Direct trans-differentiation induction composition and stem cells treated therewith

Abstract

This invention provides a composition that induces direct trans-differentiation of CtBPs through oligomerization, and applications for stem cells treated with this composition. [Solution] Benzooxazole derivatives represented by the following structural formula, stereoisomers thereof, pharmaceutically acceptable salts thereof, or solvates or hydrates thereof are provided. The compounds of the present invention can regulate the oligomerization of CtBPs, thereby directly inducing trans-differentiation of adult stem cells into neural progenitor cells, and can therefore be utilized for inducing oligomerization or direct trans-differentiation of CtBPs. JPEG2026143756000015.jpg50130

Description

[Technical Field]

[0001] This invention relates to a composition that induces direct trans-differentiation of CtBPs through oligomerization, and to applications of stem cells treated with this composition. [Background technology]

[0002] The nervous system is divided into the central nervous system and the peripheral nervous system. The central nervous system includes the brain and spinal cord, while everything else is part of the peripheral nervous system. The peripheral nervous system is divided into the somatic nervous system, which includes motor and sensory nerves, and the autonomic nervous system. The nerve cells (neurons), astrocytes, and oligodendrocytes that make up the central nervous system (brain and spinal cord) can be produced by differentiating neural stem cells (NSCs) or neural progenitor cells (NPCs) that have been differentiated into pluripotent stem cells. On the other hand, the peripheral nerves (autonomic nerves, motor nerves, and sensory nerves) and Schwann cells that make up the peripheral nervous system originate from neural crest stem cells (NCSCs) that have been differentiated into pluripotent stem cells. Nerve cells, which make up nerve tissue, transmit command signals from the central nervous system to the peripheral nervous system via dendrites and axons, or transmit information received from the peripheral nervous system to the central nervous system. Axons transmit electrical signals to nerve cells further away from the central nervous system and are surrounded by myelin, an insulating structure, to speed up signal transmission; this is called myelination. The mammalian brain becomes capable of performing complex functions by developing a systematic neural network through a series of processes including the division and differentiation of neural stem cells, their survival and death, and synapse formation. Even in adulthood, animal brain nerve cells produce many substances necessary for nerve growth, causing axons and dendrites to grow, and differentiation continues because synaptic connections and neural networks are constantly remodeled (synaptic remodeling) each time new learning and memory occur. If nerve cells do not receive target-derived survival factors, such as nerve growth factors, during the process of cell differentiation and synapse formation, cell death occurs. Cell death caused by stress and cytotoxic agents is a major cause of degenerative brain diseases.Unlike the central nervous system, the peripheral nervous system of animals regenerates axons over a long period of time after injury. The posterior axon at the site of nerve injury degenerates through a process known as Wallerian degeneration, the nerve cell body then begins axonal regrowth, and Schwann cells further differentiate, determining target nerves through survival and death after division. It then undergoes further developmental processes and regenerates.

[0003] Degenerative neurological diseases are conditions characterized by the gradual structural and functional loss of nerve cells due to diverse causes, leading to the degeneration of central nervous system functions such as cognition, motor function, and sensory function. These diseases involve progressive nerve cell degeneration in specific areas of the nervous system, resulting in symptoms such as dementia, extrapyramidal abnormalities, cerebellar abnormalities, sensory impairments, and motor impairments. Simultaneous abnormalities in various areas can also occur, leading to complex and multifaceted symptoms. Diagnosis is based on the patient's clinical presentation, but the diverse nature of symptoms and the frequent sharing of common clinical manifestations make diagnosis difficult. These degenerative neurological diseases often develop gradually and tend to occur with aging. Once the disease develops, it progresses persistently for several years or even decades until death. Fundamental treatment is difficult, resulting in a significant social burden. While genetic factors, such as familial influence, play a crucial role in the onset of these diseases, acquired factors are also known to be important. Degenerative neurological diseases are broadly classified according to their clinical symptoms into progressive dementia (such as Alzheimer's disease), neurological abnormalities (such as Pick's disease), postural and motor abnormalities (such as Parkinson's disease), progressive ataxia, muscle atrophy and weakness, and sensory and motor disorders.

[0004] Multiple sclerosis (MS) is an autoimmune, chronic inflammatory, and demyelinating central nervous system disease that occurs in genetically susceptible individuals and is associated with congenital immune responses mediated by environmental factors such as viruses, as well as immunological factors such as antibodies and complement. MS is classified as an inflammatory demyelinating disease and can occur at any age, but it is most common between the ages of 20 and 40, with women being approximately twice as likely to develop it as men. This neurological disorder is caused by damage to the myelin sheath surrounding the axons of nerve cells due to an inflammatory response within the central nervous system. Lesions appear scattered throughout the white matter of the brain and spinal cord, exhibiting the typical primary inflammatory disease phenomenon in the affected areas. Initially, the inflammation is temporary, and some repair of the damaged myelin occurs but is not sustained. However, over time, pathological changes progress due to widespread microglia activation associated with concentrated and chronic neuronal degeneration, and clinically, the disability symptoms gradually become critical.

[0005] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is the most deadly progressive neurodegenerative disease characterized by significant loss of motor neurons in the primary motor cortex, brainstem, and spinal cord. The loss of motor neurons destroys basic and fundamental movements such as breathing, generally leading to death within 2 to 5 years of diagnosis. The progressive deterioration of motor function eventually severely impairs the patient's respiratory capacity, requiring some form of respiratory support for survival. Other symptoms also include muscle weakness in the hands, arms, legs, or swallowing muscles. Some patients (e.g., frontotemporal dementia (FTD)-ALS) may also develop frontotemporal dementia. According to the ALS Association, approximately 5,600 people are diagnosed with ALS in the United States each year. The incidence of ALS is 2 per 100,000 people. Two forms of ALS have been described: one is sporadic ALS (sALS), which is the most common form of ALS in the United States, accounting for 90-95% of all diagnosed cases; the other is familial ALS (fALS), which occurs mainly in families with dominant inheritance and accounts for only about 5-10% of all cases in the United States. sALS and fALS are clinically indistinguishable. Currently, glutamate toxicity inhibitors such as riluzole and antioxidants such as edaravone are prescribed clinically, but while these drugs may slightly slow the progression of ALS, they cannot be expected to have a therapeutic effect. Another strategy for treating ALS is stem cell-based therapy. Stem cells have the potential to differentiate into motor neurons and replace degenerated motor neurons in the central nervous system of ALS patients, such as the primary motor cortex, brainstem, and spinal cord. In fact, it has been suggested that stem cells derived from various sources, including induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) (e.g., bone marrow stromal cells (BMSCs) and adipose-derived stem cells (ASCs)), and neural stem cells of neural tissue origin (e.g., fetal spinal neural stem cells (NSCs), pluripotent neural progenitor cells (NPCs)), may replace degenerated motor neurons during the progression of ALS (see, for example, Kim et al., Exp. Neurobiol., 2014, 23(3), 207-214).

[0006] Parkinson's disease, first reported by James Parkinson in 1817, is a representative degenerative brain disease characterized by loss of motor function due to aging, specifically the loss of nerve cells in the substantia nigra (SN), where dopamine (a neurotransmitter that regulates muscles) is secreted, and dopamine deficiency in the striatum. Parkinson's disease affects approximately 1% of the population aged 65 and over, and about 5% of the population aged 85 and over (Twelves et al., Mov Disord 18, 19-31). Characteristic clinical symptoms include resting tremor, bradykinesia, rigidity, and postural instability, and the disease is induced by selective loss of dopaminergic neurons in the substantia nigra. The presence of intraneuronal proteinous inclusions known as Lewy bodies is a representative pathological feature (Olanow et al.). (al., Annu Rev Neurosci 22, 123-44). The exact cause of Parkinson's disease is not fully understood. In the sporadic form, which accounts for the majority of Parkinson's disease cases, the cause is idiopathic and largely unknown, but a complex interaction between environmental factors and genetic susceptibility, which has not yet been fully elucidated, is suspected to be an important cause (Langston et al.). , Ann Neurol 44(3 Suppl 1):S45-52). Pathologically, Parkinson's disease manifests as behavioral abnormalities due to the specific loss of dopaminergic neurons and nerve fibers containing melanin pigment in the substantia nigra of the midbrain, and dopamine deficiency in the striatum. Furthermore, Lewy bodies, which are protein aggregates, are observed as a disease-marking factor in the neurons of Parkinson's disease patients. Typical pathogenesis of Parkinson's disease includes oxidative stress, mitochondrial dysfunction, ubiquitin-proteasome dysfunction, and accumulation of misfolded proteins.

[0007] Damage to the spinal nerves due to trauma or other causes can lead to paralysis of bodily functions. Although spinal nerve tissue has a simpler structure than brain tissue, regeneration is not easy. Pathological phenomena that occur after spinal cord injury can be broadly divided into primary and secondary injuries depending on the time elapsed. Primary injury occurs within minutes of the injury, primarily involving necrosis of cells at the injury site. However, the cells are destroyed so rapidly at this stage that it is almost impossible to treat with pharmacological agents. Secondary injury, following primary injury, progresses slowly over several hours to several days. Not only do cells at the injury site degenerate, but surrounding undamaged nerve cells and oligodendrocytes also gradually begin to die through apoptosis. Cell death progresses continuously, centered on the injury site, and eventually, the damaged area within the spinal cord gradually expands. Furthermore, degeneration occurs in the axons, which are the pathways for nerve signal transport, and the myelin sheath that supports axon function, ultimately forming a cystic cavity within the spinal cord. This prevents further nerve signal transmission, leading to permanent functional loss. Research has been conducted to investigate the causes of pathological effects occurring long-term after spinal nerve trauma and to study regeneration, aiming to suppress or alleviate permanent functional paralysis caused by spinal cord injury. Because the initial mechanisms of spinal cord injury progress too rapidly and are difficult to manage with pharmacological treatment, the development of therapeutic agents with pharmacological strategies that address secondary mechanisms is crucial.

[0008] To date, various pharmacological treatments have been attempted, including steroids, antioxidants, glutamate hydroxylated inhibitors, ion channel inhibitors, anti-inflammatory drugs, and nerve growth factors, but methylprednisolone is currently the only one used clinically. However, methylprednisolone has limitations; its effects can only be expected if administered within 8 hours of spinal cord injury. While it has anti-inflammatory, antioxidant, and anti-cellular self-destructive effects, reducing secondary damage and causing some recovery, it carries the risk of infection and can induce gastrointestinal complications. In effect, there are currently no effective treatments for spinal cord injury.

[0009] Until now, the focus has been solely on developing treatments to reduce the degree of secondary damage that occurs after injury, rather than on nerve regeneration. However, in recent years, research on stem cells has become active in various fields, and the potential of nerve regeneration therapy for irreversible nervous system injuries such as spinal cord injury is gradually increasing.

[0010] On the other hand, cell therapy involves administering living cells for any purpose, including, for example, regenerative medicine, organ transplantation, and for the purpose of diagnosing or preventing any condition such as cancer. Stem cells are valued for their remarkable potential in cell therapy. Stem cells are cells that can differentiate into a variety of cells that make up biological tissues, and are a general term for undifferentiated cells that are a pre-differentiation step that can be obtained in the tissues of the embryo, fetus, and adult. Unlike cells whose differentiation into specific cells is initiated by differentiation stimuli (environment) and whose cell division has stopped once differentiation is complete, stem cells are characterized by their plasticity in differentiation, as they can produce identical cells through cell division (self-renewal), proliferate (expansion), and differentiate into other cells by other environments or other differentiation stimuli. Stem cells are broadly classified into embryonic stem cells (ES cells), which are derived from the embryo and possess pluripotency (the potential to differentiate into all cell types), and adult stem cells, which are multipotency (the potential to differentiate into multiple cell types) and are obtained from various tissues. Adult stem cells include hematopoietic stem cells, mesenchymal stem cells (MSCs), neural stem cells (NSCs), intestinal stem cells, muscle stem cells, hair follicle stem cells, and endothelial stem cells. Of these, mesenchymal stem cells are well known to be present in many mature tissues, possessing self-regenerative capabilities and the potential to differentiate into diverse lineages, and can be easily obtained from adult tissues such as bone marrow, adipose tissue, placenta, umbilical cord blood, and Wharton's jelly.

[0011] In order to overcome the limitations of conventional differentiation into specific cells using induced pluripotent stem cells and cell therapy achieved through this approach, extensive research has been conducted on direct transdifferentiation (direct conversion), which is a technique that "directly" reprograms any given somatic cell into a desired specific cell without going through the process of re-differentiation after reprogramming the somatic cell back into induced pluripotent stem cells that can differentiate into all cell types. However, existing direct transdifferentiation methods have various problems. For example, while cross-differentiation methods using pluripotent stem cells formed during the induction of reprogrammed stem cells can induce a variety of tissue cells, they carry the inherent risk of teratoma formation. Meanwhile, differentiation methods based on the genetic combination of reprogrammed stem cell factors and identified specific tissue cell-specific transcription factors have a low risk of teratoma formation, but face difficulties in identifying specific factors that induce differentiation into each respective tissue cell.

Prior Art Literature

Non-Patent Literature

[0012]

Non-Patent Literature 1

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Summary of the Invention

Problem to be Solved by the Invention

[0013] An object of the present invention is to provide a composition for inducing oligomerization of CtBPs (C-terminal binding proteins).

[0014] Another object of the present invention is to provide a composition for direct transdifferentiation induction.

[0015] Another object of the present invention is to provide a method for inducing oligomerization of CtBPs .

[0016] Another object of the present invention is to provide a method for inducing direct transdifferentiation.

[0017] Another object of the present invention is to provide a stem cell treated with any one or more compounds selected from the group consisting of a compound of Chemical Formula 1, a compound of Chemical Formula 2, and a compound of Chemical Formula 3.

[0018] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a nerve injury disease, comprising the stem cell as an active ingredient.

[0019] Another object of the present invention is to provide use of a compound of Chemical Formula 1, a compound of Chemical Formula 2, or a compound of Chemical Formula 3, for use in inducing oligomerization of CtBPs.

[0020] Another object of the present invention is to provide use of a compound of Chemical Formula 1, a compound of Chemical Formula 2, or a compound of Chemical Formula 3, for use in inducing direct transdifferentiation.

[0021] Another object of the present invention is to provide use of a stem cell treated with any one or more compounds selected from the group consisting of a compound of Chemical Formula 1, a compound of Chemical Formula 2, and a compound of Chemical Formula 3, for preventing or treating a nerve injury disease.

[0022] Furthermore, an object of the present invention is to provide a method for treating a nerve injury disease. Means for Solving the Problem

[0023] To achieve the above objective, the present invention provides a composition for inducing oligomerization of CtBPs, comprising one or more compounds selected from the group consisting of compounds of chemical formula 1, compounds of chemical formula 2, and compounds of chemical formula 3.

