Therapeutic agent for acute encephalopathy and method for treating acute encephalopathy

Abstract

The purpose of the present invention is to provide a therapeutic agent and a treatment method that exhibit an excellent therapeutic effect on acute encephalopathy for which no effective treatment method has been found.　The present invention is a therapeutic agent for acute encephalopathy that contains umbilical cord-derived mesenchymal cells. Also, the therapeutic agent for acute encephalopathy is preferably for intravenous administration. Furthermore, the umbilical cord-derived mesenchymal cells are preferably cells prepared from umbilical cord tissue including amniotic membrane, blood vessels, perivascular tissues, and Wharton's jelly.

Description

Therapeutic agent for acute encephalopathy and method for treating acute encephalopathy

[0001] The present invention relates to a therapeutic agent for acute encephalopathy and a method for treating acute encephalopathy.

[0002] Acute encephalopathy is an acute brain disorder caused by a variety of factors. According to a nationwide survey conducted by the Ministry of Health, Labor, and Welfare's Acute Encephalopathy Research Group from spring 2014 to fall 2017, approximately 400 to 700 people develop the disease annually, with the majority occurring in infants aged 0 to 3 years. The average age is 3.5 years (standard deviation 3.6 years, median 2 years). It is usually caused by infectious diseases, particularly viral infections such as influenza, roseola, colds, and gastroenteritis. The mechanisms of acute encephalopathy are thought to include immune responses to bacterial or viral infections, hypercytokinemia, excitotoxicity associated with convulsions, and metabolic abnormalities. Current treatments include steroid pulse therapy, vitamins, free radical inhibitors, and hypothermia therapy, but there is currently no fundamental cure, which places a long-term burden on the individual and their family. Furthermore, acute encephalopathy associated with infectious diseases can later lead to attention-deficit hyperactivity disorder (ADHD). Therefore, there is a strong demand for the development of therapeutic agents and methods that have excellent therapeutic effects for such acute encephalopathy, for which no fundamental treatment has been found.

[0003] On the other hand, mesenchymal stromal cells (hereinafter referred to as "MSCs") are stem cells with pluripotency and self-renewal ability, and are capable of differentiating not only into mesenchymal cells such as osteoblasts, chondrocytes, adipocytes, and muscle cells, but also of differentiating beyond the germ layer into neurons, hepatocytes, etc. Furthermore, MSCs exert anti-inflammatory effects against excessive inflammation and repair of damaged tissues and cells through the paracrine effect of humoral factors they produce, and are thought to consequently exhibit therapeutic effects against various diseases.

[0004] It is known that mesenchymal cells are effective in treating brain diseases. For example, clinical studies are underway in which autologous bone marrow-derived mesenchymal cells are administered directly to patients with cerebral palsy, and their effectiveness has been reported (Non-Patent Document 1). It is also known that administration of umbilical cord-derived mesenchymal cells to treat cerebral palsy caused by cerebral hemorrhage is effective (Patent Document 1). Non-Patent Document 2 discloses results suggesting that exosomes from umbilical cord-derived mesenchymal cells are effective in treating neurodegenerative diseases. However, there is no information on whether mesenchymal cells are effective in treating acute encephalopathy, particularly acute infant encephalopathy, and nothing is known about the use of mesenchymal cells to treat acute encephalopathy.

[0005] International Publication No. 2017 / 204231

[0006] Cytotherapy 2013 Dec; 15 (12): 1549-62 Theranostics, 2019, Aug 14; 9 (20), 5956-5975

[0007] An object of the present invention is to provide a therapeutic agent and a therapeutic method that exhibit excellent therapeutic effects against acute encephalopathy, for which no effective treatment has been found.

[0008] As a result of intensive research to solve the above-mentioned problems, the present inventors discovered that intravenous administration of a cell population containing cells prepared from umbilical cord tissue including amniotic membrane, blood vessels, perivascular tissue, and Wharton's jelly in an animal model of infant acute encephalitis significantly reduced degenerated neurons in the dorsal hippocampus and lateral septal nucleus, suppressed inflammatory cytokines in the dorsal hippocampal tissue, and improved behavior, leading to the completion of the present invention.

[0009] That is, the present invention relates to the following: [1] A therapeutic agent for acute encephalopathy comprising umbilical cord-derived mesenchymal cells. [2] The therapeutic agent for acute encephalopathy according to [1], which is for intravenous administration. [3] The therapeutic agent for acute encephalopathy according to [1] or [2], wherein the umbilical cord-derived mesenchymal cells are cells prepared from umbilical cord tissue including amniotic membrane, blood vessels, perivascular tissue, and Wharton's jelly. [4] The therapeutic agent for acute encephalopathy according to [1] or [2], wherein the umbilical cord-derived mesenchymal cells are (i) positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5, and PD-L2, and (ii) negative for CD45, CD34, CD11b, CD19, and HLA-class II. [5] The therapeutic agent for acute encephalopathy according to [1] or [2], wherein the acute encephalopathy is encephalopathy triggered by an infection. [6] The therapeutic agent for acute encephalopathy according to [1] or [2], wherein the acute encephalopathy is acute infant encephalopathy. [7] A method for treating acute encephalopathy, comprising administering umbilical cord-derived mesenchymal cells that are (i) positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5, and PD-L2, and (ii) negative for CD45, CD34, CD11b, CD19, and HLA-class II. [8] The method for treating acute encephalopathy according to [7], wherein the administration is intravenous. [9] The method for treating acute encephalopathy according to [7] or [8], wherein the umbilical cord-derived mesenchymal cells are cells prepared from umbilical cord tissue including amniotic membrane, blood vessels, perivascular tissue, and Wharton's jelly.

[10] The method for treating acute encephalopathy according to [7] or [8], wherein the acute encephalopathy is encephalopathy triggered by an infection.

[11] The method for treating acute encephalopathy described in [7] or [8], wherein the acute encephalopathy is acute infant encephalopathy.

[0010] According to the present invention, it is possible to provide a therapeutic agent for acute encephalopathy and a therapeutic method for acute encephalopathy, which comprise umbilical cord-derived mesenchymal cells prepared from umbilical cord tissue including amniotic membrane, blood vessels, perivascular tissue, and Wharton's jelly, and / or are administered intravenously.

