame oil, cottonseed oil and other vegetable oils, medium-chain fatty acid triglycerides, etc .; as aqueous bases, for example, Macrogol 400. Etc .; as gel base, for example, carboxyvinyl polymer, gum quality, etc .; as surfactant, for example, polysorbate 80, hardened castor oil, glycerin fatty acid ester, sorbitan sesquioleate, etc .; Examples include sala shimitsuro, various surfactants, arabic rubber, arabic rubber powder, xanthan gum, soybean lecithin, and the like; as binders, for example, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, polyvinyl. Pyrrolidone, polyvinyl alcohol, etc .; as excipients, for example, sucrose, lactose, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, etc .; as lubricants, for example, sucrose fatty acid ester, magnesium stearate, etc. , Tarc, etc .; as disintegrants, for example, low-substituted hydroxypropyl cellulose, crospovidone, croscarmellose sodium, etc .; as effervescent agents, for example, sodium hydrogen carbonate, etc .; as fluidizers, for example, Examples thereof include sodium aluminometasilicate and light anhydrous silicic acid.

The therapeutic agent for neuropathy of the present invention can be provided in various forms, for example, various dosage forms such as solid preparations, semi-solid preparations, and liquid preparations, depending on the intended purpose. For example, solids (tablets, powders, powders, granules, capsules, etc.), semi-solids [ointments (hard ointments, ointments, etc.), creams, etc.], liquids [lotions, extracts, suspensions, etc.] Turbids, emulsions, syrups, injections (including infusions, implantable injections, continuous injections, time-prepared injections), dialysis agents, aerosols, soft capsules, drinks, etc.], patch It can be used in the form of an agent, a poultice, or the like. The neuropathy therapeutic agent of the present invention can also be used in the form of a solution or emulsion in an oily or aqueous vehicle. Furthermore, the therapeutic agent for neuropathy of the present invention can be applied to the affected area by spraying, and the therapeutic agent for neuropathy of the present invention can also be used in the form of gelling or sheeting in the affected area after spraying. The therapeutic agent for neuropathy of the present invention can also be applied to the affected area after forming the mesenchymal stem cells into a sheet or three-dimensional structure.

The neuropathy therapeutic agent of the present invention includes physiological saline, Japanese Pharmacopoeia physiological saline, 5% glucose solution, Japanese Pharmacopoeia glucose injection, Ringer's solution, Japanese Pharmacopoeia Ringer's solution, Lactobacillus ringer's solution, Ringer's acetate solution, Ringer's carbonate solution No. 1 ( Suspension or dilution using infusion fluids such as starting fluid), fluid 2 (dehydration replacement fluid), fluid 3 (maintenance fluid), fluid 4 (postoperative recovery fluid), or cell culture medium such as DMEM. It can be used by suspending or diluting with physiological saline solution, 5% glucose solution, No. 1 solution (starting solution), and more preferably 5% glucose solution, No. 1 solution (starting solution). be able to.

When the therapeutic agent for neuropathy of the present invention is a liquid preparation, the pH of the therapeutic agent for neuropathy is not particularly limited as long as it is within a pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable range. However, as an example, a range of 2.5 to 9.0, preferably 3.0 to 8.5, and more preferably 3.5 to 8.0 can be mentioned.