[0024] Furthermore, the present invention provides a composition for directly inducing trans-differentiation of adult stem cells into neural progenitor cells, comprising one or more compounds selected from the group consisting of compounds of chemical formula 1, compounds of chemical formula 2, and compounds of chemical formula 3.

[0025] Furthermore, the present invention provides a method for inducing oligomerization of CtBPs.

[0026] Furthermore, the present invention provides a method for directly inducing trans differentiation.

[0027] Furthermore, the present invention provides stem cells treated with one or more compounds selected from the group consisting of compounds of chemical formula 1, compounds of chemical formula 2, and compounds of chemical formula 3.

[0028] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of nerve injury diseases, comprising the aforementioned stem cells, stem cell cultures, or suspension cultures as active ingredients.

[0029] Furthermore, the present invention provides applications for the compound of chemical formula 1, the compound of chemical formula 2, or the compound of chemical formula 3 for use in the oligomerization induction of CtBPs.

[0030] Furthermore, the present invention provides uses for the compound of chemical formula 1, the compound of chemical formula 2, or the compound of chemical formula 3 for direct trans differentiation induction.

[0031] Furthermore, the present invention provides stem cells treated with one or more compounds selected from the group consisting of compounds of chemical formula 1, compounds of chemical formula 2, and compounds of chemical formula 3 for use in the prevention or treatment of nerve damage diseases.

[0032] Furthermore, the present invention provides a method for treating nerve injury diseases, comprising the step of transplanting stem cells treated with one or more compounds selected from the group consisting of compounds of chemical formula 1, compounds of chemical formula 2, and compounds of chemical formula 3 into an individual having a nerve injury disease. [Effects of the Invention]

[0033] According to the present invention, the compounds of the present invention can directly induce trans-differentiation of adult stem cells into neural progenitor cells by regulating the oligomerization of CtBPs. ciNSC5 cells thus directly trans-differentiated do not express neural stem cell markers or mature neuron markers, but exhibit significantly increased expression of intermediate neuronal progenitor or immature neuron marker proteins compared to adult stem cells. They also exhibit increased secretion of secretory proteins, including growth factors, tissue degradation factors, ligands involved in neuronal differentiation, neurogenesis, and axonal regeneration, and cytokines. Furthermore, they exhibit increased expression of enzymes and neurogenesis-related factors for neurogenesis, neurogenesis, neuroprotection, and glial scar degradation, and possess the ability to differentiate into dopaminergic neurons or neurons. Therefore, the compounds of the present invention can be utilized for inducing CtBP oligomerization or direct trans-differentiation. Since they showed remarkable therapeutic effects in both ALS mouse models, MS mouse models, PD Rett models, and chronic spinal cord injury Rett models, stem cells treated with the compounds of the present invention can be utilized as cell therapy agents. [Brief explanation of the drawing]

[0034] [Figure 1] This figure shows the DARTS (Drug Affinity Responsive Target Stability) analysis results for YJ101, YJ102, and YJ103. [Figure 2] This figure shows the CTBP1 oligomerization regulatory phase of YJ102, as confirmed by immunoprecipitation. [Figure 3]The molecular docking results of CtBP1 with YJ101, YJ102, and YJ103 are shown. [Figure 4] This figure shows the target gene of CtBP1-YJ102 identified using the ChIP method. [Figure 5] This figure shows the presence or absence of CtBP1-mediated differentiation of ciNSC5 by YJ102. [Figure 6] This figure shows whether or not direct trans-differentiation occurred in human UCB-MSCs treated with YJ101, YJ102, or YJ103. [Figure 7] This figure shows whether mesenchymal stem cells of diverse origins can be directly trans-differentiated by YJ102. [Figure 8] This figure shows the cytotoxicity of YJ101, YJ102, and YJ103 against UCB-MSCs. [Figure 9] This figure shows the effects of YJ101, YJ102, and YJ103 on the cell proliferation of UCB-MSCs. [Figure 10] This figure shows the expression of neuronal markers confirmed by immunofluorescence analysis in ciNSC5 cells treated with YJ102 for 5 days. [Figure 11] This figure shows the expression of neural progenitor cell marker proteins in ciNSC5 cells treated with YJ102 for 5 days, as confirmed by Western blot analysis. [Figure 12] This figure shows the expression of MSC cell surface antigen marker proteins by Western blot analysis in ciNSC5 cells treated with YJ102 for 5 days. [Figure 13] Figures 11 and 12 summarize the percentage increase or decrease in the Western blot analysis results. [Figure 14] This figure shows the increase in growth factors, tissue degradation, and neuronal differentiation / regeneration-related factors in ciNSC5 treated with YJ102 for 5 days. [Figure 15] This figure shows the results of Figure 14, categorized by the type of change. [Figure 16]This figure shows the results of Weston Block analysis of growth factors, tissue degradation, and neuronal differentiation / regeneration-related proteins in the culture medium after subculturing ciNSC5 differentiated from UCB-MSCs by YJ102 treatment and culturing them for 2 days in a medium without YJ102. [Figure 17] This figure shows the results of Figure 16, categorized by the type of change. [Figure 18] This figure shows the results of Weston Blot analysis of growth factors, tissue degradation, and neuronal differentiation / regeneration-related proteins in the culture medium after additional culture of ciNSC5 differentiated from UCB-MSCs by YJ102 treatment in a medium without YJ102 for 4 or 7 days. [Figure 19] This figure shows the results of Figure 18, categorized by the type of change. [Figure 20] This is the result of measuring the amounts of growth factors and six tissue degradation-related factors that increase with ciNSC5 in the medium after culturing ciNSC5 in a medium that does not contain YJ102 for two days, using ELISA. [Figure 21] This figure shows the ELISA result values ​​and the percentage increase observed in ciNSC5 medium compared to the amount observed in MSC medium. [Figure 22] This figure shows a heat map of the results of RNA-seq analysis of genes expressed in ciNSC5. [Figure 23] This figure shows the results of selecting and analyzing genes involved in neurogenesis, synapses, neurotransmitters, and nerve growth factors from factors whose expression changes in the culture medium on day 5, based on RNA-seq results. [Figure 24] Figure 23 shows the results of the gene ontology analysis in a table. [Figure 25] This figure shows the analysis of enzyme expression for glial scar degradation in ciNSC5 differentiated from UCB-MSCs treated with YJ102 for 5 days, performed by real-time RT-PCR. [Figure 26] This figure shows the analysis of neurogenesis-related transcription factor expression in ciNSC5 differentiated from UCB-MSCs treated with YJ102 for 5 days, performed by real-time RT-PCR. [Figure 27]This figure shows the results of real-time RT-PCR analysis of neuronal markers such as myelination, synapsis, matrix / cell adhesion, and calcium signaling in ciNSC5 differentiated from UCB-MSCs treated with YJ102 for 5 days. [Figure 28] This figure shows the analysis of neuronal receptor and channel expression in ciNSC5 differentiated from UCB-MSCs treated with YJ102 for 5 days, performed by real-time RT-PCR. [Figure 29] This figure shows the changes in cell characteristics of ciNSC5 during subculturing, as confirmed by subculturing doubling time. [Figure 30] This figure shows the changes in cell characteristics of ciNSC5 during subculturing, as confirmed by the CPDL value based on the number of subculturing cycles. [Figure 31] This figure shows the Tuj1 expression of ciNSC5 during subculturing. [Figure 32] This figure shows the confirmation of chromosomal abnormalities during subculturing of ciNSC5 via karyotyping. [Figure 33] This figure shows whether or not ciNSC5 differentiates into nerve cells. [Figure 34] This figure shows the adipogenesis during MDI treatment, as confirmed by the expression of its markers, C / EBPα and PPARγ. [Figure 35] This figure shows the lifespan of an ALS mouse model after ciNSC5 transplantation. [Figure 36] This figure shows the results of behavioral analysis (rotarod test, motor score, hanging wire test, and balance beam test) performed on an ALS mouse model after ciNSC5 transplantation. [Figure 37] This figure shows the lumbar spinal nerve loss in an ALS mouse model after ciNSC5 transplantation. [Figure 38] This figure shows the results of a behavioral analysis of an EAE mouse model after ciNSC5 transplantation. [Figure 39] This figure shows the extent and degree of demyelination in an EAE mouse model after ciNSC5 transplantation. [Figure 40]This image shows mononuclear cells that have infiltrated the spinal cord white matter of an EAE mouse model after ciNSC5 transplantation. [Figure 41] This image shows macrophages that have infiltrated the spinal white matter of an EAE mouse model after ciNSC5 transplantation. [Figure 42] This figure shows the activation of microglia and astrocytes in the spinal cord of an EAE mouse model after ciNSC5 transplantation. [Figure 43] This figure shows the activation of microglia and astrocytes in the spinal cord of an EAE mouse model after ciNSC5 transplantation. [Figure 44] This figure shows the process of fabricating a PD red model via 6-OHDA injection and the location of the stereotaxic lesion (Partial injury: AP +0.7, ML +2.6, DV -4.5 from bregma; and Complete injury: AP -2.2, ML +1.5, DV -8.0 from bregma). [Figure 45] This figure shows the recovery of motor function in the PD Rett model after ciNSC5 transplantation, confirmed by behavioral analysis (rotation test and stepping test). [Figure 46] This figure shows the disappearance of dopamine neurons in a PD Red model after ciNSC5 transplantation. [Figure 47] This figure shows the confirmation of transplantation using a human-specific antibody (STEM121) in a PD Red model after ciNSC5 transplantation. [Figure 48] This figure shows that ciNSCs differentiated into transplanted human dopamine neurons by confirming the presence of human-specific antibodies (STEM121) and dopamine neurons (TH-positive cells) at the transplantation site of cells in a PD red model after ciNSC5 transplantation. [Figure 49] This figure shows the results of behavioral analysis (BBB, gridwork, and footprint) confirming motor function recovery in a Rett model of chronic spinal cord injury after ciNSC5 transplantation. [Figure 50] This figure shows the analysis of motor function recovery using FRI in a Rett model of chronic spinal cord injury after ciNSC5 transplantation. [Figure 51] This figure shows the lesion volume and myelin loss in a Rett model of chronic spinal cord injury after ciNSC5 transplantation. [Figure 52] This figure shows the degree of gliotic injury formation at the lesion site in a Rett model of chronic spinal cord injury after ciNSC5 transplantation. [Figure 53] This figure shows axonal regeneration confirmed by BDA-preceding axonal tracking in a Rett model of chronic spinal cord injury after ciNSC5 transplantation. [Figure 54] This figure shows axonal regeneration confirmed by retrograde axon tracking using fluorogold in a Rett model of chronic spinal cord injury after ciNSC5 transplantation. [Figure 55] This figure shows the expression pattern of the axonal regeneration factor GAP43 in a chronic spinal cord injury Rett model after ciNSC5 transplantation, as confirmed by Western blotting. [Figure 56] This figure shows the results of immunohistochemical staining performed using the human-specific antibody STEM121 antibody and a neuronal cell marker to confirm whether cells in a chronic spinal cord injury Rett model after ciNSC5 transplantation had differentiated into mature neurons 6 weeks after transplantation. [Modes for carrying out the invention]

[0035] The present invention will be described in detail below with reference to the attached drawings and examples of its implementation. However, the following examples are presented as illustrative examples of the present invention, and if it is determined that a specific description of a well-known technology or configuration to those skilled in the art would unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and this will not limit the present invention. The present invention is subject to various modifications and applications within the scope of equivalents described in the claims below and analyzed therefrom.

[0036] Furthermore, the terminology used herein is intended to appropriately describe preferred embodiments of the present invention and may vary depending on the intent of the user, operator, or the conventions of the art to which the present invention pertains. Therefore, definitions of these terms must be based on the overall content of this specification. When a part of the specification "includes" a component, this means, unless otherwise stated to the contrary, that it does not exclude other components, but rather that other components may be further included.

[0037] All technical terms used herein, unless otherwise defined, are used in the sense generally understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of this invention. The contents of all publications cited herein as references are incorporated into this invention.

[0038] Throughout this specification, the percentages used to indicate the concentration of a particular substance, unless otherwise specified, refer to (w / w)% for solid / solid, (w / v)% for solid / liquid, and (v / v)% for liquid / liquid.

[0039] In one aspect, the present invention relates to benzoxazole derivatives represented by the following chemical formula 1, stereoisomers thereof, pharmaceutically acceptable salts thereof, or solvates or hydrates thereof.

[0040] [ka]

[0041] In Chemistry 1, R1 to R3 are each independently hydrogen; halogen group; C1-C4 linear or branched alkyl group; C1-C4 linear or branched alkoxy group; nitro group (-NO2); or amino group (-NH2). L is a C1-C4 alkylene group, R4 is a linear or branched alkoxy group of C1-C4; -C(=O)OR6; or - OC(=O)R7, R5 is hydrogen; halogen group; C1-C4 linear or branched alkyl group; or C1- A linear or branched alkoxy group of C4, R6 and R7 are each independently C1-C4 linear or branched alkyl groups. .

[0042] In the present invention, the stereoisomer is an isomer that arises from a spatially different arrangement of atoms or groups of atoms within a molecule, and includes all optical isomers and geometric isomers.

[0043] In the present invention, the hydrate is a compound in which water is bonded to a benzoxazole derivative, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof by non-covalent intermolecular forces, and may contain a stoichiometric or non-stoichiometric amount of water. Specifically, the hydrate may contain water in a ratio of about 0.25 moles to about 10 moles based on 1 mole of the active ingredient, and more specifically, it may contain about 0.5 moles, about 1 mole, about 1.5 moles, about 2 moles, about 2.5 moles, about 3 moles, about 5 moles, etc.

[0044] In this specification, "solvate" refers to a solvate in which a benzoxazole derivative, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof is bonded to a solvent other than water by intermolecular forces, and the solvent may be present in stoichiometric or non-stoichiometric amounts. Specifically, the solvate may contain solvent molecules in a ratio of approximately 0.25 moles to approximately 10 moles based on 1 mole of the active ingredient, and more specifically, approximately 0.5 moles, approximately 1 mole, approximately 1.5 moles, approximately 2 moles, approximately 2. It can be contained in amounts such as 5 moles, approximately 3 moles, or approximately 5 moles.

[0045] In one embodiment, the benzoxazole derivative represented by Chemical Formula 1 may be a benzoxazole derivative represented by the following Chemical Formula 2, its stereoisomer, its pharmaceutically acceptable salt, or its solvate or hydrate. .

[0046] [ka]

[0047] In Chemistry 2, The definitions of L and R1 to R5 are as defined in Formula 1 above. In one specific example, two of R1-R3 are hydrogen atoms, and the remaining one is a hydrogen halogen group. A C1-C4 linear or branched alkyl group; a C1-C4 linear or branched alkoxy group; a nitro group (-NO2); or an amino group (-NH2).