[0011] Figure 1-1 shows the surface antigens (CD105, CD73, CD45, CD34) and characteristics of umbilical cord-derived mesenchymal cells. The antigens are either negative (less than 2%) or positive (more than 95%), indicating that this cell population is generally homogeneous. Figure 1-2 shows the surface antigens (CD90, CD44, CD11b, CD19) and characteristics of umbilical cord-derived mesenchymal cells. The antigens are either negative (less than 2%) or positive (more than 95%), indicating that this cell population is generally homogeneous. Figure 1-3 shows the surface antigens (HLA-ABC, HLA-DR) and characteristics of umbilical cord-derived mesenchymal cells. The antigens are either negative (less than 2%) or positive (more than 95%), indicating that this cell population is generally homogeneous. Figure 2 shows a diagram illustrating the isolation and culture of primary microglial cells. Figure 3A shows a diagram illustrating the drug stimulation of microglial cells and coculture with MSCs. Figure 3B shows microscopic images of drug-stimulated microglial cells (control, Poly(I:C), KA, Poly(I:C), and KA) co-cultured with UC-MSCs (24 hours). Figure 3C shows microscopic images of microglial cells that became ameboid-shaped upon the addition of Poly(I:C). Figure 3D shows microscopic images of microglial cells with increased processes upon co-culture with UC-MSCs. Figure 3E shows the percentage (%) of ameboid-shaped microglial cells obtained by culture under each condition. Figure 3F shows the percentage (%) of microglial cells with processes obtained by culture under each condition. Figure 3G-1 shows the results of IL6 and TNFα expression analysis for microglial cells obtained by culture under each condition. Figure 3G-2 shows the results of IL1b and iNOS expression analysis for microglial cells obtained by culture under each condition. Figure 3G-3 shows the results of Arg1 expression analysis for microglial cells obtained by culturing under each condition. Figure 3H shows immunocytostained images of microglial cells obtained by culturing under each condition.Figure 3I shows the percentage (%) of iNos-positive cells (number of iNos-positive cells / number of DAPI-positive cells × 100 (%)) and the percentage (%) of Arg1-positive cells (number of Arg1-positive cells / number of DAPI-positive cells × 100 (%)) among microglial cells cultured under each condition. Figure 4A shows the design of the in vivo experiment. Figure 4B shows the observation areas (a, b, c) in the mouse brain where immunohistochemical staining was performed. Figure 4C shows the results of immunohistochemical staining of brain sections from each mouse. A: normal control; B: mice administered Poly(I:C) 20 mg / kg only; C: mice administered KA 2.5 mg / kg only (≠ SE); D: mice administered Poly(I:C) 20 mg / kg + KA 2.5 mg / kg (= SE). FIG. 4D-1 shows the results of immunohistochemical staining of brain sections (sections of region a in FIG. 4B) from a control mouse (Control), an SE model mouse (SE), and an SE model mouse after administration of UC-MSCs (SE+MSCs). FIG. 4D-2 shows the results obtained by quantifying the results of FIG. 4D-1 and summarizing them in a graph. FIG. 4E-1 shows the results of immunohistochemical staining of brain sections (sections of region b in FIG. 4B) from a control mouse (Control), an SE model mouse (SE), and an SE model mouse after administration of UC-MSCs (SE+MSCs). FIG. 4E-2 shows the results obtained by quantifying the results of FIG. 4E-1 and summarizing them in a graph. FIG. 4F-1 shows the results of immunohistochemical staining of brain sections (sections of region c in FIG. 4B) from a control mouse (Control), an SE model mouse (SE), and an SE model mouse after administration of UC-MSCs (SE+MSCs). Figure 4F-2 shows the results of quantifying the results of Figure 4F-1 and summarizing them in a graph. Figure 4G shows the results of comparing the expression of various cytokines in the dorsal hippocampus of control mice (Control), SE model mice (SE), and SE model mice after administration of UC-MSCs (SE + MSCs). Figure 4H shows the results of comparing the expression of NKCC1 and KCC2 in the dorsal hippocampus of control mice (Control), SE model mice (SE), and SE model mice after administration of UC-MSCs (SE + MSCs).Figure 5 shows the method and results of an open field test for control mice (Control), SE model mice (SE), and SE model mice after administration of UC-MSCs (SE+MSCs). (A) Open field apparatus, (B) Example results of the open field test for each group, (C) total distance traveled (cm), (D) total movement time (seconds), (E) center-stay rate (%). Figure 6 shows the method and results of a Y-maze test for control mice (Control), SE model mice (SE), and SE model mice after administration of UC-MSCs (SE+MSCs). (A) Y-maze apparatus, (B) percent alternation (% alternation = {number of alternations / (total arm entries - 1) × 100}, (C) total number of entries (total arm entries).

[0012] The present invention will be described in detail below. The terms used in this specification are to be interpreted in the sense commonly used in the art unless otherwise specified.

[0013] <Therapeutic Agent for Acute Encephalopathy> The therapeutic agent for acute encephalopathy of the present invention is characterized by containing umbilical cord-derived mesenchymal cells.

[0014] In the present invention, the umbilical cord refers to a white tubular tissue that connects a fetus and a placenta, and is a tissue that contains amniotic membrane, blood vessels, perivascular tissue, and Wharton's jelly, but does not contain placenta or umbilical cord blood. The umbilical cord of the present invention is not particularly limited as long as it is collected from a mammal, but is preferably an umbilical cord from a primate mammal, and more preferably a human umbilical cord.

[0015] When human umbilical cord-derived mesenchymal cells are used as a therapeutic agent for acute encephalopathy in the present invention, the umbilical cord may be an umbilical cord collected from the patient to be treated (autologous umbilical cord) or an umbilical cord collected from a non-patient to be treated (allogeneic umbilical cord). The use of an allogeneic umbilical cord has the advantage of allowing administration at an early stage of onset. Furthermore, the human umbilical cord-derived mesenchymal cells of the present invention have been confirmed to exert therapeutic effects without being eliminated by immune rejection or the like in the examples (xenogeneic umbilical cord) in which they were administered to rodents (mice) described below. Furthermore, when considering administering the therapeutic agent for acute encephalopathy of the present invention to humans, it is preferable that the umbilical cord be derived from a human.

[0016] In the present invention, the umbilical cord can be collected by appropriately cutting the umbilical cord from postpartum tissues, including the placenta and umbilical cord, delivered via vaginal delivery and cesarean section. After removing umbilical cord blood from the collected umbilical cord, it may be subjected to aseptic or bacteriostatic treatment. The aseptic or bacteriostatic treatment may include, but is not limited to, application of povidone-iodine or immersion in a medium or buffer containing one or more antibiotics and / or antifungals, such as penicillin, streptomycin, amphotericin B, gentamicin, and nystatin. If necessary, the method may also include a step of selectively lysing red blood cells. Methods for selectively lysing red blood cells include, for example, ammonium chloride lysis followed by incubation in a hypertonic or hypotonic medium, and are well known in the art.