When the therapeutic agent for neuropathy of the present invention is a liquid preparation, the osmotic pressure of the therapeutic agent for neuropathy is not particularly limited as long as it is within the range acceptable to the living body. As an example of the osmotic pressure ratio of the composition of the present invention, a range of preferably 0.7 to 5.0, more preferably 0.8 to 3.0, and further preferably 0.9 to 1.4 can be mentioned. The osmotic pressure can be adjusted by a method known in the art using inorganic salts, polyhydric alcohols, sugar alcohols, sugars and the like. The osmotic pressure ratio is the ratio of the osmotic pressure of the sample to the osmotic pressure of 286 mOsm (0.9 w / v% sodium chloride aqueous solution) based on the 15th revised Japanese Pharmacy, and the osmotic pressure is the osmotic pressure measurement method described in the Japanese Pharmacy. Measure with reference to the freezing point descent method). The standard solution for measuring the osmotic pressure ratio (0.9 w / v% sodium chloride aqueous solution) is prepared by drying sodium chloride (standard reagent of the Japanese Pharmacopoeia) at 500 to 650 ° C. for 40 to 50 minutes and then in a desiccator (silica). Allow to cool, weigh accurately 0.900 g, dissolve in purified water to prepare exactly 100 mL, or use a commercially available standard solution for measuring osmotic pressure ratio (0.9 w / v% sodium chloride aqueous solution).

The routes of administration of the therapeutic agent for neuropathy of the present invention to a subject include oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intraventricular administration, intrathecal administration, intraperitoneal administration, and sublingual administration. Examples thereof include transrectal administration, transvaginal administration, intraocular administration, nasal administration, inhalation, transdermal administration, implants, spraying on the surface of organs, and direct administration by attaching a sheet or the like. From the viewpoint of efficacy, it is preferably intraarterial administration, intravenous administration, intraventricular administration and intrathecal administration, and from the viewpoint of reducing the burden on the subject, it is more preferably intravenous administration, intramuscular administration, and nasal administration. It is an internal administration, and from the viewpoint of effect, intraventricular administration and intramuscular administration are preferable.

The dose (dose) of the therapeutic agent for neuropathy of the present invention may vary depending on the patient's condition (weight, age, symptom, physical condition, etc.), the dosage form of the therapeutic agent for neuropathy of the present invention, etc., but is sufficient. From the viewpoint of exerting the therapeutic effect of the therapeutic agent for transdisorder, a large amount tends to be preferable, while a small amount tends to be preferable from the viewpoint of suppressing the occurrence of side effects. Usually, when administered to an adult, the number of cells is 1 × 10 ³ to 1 × 10 ¹² cells / time, preferably 1 × 10 ⁴ to 1 × 10 ¹¹ cells / time, more preferably 1 × 10 ⁵ to 1 × 10 ¹⁰ pieces / time, more preferably 5 × 10 ⁶ to 1 × 10 ⁹ pieces / time. The dose per patient's body weight is 1 x 10 to 5 x 10 ¹⁰ pieces / kg, preferably 1 x 10 ^{2 to} 5 x 10 ⁹ pieces / kg, and more preferably 1 x 10 ^{3 to} 5 x 10. ⁸ pieces / kg, more preferably 1 × 10 ^{4 to} 5 × 10 ⁷ pieces / kg. When administered to a newborn baby, the number of cells is 1 × 10 ³ to 1 × 10 ¹¹ cells / time, preferably 1 × 10 ⁴ to 1 × 10 ¹⁰ cells / time, more preferably 1 × 10 ⁵ to 1 ×. 10 ⁹ pieces / time, more preferably 5 × 10 ^{5 to} 5 × 10 ⁸ pieces / time. The dose per patient's body weight is 1 x 10 to 5 x 10 ¹⁰ pieces / kg, preferably 1 x 10 ^{2 to} 5 x 10 ⁹ pieces / kg, and more preferably 1 x 10 ^{3 to} 5 x 10. ⁸ pieces / kg, more preferably 1 × 10 ^{4 to} 5 × 10 ⁷ pieces / kg. In addition, this dose may be administered as a single dose in a plurality of times, or this dose may be administered in a plurality of times.

