JP2024023760A - Mesenchymal stem cells and therapeutic agents for neurological disorders - Google Patents

Abstract

An object of the present invention is to provide a novel therapeutic agent for neurological disorders. [Solution] The present invention for solving the above problems includes HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL , BMP2, NTN1, ASCL1, and NRP2 are mesenchymal stem cells characterized by high expression of one or more of them. [Selection diagram] None

Description

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

As society continues to age, neurodegenerative diseases, which are disorders of brain nerve cells, 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 areas such as the cerebral cortex and hippocampus drop out, leading to a decline in memory ability. Additionally, in Parkinson's disease, nerve cells in a region called the substantia nigra drop out, resulting in impaired motor function. Furthermore, in addition to neurodegenerative diseases, cerebral infarction and cerebral hemorrhage due to stroke are known as diseases caused by damage to brain nerve cells.

Additionally, perinatal neonatal brain damage occurs in 1 to 2 out of 1,000 births, and is a cause of lifelong cerebral palsy. Perinatal brain disorders that cause cerebral palsy include hypoxic-ischemic encephalopathy, cerebral hemorrhage, and periventricular leukomalacia, and the main pathological condition of these is an increase in active oxygen caused by mitochondrial dysfunction. , an inflammatory condition consisting of macrophage activation and associated hypercytokinemia. Although the causes and symptoms of the above-mentioned neurodegenerative diseases, stroke, and cerebral palsy are different, it can be said that they have one thing in common: a decrease in the number of nerve cells.

Treatment drugs such as donepezil for Alzheimer's disease and levodopa for Parkinson's disease are used, but these drugs restore the information processing function of the neural network caused by the loss of nerve cells by replenishing chemical transmitter signals. The purpose of this treatment is to reduce the number of nerve cells, and it cannot prevent the loss of nerve cells itself. Furthermore, among the existing therapeutic drugs for neurodegenerative diseases and stroke, none show an action to protect nerve cells. Therefore, the development of new therapeutic agents that have the effect of protecting nerve cells is desired.

Mesenchymal stem cells are multipotent progenitor cells that were 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 fat, and mesenchymal stem cell transplantation is expected to be a new treatment method for various intractable diseases ( (See Patent Documents 1 and 2). Recently, it has been known that cells with equivalent functions exist in interstitial cells such as adipose tissue, placenta, umbilical cord, and ovarian membrane. Therefore, mesenchymal stem cells are sometimes referred to as stromal cells.

Japanese Patent Application Publication No. 2012-157263 Special Publication No. 2012-508733

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

The present invention aims to provide a new therapeutic agent for neurological disorders under the above-mentioned circumstances.

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

That is, the present invention relates to:
[1] Any one of HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, NRP2 A mesenchymal stem cell characterized by high expression of one or more of the following.
[2] The mesenchymal stem cell according to [1], which is allogeneic.
[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], which is prepared by a suspension culture method.
[5] A therapeutic agent for neurological disorders containing the mesenchymal stem cells according to any one of [1] to [4].

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

FIG. 1 is a diagram showing the results of comparing the mRNA expression levels of various factors in mesenchymal stem cells cultured in various media. FIG. 2 is a diagram showing the results of comparing the mRNA expression levels of various factors in flat culture cells (ADH) and suspension culture cells (SUS) regarding mesenchymal stem cells. FIG. 3 is a diagram showing the effect of administering mesenchymal stem cells obtained by culturing using various culture methods to a rat transient cerebral ischemia model (body weight). FIG. 2 is a diagram showing the effect of administering mesenchymal stem cells obtained by culturing using various culture methods to a rat transient cerebral ischemia model (neurological symptom score). FIG. 3 is a diagram showing the effect of administering mesenchymal stem cells obtained by culturing using various culture methods to a rat transient cerebral ischemia model (number of steps). FIG. 2 is a diagram showing the effect of administering mesenchymal stem cells obtained by culturing using various culture methods to a rat transient cerebral ischemia model (tape peeling test). This is a time-lapse image showing cell-cell interactions between neurons and mesenchymal stem cells. FIG. 2 is a diagram showing the neuronal cell death-inhibiting effect of mesenchymal stem cells obtained by culture in a serum-free medium (Rohto). FIG. 2 is a diagram showing the dose-dependent neuron activation effect of mesenchymal stem cells cultured in a serum-free medium (Rohto). FIG. 2 is a diagram showing the neuron activation effect of mesenchymal stem cells obtained by planar (ADH) or suspension (SUS) culture using a serum-free medium (Rohto) (comparison with fibroblasts). FIG. 2 is a diagram showing the neuron activation effect of mesenchymal stem cells cultured in a serum-free medium (Rohto) (comparison with cells cultured in other media). This is a cell photograph showing the neuronal cell death suppressing effect of mesenchymal stem cells cultured in a serum-free medium (Rohto) (comparison with cells cultured in other media). FIG. 2 is a diagram showing the neuronal cell death-inhibiting effect of mesenchymal stem cells cultured in a serum-free medium (Rohto) (comparison with cells cultured in other media).

Hereinafter, the mesenchymal stem cells and the therapeutic agent for neurological disorders of the present invention will be explained in detail.

[Mesenchymal stem cells]
The mesenchymal stem cells of the present invention contain 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 one or more of YRPW motif-like), BMP2 (bone morphogenetic protein 2), NTN1 (netrin 1), ASCL1 (achaete-scute family bHLH transcription factor 1), and NRP2 (neuropilin 2) It is characterized by

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 protecting neurons 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 inflammatory responses. DLL1 belongs to the DSL family of Notch ligands and is known to be involved in regulating Notch signals.

In addition, 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 were highly expressed. "Existing" includes that each mRNA expression is highly expressed, each protein is highly expressed, or both are highly expressed.

Furthermore, the mesenchymal stem cells of the present invention only need to express the above-mentioned factors at a higher level than other cells, but specifically, the mesenchymal stem cells of the present invention may be used under conventional culture conditions (e.g. , culture in MEM-α medium containing 10% FBS). Preferably, the expression level is at least twice that of mesenchymal stem cells obtained under conventional culture conditions, more preferably at least 5 times, still more preferably at least 10 times, particularly preferably at least 100 times.

Compared to fibroblasts, the mesenchymal stem cells of the present invention have the following characteristics: HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, It is sufficient that one or more of HEYL, BMP2, NTN1, ASCL1, and NRP2 are highly expressed. It is preferably expressed twice or more, more preferably 5 times or more, still more preferably 10 times or more, particularly preferably 100 times or more compared to skin fibroblasts.