[0048] In the embodiments of the present invention, the benzoxazole derivative represented by Chemical Formula 1 is the following chemical formula It may be any one of the compounds of chemical formula I, chemical formula II, or chemical formula III.

[0049] [ka]

[0050] In one aspect, the present invention relates to a composition for inducing the oligomerization of CtBPs (C-terminal Binding Proteins), comprising one or more compounds selected from the group consisting of compounds of chemical formula I, chemical formula II, and chemical formula III, as shown in Chemical Formula 3.

[0051] In one specific example, CtBPs may be either CtBP1 or CtBP2.

[0052] In one embodiment, the composition can protect CtBPs from enzymes and induce oligomerization.

[0053] In one embodiment, the oligomerization may be homooligomerization of CtBP1-CtBP1 or CtBP2-CtBP2.

[0054] In one embodiment, the compounds of chemical formula I, chemical formula II, and chemical formula III can directly bond with CtBP1, and the compound of chemical formula 2 can also directly bond with CtBP2.

[0055] In one embodiment, the compound of chemical formula I can bond to Ser100 of CtBP1, the compound of chemical formula II can bond to Ser100 and Arg266 of CtBP1, and the compound of chemical formula III can bond to Phe102, Arg184, and His236 of CtBP1.

[0056] In one embodiment, the composition can reduce the expression of HES1.

[0057] In one embodiment, the composition can increase the expression of Sox2.

[0058] In one embodiment, the composition can induce the expression of OCT4.

[0059] In one embodiment, the composition can differentiate adult stem cells into neural progenitor cells via CtBPs.

[0060] In one example, adult stem cells may be mesenchymal stem cells (MSCs), and the mesenchymal stem cells may be umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) or umbilical cord-derived mesenchymal stem cells (UC-MSCs). C) Adipose-derived mesenchymal stem cells (AD-MSC) or bone marrow-derived mesenchymal stem cells (BM- It can be MSC.

[0061] In one example, neural progenitor cells were ciNSC5 (chemically induced It may be a neural stem cell (5).

[0062] In one embodiment, the composition may be a reagent composition or a culture medium composition. In one aspect, the present invention relates to a composition for inducing direct conversion of adult stem cells to neural progenitor cells, comprising one or more compounds selected from the group consisting of compounds of chemical formula I, compounds of chemical formula II, and compounds of chemical formula III.

[0063] In one embodiment, the composition can induce CtBP oligomers from adult stem cells and induce direct trans-differentiation into neural progenitor cells via CtBPs.

[0064] In one embodiment, adult stem cells may be mesenchymal stem cells, and these mesenchymal stem cells may be umbilical cord blood-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells. Since any adult stem cell can be applied regardless of the specific tissue cell, the invention is not limited thereto. In many specific embodiments of the present invention, mesenchymal stem cells derived from umbilical cord (blood) were used, but it was confirmed that direct trans-differentiation into neural progenitor cells is also possible from adipose-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells.

[0065] In one embodiment, the neural progenitor cell may be ciNSC5.

[0066] In one embodiment, the composition can increase the expression of a neuronal marker, which may be Tuj1, TBR2, MASH1, GAP-43, or p75.

[0067] In one embodiment, the composition can increase the expression of neural progenitor cell marker proteins, which may be Tuj1 protein, CD325 (N-cadherin) protein, p75 protein, GAP-43 protein, CD54 protein, CD309 protein, CD56 (NCAM) protein, PSA-NCAM protein, CD29 protein, MASH1 protein, or TBR2 protein, and the expression of Tuj1 protein is increased by 6 times or more, preferably 6 to 8 times, and the expression of CD325 (N-cadherin) protein is increased by 7 times after induction compared to before direct trans differentiation induction. In summary, it is possible to increase the expression of the following proteins by a preferred 7-10 times, p75 protein by 4 times or more, preferably 4-8 times, GAP43 protein by 4 times or more, preferably 4-7 times, CD54 protein by 2 times or more, preferably 2-5 times, CD309 protein by 3 times or more, preferably 3-7 times, CD56(NCAM) protein by 3.5 times or more, preferably 3.5-6 times, PSA-NCAM protein by 4 times or more, preferably 4-6 times, CD29 protein by 4.5 times or more, preferably 4.5-8 times, MASH1 protein by 4 times or more, preferably 4-8 times, or TBR2 protein by 3 times or more, preferably 3-7 times.

[0068] In one embodiment, the composition can reduce the expression of adult stem cell marker proteins, which may be CD44, CD73, or CD105, and can reduce the expression of CD44 protein by 50-90%, CD73 protein by 30-70%, or CD105 protein by 50-90% after induction compared to before direct trans-differentiation induction.

[0069] In one embodiment, the composition can increase the secretion of secretory proteins, which may be growth factors, tissue degradation factors, ligands, or cytokines.

[0070] In one embodiment, the growth factor may be PlGF, NGF, BDNF, or VEGFA, and the secretion of PlGF can be increased by 1.5 to 4 times, NGF by 1.5 to 6 times, BDNF by 1.5 to 3 times, or VEGFA by 1.5 to 5 times after induction compared to before direct trans-differentiation induction.

[0071] In one embodiment, the tissue degradation factor may be MMP1, MMP2, MMP7, or TIMP2, and the secretion of MMP1 can be increased by 1.5 to 3.5 times or MMP2 by 2 to 6 times after induction compared to before direct trans differentiation induction.

[0072] In one embodiment, the ligand may be SHH or Notch1.

[0073] In one embodiment, the cytokine may be TNFSF12 or IL-16, and the secretion of TNFSF12 can be increased by 1.5 to 4 times after induction compared to before direct trans differentiation induction.

[0074] In one embodiment, the composition may be a reagent composition or a culture medium composition.

[0075] In the present invention, the mRNA expression level of the marker gene can be measured by polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive RT-PCR, RNase, S1 nuclease assay, in situ hybridization, nucleic acid microarray, Northern blotting, or DNA chip method using the nucleic acid sequence of the marker, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair, probe that specifically recognizes the nucleic acid sequence and fragments of the complementary sequence, or a primer pair and probe. The protein expression level of the marker can be measured by Western blotting, ELISA (enzyme-linked immunosorbent assay) using an antibody, antibody fragment, aptamer, avidity multimer, or peptide mimetic that specifically recognizes the full-length protein or fragments of the marker protein. It can be measured by methods such as assay, radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray.

[0076] The term "direct reprogramming (direct conversion, transdifferentiation)" used in this invention refers to a technique for inducing conversion between mature (differentiated) cells of completely different cell types in higher organisms. This technique involves directly converting any somatic cell (cell type A) into a specific cell type (cell type B) without going through the process of re-differentiation, which involves dedifferentiating it into induced pluripotent stem cells capable of differentiating into any cell type. Currently, direct reprogramming is recognized as having potential applications in disease modeling and new drug discovery, and it is expected to be applied to gene therapy and regenerative medicine in the future.

[0077] In one aspect, the present invention relates to a method for inducing oligomerization of CtBPs, comprising the step of treating a sample in vitro with one or more compounds selected from the group consisting of compounds of chemical formula I, chemical formula II, and chemical formula III.

[0078] In one example, the compound can be treated for 3 to 7 days, and most preferably for 5 days.

[0079] In one specific example, CtBPs may be either CtBP1 or CtBP2.

[0080] In one embodiment, the oligomerization may be homooligomerization of CtBP1-CtBP1 or CtBP2-CtBP2.

[0081] In one embodiment, the sample may be an adult stem cell, and in one embodiment, the adult stem cell may be a mesenchymal stem cell, and the mesenchymal stem cell is These may be umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), umbilical cord-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, or bone marrow-derived mesenchymal stem cells.

[0082] In one example, the method showed that the sample had Tuj1 and CD3 levels after treatment compared to before treatment. 25 (N-cadherin), p75, GAP-43, CD309, CD56 (NCA M) can increase the expression of PSA-NCAM, CD29, MASH1, or TBR2, and Tuj1 can be increased by 6 times or more, preferably 6 to 8 times, CD325 (N-cadherin) by 7 times or more, preferably 7 to 10 times, p75 by 4 times or more, preferably 4 to 8 times, GAP43 by 4 times or more, preferably 4 to 7 times, CD54 by 2 times or more, preferably 2 to 5 times, CD309 by 3 times or more, preferably 3 to 7 times, CD56 (NCAM) by 3.5 times or more, preferably 3.5 to 6 times, PSA-NCAM by 4 times or more, preferably 4 to 6 times, CD29 by 4.5 times or more, preferably 4.5 to 8 times, MASH1 by 4 times or more, preferably 4 to 8 times, or TBR2 expression can be increased by more than three times, preferably by three to seven times.

[0083] In one embodiment, the method can increase the secretion of growth factors, tissue degradation factors, ligands, or cytokines in the sample after treatment compared to before compound treatment, and the secretion of PlGF, NGF, BDNF, VEGFA, MMP1, MMP2, M after treatment compared to before compound treatment. It can increase the secretion of MP7, TIMP2, SHH, Notch1, TNFSF12, or IL-16, and increase the secretion of PlGF by 1.5-4 times, NGF by 1.5-6 times, BDNF by 1.5-3 times, VEGFA by 1.5-5 times, MMP1 by 1.5-3.5 times, MMP2 by 2-6 times, or TNFSF12 by 1.5-4 times. It can also decrease the expression of CD44, CD73, or CD105 in the sample after treatment compared to before treatment, reducing CD44 expression by 50-90%, CD73 expression by 30-70%, or CD105 expression by 50-90%. It can be reduced by 0%.

[0084] In one aspect, the present invention relates to a method for directly inducing trans-differentiation of adult stem cells into neural progenitor cells, comprising the step of treating adult stem cells in vitro with one or more compounds selected from the group consisting of compounds of chemical formula I, chemical formula II, and chemical formula III.

[0085] In one example, the compound can be treated for 3 to 7 days, and most preferably for 5 days.

[0086] In one embodiment, trans differentiation can be induced directly by CtBPs, and it is even more preferable that trans differentiation be induced directly through oligomerization of CtBPs.

[0087] In one example, the method showed that Tuj1 and CD3 levels in cells were increased after treatment compared to before treatment. 25 (N-cadherin), p75, GAP-43, CD309, CD56 (NCA M) can increase the expression of PSA-NCAM, CD29, MASH1 or TBR2, Tuj1 2-5 times, CD325 (N-cadherin) 3-6 times, p75 1.5-4 times, GAP43 2-6 times, CD309 1.5-3 times, CD56 (NCA M) 1.5~4.5x, PSA-NCAM 1.5~4x, CD29 1.5~4x This can increase MASH expression by 2-4 times or TBR2 expression by 5-9 times.

[0088] In one embodiment, the method can increase the secretion of growth factors, tissue degradation factors, ligands, or cytokines in cells after treatment compared to before treatment with the compound.

[0089] In one example, the method increased the secretion of PlGF, NGF, BDNF, VEGFA, MMP1, MMP2, MMP7, TIMP2, SHH, Notch1, TNFSF12, or IL-16 in cells after treatment compared to before compound treatment, and increased PlGF by 1.5 to 4 times. This can increase the secretion of NGF by 1.5 to 6 times, BDNF by 1.5 to 3 times, VEGFA by 1.5 to 5 times, MMP1 by 1.5 to 3.5 times, MMP2 by 2 to 6 times, or TNFSF12 by 1.5 to 4 times.

[0090] In one embodiment, the method can reduce the expression of CD44, CD73, or CD105 in cells after treatment compared to before compound treatment, reducing CD44 expression by 50-90%, CD73 expression by 30-70%, or CD105 expression by 40-80%.

[0091] Direct trans differentiation induced by the direct trans differentiation composition and direct trans differentiation method of the present invention enables direct differentiation induction from adult stem cells to neural progenitor cells, which can then be further differentiated into neurons and / or dopaminergic neurons. In this case, the direct trans differentiation method may further include the step of differentiating neural progenitor cells obtained by incorporating one or more compounds selected from the group consisting of compounds of chemical formula I, chemical formula II, and chemical formula III of the present invention, or a direct trans differentiation composition containing these compounds, into adult stem cells into neurons or dopaminergic neurons, where the step of differentiating mesenchymal stem cells into neurons or dopaminergic neurons may be carried out by applying general differentiation techniques.

[0092] In one aspect, the present invention relates to stem cells treated with one or more compounds selected from the group consisting of compounds of chemical formula I, compounds of chemical formula II, and compounds of chemical formula III.

[0093] In one embodiment, the stem cells treated with the compound may be adult stem cells, and 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-MSCs) or umbilical cord-derived mesenchymal stem cells (UC -MSC), adipose-derived mesenchymal stem cells (AD-MSC) or bone marrow-derived mesenchymal stem cells, It can be BM-MSC.

[0094] In one embodiment, the stem cells of the present invention may be stem cells treated in vitro for 3 to 7 days with one or more compounds selected from the group consisting of compounds of chemical formula I, chemical formula II, and chemical formula III, and may be stem cells produced by a method comprising the steps of treating stem cells with one or more compounds selected from the group consisting of compounds of chemical formula I, chemical formula II, and chemical formula III; and culturing the compound-treated stem cells for 3 to 7 days.

[0095] In one embodiment, the stem cells of the present invention are prepared by dissolving the compound in DMSO at 30 mM and then 5... Stem cells treated with a concentration of 100 μM are acceptable.

[0096] In one embodiment, the stem cells of the present invention may be neural progenitor cells that have been directly converted into adult stem cells by compound treatment, and the neural progenitor cells may be ciNSC5 (chemically induced neural stem cell 5).

[0097] In one embodiment, the stem cells of the present invention can express increased neuronal markers compared to stem cells not treated with the compound, and these neuronal markers are Tuj1, TBR2, and MASH1. It may be GAP-43 or p75.

[0098] In one embodiment, the stem cells of the present invention can express increased neural progenitor cell marker proteins compared to stem cells not treated with the compound. These neural progenitor cell marker proteins include Tuj1 protein, CD325 (N-cadherin) protein, p75 protein, GAP-43 protein, CD54 protein, CD309 protein, CD56 (NCAM) protein, PSA-NCAM protein, CD29 protein, and MA. It may be either the SH1 protein or the TBR2 protein.

[0099] In one embodiment, the stem cells of the present invention are Tuj compared to stem cells that have not been treated with the compound. 1 Protein is increased by 6 times or more, preferably 6 to 8 times; CD325 (N-cadherin) protein is increased by 7 times or more, preferably 7 to 10 times; p75 protein is increased by 4 times or more, preferably 4 to 8 times; GAP43 protein is increased by 4 times or more, preferably 4 to 7 times; CD54 protein is increased by 2 times or more, preferably 2 to 5 times; CD309 protein is increased by 3 times or more, preferably 3 to 7 times; CD56 (NCAM) protein is increased by 3.5 times or more, preferably 3.5 to 6 times; PSA -NCAM protein is increased by 4 times or more, preferably 4 to 6 times; CD29 protein is increased by 4.5 times or more, preferably 4.5 to 8 times; MASH1 protein is increased by 4 times or more, preferably 4 to 8 times. Alternatively, the expression of the TBR2 protein can be increased by more than 3 times, preferably 3 to 7 times. .