[0017] The umbilical cord-derived mesenchymal cells of the present invention refer to a cell population prepared using an umbilical cord as a raw material, and examples of the preparation method include a method comprising the steps of (1) cutting the umbilical cord, (2) culturing umbilical cord sections, and (3) passaging the umbilical cord-derived mesenchymal cells. Other examples of the preparation method for the cells include a method comprising the steps of (A) dissociating the tissue by cutting the umbilical cord or by enzymatic treatment, or both, (B) culturing the umbilical cord tissue, and (C) passaging the umbilical cord-derived mesenchymal cells. The umbilical cord-derived mesenchymal cells may be a heterogeneous cell population rather than a homogeneous cell population. The umbilical cord-derived mesenchymal cells of the present invention may also be defined as a cell population having the following properties: (a) they exhibit adhesion to plastic when cultured in a standard medium, and (b) they are positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5, and PD-L2, and negative for CD45, CD34, CD11b, CD19, and HLA-class II.

[0018] In the present invention, "positive" means that the detection level is higher than that of a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen, using an analytical method such as flow cytometry that utilizes an antigen-antibody reaction. Similarly, "negative" means that the detection level is equal to or lower than that of a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen.

[0019] In the present invention, HLA-class I means HLA-A, B, or C, and HLA-class II means HLA-DR, DQ, or DP.

[0020] In the step (1) of cutting the umbilical cord of the present invention, the umbilical cord obtained by the above-described method can be cut by mechanical force (chopping force or shearing force) while still containing the amniotic membrane, blood vessels, perivascular tissue, and Wharton's jelly. Although not particularly limited, the umbilical cord sections obtained by cutting have a length of 1 to 10 mm. ３ , 1 to 5 mm ３ , 1 to 4 mm ３ , 1 to 3 mm ３ or 1 to 2 mm ３In the enzymatic treatment step (A) of the present invention, the umbilical cord obtained by the above-described method may be treated with an enzyme to dissociate the tissues while still containing the amniotic membrane, blood vessels, perivascular tissue, and Wharton's jelly. Examples of enzymatic treatment include, but are not limited to, collagenase, dispase, hyaluronidase, and the like.

[0021] The step (2) (B) of culturing the umbilical cord section of the present invention can be performed by seeding the cut umbilical cord section in a culture vessel and culturing it in a culture medium suitable for umbilical cord-derived mesenchymal cells. In a preferred embodiment of this step (2), the umbilical cord section is not treated with a digestive enzyme.

[0022] In the present invention, the culture vessel is not particularly limited as long as it has a solid surface, but the term "solid surface" refers to any material that allows binding with the umbilical cord-derived mesenchymal cells of the present invention. In a specific embodiment, such a material is a plastic material that has been treated to promote binding of mammalian cells to its surface (e.g., treated to increase hydrophilicity). The shape of the culture vessel having a solid surface is not particularly limited, but a petri dish, flask, or the like is preferably used.

[0023] In the present invention, a culture medium suitable for umbilical cord-derived mesenchymal cells may be prepared by adding serum to a basal medium and / or one or more serum substitutes such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. These media may further contain, as necessary, substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antifungals, antioxidants, pyruvic acid, buffers, inorganic salts, etc. Examples of basal media include Dulbecco's Modified Eagle's Medium (DMEM) (high glucose or low glucose), Alpha Modified Eagle's Minimum Essential Medium (α-MEM), improved DMEM, DMEM / MCDB 201, Eagle's basal medium, Ham's F10 medium (F10), Ham's F-12 medium (F12), Iscove's Modified Dulbecco's Medium (IMDM), Fischer's medium, mesenchymal stem cell growth medium (MSCGM), DMEM / F12, RPMI 1640, CELL-GRO-FREE, and mixed media thereof.

[0024] Examples of serum include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, porcine serum, sheep serum, rabbit serum, and rat serum. When serum is used, it may be added to the basal medium at 5 to 20% (v / v), preferably 10% (v / v). Examples of fatty acids include, but are not limited to, linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoylic 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, particular examples include, but are not limited to, hyaluronic acid and heparan sulfate. Examples of growth factors include, but are not limited to, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), leukocyte inhibitory factor (LIF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), and erythropoietin. Examples of antibiotics and / or antifungal agents include penicillin G, streptomycin sulfate, amphotericin B, gentamicin, nystatin, and mixtures thereof.

[0025] In steps (2) and (B), it is desirable to hold down the seeded umbilical cord sections with a plate or the like during the culture period to prevent the sections from floating in the culture medium. An example of such a plate is the plate described in JP 2015-70824 A. Furthermore, in steps (2) and (B), the umbilical cord sections are cultured in a bronchial atmosphere containing 0 to 5% CO ２ Preferably, the medium is cultured under 2-25% O ２More preferably, 5 to 20% O ２ Preferably, the culture is carried out at 25 to 40°C, more preferably at 37°C.

[0026] In step (2) (B), cells are preferably cultured until they migrate from the umbilical cord section and reach 50%, 60%, 70%, 80% or more confluence in the culture vessel. After culture, the cells are washed to remove unbound cells and cell debris, and then detached with a detachment agent containing trypsin, EDTA solution, collagenase, dispase, hyaluronidase, or a mixture thereof. The detachment solution containing the cells and umbilical cord section is filtered using a cell strainer or the like, thereby obtaining only the cells as umbilical cord-derived mesenchymal cells. The obtained umbilical cord-derived mesenchymal cells can be seeded into the above-mentioned culture vessel and cultured in the above-mentioned culture medium.

[0027] Subsequently, in step (3) (C), the umbilical cord-derived mesenchymal stem cells are passage-cultured, thereby allowing the umbilical cord-derived mesenchymal cells to be expanded appropriately to the required number. For passage, the cells may be detached using a detachment agent containing trypsin, EDTA solution, collagenase, dispase, hyaluronidase, or a mixture thereof, and then seeded at an appropriate cell density in a separately prepared culture vessel for continued culture. When seeding the cells, a typical cell density is 100 cells / cm. ２ ~100,000 cells / cm ２ , 500 cells / cm ２ ~50,000 cells / cm ２ , 1,000-10,000 cells / cm ２ , 2,000-10,000 cells / cm ２ In a specific embodiment, the cell density is 2,000 to 10,000 cells / cm. ２ The period required to reach an appropriate confluency is preferably adjusted to 3 to 7 days. During the culture, the medium may be replaced as needed.

[0028] The umbilical cord-derived mesenchymal stem cells may be passaged in step (3) (C) until they reach senescence, at which point cell division ceases. From the viewpoint of therapeutic use, they are preferably passaged 3 to 25 times, more preferably 4 to 12 times.

[0029] To confirm that the cells obtained by the above steps are the umbilical cord-derived mesenchymal cells of the present invention, surface antigens and the like may be analyzed by conventional methods such as flow cytometry. Alternatively, the cells may be evaluated by measuring the amounts of various proteins produced by the cells.

