Cell therapeutic agent for treating central nervous system diseases

Abstract

The present invention relates to glial-like cells directly transdifferentiated from adult stem cells and to a cell therapeutic agent for treating central nervous system diseases comprising same. The glial-like cells directly transdifferentiated from adult stem cells by a method of the present invention reduce behavioral deficits including anxiety disorders, repetitive behaviors, social impairments, and memory decline, and restore decreased expression of DCX and PSD95 in the hippocampus to ameliorate neuronal or synaptic developmental damage, thereby providing an effect in which the cells can be utilized as a therapeutic agent for central nervous system diseases including autism spectrum disorders (ASD).

Description

Cell therapy for treating central nervous system diseases

[0001] The present invention relates to glial-like cells directly transdifferentiated from adult stem cells and a cell therapy agent for treating central nervous system diseases containing the same.

[0002] Central nervous system (CNS) disorders affect a wide range of populations with varying degrees of severity. Among them, neurological disorders, including schizophrenia (SCZ), autism spectrum disorders (ASD), depression, bipolar disorder, and anxiety disorders, affect a person's thoughts, mood, behavior, and social interactions and can significantly impair daily functioning (Diagnostic and Statistical Manual of Mental Disorders, 4th Ed., American Psychiatric Association (2000) and Diagnostic and Statistical Manual of Mental Disorders, 5th Ed., American Psychiatric Association (2013)).

[0003] Autism Spectrum Disorder (ASD), a neurological disorder, refers to a neurodevelopmental disorder characterized by primary symptoms such as repetitive behaviors, restricted interests, impaired verbal and nonverbal communication, or reduced understanding of social interactions. Currently, diagnostic methods for ASD based on molecular or pathological indicators have not yet been established, and there are no treatments available to address the underlying causes. While general pharmacological treatments are provided for comorbid symptoms such as epilepsy, self-harm, aggressive behavior, anxiety, or emotional disorders, there are currently no treatments available to address the core symptoms of social deficits and repetitive behaviors. Acute attacks in autistic patients are associated with inflammation within brain neural tissue, and research is underway to suppress brain inflammation by regulating glial cells. However, drug therapies that convert pro-inflammatory glial cells into a pro-inflammatory state can limit glial cell function and cause side effects, while astrocyte transplantation carries the risk of increasing the potential for inflammation. Furthermore, since the differentiation method based on pluripotent stem cells is the only way to obtain astrocytes, there is a disadvantage in that it requires ensuring safety when using allogeneic cells or a long differentiation period when using autologous cells. Additionally, schizophrenia, a representative psychiatric disorder, is a collection of various psychotic symptoms characterized by complex manifestations in diverse domains such as speech, behavior, emotion, and cognition, stemming from thought disorders as the primary pathology. Conventional antipsychotic drugs used to alleviate the symptoms of schizophrenia improve patients' quality of life to some extent, but they do not induce a complete cure, and their use is limited due to side effects. Depression includes major depressive disorder and hypothymia, and is associated with a depressed mood (sadness), impaired concentration, insomnia, fatigue, abnormal appetite, excessive guilt, and suicidal thoughts.Anxiety disorders are conditions characterized by fear, worry, and anxiety, which are typically generalized and aimless as an overreaction to situations. Anxiety disorders differ in the situations or types of objects that trigger fear, anxiety, or avoidance behaviors, as well as in the associated cognitive concepts. Anxiety differs from fear in that it is an emotional response to a perceived future threat, whereas fear is associated with a perceived or actual immediate threat. Anxiety and fear also differ in the context of associated thoughts or beliefs.

[0004] Meanwhile, glial cells are the major constituent cells of the nervous system responsible for maintaining homeostasis, including neuronal development, metabolism, function, and immunity. In the central nervous system, astrocytes, oligodendrons, and microglia function, while in the peripheral nervous system, Schwann cells and satellite glial cells do the same. They maintain nervous system homeostasis by responding to pathophysiological changes in the nervous system caused by genetic, developmental, and environmental factors. Since neuronal damage can be induced or repaired depending on the variable responses of glial cells in the early stages of various diseases, glial cells hold potential as cell therapies for the treatment of neurological disorders. Glial cells maintain neuronal function through neurofibrillary growth, synapse formation, inflammatory and immune responses, metabolic support, myelin formation, and acting as structural supports. While each glial cell possesses a distinct function to support the structure and function of the nervous system, Schwann cells of the peripheral nervous system are known to possess all of these diverse functions. In particular, they play an important role in inducing nerve regeneration through morphological and functional plasticity in the event of nerve damage, and are therefore evaluated as suitable cells for treating nerve damage as single-cell therapies.

[0005] The object of the present invention is to provide a method for producing glial-like cells.

[0006] In addition, the objective of the present invention is to provide glial cell-like cells.

[0007] In addition, the objective of the present invention is to provide a cell therapy composition for treating central nervous system diseases.

[0008] In addition, the objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of central nervous system diseases.

[0009] In addition, the objective of the present invention is to provide a use for glial cell-like cells for the treatment of central nervous system diseases.

[0010] In addition, the objective of the present invention is to provide a method for treating central nervous system diseases.

[0011] To solve the above problem, the present invention provides a method for producing glial cell-like cells.

[0012] In addition, the present invention provides glial cell-like cells produced by the above method.

[0013] In addition, the present invention provides a cell therapy composition for treating central nervous system diseases comprising the glial cell-like cells.

[0014] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of central nervous system diseases comprising the glial cell-like cell or cell therapeutic composition.

[0015] In addition, the present invention provides a use for glial cell-like cells for the treatment of central nervous system diseases.

[0016] In addition, the present invention provides a method for treating a central nervous system disease comprising the step of administering the glial cell-like cell or the cell therapeutic composition to an individual suffering from a central nervous system disease.

[0017] Glial cell-like cells directly transdifferentiated from adult stem cells by the method of the present invention have the effect of reducing behavioral deficits, including anxiety disorders, repetitive behaviors, social disorders, and memory decline, and improving neural or synaptic developmental damage by restoring the reduced expression of DCX and PSD95 in the hippocampus; therefore, they can be utilized as a therapeutic agent for central nervous system diseases, including autism spectrum disorders (ASD).

[0018] Figure 1 is a schematic diagram of direct transdifferentiation of mesenchymal stem cells isolated from an autistic patient into Magic cells (Mesenchymal-derived Anti-inflammatory Glial-like Induced Cells), which are glial-like cells.

[0019] Figure 2 is a diagram showing the process of differentiating mesenchymal stem cells from an autistic patient into Magic cells, which are glial cell-like cells.

[0020] Figure 3 is a figure confirming the morphological changes of Magic cells according to the differentiation period.

[0021] Figure 4 is a figure showing the changes in CD44, CD73, CD90, and CD105 expression levels in Magic cells analyzed by qRT-PCR.

[0022] Figure 5 is a figure showing the changes in NGFR, CD49d, S100b, and MPZ expression levels in Magic cells analyzed by qRT-PCR.

[0023] Figure 6 is a figure showing the change in the expression transcript of Magic cells analyzed by RNA sequencing.

[0024] Figure 7 shows transcripts whose expression changed by more than twofold in Magic cells compared to control mesenchymal stem cells.

[0025] Figure 8 shows the results of a DAVID ontology analysis performed on transcripts whose expression changed by more than twofold in Magic cells compared to control mesenchymal stem cells.

[0026] Figure 9 is a schematic diagram showing the manufacturing process of the autism model mouse and the stem cell administration schedule of the present invention.

[0027] Figure 10 shows the elevated plus maze test (A) and open field test (B) methods for evaluating anxiety behavior.

[0028] Figure 11 shows the open arm duration time evaluated by the elevated plus maze test:

[0029] ICV set: Intraventricular administration group of stem cells;

[0030] IN set: Intranasal administration group of stem cells;

[0031] Normal: Normal mouse group;

[0032] Autism: Autism mouse group induced by VPA administration;

[0033] STL: Low concentration of stem cells (total number of cells 2.5 x 10⁶ 4 ) administration group; and

[0034] STH: High concentration of stem cells (total number of cells 2.5 x 10⁶ 5 ) administration group; and

[0035] PC: Positive control group administered risperidone.