[0100] In one embodiment, the stem cells of the present invention can exhibit reduced expression of CD44, CD73, or CD105 compared to stem cells not treated with the compound, with CD44 protein expression reduced by 50-90%, CD73 protein expression by 30-70%, or CD105 protein expression by 50-90%.

[0101] In one embodiment, the stem cells of the present invention can secrete more secretory proteins compared to stem cells that have not been treated with the compound, and the secretory proteins may be growth factors, tissue degradation factors, ligands, or cytokines.

[0102] In one embodiment, the growth factor may be PlGF, NGF, BDNF, or VEGFA, and the stem cells of the present invention secrete 1.5 to 4 times more PlGF, 1.5 to 6 times more NGF, and 1.5 to 3 times more BDNF than stem cells not treated with the compound. The secretion of FA can be increased by 1.5 to 5 times.

[0103] In one embodiment, the tissue degradation factor may be MMP1, MMP2, MMP7, or TIMP2, and the stem cells of the present invention can secrete 1.5 to 3.5 times more MMP1 or 2 to 6 times more MMP2 compared to stem cells that have not been treated with the compound.

[0104] In one embodiment, the ligand may be SHH or Notch1.

[0105] In one embodiment, the cytokine may be TNFSF12 or IL-16, and the stem cells of the present invention secrete 1% more TNFSF12 than stem cells not treated with the compound. It can be increased by 5 to 4 times.

[0106] In one embodiment, the stem cells of the present invention are HES compared to stem cells that have not been treated with the compound. 1. The expression of Sox2 or OCT4 may be increased.

[0107] In one embodiment, the stem cells of the present invention can be subjected to treatment with a compound to induce oligomerization of CtBPs (C-terminal binding proteins). The compound can induce oligomerization by protecting CtBPs from enzymes. .

[0108] In one specific example, CtBPs may be either CtBP1 or CtBP2.

[0109] In one embodiment, the oligomerization may be homooligomerization of CtBP1-CtBP1 or CtBP2-CtBP2.

[0110] In one embodiment, the compound of chemical formula I, the compound of chemical formula II, and the compound of chemical formula III can directly bond with CtBP1, and the compound of chemical formula II can also directly bond with CtBP2.

[0111] In one embodiment, the compound of chemical formula I can bond to Ser100 of CtBP1, the compound of chemical formula II can bond to Ser100 and Arg266 of CtBP1, and the compound of chemical formula III can bond to Phe102, Arg184, and His236 of CtBP1.

[0112] In one embodiment, the compound can differentiate adult stem cells into neural progenitor cells via CtBPs.

[0113] In one embodiment, the compound can induce CtBP oligomers from adult stem cells and induce direct trans-differentiation into neural progenitor cells via CtBPs.

[0114] In one example, adult stem cells may be mesenchymal stem cells, and the mesenchymal stem cells may be umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). These may be adipose-derived mesenchymal stem cells or bone marrow-derived mesenchymal stem cells, and are not limited to adult stem cells, as they can be applied regardless of the specific tissue cell. In many specific examples of the present invention, mesenchymal stem cells derived from umbilical cord (blood) were used, but it was confirmed that direct trans-differentiation into neural progenitor cells is also possible from adipose-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells.

[0115] In one embodiment, the stem cells of the present invention are MAP2, NSE, NeuN, doublecortin, NeuroD1 (Neurogenic differentiation). 1) Expression of Nestin, Mussashi 1, or GFAP is not required.

[0116] In one embodiment, the stem cells of the present invention can increase the expression of neurogenesis-inducing genes, inducible neurotrophic factor secretion genes, neurogenesis-related genes, neuron marker genes, neuron receptor genes, and channel-related genes compared to stem cells that have not been treated with the compound.

[0117] In one embodiment, the stem cells of the present invention can express enzymes for glial scar degradation in an increased manner compared to stem cells that have not been treated with the compound.

[0118] In one embodiment, the stem cells of the present invention can differentiate into dopaminergic neurons, neurons, or mature neurons.

[0119] In one embodiment, the stem cells of the present invention do not need to induce adipogenesis.

[0120] As used in this invention, the term "expression" refers to the process by which nucleic acids are transcribed from a DNA template (for example, into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.

[0121] In the present invention, the mRNA expression level of the gene can be measured by polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive RT-PCR, RNase, S1 nuclease assay, in situ hybridization, nucleic acid microarray, Northern blotting, or DNA chip method using the nucleic acid sequence of the marker, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair, probe that specifically recognizes the nucleic acid sequence and fragments of the complementary sequence, or a primer pair and probe. The protein expression level of the gene can be measured by Western blotting, ELISA (enzyme-linked immunosorbent assay) or other methods using an antibody, antibody fragment, aptamer, avidity multimer, or peptide mimetic that specifically recognizes the full-length protein or fragments thereof. It can be measured by methods such as assay, radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray.

[0122] The term "direct reprogramming (direct conversion, transdifferentiation)" as used in this invention refers to a process in which, in higher organisms, conversion is induced between mature (differentiated) cells of completely different cell types, without going through the process of dedifferentiating any somatic cell (cell type A) into induced pluripotent stem cells capable of differentiating into all cell types and then redifferentiating them (re-differentiation), but "directly" into a desired specific cell (cell type This is a technology that converts to B).

[0123] Stem cells treated with the compound of the present invention can be directly trans-differentiated into neural progenitor cells, where they can be further differentiated into mature neurons and / or dopaminergic neurons. In this case, the present invention may further include a configuration for differentiating the compound-treated stem cells into neurons or dopaminergic neurons, where the step of differentiating the mesenchymal stem cells into neurons or dopaminergic neurons may be carried out by applying general differentiation techniques.

[0124] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of nerve injury diseases, comprising the stem cells, stem cell cultures, or suspension cultures of the present invention as an active ingredient.

[0125] In one example, the nerve injury disease may be an injury to the central or peripheral nervous system, or a neurodegenerative disease, and may be a CNS (central nervous system) injury disease.

[0126] In one specific case, damage to the central or peripheral nervous system was classified as spinal cord injury (SCI), traumatic brain injury ( This may include TBI, peripheral nerve injury, stroke, or brain cancer.

[0127] In one instance, spinal cord injury may be induced by trauma or inflammation, and may be induced by one or more selected conditions from the group consisting of acute transverse myelitis, acute seed-sowing myelitis, spinal cord disease, non-Hodgkin lymphoma, hydrocephalus, hereditary ataxia, neurosyphilis, Minamata disease, Lou Gehrig's disease, and multiple sclerosis.

[0128] In one embodiment, the neurodegenerative disease may be Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), multiple sclerosis (MS), or multiple system atrophy, and it is more preferable that it be Parkinson's disease, amyotrophic lateral sclerosis (ALS), or multiple sclerosis (MS). .

[0129] In one embodiment, the composition of the present invention can achieve a preventive or therapeutic effect on Lou Gehrig's disease by significantly suppressing the death of motor nerve cells in the spinal cord, delaying the progression of Lou Gehrig's disease symptoms accompanied by motor dysfunction, and extending lifespan.

[0130] In one embodiment, the composition of the present invention can achieve a preventive or therapeutic effect on multiple sclerosis (MS) by suppressing demyelination of the central nervous system (CNS), inhibiting the penetration of mononuclear cells or macrophages into the spinal white matter, and suppressing the activation of microglial cells or astrocytes.

[0131] In one embodiment, the composition of the present invention can suppress the loss of dopamine neurons, and the transplanted stem cells of the present invention can differentiate into dopamine neurons, thereby achieving a preventive or therapeutic effect on Parkinson's disease (PD).

[0132] In one embodiment, the composition of the present invention can achieve a preventive or therapeutic effect on chronic spinal cord injury by reducing myelin loss, reducing gliotic scar formation, increasing axons, and differentiating transplanted stem cells of the present invention into mature nerve cells.

[0133] As used in this invention, the term "prevention" means all actions that suppress or delay the onset, development, and recurrence of nerve damage diseases by administering the compositions according to the present invention.

[0134] As used in this invention, the term "treatment" means all actions that improve or favorably alter the symptoms of nerve damage diseases and their complications through the administration of the composition according to the present invention. A person with ordinary skill in the art to which this invention belongs should be able to determine the precise criteria for diseases to which the composition of this application is effective and to judge the degree of improvement, enhancement, and treatment by referring to materials presented by the Korean Medical Association and other organizations.

[0135] As used herein, the term “treatment” means an approach to obtain a favorable or desirable clinical outcome. For the purposes of the present invention, a favorable or desirable clinical outcome includes, but is not limited to, relief of symptoms, reduction of disease extent, stabilization of the disease state (i.e., no worsening), delay or reduction in the rate of disease progression, improvement or temporary relief and reduction of the disease state (partial or overall), whether detectable or undetectable.

[0136] Furthermore, “treatment” can also mean extending the survival rate compared to the survival rate expected if no treatment is received. “Treatment” refers to all therapeutic treatments and preventive or protective measures. Such treatments include not only the preventive treatment of the disability but also the treatment required for a disability that has already occurred. “Palliating” a disease means reducing the extent of the disease state and / or the undesirable clinical signs, and / or slowing or lengthening the time course of its progression, compared to the case of no treatment.

[0137] 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 individual's condition.

[0138] The pharmaceutical compositions of the present invention are administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used in the present invention means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, without causing side effects. The effective dose level can be determined by factors including the individual's health condition, the type and severity of the neurological injury, the activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, and other factors well known in the medical field. The compositions of the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that provides the maximum effect with the minimum amount without side effects, which can be readily determined by those skilled in the art.

[0139] Unless otherwise specified, the aforementioned treatment means reversing, alleviating, inhibiting the progression of, or preventing the disease or illness to which the terms apply, or one or more symptoms of the aforementioned disease or illness, and the term "treatment" as used in this application means the act of treating, when “to treat” is defined as above. Accordingly, in mammals, the treatment or therapeutic therapy for autoimmune diseases may include one or more of the following: To inhibit the growth of nerve damage, that is, to stop its development. To prevent the spread of nerve damage, that is, to prevent metastasis. It reduces nerve damage. To prevent recurrence of nerve injury, and To alleviate the symptoms of nerve damage (palliating).

[0140] If a beneficiary animal can tolerate the administration of the composition, or if the administration of the composition to that animal is appropriate, the composition is indicated as "pharmaceutically or physiologically acceptable." If the administered amount is physiologically significant, the formulation can be said to have been administered in a "therapeutably effective dose." If the presence of the formulation causes a physiologically detectable change in the transplant patient, the formulation is physiologically significant.

[0141] As used herein, “effective dose” is an appropriate amount that is advantageous or has a favorable clinical or biochemical effect. The effective dose may be administered once or more times. For the purposes of the present invention, the effective dose of the stimulant temporarily alleviates the progression of the associated disease state. It is the appropriate amount to improve, stabilize, reverse, slow down, or delay.

[0142] The therapeutically effective amount of the composition of the present invention may vary depending on many factors, such as the method of administration, the site of administration, and the patient's condition. Therefore, the dosage for use in humans must be determined at an appropriate level, taking into account both safety and efficiency. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal experiments. Such factors to be considered when determining the effective amount are, for example, 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). It is described in Mack Publishing Co.

[0143] The pharmaceutical compositions of the present invention are administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, without causing side effects. The effective dose level can be determined by factors including the patient's health status, the type and severity of the disease, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and elimination rate, the duration of treatment, and other factors well known in the medical field, including drugs that are compounded or used concurrently. The compositions of the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, can be administered sequentially or concurrently with conventional therapeutic agents, and can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that provides the maximum effect with the minimum amount without side effects, which can be readily determined by those skilled in the art.

[0144] The pharmaceutical compositions of the present invention may include carriers, diluents, excipients, or combinations of two or more of these commonly used in biological formulations. The term "pharmaceutically acceptable" as used in the present invention means that the composition is non-toxic to cells or humans exposed to it. The carrier is not particularly limited as long as it is suitable for intra vivo delivery of the composition, and can be, for example, a mixture of one or more of the compounds listed in the Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Diluents, dispersants, surfactants, binders, and lubricants may be added to further formulations in primary dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or purified forms. Furthermore, these formulations can be preferably formulated according to the disease or component using appropriate methods in this field, or using methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0145] In one embodiment, the pharmaceutical composition may be one or more dosage forms selected from the group including oral dosage forms, topical preparations, suppositories, sterile injection solutions, and sprays.

[0146] The compositions of the present invention may also include carriers, diluents, excipients, or combinations of two or more of these commonly used in biological formulations. The pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for the biotransduction of the composition. For example, compounds listed in Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be used in combination, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Diluents, dispersants, surfactants, binders, and lubricants may be added additionally to formulate the compositions in preferred dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or purified forms. Furthermore, the compositions may be preferably formulated according to each disease or component using appropriate methods in the art or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0147] The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions.

[0148] The pharmaceutical composition of the present invention may further contain pharmaceutically acceptable additives, in which case the following may be used: starch, gelatinized starch, amorphous cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, candy, gum arabic, pre-gelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opa-dry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additives according to the present invention are preferably contained in amounts of 0.1 to 90 parts by weight relative to the composition, but are not limited thereto.

[0149] The composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) or orally, depending on the intended method. The dosage ranges widely depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The daily dose of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and is more preferably administered in one to several divided doses per day.

[0150] Liquid formulations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, fragrances, and preservatives, can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, and emulsions. This includes freeze-dried preparations, suppositories, etc.

[0151] In one aspect, the present invention relates to the use of compounds of chemical formula I, chemical formula II, or chemical formula III for the oligomerization induction of CtBPs.

[0152] In one aspect, the present invention relates to a compound of chemical formula I for use in direct trans differentiation induction. This relates to the uses of compounds of chemical formula II or chemical formula III.

[0153] In one aspect, the present invention relates to the use of stem cells treated with one or more compounds selected from the group consisting of compounds of chemical formula I, compounds of chemical formula II, and compounds of chemical formula III for the prevention or treatment of nerve damage diseases.

[0154] In one aspect, the present invention relates to a method for treating nerve injury, comprising the step of transplanting stem cells treated with one or more compounds selected from the group consisting of compounds of chemical formula I, compounds of chemical formula II, and compounds of chemical formula III into an individual having a nerve injury disease.