[0030] The obtained umbilical cord-derived mesenchymal cells may be prepared for therapeutic use as is, or may be cryopreserved. Cryopreservation is performed by suspending the cells in a cryopreservation solution that can adequately preserve the umbilical cord-derived mesenchymal cells and storing them at -80°C to -196.5°C. The cryopreservation solution is not particularly limited, but examples include aqueous solutions containing a cryoprotectant and glucose. Examples of cryoprotectants include dimethyl sulfoxide (hereinafter referred to as DMSO), dextran, glycerol, propylene glycol, and 1-methyl-2-pyrrolidone. Preferred cryoprotectants are DMSO and propylene glycol, with DMSO being particularly preferred. The cryoprotectant is preferably contained in the cryopreservation solution at 1.0 to 15.0 v / v%, more preferably 5.0 to 15.0 v / v%, even more preferably 5.0 to 12.0 v / v%, and particularly preferably 8.0 to 11.0 v / v%.

[0031] The glucose content of the cryopreservation solution is preferably 0.5 to 10.0 w / v%, more preferably 1.0 to 10.0 w / v%, even more preferably 2.0 to 8.0 w / v%, and particularly preferably 2.0 to 5.0 w / v%.

[0032] The cryopreservation solution may further contain other components. Examples of such components include pH adjusters and thickeners. Examples of pH adjusters include sodium bicarbonate, HEPES, phosphate buffer, ACD-A solution, and Ringer's solution. Furthermore, when a phosphate buffer is not added to Basic Stock Solution (BSS), a solution containing sodium chloride, which provides buffering capacity at a pH near that suitable for umbilical cord-derived mesenchymal cells, can also be used. Among these, the use of a phosphate buffer is particularly preferred. A pH adjuster is preferably used appropriately to adjust the pH of the cryopreservation solution to approximately 6.5 to 9.0, preferably 7.0 to 8.5. The phosphate buffer used in the present invention refers to sodium chloride, monosodium phosphate (anhydrous), monopotassium phosphate (anhydrous), disodium phosphate (anhydrous), trisodium phosphate (anhydrous), potassium chloride, and potassium dihydrogen phosphate (anhydrous), among others. The use of sodium chloride, monosodium phosphate (anhydrous), potassium chloride, or potassium dihydrogen phosphate (anhydrous) is particularly preferred.

[0033] The cryopreservation solution may or may not contain natural animal-derived components. Examples of natural animal-derived components include serum and basal medium, as described above. It is preferable that the cryopreservation solution does not contain natural animal-derived components. A cryopreservation solution that does not contain natural animal-derived components does not cause problems with quality differences between lots of natural animal-derived components, and can avoid the risk of changes in the properties of cells in umbilical cord tissue due to components that are not originally necessary for cell preservation, such as various cytokines, growth factors, and hormones contained in serum. Furthermore, it can also avoid the effects of components of unknown origin contained in basal medium. Therefore, a cryopreservation solution that does not contain natural animal-derived components is very useful, especially for clinical use.

[0034] The cryopreservation solution may further contain a thickener. The thickener is not particularly limited as long as it can constitute a cryopreservation solution capable of adequately preserving umbilical cord tissue. Examples of thickeners include carboxymethylcellulose (hereinafter referred to as CMC), sodium carboxymethylcellulose (hereinafter referred to as CMC-Na), organic acid polymers, propylene glycol alginate, and sodium alginate. Preferred thickeners are CMC and CMC-Na, with CMC-Na being particularly preferred. Among organic acid polymers, sodium polyacrylate is preferred. The thickener is preferably contained in the cryopreservation solution at 0.1 to 1.0 w / v %, more preferably 0.1 to 0.5 w / v %, and even more preferably 0.2 to 0.4 w / v %.

[0035] The cryopreservation solution is preferably an aqueous solution. In order to maintain its performance as a preservation solution, the osmotic pressure of the cryopreservation solution is preferably 1000 mOsm or more, and more preferably 1000 to 2700 mOsm.

[0036] A preferred cryopreservation solution is an aqueous solution containing a thickener, a cryoprotectant, and glucose, and free of natural animal-derived components. A more preferred example is an aqueous solution containing CMC-Na, DMSO, and glucose, and free of natural animal-derived components. In a further preferred example, the cryopreservation solution is an aqueous solution containing 0.1 to 1.0 w / v % CMC-Na, 1.0 to 15.0 w / v % DMSO, and 0.5 to 10.0 w / v % glucose, and free of natural animal-derived components.

[0037] The umbilical cord-derived mesenchymal cells obtained by the above method can be mixed with an infusion preparation to prepare a therapeutic agent for acute encephalopathy. Meanwhile, cryopreserved umbilical cord-derived mesenchymal cells can be suspended in a cryopreservation solution and thawed to be used as a therapeutic agent for acute encephalopathy directly, or they can be mixed with an infusion preparation to prepare a therapeutic agent for acute encephalopathy. When mixing, the culture medium or cryopreservation solution in which the cells are suspended can be mixed with an infusion preparation, or the cells can be separated from the solvent by centrifugation or the like, and then mixed with the infusion preparation alone. To avoid the complexity of the procedure, it is preferable not to include a step of culturing the frozen cells after thawing, or to directly mix the cryopreservation solution in which the thawed cells are suspended with the infusion preparation.

[0038] The "infusion preparation" of the present invention is not particularly limited as long as it is a solution used in treating humans, and examples thereof include physiological saline, 5% glucose solution, bicarbonate Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, No. 1 solution, No. 2 solution, No. 3 solution, No. 4 solution, etc.

[0039] The therapeutic agent for acute encephalopathy of the present invention also includes a kit combining umbilical cord-derived mesenchymal cells and an infusion preparation.

[0040] The infusion preparation containing cells may further contain a pharmaceutically acceptable carrier. In the present invention, the carrier may be a suspending agent, solubilizing agent, stabilizer, isotonicity agent, preservative, anti-adsorption agent, surfactant, diluent, vehicle, pH adjuster, soothing agent, buffer, sulfur-containing reducing agent, antioxidant, etc., which are appropriately added for administering the therapeutic agent.