[0036] Figure 12 shows the track length, center duration time, and number of center entries (hyperactivity) evaluated by an open field test:

[0037] ICV set: Intraventricular administration group of stem cells;

[0038] IN set: Intranasal administration group of stem cells;

[0039] Normal: Normal mouse group;

[0040] Autism: Autism mouse group induced by VPA administration;

[0041] STL: Low concentration of stem cells (total number of cells 2.5 x 10⁶ 4 ) administration group; and

[0042] STH: High concentration of stem cells (total number of cells 2.5 x 10⁶ 5 ) administration group.

[0043] Figure 13 is a figure showing a self-grooming test picture for evaluating repetitive behavior.

[0044] Figure 14 shows the grooming time confirmed by the self-grooming test:

[0045] ICV set: Intraventricular administration group of stem cells;

[0046] IN set: Intranasal administration group of stem cells;

[0047] Normal: Normal mouse group;

[0048] Autism: Autism mouse group induced by VPA administration;

[0049] STL: Low concentration of stem cells (total number of cells 2.5 x 10⁶ 4 ) administration group; and

[0050] STH: High concentration of stem cells (total number of cells 2.5 x 10⁶ 5 ) administration group.

[0051] Figure 15 is a diagram showing the three-chamber test method for social skills assessment.

[0052] Figure 16 shows the stranger mouse exploration time and sociability index evaluated by the three-chamber test method:

[0053] ICV set: Intraventricular administration group of stem cells;

[0054] IN set: Intranasal administration group of stem cells;

[0055] Normal: Normal mouse group;

[0056] Autism: Autism mouse group induced by VPA administration;

[0057] STL: Low concentration of stem cells (total number of cells 2.5 x 10⁶ 4 ) administration group; and

[0058] STH: High concentration of stem cells (total number of cells 2.5 x 10⁶ 5 ) administration group; and

[0059] PC: Positive control group administered risperidone.

[0060] Figure 17 is a figure showing social disorder confirmed by evaluating the social preference index using the three-chamber test method:

[0061] ICV set: Intraventricular administration group of stem cells;

[0062] IN set: Intranasal administration group of stem cells;

[0063] Normal: Normal mouse group;

[0064] Autism: Autism mouse group induced by VPA administration;

[0065] STL: Low concentration of stem cells (total number of cells 2.5 x 10⁶ 4 ) administration group;

[0066] STH: High concentration of stem cells (total number of cells 2.5 x 10⁶ 5 ) administration group; and

[0067] PC: Positive control group administered risperidone.

[0068] Figure 18 shows a novel object recognition test to evaluate memory decline.

[0069] Figure 19 shows the discrimination index derived from the novel object recognition test:

[0070] ICV set: Intraventricular administration group of stem cells;

[0071] IN set: Intranasal administration group of stem cells;

[0072] Normal: Normal mouse group;

[0073] Autism: Autism mouse group induced by VPA administration;

[0074] STL: Low concentration of stem cells (total number of cells 2.5 x 10⁶ 4 ) administration group;

[0075] STH: High concentration of stem cells (total number of cells 2.5 x 10⁶ 5 ) administration group; and

[0076] PC: Positive control group administered risperidone.

[0077] Figure 20 shows the confirmation of DCX expression in the mouse hippocampus and the quantification of DCX-positive cells:

[0078] ICV set: Intraventricular administration group of stem cells;

[0079] IN set: Intranasal administration group of stem cells;

[0080] Normal: Normal mouse group;

[0081] Autism: Autism mouse group induced by VPA administration;

[0082] STL: Low concentration of stem cells (total number of cells 2.5 x 10⁶ 4 ) administration group; and

[0083] STH: High concentration of stem cells (total number of cells 2.5 x 10⁶ 5 ) administration group.

[0084] Figure 21 shows the confirmation of PSD95 expression in the mouse hippocampus and the quantification of PSD95-positive cells:

[0085] ICV set: Intraventricular administration group of stem cells;

[0086] IN set: Intranasal administration group of stem cells;

[0087] Normal: Normal mouse group;

[0088] Autism: Autism mouse group induced by VPA administration;

[0089] STL: Low concentration of stem cells (total number of cells 2.5 x 10⁶ 4 ) administration group; and

[0090] STH: High concentration of stem cells (total number of cells 2.5 x 10⁶ 5 ) administration group.

[0091] Figure 22 is a diagram analyzing the biodistribution of Magic cells injected into the ventricles:

[0092] Normal: Normal mouse group;

[0093] Autism: A group of autism mice induced by VPA administration; and

[0094] STH: High concentration of stem cells (total number of cells 2.5 x 10⁶ 5 ) administration group.

[0095] Figure 23 is a figure analyzing the neuroinflammatory inhibitory effect following the intraventricular injection of Magic cells:

[0096] Normal: Normal mouse group;

[0097] Autism: Autism mouse group induced by VPA administration;

[0098] STL: Low concentration of stem cells (total number of cells 2.5 x 10⁶ 4 ) administration group;

[0099] STH: High concentration of stem cells (total number of cells 2.5 x 10⁶ 5 ) administration group; and

[0100] PC: Positive control group administered risperidone.

[0101] Figure 24 is a figure analyzing the neurodevelopmental promotion effect following the injection of Magic cells into the ventricles:

[0102] Normal: Normal mouse group;

[0103] Autism: Autism mouse group induced by VPA administration;

[0104] STL: Low concentration of stem cells (total number of cells 2.5 x 10⁶ 4 ) administration group;

[0105] STH: High concentration of stem cells (total number of cells 2.5 x 10⁶ 5 ) administration group; and

[0106] PC: Positive control group administered risperidone.

[0107] Hereinafter, the present invention will be described in detail with reference to the attached drawings for embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0108] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0109] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

[0110] Throughout this specification, '%' used to indicate the concentration of a particular substance is (w / w) % for solid / solid, (w / v) % for solid / liquid, and (v / v) % for liquid / liquid, unless otherwise noted.

[0111] In one aspect, the present invention relates to a method for producing glial-like cells comprising the step of directly converting adult stem cells isolated from a patient with a central nervous system disease.

[0112] In one embodiment, the central nervous system disorder may be a neurological disorder, and the neurological disorder may be autism spectrum disorders (ASD).

[0113] In one embodiment, the central nervous system disease may be a degenerative brain disease, a psychiatric disease, or a neurodevelopmental disorder.

[0114] In one embodiment, the adult stem cells may be mesenchymal stem cells (MSCs), and the mesenchymal stem cells may be umbilical cord blood-derived mesenchymal stem cells (UCB-MSC), umbilical cord-derived mesenchymal stem cells (UC-MSC), adipose-derived mesenchymal stem cells (AD-MSC), or bone marrow-derived mesenchymal stem cells (BM-MSC).

[0115] In one embodiment, the adult stem cells may be directly transdifferentiated through the steps of: 1) culturing in a medium containing a histone methyltransferase inhibitor and a histone deacetylase (HDAC) inhibitor; 2) culturing in a medium containing a bone morphogenetic protein (BMP) inhibitor and an ALK-5 kinase inhibitor; and 3) culturing in a medium containing a GSK3β inhibitor, Neuregulin (Nrg), bFGF (basic fibroblast growth factor), PDGF-AA (Platelet-Derived Growth Factor AA), and retinoic acid.

[0116] In one embodiment, the histone methyltransferase inhibitor is BIX01294 (2-(Hexahydro-4-methyl-1H-1,4-diazepin-1-yl)-6,7-dimethoxy-N-[1-(phenylmethyl)-4-piperidinyl]-4-quinazolinamine), 5-AZA-2'-deoxycytidine (5-aza-2'-deoxycytidine:DAC), Zebularine, 3'-Deazaneplanocin A hydrochloride, Lomeguatrib, Chaetocin, It may be 2,2',3S,3'S,5aR,5'aR,6,6'-octahydro-3,3'-bis(hydroxymethyl)-2,2'-dimethyl-[10bR,10'bR(11aS,11'aS)-bi-3,11a-epidithio-11aH-pyrazino[1',2':1,5]pyrrolo[2,3-b]indole]-1,1',4,4'-tetrone) or decitabine (5-aza-2'-deoxycytidine).