[0155] The present invention will be described in more detail through the following embodiments. However, the following embodiments are merely for the purpose of embodying the content of the present invention and do not limit the present invention thereto. [Examples]

[0156] Example 1. CTBP1 oligomerization of the compound of the present invention. )adjustment 1-1. Identification of YJ101, YJ102, and YJ103 binding proteins via DART analysis To identify proteins that bind to the compounds YJ101 (Chemical Formula I), YJ102 (Chemical Formula II), and YJ103 (Chemical Formula III) of the present invention in human UCB-MSCs (umbilical cord blood-derived mesenchymal stem cells), DART analysis was performed. Specifically, UCB-MSCs (Kang Stem Cells, Passage #6) were dispensed into 100 mM culture dishes and 10% KSB-3 medium (Cat.K3901) containing FBS (Cat.16000044) After culturing in Kang Stem Cells, the cells were collected and washed with PBS. Next, they were treated with IP50 Lysis Buffer [50mM Tris-H]. Cells were sonicated with Cl (pH 7.4), 150 mM NaCl, 2 mM MgCl2, and 0.1% NP40, and centrifuged at 13,000 × g for 10 minutes at 4°C to obtain the supernatant. 1 / 10 volume of 10X TNC buffer [500 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 100 mM CaCl2] was added to the supernatant, and the protein concentration was measured by the Bradford method. Next, 9 μl of cell disrupted cells (lysate) and 1 μl of YJ101 (20 or 40 μM), YJ102 (20 or 30 μM), or YJ103 (20 or 40 μM) were placed in a microcentrifuse tube and mixed, and incubated at room temperature for 1 hour. The control group was treated with 1 μl of DMSO. After incubation, 1 μl (1 mg / ml) of pronase (Cat.10 165 921 001, Roche), diluted to 1 mg / ml using 1×TNC buffer, was added to the microcentrifuse tubes and incubated at 37°C for 15 minutes. After incubation, 1 / 5 volume of 5X SDS-PAGE loading buffer was added, and the tubes were heated at 100°C for 5 minutes, followed by SDS-PAGE and coomassie blue staining. Additionally, 4 × 10⁶ UCB-MSCs were placed in 60 mM culture dishes. 5 Cells were aliquoted at cells / ml and transfected with 4ug of FLAG-CtBP1 or FLAG-CtBP2 DNA using 4μl of jetPRIME after 24 hours. Cells were collected after 48 hours and proteins were extracted and quantified using the same method as described above. DARTS analysis was performed on FLAG-labeled CtBP1 cell disruptions and Y102, and Western blot analysis was performed with flag antibody (Cat.F3165, Sigma).

[0157] As a result, after DARTS analysis, a band protected by YJ102 (*) was identified and identified as CtBP1 (C-terminal Binding Proteins) (Figure 1A). It was shown that YJ102 protects CtBP1 from degradation by pronase (Figure 1B), that overexpressed CtBP1 is protected by YJ102 from degradation by pronase (Figure 1C), and that overexpressed CtBP1 (flag-CtBP1) is protected by YJ101, YJ102, and YJ103 from degradation by pronase (Figure 1D). Furthermore, it was shown that YJ102 also protects CtBP2 from degradation by pronase (Figure 1E), confirming that overexpressed CtBP2 is also protected by YJ102 from degradation by pronase (Figure 1F).

[0158] Through this, YJ101, YJ102, and YJ103 all bond with CtBP1, Y We confirmed that J102 binds to CtBP1 and CtBP2 in human UCB-MSCs. .

[0159] 1-2. Confirmation of CTBP1 oligomerization regulation of YJ102 To confirm the effect of YJ102 on the regulation of CtBPs (C-terminal Binding Proteins) oligomerization in human UCB-MSCs, immunoprecipitation analysis was performed. Specifically, UCB-MSCs were cultured in 60 mM culture dishes in 4 × 10⁶ units. 5 Dispense at cells / ml, and the next day, FLAG-CtBP1, HA-CtBP1, FLAG- Cells were transfected with 4 μl of CtBP2 and HA-CtBP1 using jetPRIME. Next, cells were treated with 20 μM and 300 μM of YJ102 (stock 30 mM in DMSO) and MTOB (stock 300 mM in DMSO), respectively (Table 1). 48 hours after transfection, proteins were extracted using the same method as in Example 1-1. The Protein G agarose slurry was washed with PBS, blocked with 1 ml of 1% BSA, washed with PBS again, and 1 μg of anti-HA antibody diluted in 1.5 ml of PBS was added and incubated at 4°C for 2 hours. Next, the supernatant was removed by centrifugation at 3,000 × g for 2 minutes, washed twice with IP150 buffer, and 1 μg of the extracted protein was added and incubated overnight at 4°C. After incubation, the supernatant was removed by centrifugation at 3,000×g for 2 minutes, washed three times with PBS, and 50 µl of 1 mg / ml HA peptide diluted with PBS was added. The mixture was incubated at room temperature for 10 minutes. After centrifugation at 3,000×g for 2 minutes, the supernatant was transferred to a new microcentrifuse tube, 10 µl of 5X sample buffer was added, and the mixture was heated at 100°C for 5 minutes. Western blot analysis was then performed using FLAG antibody or HA antibody.

[0160] [Table 1]

[0161] As a result, it was shown that YJ102 induces oligomerization of CtBP1, and that this oligomerization is suppressed by MTOB (Figure 2). On the other hand, CtBP1 It was shown that YJ102 does not affect CtBP2 heteromerization. MTOB is a substrate of the dehydrogenease present in CtBP and is known to act as an inhibitor that suppresses CtBP transcriptional activity at high concentrations, thereby inhibiting oligomerization.

[0162] 1-3. Confirmation of interaction sites between YJ101, YJ102, and YJ103 and CtBP1 Molecular docking was performed to identify the sites where compounds YJ101, YJ102, and YJ103 of the present invention interact with CtBP1. Specifically, protein-ligand docking was performed using Glide (Grid based Ligand Docking with Energetics) docking application of the Schrodinger litus rogram (Schrodinger LC, Y, SA), with CtBP1 protein (PDB ID: 6CDF) and YJ103. The three-dimensional structure of 102 was fabricated using Schrodinger sites. Furthermore, the receptor grid for CtBP1 was generated by identifying the binding (active) site residues and confirmed using SiteMap. After receptor grid generation, the ligand was docked to CtBP1 using the Glide docking protocol, and the docked conformational isomers were evaluated using the Glide(G)Score. The G Score was calculated using the following formula:

[0163]

number

[0164] As a result, it was shown that YJ101, YJ102, and YJ103 all interact with the NAD(H) binding domain of CtBP1, and the ΔG values ​​were -4.980 kcal / mol for YJ101, -5.458 kcal / mol for YJ102, and -3.767 kcal / mol for YJ103 (Figure 3A). Furthermore, it was predicted that Ser100 of CTBP1 is important for the interaction between YJ101 and CTBP1, that Ser100 and Arg266 of CTBP1 are important for the interaction between YJ102 and CtBP1, and that Phe102, Arg184, and His236 of CTBP1 play an important role in the interaction between YJ103 and CTBP1 (Figure 3B).

[0165] 1-4. Identification of target genes for CTBP-YJ102 To identify the target gene of CtBP1-YJ102 in human UCB-MSCs, chromatin immunoprecipitation (ChIP) was performed. The analysis was carried out. Specifically, human UCB-MSCs were cultured in 2x1 100mm culture dishes. Six cells were dispensed and, the following day, treated with 10 μl of YJ102 (30 mM) or DMSO (control group) and cultured for 2 days. For cross-binding and DNA sharing, formaldehyde was added to the culture dish containing the UCB-MSCs to a concentration of 1%, and the cells were fixed by incubation at 37°C for 10 minutes. The culture medium was removed, and the cells were collected by washing twice with PBS containing a protease inhibitor. The cells were centrifuged at 3000 rpm at 4°C for 5 minutes, the supernatant was removed, and the cells were incubated on ice for 10 minutes with SDS disruption buffer containing a protease inhibitor and sonicated to disrupt the cells. The cells were centrifuged at 13,000 rpm at 4°C for 10 minutes, the supernatant was transferred to a new microcentrifuse tube, and 1 / 10 volume of ChIP Dilution Buffer was added. Next, the DNA was purified and agarose gel electrophoresis was performed to confirm that the DNA was sheared at 200-1000 bp intervals. Furthermore, for chromatin immunoprecipitation, the Protein G agarose slurry was washed with PBS, blocked with 1 ml of 1% BSA, washed again with PBS, and the cells were sonicated to obtain the lysates. Primary antibodies used were anti-CtBP1 antibody (Cat.07-306, Millipore), anti-LSD1 antibody (Cat.A1156, Abclonal), and Normal rabbit IgG (Cat.12-370, Each sample was inoculated at 4°C overnight using a rotator, with 1 ug of Merck Millipore (control group) added to each sample.

[0166] The next day, after centrifugation and removal of the supernatant, the DNA-antibody-agarose beads were placed in Low The DNA-antibody-agarose beads were washed once with Salt Immune Complex Wash Buffer, High Salt Immune Complex Wash Buffer, and LiCl Immune Complex Wash Buffer in that order, and then washed twice with 1X TE. 250 μl of elution buffer (1%) was added to the DNA-antibody-agarose beads. SDS (0.1M NaHCO3) was added, and the mixture was incubated at room temperature for 15 minutes using a rotator. After centrifugation, the supernatant was transferred to a new microcentrifuse tube. The elution process was repeated once more, and the eluted products were mixed. 20 μl of 5M NaCl was added to the eluted products, and the mixture was heated at 65°C for 4 hours. Then, 10 μl of 0.5M EDTA, 20 μl of 1M Tris-HCl (pH 6.5), and 2 μl of proteinase K (10 mg / ml) were added, and the mixture was incubated at 45°C for 1 hour. Phenol / chloroform extraction and ethanol precipitation were performed, and the DNA pellet was resuspended in DW. Real-time quantitative PCR was then performed using the primers shown in Table 2 below.

[0167] [Table 2]

[0168] The results showed that YJ102 increased the amount of CtBP1 binding to the HES1 gene promoter, decreasing HES1 expression (Figure 4A), and that YJ102 increased Sox2 expression by causing the CtBP1-LSD1 complex to bind to the Sox2 gene promoter (Figure 4B). Furthermore, YJ102 increased the amount of the CtBP1-LSD1 complex binding to the OCT4 gene promoter, inducing OCT4 expression (Figure 4C), and that YJ102 increased the amount of CtBP1 binding to the HES1 gene promoter and increased the amount of the CtBP1-LSD1 complex binding to the Sox2 and Oct4 gene promoters (Figure 4D).

[0169] Example 2. Direct trans differentiation to ciNSC5 by the compound of the present invention. 2-1. Confirmation of differentiation of UCB-MSCs into ciNSC5 by YJ102 To confirm the presence or absence of CtBP1-mediated ciNSC5 differentiation by YJ102 in human UCB-MSCs, the CTBP1 mechanism was analyzed by immunochemical analysis using NSC95397 or MTOB (4-methylthio-2-oxobutanoate) treatment. Specifically, 2.1 × 10⁶ UCB-MSCs were placed in a 12-well plate containing 18mm circular coverslips. 4 Dispensing was done in cells / well (DMSO control group: 3.5 × 10 3 Cells were dispensed in cells / well. The day after dispensing, cells were treated with 30 μM YJ102, 10 μM NSC95397 (Cat.93718-83-3, Merck), or 300 μM MTOB (Cat.K6000, Merck) alone or in combination (the control group was treated with DMSO), and incubated at 37°C for 5 days. After removing the culture medium and washing with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes. After washing three times with PBS, the cells were permeabilized with PBS-T containing 0.5% Triton X-100 for 10 minutes, washed three times with PBS-T for 10 minutes each, and then blocked for 1 hour with a blocking solution (PBS-T containing 5% BSA and 5% goat serum). Next, the cells were treated with anti-Tuj1 antibody (Cat.801201, BioLegend) diluted 1 / 5000 with blocking solution and incubated overnight at 4°C. The following day, after washing 10 times for 10 minutes each with PBS-T, the cells were treated with anti-mouse IgG-cy3 (Cat.111-165-003, Jackson ImmunoResearch) diluted 1 / 2000 with blocking solution and incubated in the dark for 1 hour. Subsequently, the cells were washed 10 times for 10 minutes each with PBS-T, mounted in a mounting solution containing DAPI, and then observed under a confocal microscope.

[0170] As a result, YJ102-treated ciNS were separated from UCB-MSC by YJ102. Tuj1 expression in C5 was shown to be reduced by NSC95397, which inhibits the binding of PxDLS motif proteins to CtBP (Figure 5A), and Tuj1 expression was also shown to be reduced by MTOB, which suppresses the transcriptional activity of CtBP (Figure 5B).

[0171] Through this, we confirmed that YJ102 differentiates UCB-MSCs into ciNSC5, a neural progenitor cell, through CtBP1 regulation.

[0172] 2-2. Confirmation of direct transdifferentiation of UCB-MSC to ciNSC5 by YJ101, YJ102, or YJ103 To confirm whether UCB-MSCs are directly trans-differentiated into Tuj1-positive neural progenitor cells, ciNSC5, by the compounds YJ101, YJ102, and YJ103 of the present invention, immunochemical analysis was performed. Specifically, 2.1 × 10⁶ UCB-MSCs were placed in a 12-well plate with 18 mm coverslips. 4 Dispensing was done in cells / well (DMSO control group: 3.5 × 10 3(Dispensed in cells / well). YJ101, YJ102, and YJ103 were each dissolved in 30 mM DMSO, and then treated with each at a concentration of 30 μM (control group was treated with DMSO), and cultured at 37°C for 5 days. After removing the culture medium and washing with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes. After washing three times with PBS, the cells were permeabilized with PBS-T containing 0.5% Triton X-100 for 10 minutes, washed three times with PBS-T for 10 minutes each, and then blocked for 1 hour with a blocking solution (PBS-T containing 5% BSA and 5% goat serum). Next, the cells were treated with anti-Tuj1 antibody (Cat.801201, BioLegend) diluted 1 / 5000 with the blocking solution and incubated overnight at 4°C. The following day, the cells were washed 10 times for 10 minutes each with PBS-T, then treated with anti-mouse IgG-FITC diluted 1 / 500 with blocking solution and incubated in the dark for 1 hour. Next, they were washed 10 times for 10 minutes each with PBS-T, mounted in a mounting solution containing DAPI, and then observed under a confocal microscope.

[0173] As a result, it was shown that Tuj1 expression was increased in ciNSC5 differentiated from UCB-MSC by YJ101, YJ102, and YJ103 (Figure 6), and YJ101, YJ We confirmed that both 102 and YJ103 directly induce trans-differentiation from UCB-MSCs to ciNSC5s.