In the present invention, mesenchymal stem cells refer to mesenchymal stem cells that have the ability to differentiate into one or more types of mesenchymal cells (bone cells, cardiomyocytes, chondrocytes, tendon cells, adipocytes, etc.) and that maintain this ability. Refers to cells that can proliferate. The term mesenchymal stem cells used in the present invention means the same cells as stromal cells, and there is no particular distinction between the two. In addition, they are sometimes simply referred to as mesenchymal cells. Tissues containing mesenchymal stem cells include, for example, adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amniotic membrane, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, Examples include 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 referred to as adipose tissue-derived stromal cells. Among these, from the viewpoint of effectiveness for the treatment of neuropathic diseases and ease of availability, there are three types of mesenchymal stem cells: adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and placenta-derived mesenchymal stem cells. 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 most preferred.

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

Mesenchymal stem cells are less susceptible to rejection even in allogeneic subjects, so pre-prepared donor cells are expanded and cryopreserved and used as mesenchymal stem cells in the disease therapeutic agent of the present invention. Can be used as stem cells. Therefore, compared to the case where one's own mesenchymal stem cells are prepared and used, the mesenchymal stem cells in the present invention are easier to commercialize and can stably obtain a certain effect. More preferably, it is allogeneic.

In the present invention, mesenchymal stem cells refer to any cell population containing mesenchymal stem cells. The cell population is 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.

In the present invention, the umbilical cord is a white tubular tissue that connects the fetus and placenta, and is composed of the umbilical vein, umbilical artery, glue-like tissue (Wharton's Jelly), and the umbilical cord matrix itself, and contains many mesenchymal stem cells. include. The umbilical cord is preferably obtained from an animal of the same species as the subject (administration target) to which the disease therapeutic agent of the present invention is to be used, and more preferably from a human animal in view of administering the disease therapeutic agent of the present invention to humans. It is the umbilical cord.

In the present invention, adipose tissue refers to a tissue containing adipocytes and interstitial cells including microvascular cells, and is, for example, a tissue obtained by surgically removing or suctioning subcutaneous fat of a mammal. Adipose tissue can be obtained from subcutaneous fat. It is preferable that the mesenchymal stem cells derived from adipose tissue, which will be described later, be obtained from an animal of the same species as the subject to whom the adipose tissue-derived mesenchymal stem cells are administered, and in consideration of administration to humans, human subcutaneous fat is more preferable. Although the subcutaneous fat supplying individual may be alive or dead, the adipose tissue used in the present invention is preferably tissue collected from a living individual. When harvesting from an individual, examples of liposuction include PAL (power assisted) liposuction, Erconia laser liposuction, or body jet liposuction. It is preferable not to use.

In the present invention, bone marrow refers to soft tissue that fills the inner cavity of bones, and is a hematopoietic organ. Bone marrow fluid exists in the bone marrow, and the cells present within it are called bone marrow cells. Bone marrow cells include erythrocytes, granulocytes, megakaryocytes, lymphocytes, adipocytes, etc., 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 bones, or other bones.

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

Mesenchymal stem cells in the present invention include HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 , In addition to highly expressing any one or more of NRP2, for example, growth characteristics (e.g., population doubling ability from passage to senescence, doubling time), karyotype analysis (e.g., normal karyotype, maternal or neonatal strains), 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 arrays; polymerase chain reactions such as reverse transcription PCR, real-time PCR, conventional PCR), miRNA expression profiling, protein arrays, protein secretion such as cytokines (e.g. plasma coagulation analysis, ELISA, cytokine arrays), metabolites (metabolomic analysis), books It may also be characterized by other methods known in the art.

(Mesenchymal stem cell preparation method)
One of 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 that highly express two or more is not particularly limited, but can be prepared, for example, as follows. That is, mesenchymal stem cells are isolated and cultured from tissues such as fat, umbilical cord, bone marrow, etc. according to methods 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, It can be obtained by separating cells that highly express CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or NRP2 using a cell sorter, magnetic beads, etc. In addition, HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, 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 Mesenchymal stem cells highly expressing , HEYL, BMP2, NTN1, ASCL1 or NRP2 can also be obtained. In the cell population obtained by this induction, more than 50% of the cell population contains HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, It is preferable that HEYL, BMP2, NTN1, ASCL1 or NRP2 is highly expressed, and 70% or more is HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, It is more preferable that GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or NRP2 is highly expressed, and 80% or more is HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3. , PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or NRP2 is more preferably highly expressed, and 90% or more is HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, It is particularly preferable that CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or NRP2 is highly expressed, and 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, during high 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 The method for preparing leaf stem cells will be specifically explained.

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

The umbilical cord can be recovered by appropriately removing the placenta from postpartum tissues including the umbilical cord and the placenta delivered by vaginal delivery or caesarean section. After removing umbilical cord blood from the collected umbilical cord, sterilization or antibacterial treatment may be performed. Removal of cord blood is accomplished by rinsing with an anticoagulant solution, such as a heparin-containing solution. Aseptic or antibacterial treatments include, but are not limited to, application of povidone-iodine, or media supplemented with one or more antibiotics and/or antifungals, such as penicillin, streptomycin, amphotericin B, gentamicin, and nystatin. Alternatively, it may be immersed in a buffer. Furthermore, if necessary, the method may include a step of selectively lysing red blood cells. Methods well known in the art can be used to selectively lyse red blood cells, such as incubation in hypertonic or hypotonic media by lysis with ammonium chloride.

The umbilical cord-derived cells of the present invention refer to a cell population prepared using the umbilical cord as a raw material, and may be obtained by any known production method, for example, by a method including the following steps (i) to (iii). can do:
(i) cutting the umbilical cord;
(ii) a step of culturing the umbilical cord obtained in step (i); and (iii) a step of subculture.

Another method for preparing the cells may include (i') dissociating the tissue by treating the umbilical cord with an enzyme instead of (i) cutting the umbilical cord. Furthermore, in addition to the step of (i) cutting the umbilical cord, the method may also include the step of (i') dissociating tissue by treating the umbilical cord with an enzyme.

In the step (i) of cutting the umbilical cord of the present invention, the umbilical cord obtained by the above method is cut by mechanical force (shredding force or shearing force) in a state containing amniotic membrane, blood vessels, perivascular tissue, and Walton's jelly. This can be done by Although not particularly limited, examples of the size of the umbilical cord section obtained by cutting are 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 (i') of dissociating tissue by enzymatically treating the umbilical cord of the present invention, the umbilical cord obtained by the above method is subjected to enzyme treatment in a state containing amniotic membrane, blood vessels, perivascular tissue, and Walton's jelly. This can be done in a step of dissociating the tissue. Although not particularly limited, examples of the enzyme treatment include enzyme treatment using one or more enzymes such as collagenase, dispase, and hyaluronidase.

(ii) 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 culture medium. Culture at density and culture conditions.

The medium used in this step is not particularly limited as long as it is a medium that can culture mesenchymal stem cells, but such a medium may be a basal medium with serum added, and/or albumin, transferrin, fatty acids, insulin. , sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. may be added. Substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts are added to these media as necessary. It's okay.