[0041] Examples of suspending agents include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, powdered tragacanth, sodium carboxymethylcellulose, and polyoxyethylene sorbitan monolaurate. Examples of solution adjuvants include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, and castor oil fatty acid ethyl esters. Examples of stabilizers include dextran 40, methylcellulose, gelatin, sodium sulfite, and sodium metasulfate. Examples of isotonicity agents include D-mannitol and sorbitol. Examples of preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol. Examples of adsorption inhibitors include human serum albumin, lecithin, dextran, ethylene oxide / propylene oxide copolymer, hydroxypropyl cellulose, methylcellulose, hydrogenated castor oil, and polyethylene glycol. Examples of the reducing agent include those having a sulfhydryl group, such as N-acetylcysteine, N-acetylhomocysteine, thiochitic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atoms. Examples of the antioxidant include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate, and chelating agents such as sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.Furthermore, commonly added components such as inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate, organic salts such as sodium citrate, potassium citrate, and sodium acetate, and sugars such as glucose may be appropriately added. Furthermore, as an anticoagulant and pH adjuster, for example, ACD-A solution (composed of sodium citrate hydrate, citric acid hydrate, glucose, etc.) may be added.

[0042] For local administration, the infusion preparation containing the cells may be mixed with organic substances such as biopolymers, or inorganic substances such as hydroxyapatite, specifically, collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof.

[0043] The method of administration of the acute encephalopathy therapeutic agent of the present invention can be exemplified by intravenous administration, intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, etc., but intravenous administration, the effectiveness of which is demonstrated in the following examples, is particularly preferred because it can be administered safely and stably without depending on the skill of the administerer.

[0044] The dose of the therapeutic agent for acute encephalopathy of the present invention is the amount of cells that, when administered to a subject, can provide a therapeutic effect against the disease compared to a non-administered subject. The specific dose can be appropriately determined depending on the age, weight, extent of ischemia, symptoms, etc. of the subject. For example, the number of umbilical cord-derived mesenchymal cells per administration is 10 ４ ~10 ９ pieces / kg weight, 10 ４ ~10 ８ pieces / kg weight, 10 ４ ~10 ７ More preferably, 10 ４ ~10 ８ per kg of body weight.

[0045] The therapeutic agent for acute encephalopathy of the present invention may be administered multiple times, for example, two, three, four, five, or more times, and the number of administrations may be appropriately determined while confirming the therapeutic effect on the subject. When administered multiple times, the administration interval may be appropriately determined while confirming the therapeutic effect on the subject, and examples include once a day, three times a week, twice a week, once a week, once every two weeks, once a month, once every three months, and once every six months. The administration period may be one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, or six months. From the viewpoint of the therapeutic effect of the therapeutic agent for acute encephalopathy of the present invention, it is preferable to administer the agent one to three times a week for one week to two months, and more preferably twice a week for two to four weeks. Furthermore, considering the early onset of cytokine storm, administration of the therapeutic agent for acute encephalopathy of the present invention should be initiated within three days, preferably within one day, after the onset of convulsions.

[0046] In the present invention, acute encephalopathy refers to acute brain disorders caused by various factors, and refers to a disease in which widespread brain dysfunction occurs due to edema of the central nervous system, resulting in the sudden onset of delirium, impaired consciousness, convulsions, etc., despite the absence of direct invasion of pathogens into the central nervous system and the absence or suspicion of clear pathologies such as vascular disorders. Recently, three main pathologies have been considered: (i) metabolic disorders (particularly mitochondrial energy production), (ii) systemic inflammatory responses (so-called cytokine storms), and (iii) excitotoxicity (neuronal cell death triggered by status epilepticus). Acute encephalopathy can occur at any age, but is most common in childhood, particularly in infancy, and most cases develop during the course of an infectious disease.

[0047] Considering the more effective effect of the therapeutic agent provided by the present invention, the acute encephalopathy in the present invention is preferably acute encephalopathy caused by an infectious disease such as a viral infection, a bacterial infection, or a mycoplasma infection, and more preferably acute infant encephalopathy.

[0048] The target age for the therapeutic agent for acute encephalopathy of the present invention is not particularly limited, but it is preferably used for acute encephalopathy in children (acute pediatric encephalopathy), and more preferably for acute encephalopathy in infants (acute infant encephalopathy). In the present invention, an infant refers to a child 28 days or older but less than one year old, and a toddler refers to a child 1 year old or older but less than six years old. Furthermore, a toddler refers to a child under 15 years old. Therefore, when referring to an infant, it generally refers to a child aged from one month to six years old.

[0049] In the present invention, the treatment of acute encephalopathy includes not only restoring the above-mentioned pathological condition to a normal state, but also alleviating symptoms, delaying progression, delaying onset, or preventing the disease. It is known that acute encephalopathy can later cause attention-deficit hyperactivity disorder (ADHD). In the present invention, the concept of treating acute encephalopathy also encompasses the prevention and / or treatment of attention-deficit hyperactivity disorder (ADHD). Therefore, the present invention can be said to encompass within its scope agents for the prevention and / or treatment of attention-deficit hyperactivity disorder (ADHD), including umbilical cord-derived mesenchymal cells.

[0050] The therapeutic agent for acute encephalopathy of the present invention may be used in combination with other therapeutic agents or may be used as a single agent.

[0051] <Method for Treating Acute Encephalopathy> The present invention also includes a method for treating acute encephalopathy using umbilical cord-derived mesenchymal cells. Specifically, the method for treatment of acute encephalopathy of the present invention includes a method for treating acute encephalopathy using an agent for treating acute encephalopathy comprising the umbilical cord-derived mesenchymal cells of the present invention, as described above. The above description is applicable to specific examples. Among these, the method for treatment of the present invention is preferably a method for intravenously administering umbilical cord-derived mesenchymal cells. Furthermore, the umbilical cord-derived mesenchymal cells are preferably cells prepared from umbilical cord tissue including amniotic membrane, blood vessels, perivascular tissue, and Wharton's jelly. Furthermore, the umbilical cord-derived mesenchymal cells are preferably (i) positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5, and PD-L2, and (ii) negative for CD45, CD34, CD11b, CD19, and HLA-class II. The therapeutic method of the present invention is particularly effective when the acute encephalopathy is encephalopathy in infants or young children, particularly acute encephalopathy caused by an infectious disease such as a viral infection. As described above, the present invention can also be said to include a method for preventing and / or treating attention deficit hyperactivity disorder (ADHD) using umbilical cord-derived mesenchymal cells.

[0052] 1. Preparation of Umbilical Cord-Derived Mesenchymal Cells Umbilical cord-derived mesenchymal cells were collected using the method described in Cytotherapy, 18, 229-241, 2016. Briefly, after approval by the Ethics Committee of the Institute of Medical Science, The University of Tokyo, and with the consent of the donor, all tissue elements of the umbilical cord (including amnion, blood vessels, perivascular tissue, and Wharton's jelly) were collected and cut into 1-2 mm pieces. ３ The cells were then minced into small pieces, seeded on a culture dish, covered with Cell Amigo (Tsubakimoto Chain Co., Ltd.), and cultured at 37°C and 5% CO in α-minimal essential medium (αMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. ２ P4 cells were obtained by a modified explant method in which they were cultured in the presence of ATP. P4 cells cultured up to the fourth passage were cryopreserved in STEM-CELLBANKER (Xenogen Pharma Co., Ltd.) and thawed immediately before use in experiments. The cells are plastic-adherent.

