Mesenchymal stem cells and neurological agents

Abstract

An object of the present invention is to provide a novel therapeutic agent for neurological disorders. The present invention, which has been made to achieve the above object, provides mesenchymal stem cells characterized by high expression of one or more of HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, and NRP2.

Description

[Technical Field]

[0001] This invention relates to mesenchymal stem cells and agents for treating neurological disorders. [Background technology]

[0002] With the aging of society, neurodegenerative diseases, which are disorders of nerve cells in the brain, are increasing year by year. Examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and spinocerebellar degeneration. In Alzheimer's disease, nerve cells in areas such as the cerebral cortex and hippocampus are lost, leading to a decline in memory ability. In Parkinson's disease, nerve cells in an area called the substantia nigra are lost, resulting in impaired motor function. In addition to neurodegenerative diseases, other diseases caused by damage to nerve cells in the brain include cerebral infarction and cerebral hemorrhage due to stroke.

[0003] Furthermore, perinatal neonatal brain damage occurs at a frequency of 1 to 2 per 1,000 births and can lead to lifelong cerebral palsy. Perinatal brain damage that causes cerebral palsy includes hypoxic-ischemic encephalopathy, cerebral hemorrhage, and periventricular leukomalacia. The main pathological conditions in these conditions are inflammatory states resulting from mitochondrial dysfunction, such as increased reactive oxygen species, activation of macrophages, and the accompanying hypercytokinemia. Although the causes and symptoms of the aforementioned neurodegenerative diseases, stroke, and cerebral palsy differ, they can be said to share one commonality: a reduction in the number of nerve cells.

[0004] While Alzheimer's disease is treated with donepezil and Parkinson's disease with levodopa, these drugs aim to restore the information processing function of neural networks, which is affected by the decrease in nerve cells, by supplementing chemical neurotransmitter signals. They cannot prevent the decrease in nerve cells themselves. Furthermore, none of the existing drugs for neurodegenerative diseases or stroke exhibit any effect that protects nerve cells. Therefore, the development of new drugs that protect nerve cells is desired.

[0005] Mesenchymal stem cells are pluripotent progenitor cells first isolated from bone marrow by Friedenstein in 1982 (see Non-Patent Document 1). Mesenchymal stem cells have been shown to exist in various tissues such as bone marrow, umbilical cord, and adipose tissue, and mesenchymal stem cell transplantation is expected as a new treatment method for various intractable diseases (see Patent Documents 1 and 2). Recently, it has been known that cells having the same functions as stromal cells exist in adipose tissue, placenta, umbilical cord, egg membrane, etc. Therefore, mesenchymal stem cells are sometimes referred to as mesenchymal stromal cells.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a novel therapeutic agent for neuropathy in the above-described situation.

Means for Solving the Problems

[0009] As a result of diligent research to solve the above problems, the inventors have discovered that mesenchymal stem (stromal) cells (MSCs) that highly express one or more of the following: HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, and NRP2 are effective in treating neurological disorders, and have completed the present invention. According to the present invention, an effective therapeutic agent for treating neurological disorders can be provided. In other words, the gist of the present invention is as follows.

[0010] In other words, the present invention relates to the following: [1] Mesenchymal stem cells characterized by high expression of one or more of the following: HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, NRP2. [2] Allogeneic mesenchymal stem cells as described in [1]. [3] Mesenchymal stem cells according to [1] or [2], derived from umbilical cord tissue or adipose tissue. [4] Mesenchymal stem cells according to any one of [1] to [3], prepared by suspension culture. A neurological agent containing mesenchymal stem cells as described in any of [5], [1], to [4]. [Effects of the Invention]

[0011] According to the present invention, a novel therapeutic agent for neurological disorders can be provided. [Brief explanation of the drawing]

[0012] [Figure 1]Figure 1 shows the results of comparing the mRNA expression levels of various factors in mesenchymal stem cells obtained by culturing in various media. [Figure 2] Figure 2 shows 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. [Figure 3] This figure shows the effects of administering mesenchymal stem cells obtained by various culture methods to a rat transient cerebral ischemia model (body weight). [Figure 4] This figure shows the effects of administering mesenchymal stem cells obtained by various culture methods to a rat transient ischemia model (neurological symptom score). [Figure 5] This figure shows the effects of administering mesenchymal stem cells obtained by various culture methods to a rat transient cerebral ischemia model (number of steps). [Figure 6] This figure shows the effects of administering mesenchymal stem cells obtained by various culture methods to a rat transient cerebral ischemia model (tape peel test). [Figure 7] This is a time-lapse image showing the intercellular interactions between nerve cells and mesenchymal stem cells. [Figure 8] This figure shows the inhibitory effect on neuronal cell death of mesenchymal stem cells obtained by culturing in serum-free medium (Rohto). [Figure 9] This figure shows the dose-dependent neuronal cell activation effect of mesenchymal stem cells cultured in serum-free medium (Rohto). [Figure 10] This figure shows the neuronal cell activation effect of mesenchymal stem cells obtained by planar (ADH) or suspension (SUS) culture using serum-free medium (Rohto) (compared to fibroblasts). [Figure 11] This figure shows the neuronal cell activation effect of mesenchymal stem cells obtained by culturing in serum-free medium (Rohto) (compared to cells cultured in other media). [Figure 12] This is a cell image showing the inhibitory effect on neuronal cell death of mesenchymal stem cells obtained by culturing in serum-free medium (Rohto) (compared to cells cultured in other media). [Figure 13] This figure shows the inhibitory effect on neuronal cell death of mesenchymal stem cells cultured in serum-free medium (Rohto) (compared to cells cultured in other media). [Modes for carrying out the invention]

[0013] The mesenchymal stem cells and neuropathic agents of the present invention will be described in detail below.

[0014] [Mesenchymal stem cells] The mesenchymal stem cells of the present invention include 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 YRPW motif-like), This gene is characterized by high expression of one or more of the following: BMP2 (bone morphogenetic protein 2), NTN1 (netrin 1), ASCL1 (achaete-scute family bHLH transcription factor 1), and NRP2 (neuropilin 2).

[0015] HGF is a type of growth factor and is known to have neuroprotective effects. 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, a type of inflammatory cytokine, and is known to be involved in the inflammatory response. DLL1 belongs to the DSL family of Notch ligands and is known to be involved in the regulation of Notch signaling.

[0016] Furthermore, high expression of the above HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, and NRP2 includes high expression of each mRNA, high expression of each protein, or high expression of both.

[0017] Furthermore, the mesenchymal stem cells of the present invention only need to express the above factors at a higher level than other cells. Specifically, the mesenchymal stem cells of the present invention only need to express the above factors at a higher level than mesenchymal stem cells obtained under conventional culture conditions (for example, culture in MEM-α medium containing 10% FBS). Preferably, the expression is 2 times or more, more preferably 5 times or more, even more preferably 10 times or more, and particularly preferably 100 times or more compared to mesenchymal stem cells obtained under conventional culture conditions.

[0018] The mesenchymal stem cells of the present invention may express one or more of the following compared to fibroblasts: HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, and NRP2. Preferably, the expression level is 2 times or more, more preferably 5 times or more, even more preferably 10 times or more, and particularly preferably 100 times or more compared to dermal fibroblasts.

[0019] In this invention, mesenchymal stem cells refer to cells that have the ability to differentiate into one or more cells belonging to the mesenchyme (such as osteocytes, cardiomyocytes, chondrocytes, tendon cells, and adipocytes) and that can proliferate while maintaining that ability. In this invention, the term mesenchymal stem cells refers to the same cells as stromal cells, and there is no particular distinction between the two. They may also be simply referred to as mesenchymal cells. Examples of tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, and tooth germ. For example, adipose tissue-derived mesenchymal stem cells refer to mesenchymal stem cells contained in adipose tissue and may also be called adipose tissue-derived stromal cells. Of these, from the standpoint of effectiveness in treating neurological disorders and ease of availability, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells are preferred, adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells are more preferred, and umbilical cord-derived mesenchymal stem cells are the most preferred.