The therapeutic agent for neuropathy of the present invention may be administered together with one or more other agents. Other agents include any agent that can be used as a therapeutic agent for neuropathy, such as anti-Parkinson's disease agents such as levodopa, amantadine, carvidopa, and dopaminergic agents such as bromocryptin, purgoride, ropinilol, pramipexol. Drugs, selective B-type monoamine oxidase inhibitors (MAO-B) such as selegiline and lasalidine, catechol-O-methyltransferase (COMT) inhibitors such as enteracapon and tolucapon, and anticholinergic agents such as benztropon and trihexyphenidyl. Antihistamines such as diphenhydramine and orphenadrine, cholinesterase inhibitors such as donepezil, rivastigmin, galantamine and taclin, N-methyl-D-aspartate receptor antagonists such as memantin, haloperidol, thioridine, thiothixene, olanzapine, lisperidone, Antipsychotics such as quetiapine and clozapine, β-blockers such as propranolol, sedatives such as benzodiazepine, anticoagulants such as heparin, low molecular weight heparin, and warfarin, anticonvulsants such as carbamatepine, gabapentin, phenitoin, pregavalin, valprosan, and lamotridine. Drugs, tricyclics, antidepressants such as benrafaxin, bupropion, amitriptilin, desipramine, paroxetine, central α-2 adrenergic agonists such as chronidine and tizanidine, corticosteroids such as dexamethasone and prednisolone, amantadine and dextri NMDA receptor antagonists such as metrophans, local anesthetics such as codeine, mexiretin, capsaicin, etodrac, indomethasine, thrin consent, tolmetatin, nabmeton, pyroxicum, acetaminophen, phenobipron, flurubipron, ibuprofen, ketoprofen, naproxene, naproxene sodium , Oxaprosin, aspirin, cholinemagnesium trisalicylic acid, diflunisal, meclophenamate, mephenamic acid, phenylbutazone, ketrolac, selecoxib, codeine, hydrocodeine, propoxyphene, fentanyl, hydromorphone, levofanol, meperidine, mesadone, morphine, oxycodon, oxymorphone , Buprenorfin, butorphanol, nalbufin, pentazocin and other analgesics, propofol and other radical scavengers, anti-inflammatory agents, serotonin, norepinephrine, NSAID, ginkgo leaf extract and the like. In addition to the administration of the neuropathy therapeutic agent of the present invention, hypothermia therapy such as hypothermia therapy, cerebral hypothermia therapy, and cerebral hypothermia therapy can be combined.

The mesenchymal stem cells of the present invention can be used for various neuropathy, and specific diseases include autonomic neuropathy, Hornel syndrome, multisystem ataxia, autonomic neuropathy such as pure autonomic neuropathy, and chronic pain. , Neuropathic pain, pain such as complex local pain syndrome, ischemic stroke, transient cerebral ischemic attack, hypoxia-ischemia, intracranial bleeding such as intracerebral bleeding / intraventricular bleeding, subepithelial bleeding, etc. Stroke (cerebellar vascular accident), Alzheimer's disease, cerebrovascular dementia, Levi body dementia, HIV-related dementia, dementia such as frontotemporal dementia, spasmodic syndrome, atetose or dyskinesia syndrome and ataxia syndrome Cerebral paralysis syndrome, hypoglycemia, hypernatremia, hyponatremia, hypomagnesemia, congenital metabolic disorders, etc., neonatal spasmodic disease, demyelinating disease such as multiple sclerosis, Kiran-Valley syndrome, inheritance Sexual neuropathy, motor neuron disease including muscular atrophic lateral sclerosis (ALS), severe myasthenia, mononeuropathy, multiple neuropathy, peripheral nervous system disorders such as neuropathy, acute transverse myelitis, arteriovenous malformations, Examples thereof include spinal disorders such as spinal cord infarction (ischemic spinal disorder), cerebral diseases such as spinocerebellar ataxia and spinocerebellar degeneration, brain tumors, encephalitis, meningitis, Parkinson's disease and the like. It can also be used for perinatal encephalopathy, neonatal encephalopathy, cerebral palsy, etc. for newborns. Of these, ischemic stroke, transient cerebral ischemic attack, hypoxia-ischemia, intracranial hemorrhage such as intracerebral hemorrhage / intraventricular hemorrhage, stroke (cerebrovascular accident) such as subarachnoid hemorrhage, and perinatal brain Disorders, neonatal encephalopathy, cerebral palsy and the like are preferred.