[0053] The surface antigens of the umbilical cord-derived mesenchymal cells were positive for CD73, CD105, CD90, and CD44 (Figure 1-1), and negative for CD34, CD45, CD19, CD80, CD86, CD40, and CD11b (Figure 1-2). The cells were positive for HLA-class I (HLA-ABC) and negative for HLA-class II (HLA-DR). The presence or absence of surface antigen expression was confirmed by FACS analysis using specific antibodies against each antigen. The results are shown in Figures 1-1 to 1-3.

[0054] 2. Isolation and culture of primary microglial cells. B6 albino mice (B6N-Tyrc-Brd / BrdCrCrl, Charles River Laboratories International, Japan) aged 0 to 2 days were euthanized, and cerebral tissue was collected under a microscope, minced, and dispersed in an enzyme solution containing 0.01% papain (Sigma-Aldrich), 0.1% collagenase (Sigma-Aldrich), and 0.5% DNase I (Sigma-Aldrich) in Hanks buffer solution (HBSS; Wako Pure Chemical Industries), and then filtered. The obtained mixed glial cells were cultured in Dulbecco's modified Eagle's medium (DMEM / F12; Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum, antibiotic-antimycotic (100X; Life Technologies, USA), and recombinant mouse macrophage colony-stimulating factor (M-CSF; Wako Pure Chemical Industries) at 37°C and 5% CO. ２ The cells were cultured for 10 to 14 days under 5% CO₂. The cultured cells were collected, the pellet was loosened, and mixed with CD11b microbeads (Miltenyi Biotech). CD11b cells were selected and collected using an MS column (Miltenyi Biotech) on a MiniMACS™ cell separator (Miltenyi Biotech). The purified microglial cells were plated at 1.0 × 10 cells per well onto a poly-L-lysine (Sigma-Aldrich)-coated 6-well plate (Corning Japan). ５ Primary microglia were cultured in M-CSF-containing medium at 37°C and 5% CO at a density of 1000 cells / ml. ２The cells were cultured under 5% CO₂ for 5 to 7 days, with the medium changed twice a week. The scheme is shown in Figure 2.

[0055] 3. Co-culture of microglial cells with umbilical cord-derived mesenchymal cells (UC-MSCs) (in vitro) 3-1. Drug stimulation of microglial cells and co-culture with MSCs The experimental scheme is shown in Figure 3A. Primary microglia cultured in normal medium were treated with 10 μg / ml Poly(I:C) (TOCRIS), 100 μM kainic acid (KA) (TOCRIS), or both. For the UC-MSC co-culture group, a 6-well transwell system (FALCON cell culture insert, Corning Inc.) equipped with a 0.4 μm filter membrane was used, and co-culture with UC-MSCs was initiated 3 hours after drug stimulation. 6 hours after drug stimulation, the medium was replaced with normal medium for all cultured cell groups. UC-MSCs were cultured at a concentration of 5 × 10 ５ Plate cells / well into the upper chamber and incubate in 5% CO ２ The mixture was incubated at 37°C for 24 hours (overnight) in the presence of HCl.

[0056] 3-2. Morphological Analysis of Microglial Cells Morphological analysis of the cells obtained in 3-1 above was performed 24 hours after co-culture with UC-MSCs. Confocal images were obtained using an all-in-one fluorescence microscope (BZ-X800, KEYENCE, Osaka, Japan) (Figures 3B, 3C, and 3D). Referring to previous reports, the following three distinct morphological categories were determined. Five to seven images were randomly selected from 20 fields, and at least 200 cells per group were analyzed and classified in three independent experiments (Figures 3E and 3F). (1) Rod-shaped microglia: elongated cells with a few processes; (2) Ameboid microglia: round cells with one or fewer processes; and (3) Branched microglia: branched cells with two or more processes.

[0057] As shown in Figures 3B-3F, microglial cells became ameboid-like upon addition of Poly(I:C). Co-culture with UC-MSCs significantly reduced the number of ameboid-like cells (Figure 3E), while significantly increased the number of cell processes (Figure 3F).

[0058] 3-3. Analysis of Expression of Inflammatory Cytokines, etc. in Microglial Cells Total RNA was extracted from the cells obtained in 3-1 above using a NucleoSpin® RNA column (TaKaRa Corporation). Single-stranded cDNA was generated by reverse transcription using a PrimeScript RT Reagent Kit (TaKaRa Corporation). Real-time PCR was performed using TB Green® Premix Ex Taq™ II (TaKaRa Corporation). Expression levels of cytokines, etc. (IL6, TNFα, IL1β, iNOS, Arg1) were analyzed under the following reaction conditions: initial denaturation at 95°C for 30 minutes, followed by 40 cycles of denaturation at 95°C for 5 seconds and annealing at 60°C for 30 seconds. Relative mRNA expression was normalized by GAPDH expression and determined using the 2-ΔΔCT method. The primer sequences used in the reaction are shown in Table 1. The results are shown in Figure 3G.

[0059]

[0060] As shown in Figure 3G, in microglia, the Poly(I:C)-added groups (Poly(I:C) or Poly(I:C) + KA groups) showed elevated levels of inflammatory cytokines (IL-6, IL-1β) and tissue-damaging cytokines (TNF-α), but coculture with UC-MSCs suppressed the expression of these cytokines. Meanwhile, iNOS, a marker of activated M1 macrophages, was elevated in the Poly(I:C)-added group, but Arg1 expression, an indicator of M2 macrophages thought to play a role in immunosuppression and tissue repair, was elevated by coculture with UC-MSCs. These morphological changes were not significantly different in the control group or the KA group, regardless of whether or not they were cocultured with UC-MSCs.

[0061] 3-4. Immunocytochemistry. Microglia were cultured on poly-L-lysine-coated glass coverslips (Matsunami Glass) and co-cultured with UC-MSCs using a 6-well plate transwell system. The coverslips were harvested, and the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. The samples were then blocked with 5% goat serum in 0.3% Triton-X 100 (Sigma-Aldrich) and then incubated overnight at 4°C with primary antibodies. The primary antibodies used were rabbit anti-Iba-1 (1:1000, Wako), rabbit anti-inducible nitric oxide synthase (iNOS) (1:1000, Abcam), and mouse anti-arginase 1 (Arg1) (GFP) (1:200, Abcam). The secondary antibodies used were donkey anti-mouse IgG heavy and light chain-specific antibodies (Alexa Fluor® 488) (1:1,000, Abcam) and donkey anti-rabbit IgG (Alexa Fluor® 594) (1:1,000, Abcam). Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Five images from each experimental group were taken with the same exposure time using an all-in-one fluorescence microscope (BZ-X800, KEYENCE, Osaka, Japan). The number of iNOS-positive and Arg1-positive cells / total cells was counted and quantified in three independent experiments. The results are shown in Figures 3H and 3I.