[0020] The mesenchymal stem cells in this invention may be of the same species as the subject being treated (test subject) or of a different species. Examples of mesenchymal stem cell species in this invention include human, horse, cattle, sheep, pig, dog, cat, rabbit, mouse, and rat, and are preferably cells of the same species as the subject being treated (test subject). The mesenchymal stem cells in this invention may be derived from the subject being treated (test subject), i.e., autologous cells, or from another subject of the same species, i.e., allogeneic cells. They are preferably allogeneic cells.

[0021] Since mesenchymal stem cells are less likely to cause rejection reactions even to allogeneic subjects, cells prepared in advance from a donor, cultured and cryopreserved, can be used as mesenchymal stem cells in the disease treatment agent of the present invention. Therefore, compared to using a patient's own mesenchymal stem cells, it is easier to commercialize and easier to obtain a stable and consistent effect, and from this viewpoint, it is more preferable that the mesenchymal stem cells in the present invention be allogeneic.

[0022] In the present invention, mesenchymal stem cells mean any cell population that includes mesenchymal stem cells. In this cell population, at least 20%, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98%, or 99% or more are mesenchymal stem cells.

[0023] In the present invention, the umbilical cord is a white, tubular tissue that connects the fetus to the placenta, and is composed of the umbilical vein, umbilical artery, gelatinous tissue (Wharton's Jelly), the umbilical cord matrix itself, etc., and contains a large amount of mesenchymal stem cells. The umbilical cord is preferably obtained from an animal of the same species as the subject (target of administration) using the disease treatment agent of the present invention, and more preferably from a human umbilical cord, considering that the disease treatment agent of the present invention will be administered to humans.

[0024] In the present invention, adipose tissue refers to tissue containing adipocytes and stromal cells including microvascular cells, and is, for example, tissue obtained by surgically excising or aspirating subcutaneous fat of a mammal. Adipose tissue can be obtained from subcutaneous fat. It is preferable to obtain it from the same species of animal as the recipient of the adipose tissue-derived mesenchymal stem cells described later, and more preferably from human subcutaneous fat when considering administration to humans. The individual supplying the subcutaneous fat may be alive or dead, but the adipose tissue used in the present invention is preferably tissue collected from a living individual. When collecting from an individual, liposuction can be performed using methods such as PAL (power-assisted) liposuction, Erchonia laser liposuction, or BodyJet liposuction, and it is preferable not to use ultrasound from the viewpoint of maintaining the state of the cells.

[0025] In this invention, bone marrow refers to the soft tissue that fills the cavity of bone and is a hematopoietic organ. Bone marrow fluid is present in the bone marrow, and the cells present in it are called bone marrow cells. Bone marrow cells include red blood cells, granulocytes, megakaryocytes, lymphocytes, adipocytes, as well as mesenchymal stem cells, hematopoietic stem cells, vascular endothelial progenitor cells, etc. Bone marrow cells can be collected, for example, from human ilium, long bones, or other bones.

[0026] In the present invention, each tissue-derived mesenchymal stem cell, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells, refers to any cell population that includes each tissue-derived mesenchymal stem cell, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells. In this cell population, at least 20%, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98%, or 99% or more are tissue-derived mesenchymal stem cells, such as adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells.

[0027] The mesenchymal stem cells in this 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 overexpression of one or more NRP2s, the organism may be characterized by, for example, growth characteristics (e.g., population doubling ability and doubling time from passage to aging), karyotype analysis (e.g., normal karyotype, maternal or neonatal lineage), surface marker expression by flow cytometry (e.g., FACS analysis), immunohistochemistry and / or immunocytochemistry (e.g., epitope detection), gene expression profiling (e.g., gene chip array; polymerase chain reaction such as reverse transcription PCR, real-time PCR, and conventional PCR), miRNA expression profiling, protein arrays, protein secretion such as cytokines (e.g., plasma coagulation analysis, ELISA, cytokine arrays), metabolites (metabolome analysis), and other methods known in this field.

[0028] (Method for preparing mesenchymal stem cells) The method for preparing mesenchymal stem cells that highly express one or more of the following HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, and NRP2 is not particularly limited, but they can be prepared as follows, for example. In other words, mesenchymal stem cells can be isolated and cultured from tissues such as fat, umbilical cord, and bone marrow according to methods known to those skilled in the art, and cells that highly express HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, or NRP2 can be obtained by separating them using a cell sorter, magnetic beads, etc., with antibodies that specifically bind to HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, or NRP2. Furthermore, by culturing using a specific culture medium, it is possible to 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 in mesenchymal stem cells, thereby obtaining mesenchymal stem cells that highly express HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, or NRP2 in mesenchymal stem cells.In the cell population obtained by this induction, it is preferable that 50% or more of the cell population highly express HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, or NRP2, more preferably that 70% or more highly express HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, or NRP2, and more preferably that 80% or more highly express HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, It is even more preferable that IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, or NRP2 are highly expressed, and it is particularly preferable that 90% or more are highly expressed as HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, or NRP2, and it is even more preferable that substantially HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, Most preferably, the cells are a homogeneous population with high expression of DLL1, HEYL, BMP2, NTN1, ASCL1, or NRP2.The following describes in detail how to prepare mesenchymal stem cells that highly express HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, or NRP2.

[0029] Mesenchymal stem cells can be prepared by methods well known to those skilled in the art. Below, as examples, methods for preparing umbilical cord tissue-derived mesenchymal stem cells and adipose tissue-derived mesenchymal stem cells are described.

[0030] The umbilical cord can be collected from the postpartum tissue, including the placenta and umbilical cord, delivered via vaginal delivery or cesarean section, after appropriately removing the placenta. After removing the umbilical cord blood from the collected umbilical cord, sterilization or bacteriostatic treatment may be performed. Removal of umbilical cord blood is performed by rinsing with an anticoagulant solution such as a heparin-containing solution. Sterilization or bacteriostatic treatment is not particularly limited, but may include application of povidone-iodine or immersion in a culture medium or buffer containing one or more antibiotics and / or antifungal agents such as penicillin, streptomycin, amphotericin B, gentamicin, and nystatin. It may also include a step of selectively lysing red blood cells, if necessary. As a method for selectively lysing red blood cells, methods well known in the art, such as incubation in hypertonic or hypotonic media by dissolution with ammonium chloride, can be used.

[0031] The umbilical cord-derived cells of the present invention refer to a cell population prepared using umbilical cord as a raw material, and can be obtained by a known manufacturing method, for example, by a method including the following steps (i) to (iii): (i) The process of cutting the umbilical cord; (ii) A step of culturing the umbilical cord obtained in step (i); and (iii) The process of subculturing.

[0032] Furthermore, as an alternative method for preparing the cells, (i) instead of cutting the umbilical cord, (i') the step of dissociating the tissue by enzymatic treatment of the umbilical cord may be included. In addition, (i) in addition to cutting the umbilical cord, (i') the step of dissociating the tissue by enzymatic treatment of the umbilical cord may be included.

[0033] In the present invention, (i) the step of cutting the umbilical cord may be performed by cutting the umbilical cord obtained by the method described above, including the amnion, blood vessels, perivascular tissue, and Wharton's jelly, by mechanical force (shredding force or shearing force). The umbilical cord section obtained by cutting is not particularly limited, but is 1 to 10 mm in size. 3 , 1 to 5 mm 3 , 1 to 4 mm 3 , 1 to 3 mm 3 or 1 to 2 mm 3 The size of the umbilical cord is illustrated. In the (i') step of dissociating tissue by enzymatic treatment of the umbilical cord according to the present invention, the umbilical cord obtained by the above method may be dissociated by enzymatic treatment while containing the amniotic membrane, blood vessels, perivascular tissue and Wharton's jelly. While not particularly limited, examples of enzymatic treatment include enzymatic treatment using one or more enzymes such as collagenase, dispase and hyaluronidase.