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples, but the present invention is not limited to these Examples and the like.

[Preparation and culture of umbilical cord-derived mesenchymal stem cells]
Umbilical cord-derived cells were collected by the method described in Cytotherapy, 18, 229-241, 2016. Briefly, with the approval of the Institutional Review Board of the Institute of Medical Sciences, the University of Tokyo, the umbilical cord collected with the consent of the donor ^{is cut into 1 to 2 mm 3} pieces and seeded on a culture dish. Then, cover with cell amigo (Tsubakimoto Chain Co., Ltd.) and incubate in α-minimal essential medium (MEM-α) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Leaf stem cells (hereinafter referred to as "UCMSC") were obtained. In addition, the cells obtained from the umbilical cord tissue by the above-mentioned improved explant method are designated as the first passage cells (P1), and the number of passages increases by performing the passage, and the cells are described as the second passage cells (P2). To do.

The obtained UCMSC was exfoliated using trypsin (TrypLE Select (1X)), transferred to a centrifuge tube, and centrifuged at 400 × g for 5 minutes to obtain a cell precipitate. After removing the supernatant, an appropriate amount of cell cryopreservation solution (STEM-CELLBANKER) was added and suspended. The cell suspension solution was dispensed into cryotubes and stored in a freezer at −80 ° C. After that, it was transferred to the gas phase on liquid nitrogen and storage was continued.

[MRNA expression]
Supplement mix of UCMSC from P2 to P4 to serum-free medium for mesenchymal stem cells (Rohto, serum-free medium) and PromoCell medium (Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009, Lot.435M415), respectively. (Promot Cell, C-39809, Lot.435M126) was added) and MEM-α medium (Thermo Fisher, # 12571-063, Lot.18997009 was added with serum to 10%), and frozen. Stock was made. Fibroblasts were cultured in MEM-α medium, and then frozen stock was prepared. Each P4 cell and fibroblast were put to sleep ^{, seeded on a 6-well plate at 15,000 cells / cm 2} , and cultured in each of the three media for 1 day, and then Total RNA was collected. The mRNA expressions of HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR and APOE were detected by quantitative PCR, respectively.

It was found that the mRNA expression of HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE was significantly higher in the cells cultured in the serum-free medium than in the MEM-α medium. It was also found that the cells cultured in PromoCell medium had significantly higher mRNA expression levels of VEGFA, NEUROG1, GRPR, CRHR2, and CCKAR than the cells cultured in MEM-α medium (Fig. 1).

[Preparation of suspended cultured cells (SUS) and planar cultured cells (ADH)]
The above-mentioned frozen cells derived from umbilical cord tissue were put to sleep, seeded in a cell culture flask, and cultured in a serum-free medium for mesenchymal stem cells (Rohto). The cells on the 4th day of culture were collected, a cell suspension containing the required number of cells and microcarriers were mixed, added to a culture tank, and stirring culture was started. On the 3rd or 4th day, a part of the cell / microcarrier suspension is taken, a new microcarrier is added, and subculture is carried out for 3 or 4 days for 2 weeks. Obtained. The above-mentioned frozen cells derived from umbilical cord tissue were put to sleep, seeded in a cell culture flask, and cultured in a serum-free medium for mesenchymal stem cells (Rohto). Subculture was performed once every 3 or 4 days, and the cells were cultured for a total of 2 weeks to obtain planar cultured cells (hereinafter referred to as "ADH").

[Comparison of planar cultured cells (ADH) and suspended cultured cells (SUS)]
Total RNA was recovered from the frozen stock of planar cultured cells (ADH) or suspended cultured cells (SUS) obtained by the above method. The mRNA expressions of PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, NRP2, VEGFA and APOE were detected by quantitative PCR.