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, MCDB201. Examples include a medium and a mixed medium thereof.

Examples of the above 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, etc. . When using serum, it may be added in an amount of 5 v/v% to 15 v/v%, preferably 10 v/v%, to the basal medium.

Examples of the fatty acids include, but are not limited to, linoleic acid, oleic acid, linoleic acid, arachidonic acid, myristic acid, palmitoic acid, palmitic acid, and stearic acid. Examples of lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and the like. 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, etc. . Examples of proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of the polysaccharide include glycosaminoglycans, and among the glycosaminoglycans, hyaluronic acid, heparan sulfate, and the like are particularly exemplified, but the polysaccharide is not limited thereto. Growth factors include, for example, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), and epidermal growth factor (EGF). Examples include, but are not limited to, connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), and the like. 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 that does not contain xenogeneic components such as serum (xeno-free). Such media are provided as pre-prepared media for mesenchymal stem cells (stromal cells) by, for example, PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, Corning, and Rohto. has been done.

In the present invention, the term "solid surface" refers to any material that enables binding and adhesion of adipose tissue-derived mesenchymal stem cells in the present invention. In certain embodiments, such materials are plastic materials that have been treated to promote binding and adhesion of mammalian cells to their surfaces. Although the shape of the culture container having a solid surface is not particularly limited, petri dishes, flasks, etc. are preferably used. Cells are washed after incubation to remove unbound cells and cell debris.

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

The umbilical cord tissue-derived mesenchymal stem cells of the present invention can also be produced using a suspension culture production method. Examples of suspension culture production methods include a method in which cells are aggregated and cultured with stirring as a cell mass on a sphere, and a method in which cells are adhered to microcarriers and cultured by stirring the microcarriers. Note that stirring can be done by rotating a stirring blade in a container with a stirrer, or by placing a bag containing the culture solution and cells on a shaker and shaking the bag to suspend the culture solution. Further, the medium used in the floating culture production method is not particularly limited as long as it is a medium that can culture 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, but examples include polyester, polystyrene, glass, and dextran.

Adipose tissue-derived mesenchymal stem cells may be obtained, for example, by the production method described in US Pat. No. 6,777,231, and may be produced by a method including the following steps (i) to (iii). can:
(i) obtaining a cell suspension by enzymatic digestion of adipose tissue;
(ii) sedimenting the 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.

The adipose tissue used in step (i) is preferably washed. Washing may be performed using a physiologically compatible saline solution, such as phosphate buffered saline (PBS), by sedimentation with vigorous agitation. This is to remove impurities (also called debris, such as damaged tissue, blood, red blood cells, etc.) contained in the fat tissue from the tissue. Therefore, washing and sedimentation are generally repeated until the supernatant is thoroughly cleared of debris. The remaining cells exist as clumps of various sizes, so in order to dissociate them while minimizing damage to the cells themselves, the washed cell clumps are treated with enzymes that weaken or break intercellular bonds (e.g. Treatment with collagenase, dispase, trypsin, etc.) is preferred. The amount of such enzymes and duration of treatment will vary depending on the conditions used, but are known in the art. Instead of or in conjunction with such enzymatic treatment, cell clumps can be broken down by other treatments such as mechanical agitation, ultrasonic energy, thermal energy, etc., but with minimal damage to the cells. In order to suppress this, it is preferable to use only enzyme treatment. When using an enzyme, it is desirable to deactivate the enzyme using a medium or the like after an appropriate period of time in order to minimize harmful effects on cells.

The cell suspension obtained by step (i) contains a slurry or suspension of aggregated cells, as well as various contaminant cells, such as red blood cells, smooth muscle cells, endothelial cells, and fibroblasts. Therefore, the aggregated cells and these contaminant cells may be separated and removed subsequently, but since they can be removed by adhesion and washing in step (iii) described below, such separation and removal are omitted. It's okay. Separation and removal of contaminant cells can be achieved by centrifugation that forcibly separates the cells into a supernatant and a precipitate. The resulting precipitate containing contaminant cells is suspended in a physiologically compatible solvent. The suspended cells may contain red blood cells, but red blood cells are excluded by selection based on adhesion to the solid surface, which will be described later, so a lysing step is not necessarily necessary. Methods well known in the art can be used to selectively lyse red blood cells, such as incubation in hypertonic or hypotonic media by lysis with ammonium chloride. After lysis, the lysate may be separated from the desired cells, eg, by filtration, centrifugation, or density fractionation.

In step (ii), in order to increase the purity of the mesenchymal stem cells in the suspended cells, they may be washed once or multiple times in succession, centrifuged, and resuspended in a medium. 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 is a medium that can culture mesenchymal stem cells, but such a medium may be a basal medium supplemented with serum and/or albumin, transferrin, fatty acids, It may be made by adding one or more serum substitutes such as insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. Substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts are added to these media as necessary. It's okay.

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, MCDB201. Examples include a medium and a mixed medium thereof.

Examples of the above 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, etc. . When using serum, it may be added in an amount of 5 v/v% to 15 v/v%, preferably 10 v/v%, to the basal medium.

Examples of the fatty acids include, but are not limited to, linoleic acid, oleic acid, linoleic acid, arachidonic acid, myristic acid, palmitoic acid, palmitic acid, and stearic acid. Examples of lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and the like. 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 proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of the polysaccharide include glycosaminoglycans, and among the glycosaminoglycans, hyaluronic acid, heparan sulfate, and the like are particularly exemplified, but the polysaccharide is not limited thereto. Growth factors include, for example, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), and epidermal growth factor (EGF). Examples include, but are not limited to, connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), and the like. 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 that does not contain xeno-derived components such as serum (xeno-free). Such media are provided as pre-prepared media for mesenchymal stem cells (stromal cells) by, for example, PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, Corning, and Rohto. has been done.

Subsequently, in step (iii), the cells in the cell suspension obtained in step (ii) are grown on a solid surface without differentiation to an appropriate cell density and using an appropriate cell culture medium as described above. Cultivate under culture conditions. In the present invention, the term "solid surface" refers to any material that enables binding and adhesion of adipose tissue-derived mesenchymal stem cells in the present invention. In certain embodiments, such materials are plastic materials that have been treated to promote binding and adhesion of mammalian cells to their surfaces. Although the shape of the culture container having a solid surface is not particularly limited, petri dishes, flasks, etc. are preferably used. Cells are washed after incubation to remove unbound cells and cell debris.

In the present invention, cells that ultimately remain bound and adhered to a 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 according to the present invention, surface antigens may be analyzed using conventional methods such as flow cytometry. Additionally, the ability to differentiate into each cell lineage may be tested, and such differentiation can be performed using conventional methods.