[0062] As shown in Figure 3H, in the Poly(I:C)-added group, the number of iNOS-positive cells (red) increased in microglia, indicating an M1 type, whereas co-culture with UC-MSCs increased the number of Arg1-positive cells (green), an indicator of M2 macrophages.

[0063] 4. Inflammation-Associated Status Epilepticus (SE) Model and UC-MSC Administration (In Vivo) 4-1. Administration of UC-MSCs to SE Model Mice Figure 4A shows the in vivo experimental design. Postnatal day 15 (P15) mice were administered 30 mg / kg poly(I:C) followed 3 hours later by 2.5 mg / kg kainic acid (KA) to induce status epilepticus, a condition associated with systemic inflammation during infant brain development. These drugs were administered intraperitoneally. Control pups received an equal volume of saline. Mice were continuously observed for up to 2 hours after KA administration. Seizure severity was assessed using a modified Rassin scale, adapted for age at P15. SE was defined as a grade 3 or higher seizure lasting at least 30 minutes and was analyzed as the SE group (Table 2). Mice that did not die or develop SE were excluded from the experiment. Six hours after KA administration, 1 × 10 frozen-thawed UC-MSCs were ５ Cells (in 50 μl of STEM-CELLBANKER) or cryoprotectant alone were administered via the tail vein. After treatment, the mice were returned to their original cages.

[0064]

[0065] Immunohistological analysis and Fluoro-Jade C (FJC) double labeling Immunohistochemical analysis and subsequent FJC staining were performed using free-floating tissue sections. 72 hours after UC-MSC administration (P18), SE mice were treated with CO ２Mice were euthanized by asphyxiation and perfused transcardially with phosphate-buffered saline (PBS) at pH 7.4, followed by 4% paraformaldehyde (PFA) in PBS. The extracted brain tissue was fixed overnight in 4% PFA and placed in sucrose-containing PBS at 4°C with increasing sucrose concentrations (10%, 20%, and 30%) every 24 hours. The brain tissue was then microtomed into 40-μm-thick free-floating sections. After washing, the sections were immersed in 5% goat serum in 0.3% Triton-X 100 (Sigma-Aldrich)-PBS for blocking and permeabilization, and then incubated overnight in rabbit anti-Iba1 antibody (1:1000) at 4°C. The sections were then washed in PBS and incubated with donkey anti-rabbit IgG (Alexa Fluor® 594) (1:1,000, Abcam) for 1 hour at room temperature. The fluorescently labeled sections were then attached to hydrophilic silanized slides. Using an FJC staining kit (Biosensys, Thebarton, South Australia), the sections were washed in distilled water for 2 minutes, immersed in 1% sodium hydroxide solution and 80% ethanol for 5 minutes, then washed in 70% ethanol for 2 minutes, and immersed in distilled water for 2 minutes. After incubation in potassium permanganate solution for 5 minutes, the slides were transferred to FJC staining solution and stained with DAPI for 10 minutes. For histological analysis, three brain regions (a: Bregma 1.00 to 0.00 mm, b: Bregma -1.50 to -2.50 mm, c: Bregma -2.50 to -3.50 mm) were stained per mouse, with three sections stained for each region. Five random, non-overlapping fields were acquired from each region for quantitative analysis. Images were taken with an all-in-one fluorescence microscope, BZ-X800 (KEYENCE). Cell counting and quantification were performed using Image J Fiji software, and the results are shown in Figures 4C, 4D, 4E, and 4F. In Figure 4C, A represents a normal control, B represents administration of 20 mg / kg of Poly(I:C) alone, C represents administration of 2.5 mg / kg of KA alone (≠SE), and D represents administration of 20 mg / kg of Poly(I:C) + 2.5 mg / kg of KA (=SE).

[0066] Figure 4B shows the areas of the mouse brain where significant changes were observed, and the locations where immunohistochemical staining was performed. As shown in Figure 4C, only the group administered poly(I:C) followed by KA 3 hours later (poly(I:C) + KA group) exhibited seizures of grade 3 or higher lasting 30 minutes or longer on the modified Rasin scale, and an increase in microglia (Iba1-positive cells: red) was observed in the CA1 region of the dorsal hippocampus. The poly(I:C) + KA group was used as a SE model mouse and the following analysis was performed.

[0067] Figures 4D-1 and 4D-2 are cross sections of region a in Figure 4B. In the lateral septum nucleus (LSN) region, SE model mice showed an increase in microglia (Iba1-positive cells: red) and a significant increase in degenerated neurons (FjC-positive cells: green). However, administration of UC-MSCs resulted in a decrease in degenerated neurons (P<0.001). The number of microglia (Iba1-positive cells: red) did not change with administration of UC-MSCs. Figures 4E-1 and 4E-2 are cross sections of region b in Figure 4B. In the CA1 region of the dorsal hippocampus of SE model mice, an increase in microglia (Iba1-positive cells: red) and a significant increase in degenerated neurons (FjC-positive cells: green) were observed. Administration of UC-MSCs resulted in a decrease in degenerated neurons (P<0.01). The number of microglia (Iba1-positive cells: red) did not change with UC-MSC administration (Figure 4E-2). Figures 4F-1 and 4F-2 are cross-sections of area c in Figure 4B, showing the CA1 region of the dorsal hippocampus. Similarly, an increase in microglia (Iba1-positive cells: red) was observed, along with a significant increase in degenerated neurons (FjC-positive cells: green). A decrease in degenerated neurons was observed with UC-MSC administration (P<0.01). The number of microglia (Iba1-positive cells: red) did not change with UC-MSC administration.

[0068] 4-3. Analysis of Inflammatory Cytokine Production in the Dorsal Hippocampus of SE Model Mice. 24 hours after administration of UC-MSCs, the dorsal hippocampus of SE model mice was collected, and total RNA was extracted using a NucleoSpin® RNA column (TaKaRa Corporation). Single-stranded cDNA was generated by reverse transcription using a PrimeScript RT Reagent Kit (TaKaRa Corporation). Real-time PCR was performed using TB Green® Premix Ex Taq™ II (TaKaRa Corporation). Expression levels of cytokines (IL6, TNF-α, iNOS) were measured under the following reaction conditions: initial denaturation at 95°C for 30 minutes, followed by 40 cycles of 95°C for 5 seconds and annealing at 60°C for 30 seconds. Relative mRNA expression was normalized by GAPDH expression and determined using the 2-ΔΔCT method. The primer sequences used in the reaction are shown in Table 1. The results are shown in Figure 4G.