The mRNA expression levels of PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, NRP2, VEGFA, APOE were significantly higher in suspension-cultured cells (SUS) than in planar cultured cells (ADH). Was found to be high (Fig. 2).

[Preparation of adipose-derived mesenchymal stem cells]
After obtaining the consent of the human donor, the subcutaneous adipose tissue obtained by the liposuction method was washed with physiological saline. Collagenase (solvent is saline) was added and shaken at 37 ° C. for 90 minutes to disperse to achieve extracellular matrix disruption and cell isolation. Subsequently, the suspension was centrifuged at 800 g for 5 minutes to obtain a precipitate of stromal vascular cells. A serum-free medium for mesenchymal stem cells (Rohto) was added to the above-mentioned cell precipitate, the cell suspension was centrifuged at 400 g for 5 minutes, and after removing the supernatant, a serum-free medium for mesenchymal stem cells (Rohto) was used. The cells were seeded in a flask after resuspension. Cells were cultured at 37 ° C. in 5% CO ₂ for several days. A few days later, the culture was washed with PBS to remove residual blood cells and adipose tissue contained in the culture medium, and the mesenchymal stem cells adhered to the plastic container (hereinafter referred to as "ADMSC"). Got

[Cyropreservation of adipose tissue-derived mesenchymal stem cells]
The obtained ADMSC was exfoliated with trypsin, transferred to a centrifuge tube, and centrifuged at 400 × g for 5 minutes to obtain a cell precipitate. After removing the supernatant, an appropriate amount of cell cryopreservation solution (STEM-CELLBANKER) was added and suspended. The cell suspension solution was dispensed into cryotubes and stored in a freezer at −80 ° C. After that, it was transferred to the gas phase on liquid nitrogen and storage was continued.

[Confirmation of therapeutic effect using rat transient ischemic model (Koizumi model)]
The middle cerebral artery (MCA) of a rat (Wistar, Charles River, Japan) was occluded, and ADMSC, ADH and SUS were suspended in HBSS (Hank's Balanced Salt solution) 23.3 to 27.8 hours later, and the tail vein. It was administered from the inner ^{(8 × 10 6 cells / kg} ). As a comparison target, an animal to which only HBSS was administered without administering cells was provided. Before and 14 days after surgery, body weight and neurological symptoms were observed, and a step test and a tape peeling test were performed.

Before and 14 days after surgery, a step test was conducted in which the rat's trunk, hind limbs, and right forelimb were held and lifted, and only the left forelimb touched the laboratory table, and the horizontal plane was moved 30 cm in the opposite direction in about 5 seconds. did. The number of steps on the left forelimb at that time was recorded. It was repeated 3 times, and the average value of the number of steps was used as the measured value (step test). In addition, neurological symptoms were observed with reference to the report by Kimata et al. (Pharmacology and Treatment, 1991; 19: 4491-4503).

A tape peeling test was performed based on the measurement method reported by Leong et al. (Leong et al. Stem Cells Translational Medicine, 2012; 1: 177-187). ^{A 15 mm 2} tape was applied to the back of the right (paralyzed side) forelimb, placed in a cage, and the time until the tape was to be peeled off was measured (Cut off was 120 seconds, tape peeling test).

It was revealed that administration of ADMSC, ADH and SUS reduced the degree of weight loss (Fig. 3). It was also revealed that administration of ADMSC, ADH and SUS reduced the neurological symptom score (Fig. 4). It was revealed that the number of steps was increased by administering ADMSC, ADH, and SUS (Fig. 5). It was revealed that administration of ADMSC, ADH, and SUS shortened the time required to remove the tape (Fig. 6).

From the above results, the improvement effect on nerve injury after cerebrovascular accident was shown.