Mesenchymal stem cells in the present invention can be prepared as described above, but may also be defined as cells having the following characteristics;
(1) Adhesive to plastic under culture conditions in standard medium;
(2) Positive for surface antigens CD73 and CD90, negative for CD45, and (3) Able to differentiate into osteocytes, 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, HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1 can be isolated by selectively separating cells that highly express NTN1, ASCL1, or NRP2 proteins using immunological methods using cell sorters, magnetic beads, etc. Mesenchymal stem cells highly expressing , CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1 or NRP2 proteins can be obtained. Also, specific drugs capable of inducing 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 induces HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL It is also possible to obtain mesenchymal stem cells that highly express BMP2, NTN1, ASCL1, or NRP2. As an example, a specific method for selective separation by immunological techniques using a cell sorter will be described below.

The mesenchymal stem cells prepared above are treated with a trypsin/EDTA solution and the resulting cell suspension is centrifuged (room temperature, 400 G, 5 minutes) to remove the supernatant. Add Staining Buffer (1% BSA-PBS) to the cells and adjust to 1 x 10 ⁶ cells/500 μL. After making the cell suspension concentration uniform by pipetting, transfer 50 μL to a new 1.5 mL microtube. Dispense in portions. 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 kept in the refrigerator for 30 minutes to 1 hour, protected from light. Make it react. After washing 3 times with 1 mL of Staining Buffer, add Staining Buffer to make 50 μL, add secondary antibody (Anti Mouse IgG alexar488, manufactured by Thermofisher Scientific, A21202) at a concentration of 1 to 10 μg/mL, and suspend. , incubate for 30 minutes to 1 hour under light-shielded and refrigerated conditions. After washing three times with 1 mL of Staining Buffer, add 300 μL of PI Buffer (prepared by adding 28.8 μL of Propidium iodide solution (manufactured by SIGMA, P4864) to 14.4 mL of Staining Buffer), suspend well, and place in a cell strainer. It can be passed through a tube with an attached tube and separated using fluorescence activated cell sorting (FACS).

(Cryopreservation of mesenchymal stem cells)
The mesenchymal stem cells in the present invention may be cells that have been cryopreserved and thawed repeatedly, 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 suspension. Suspension can be performed by detaching the cells with a detachment agent such as trypsin, transferring the cells to a cryopreservation container, treating the cells as appropriate, and then adding a cryopreservation solution.

The cryopreservation solution may contain DMSO (dimethyl sulfoxide) as a cryoprotectant, but since DMSO has the property of inducing differentiation of mesenchymal stem cells in addition to cytotoxicity, the DMSO content is It is preferable to reduce Glycerol, propylene glycol or polysaccharides are exemplified as substitutes for DMSO. If 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, additives described in WO2007/058308 may also be included. Examples of such cryopreservation solutions include cryopreservation solutions provided by BioVerde, Nippon Genetics, Reprocell, Zenoac, Cosmo Bio, Kojin Bio, and Thermo Fisher Scientific. A liquid may also 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 any freezer that can achieve this temperature. obtain. Although not particularly limited, in order to avoid sudden temperature changes, a programmable freezer may be used to appropriately control the cooling rate. The cooling rate may be appropriately selected depending on the components of the cryopreservation solution, and may be performed according to the instructions of the cryopreservation solution manufacturer.

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, Examples include 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, it may be stored in a gas phase above liquid nitrogen (approximately below -150°C to above -180°C). When storing in the gas phase on liquid nitrogen, storage containers well known to those skilled in the art can be used. Although not particularly limited, for example, when storing for two weeks or more, it is preferable to store in a gas phase over liquid nitrogen.

The thawed mesenchymal stem cells may be cultured as appropriate before the next cryopreservation. Cultivation of mesenchymal stem cells is carried out using the above-mentioned medium capable of culturing mesenchymal stem cells, and is not particularly limited, at a culture temperature of about 30 to 40°C, preferably about 37°C, and in an atmosphere containing _CO2. It may also be carried out in an atmosphere. The CO ₂ concentration is about 2-10%, preferably about 5-10%. During culture, after reaching an appropriate confluency for the culture vessel (for example, cells occupy 50% to 80% of the culture vessel), cells are detached using a detachment agent such as trypsin. However, the cells may be seeded at an appropriate cell density in a separately prepared culture container and culture may be continued. Typical cell densities for seeding cells 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 2,000 to 10,000 cells/cm ² , and 2,000 to 10,000 cells/cm ² . In certain embodiments, the cell density is between 2,000 and 10,000 cells/cm ² . It is preferable to adjust the period until appropriate confluency is reached from 3 days to 7 days. During culture, the medium may be replaced as necessary.

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

The mesenchymal stem cells of the present invention may be cells in any state, such as cells recovered by detaching cells in culture, or cells frozen in a cryopreservation solution. good. It is preferable to use cells of the same lot obtained through expansion culture that are subdivided and cryopreserved because similar effects can be stably obtained and they are easy to handle. Mesenchymal stem cells in a cryopreserved state may be thawed immediately before use, and suspended in a cryopreservation solution and directly mixed into an infusion solution or a solution such as a culture medium. Alternatively, the frozen preservation solution may be removed by a method such as centrifugation, and then suspended in a solution such as an infusion solution or a culture medium. Here, the "infusion" in the present invention refers to a solution used in human treatment, and is not particularly limited, but includes, for example, physiological saline, JP physiological saline, 5% glucose solution, JP Glucose injection solution, Ringer's solution, Ringer's solution JP, Lactated Ringer's solution, Ringer's acetate solution, Solution No. 1 (starting solution), Solution No. 2 (dehydration replenishment solution), Solution No. 3 (maintenance solution), Solution No. 4 (post-operative recovery solution) etc.

[Treatment for neuropathy]
The neuropathy therapeutic agent of the present invention includes HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, APOE, PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL of the present invention described above. Contains mesenchymal stem cells that highly express one or more of BMP2, NTN1, ASCL1, and NRP2. According to the therapeutic agent for neuropathy of the present invention, neuropathy can be effectively protected. Regarding mesenchymal stem cells containing the neuropathy therapeutic agent of the present invention, the explanation in the section on mesenchymal stem cells above can be applied.

In addition to the mesenchymal stem cells mentioned above, the therapeutic agent for neuropathy of the present invention may be prepared using pharmaceutically acceptable carriers and additives according to the usage and form, as long as the effects of the present invention are not impaired. It may also contain substances. Such carriers and additives include, for example, isotonic agents, thickeners, sugars, sugar alcohols, preservatives, bactericidal or antibacterial agents, pH regulators, stabilizers, and chelating agents. , oil base, gel base, surfactant, suspending agent, binder, excipient, lubricant, disintegrant, foaming agent, fluidizing agent, dispersing agent, emulsifier, buffering agent, solubilizing agent , antioxidants, sweeteners, acidulants, coloring agents, flavoring agents, fragrances, cooling agents, etc., but are not limited to these. Typical components include, for example, the following carriers and additives.