[0174] 2-3. Confirmation of direct trans-differentiation of mesenchymal stem cells of different origins using YJ101, YJ102, or YJ103. When human UCB-MSCs were treated with YJ101, YJ102, or YJ103 for 5 days, the phenomenon of direct trans-differentiation into ciNSC5, a neural progenitor cell, occurred similarly in MSCs of different origins. To confirm this, human AD-MSCs (adipose-derived mesenchymal stem cells) and human BM-MSCs (bone marrow-derived mesenchymal stem cells; A) were used. The expression of neuronal markers was confirmed by immunochemical analysis in ciNSC5 differentiated using TCCs and human UCB-MSCs (umbilical cord blood-derived mesenchymal stem cells; Promo Cells). Specifically, AD-MSCs (StemPro Human Adipose-Derived Stem Cells, Gibco, Passage #6) were used in MesenPR In ORS medium, BM-MSCs (ATCC, Passage # 6) are classified as Mesenchymal Stem Cell Basal Medium for Adipose , in Umbilical and Bone Marrow-derived MSC (Cat.PCS-500-030, ATCC) medium, and UCB-MSC (Promo Cell, Passage #6) is CellCor CD MSC (Cat.YSP0 01. After culturing in Xell Therapeutics medium, transfer 2.1 × 10⁶ cells to a 12-well plate containing an 18mm circular coverslip. 4 Dispensing was done in cells / well (DMSO control group: 3.5 × 10 3 (Dispensing by cells / well) After dissolving YJ101, YJ102, and YJ103 in DMSO at 30 mM each. The cells were treated with concentrations of 10, 20, or 30 μM, respectively (the control group was DMSO-treated UC). B-MSCs were cultured at 37°C for 5 days. After removing the culture medium and washing with PBS, they were fixed with 4% paraformaldehyde at room temperature for 10 minutes. After washing three times with PBS, 0.5% Cells were permeabilized with PBS-T containing Triton X-100 for 10 minutes, washed three times with PBS-T for 10 minutes each, and then blocked for 1 hour with a blocking solution (PBS-T containing 5% BSA and 5% goat serum). Next, they were treated with anti-Tuj1 antibody (Cat.801201, BioLegend) diluted 1 / 5000 with the blocking solution and incubated overnight at 4°C. The following day, after washing 10 times with PBS-T for 10 minutes each, they were treated with anti-mouse IgG-cy3 diluted 1 / 2000 with the blocking solution and incubated in the dark for 1 hour. Next, they were washed 10 times with PBS-T for 10 minutes each, mounted with a mounting solution containing DAPI, and then observed under a confocal microscope.

[0175] As a result, it was shown that Tuj1 expression was increased in ciNSC5 differentiated from AD-MSC by YJ101, YJ102, and YJ103 (Figure 7A), and YJ101, YJ Increased Tuj1 expression was observed in ciNSC5 differentiated from BM-MSCs by 102 and YJ103 (Figure 7B), and also in ciNSC5 differentiated from UCB-MSCs by YJ101, YJ102, and YJ103 (Figure 7C).

[0176] 2-4. Confirmation of cytotoxicity of the compound The cytotoxicity of YJ101, YJ102, and YJ103 was confirmed in human UCB-MSCs using the Caspase-Glo® 3 / 7 Assay System (Cat.G8091, Promega). Specifically, 100 μl (6,000 cells) of UCB-MSCs were dispensed into each well of a 96-well plate. YJ101, YJ102, and YJ103 were dissolved in 30 mM DMSO, respectively, and then treated with concentrations of 10, 20, or 40 μM (the control group was treated with DMSO). The cells were then cultured at 37°C for 3 or 5 days. Compound plates were prepared in 96-well plates by serial dilution starting from 40 μM. The highest doses were plated in rows 4, 8, and 12, while DMSO (excipient control) was plated in rows 1, 5, and 9 (Table 3). The compound plate was mixed in a shaker for 1 minute, the medium from the 96-well plate containing the dispensed UCB-MSCs was removed, and then 100 μl of the compound solution was added. The plates were then incubated at 37°C for 3 and 5 days. Next, 100 μl of Caspase-Glo 3 / 7 reagent, prepared by mixing Place Caspase-Glo 3 / 7 Buffer with Caspase-Glo 3 / 7 substrate, was added to each well of the 96-well plate. After covering the plate, the mixture was shaken at 300-500 rpm for 30 seconds. The plates were then incubated at room temperature for 30 minutes, the luminescence was measured using a plate-reading luminometer, and the floating stage was calculated using Equation 2 below.

[0177] [Table 3]

[0178]

number

[0179] As a result, it was shown that none of the compounds YJ101, YJ102, and YJ103 of the present invention induce apoptosis in UCB-MSC cells (Figure 8).

[0180] 2-5. Confirmation of the effects of compounds on MSC proliferation To confirm the effects of YJ101, YJ102, and YJ103 on MSC proliferation UCB-MSCs were dispensed into each well of a 96-well plate at a concentration of 100 μl (6,000 cells). YJ101, YJ102, and YJ103 were dissolved in DMSO at a concentration of 30 mM, respectively, and then the cells were treated with these compounds at concentrations of 10, 20, or 40 μM (the control group was treated with DMSO). The cells were then cultured at 37°C for 3 or 5 days. The compound plate was the same as in Example 2. The same method as in -4 was used for preparation, and after removing the culture medium from the 96-well plate containing the dispensed UCB-MSCs, the plate was treated with 100 μl of compound solution and then incubated at 37°C for 3 and 5 days. Next, 100 μl of CellTiter-Glo reagent, prepared by mixing CellTiter-Glo Buffer with CellTiter-Glo substrate, was added to each well of a 96-well plate, the plate lid was closed, and the plate was incubated at room temperature for 30 minutes. Then, the luminescence was measured using a plate-reading luminometer, and the floating was calculated using Equation 3 below.

[0181]

number

[0182] As a result, compounds YJ101, YJ102, and YJ103 of the present invention all reduced cell proliferation in a concentration-dependent manner (Figure 9). Generally, cell proliferation decreases during cell differentiation.

[0183] Example 3. Properties of ciNSC5 directly trans-fractionated by the compound of the present invention 3-1. Neuronal Marker Expression Analysis of ciNSC5 YJ102 was treated by the method of Example 2 for 5 days, and the expression of neural markers in ciNSC5 (chemical induced neural stem cell 5) directly transdifferentiated from human UCB-MSC was confirmed by immunochemical analysis and Western blot analysis. Specifically, for immunochemical analysis, UCB-MSC was plated at 2.1×10 4 cells / well in a 12-well plate containing 18 mm cover glasses (the DMSO control group was plated at 3.5×10 3 cells / well). The day after plating, YJ1 02 was dissolved in DMSO at 30 mM, then the cells were treated at a concentration of 30 uM (the control group was treated with DMSO), and cultured at 37°C for 5 days. After removing the culture medium and washing with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes. After washing 3 times with PBS, the cells were permeabilized with PBS-T containing 0.5% Triton X-100 for 10 minutes, and then washed with PBS-T 3 times for 10 minutes each, then blocked with a blocking solution (PBS-T containing 5% BSA and 5% goat serum) for 1 hour. Next, as primary antibodies diluted in the blocking solution, anti-Tuj1 antibody, anti-TBR2 antibody (Cat. Ab23345, abcam), anti-MASH1 antibody (Cat. Ab74065, abcam), anti-GAP4 3 antibody (Cat.Ab16053, abcam), anti-p75 antibody (Cat.G323A, Promega), anti-MAP2 antibody (Cat.4542, Cell signaling), anti-NSE antibody (Cat.AB951, Chemicon), anti-NeuN antibody (Cat.MAB377, Chemicon), anti-Calretinin antibody (Cat.180211, Invitrogen), Anti-Doublecortin antibody (Cat.Ab18723, abcam), anti-NeuroD1 antibody (Cat.Ab213725, abcam), anti-Nestin antibody (Cat.Ab18102, abcam), anti-Musashi Cells were treated with one antibody (Cat.AB5977, Chemicon) and an anti-GFAP antibody (Cat.G3893, Sigma), respectively, and incubated overnight at 4°C. The following day, after washing 10 times for 10 minutes each with PBS-T, the cells were treated with anti-mouse IgG-cy3 or anti-rabbit IgG-cy3 (Cat.111-165-003, Jackson ImmunoResearch) diluted in blocking solution and incubated in the dark for 1 hour. Subsequently, the cells were washed 10 times for 10 minutes each with PBS-T, mounted in a mounting solution containing DAPI, and then observed under a confocal microscope. Furthermore, for Western blot analysis, the culture medium was removed and the cells were washed with PBS. Then, disruption buffer (1% Nonidet® P-40, 20 mM Tris (pH 8.0), 137 mM NaCl, 0.5 mM EDTA, 10% glycerol, 10 mM Na2P2O7, 10 mM NaF, 1 μg / ml aprotinin, 10 μg / ml leupeptin, 1 mM vanadate, and 1 mM PMSF) was added, and the cells were collected using a scraper. After rocking at 4°C for 20 minutes, the cells were centrifuged at 12,000 rpm for 30 minutes. After centrifugation, the supernatant was transferred to another e-tube, and the protein was quantified using a BCA analysis kit. The same amount of protein was loaded onto an SDS-PAGE and subjected to electrophoresis.Next, the antibodies were transferred to a nitrocellulose membrane, and Western blotting was performed according to the protocols provided in the antibody data sheets for each primary antibody: anti-CD325 antibody (Cat.10-0224, Invitrogen), anti-p75 antibody, anti-GAP43 antibody, anti-CD54 antibody (Cat.67836, Cell signaling), anti-CD309 antibody (Cat.MA5-15157, Invitrogen), anti-CD56 antibody (Cat.A7913, Abclonal), anti-PSA-NCAM antibody (Cat.5324, Millipore), anti-CD29 antibody (Cat.Ab183666, abcam), anti-MASH1 antibody, anti-TBR2 antibody, anti-CD44 antibody, anti-CD73 antibody, and anti-CD105 antibody.

[0184] Immunoanalysis results showed that ciNSC5 directly trans-differentiated from human UCB-MSCs after being treated with YJ102 for 5 days expressed the neural progenitor markers Tuj1, TBR2, MASH1, GAP-43, and p75, but did not express the mature neuronal markers MAP2, NSE, and NeuN. It also did not express doublecortin and NeuroD1 (Neurogenic differentiation 1), which are known to be expressed during the early differentiation process of nerve cells, nor did it express the early neural stem cell markers Nestin, Mussashi 1, and GFAP (Figure 10). Furthermore, Western blot analysis of neural progenitor cell marker proteins showed that compared to MSCs, ciNSC5 had more than 6 times (approximately 6-8 times) Tuj1 protein, more than 7 times (approximately 7-10 times) CD325 (N-cadherin) protein, more than 4 times (approximately 4-8 times) p75 protein, more than 4 times (approximately 4-7 times) GAP43 protein, more than 2 times (approximately 2-5 times) CD54 protein, more than 3 times (approximately 3-7 times) CD309 protein, and more than 3.5 times (approximately) CD56 (NCAM) protein. The following proteins increased significantly: PSA-NCAM protein increased by more than 4 times (approximately 4-6 times), CD29 protein increased by more than 4.5 times (approximately 4.5-8 times), MASH1 protein increased by more than 4 times (approximately 4-8 times), and TBR2 protein increased by more than 3 times (approximately 3-7 times) (Figures 11 and 13). On the other hand, the protein expression of the MSC markers CD44, CD73, and CD105 was shown to decrease by more than 90%, 50%, and 80%, respectively, in ciNSC5 compared to MSCs (Figures 12 and 13).

[0185] 3-2. Analysis of secreted proteins of ciNSC5 YJ102 was treated for 5 days using the method described in Example 2, and Western blot and ELISA analysis were performed to confirm the therapeutic effect of ciNSC5 directly trans-differentiated from human UCB-MSCs through the secretion of growth factors, tissue degradation, and neuronal differentiation / regeneration-related factors. Specifically, UCB-MSCs were cultured in 100 mm culture dishes in a 3 × 10⁶ format. 5 Dispense into cells / well (DMSO control group: 5 × 10 4 Cells were dispensed into cells / wells, and the following day, YJ102 was dissolved in 30 mM DMSO, and the cells were treated with a 30 μM solution (the control group was treated with DMSO). Cell lysates of ciNSC5 differentiated from UCB-MSCs were obtained. The culture medium was removed using the ciNSC5 differentiated from UCB-MSCs, washed with PBS, and then subcultured in fresh culture medium without YJ102. Cells were cultured at 37°C for 2, 4, or 7 days. Subsequently, each cell was lysed and Western blot analysis was performed. Western blot analysis was then performed on the ciNSC5 cell lysates and culture medium. The antibody information used during the analysis is shown in Tables 4 and 5.

[0186] [Table 4]

[0187] [Table 5]

[0188] As a result, it was shown that ciNSC5 differentiated from UCB-MSCs by YJ102 treatment showed increased levels of growth factors PlGF and VEGFA (VEGF-A) compared to MSCs, as well as increased levels of tissue degradation factors MMP1, MMP2, MMP7, and TIMP2. Furthermore, SHH and Notch1, ligands involved in neural differentiation, neurogenesis, and axon regeneration, were shown to increase (Figures 14 and 15). Furthermore, when ciNSC5 differentiated from UCB-MSCs by YJ102 treatment was subcultured and cultured for 2 days in a medium without YJ102, the growth factors that increased in the ciNSC5 culture medium compared to MSCs were PlGF, NGF, BDNF, and VEGFA (PlGF: 2.5 times, NGF: 3.8 times, BDNF: 2 times, and VEGFA: 2.1 times), and the cytokines TNFSF12 and IL-16 were shown to increase (Figures 16 and 17). In addition, tissue degradation factors MMP1 and MMP2 increased, and MMP7 and TIMP2 also increased, and among ligands involved in neuronal differentiation, neurogenesis, and axonal regeneration, SHH and Notch1 were shown to increase (Figures 16 and 17). Furthermore, after culturing ciNSC5 for an additional 4 or 7 days in a medium without YJ102, the proteins in the medium were examined using Western blot analysis. The results showed that the secretion of growth factors, tissue degradation, and neuronal differentiation / regeneration-related proteins changed, as shown in Figures 18 and 19. In addition, the amounts of VEGFA, MMP-2, NGF, MMP1, PlGF, and TNFSF12, which were shown to increase in ciNSC5 in the aforementioned Western blot analysis, were examined in the medium in which ciNSC5 was cultured for 2 days in a medium without YJ102. The results showed that the amounts of VEGFA, MMP-2, NGF, MMP1, PlGF, and TNFSF12 secreted into the ciNSC5 medium were significantly increased compared to the amounts observed in the MSC medium (Figures 20 and 21).