[0117] In one embodiment, the HDAC inhibitor may be valproate, tricostatin A, phenylbutylate, sodium butylate, suberoylanilide hydroxamic acid (SAHA), or suberohydroxamic acid (SBHA), and the valproate may be valproic acid (VPA), sodium valproate, or divalproex sodium.

[0118] In one embodiment, the bone morphogenetic protein inhibitor may be LDN193189, Dorsomorphin, or Noggin.

[0119] 일 구현예에서, 상기 ALK-5 키나아제 억제제는 RepSox (1,5-Naphthyridine, 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]), SB525334 (6-(2-tert-butyl-4-(6-methylpyridin-2-yl)-1H-imidazol-5-yl)quinoxaline), GW788388 (4-(4-(3)-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)benzamide), SD-208 (2-(5-chloro-2-fluorophenyl)-N-(pyridin-4-yl)pteridin-4-amine), Galunisertib (LY2157299, 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide), EW-7197 (N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2-methanamine), LY2109761 (7-(2-morpholinoethoxy)-4-(2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline), SB505124 (2-(4-(benzo[d][1,3]dioxol-5-yl)-2-tert-butyl-1H-imidazol-5-yl)-6-methylpyridine), LY364947 (Quinoline, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl])l SB431542 (4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide)l K02288 (3)-[(6-Amino-5-(3),4,5-trimethoxyphenyl)-3-pyridinyl]phenol] or LDN-212854 (Quinoline, 5-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]).

[0120] In one embodiment, the GSK3 inhibitor may be CHIR99021 or BIO.

[0121] In one embodiment, the glial-like cell of the present invention relates to a glial-like cell having morphological characteristics similar to Schwann cells and increased expression of Schwann cell marker genes by stimulating the mesenchymal stem cell with an early neuroectodermal lineage differentiation signal, a peripheral neuronal differentiation signal, and a Schwann cell differentiation signal before the mesenchymal stem cell loses its early dividing ability.

[0122] In one embodiment, the glial cell-like cells of the present invention may be placed in the CA1 and CA3 regions of the hippocampus when administered into the ventricles.

[0123] In one embodiment, the glial cell-like cell of the present invention can reduce the activation of astrocytes or microglia, or the expression of TGFβ-1, which is increased by the onset of a central nervous system disease, and thereby have the effect of suppressing neuroinflammation.

[0124] In one embodiment, the glial cell-like cell of the present invention can increase the expression of neurotrophic factors (e.g., Trk R or BDNF) that are reduced by the onset of central nervous system disease, and thereby have the effect of promoting neurodevelopment.

[0125] As used in this invention, the term "stem cell" refers to undifferentiated cells capable of differentiating into various cells constituting biological tissues and capable of regenerating without limitation to form specialized cells of tissues and organs. Stem cells are pluripotent or multipotent cells capable of development. Stem cells proliferate into mature and complete forms of cells of tissues.

[0126] As used in the present invention, the term "mesenchymal stem cell" refers to an undifferentiated cell having multipotency derived from adult cells of mammals, including humans, preferably humans, and means a stem cell having multipotency capable of differentiating into adipocytes, osteocytes, chondrocytes, muscle cells, neurons, and cardiomyocytes. Mesenchymal stem cells may be derived from various adult cells, such as, for example, bone marrow, blood, brain, skin, fat (i.e., adipose tissue or adipocytes), umbilical cord blood, and Wharton's jelly of the umbilical cord.

[0127] The term "differentiation" as used in this invention refers to the phenomenon in which less specialized cells develop into specific cells, resulting in changes in cell size, shape, membrane potential, metabolic activity, and response to signals that characterize a particular type of cell. Differentiation is defined as the emergence of qualitative differences between parts of a biological system that were initially nearly homogeneous, or as a result, the state in which a system is divided into qualitatively distinguishable subsystems. In particular, stem cell differentiation refers to the phenomenon in which stem cells develop in a directional manner into cells possessing specific functions.

[0128] The term "Direct reprogramming, Direct conversion, Transdifferentiation" as used in this invention refers to a process in which mature (fully differentiated) cells of completely different cell types in higher organisms are induced to convert. It is a technology that converts arbitrary somatic cells (cell type A) into a specific desired cell type (cell type B) 'directly' without undergoing the process of redifferentiation, which involves dedifferentiating them into induced pluripotent stem cells capable of differentiating into all cell types.

[0129] In one aspect, the present invention relates to glial cell-like cells produced by the method of the present invention.

[0130] In one embodiment, the glial cell-like cell may have reduced expression of the CD44, CD73, or CD105 genes compared to the undifferentiated adult stem cell.

[0131] In one embodiment, the glial cell-like cell may have increased expression of CD90, NGFR, CD49d, S100b, or MPZ genes compared to undifferentiated adult stem cells.

[0132] In one embodiment, the gyosepher-like cells may have (i) reduced expression of CD44, CD73, and CD105 genes compared to undifferentiated adult stem cells, and (ii) increased expression of CD90, NGFR, CD49d, S100b, and MPZ genes compared to undifferentiated adult stem cells.

[0133] In one embodiment, the expression of the CD44, CD73, and CD105 genes may each be reduced by at least about 10% compared to the pre-differentiation adult stem cells.

[0134] In one embodiment, the expression of the CD90, NGFR, CD49d, S100b, and MPZ genes may each be increased by at least about 2 times compared to the pre-differentiation adult stem cells.

[0135] In one embodiment, the glial cell-like cells may be distributed in the CA1 and CA3 regions of the hippocampus when administered into the ventricles.

[0136] As used in the present invention, the term "expression" generally refers to a cellular process in which a biologically active polypeptide is generated from a DNA sequence and exhibits biological activity in a cell. In this sense, gene expression includes not only transcription and translation processes, but also post-transcriptional and post-translational processes that may affect the biological activity of the gene or gene product. These processes include, but are not limited to, RNA synthesis, processing, and transport, as well as polypeptide synthesis, transport, and post-translational modification of the polypeptide.

[0137] In the present invention, the expression can be confirmed by measuring the expression level of a gene or mRNA using a polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive RT-PCR, nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, or DNA chip method using a nucleic acid sequence, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair, a probe, or a primer pair and a probe that specifically recognize the nucleic acid sequence and a fragment of the sequence complementary to the nucleic acid sequence, and using an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetics that specifically recognize the entire length of the corresponding protein or its fragment using Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Protein expression levels can be confirmed by measuring them using radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray methods.

[0138] In one aspect, the present invention relates to a cell therapy composition for treating central nervous system diseases, comprising the glial cell-like cell of the present invention as an active ingredient.

[0139] In one embodiment, the composition of the present invention can improve neuronal or synaptic developmental impairment by restoring the reduced expression of DCX and PSD95 in the hippocampus.

[0140] In one embodiment, the composition of the present invention can improve anxiety disorders, repetitive behaviors, social disorders, or memory loss behaviors.

[0141] In one embodiment, the central nervous system disease may be a degenerative brain disease, a psychiatric disease, or a neurodevelopmental disorder.

[0142] In one embodiment, the central nervous system disorder may be a neurological disorder, and the neurological disorder may be schizophrenia (SCZ), autism spectrum disorders (ASD), depression, bipolar disorder, anxiety disorder, neurodegenerative disorder, neurodevelopmental disorder, post-traumatic stress disorder (PTSD), hearing disorder, panic disorder, or attention deficit / hyperactivity disorder (ADHD).