Examples of carriers include aqueous carriers such as water and aqueous ethanol; examples of tonicity agents (inorganic salts) include sodium chloride, potassium chloride, calcium chloride, and magnesium chloride; examples of polyhydric alcohols include, for example. , glycerin, propylene glycol, polyethylene glycol, etc.; as thickeners, for example, carboxyvinyl polymer, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, alginic acid, polyvinyl alcohol (fully or partially saponified), polyvinyl pyrrolidone, macrogol Examples of sugars include cyclodextrin, glucose, etc.; Examples of sugar alcohols include xylitol, sorbitol, mannitol, etc. (these may be d-, l-, or dl-isomers); preservatives; Examples of bactericidal or antibacterial agents include dibutylhydroxytoluene, butylhydroxyanisole, alkyldiaminoethylglycine hydrochloride, sodium benzoate, ethanol, benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, sorbic acid, and sorbin. potassium acid, trometamol, sodium dehydroacetate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, oxyquinoline sulfate, phenethyl alcohol, benzyl alcohol, biguanide compounds (specifically, polyhexanide hydrochloride ( (polyhexamethylene biguanide), Glokill (trade name manufactured by Rhodia), etc.; Examples of pH regulators include hydrochloric acid, boric acid, aminoethylsulfonic acid, epsilon-aminocaproic acid, citric acid, acetic acid, and sodium hydroxide. , potassium hydroxide, calcium hydroxide, magnesium hydroxide, sodium bicarbonate, sodium carbonate, borax, triethanolamine, monoethanolamine, diisopropanolamine, sulfuric acid, magnesium sulfate, phosphoric acid, polyphosphoric acid, propionic acid, sulfur acids, gluconic acid, fumaric acid, lactic acid, tartaric acid, malic acid, succinic acid, gluconolactone, ammonium acetate, etc.; as stabilizers, for example, dibutylhydroxytoluene, trometamol, sodium formaldehyde sulfoxylate (Rongalit), Tocopherol, sodium pyrosulfite, monoethanolamine, aluminum monostearate, glyceryl monostearate, sodium bisulfite, sodium sulfite, etc.; oil bases include, for example, olive oil, corn oil, soybean oil, sesame oil, cottonseed oil, etc. Vegetable oils, medium chain fatty acid triglycerides, etc.; aqueous bases include, for example, macrogol 400; gel bases include, for example, carboxyvinyl polymers, gums, etc.; surfactants include, for example, polysorbate 80 , hydrogenated castor oil, glycerin fatty acid ester, sorbitan sesquioleate, etc.; As a suspending agent, for example, white beeswax, various surfactants, gum arabic, gum arabic powder, xanthan gum, soybean lecithin, etc.; as a binder Examples of excipients include hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol; examples of excipients include sucrose, lactose, starch, corn starch, crystalline cellulose, and light anhydrous silica. Examples of lubricants include sucrose fatty acid ester, magnesium stearate, talc, etc.; Examples of disintegrants include low-substituted hydroxypropyl cellulose, crospovidone, croscarmellose sodium, etc.; Examples of the agent include sodium hydrogen carbonate; examples of the fluidizing agent include sodium aluminate metasilicate, light anhydrous silicic acid, and the like.

The therapeutic agent for neuropathy of the present invention can be provided in various forms depending on the purpose, for example, various dosage forms such as solid preparations, semi-solid preparations, and liquid preparations. For example, solid preparations (tablets, powders, powders, granules, capsules, etc.), semi-solid preparations [ointments (plaques, ointments, etc.), creams, etc.], liquid preparations [lotion, extracts, suspensions, etc.] Cloudy agents, emulsions, syrups, injections (including infusions, implantable injections, long-acting injections, ready-to-use injections), dialysis agents, aerosols, soft capsules, drinks, etc.], patches It can be used in the form of tablets, poultices, etc. The therapeutic agent for neuropathy of the present invention can also be used in the form of a solution or emulsion in an oily or aqueous vehicle. Furthermore, the neuropathy therapeutic agent of the present invention can be applied to the affected area by spraying, and the neuropathy therapeutic agent of the present invention can also be used in the form of being gelled or sheeted on the affected area after being sprayed. The therapeutic agent for neurological disorders 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 therapeutic agent for neuropathy of the present invention includes physiological saline, JP physiological saline, 5% glucose solution, JP glucose injection, Ringer's solution, JP Ringer's solution, lactated Ringer's solution, acetic acid Ringer's solution, bicarbonate Ringer's solution, No. 1 solution ( Suspension or dilution using an infusion solution such as starting solution), Solution No. 2 (dehydration replenishment solution), Solution No. 3 (maintenance solution), Solution No. 4 (post-operative recovery solution), or a cell culture medium such as DMEM. It can be used by suspending or diluting it, preferably in physiological saline, 5% glucose solution, No. 1 solution (starting solution), more preferably in 5% glucose solution, No. 1 solution (starting solution). be able to.

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

When the neuropathy therapeutic agent of the present invention is a liquid preparation, the osmotic pressure of the neuropathy therapeutic agent is not particularly limited as long as it is within a range acceptable to living organisms. An example of the osmotic pressure ratio of the composition of the present invention is a range of preferably 0.7 to 5.0, more preferably 0.8 to 3.0, and still more preferably 0.9 to 1.4. Osmotic pressure can be adjusted by methods 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 edition of the Japanese Pharmacopoeia, and the osmotic pressure is determined by the osmotic pressure measurement method ( Measure using the freezing point depression method) as a reference. The standard solution for osmotic pressure ratio measurement (0.9 w/v% sodium chloride aqueous solution) is prepared by drying sodium chloride (Japanese Pharmacopoeia standard reagent) at 500 to 650°C for 40 to 50 minutes, and then drying it in a desiccator (silica gel). Allow to cool, then accurately weigh 0.900 g and dissolve in purified water to make exactly 100 mL, or use a commercially available standard solution for osmotic pressure ratio measurement (0.9 w/v % sodium chloride aqueous solution).

The administration route of the therapeutic agent for neuropathy of the present invention to a subject includes oral administration, subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intraventricular administration, intrathecal administration, intraperitoneal administration, sublingual administration, Examples include transrectal administration, intravaginal administration, intraocular administration, nasal administration, inhalation, transdermal administration, implant, direct administration by spraying on the surface of an organ, pasting a sheet, etc., and the therapeutic agent for neuropathy of the present invention. From the viewpoint of effectiveness, intraarterial administration, intravenous administration, intraventricular administration, and intrathecal administration are preferable, and from the viewpoint of reducing the burden on the subject, intravenous administration, intramuscular administration, and intranasal administration are more preferable. From the viewpoint of effectiveness, intraventricular administration and intrathecal administration are preferred.