[0189] 3-3. Gene analysis of genes expressed in ciNSC5 using RNA-seq YJ102 was treated for 5 days to directly trans-fractionate ciNS from human UCB-MSCs. The expression profile of C5 was analyzed by genome-wide RNA-seq. Specifically, 1 × 10⁶ human UCB-MSCs were cultured in a 60 mM culture dish. 5 After individual dispensing (control group: 1.67 × 10) 4 (Individually dispensed) YJ102 dissolved in 30 mM DMSO is injected into cells 3 times The cells were treated with 0 μM (the control group was treated with DMSO) and cultured at 37°C for 2, 3, or 5 days. Next, the culture medium was removed and washed with PBS, and the cells were collected with a scraper and transferred to a microcentrifuse tube. The PBS was removed by centrifugation, and 500 μl of Tri-RNA Reagent was added and thoroughly mixed by pipetting. 100 μl of chloroform was added and the tube was vortexed for 15 seconds to mix, then the mixture was centrifuged at 14,000 rpm for 20 minutes at 4°C. Next, the upper aqueous phase was transferred to a new microcentrifuse tube using a pipette, and 550 μl of isopropanol was added and mixed. The mixture was centrifuged at 14,000 rpm for 20 minutes at 4°C, the supernatant was discarded, and the pellet was washed with 1 ml of 70% ethanol. After centrifugation at 14,000 rpm for 2 minutes, the supernatant was discarded. After air-drying the pellet, the RNA was lysed in 50 μl of RNase-free DIW. 1 μl of DNase was added and incubated at 37°C for 30 minutes. Next, the same volume of 8 M LiCl was added and the sample was placed on ice for 1 hour. After centrifugation at 14,000 rpm at 4°C for 10 minutes, the sample was washed with 70% ethanol and then air-dried. The pellet was resuspended in 100 μl of RNase-free DIW, centrifuged at 14,000 rpm at 4°C for 10 minutes, the supernatant was removed, and the pellet was air-dried. RNA concentration was then quantified using nanodrop. Next, RNA sequencing was performed using Theragen Bio, and the expression levels of genes between samples were compared using FPKM (Fragments Per Kilobase of transcript per Million). For accuracy verification, a significant gene ontology classification was selected using Fisher's method based on genes with a p-value of less than 0.05. Functional analysis was performed on genes that increased more than twice or decreased to 0.5 times compared to DMSO-treated UCB-MSCs.The gene expression program file is presented as a heat map with three RGB (red, green, black) colors based on expression values (Figure 22). Trend analysis was performed after confirming Gene Ontology (GO) using Reactome, a gene function database.

[0190] As a result of RNA-seq, the expression of cell division-related genes decreased and the expression of nervous system-related genes increased in UCB-MSCs treated with YJ102 for 3 days. Based on this, among factors whose expression changes in the medium on day 5, genes involved in neurogenesis, synapses, neurotransmitters, nerve growth factors and the like were selected for analysis. The results showed that in ciNSC5, the expression of genes that induce neurogenesis (transcription factors, ligands and receptors) and genes involved in the secretion of brain-derived neurotrophic factor for nerve regeneration and neuroprotection was prominent (Figures 23 and 24).

[0191] 3-4. Analysis of genes expressed in ciNSC5 using real time RT-PCR The expression of genes selected based on the RNA-seq analysis results of Example 3-3 above was confirmed by real time RT-PCR, and the gene expression patterns in MSCs and ciNSC5 were confirmed. Specifically, human UCB-MSCs were placed in a 60 mM culture dish at 1×10 5 cells were aliquoted (the control group had 1.67×10 4 cells aliquoted), then YJ102 dissolved in DMSO at 30 mM was applied to the cells at a concentration of 30 uM (the control group was treated with DMSO), and cultured at 37°C for 5 days. Subsequently, RNA was extracted by the same method as in the above example and then quantified.

[0192] The results showed that the expression of enzymes for glial scar degradation was significantly increased in ciNSC5 differentiated from UCB-MSCs treated with YJ102 for 5 days compared with the control group (Figure 25), and the expression of transcription factors related to neurogenesis was also increased compared with the control group (Figure 26). It was also shown that the expression of neuronal markers such as myelination, synapsis, matrix / cell adhesion, and calcium signaling was mostly increased compared with the control group (Figure 27), and the expression of neuronal receptors and channels was also mostly increased compared with the control group (Figure 28).

[0193] Through this, it was found that YJ102 treatment induces diverse neurogenic gene programs, and also induces secreted molecules related to neurogenesis and axon regeneration. It was also confirmed that YJ102 can increase the expression of enzymes that enable glial scar degradation in ciNSC5.

[0194] 3-5. Confirmation of changes in cell characteristics due to passage of ciNSC5 The presence or absence of changes in cell characteristics due to passage of ciNSC5 differentiated into human UCB-MSCs by YJ102 was confirmed by immunochemical analysis against the Tuj1 antibody. Specifically, 2×10 6 cells were aliquoted into 100 mm culture dishes, and passage was performed every 2 days. After culturing up to a total of 6 passages, the culture medium was removed, washed with PBS, and fixed with 4% paraformaldehyde at room temperature for 10 minutes. After washing 3 times with PBS, cells were permeabilized with PBS-T containing 0.5% Triton X-100 for 10 minutes, washed 3 times for 10 minutes each with PBS-T, then blocked with blocking solution (PBS-T containing 5% BSA and 5% goat serum) for 1 hour. Next, anti-Tuj1 antibody diluted 1 / 5000 in blocking solution was applied and incubated overnight at 4°C. The next day, after washing 10 times for 10 minutes each with PBS-T, 1 / Cells were treated with anti-mouse IgG-cy3 diluted 2000 times and incubated in the dark for 1 hour. They were then washed 10 times with PBS-T for 10 minutes each, mounted in a mounting solution containing DAPI, and observed under a confocal microscope.

[0195] As a result, it was shown that the doubling time was maintained even after passage of ciNSC5 (Figure 29), and that the CPDL (cumulative population doubling level) value was also maintained with respect to the number of passages (Figure 30). Furthermore, Tuj1 expression was maintained even after passage of ciNSC5 (Figure 31), and it was shown that there were no chromosomal abnormalities in ciNSC5 differentiated by YJ102 (Figure 32).

[0196] Through this process, it was found that the cellular characteristics of ciNSC5 are maintained even after subculturing.

[0197] 3-6. Confirmation of differentiation of ciNSC5 into nerve cells We investigated whether ciNSC5 differentiated into human UCB-MSCs by YJ102 could differentiate into nerve cells, and examined whether adipogenesis occurred by MDI (Methylisobutylxanthine, Dexamethasone, Insulin) treatment to investigate the differentiation stability of ciNSC5. Specifically, UC Place B-MSC in a 100mm culture dish in a 3x10 5 Dispense into cells / well ( The DMSO control group was 5 times 10 4 Cells were dispensed into a single well, and the following day, YJ102 was dissolved in DMSO at a concentration of 30 mM, and the cells were treated with this solution at a concentration of 30 μM (the control group consisted of DMSO-treated UCB-MSCs). The cells were then cultured at 37°C for 5 days to obtain ciNSC5 differentiated from the UCB-MSCs. Next, to differentiate the ciNSC5 into dopaminergic neurons or nerve cells, 5 × 10⁶ cells were added to each of the control group's MSCs. 5Cells / dish cells were dispensed into 60mm culture dishes, and the culture medium was changed every 3 days to observe morphological changes. In addition, to confirm whether ciNSC5 can differentiate into adipocytes, 5 × 10⁶ MSCs and ciNSC5 cells were cultured. 5Cells were dispensed into 60 mm culture dishes using cells / dish and cultured for 3 days in medium supplemented with MDI (0.5 mM of 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 10 μg / ml insulin) before being harvested. For immunochemical analysis, the culture medium was removed, washed with PBS, and fixed with 4% paraformaldehyde at room temperature for 10 minutes. After washing three times with PBS, the cells were permeabilized with PBS-T containing 0.5% Triton X-100 for 10 minutes, washed three times with PBS-T for 10 minutes each, and then blocked for 1 hour with a blocking solution (PBS-T containing 5% BSA and 5% goat serum). Subsequently, the cells were treated with anti-TH antibody (Cat. p40101-150, Pel-Freez) and anti-MAP2 antibody (Cat. 4542, cell signaling), respectively, diluted with the blocking solution, and incubated overnight at 4°C. The following day, after washing 10 times with PBS-T for 10 minutes each, the cells were treated with anti-rabbit IgG-cy3 diluted in blocking solution and incubated in the dark for 1 hour. Next, the cells were washed 10 times with PBS-T for 10 minutes each, mounted in mounting solution containing DAPI, and observed under a confocal microscope. To extract RNA, the culture medium was removed, washed with PBS, and the cells were collected with a scraper and transferred to a microcentrifuse tube. The PBS was removed by centrifugation, 500 μl of Tri-RNA Reagent was added, and the mixture was thoroughly mixed by pipetting. 100 μl of chloroform was added, the tube was vortexed for 15 seconds to mix, and then the mixture was centrifuged at 14,000 rpm for 20 minutes at 4°C. Next, the upper aqueous phase was transferred to a new microcentrifuse tube using a pipette, and 550 μl of isopropanol was added and mixed. The pellet was centrifuged at 14,000 rpm for 20 minutes at 4°C, and after discarding the supernatant, it was washed with 1 ml of 70% ethanol. After centrifuging at 14,000 rpm for 2 minutes, the supernatant was discarded. After air-drying the pellet, the RNA was lysed with 50 μl of RNase-free DIW. 1 μl of DNase was added and the mixture was incubated at 37°C for 30 minutes.Next, the pellet was inoculated with the same volume of 8M LiCl and left on ice for 1 hour. After centrifugation at 14,000 rpm for 10 minutes at 4°C, it was washed with 70% ethanol and then air-dried. The pellet was resuspended in 100 μl RNase-free DIW, centrifuged at 14,000 rpm for 10 minutes at 4°C to remove the supernatant, air-dried, and the RNA concentration was quantified using nanodrop. Next, 500 ng of RNA was mixed with 2 μl of PrimeScript RT Master Mix, and the total volume was adjusted to 10 μl using DW. cDNA was synthesized by reverse transcription at 37°C for 15 minutes, and the reverse transcriptase was inactivated by reacting at 85°C for 5 seconds. The expression of C / EBPα and PPARγ, markers of adipogenesis, was confirmed using real-time PCR with the synthesized cDNA.

[0198] The results showed that ciNSC5 differentiates into nerve cells when cultured in differentiation medium. (Figure 33) Unlike MSCs, it did not respond to the induction of adipogenesis (Figure 34).

[0199] Example 4. Confirmation of the therapeutic effect of ciNSC5 directly trans-fractionated by the compound of the present invention on Lou Gehrig's disease. 4-1. Confirmation of increased lifespan in ALS model mice To confirm the efficacy of ciNSC5 against Lou Gehrig's disease, ALS model mice were created, and after transplanting ciNSC5 into the ALS model mice, the lifespan of the mice was checked. Specifically, transgenic ALS mice (B6SJL-Tg(SOD1-G93A)) were used. 1Gur / J over-expressing human SOD1 containing the Gly93→Ala mutation (The Jackson Laboratory, Bar Harbor, ME, USA) was crossed with female mice with a similar background (B6 / SJLF1) to develop SOD1. G93AA transgenic ALS mouse model was constructed. The genotype of its offspring was analyzed using PCR assays for tail DNA. At 60 days postnatal, mice were randomly grouped and then anesthetized with chloral hydrate (500 mg / kg, intraperitoneally). After treatment with 10 μl of MSCs suspended in saline or YJ102 for 5 days, ciNSC5 (1 × 10⁻¹) were differentiated into human UCB-MSCs. 6 Cells were injected into the cisterna magna. The control group received the same amount of saline solution in the cisterna magna. Next, the lifespan of each group was checked. As a result, both the MSC-transplantation group and the ciNSC5-transplantation group showed increased lifespan compared to the saline-treated group. In particular, the lifespan of the ciNSC5-transplanted group was extended by 12 days compared to the saline-transplantation group and by 9 days compared to the MSC-transplantation group. (Figure 35).

[0200] 4-2. Confirmation of ALS onset and delayed progression of motor symptoms After transplanting ciNSC5 into the aforementioned ALS model mice, behavioral tests were performed every 5 days starting from day 75 postnatal to assess the mice's motor function. The behavioral tests included the Rotarod test, motor score, hanging wire test, and balance beam test. For the Rotarod test, which evaluates the mice's general motor coordination, strength, and balance, the mice were trained on a Rotarod device (4-40 rpm, 180 sec) starting 3 days before the test. During the test, each mouse exercised on the Rotarod for 180 seconds, and the time it took for the mouse to fall off the Rotarod during the exercise was recorded. Each mouse underwent a total of three tests, with a rest interval of at least 10 minutes between tests. The average of the three records was used to determine each mouse's score. Furthermore, the motor score was determined by observing the condition of the hind limbs when the mouse's tail was lifted, and assigning a score from 0 to 4 based on the following criteria: [4 points = normal; 3.5 points = onset of hind limb splaying defect; 3 points = abnormal gait; 2 points = partial hind limb paralysis (first sign of dragging); 1 point = hind limb paralysis and forelimb weakness; 0 points = forelimb paralysis of a similar degree to that of the hind limbs]. In addition, to perform the hanging wire test to evaluate the neuromuscular strength of the legs maintained against gravity, each mouse was placed on a wire lead in a normal housing case and the lead was flipped back and forth. The test was conducted for a total of 180 seconds, and the time until the mouse, suspended from the wire lead, fell to the floor was measured. The test was conducted a total of three times per mouse, with a rest interval of at least 10 minutes between tests. The average of the three records was used to determine each mouse's score.In addition, for the balance beam test, mice were placed on a round wooden beam (0.5 cm diameter, 100 cm long, and 60 cm high) and scored from 0 to 6 based on the following criteria: [0 points = mouse cannot stand on the beam for 30 seconds; 1 point = mouse can stand on the upper right of the beam for 30 seconds; 2 points = mouse cannot walk but can rotate to the left or right on the beam; 3 points = mouse can rotate to the left or right on the beam and can take one or more steps; 4 points = mouse can cross the beam showing a foot slide of 50% or more of the affected hind limb; 5 points = mouse can cross the beam showing a foot slide of 50% or less of the affected hind limb; 6 points = mouse can cross the beam with one foot slide or less].

[0201] Rotarod test results showed no significant difference between the saline-treated group and the MSC-transplant group. However, the ciNSC5-transplant group showed a considerably delayed decline in motor function compared to both the saline-transplant and MSC-transplant groups (12.8±5.8 for the saline group, 21.8±9.0 for the MSC group, and 107.4±22.7 for the ciNSC5 group at 120 days) (Figure 36A). Furthermore, when motor scores were examined, the motor score in SOD1G93A transgenic mice began to decrease from day 90 and continued to decrease until the end of the experiment. While the MSC-transplant mice showed slightly higher scores than the saline-treated mice, the difference was not significant. In contrast, the ciNSC5-transplant mouse group showed a decrease in motor score after 120 days, and their motor score during the experiment was significantly higher than both the saline and MSC groups (2.1±0.4 for the saline group, 2.3±0.4 for the MSC group, and 3.7±0.2 for the ciNSC5 group at 120 days) (Figure 36B). Furthermore, in the hanging wire test results, mice in the saline-transplant group and the MSC-transplant group began to fall from the wire from day 80, but in the mice transplanted with ciNSC5, the onset of the disease was delayed to the point that some mice were observed to fall from the wire after day 95. In addition, the ciNSC5 transplant group showed significantly higher scores than the other two groups throughout the entire process of the hanging wire test (120-day saline group 6.4±2.8, MSC group 22.9±15.14, and ciNSC5 group 113.1±18.2) (Figure 36C). Moreover, in the balance beam test results, the ciNSC5 transplant group showed significantly higher scores than the other two groups from day 85 after birth until the end of the experiment (120-day saline group 1.5±0.32, MSC group 2.3±0.5, and ciNSC5 group 4.0±0.5) (Figure 36D).