[0034] The step of culturing the umbilical cord obtained in step (i) of the present invention involves culturing the umbilical cord obtained in step (i) on a solid surface using an appropriate cell medium at an appropriate cell density and culture conditions.

[0035] The culture medium used in this process is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells. Such a medium may be prepared by adding serum to a basal medium and / or by adding one or more serum substitutes such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. These media may also be further supplemented with substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts as needed.

[0036] Examples of the above-mentioned basal media 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, MCDB201 medium, and mixed media of these.

[0037] Examples of the serums mentioned above 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, and rat serum. When using serum, 5 v / v% to 15 v / v%, preferably 10 v / v%, may be added to the basal medium.

[0038] Examples of fatty acids include, but are not limited to, linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. Examples of lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Examples of amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, and L-glycine. Examples of proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of polysaccharides include glycosaminoglycans, and among glycosaminoglycans, hyaluronic acid and heparan sulfate are particularly exemplified, but are not limited to these. Examples of growth factors include, but are not limited to, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), epidermal growth factor (EGF), connective tissue growth factor (CTGF), and vascular endothelial growth factor (VEGF). 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 xeno-free medium that does not contain heterologous components such as serum. Such media are provided as pre-prepared media for mesenchymal stem cells (stromal cells) by companies such as PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, Corning, and Rohto.

[0039] In this invention, "solid surface" means any material that enables the binding and adhesion of adipose tissue-derived mesenchymal stem cells in this invention. In certain embodiments, such a material is a plastic material treated to promote 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 petri dishes and flasks are preferably used. After incubation, the cells are washed to remove unbound cells and cell debris.

[0040] In this invention, cells that ultimately remain bound and adhered to a solid surface can be selected as a population of umbilical cord tissue-derived mesenchymal stem cells.

[0041] The umbilical cord tissue-derived mesenchymal stem cells of the present invention can also be produced using a suspension culture method. Suspension culture methods include methods of agglomerating cells to form cell aggregates on spheres and culturing them by stirring, and methods of adhering cells to microcarriers and culturing them by stirring the microcarriers. Stirring can be done by rotating the stirring blades in a container with a stirrer, or by placing a bag containing the culture medium and cells on a shaker and shaking the bag to suspend the culture medium. The culture medium used in the suspension culture method is not particularly limited as long as it is a medium that can culture mesenchymal stem cells, and the above-mentioned media are examples. The microcarriers are not particularly limited as long as they can be used in suspension culture, but examples include polyester, polystyrene, glass, and dextran.

[0042] Adipose tissue-derived mesenchymal stem cells may be obtained by a manufacturing method described, for example, U.S. Patent No. 6,777,231, which can be manufactured by a method comprising the following steps (i) to (iii): (i) A process of obtaining cell suspensions by enzymatic digestion of adipose tissue; (ii) the step of settling the cells and resuspending the cells in a suitable culture medium; and (iii) A step of culturing cells on a solid surface and removing cells that do not show binding to the solid surface.

[0043] In step (i), it is preferable to use washed adipose tissue. Washing can be performed by vigorously agitating and settling the tissue using a physiologically suitable saline solution (e.g., phosphate-buffered saline (PBS)). This is to remove impurities (also called debris, e.g., damaged tissue, blood, red blood cells, etc.) contained in the adipose tissue. Therefore, washing and settling are generally repeated until the debris is completely removed from the supernatant. Since the remaining cells exist as clumps of various sizes, it is preferable to treat the washed cell clumps with an enzyme that weakens or destroys intercellular junctions (e.g., collagenase, dispase, or trypsin) in order to dissociate them while minimizing damage to the cells themselves. The amount and duration of such enzymes vary depending on the conditions used, but are known in the art. Instead of, or in combination with, such enzymatic treatment, the cell clumps can be broken down by other methods such as mechanical agitation, ultrasonic energy, or thermal energy, but it is preferable to perform the treatment with enzymes alone in order to minimize cell damage. When using enzymes, it is desirable to inactivate them using a culture medium or similar method after an appropriate period of time to minimize harmful effects on cells.

[0044] The cell suspension obtained in step (i) includes a slurry or suspension of aggregated cells, as well as various contaminating 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 this separation and removal can be omitted as they can be removed by adhesion and washing in step (iii) described later. If contaminating cells are to be separated and removed, this can be achieved by centrifugation, which forces the cells to separate into supernatant and precipitate. The resulting precipitate containing contaminating cells is suspended in a physiologically suitable solvent. Although the suspended cells may contain erythrocytes, the lysis step is not necessarily required because erythrocytes are excluded by selection by adhesion to the solid surface described later. As a method for selectively lysing erythrocytes, methods well known in the art, such as lysis with ammonium chloride and incubation in hypertonic or hypotonic medium, can be used. After lysis, the lysate may be separated from the desired cells by, for example, filtration, centrifugation, or density fractionation.

[0045] In step (ii), the suspended cells may be washed once or multiple times in succession to increase the purity of the mesenchymal stem cells, then centrifuged and resuspended in the culture medium. Alternatively, the cells may be separated based on the cell surface marker profile or based on cell size and granularity.

[0046] The culture medium used in resuspension is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells. Such a medium may be prepared by adding serum to a basal medium and / or by adding one or more serum substitutes such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. These media may also be further supplemented with substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts, as needed.

[0047] Examples of the above-mentioned basal media 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, MCDB201 medium, and mixed media of these.

[0048] Examples of the serums mentioned above 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, and rat serum. When using serum, 5 v / v% to 15 v / v%, preferably 10 v / v%, may be added to the basal medium.

[0049] Examples of fatty acids include, but are not limited to, linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. Examples of lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Examples of amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, and L-glycine. Examples of proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of polysaccharides include glycosaminoglycans, and among glycosaminoglycans, hyaluronic acid and heparan sulfate are particularly exemplified, but are not limited to these. Examples of growth factors include, but are not limited to, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), epidermal growth factor (EGF), connective tissue growth factor (CTGF), and vascular endothelial growth factor (VEGF). 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 xeno-free medium that does not contain heterologous components such as serum. Such media are provided as pre-prepared media for mesenchymal stem cells (stromal cells) by companies such as PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, Corning, and Rohto.

[0050] Next, in step (iii), the cells in the cell suspension obtained in step (ii) are cultured on a solid surface without differentiation using the appropriate cell medium described above, at an appropriate cell density and culture conditions. In the present invention, "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 promote 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 petri dishes and flasks are preferably used. After incubation, the cells are washed to remove unbound cells and cell debris.

[0051] In this invention, cells that ultimately remain bound and adhered to a solid surface can be selected as a population of adipose tissue-derived mesenchymal stem cells.

[0052] To confirm that the selected cells are mesenchymal stem cells according to the present invention, surface antigens may be analyzed using conventional methods such as flow cytometry. Furthermore, the ability to differentiate into each cell lineage may be examined, and such differentiation can be performed using conventional methods.

[0053] The mesenchymal stem cells in this invention can be prepared as described above, but may also be defined as cells having the following characteristics: (1) Under standard culture conditions, the following conditions are observed: (2) The surface antigens CD73 and CD90 are positive, and CD45 is negative, and (3) Can differentiate into osteocytes, adipocytes, and chondrocytes under the culture conditions.

[0054] By selectively separating cells that highly express HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, or NRP2 protein from the mesenchymal stem cells obtained through the above process using immunological methods such as cell sorters and magnetic beads, mesenchymal stem cells that highly express HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, or NRP2 protein can be obtained. Furthermore, by culturing in a specific medium 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 in mesenchymal stem cells, 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 can be efficiently induced, thereby enabling the efficient induction of HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, It is also possible to obtain mesenchymal stem cells that highly express APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, or NRP2. As an example, a specific method of selective isolation using immunological techniques with a cell sorter is described below.