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

The therapeutic agent for neuropathy of the present invention may be administered together with one or more other drugs. Other drugs include any drug that can be used as a treatment for neurological disorders, such as antiparkinsonian drugs such as levodopa, amantadine, and carbidopa, and dopamine agonists such as bromocriptine, purgolide, ropinirole, and pramipexole. drugs, selective B-type monoamine oxidase inhibitors (MAO-B) such as selegiline and lasalidine, catechol-O-methyltransferase (COMT) inhibitors such as entacapone and tolcapone, anticholinergic drugs such as benztropon and trihexyphenidyl, Antihistamines such as diphenhydramine and orphenadrine, cholinesterase inhibitors such as donepezil, rivastigmine, galantamine, and tacrine, N-methyl-D-aspartate receptor antagonists such as memantine, haloperidol, thioridazine, thiothixene, olanzapine, risperidone, Antipsychotics such as 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. Drugs, tricyclics, antidepressants such as venlafaxine, bupropion, amitriptyline, desipramine, paroxetine, central alpha-2 adrenergic agonists such as clonidine, tizanidine, corticosteroids such as dexamethasone, prednisolone, amantadine, dextrin NMDA receptor antagonists such as Metrophan, local anesthetics such as lidocaine, mexiletine, and capsaicin, etodolac, indomethacin, sulinochloride, tolmethatin, nabumetone, piroxicam, acetaminophen, fenobiprone, flurbiprone, ibuprofen, ketoprofen, naproxen, naproxen sodium , oxaprosin, aspirin, choline magnesium trisalicylate, diflunisal, meclofenamate, mefenamic acid, phenylbutazone, ketorolac, celecoxib, codeine, hydrocodeine, propoxyphene, fentanyl, hydromorphone, levophanol, meperidine, methadone, morphine, oxycodone, oxymorphone , analgesics such as buprenorphine, butorphanol, nalbuphine, and pentazocine, radical scavengers such as propofol, anti-inflammatory agents, serotonin, norepinephrine, NSAIDs, and ginkgo biloba extract. Further, in addition to the administration of the therapeutic agent for neurological disorders of the present invention, low-temperature therapy such as hypothermia therapy, cerebral hypothermia therapy, and cerebral hypothermia therapy can also be performed together.

The mesenchymal stem cells of the present invention can be used for various neurological disorders, but specific diseases include autonomic nervous system disorders such as autonomic neuropathy, Horner's syndrome, multiple system atrophy, pure autonomic failure, and chronic pain. , neuropathic pain, pain such as complex regional pain syndrome, ischemic stroke, transient ischemic attack, hypoxia-ischemia, intracranial hemorrhage such as intracerebral hemorrhage/intraventricular hemorrhage, subarachnoid hemorrhage, etc. Stroke (cerebrovascular accident), Alzheimer's disease, cerebrovascular dementia, Lewy body dementia, HIV-related dementia, dementia such as frontotemporal dementia, convulsive syndrome, athetosis or dyskinesia syndrome, and ataxic syndrome Cerebral palsy syndrome such as, hypoglycemia, hypernatremia, hyponatremia, hypomagnesemia, neonatal convulsive disease due to inborn metabolic abnormalities, demyelinating diseases such as multiple sclerosis, Killan-Barre syndrome, genetic peripheral nervous system disorders such as sexual neuropathy, amyotrophic lateral sclerosis (ALS), myasthenia gravis, mononeuropathy, polyneuropathy, and plexopathy, acute transverse myelitis, arteriovenous malformations, Examples include spinal cord disorders such as spinal cord infarction (ischemic spinal cord disorder), cerebellar diseases such as spinocerebellar ataxia and spinocerebellar degeneration, brain tumors, encephalitis, meningitis, and Parkinson's disease. It can also be used for perinatal brain disorders, neonatal encephalopathy, cerebral palsy, etc. in newborns. Among these, ischemic stroke, transient ischemic attack, hypoxia-ischemia, intracranial hemorrhage such as intracerebral hemorrhage and intraventricular hemorrhage, stroke (cerebrovascular accident) such as subarachnoid hemorrhage, and perinatal cerebral hemorrhage. disorders, neonatal encephalopathy, cerebral palsy, etc. are preferred.

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

[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, after obtaining approval from the Ethics Committee of the Institute of Medical Science, the University of Tokyo, and with consent from the donor, the umbilical cord was collected, cut into pieces of 1 to ² mm, and seeded onto culture dishes. The cells derived from the umbilical cord were then 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. Leaf stem cells (hereinafter referred to as "UCMSCs") were obtained. In addition, the cells obtained from the umbilical cord tissue by the above-mentioned modified explant method are referred to as first passage cells (P1), and the passage number advances by passage, and is described as second passage cells (P2). do.

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 a cell pellet. After removing the supernatant, an appropriate amount of cell cryopreservation solution (STEM-CELLBANKER (Zenoac)) was added and suspended. The cell suspension solution was dispensed into cryotubes and stored at -80°C in a freezer. Thereafter, it was transferred to a gas phase above liquid nitrogen and continued storage.

[mRNA expression]
UCMSCs were added to mesenchymal stem cell serum-free medium (Rohto, serum-free medium) and PromoCell medium (Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009, Lot.435M415) from P2 to P4 with Supplement mix ( PromoCell, C-39809, Lot.435M126) and MEM-α medium (Thermo Fisher, #12571-063, Lot.18997009 with serum added to 10%) and frozen. A stock was made. After culturing fibroblasts in MEM-α medium, frozen stocks were prepared. Each P4 cell and fibroblast were woken up, seeded at 15,000 cells/cm ² in a 6-well plate, and cultured for 1 day in each of the three types of media, and then total RNA was collected. The mRNA expression of HGF, SHH, OLIG2, VEGFA, NEUROG1, GRPR, IL1R1, CRHR2, CCKAR, and APOE was detected using quantitative PCR.

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

[Preparation of suspension culture cells (SUS) and planar culture cells (ADH)]
The frozen cells derived from the umbilical cord tissue described above were awakened, seeded in a cell culture flask, and cultured using a serum-free medium for mesenchymal stem cells (Rohto). Cells were collected on the fourth day of culture, and a cell suspension containing the required number of cells and microcarriers were mixed, added to a culture tank, and agitation culture was started. On the 3rd or 4th day, remove a portion of the cell/microcarrier suspension, add new microcarriers and culture for 3 or 4 days. Obtained. The frozen cells derived from the umbilical cord tissue described above were awakened, seeded in a cell culture flask, and cultured using a serum-free medium for mesenchymal stem cells (Rohto). The cells were subcultured once every 3 or 4 days and cultured for a total of 2 weeks to obtain flat cultured cells (hereinafter referred to as "ADH").