[Interaction between nerve cells and mesenchymal stem cells]
SH-SY5Y (human-derived neuroblastoma; ECACC, Lot.16E028, Acc Nc: 94030304) subjected to DiD fluorescence staining with Vybrant DiO Cell-labeling solution (Thermo Fisher, # V22886) into a 12-well plate. Seed in DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium containing 10% FBS (Thermo Fisher, # 10437-028, Lot.1658423) and Pen-Strep at 37 ° C. The cells _{were cultured under O 2} : 20% and CO ₂ : 5% conditions. One day after culturing, the cells in each well were washed twice with D-PBS (-), and in DMEM, no glucose (Thermo Fisher, # 11966025) medium containing Pen-Strep, 37 ° C., N ₂ : 95%, O ₂ : Incubated for 2 hours under 5% conditions. Then, UCMSC prepared in the same manner as above, cultured in serum-free medium for mesenchymal stem cells (Rohto), and DiO fluorescently stained UCMSC was added to a 12-well plate, and 10% FBS (Thermo Fisher, # 10437-). Incubate in DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium containing 028, Lot.1658423) and Pen-Strep under the conditions of _{37 ° C., O 2} : 20%, CO _{2: 5%.} After each time, a time-lapse image was taken. In each photograph, green indicates UCMSC (cells pointed by the arrow pointing downward to the left (dotted line in the handle)), and red indicates SH-SY5Y (cells indicated by the arrow pointing to the upper right). (Fig. 7).

It was observed that UCMSC and SH-SY5Y mutually extend protrusions and interact with each other.

SH-SY5Y (human-derived neuroblastoma; ECACC, Lot.16E028, Acc Nc: 94030304) was seeded on a 96-well plate and 5% FBS (Thermo Fisher, # 10437-028, Lot.1658423). And Pen-Strep were cultured in DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium at 37 ° C. under the conditions of _{O 2} : 20% and CO _{2: 5%.} One day after culturing, the medium in Well was removed, and the medium was DMEM, No glucose (Thermo Fisher, # 11966025) containing Pen-Strep at 37 ° C., N ₂ : 95%, O ₂ : 5% (hereinafter, OGD). Cultivated under (Conditions) for 24 hours (OGD treatment group). Similarly, one day after culturing, the medium in the Well where the SH-SY5Y cells are cultured is removed, and the umbilical cord-derived mesenchymal stem cells prepared by the above method are used in a serum-free medium for mesenchymal stem cells (Rohto). The cultured UCMSC was added at 1,000 cells / well and cultured for 24 hours under OGD conditions (MSC group). For comparison, similarly, 1 day after culturing, the medium in Well was removed, and DMEM / F12 containing new 5% FBS (Thermo Fisher, # 10437-028, Lot.1658423) and Pen-Strep was added. (Thermo Fisher, # 11320-033, Lot.1930023) A control group (Control group) was obtained by culturing in a _{medium under the conditions of 37 ° C., O 2} : 20%, and CO _{2: 5% for 24 hours.} After culturing, the lactate dehydrogenase (LDH) activity in each culture supernatant was measured (FIG. 8).

By culturing under OGD (Oxygen and glucose deprivation) conditions, the LDH activity of SH-SY5Y cells increases, but by co-culturing with UCMSC, the increase in LDH activity is suppressed and nerve cell death due to ischemia occurs. It was confirmed that it was suppressed.