[0055] The mesenchymal stem cells prepared above are treated with trypsin-EDTA solution, etc., and the resulting cell suspension is centrifuged (room temperature, 400G, 5 minutes) and the supernatant is removed. Staining buffer (1% BSA-PBS) is added to the cells, and 1 × 10⁶ 6 Prepare a cell suspension to a concentration of 500 μL per cell, and after homogenizing the cell suspension concentration by pipetting, dispense 50 μL into new 1.5 mL microcentrifuge tubes. Add the primary antibody (Mouse anti human TFPI, Sekisui Diagnostics, ADG4903) to the dispensed cell suspension at a concentration of 5-20 μg / mL and suspend, then react for 30 minutes to 1 hour under light-shielded and refrigerated conditions. Wash three times with 1 mL of Staining Buffer, then add Staining Buffer to make a total volume of 50 μL, add the secondary antibody (Anti Mouse IgG alexar488, Thermofisher Scientific, A21202) at a concentration of 1-10 μg / mL and suspend, then react for 30 minutes to 1 hour under light-shielded and refrigerated conditions. After washing three times with 1 mL of Staining Buffer, 300 μL of PI Buffer (prepared by adding 28.8 μL of Propidium iodide solution (SIGMA, P4864) to 14.4 mL of Staining Buffer) is added and the cells are thoroughly suspended. The cells are then passed through a tube equipped with a cell strainer and separated by fluorescence activated cell sorting (FACS).

[0056] (Cryopreservation of mesenchymal stem cells) The mesenchymal stem cells in this invention may be cells that have been cryopreserved and thawed repeatedly as appropriate, provided they possess disease-treating efficacy. In this invention, cryopreservation can be performed by suspending mesenchymal stem cells in a cryopreservation solution well known to those skilled in the art and then cooling them. Suspension can be performed by detaching the cells with a detaching agent such as trypsin as needed, transferring them to a cryopreservation container, processing them as appropriate, and then adding the cryopreservation solution.

[0057] The cryopreservation solution may contain DMSO (Dimethyl sulfoxide) as a cryoprotective agent. However, since DMSO has cytotoxic properties and also induces differentiation of mesenchymal stem cells, it is preferable to reduce the DMSO content. Examples of substitutes for DMSO include glycerol, propylene glycol, or polysaccharides. When DMSO is used, it should be contained at a concentration of 5% to 20%, preferably 5% to 10%, and more preferably 10%. In addition, additives described in WO2007 / 058308 may also be included. As such a cryopreservation solution, cryopreservation solutions provided by companies such as Bioverde, Nippon Genetics Co., Ltd., Reprocell, Zenoac, Cosmo Bio, Kojin Bio, Thermo Fisher Scientific, etc. may be used.

[0058] When freezing the suspended cells described above, freezing at a temperature between -80°C and -100°C (for example, -80°C) is sufficient and can be done using any freezer capable of achieving that temperature. While not particularly limited, a programmable freezer may be used to appropriately control the cooling rate in order to avoid rapid temperature changes. The cooling rate may be appropriately selected depending on the components of the cryopreservation solution and may be carried out according to the manufacturer's instructions for the cryopreservation solution.

[0059] The storage period is not particularly limited, as long as the cells frozen and stored under the above conditions retain the same properties as before freezing after thawing. Examples include 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 longer. Cell damage can be suppressed by storing at a lower temperature, so the cells may be stored in the gas phase over liquid nitrogen (approximately -150°C or below to -180°C or above). When storing in the gas phase over liquid nitrogen, storage containers well known to those skilled in the art can be used. Although not particularly limited, for example, when storing for 2 weeks or more, storage in the gas phase over liquid nitrogen is preferable.

[0060] The thawed mesenchymal stem cells may be appropriately cultured until the next cryopreservation. The culture of mesenchymal stem cells is carried out using a medium capable of culturing the above-described mesenchymal stem cells, and is not particularly limited. However, it may be carried out at a culture temperature of about 30 to 40 °C, preferably about 37 °C, in an atmosphere of air containing CO2. The CO2 concentration is about 2 to 10%, preferably about 5 to 10%. In the culture, after reaching an appropriate confluence (for example, the cells occupy 50% to 80% of the culture vessel) with respect to the culture vessel, the cells are detached by a detachment agent such as trypsin, and seeded at an appropriate cell density in a separately prepared culture vessel to continue the culture. When seeding the cells, typical cell densities include 100 cells / cm 2 ~100,000 cells / cm 2 、500 cells / cm 2 ~50,000 cells / cm 2 、1,000 to 10,000 cells / cm 2 、2,000 to 10,000 cells / cm 2 etc. are exemplified. In a specific embodiment, the cell density is 2,000 to 10,000 cells / cm 2 . It is preferable to adjust the period until reaching an appropriate confluence to be 3 to 7 days. During the culture, the medium may be appropriately replaced as necessary.

[0061] The thawing of the cryopreserved cells can be carried out by a method well known to those skilled in the art. For example, a method of performing it by standing or shaking in a constant temperature bath at 37 °C or in a hot water bath is exemplified.

[0062] The mesenchymal stem cells of the present invention may be cells in any state, for example, cells detached and collected from cultured cells, or cells frozen in a cryopreservation solution. Using cells from the same lot obtained by large-scale culture, divided into smaller portions and cryopreserved, is preferable because it provides stable and similar effects and is easy to handle. Cryopreserved mesenchymal stem cells may be thawed immediately before use and directly mixed with an infusion solution or culture medium while suspended in the cryopreservation solution. Alternatively, the cryopreservation solution may be removed by methods such as centrifugation before being suspended in an infusion solution or culture medium. Here, "infusion solution" in the present invention refers to a solution used in the treatment of humans and is not particularly limited, but examples include physiological saline, Japanese Pharmacopoeia physiological saline solution, 5% glucose solution, Japanese Pharmacopoeia glucose injection solution, Ringer's solution, Japanese Pharmacopoeia Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, Solution No. 1 (starting solution), Solution No. 2 (dehydration replenishment solution), Solution No. 3 (maintenance solution), Solution No. 4 (postoperative recovery solution), etc.

[0063] [Neuropathic treatment agent] The neuropathic agent of the present invention contains mesenchymal stem cells that highly express one or more of the following HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, and NRP2 as described above. The neuropathic agent of the present invention can effectively protect against neuropathies. The description in the section on mesenchymal stem cells above can be applied to the mesenchymal stem cells containing the neuropathic agent of the present invention.

[0064] The neuropathic agent of the present invention may contain, in addition to the mesenchymal stem cells described above, pharmaceutically acceptable carriers and additives in accordance with conventional methods, depending on its use and form, as long as the effects of the present invention are not impaired. Examples of such carriers and additives include, but are not limited to, isotonic agents, thickeners, sugars, sugar alcohols, preservatives, bactericidal or antibacterial agents, pH adjusters, stabilizers, chelating agents, oily bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersants, emulsifiers, buffers, solubilizers, antioxidants, sweeteners, acidulants, colorants, flavoring agents, fragrances or cooling agents. Typical components include, for example, the following carriers and additives.