[0143] In one embodiment, the central nervous system disorder may be schizophrenia (SCZ), affective disorder, mental disorder, mood disorder, bipolar disorder, mania, depression, dysthymia, cyclothymic disorder, panic disorder, agoraphobia, social phobia, obsessive-compulsive disorder, post-traumatic stress disorder, anxiety disorder, somatoform disorder, hypochondriasis, dissociative disorder, sexual disorder, eating disorder, sleep disorder, regulation disorder, substance-related disorder, anhedonia, delirium, cognitive disorder, Alzheimer's disease, Parkinson's disease, mental retardation, autism spectrum disorder (ASD), Tourette's disorder, tic disorder, or attention deficit hyperactivity disorder.

[0144] In one embodiment, the glial cell-like cells of the present invention may be placed in the CA1 and CA3 regions of the hippocampus when administered into the ventricles.

[0145] In one embodiment, the glial cell-like cell of the present invention can reduce the activation of astrocytes or microglia, or the expression of TGFβ-1, which is increased by the onset of a central nervous system disease, and thereby have the effect of suppressing neuroinflammation.

[0146] In one embodiment, the glial cell-like cell of the present invention can increase the expression of neurotrophic factors (e.g., Trk R or BDNF) that are reduced by the onset of central nervous system disease, and thereby have the effect of promoting neurodevelopment.

[0147] In the present invention, the term "cell therapeutic agent" refers to a pharmaceutical product used for the purposes of treatment, diagnosis, and prevention, comprising living autologous, allogenic, or xenogenic cells and tissues produced by isolation, culture, and special processing from a human being. It refers to a pharmaceutical product used for the purposes of treatment, diagnosis, and prevention through a series of actions such as proliferating, selecting, or otherwise altering the biological characteristics of living autologous, allogenic, or xenogenic cells in vitro to restore the function of cells or tissues.

[0148] The cell therapeutic agent according to the present invention may be injected into the body of an individual, for example, by using the clinical method published by Lindvall et al. (1989, Arch. Neurol. 46: 615-31) or Douglas Kondziolka (Pittsburgh, 1998). The formulation may include a pharmaceutically acceptable conventional carrier in addition to the active ingredient, a mature cardiac tissue structure or organoid, and in the case of an injectable formulation, may include a preservative, an analgesic, a solubilizing agent, or a stabilizer, and in the case of a formulation for local administration, may include a base, an excipient, a lubricant, or a preservative.

[0149] The cell therapeutic agent according to the present invention may be manufactured in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person with ordinary knowledge in the ordinary art. The pharmaceutically acceptable carrier included in the cell therapeutic agent of the present invention is one that is commonly used in formulation and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. The cell therapeutic agent composition of the present invention may additionally include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., in addition to the above components.

[0150] The cell therapy product according to the present invention can be administered parenterally, intravenously, subcutaneously, intraperitoneally, or topically. Suitable dosages of the cell therapy product composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, diet, time of administration, route of administration, excretion rate, and response responsiveness.

[0151] In the present invention, the terms “administering,” “introducing,” and “implanting” are used interchangeably and may refer to the placement of a composition according to one embodiment into an individual by a method or route that results in at least partial localization of the composition according to one embodiment to a desired site. At least a portion of the cells or cellular components of the composition according to one embodiment may be administered by any suitable route to deliver them to a desired location within a living individual.

[0152] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of central nervous system diseases, comprising a glial cell-like cell or cell therapeutic composition of the present invention as an active ingredient.

[0153] In one embodiment, the central nervous system disorder may be schizophrenia (SCZ), affective disorder, mental disorder, mood disorder, bipolar disorder, mania, depression, dysthymia, cyclothymic disorder, panic disorder, agoraphobia, social phobia, obsessive-compulsive disorder, post-traumatic stress disorder, anxiety disorder, somatoform disorder, hypochondriasis, dissociative disorder, sexual disorder, eating disorder, sleep disorder, regulation disorder, substance-related disorder, anhedonia, delirium, cognitive disorder, Alzheimer's disease, Parkinson's disease, mental retardation, autism spectrum disorder (ASD), Tourette's disorder, tic disorder, or attention deficit hyperactivity disorder.

[0154] In one embodiment, the central nervous system disorder may be a neurological disorder, and the neurological disorder may be schizophrenia (SCZ), autism spectrum disorders (ASD), depression, bipolar disorder, anxiety disorder, neurodegenerative disorder, neurodevelopmental disorder, post-traumatic stress disorder (PTSD), hearing disorder, panic disorder, or attention deficit / hyperactivity disorder (ADHD).

[0155] In one embodiment, the composition may be an oral formulation, a parenteral formulation, or a topical formulation, and may be administered intravenously, intramuscularly, subcutaneously, into the spinal canal, ventricles, brain, or nasal cavity.

[0156] As used in the present invention, the term "prevention" refers to any act of suppressing or delaying the occurrence, development, and recurrence of central nervous system diseases by administering a composition according to the present invention.

[0157] As used in this invention, the term "treatment" refers to any act of improving or beneficially altering the symptoms of central nervous system diseases and complications arising therefrom through the administration of a composition according to this invention. A person skilled in the art to which this invention pertains would be able to determine the precise criteria for diseases to which the composition of this invention is effective, and to assess the degree of improvement, enhancement, and treatment, by referring to materials provided by organizations such as the Korean Medical Association.

[0158] As used herein, the term “treatment” refers to an approach to obtain beneficial or desirable clinical results. For the purposes of the invention, beneficial or desirable clinical results include, without limitation, relief of symptoms, reduction of disease range, stabilization of disease state (i.e., no worsening), delay or reduction of the rate of disease progression, improvement or temporary relief and alleviation of disease state (partially or wholly), and whether or not detectable.

[0159] Furthermore, "treatment" may also mean increasing the survival rate compared to the survival rate expected without treatment. "Treatment" refers to both therapeutic treatments and preventive or preventive measures. Such treatments include those required for disabilities that have already occurred as well as those that are prevented. "Palliating" a disease means reducing the extent of the disease state and / or undesirable clinical signs and / or slowing or prolonging the time course of progression compared to the case without treatment.

[0160] The therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the target site, the condition of the individual, etc.

[0161] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects. The effective dose level may be determined based on factors including the individual's health status, the type and severity of central nervous system disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Considering all of the above factors, it is important to administer an amount that obtains maximum effect with a minimum amount without adverse effects, and this can be easily determined by a person skilled in the art.

[0162] If the recipient animal can tolerate the administration of the composition or if the administration of the composition to the animal is appropriate, the composition indicates "pharmaceutical or physiologically acceptable." If the administered amount is physiologically significant, the said preparation may be said to have been administered at a "therapeutically effective amount." If the presence of the said preparation causes a physiologically detectable change in the recipient patient, the said preparation is physiologically significant.

[0163] As used herein, the "effective amount" is an appropriate amount that influences beneficial or desirable clinical or biochemical outcomes. The effective amount may be administered once or more. For the purposes of the present invention, the effective amount of the accelerator is an appropriate amount to temporarily alleviate, improve, stabilize, reverse, slow down, or delay the progression of the associated disease state.

[0164] The therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the target site, and the patient's condition. Therefore, when used in humans, the dosage should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations for determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0165] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects. The effective dose level may be determined based on factors including the patient's health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Considering all of the above factors, it is important to administer an amount that obtains maximum effect with a minimum amount without adverse effects, and this can be easily determined by a person skilled in the art.

[0166] The pharmaceutical composition of the present invention may include a carrier, a diluent, an excipient, or a combination of two or more of these commonly used in biological preparations. As used in the present invention, the term "pharmaceutical acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and may be used, for example, compounds listed in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into primary formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component using appropriate methods in the field or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0167] In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral formulations, topical preparations, suppositories, sterile injectable solutions, and sprays.

[0168] The composition of the present invention may also include carriers, diluents, excipients, or combinations of two or more thereof that are commonly used in biological preparations. Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for in vivo delivery of the composition, and may be used, for example, compounds listed in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into primary formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component using appropriate methods in the field or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0169] The composition of the present invention may additionally contain one or more active ingredients exhibiting the same or similar functions.