SH-SY5Y (human neuroblastoma; ECACC made, Lot.16E028, Acc Nc: 94030304) were seeded by 12 well plate Nizorezore 0.038 × 10 ⁶ cells / well and, 10% FBS ( DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium containing Thermo Fisher # 10437-028, Lot.1658423) and Pen-Strep at 37 ° C, O ₂ : 20%, CO ₂ : Cultured under 5% conditions. One day after culturing, the cells in each well were washed twice with D-PBS (-) and then cultured for 2 hours under the same OGD conditions as described above (cell-free group). Similarly, one day after culturing, the medium in Well was removed, and the suspension cultured cells (SUS) prepared by the above method were used in half the amount (half amount group), the same amount (same amount group), and the same amount of seeded SH-SY5Y cells. A transwell insert seeded in a double amount (double amount group) was placed and cultured for 2 hours under OGD conditions (MSC group). Then, in DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium containing 10% FBS (Thermo Fisher, # 10437-028, Lot.1658423) and Pen-Strep, 37 ° C., O ₂ The cells were cultured for 48 hours under the conditions of: 20% and CO _{2: 5%.} Similarly, as a comparison target, after 1 day of culturing, the cells in each well were washed twice with D-PBS (-), and then a new 10% FBS (manufactured by Thermo Fisher, # 10437-028, Lot.1658423). ) And Pen-Strep in DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium _{under the conditions of 37 ° C., O 2} : 20%, CO ₂ : 5% (Control). group). After culturing, the cell activity of each SH-SY5Y was evaluated by WST-8 (Cell Counting Kit-8) (Fig. 9).

It was clarified that the cell activity of nerve cells was significantly reduced by culturing under OGD conditions, but the decrease of cell activity could be suppressed by co-culturing with MSC.

SH-SY5Y (human neuroblastoma; ECACC made, Lot.16E028, Acc Nc: 94030304) were seeded by each 0.038 × 10 ⁶ cells / well in 12 well plate the, 10% FBS (Thermo Fisher Inc. Made in DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium containing # 10437-028, Lot.1658423) and Pen-Strep, 37 ° C, O ₂ : 20%, CO ₂ : 5%. It was cultured under the conditions. One day after culturing, the cells in each well were washed twice with D-PBS (-) and then cultured for 2 hours under the same OGD conditions as described above (cell-free group). Similarly, one day after culturing, the medium in Well was removed, and suspended cultured cells (SUS, SUS group), planar cultured cells (ADH, ADH group) and fibroblasts (Fibroblast, Fibroblast group) prepared by the above method were removed. Was cultivated for 2 hours under OGD conditions with a transwell insert seeded in the same amount as SH-SY5Y. Then, in DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium containing 10% FBS (Thermo Fisher, # 10437-028, Lot.1658423) and Pen-Strep, 37 ° C., O ₂ The cells were cultured for 48 hours under the conditions of: 20% and CO _{2: 5%.} Similarly, as a comparison target, after 1 day of culturing, the cells in each well were washed twice with D-PBS (-), and then a new 10% FBS (manufactured by Thermo Fisher, # 10437-028, Lot.1658423). ) And Pen-Strep in DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium under 37 ° C., O ₂ : 20%, CO ₂ : 5%% conditions for 48 hours ( Control group). After culturing, the cell activity of each SH-SY5Y was evaluated by WST-8 (Cell Counting Kit-8) (Fig. 10).

By co-culturing with MSC, it is possible to suppress the decrease in cell activity of nerve cells by culturing under OGD conditions, but it is clear that this effect is not present depending on fibroblasts and is an MSC-specific effect. It became.