[0065] Examples of carriers include aqueous carriers such as water and aqueous ethanol; examples of isotonic agents (inorganic salts) include sodium chloride, potassium chloride, calcium chloride, and magnesium chloride; examples of polyhydric alcohols include glycerin, propylene glycol, and polyethylene glycol; examples of thickeners include carboxyvinyl polymer, hydroxyethylcellulose, hydroxypropyl methylcellulose, methylcellulose, alginic acid, polyvinyl alcohol (complete or partially saponified), polyvinylpyrrolidone, and macrogol; examples of sugars include cyclodextrin and glucose; examples of sugar alcohols include xylitol, sorbitol, and mannitol (these may be d-isomers, l-isomers, or dl-isomers); examples of preservatives, bactericides, or antibacterial agents include dibutylhydroxytoluene, butylhydroxyanisole, alkyldiaminoethylglycine hydrochloride, sodium benzoate, ethanol, benzalkonium chloride, and benzethonium chloride. , chlorhexidine gluconate, chlorobutanol, sorbic acid, potassium sorbate, trometamol, sodium dehydroacetate, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, oxyquinoline sulfate, phenethyl alcohol, benzyl alcohol, biguanide compounds (specifically, polyhexanide hydrochloride (polyhexamethylene biguanide), etc.), Glokill (a trade name of Rhodia Corporation), etc.; as pH adjusters, for example For example, hydrochloric acid, boric acid, aminoethylsulfonic acid, epsilon-aminocaproic acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, sodium bicarbonate, sodium carbonate, borax, triethanolamine, monoethanolamine, diisopropanolamine, sulfuric acid, magnesium sulfate, phosphoric acid, polyphosphate, propionic acid, oxalic acid, gluconic acid, fumaric acid, lactic acid, tartaric acid, malic acid, succinic acid, gluconolactone, ammonium acetate, etc.Examples of stabilizers include dibutylhydroxytoluene, trometamol, sodium formaldehyde sulfoxylate (longalit), tocopherol, sodium pyrosulfite, monoethanolamine, aluminum monostearate, glyceryl monostearate, sodium bisulfite, sodium sulfite, etc.; examples of oily bases include vegetable oils such as olive oil, corn oil, soybean oil, sesame oil, and cottonseed oil, and medium-chain triglyceride compounds; examples of aqueous bases include macrogol 400; examples of gel bases include carboxyvinyl polymers and gums; examples of surfactants include polysorbate 80, hydrogenated castor oil, glycerin fatty acid esters, and sorbitan sesquioleate; examples of suspending agents include bleached beeswax and various surfactants, etc. Examples of binders include gum, gum arabic powder, xanthan gum, and soy lecithin; examples of binders include hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol; examples of excipients include sucrose, lactose, starch, corn starch, crystalline cellulose, and light anhydrous silicic acid; examples of lubricants include sucrose fatty acid esters, magnesium stearate, and talc; examples of disintegrants include low-substituted hydroxypropylcellulose, crospovidone, and croscarmellose sodium; examples of foaming agents include sodium bicarbonate; and examples of fluidizing agents include sodium aluminometasilicate and light anhydrous silicic acid.

[0066] The neuropathic agent of the present invention can be provided in various forms depending on the purpose, such as solid preparations, semi-solid preparations, and liquid preparations. For example, it can be used in the form of solid preparations (tablets, powders, powders, granules, capsules, etc.), semi-solid preparations [ointments (hard ointments, soft ointments, etc.), creams, etc.], liquid preparations [lotions, extracts, suspensions, emulsions, syrups, injectable preparations (including intravenous solutions, implantable injections, sustained-release injections, and injections prepared on the day of use), dialysis preparations, aerosols, soft capsules, drinks, etc.], patches, poultices, etc. The neuropathic 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 neuropathic agent of the present invention can be applied to the affected area by spraying, and can also be used in a form that gels or forms a sheet on the affected area after spraying. The neuropathic agent of the present invention can also be applied to the affected area after the above-mentioned mesenchymal stem cells have been formed into a sheet or three-dimensional structure.

[0067] The neuropathic agent of the present invention can be used by suspending or diluting it with intravenous fluids such as physiological saline, Japanese Pharmacopoeia physiological saline solution, 5% glucose solution, Japanese Pharmacopoeia glucose injection solution, Ringer's solution, Japanese Pharmacopoeia Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, Solution No. 1 (initial solution), Solution No. 2 (dehydration replenishment solution), Solution No. 3 (maintenance solution), Solution No. 4 (postoperative recovery solution), or cell culture media such as DMEM. Preferably, it can be used by suspending or diluting it with physiological saline solution, 5% glucose solution, or Solution No. 1 (initial solution), and more preferably with 5% glucose solution or Solution No. 1 (initial solution).

[0068] When the neuropathic agent of the present invention is a liquid formulation, the pH of the neuropathic agent is not particularly limited as long as it is within a range that is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable. 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 is given.

[0069] When the neuropathic agent of the present invention is a liquid formulation, the osmotic pressure of the neuropathic agent is not particularly limited as long as it is within a range acceptable to the body. An example of the osmotic pressure ratio of the composition of the present invention is preferably in the range of 0.7 to 5.0, more preferably 0.8 to 3.0, and even more preferably 0.9 to 1.4. The osmotic pressure can be adjusted using inorganic salts, polyhydric alcohols, sugar alcohols, sugars, etc., by methods known in the art. 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 edition of the Japanese Pharmacopoeia, and the osmotic pressure is measured with reference to the osmotic pressure measurement method (freezing point depression method) described in the Japanese Pharmacopoeia. To prepare the standard solution for osmotic pressure ratio measurement (0.9 w / v% sodium chloride aqueous solution), sodium chloride (Japanese Pharmacopoeia standard reagent) is dried at 500-650°C for 40-50 minutes, then cooled in a desiccator (silica gel). 0.900g of this solution is accurately weighed and dissolved in purified water to make exactly 100mL. Alternatively, a commercially available standard solution for osmotic pressure ratio measurement (0.9 w / v% sodium chloride aqueous solution) can be used.

[0070] The routes of administration of the neuropathic agent of the present invention to the target subject include oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intra-arterial administration, intracerebroventricular administration, intrathecal administration, intraperitoneal administration, sublingual administration, transrectal administration, transvaginal administration, intraocular administration, transnasal administration, inhalation, transdermal administration, implantation, direct administration by spraying onto organ surfaces and application of sheets, etc. However, from the viewpoint of the efficacy of the neuropathic agent of the present invention, intra-arterial administration, intravenous administration, intracerebroventricular administration and intrathecal administration are preferred, from the viewpoint of reducing the burden on the target subject, intravenous administration, intramuscular administration and intranasal administration are more preferred, and from the viewpoint of efficacy, intracerebroventricular administration and intrathecal administration are preferred.

[0071] In the neuropathic agent of the present invention, the dose (amount administered) may vary depending on the patient's condition (weight, age, symptoms, physical condition, etc.) and the dosage form of the neuropathic agent of the present invention. However, from the viewpoint of achieving sufficient therapeutic effect of the neuropathic agent, a larger dose tends to be preferable, while from the viewpoint of suppressing the occurrence of side effects, a smaller dose tends to be preferable. Typically, when administered to adults, the cell count is 1 × 10⁶. 3~1 × 10 12 pieces / time, preferably 1 x 10 4 ~1 × 10 11 1 piece / time, comfortable 1 x 10 5 ~1 × 10 10 pieces / time, more preferably 5 x 10 6 ~1 × 10 9 The dosage is per dose. The dosage per patient's body weight is 1 × 10 to 5 × 10 10 pieces / kg, preferably 1 x 10 2 ~5×10 9 pieces / kg, comfortable 1 x 10 3 ~5×10 8 pieces / kg, more preferably 1 x 10 4 ~5×10 7 The cell count is cells / kg. When administered to newborns, the cell count is 1 × 10⁶. 3 ~1 × 10 11 pieces / time, preferably 1 x 10 4 ~1 × 10 10 1 piece / time, comfortable 1 x 10 5 ~1 × 10 9 pieces / time, more preferably 5 x 10 5 ~5×10 8 The dosage is per dose. The dosage per patient's body weight is 1 × 10 to 5 × 10 10 pieces / kg, preferably 1 x 10 2 ~5×10 9 pieces / kg, comfortable 1 x 10 3 ~5×10 8 pieces / kg, more preferably 1 x 10 4 ~5×10 7 The dose is per kg. This dose may be administered as a single dose multiple times, or divided into multiple doses.