[0170] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0171] The composition of the present invention may be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) or orally depending on the intended method, and the dosage varies depending on the patient's body weight, age, gender, health condition, diet, time of administration, method of administration, excretion rate, and severity of the disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and it is more preferable to administer it once or several times a day.

[0172] Liquid formulations for oral administration of the composition of the present invention include suspensions, liquid formulations, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, etc.

[0173] In one aspect, the present invention relates to the use of glial cell-like cells for the treatment of central nervous system diseases.

[0174] In one aspect, the present invention relates to a method for treating a central nervous system disease comprising the step of administering a glial cell-like cell of the present invention or a cell therapeutic composition of the present invention to an individual suffering from a central nervous system disease.

[0175] The therapeutic method of the present invention comprises administering the glial cell-like cells of the present invention or the cell therapeutic composition of the present invention to an individual suffering from a central nervous system disease. It is preferable to apply a specific therapeutic effective dose for a specific individual differently depending on various factors, including the specific composition (such as the type and degree of the response to be achieved and whether other agents are used in some cases), the individual's age, body weight, general health status, gender and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and drugs used together or concurrently with the specific composition, as well as similar factors well known in the pharmaceutical field. The daily dosage is 0.0001 to 100 mg / kg based on the amount of the pharmaceutical composition of the present invention, preferably 0.01 to 100 mg / kg, and may be administered 1 to 6 times a day. However, it is obvious to those skilled in the art that the dosage or administration amount of each active ingredient must be such that it does not cause side effects by containing an excessively high content of each active ingredient. Therefore, it is preferable to determine the effective dose of the composition suitable for the purpose of the present invention by considering the aforementioned matters.

[0176] The above-mentioned individual is applicable to any mammal, and said mammal includes not only humans and primates, but also livestock such as cattle, pigs, sheep, horses, dogs, and cats.

[0177] The glial cell-like cells of the present invention or the cell therapy composition of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration may be anticipated, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intradural, or intracerebroventricular injection.

[0178] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0179] Example 1. Preparation of patient-derived glial-like cells

[0180] Mesenchymal-derived anti-inflammatory glial-like induced cells (MAGCs) were prepared by directly transdifferentiating mesenchymal stem cells isolated from patients with autism spectrum disorders (ASD) into glial-like cells (Fig. 1). Specifically, 0.5 ml / well of 0.1% gelatin solution was coated onto a 24-well plate at 37°C for 30 minutes, and mesenchymal stem cells isolated from ASD patients were coated 2×10 4Dispensed at a density of / well and cultured in 1 ml of alpha-MEM containing 10% FBS and 1% penicillin / streptomycin. After 24 hours of incubation, remove the existing culture medium, mix 800 ml of alpha-MEM containing 10% FBS and 1% penicillin / streptomycin with 200 ml of Neurobasal medium (Thermo Fisher, 21103049) containing 1% N-2 Supplement (Thermo Fisher, 17502001), 2% B-27 Supplement (Thermo Fisher, 12587001), 1% GlutaMAX (Thermo Fisher, 35050061), and 1% penicillin / streptomycin, and add 1 µM 5-Aza-2'-deoxycytidine (Sigma Aldrich, A3656), 50 µm valproic acid (Sigma Aldrich, 1708707), and 1 mM 1-Mercaptoethanol (VWR, 97604-590). The culture was performed with the added culture medium (Day 0). The existing culture medium was removed, and 600 ml of alpha-MEM containing 10% FBS and 1% penicillin / streptomycin was mixed with 400 ml of Neurobasal medium containing 1% N-2 Supplement, 2% B-27 Supplement, 1% GlutaMAX and 1% penicillin / streptomycin, and the culture was performed with the addition of 100 nM LDN193189 (Tocris, 6053) and 10 uM SB431542 (Tocris, 1614) (Day 1).The existing culture medium was removed, and 400 ml of alpha-MEM containing 10% FBS and 1% penicillin / streptomycin was mixed with 600 ml of Neurobasal medium containing 1% N-2 Supplement, 2% B-27 Supplement, 1% GlutaMAX and 1% penicillin / streptomycin, and cultured with the added culture medium containing 100 nM LDN193189 and 10 uM SB431542 (Day 2). The existing culture medium was removed, and 400 ml of alpha-MEM containing 10% FBS and 1% penicillin / streptomycin was mixed with 600 ml of Neurobasal medium containing 1% N-2 Supplement, 2% B-27 Supplement, 1% GlutaMAX and 1% penicillin / streptomycin, and cultured in the medium supplemented with 100 nM LDN193189, 10 uM SB431542 and 3 uM CHIR99021 (Tocris, 4423) (Day 3). The existing culture medium was removed, and 200 ml of alpha-MEM containing 10% FBS and 1% penicillin / streptomycin was mixed with 800 ml of Neurobasal medium containing 1% N-2 Supplement, 2% B-27 Supplement, 1% GlutaMAX and 1% penicillin / streptomycin, and cultured with the added culture medium containing 100 nM LDN193189, 10 uM SB431542 and 3 uM CHIR99021 (Day 4).The existing culture medium was removed, and 200 ml of alpha-MEM containing 10% FBS and 1% penicillin / streptomycin was mixed with 800 ml of Neurobasal medium containing 1% N-2 Supplement, 2% B-27 Supplement, 1% GlutaMAX and 1% penicillin / streptomycin. The culture was then incubated with the added medium containing 3 uM CHIR99021, 200 ng / ml Neuregulin (Peprotech, 100-03), 10 ng / ml basic FGF (Peprotech, 100-18C), 5 uM Retinoic acid (Sigma Aldrich, R2625), and 10 ng / ml PDGF-AA (Peprotech, 100-13A) (Day 5). 500 ml of the existing culture medium was removed, and 1 ml of Neurobasal medium containing 3 uM CHIR99021, 200 ng / ml Neuregulin, 10 ng / ml basic FGF, 5 uM Retinoic acid, 10 ng / ml PDGF-AA, 1% N-2 Supplement, 2% B-27 Supplement, 1% GlutaMAX, and 1% penicillin / streptomycin was added for incubation (Day 6). 500 ml of the existing culture medium was removed, and 500 ml of Neurobasal medium containing 1% N-2 Supplement, 2% B-27 Supplement, 1% GlutaMAX, and 1% penicillin / streptomycin was added. Based on 1 ml of culture medium, 3 uM CHIR99021, 200 ng / ml Neuroregulin, 10 ng / ml basic FGF, 5 uM Retinoic acid, and 10 ng / ml PDGF-AA were added and cultured (Day 9).500 ml of the existing culture medium was removed, and 500 ml of Neurobasal medium containing 1% N-2 Supplement, 2% B-27 Supplement, 1% GlutaMAX, and 1% penicillin / streptomycin was added. Then, based on 1 ml of culture medium, 3 uM CHIR99021, 200 ng / ml Neuroregulin, 10 ng / ml basic FGF, 5 uM Retinoic acid, and 10 ng / ml PDGF-AA were added and cultured (Day 12). Single cells were isolated by treating each well with 500 µl / well of Accutase (Sigma Aldrich, A6964) at 37°C for 30 minutes, and then diluted by adding 500 µl / well of Neurobasal medium containing N-2 Supplement, 2% B-27 Supplement, 1% GlutaMAX, and 1% penicillin / streptomycin. The cells were collected by centrifugation at 1,000 rpm at room temperature with the break-off state for 3 minutes (Day 15 or Day 21) (Fig. 2). The collected cells were washed once with PBS and diluted to an appropriate concentration for analysis or transplantation.

[0181] Example 2. Analysis of differentiation degree of Magic cells

[0182] 2-1. Morphological Analysis

[0183] When the morphological changes of the patient's mesenchymal stem cells were directly transdifferentiated and compared with mesenchymal stem cells of the same culture period to which the differentiation process was not applied, a change to a spindle shape of the cells was observed on days 7 and 14 of differentiation (Fig. 3).