SH-SY5Y (human neuroblastoma; ECACC made, Lot.16E028, Acc Nc: 94030304) were seeded by each 0.038 × 10 ⁶ cells / well in 12 well plate the, 10% FBS (Thermo Fisher Inc. Made in DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium containing # 10437-028, Lot.1658423) and Pen-Strep, 37 ° C, O ₂ : 20%, CO ₂ : 5%. It was cultured under the conditions. One day after culturing, the cells in each well were washed twice with D-PBS (-) and then cultured for 2 hours under the same OGD conditions as described above (cell-free group). Similarly, 1 day after culturing, the medium in Well was removed, and umbilical cord-derived mesenchymal stem cells (manufactured by PromoCell) were used as serum-free medium for mesenchymal stem cells (Rohto, serum-free group) and PromoCell medium (Mesenchymal Stem Cell). Supplement mix (PromoCell, C-39809, Lot.435M126) added to Growth Medium 2 (PromoCell, C-28009, Lot.435M415), PromoCell group) and MEM-α (Thermo Fisher, # UCMSC and fibroblasts (Fibroblast, Fibroblast group) cultured in 12571-063, Lot.18997009, MEM-α group, respectively, were placed on a transwell insert cultured in the same amount as SH-SY5Y, and cultured for 2 hours under OGD conditions. .. Then, in DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium containing 10% FBS (Thermo Fisher, # 10437-028, Lot.1658423) and Pen-Strep, 37 ° C., O ₂ The cells were cultured for 48 hours under the conditions of: 20% and CO _{2: 5%.} Similarly, as a comparison target, after 1 day of culturing, the cells in each well were washed twice with D-PBS (-), and then a new 10% FBS (manufactured by Thermo Fisher, # 10437-028, Lot.1658423). ) And Pen-Strep in DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium _{under the conditions of 37 ° C., O 2} : 20%, CO ₂ : 5% (Control). group). After culturing, the cell activity of each SH-SY5Y was evaluated by WST-8 (Cell Counting Kit-8) (Fig. 11).

By co-culturing with MSC, it is possible to suppress the decrease in cell activity of nerve cells by culturing under OGD conditions, but it was clarified that the effect is more remarkable in MSC cultured in serum-free medium.

SH-SY5Y (human neuroblastoma; ECACC made, Lot.16E028, Acc Nc: 94030304) were seeded by each 0.038 × 10 ⁶ cells / well in 12 well plate the, 10% FBS (Thermo Fisher Inc. Made in DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium containing # 10437-028, Lot.1658423) and Pen-Strep, 37 ° C, O ₂ : 20%, CO ₂ : 5%. It was cultured under the conditions. One day after culturing, the cells in each well were washed twice with D-PBS (-) and then cultured for 2 hours under the same OGD conditions as described above (cell-free group). Similarly, 1 day after culturing, the medium in Well was removed, and umbilical cord-derived mesenchymal stem cells (PromoCell, C-12971, Lot.427Z021) were used as serum-free medium for mesenchymal stem cells (Rohto, serum-free group). ), Supplement mix (PromoCell, C-39809, Lot.435M126) added to PromoCell medium (Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009, Lot.435M415), PromoCell group) and MEM-α UCMSC and fibroblast (fibroblast, fibroblast group) cultured in (Thermo Fisher, # 12571-063, Lot.18997009, MEM-α group) were placed in the same amount as SH-SY5Y. , Cultivated for 2 hours under OGD conditions. Then, in DMEM / F12 (Thermo Fisher, # 11320-033, Lot.1930023) medium containing 10% FBS (Thermo Fisher, # 10437-028, Lot.1658423) and Pen-Strep, 37 ° C., O ₂ The cells were cultured for 24-48 hours under the conditions of: 20% and CO _{2: 5%.} After culturing for 48H, cell images were acquired (Fig. 12). EthD-III was used for staining of dead cells. After measuring the number of dead cells in each image, the dead cell rate was calculated by "number of dead cells / absorbance of WST-8" (FIG. 13).

It was revealed that the cell proliferation activity was higher in the serum-free group than in the cell-free group, the fibroblast group, the PromoCell group and the MEM-α group. In addition, it was revealed that the number of dead cells in the serum-free group was smaller than the total number of cells in the cell-free group, the PromoCell group, and the MEM-α group.

According to the present invention, it is possible to provide a novel therapeutic agent for neuropathy.

Claims (5)

HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, NRP2, or Mesenchymal stem cells characterized by high expression of two or more. The mesenchymal stem cell according to claim 1, which is derived from an allogeneic family. The mesenchymal stem cell according to claim 1 or 2, which is derived from umbilical cord tissue or adipose tissue. The mesenchymal stem cell according to any one of claims 1 to 3, which is prepared by a suspension culture method. A neuropathy therapeutic agent containing mesenchymal stem cells according to any one of claims 1 to 4.

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