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

The mRNA expression levels of PAX3, PAX5, EGF, CXCL1, GDNF, NRCAM, DLL1, HEYL, BMP2, NTN1, ASCL1, NRP2, VEGFA, and APOE were significantly lower in suspension culture cells (SUS) than in flat culture cells (ADH). was found to be high (Figure 2).

[Preparation of adipose-derived mesenchymal stem cells]
After consent was obtained from the human donor, the subcutaneous adipose tissue obtained by liposuction was washed with physiological saline. To achieve destruction of the extracellular matrix and isolation of cells, collagenase (solvent: physiological saline) was added and dispersed by shaking at 37° C. for 90 minutes. Subsequently, this suspension was centrifuged at 800 g for 5 minutes to obtain a precipitate of interstitial vascular cells. Add serum-free medium for mesenchymal stem cells (Rohto) to the above cell precipitate, centrifuge the cell suspension at 400g for 5 minutes, remove the supernatant, and then remove serum-free medium for mesenchymal stem cells (Rohto). The cells were resuspended and seeded into flasks. Cells were cultured for several days at 37 °C in 5% _CO2 . After a few days, the culture was washed with PBS to remove residual blood cells and adipose tissue contained in the culture solution, and the mesenchymal stem cells (hereinafter referred to as "ADMSC") adhering to the plastic container were collected. I got it.

[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)) was added and suspended. The cell suspension solution was dispensed into cryotubes and stored at -80°C in a freezer. Thereafter, it was transferred to a gas phase above liquid nitrogen and continued storage.

[Confirmation of therapeutic effect using rat transient cerebral ischemia model (Koizumi model)]
The middle cerebral artery (MCA) of rats (Wistar, Charles River Japan) was occluded, and 23.3 to 27.8 hours later, ADMSCs, ADH, and SUS were suspended in HBSS (Hank's Balanced Salt solution) and injected into the tail vein. The cells were administered internally (8×10 ⁶ cells/kg). In addition, as a comparison, animals were prepared in which only HBSS was administered without administering cells. Before surgery and 14 days after surgery, body weight and neurological symptoms were observed, and a step test and tape peeling test were performed.

Before surgery and 14 days after surgery, a step test was performed in which the trunk, hind limbs, and right forelimb of the rat were restrained and lifted, and only the left forelimb touched the experimental table, and the rat was moved 30 cm in the opposite direction on a horizontal plane in about 5 seconds. did. The number of steps taken with the left forelimb was recorded. The test was repeated three times, and the average value of the number of steps was taken 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 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 15 mm ² tape was applied to the back of the right (paralyzed side) forelimb, and the animal was placed in a cage and the time taken to remove the tape was measured (cut off was 120 seconds, tape peeling test).

It became clear that the degree of weight loss was reduced by administering ADMSC, ADH, and SUS (Figure 3). Furthermore, it was revealed that the neurological symptom score decreased by administering ADMSC, ADH, and SUS (FIG. 4). It became clear that the number of steps increased by administering ADMSC, ADH, and SUS (Figure 5). It became clear that the time required to peel off the tape was shortened by administering ADMSC, ADH, and SUS (Figure 6).

The above results demonstrated an improvement effect on nerve injury after cerebrovascular accident.

[Interaction between nerve cells and mesenchymal stem cells]
SH-SY5Y (human-derived neuroblastoma; manufactured by ECACC, Lot.16E028, Acc Nc:94030304) was stained with DiD fluorescence using Vybrant DiO Cell-labeling solution (Thermo Fisher, #V22886) in a 12-well plate. Seed and incubate at 37°C in DMEM/F12 (Thermo Fisher, #11320-033, Lot.1930023) medium containing 10% FBS (Thermo Fisher, #10437-028, Lot.1658423) and Pen-Strep. Culture was performed under the conditions of 20% O ₂ and 5% CO ₂ . After one day of culture, the cells in each well were washed twice with D-PBS(-) and incubated in DMEM, no glucose (Thermo Fisher, # 11966025) medium containing Pen-Strep at 37°C, _N2 : 95%, The cells were cultured for 2 hours under O ₂ :5% condition. Then, UCMSCs prepared in the same manner as above, cultured in serum-free medium for mesenchymal stem cells (Rohto), and stained with DiO fluorescence were added to a 12-well plate, and 10% FBS (Thermo Fisher, #10437- 028, Lot. 1658423) and Pen-Strep in DMEM/F12 (Thermo Fisher, #11320-033, Lot. 1930023) medium at 37°C, O ₂ : 20%, CO ₂ : 5%. After each time period, time-lapse images were taken. In each photo, green indicates UCMSC (cells pointed to by the arrow pointing downward to the left (the handle is a dotted line)), and red indicates SH-SY5Y (cells pointed to by the arrow pointing upward to the right). (Figure 7).

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

SH-SY5Y (human-derived neuroblastoma; manufactured by ECACC, Lot. 16E028, Acc Nc: 94030304) was seeded in a 96 well plate, and 5% FBS (manufactured by Thermo Fisher, #10437-028, Lot. 1658423) was seeded. The cells were cultured in DMEM/F12 (Thermo Fisher, #11320-033, Lot.1930023) medium containing Pen-Strep at 37° C. under the conditions of 20% O ₂ and 5% CO ₂ . After one day of culture, the medium in the well was removed and cultured in DMEM, No glucose (Thermo Fisher, # 11966025) medium containing Pen-Strep at 37°C under the conditions of 95% _N2 , ₅ % O2 (hereinafter referred to as OGD). (OGD treatment group). Similarly, after one day of culture, the medium in the well in which the SH-SY5Y cells were cultured was removed, and the umbilical cord-derived mesenchymal stem cells prepared as described above were placed in a serum-free medium for mesenchymal stem cells (Rohto). Cultured UCMSCs were added at 1,000 cells/well and cultured under OGD conditions for 24 hours (MSC group). For comparison, after one day of culture, the medium in the well was removed and fresh DMEM/F12 containing 5% FBS (manufactured by Thermo Fisher, #10437-028, Lot.1658423) and Pen-Strep was added. (Thermo Fisher, #11320-033, Lot. 1930023) culture medium for 24 hours at 37° C. under the conditions of 20% O ₂ and 5% CO ₂ was used as a control group. After culturing, lactate dehydrogenase (LDH) activity in each culture supernatant was measured (FIG. 8).

Culturing under OGD (Oxygen and glucose deprivation) conditions increases the LDH activity of SH-SY5Y cells, but co-culture with UCMSCs suppresses the increase in LDH activity and prevents neuronal death due to ischemia. It was confirmed that it was suppressed.