[0072] The neuropathic agent of the present invention may be administered together with one or more other agents.Other drugs include any drugs that can be used to treat neurological disorders, such as antiparkinson's disease drugs like levodopa, amantadine, and carbidopa; dopamine agonists like bromocriptine, pulgolide, ropinirole, and pramipexole; selective monoamine oxidase inhibitors (MAO-B) like selegiline and rasaridine; catechol-O-methyltransferase (COMT) inhibitors like entacapone and tolcapone; anticholinergics like benztropone and trihexyphenidyl; and antihistamines like diphenhydramine and orphenadrine. Cholinesterase inhibitors such as donepezil, rivastigmine, galantamine, and tacrine; N-methyl-D-aspartate receptor antagonists such as memantine; antipsychotics such as haloperidol, thioridazine, thiothixen, olanzapine, risperidone, quetiapine, and clozapine; beta-blockers such as propranolol; sedatives such as benzodiazepines; anticoagulants such as heparin, low molecular weight heparin, and warfarin; anticonvulsants such as carbamazepine, gabapentin, phenytoin, pregabalin, valprosan, and lamotrigine; tricyclic antidepressants, venlafaxine, and bupropio Antidepressants such as amitriptyline, desipramine, and paroxetine; central α-2 adrenergic agonists such as clonidine and tizanidine; corticosteroids such as dexamethasone and prednisolone; NMDA receptor antagonists such as amantadine and dextrimetrophane; local anesthetics such as lidocaine, mexiletine, and capsaicin; etodolac, indomethacin, thrinoxine, tolmetatin, nabumetone, piroxicam, acetaminophen, phenobipron, flurbipron, ibuprofen, ketoprofen, naproxen, naproxen sodium, oxaloxine Examples include analgesics such as prosin, aspirin, choline magnesium trisalicylic acid, diflunisal, meclofenamate, mefenamic acid, phenylbutazone, ketorolac, celecoxib, codeine, hydrocodeine, propoxyfen, fentanyl, hydromorphone, levofanol, meperidine, methadone, morphine, oxycodone, oxymorphone, buprenorphine, butorphanol, nalbuffine, and pentazocine; radical scavengers such as propofol; anti-inflammatory agents; serotonin, norepinephrine, NSAIDs, and ginkgo biloba extract.Furthermore, along with the administration of the neurological disorder treatment agent of the present invention, low-temperature therapies such as hypothermia therapy, cerebral hypothermia therapy, and cerebral hypothermia therapy can also be performed in combination.

[0073] The mesenchymal stem cells of the present invention can be used for a variety of neurological disorders, including autonomic nervous system disorders such as autonomic neuropathy, Horner's syndrome, multiple system atrophy, and pure autonomic neuropathy; chronic pain, neuropathic pain, and complex regional pain syndrome; ischemic stroke, transient ischemic attack, hypoxia-ischemia, intracranial hemorrhage such as intracerebral hemorrhage and intraventricular hemorrhage, and other strokes (cerebrovascular accidents) such as subarachnoid hemorrhage; dementia such as Alzheimer's disease, vascular dementia, Lewy dementia, HIV-related dementia, and frontotemporal dementia; convulsive syndromes, athetosis or other movement disorder syndromes, and ataxia syndromes. Examples of conditions for which this treatment is indicated include cerebral palsy syndrome, neonatal convulsive disorder due to hypoglycemia, hypernatremia, hyponatremia, hypomagnesemia, congenital metabolic disorders, demyelinating diseases such as multiple sclerosis, Killan-Barré syndrome, hereditary neuropathy, motor neuron diseases including amyotrophic lateral sclerosis (ALS), myasthenia gravis, mononeuropathy, polyneuropathy, peripheral nervous system disorders such as nerve plexus disorders, acute transverse myelitis, arteriovenous malformations, spinal cord disorders such as spinal cord infarction (ischemic spinal cord injury), cerebellar diseases such as spinocerebellar ataxia and spinocerebellar degeneration, brain tumors, encephalitis, meningitis, and Parkinson's disease. It can also be used in neonates for perinatal brain injury, neonatal encephalopathy, and cerebral palsy. Of these, ischemic stroke, transient ischemic attack, hypoxia-ischemia, intracranial hemorrhage such as intracerebral hemorrhage and intraventricular hemorrhage, stroke (cerebrovascular accident) such as subarachnoid hemorrhage, perinatal brain injury, neonatal encephalopathy, and cerebral palsy are preferred. [Examples]

[0074] The present invention will be described in detail below with reference to examples and test examples, but the present invention is not limited to these examples.

[0075] [Preparation and culture of umbilical cord-derived mesenchymal stem cells] Umbilical cord-derived cells were collected using the method described in Cytotherapy, 18, 229-241, 2016. In short, umbilical cords were collected with the consent of the donor and approved by the Ethics Committee of the Institute of Medical Science, University of Tokyo, and were 1 to 2 mm in size. 3 Umbilical cord-derived mesenchymal stem cells (hereinafter referred to as "UCMSCs") were obtained by a modified Explant method, in which the cells were shredded into fragments, seeded on a culture dish, covered with Cell Amigo (Tsubakimoto Chain Co., Ltd.), and cultured in α-minimal essential medium (MEM-α) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cells obtained from umbilical cord tissue by the aforementioned modified Explant method are designated as first passage cells (P1), and as the passage number increases with subsequent passages, they are designated as second passage cells (P2), and so on.

[0076] The obtained UCMSCs were detached using trypsin (TrypLE Select (1X)), transferred to a centrifuge tube, and centrifuged at 400×g for 5 minutes to obtain cell precipitate. After removing the supernatant, an appropriate amount of cell cryopreservation solution (STEM-CELLBANKER (Zenoac Co.)) was added to suspend the cells. The cell suspension solution was dispensed into cryotubes and stored in a freezer at -80°C. Subsequently, it was transferred to the gas phase over liquid nitrogen and storage was continued.

[0077] [mRNA expression] UCMSCs were cultured in serum-free medium for mesenchymal stem cells (Rohto, serum-free medium), PromoCell medium (Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009, Lot.435M415) with added Supplement mix (PromoCell, C-39809, Lot.435M126)) and MEM-α medium (Thermo Fisher, #12571-063, Lot.18997009 with added serum to 10%), respectively, and frozen stocks were prepared. Fibroblasts were cultured in MEM-α medium and then frozen stocks were prepared. Each P4 cell and fibroblast cell was induced dormancy and stored in 6-well plates at 15,000 cells / cm².2 The cells were seeded and cultured for one day in each of the three culture media, after which total RNA was recovered. mRNA expression of HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, and APOE was detected using quantitative PCR.

[0078] Cells cultured in serum-free medium showed significantly higher mRNA expression of HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, and APOE compared to cells cultured in MEM-α medium. Furthermore, cells cultured in PromoCell medium showed significantly higher mRNA expression levels of VEGFA, NEUROG1, GRPR, CRHR2, and CCKAR compared to cells cultured in MEM-α medium (Figure 1).

[0079] [Preparation of suspension cultured cells (SUS) and planar cultured cells (ADH)] The aforementioned umbilical cord tissue-derived frozen cells were induced to dormancy, seeded in cell culture flasks, and cultured using serum-free medium for mesenchymal stem cells (Rohto). After 4 days of culture, the cells were harvested, mixed with a cell suspension containing the required number of cells and microcarriers, added to a culture vessel, and stirred culture was started. On the 3rd or 4th day, a portion of the cell / microcarrier suspension was taken, new microcarriers were added, and the cells were subcultured for 3 or 4 days for 2 weeks to obtain suspension culture cells (hereinafter referred to as "SUS"). The aforementioned umbilical cord tissue-derived frozen cells were induced to dormancy, seeded in cell culture flasks, and cultured using 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 culture cells (hereinafter referred to as "ADH").