[0184] 2-2. Analysis of Reduced Expression of Mesenchymal Stem Cell Marker Genes

[0185] Changes in the expression levels of mesenchymal stem cell marker genes CD44, CD73, and CD105, and the Schwann cell marker gene CD90 following direct transdifferentiation of mesenchymal stem cells into Magic cells were confirmed by qRT-PCR. At this time, mesenchymal stem cells with passage number 1 to 4 that had not undergone direct transdifferentiation (pMSC-unconverted) were used as the positive control, and Schwann cells differentiated from human embryonic stem cells (hESC-Schwann cell) were used as the negative control.

[0186] As a result, after direct transdifferentiation, the marker genes of mesenchymal stem cells, CD44, CD73, and CD105, decreased by more than 10%, 40%, and 20%, respectively, in Magic cells compared to the positive control group, while the expression level of CD90 was found to increase (Fig. 4). The gene expression patterns of all Magic cells were similar to those of the negative control group, human embryonic stem cell-derived Schwann cells.

[0187] 2-3. Analysis of Increased Expression of Positive Marker Genes Following Schwann Cell Differentiation

[0188] qRT-PCR was performed to increase the expression of Schwann cell marker genes NGFR, CD49d, S100b, and MPZ in Magic cells. In this case, Schwann cells differentiated from human embryonic stem cells (hESC-Schwann cells) were used as a positive control, and unconverted mesenchymal stem cells with passage number 1 to 4 were used as a negative control.

[0189] As a result of qRT-PCR analysis, the expression levels of GFR, CD49d, S100b, and MPZ in Magic cells increased by more than 1000%, 300%, 1200%, and 200%, respectively, compared to mesenchymal stem cells, which were the negative control, and changed to a pattern close to the expression levels of Schwann cells derived from human embryonic stem cells, which were the positive control (Fig. 5).

[0190] Example 3. Analysis of gene expression patterns in Magic cells

[0191] 3-1. Analysis of Changes in Expressed Transcripts

[0192] To confirm changes in the expression transcripts of Magic cells, RNA sequencing was performed on Magic cells (Converted cells), undifferentiated mesenchymal stem cells (MSCs d0), mesenchymal stem cells cultured in mesenchymal stem cell culture medium for the same period as differentiation (MSCs d15), and human embryonic stem cell-derived Schwann cells (Differentiated Schwann cells), and unbiased transcriptome analysis was performed.

[0193] As a result, the gene expression pattern of Magic cells was found to have changed to be similar to the gene expression pattern of human embryonic stem cell-derived Schwann cells (Fig. 6).

[0194] 3-2. Gene Ontology Analysis

[0195] Gene ontology analysis was performed using the DAVID algorithm with transcripts (Fig. 7) that showed a decrease or increase in expression of more than twofold in Magic cells (Converted cells) based on the transcript expression of control mesenchymal stem cells (MSCs D15) in Example 3-1 above.

[0196] As a result, a decrease in the expression of genes related to blood vessel, muscle, and bone development in mesenchymal stem cells and an increase in the gene group related to neuronal development were confirmed in Magic cells (Fig. 8).

[0197] Example 4. Confirmation of the anti-ASD effect of Magic cells

[0198] 4-1. Administration of Magic Cells

[0199] Valproic acid (VPA 500 mg / kg) was administered once intraperitoneally to ICR mice on day 12.5 of gestation. Subsequently, using an ASD mouse model with social deficits among the male pups born thereafter, Magic cells prepared in Example 1 (added to sterile physiological saline as an excipient) and an immunosuppressant (cyclosporine 15 mg / kg) were injected once at week 5. The Magic cells were at a low concentration (total cell count 2.5 x 10⁶ 4 ) or high concentration (total number of cells 2.5x10 5 At a dose of ), a total of 5 μL is surgically injected once into the ventricles (AP -0.58, mL 1.1, DV -3.0) at a rate of 0.5 μL / min (ICV), or at a high concentration (total cell count 2.5 x 10⁶ 5 Hyaluronidase solution was administered intranasally by dropping 1 μL into each nostril (5 μL in each side, for a total of 10 μL) at the dose of ) and, once absorption into the nose was complete, 5 μL was injected into each nostril in the same manner to administer a total of 10 μL intranasally (IN) (Table 1). At this time, the excipients were administered to the normal control group (Normal) and the disease group (Autism) in the same manner, while risperidone was administered intraperitoneally once daily for 3 weeks to the positive control group (PC). In the case of the immunosuppressant, it was administered intraperitoneally once on the day of Magic cell administration (Fig. 9).

[0200] Injection route (stem cell)GroupNDrugImmunosuppressantICVNormal14VehicleVehicleAutism12VehicleVehicleST L12Magic stem cell 2.5 x 10 4 cellsCyclosporine 15 mg / kgST H12Magic stem cell 2.5 x 10 5cellsCyclosporine 15 mg / kgPC10Risperidone 0.2 mg / kgVehicleINNormal3VehicleVehicleAutism2VehicleVehicleST H2Stem cell 2.5 x 10 5 cellsCyclosporine 15 mg / kg

[0201] 4-2. Behavioral Assessment

[0202] 4-2-1. Assessment of Anxiety Behaviors

[0203] To evaluate the anti-ASD efficacy of Magic cells, elevated plus maze and open field tests were performed 2 weeks after administration. Specifically, for the elevated plus maze experiment, mice were placed in the middle area of ​​an elevated cross maze consisting of two closed arms and an open arm at a height of 60 cm, and their movements were recorded for 5 minutes to measure the time spent on the open arm (Fig. 10A). Additionally, for the open field test, mice were acclimatized to the environment in an open box (length x width x Height = 45 x 45 x 45 cm) for 5 minutes, and their voluntary movements were recorded for 25 minutes (Fig. 10B). Using a Viewer program, the track length, time spent in the center zone, and number of entries were measured.

[0204] The efficacy of Magic cells for anxiety disorders caused by ASD was evaluated using the Elevated plus maze test. As a result, the open arm duration time in the Autism group was significantly reduced compared to the Normal group, confirming the presence of anxiety disorders (#p<0.05; compared with Normal group) (Fig. 11). When Magic cells were administered via ICV, the open arm duration time in the high-concentration group, which had been reduced by VPA administration, increased to a level similar to that of the positive control group, confirming that it improved anxiety induced by VPA administration (*p<0.05 and **p<0.01; compared with Autism group) (Fig. 11). Furthermore, the results of the open field test showed that VPA administration led to an increase in track length, center duration time, and center entry count in the Autism group compared to the Normal group, indicating a tendency toward hyperactivity due to VPA administration (Fig. 12). Although no significant improvement in hyperactivity was observed with the administration of Magic cell ICV, the administration of Magic cell IN showed a tendency to reduce the increase in track length and center duration time caused by VPA administration (Fig. 12).

[0205] 4-2-2. Evaluation of Repetitive Behaviors

[0206] To evaluate the anti-ASD efficacy of Magic cells, a self-grooming test was performed 3 weeks after administration. Specifically, mice were placed in a 1 L beaker, and their movements were recorded for 15 minutes to measure the grooming time as shown in Fig. 13.

[0207] As a result, a self-grooming test was performed to evaluate the efficacy of Magic cells on repetitive behavior. As a result, due to VPA administration, grooming time in the Autism group increased compared to the Normal group, and the high concentration group showed a tendency to reduce the increased grooming time (Fig. 14).

[0208] 4-2-3. Assessment of Social Disorders

[0209] To evaluate the anti-ASD efficacy of Magic cells, an assessment of social disorder was performed 3 weeks after administration. Specifically, in a chamber consisting of three compartments—left, middle, and right—experimental mice were placed in the middle compartment and allowed to adapt for 5 minutes. Then, a stranger mouse 1, which had the same age and sex as the experimental group but had no prior exposure, was placed in the wire cage in the right compartment, and the movements of the experimental group were recorded for 10 minutes (Fig. 15A). The sociability index was calculated using the following Equation 1.