[0069] As shown in Figure 4G, SE model mice showed a significant increase in inflammatory cytokines, which was significantly suppressed by administration of UC-MSCs. Furthermore, there was little difference in NKCC1 RNA expression between control and SE model mice, but KCC2 expression was significantly decreased in SE model mice and was restored by administration of UC-MSCs.

[0070] 4-4. Changes in NKCC1 and KCC2 during the acute phase We investigated NKCC1 and KCC2, which are cation-chloride cotransporters (CCCs) that are expressed in neurons and regulate intracellular Cl- concentrations, thereby controlling GABA (gamma amino butyric acid) signaling, which normally functions as an inhibitory system.

[0071] As shown in Figure 4H, qRT-PCR of dorsal hippocampal tissue performed using the same tissue as in 4-3 showed no significant changes in NKCC1 (Figure 4H). On the other hand, the SE group showed a significant suppression of KCC2 expression compared to the control group, but the UC-MSCs group showed a significant improvement.

[0072] 4-5. Open Field Test (Behavioral Assessment) An open field test was performed at ages 28-30. The open field box (40 × 40 × 40 cm [L × W × H]) consisted of a gray PVC floor and walls. Each mouse was recorded for 10 minutes on a video monitor. The body center and the four corners of the box were tracked using DeepLabCut (DLC), an open-access deep learning software. The deep learning network was trained using at least 200 manually labeled frames from multiple videos for 2 million iterations. Total movement time (seconds), total distance traveled (cm), and percent time spent in the center were then measured, and hyperactivity and anxiety were assessed using a custom R script available online (https: / / github.com / ETHZ-INS / DLCAnalyzer). The results are shown in Figure 5. (Control group = 9 animals, SE group = 9 animals, SE+ MSCs group = 9 animals, mean ± SD, *P < 0.05)

[0073] As shown in Figure 5A-E, the total distance traveled increased in the SE group compared to the control group (p = 0.072), and tended to improve with UC-MSC administration (p = 0.068). The total travel time tended to increase in the SE group compared to the control group (p = 0.099), but no improvement was observed with UC-MSC administration. On the other hand, the center-dwell rate was significantly increased in the SE group compared to the control group (p < 0.05), and significantly improved in the UC-MSC group compared to the SE group (p < 0.05).

[0074] In the above tests, each value is expressed as the mean ± standard deviation (SD) from three independent experiments. Differences between groups were analyzed using "R software" (https: / / cran.r-project.org / ). Multiple groups were compared using two-way and one-way analysis of variance (ANOVA), followed by Turkey's test. A P value of 0.05 or less was considered statistically significant.

[0075] 4-6. Y-maze Test (Behavioral Assessment) The Y-maze test was performed at ages 28-30. The Y-maze consisted of three gray PVC arms (40 × 3 × 20 cm [L × W × H]). Mice were placed in one arm of the apparatus, and their exploratory behavior in the maze was recorded for 10 minutes on a video monitor. Alternative behavior was defined as a complete entry of all four limbs into a different arm and was counted manually. The percentage of alternation was attributed to spatial working memory. The number of arm entries was also measured to reflect the level of hyperactivity. Figure 6A shows the counting method for the Y-maze. Figure 6B shows the alternation rate. Analysis of the alternation rate allows us to assess spontaneous locomotor activity and spatial working memory. If a mouse repeatedly enters the same arm, it is judged that it does not remember its own exploratory behavior, i.e., that it has poor spatial working memory. Figure 6C shows the total number of arm entries (measurement of hyperactivity). (Control group = 9 mice, SE group = 9 mice, SE + MSCs group = 9 mice, mean ± SD, *P < 0.05)

[0076] As shown in Figure 6C, no significant difference was observed in the alternation response rate between any of the groups. However, the SE group showed a significant increase in the total number of arm entries compared to the control group, and the UC-MSCs-administered group showed a decrease in the number of entries compared to the SE group. In other words, hyperactivity was observed in the SE group, but hyperactivity was significantly improved in the UC-MSCs-administered group. In SE model mice, behavioral evaluation using open-field and Y-maze tests revealed findings consistent with attention-deficit hyperactivity disorder (ADHD) rather than a decline in memory in the dorsal hippocampal CA1 region, and these findings were improved by early administration of UC-MSCs. The results of the open field and Y-maze tests showed that the SE model mice did not have any obvious motor dysfunction, and the cognitive dysfunction suggested attention-deficit hyperactivity disorder (ADHD) rather than a decline in working memory in the hippocampal CA1 region, and administration of UC-MSCs was shown to have an ameliorating effect.

Claims

1. A therapeutic agent for acute encephalopathy comprising umbilical cord-derived mesenchymal cells.

2. The therapeutic agent for acute encephalopathy according to claim 1, which is for intravenous administration.

3. The therapeutic agent for acute encephalopathy according to claim 1 or 2, wherein the umbilical cord-derived mesenchymal cells are cells prepared from umbilical cord tissue including amniotic membrane, blood vessels, perivascular tissue and Wharton's jelly.

4. The therapeutic agent for acute encephalopathy according to claim 1 or 2, wherein the umbilical cord-derived mesenchymal cells are (i) positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5 and PD-L2, and (ii) negative for CD45, CD34, CD11b, CD19 and HLA-class II.

5. The therapeutic agent for acute encephalopathy described in claim 1 or 2, wherein the acute encephalopathy is acute encephalopathy triggered by an infectious disease.

6. The therapeutic agent for acute encephalopathy according to claim 1 or 2, wherein the acute encephalopathy is acute infant encephalopathy.

7. A method for treating acute encephalopathy, comprising administering umbilical cord-derived mesenchymal cells that are (i) positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5, and PD-L2, and (ii) negative for CD45, CD34, CD11b, CD19, and HLA-class II.

8. The method for treating acute encephalopathy according to claim 7, wherein said administration is intravenous administration.

9. The method for treating acute encephalopathy according to claim 7 or 8, wherein the umbilical cord-derived mesenchymal cells are cells prepared from umbilical cord tissue including amniotic membrane, blood vessels, perivascular tissue and Wharton's jelly.

10. A method for treating acute encephalopathy described in claim 7 or 8, wherein the acute encephalopathy is acute encephalopathy triggered by an infectious disease.

11. The method for treating acute encephalopathy described in claim 7 or 8, wherein the acute encephalopathy is acute infant encephalopathy.

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