[0080] [Comparison of planar cultured cells (ADH) and suspension cultured cells (SUS)] Total RNA was recovered from frozen stocks of planar cultured cells (ADH) or suspension cultured cells (SUS) obtained by the above method. mRNA expression of PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, NRP2, VEGFA, and APOE was detected using quantitative PCR.

[0081] Compared to planar cultured cells (ADH), suspension cultured cells (SUS) showed significantly higher mRNA expression levels for PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, NRP2, VEGFA, and APOE (Figure 2).

[0082] [Preparation of adipose-derived mesenchymal stem cells] After obtaining consent from a human donor, subcutaneous adipose tissue obtained by liposuction was washed with physiological saline. To achieve disruption of the extracellular matrix and isolation of cells, collagenase (with physiological saline as the solvent) was added, and the mixture was shaken at 37°C for 90 minutes to disperse. Subsequently, this suspension was centrifuged at 800g for 5 minutes to obtain a precipitate of stromal vascular cells. Serum-free medium for mesenchymal stem cells (Rohto) was added to the cell precipitate, and the cell suspension was centrifuged at 400g for 5 minutes. After removing the supernatant, the cells were resuspended in serum-free medium for mesenchymal stem cells (Rohto), and the cells were seeded in a flask. The cells were cultured at 37°C in 5% CO2 for several days. After several days, the culture was washed with PBS to remove any remaining blood cells or adipose tissue in the culture medium, and mesenchymal stem cells (hereinafter referred to as "ADMSCs") adhered to a plastic container were obtained.

[0083] [Cryopreservation of adipose tissue-derived mesenchymal stem cells] The obtained ADMSCs were detached using 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 (Zenoac Co.)) was added and the cells were suspended. The cell suspension was dispensed into cryotubes and stored in a freezer at -80°C. Subsequently, it was transferred to the gas phase over liquid nitrogen and storage was continued.

[0084] [Confirmation of therapeutic effects using a rat transient ischemic attack model (Koizumi model)] In rats (Wistar, Charles River Co., Ltd. Japan), the middle cerebral artery (MCA) was occluded, and 23.3 to 27.8 hours later, ADMSC, ADH, and SUS were suspended in HBSS (Hank's Balanced Salt solution) and administered intravenously (8 × 10⁻¹⁰). 6 (cells / kg). For comparison, animals that received HBSS only without cell administration were included. Body weight and neurological symptoms were observed before surgery and 14 days after surgery, and a step test and tape peel test were performed.

[0085] Before surgery and 14 days after surgery, rats were held up by their trunk, hind limbs, and right forelimb, and a step test was performed in which only the left forelimb touched the experimental table, and the rats were moved 30 cm in the opposite direction on a horizontal plane in approximately 5 seconds. The number of steps taken by the left forelimb was recorded. The test was repeated three times, and the average number of steps was used as the measurement (step test). In addition, neurological symptoms were observed, referring to the report by Kimata et al. (Pharmacology and Therapeutics, 1991;19:4491-4503).

[0086] The tape peel 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 15mm tape was applied to the underside of the right (paretic) forelimb. 2 We applied tape to the cage and measured the time it took for the animal to try to remove the tape (Cut-off was 120 seconds, tape removal test).

[0087] It was found that administering ADMSC, ADH, and SUS reduced the degree of weight loss (Figure 3). It was also found that administering ADMSC, ADH, and SUS reduced the neurological symptom score (Figure 4). It was found that administering ADMSC, ADH, and SUS increased the number of steps taken (Figure 5). It was found that administering ADMSC, ADH, and SUS shortened the time it took to remove the tape (Figure 6).

[0088] These results demonstrate an improvement in nerve damage following cerebrovascular accidents.

[0089] [Interaction between nerve cells and mesenchymal stem cells] SH-SY5Y (human neuroblastoma; ECACC, Lot.16E028, Acc Nc:94030304), stained with DiD fluorescence using Vybrant DiO cell-labeling solution (Thermo Fisher, #V22886), was seeded in a 12-well plate and cultured 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, O2:20%, CO2:5%. After 1 day of culture, the cells in each well were washed twice with D-PBS(-) and cultured for 2 hours at 37°C under conditions of N2:95% and O2:5% in DMEM, no glucose (Thermo Fisher, #11966025) medium containing Pen-Strep. Subsequently, UCMSCs prepared in the same manner as above and cultured in serum-free medium for mesenchymal stem cells (Rohto) and stained with DiO2 fluorescence were added to a 12-well plate and cultured at 37°C under conditions of O2:20% and CO2:5% in DMEM / F12 medium (Thermo Fisher, #11320-033, Lot.1930023) containing 10% FBS (Thermo Fisher, #10437-028, Lot.1658423) and Pen-Strep. Time-lapse images were taken at each time interval. In each photograph, green indicates UCMSC (cells indicated by a downward-left arrow (with a dotted line on the handle)), and red indicates SH-SY5Y (cells indicated by an upward-right arrow) (Figure 7).

[0090] It was observed that UCMSC and SH-SY5Y cells extend their processes to interact with each other, resulting in cell-to-cell interactions.

[0091] SH-SY5Y (human neuroblastoma; ECACC, Lot.16E028, Acc Nc:94030304) was seeded in a 96-well plate and cultured in DMEM / F12 medium containing 5% FBS (Thermo Fisher, #10437-028, Lot.1658423) and Pen-Strep (Thermo Fisher, #11320-033, Lot.1930023) at 37°C, O2:20%, CO2:5%. After 1 day of culture, the medium in the wells was removed and the cells were cultured for 24 hours in DMEM, No glucose medium containing Pen-Strep (Thermo Fisher, #11966025) at 37°C, N2:95%, O2:5% (hereinafter referred to as OGD conditions) (OGD treatment group). Similarly, after 1 day of culture, the culture medium in the well containing the SH-SY5Y cells was removed, and 1,000 cells / well of UCMSCs (Umbilical Cord Derived Mesenchymal Stem Cells) prepared using the method described above and cultured in serum-free medium for mesenchymal stem cells (Rohto) were added and cultured for 24 hours under OGD conditions (MSC group). For comparison, the culture medium in the well was removed after 1 day of culture, and the cells were cultured for 24 hours in fresh 5% FBS (Thermo Fisher, #10437-028, Lot.1658423) and DMEM / F12 (Thermo Fisher, #11320-033, Lot.1930023) medium containing Pen-Strep at 37°C, O2:20%, CO2:5% under the conditions of 37°C, 5% CO2, for a control group. After culturing, lactate dehydrogenase (LDH) activity was measured in each culture supernatant (Figure 8).

[0092] It was confirmed that culturing SH-SY5Y cells under OGD (Oxygen and glucose deprivation) conditions increased their LDH activity, but co-culturing them with UCMSCs suppressed this increase in LDH activity and inhibited neuronal cell death due to ischemia.

[0093] SH-SY5Y (human neuroblastoma; manufactured by ECACC, Lot. 16E028, Acc Nc: 94030304) was placed in 12-well plates, each measuring 0.038 × 10⁶. 6Cells were seeded one cell per well and cultured in DMEM / F12 medium containing 10% FBS (Thermo Fisher, #10437-028, Lot. 1658423) and Pen-Strep (Thermo Fisher, #11320-033, Lot. 1930023) at 37°C, O2: 20%, CO2: 5%. After 1 day of culture, the cells in each well were washed twice with D-PBS(-) and cultured for 2 hours under the same OGD conditions as described above (cell-free group). Similarly, after 1 day of culture, the medium in the wells was removed, and suspension cultured cells (SUS) prepared using the method described above were placed on transwell inserts seeded with half the amount (half-volume group), the same amount (same-volume group), and twice the amount (double-volume group) of seeded SH-SY5Y cells, and cultured for 2 hours under OGD conditions (MSC group). Subsequently, the cells were cultured for 48 hours at 37°C, O2: 20%, and CO2: 5% in DMEM / F12 medium containing 10% FBS (Thermo Fisher, #10437-028, Lot. 1658423) and Pen-Strep (Thermo Fisher, #11320-033, Lot. 1930023). For comparison, the cells in each well were similarly washed twice with D-PBS(-) after 1 day of culture, and then cultured for 48 hours at 37°C, O2: 20%, and CO2: 5% in fresh DMEM / F12 medium containing 10% FBS (Thermo Fisher, #10437-028, Lot. 1658423) and Pen-Strep (Thermo Fisher, #11320-033, Lot. 1930023) (Control group). After culturing, the cell activity of each SH-SY5Y cell was evaluated using WST-8 (Cell Counting Kit-8) (Figure 9).