[0210]

[0211] Afterwards, another stranger mouse 2 was placed in the empty cage in the left compartment. The existing stranger mouse 1 became a familiar mouse, and the social preference of the experimental population was measured for 10 minutes (Fig. 15B). The social preference index was derived using the following Equation 2.

[0212]

[0213] As a result, VPA administration significantly reduced the stranger mouse search time in the Autism group compared to the Normal group, showed no difference between the search times for stranger mice and empty cages (time investigating graph), and significantly reduced the sociability index (p<0.01 and p<0.0001; compared with Normal) (Fig. 16). On the other hand, ICV administration of Magic cells showed an effect of improving social disorders starting from the low-concentration group, and the high-concentration group confirmed a significant improvement effect similar to that of the positive control group (p<0.0001; compared with Autism group) (Fig. 16). Additionally, the group administered Magic cells IN showed a tendency to increase the reduction in stranger mouse search time and sociability index caused by Autism (Fig. 16). In addition, VPA administration significantly reduced the stranger mouse search time in the Autism group compared to the Normal group, showed no difference between the stranger mouse search time and the familiar mouse search time (time investigating graph), and significantly reduced the social preference index (###p<0.001; compared with Normal) (Fig. 17). Upon administration of Magic cell ICV, an effect of improving such social disorders was observed starting from the low-concentration group, and the high-concentration group confirmed a significant improvement effect similar to that of the positive control group (**p<0.01 and ****p<0.0001; compared with Autism group) (Fig. 17). Furthermore, the Magic cell IN administration group showed a tendency to increase the reduction in stranger mouse search time and the social preference index caused by Autism (Fig. 17).

[0214] 4-2-4. Assessment of Memory Decline

[0215] To evaluate the anti-ASD efficacy of Magic cells, a novel object recognition test was performed 4 weeks after administration. Specifically, mice were placed in an open box (length x width x Height = 45 x 45 x 45 cm) and allowed to adapt for 4 minutes, after which two identical objects were placed and the experimental population was given 4 minutes to explore them (Fig. 18). After 24 hours, one of the two objects was replaced with a new object, and the exploration time for both objects was measured for 4 minutes. The discrimination index, a memory indicator, was calculated using the following mathematical formula 3.

[0216]

[0217] As a result, it was confirmed that memory decline was induced by the administration of VPA, as the discrimination index in the Autism group was significantly reduced compared to the Normal group (###p<0.001; compared with Normal group) (Fig. 19). Upon administration of Magic cell ICV, a significant improvement in the discrimination index was observed starting from the low-dose group, and in particular, the effect in the high-dose group was similar to that of the positive control group (***p<0.001; compared with Autism group) (Fig. 19). Additionally, upon administration of Magic cell IN, a tendency was observed to improve the reduction in the discrimination index seen in the Autism group (Fig. 19).

[0218] 4-3. Histopathological Analysis

[0219] 4-3-1. Analysis of Neurodevelopmental Impairment

[0220] Since the neuropathology of ASD is typically characterized by defects in neuronal and synaptic development and function, we evaluated whether Magic cells could improve neurodevelopmental impairment by analyzing DCX expression, which plays a major role in neuronal development, through histological analysis. Specifically, 5 weeks after Magic cell administration, the thorax of mice was laparotized, perfused with 0.05 M phosphate buffered saline (PBS), and the brains were extracted using 4% PFA fixation. The extracted brains were fixed with 4% PFA and dehydrated with a 30% sucrose solution at 4°C for 5 days. Subsequently, the brain tissue was frozen at -80°C using an OCT compound, and coronal sections of the frozen tissue were performed to a thickness of 25 μm using a cryostat microtome. Among the obtained brain tissue sections, hippocampal sections were washed three times with PBS. They were reacted with a 1% H2O2 solution for 15 minutes to remove endogenous peroxidase and washed three more times with PBS. Washed tissue was reacted with anti-DCX antibody (1:200) at 4°C for at least 16 hours, then washed with PBS, and reacted with biotinylated-anti-goat IgG secondary antibody (1:250) at room temperature for 1 hour. After washing with PBS, color development was performed using 3,3-diaminobenzidine tetrahydrochloride, the stained tissue was attached to a slide and dried, then dehydrated using EtOH, and the dried slide was reacted with Xylene and mounted. Tissue images were acquired by taking photos with a K1-Fluo confocal microscope, and the images were quantified using Image J software.

[0221] As a result, DCX-positive cells in the hippocampus were found to be reduced in the Autism group compared to the Normal group (##p<0.01; compared with Normal group). On the other hand, administration of Magic cells via ICV showed a significant increase in DCX-positive cells, which had been reduced by VPA administration, starting from low doses (**p<0.01 and ***p<0.001; compared with Autism group) (Fig. 20). In addition, administration of Magic cells via IN also showed a tendency to improve the reduction of DCX-positive cells (Fig. 20).

[0222] 4-3-2. Analysis of Neurodevelopmental Impairment

[0223] To determine whether defects in synaptic development and function in ASD are improved by the administration of Magic cells, changes in the expression of PSD95 in brain tissue, a major factor located in excitatory synapses that regulates synaptic stabilization, were analyzed using an anti-PSD95 antibody (1:1000) and a biotinylated-anti-rabbit IgG secondary antibody (1:500) as in Example 5-3-1 above.

[0224] As a result, it was confirmed that PSD95 expression in the hippocampus was reduced in the Autism group compared to the Normal group (###p<0.001; compared with Normal group). On the other hand, administration of Magic cells ICV was shown to significantly increase PSD95 expression, which had been reduced by VPA administration, starting from low concentrations (Fig. 21). In addition, administration of Magic cells IN was also shown to significantly increase PSD95 expression, which had been reduced by VPA administration (**p<0.01 and ***p<0.001; compared with Autism group) (Fig. 21).

[0225] Based on the above results, it was confirmed that both intraventricular and intranasal administration of Magic cells restored the reduced expression of DCX and PSD95 in the hippocampus of a VPA autism spectrum disorder mouse model, thereby improving neuronal and synaptic developmental impairments and alleviating anxiety disorders, repetitive behaviors, social disorders, and memory impairment.

[0226] Example 5. Analysis of biodistribution following intraventricular injection of MAGIC cells

[0227] In order to analyze the biodistribution when the Magic cells of the present invention are injected into the ventricles, hippocampal tissue sections were obtained from normal control (Normal), disease group (Autism), and Magic cell high-dose administration group (STH) mice, and immunohistochemical analysis was performed using hNuclear antibody (1:200), GFAP antibody (1:1000), DAPI (1:1000), and fluorescently labeled secondary antibody that specifically react with human-derived nuclei.

[0228] As a result, unlike the normal control group (Normal) and disease group (Autism) in which no fluorescence expression of hNuclear was observed, fluorescence expression of hNuclear was clearly observed in the group administered high concentrations of Magic cells, and the fluorescence signal of hNuclear was detected in GFAP-positive cells (Fig. 22). This indicated that Magic cells can be distributed in the CA1 and CA3 regions of the hippocampus upon ICV administration.

[0229] Example 6. Analysis of the neuroinflammatory effect of intraventricular injection of MAGIC cells

[0230] To analyze the neuroinflammatory inhibitory efficacy after intraventricular injection of Magic cells, hippocampal tissue sections were obtained from normal control (Normal), disease group (Autism), Magic cell low-dose administration group (STL), Magic cell high-dose administration group (STH), and positive control (PC) mice, and immunohistochemical analysis was performed using antibodies against the astrocyte marker (GFAP) (1:1000) and the microglia marker (Iba-1) (1:1000). In addition, to analyze the anti-inflammatory cytokine (TGFβ-1), RNA was extracted from the hippocampus using trizol solution, synthesized into cDNA using TOPscript RT DryMIX, and the mRNA expression level of TGFβ-1 was analyzed via qRT-PCR.