SH-SY5Y (human-derived neuroblastoma; manufactured by ECACC, Lot.16E028, Acc Nc:94030304) was seeded in 12 well plates at 0.038 x 10 ⁶ cells/well, and 10% FBS ( Thermo Fisher, #10437-028, Lot.1658423) and Pen-Strep in DMEM/F12 (Thermo Fisher, #11320-033, Lot.1930023) medium at 37°C, _O2 : 20%, CO2 _. : Cultured under 5% conditions. After one 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 one day of culture, the medium in the well was removed, and the suspended culture cells (SUS) prepared in the manner described above were added to half the amount (half amount group), the same amount (same amount group), and the same amount (same amount group) of the seeded SH-SY5Y cells. A transwell insert seeded with twice the amount (double amount group) was placed and cultured under OGD conditions for 2 hours (MSC group). Thereafter, 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 and _O2. : 20% and CO ₂ :5% for 48 hours. As a comparison, after one day of culture, the cells in each well were washed twice with D-PBS(-), and then fresh 10% FBS (Thermo Fisher, #10437-028, Lot.1658423) was added. ) and Pen-Strep in DMEM/F12 (Thermo Fisher, #11320-033, Lot.1930023) medium at 37°C, O ₂ : 20%, CO ₂ : 5% for 48 hours (Control group). After culturing, the cell activity of each SH-SY5Y was evaluated using WST-8 (Cell Counting Kit-8) (FIG. 9).

Although the cell activity of neurons is significantly reduced by culturing under OGD conditions, it has been revealed that co-culturing with MSCs can suppress the decrease in cell activity.

SH-SY5Y (human-derived neuroblastoma; manufactured by ECACC, Lot. 16E028, Acc Nc: 94030304) was seeded in a 12-well plate at 0.038 x 10 ⁶ cells/well, and 10% FBS (Thermo Fisher Thermo Fisher, #10437-028, Lot.1658423) and Pen-Strep in DMEM/F12 (Thermo Fisher, #11320-033, Lot.1930023) medium at 37°C, _O2 : 20%, _CO2 : 5%. It was cultured under the following conditions. After one 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 one day of culture, the medium in the well was removed, and the suspension cultured cells (SUS, SUS group), flat cultured cells (ADH, ADH group), and fibroblasts (Fibroblast, Fibroblast group) were prepared using the method described above. A transwell insert in which the same amount of SH-SY5Y and SH-SY5Y were seeded was placed and cultured under OGD conditions for 2 hours. Thereafter, 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 and _O2. : 20% and CO ₂ :5% for 48 hours. As a comparison, after one day of culture, the cells in each well were washed twice with D-PBS(-), and then fresh 10% FBS (Thermo Fisher, #10437-028, Lot.1658423) was added. ) and Pen-Strep in DMEM/F12 (Thermo Fisher, #11320-033, Lot.1930023) medium at 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 using WST-8 (Cell Counting Kit-8) (FIG. 10).

By co-culturing with MSCs, it is possible to suppress the decrease in neuronal cell activity caused by culturing under OGD conditions, but this effect is not present in some fibroblasts, and it is clear that this effect is specific to MSCs. It became.

SH-SY5Y (human-derived neuroblastoma; manufactured by ECACC, Lot. 16E028, Acc Nc: 94030304) was seeded in a 12-well plate at 0.038 x 10 ⁶ cells/well, and 10% FBS (Thermo Fisher Thermo Fisher, #10437-028, Lot.1658423) and Pen-Strep in DMEM/F12 (Thermo Fisher, #11320-033, Lot.1930023) medium at 37°C, _O2 : 20%, _CO2 : 5%. It was cultured under the following conditions. After one 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 one day of culture, the medium in the well was removed and the umbilical cord-derived mesenchymal stem cells (manufactured by PromoCell) were grown in serum-free medium for mesenchymal stem cells (Rohto, serum-free group) and PromoCell medium (Mesenchymal Stem Cell). Supplement mix (manufactured by PromoCell, C-39809, Lot.435M126) was added to Growth Medium 2 (manufactured by PromoCell, C-28009, Lot.435M415), PromoCell group) and MEM-α (manufactured by Thermo Fisher, # 12571-063, Lot.18997009, MEM-α group) and fibroblast cells (Fibroblast, Fibroblast group) were placed on a transwell insert in which the same amount of SH-SY5Y was cultured and cultured under OGD conditions for 2 hours. . Thereafter, 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 and _O2. : 20% and CO ₂ :5% for 48 hours. As a comparison, after one day of culture, the cells in each well were washed twice with D-PBS(-), and then fresh 10% FBS (Thermo Fisher, #10437-028, Lot.1658423) was added. ) and Pen-Strep in DMEM/F12 (Thermo Fisher, #11320-033, Lot.1930023) medium at 37°C, O ₂ : 20%, CO ₂ : 5% for 48 hours (Control group). After culturing, the cell activity of each SH-SY5Y was evaluated using WST-8 (Cell Counting Kit-8) (FIG. 11).

By co-culturing with MSCs, it was possible to suppress the decline in neuronal cell activity caused by culturing under OGD conditions, but it became clear that this effect was more pronounced with MSCs cultured in serum-free medium.

SH-SY5Y (human-derived neuroblastoma; manufactured by ECACC, Lot. 16E028, Acc Nc: 94030304) was seeded in a 12-well plate at 0.038 x 10 ⁶ cells/well, and 10% FBS (Thermo Fisher Thermo Fisher, #10437-028, Lot.1658423) and Pen-Strep in DMEM/F12 (Thermo Fisher, #11320-033, Lot.1930023) medium at 37°C, _O2 : 20%, _CO2 : 5%. It was cultured under the following conditions. After one 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 one day of culture, the medium in the well was removed and the umbilical cord-derived mesenchymal stem cells (manufactured by PromoCell, C-12971, Lot.427Z021) were transferred to a serum-free medium for mesenchymal stem cells (Rohto, serum-free group). ), PromoCell medium (Supplement mix (manufactured by PromoCell, C-39809, Lot. 435M126) added to Mesenchymal Stem Cell Growth Medium 2 (manufactured by PromoCell, C-28009, Lot. 435M415), PromoCell group) and MEM-α (manufactured by Thermo Fisher, #12571-063, Lot.18997009, MEM-α group) A transwell insert was placed in which the same amount of UCMSCs and fibroblasts (Fibroblast, fibroblast group) were cultured as SH-SY5Y. , and cultured for 2 hours under OGD conditions. Thereafter, 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 and _O2. :20% and CO ₂ :5% for 24 to 48 hours. After culturing for 48 hours, cell images were obtained (FIG. 12). Note that EthD-III was used to stain 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" (FIG. 13).

It was revealed that the serum-free group had higher cell proliferation activity than the cell-free group, fibroblast group, PromoCell group, and MEM-α group. Furthermore, it was revealed that the number of dead cells in the serum-free group was smaller than the total number of cells compared to the no-cell administration group, the PromoCell group, and the MEM-α group.

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

Claims (5)

One of 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 allogeneic. 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 therapeutic agent for neurological disorders containing the mesenchymal stem cells according to any one of claims 1 to 4.

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