[0094] While culturing nerve cells under OGD conditions significantly reduces their cellular activity, it was found that co-culturing them with MSCs can suppress this decrease in cellular activity.

[0095] SH-SY5Y (human neuroblastoma; manufactured by ECACC, Lot. 16E028, Acc Nc: 94030304) was placed in 0.038 × 10⁶ wells on a 12-well plate. 6Cells were seeded one cell per well and cultured in DMEM / F12 medium containing 10% FBS (Thermo Fisher, #10437-028, Lot. 1658423) and Pen-Strep (Thermo Fisher, #11320-033, Lot. 1930023) at 37°C, O2: 20%, CO2: 5%. After 1 day of culture, the cells in each well were washed twice with D-PBS(-) and cultured for 2 hours under the same OGD conditions as described above (cell-free group). Similarly, after 1 day of culture, the medium in the wells was removed, and transwell inserts seeded with the same amount of suspension cultured cells (SUS, SUS group), planar cultured cells (ADH, ADH group), and fibroblasts (Fibroblast, Fibroblast group) as SH-SY5Y were placed on top and cultured for 2 hours under OGD conditions. Subsequently, the cells were cultured for 48 hours at 37°C, O2: 20%, and CO2: 5% in DMEM / F12 medium containing 10% FBS (Thermo Fisher, #10437-028, Lot. 1658423) and Pen-Strep (Thermo Fisher, #11320-033, Lot. 1930023). For comparison, the cells in each well were similarly washed twice with D-PBS(-) after 1 day of culture, and then cultured for 48 hours at 37°C, O2: 20%, and CO2: 5% in fresh DMEM / F12 medium containing 10% FBS (Thermo Fisher, #10437-028, Lot. 1658423) and Pen-Strep (Thermo Fisher, #11320-033, Lot. 1930023) under the same conditions (Control group). After culturing, the cell activity of each SH-SY5Y cell was evaluated using WST-8 (Cell Counting Kit-8) (Figure 10).

[0096] Co-culturing with MSCs can suppress the decrease in neuronal activity caused by culturing under OGD conditions, but this effect was not observed with fibroblasts, revealing that it is an MSC-specific effect.

[0097] SH-SY5Y (human neuroblastoma; manufactured by ECACC, Lot. 16E028, Acc Nc: 94030304) was placed in 0.038 × 10⁶ wells on a 12-well plate. 6Cells were seeded one cell per well and cultured in DMEM / F12 medium containing 10% FBS (Thermo Fisher, #10437-028, Lot. 1658423) and Pen-Strep (Thermo Fisher, #11320-033, Lot. 1930023) at 37°C, O2: 20%, CO2: 5%. After 1 day of culture, 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, after 1 day of culture, the culture medium in the well was removed, and transwell inserts containing UCMSCs cultured in serum-free medium for mesenchymal stem cells (PromoCell, Inc.) (Rohto, serum-free group), PromoCell medium (Mesenchymal Stem Cell Growth Medium 2 (PromoCell, Inc., C-28009, Lot.435M415) with added Supplement mix (PromoCell, Inc., C-39809, Lot.435M126), PromoCell group), and MEM-α (Thermo Fisher, Inc., #12571-063, Lot.18997009, MEM-α group), as well as fibroblasts (Fibroblast group) cultured in the same amount as SH-SY5Y, were placed on top and cultured for 2 hours under OGD conditions. Subsequently, the cells were cultured for 48 hours at 37°C, O2: 20%, and CO2: 5% in DMEM / F12 medium containing 10% FBS (Thermo Fisher, #11320-033, Lot. 1930023) and Pen-Strep. For comparison, the cells in each well were similarly washed twice with D-PBS(-) after 1 day of culture, and then cultured for 48 hours at 37°C, O2: 20%, and CO2: 5% in fresh DMEM / F12 medium containing 10% FBS (Thermo Fisher, #10437-028, Lot. 1658423) and Pen-Strep (Thermo Fisher, #11320-033, Lot. 1930023) at 37°C, O2: 20%, and CO2: 5% (Control group). After culturing, the cell activity of each SH-SY5Y cell was evaluated using WST-8 (Cell Counting Kit-8) (Figure 11).

[0098] Co-culturing with MSCs can suppress the decrease in neuronal activity caused by culturing under OGD conditions, and this effect was found to be even more pronounced with MSCs cultured in serum-free medium.

[0099] SH-SY5Y (human neuroblastoma; manufactured by ECACC, Lot. 16E028, Acc Nc: 94030304) was placed in 0.038 × 10⁶ wells on a 12-well plate. 6Cells were seeded one cell per well and cultured in DMEM / F12 medium containing 10% FBS (Thermo Fisher, #10437-028, Lot. 1658423) and Pen-Strep (Thermo Fisher, #11320-033, Lot. 1930023) at 37°C, O2: 20%, and CO2: 5%. After 1 day of culture, 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, after 1 day of culture, the culture medium in the well was removed, and transwell inserts containing UCMSCs and fibroblasts (fibroblast group) cultured in the same amount as SH-SY5Y were placed on top of transwell inserts cultured in serum-free medium for mesenchymal stem cells (PromoCell, C-12971, Lot.427Z021), serum-free group, PromoCell medium (Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009, Lot.435M415) with added Supplement mix (PromoCell, C-39809, Lot.435M126), and MEM-α (Thermo Fisher, #12571-063, Lot.18997009, MEM-α group), respectively, and cultured under OGD conditions for 2 hours. Subsequently, cells were cultured for 24-48 hours at 37°C, O2: 20%, and CO2: 5% in DMEM / F12 medium containing 10% FBS (Thermo Fisher, #10437-028, Lot. 1658423) and Pen-Strep (Thermo Fisher, #11320-033, Lot. 1930023). After 48 hours of culture, cell images were acquired (Figure 12). EthD-III was used for staining dead cells. After counting the number of dead cells in each image, the dead cell rate was calculated as "number of dead cells / absorbance of WST-8" (Figure 13).

[0100] The serum-free group showed higher cell proliferation activity compared to the cell-free group, the fibroblast group, the PromoCell group, and the MEM-α group. Furthermore, the serum-free group had a lower number of dead cells relative to the total cell count compared to the cell-free group, the PromoCell group, and the MEM-α group. [Industrial applicability]

[0101] According to the present invention, a novel therapeutic agent for neurological disorders can be provided.

Claims

1. A neuropathogenic agent containing mesenchymal stem cells, wherein the mesenchymal stem cells express one or more of VEGFA, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, and NRP2 at a level at least twice as high as mesenchymal stem cells obtained by planar culture using serum-free medium derived from umbilical cord tissue, are derived from umbilical cord tissue, are prepared by suspension culture using serum-free medium, and the suspension culture method is characterized by a method of culturing by adhering cells to a microcarrier and agitating the microcarrier, and the microcarrier is based on a material selected from polyester, polystyrene, glass, or dextran.

2. The neuropathological agent according to claim 1, wherein the mesenchymal stem cells are of allogeneic origin.

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