[0231] As a result, VPA administration significantly increased the activation of astrocytes and microglia in the disease group compared to the normal control group (###p<0.001 and ####p<0.0001; compared with Autism), and the activation of astrocytes and microglia caused by VPA administration was significantly reduced in the low-concentration Magic cell administration group and the high-concentration Magic cell administration group to a degree similar to that of the positive control group (**p<0.01, ***p<0.001, and ****p<0.0001; compared with Autism) (Fig. 23). In addition, TGFβ-1 expression was significantly reduced in the disease group ($$p<0.01; compared with Autism, unpaired t-test), and the groups administered with low concentrations of stem cells and positive control showed a tendency to increase TGFβ-1 expression. In particular, the group administered with high concentrations of Magic cells showed a significant effect in restoring TGFβ-1 expression reduced by VPA administration (**p<0.01; compared with Autism) (Fig. 23), confirming that Magic cells showed an improvement effect on neuroinflammation occurring in the disease group.

[0232] Example 7. Analysis of the neurodevelopmental promotion effect following intraventricular injection of MAGIC cells

[0233] To analyze changes in neurotrophic factors that promote neurodevelopment after intraventricular Magic cell injection, mRNA levels of Trk receptor (Trk R) and BDNF in the hippocampus isolated from normal control (Normal), disease group (Autism), low concentration Magic cell administration group (STL), high concentration Magic cell administration group (STH), and positive control (PC) mice were analyzed by qRT-PCR as in Example 7 above.

[0234] As a result, the mRNA expression of Trk R in the disease group was significantly reduced compared to the normal control group due to VPA administration (#p<0.05; compared with Autism), and the Magic cell high-concentration administration group and the positive control group showed a tendency to restore the Trk R expression reduced by VPA administration (Fig. 24). In addition, BDNF mRNA expression showed a significantly increased effect in the Magic cell high-concentration administration group compared to the disease group (*p<0.05; compared with Autism) (Fig. 24), confirming that Magic cells promote the production of neurotrophic factors.

Claims

1. A method for producing glial-like cells comprising the step of directly transdifferentiating adult stem cells isolated from a patient with a central nervous system disease.

2. In Paragraph 1, The step of directly transdifferentiating the above adult stem cells is: 1) A step of culturing in a medium containing a histone methyltransferase inhibitor and an HDAC (histone deacetylase) inhibitor; 2) a step of culturing in a medium containing a bone morphogenetic protein (BMP) inhibitor and an ALK-5 kinase inhibitor; and 3) A method for producing glial cell-like cells comprising the step of culturing in a medium containing a GSK3β inhibitor, Neuregulin (Nrg), bFGF (basic fibroblast growth factor), PDGF-AA (Platelet-Derived Growth Factor AA), and retinoic acid.

3. In Paragraph 2, The above-mentioned histone methyltransferase inhibitors are BIX01294 (2-(Hexahydro-4-methyl-1H-1,4-diazepin-1-yl)-6,7-dimethoxy-N-[1-(phenylmethyl)-4-piperidinyl]-4-quinazolinamine), 5-AZA-2'-deoxycytidine (5-aza-2'-deoxycytidine:DAC), Zebularine, 3'-Deazaneplanocin A hydrochloride, Lomeguatrib, Chaetocin, A method for preparing glial cell-like cells, wherein the substance is 2,2',3S,3'S,5aR,5'aR,6,6'-octahydro-3,3'-bis(hydroxymethyl)-2,2'-dimethyl-[10bR,10'bR(11aS,11'aS)-bi-3,11a-epidithio-11aH-pyrazino[1',2':1,5]pyrrolo[2,3-b]indole]-1,1',4,4'-tetrone) or decitabine (Decitabine, 5-aza-2'-deoxycytidine).

4. In Paragraph 2, A method for producing glial cell-like cells, wherein the above HDAC inhibitor is valproate, tricostatin A, phenylbutylate, sodium butylate, suberoylanilide hydroxamic acid (SAHA), or suberohydroxamic acid (SBHA).

5. In Paragraph 2, A method for producing glial cell-like cells, wherein the above-mentioned bone morphogenetic protein inhibitor is LDN193189, Dorsomorphin, or Noggin.

6. In Paragraph 2, 상기 ALK-5 키나아제 억제제는 RepSox (1,5-Naphthyridine, 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]), SB525334 (6-(2-tert-butyl-4-(6-methylpyridin-2-yl)-1H-imidazol-5-yl)quinoxaline), GW788388 (4-(4-(3)-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)benzamide), SD-208 (2-(5-chloro-2-fluorophenyl)-N-(pyridin-4-yl)pteridin-4-amine), Galunisertib (LY2157299, 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide), EW-7197 (N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2-methanamine), LY2109761 (7-(2-morpholinoethoxy)-4-(2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline), SB505124 (2-(4-(benzo[d][1,3]dioxol-5-yl)-2-tert-butyl-1H-imidazol-5-yl)-6-methylpyridine), LY364947 (Quinoline, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl])l SB431542 (4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide)l K02288 (3)-[(6-Amino-5-(3),4,A method for preparing glial cell-like cells, wherein the cell is 5-trimethoxyphenyl)-3-pyridinyl]phenol] or LDN-212854 (Quinoline, 5-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]).

7. In Paragraph 2, A method for producing glial cell-like cells, wherein the above-mentioned GSK3 inhibitor is CHIR99021 or BIO.

8. Glial cell-like cells produced by the method of paragraph 1.

9. In Paragraph 8, The glial cell-like cell mentioned above is, (i) Compared to undifferentiated adult stem cells, the expression of CD44, CD73, and CD105 genes is reduced, and (ii) Glial cell-like cells characterized by increased expression of CD90, NGFR, CD49d, S100b, and MPZ genes compared to undifferentiated adult stem cells.

10. In Paragraph 9, Glial cell-like cells in which the expression of the CD44, CD73, and CD105 genes is reduced by at least about 10% each compared to pre-differentiated adult stem cells.

11. In Paragraph 9, Glial cell-like cells in which the expression of the CD90, NGFR, CD49d, S100b, and MPZ genes is each increased by at least about 2 times compared to pre-differentiated adult stem cells.

12. In any one of paragraphs 9 through 11, The glial cell-like cell is characterized by being distributed to the CA1 and CA3 regions of the hippocampus when administered into the ventricles.

13. A cell therapy composition for treating central nervous system diseases comprising the glial cell-like cells of claim 8 as an active ingredient.

14. In Paragraph 13, A cell therapy composition for treating central nervous system diseases, wherein the central nervous system disease is a degenerative brain disease, a psychiatric disease, or a neurodevelopmental disorder.

15. In Paragraph 13, A cell therapy composition for treating central nervous system diseases, wherein the above central nervous system disease is schizophrenia (SCZ), affective disorder, mental disorder, mood disorder, bipolar disorder, mania, depression, dysthymia, cyclothymic disorder, panic disorder, agoraphobia, social phobia, obsessive-compulsive disorder, post-traumatic stress disorder, anxiety disorder, somatoform disorder, hypochondriasis, dissociative disorder, sexual disorder, eating disorder, sleep disorder, regulation disorder, substance-related disorder, anhedonia, delirium, cognitive impairment, Alzheimer's disease, Parkinson's disease, mental retardation, autism spectrum disorder (ASD), Tourette's disorder, tic disorder, or attention deficit hyperactivity disorder.

16. A pharmaceutical composition for the prevention or treatment of central nervous system diseases comprising, as an active ingredient, the glial cell-like cell of claim 8 or the cell therapeutic agent composition of claim 13.

17. In Paragraph 16, A pharmaceutical composition for the prevention or treatment of central nervous system diseases, wherein the above pharmaceutical composition is an oral formulation, a parenteral formulation, or a topical formulation.

18. In Paragraph 16, The above pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of central nervous system diseases, administered intravenously, intramuscularly, subcutaneously, into the spinal canal, ventricles, brain, or nasal cavity.

19. Uses of glial-like cells for the treatment of central nervous system diseases.

20. A method for treating a central nervous system disease comprising the step of administering the glial cell-like cell of claim 8 or the cell therapeutic agent composition of claim 13 to an individual suffering from a central nervous system disease.

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