[0202] 4-3. Confirmation of the effect of suppressing the loss of motor neurons in the spinal cord. To determine whether ciNSC5 transplantation alleviates lumbar spinal nerve loss associated with the clinical symptoms of ALS, Cresyl violet staining was performed on day 110 after transplantation, and the number of motor neurons in the lumbar spinal cord was counted.

[0203] As a result, compared to a mean of 7711.3 ± 193.8 motor neurons in wild-type mice, saline-treated SOD1 G93A Transgenic mice showed a significant decrease in the number of motor neurons (3875.1 ± 465), while MSC-transplanted mice showed a slight increase in the number of motor neurons compared to saline-treated mice, but the difference was not statistically significant. However, the number of motor neurons in the ciNSC5-transplanted mouse group was significantly increased compared to the saline-treated or MSC-transplanted groups (Figure 37).

[0204] Through this, ciNSC5 is SOD1 G93A We confirmed that in a transgenic ALS mouse model, saline-treated groups significantly suppressed spinal motor neuron death compared to both saline-treated and MSC-treated groups, delayed the progression of Lou Gehrig's disease symptoms accompanied by motor dysfunction, and extended the lifespan of ALS mice.

[0205] Example 5. Confirmation of the therapeutic effect of ciNSC5 directly trans-fractionated by the compound of the present invention on multiple sclerosis. 5-1. Confirmation of improvement in behavioral symptoms in EAE mice The efficacy of ciNSC5 was confirmed in an experimental autoimmune encephalomyelitis (EAE) mouse model, which is a standard model of multiple sclerosis (MS). Specifically, adult female C57BL / 6NTac mice (8-9 weeks old; 19-21g) were purchased from Narabiotec Co., Ltd. (Seoul, Republic of Korea), and 20 mg / ml of ciNSC5 was added to Complete Freund's Adjuvant (Sigma-Aldrich) containing 4 mg / ml of sterile Mycobacterium tuberculosis (Difco). 0ng MOG35-55 peptide (Sigma-Aldrich, St. Louis, EAEs were induced by subcutaneous immunization with MO (USA). On days 0 and 2, 200 ng pertussis toxin (Sigma-Aldrich), which plays a major role in stimulating the immune response, was intraperitoneally injected (Figure 38A). On day 8 after MOG injection, mice were anesthetized with chloral hydrate (500 mg / kg, intraperitoneally) and 10 μl of MSCs or ciNSC5 (1 × 10⁶) suspended in saline were injected. 6 Cells were injected into the cisterna magna. The control group received the same amount of saline solution at the same time point. Subsequently, the behavioral symptoms of the EAE mouse model were evaluated daily using the following standard score range of 0 to 7: score 0 = no symptoms; score 1 = partial tail drooping; score 2 = moderate hindlimb weakness (waddling gait); score 3 = moderately severe hindlimb weakness; score 4 = bilateral lower limb paralysis; score 5 = bilateral lower limb paralysis with moderate forelimb weakness; score 6 = quadriplegia, mortally ill; score 7 = death. The behavioral analysis results were analyzed using repeated ANOVA (time vs treatment) evaluations.

[0206] As a result, the mice in the saltwater-treated control group began to show typical behavioral symptoms, including tail drooping and leg paralysis, which demonstrate the progression of paralysis, 8-9 days after EAE induction (initiation step), reaching their peak at 18-20 days and persisting until the end of the experiment (Figure 38B). In the groups transplanted with MSCs or ciNSC5s, EAE behavioral symptoms were significantly alleviated even at the initiation step, and in particular, the clinical score in the ciNSC5 transplant group was significantly lower than that of the MSC transplant group (saline group 2.7±0.2, MSC group 2.1±0.2, and ciNSC5 group 1.5±0.2 at 9 days post-transplant) (Figure 38B and D). There was no significant difference in body weight between the experimental groups during the experimental period (Figure 38C).

[0207] 5-2. Confirmation of the effect of suppressing CNS demyelination. 30 days after MOG injection, mice were anesthetized with chloral hydrate (500 mg / kg), and 0 The animals were perfused via cardiac puncture with 0.1M PBS (pH 7.4) and then with 4% paraformaldehyde in 0.1M PBS (pH 7.4). Lumbar segments of the spinal cord were incised and post-fixed in the same fixative for 6 hours, then placed in 30% sucrose in 0.1M PBS (pH 7.4). The segments were placed in OCT to prepare frozen sections, and the transverse sections were cut to 16 μm using cryostat (Leica, Germany). For molecular studies, the animals were perfused with PBS, and sections of the lumbar spinal cord (L4-L5) were isolated and frozen at -80°C until use. The frozen sections were analyzed by immunohistochemistry using antibodies against GFAP (DAKO, Santa Clara, CA, USA), CD11b (Millipore, Billerica, MA), and CD68 (Millipore). For dual labeling, FITC or cy3-conjugated secondary antibodies were used (Jackson ImmunoResearch, West Grove, PA). Nuclei were also labeled with DAPI (Molecular Probes, Eugene, OR). Immunostaining control studies were performed by replacing the primary antibody with non-immune and control antibodies, pre-adsorbing them with excess amounts (10 μg / ml) of each antigen, and removing the primary antibody. Fluorescence intensities above titer were quantified and averaged using MetaMorph software (Molecular Devices, Sunnyvale, CA). Titer values ​​were at least three background values, and the background was quantified and averaged for the primary antibody removal control. In addition, for Western blot analysis, the frozen spinal cord tissue was homogenized with a disruption buffer (1% Nonidet® P-40, 20 mM Tris (pH 8.0), 137 mM NaCl, 0.5 mM EDTA, 10% glycerol, 10 mM Na2P2O7, 10 mM NaF, 1 μg / ml aprotinin, 10 μg / ml leupeptin, 1 mM vanadate, and 1 mM PMSF).After collecting this in an e-tube, it was rocked at 4°C for 20 minutes and centrifuged at 12,000 rpm for 30 minutes. The supernatant was transferred to another e-tube, and protein quantification was performed using a BCA assay kit. An equal amount of protein was loaded onto SDS-PAGE and subjected to electrophoresis. Subsequently, the protein was transferred onto a nitrocellulose membrane, and Western blot analysis was performed according to the protocol provided in the corresponding antibody data sheet. The obtained data are presented as mean ± SEM. Multiple comparisons between groups were performed by one-way ANOVA. Tukey's multiple comparison was used for post hoc analysis. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 15.0 (SPSS Science, Chicago, IL).

[0208] The results showed that demyelination, evaluated by luxol fast blue staining, was significantly increased in the white matter of the spinal cord of EAE mice 30 days after immunization (Figure 39A, Saline), while demyelination in the spinal cord was significantly reduced in ciNSC5-transplanted EAE mice (Figures 38A and B). In addition, ciNSC5-transplanted EAE mice also showed a significantly better demyelination-reducing effect compared with MSC-transplanted EAE mice. To re-verify the demyelination-inhibiting efficacy of ciNSC5, the expression of myelin basic protein (MBP) was confirmed by immunostaining and Western blot. The results showed that compared with the sham group, MBP expression was significantly decreased in EAE mice administered saline, and in the spinal cord of ciNSC5-transplanted mice the reduction of MBP was significantly inhibited compared with saline-treated or MSC-transplanted mice (Figures 39C and D). Western blot results also similarly showed that MBP expression was significantly higher in ciNSC5-transplanted mice compared with the other two groups (saline-transplanted group or MSC-transplanted group) (Figures 39E and F). Through these results, it was confirmed that ciNSC5 suppresses CNS demyelination, which is known as a typical pathological feature of MS patients, and alleviates its scope and severity.

[0209] 5-3. Confirmation of the inhibitory effect on mononuclear cell and macrophage penetration into spinal white matter. Thirty days after MOG immunization, the infiltration of mononuclear cells around small blood vessels in the spinal cord was detected by cresyl violet (Cat.C5042, Sigma) staining. Additionally, CD68-positive macrophages were confirmed by immunohistochemical staining on the same day.

[0210] As a result, EAE mice showed concentrated mononuclear cell infiltration around the white matter of the spinal cord (Figure 40A and B, saline), whereas the MSC or ciNSC5-treated groups showed reduced mononuclear cell infiltration into the spinal cord white matter in both thoracic (A) and lumbar (B) sections (Figure 4 0) We confirmed that ciNSC5 transplantation mitigates EAE-induced mononuclear cell infiltration into spinal cord white matter. Furthermore, while D68-positive macrophages were observed in the white matter of saline-treated EAE mice, the number of infiltrated macrophages was significantly reduced in the MSC or ciNSC5 transplantation groups (Figure 41).

[0211] 5-4. Confirmation of inhibitory effect on activation of microglia and astrocytic cells. To confirm the effect of ciNSC5 on the activation of glial cells such as microglia and astrocytes in the spinal cord, immunohistochemical analysis against CD11b (microglia marker) or GFAP (astroglial marker) antibodies was performed 30 days after immunization.

[0212] As a result, very strong GFAP immunoreactivity was observed in the spinal cord of EAE mice treated with saline, confirming activation of spinal cord astrocytes (Figure 42). On the other hand, in EAE mice transplanted with MSCs or ciNSC5, activation of spinal cord astrocytes was significantly reduced, and quantification of GFAP fluorescence intensity showed that both the MSC-transplanted group and the ciNSC5-transplanted group showed significantly reduced GFAP activity. FAP intensity decreased. In particular, ciNSC5 showed a significant inhibitory effect on GFAP expression compared to MSCs (Figure 42D). This indicates that ciNSC5 suppresses astroglial cell activation in EAE mice. Furthermore, immunostaining with CD11b revealed that microglia in the spinal cord of EAE mice showed the typical activation pattern with cytoplasmic hypertrophy and shortened branches (Figure 43A), whereas the activation pattern of microglia was significantly reduced in EAE mice transplanted with MSCs or ciNSC5. In particular, many resting forms of microglia with elongated branches were observed in ciNSC5-transplanted mice (Figure 43). These results indicate that microglia activation was suppressed by ciNSC5 transplantation in EAE mice.

[0213] Example 6. Confirmation of the therapeutic effect of ciNSC5 directly trans-fractionated by the compound of the present invention on Parkinson's disease. 6-1. Confirmation of the effect on improving motor function recovery To confirm the effect of ciNSC5 on functional recovery in the chronic step of a PD animal model via 6-OHDA infusion, the body weight of adult Sprague-Dawley male letts (230-250g, Sam:TacN(SD)BR, Samtako, Osan, Korea) was measured, and the letts were anesthetized with chloral hydrate (500mg / kg, intraperitoneal injection), fixed stereotaxically, and held in a fixed position. Partial injury model: AP+0.7, ML+2.6, DV-4.5 from bregma; and Complete injury model: AP-2.2. 8 µg of 6-OHDA was dissolved in 4 μl of saline solution and injected into ML+1.5, DV-8.0 (from bregma) using a 33-gauge Hamilton syringe (Figure 44). Four weeks after OHDA injection, Rett was anesthetized with chloral hydrate (500 mg / kg, ip), then stereotaxically fixed, and 10 μl of MSC or ciNSC5 (1 × 10⁶) suspended in saline was placed in the right striatum (AP: +1.0, ML: -3.0, DV: -5.0). 6Cells were transplanted. Next, apomorphine (0.05 mg / kg) was injected subcutaneously and placed in a 40 cm diameter transparent cylinder. After 10 minutes of adaptation, if the patient rotated more than 4 times per 10 minutes, it was determined that the Parkinson's disease model was complete. To evaluate the efficacy of ciNSC5 in improving motor function in the partial injury model, rotation tests were performed every two weeks after cell transplantation. In the complete injury model, stepping tests were performed once a week starting from the second week after transplantation to confirm motor function recovery. Each forelimb of the patient was placed on a treadmill, and a 1-minute video was recorded and observed. The number of steps in the contralateral direction (where gait impairment occurred on the opposite side of the side to which 6-OHDA was injected) and the ipsilateral direction (where normal gait was possible) were counted, and the step-pin percentage (%) was calculated by dividing the contralateral steps by the total steps. Normal patients showed a step-pin percentage of 50%, while patients with induced Parkinson's disease showed a value of less than 10%.

[0214] As a result, in the partial injury model, the number of rotations decreased in Retts who received MSCs and ciNSC5s as time passed after transplantation, and the ciNSC5-transplanted Retts showed a significant improvement in motor function compared to the saline transplantation group from week 2 to week 6 post-transplantation (Figure 45A). In the complete injury model, the saline administration group showed no change compared to pre-transplantation, while the ciNSC5 transplanted group showed a significant increase in steppin frequency from week 2 post-transplantation compared to the saline group, and this effect persisted until week 6 post-transplantation (Figure 45B).

[0215] 6-2. Confirmation of the effect of suppressing the loss of dopamine neurons. Cells were transplanted into 6-OHDA-injected PD animal models, and after 4 or 6 weeks, Rett was anesthetized with chloral hydrate (500 mg / kg) and perfused via cardiac puncture with 0.1 M PBS (pH 7.4) and then 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brain was excised and post-fixed by impregnation in the same fixative for 24 hours, and then fixed with 0.1 MP. The tissue was placed in 30% sucrose in BS (pH 7.4). The tissue embedded with the OCT compound was cut to 40 μm using cryostat (Leica, Germany), and the tissue containing Substantia nigra was immunostained with an antibody against TH, a dopamine neuron marker, and immunohistochemical staining was performed.

[0216] The results showed that tissues transplanted with ciNSC5 retained a significantly higher number of dopamine neurons than tissues transplanted with saline or MSCs (Figure 46).

[0217] 6-3. Confirmation of differentiation effect on TH-positive dopamine neurons Immunostaining of Rett's striatum at 6 weeks post-transplant in a partial injury model using the human-specific antibody STEM121 revealed that no STEM121-positive cells were observed in the MSC transplantation group (data not shown), but STEM121-positive cells were confirmed in the ciNSC5 transplantation group (Figure 47). This indicates that the transplanted ciNSC5s were still viable in the tissue at 6 weeks post-transplantation. Furthermore, double immunostaining using STEM121 antibody and TH antibody revealed that ciNSC5s were present in the stigma tissue within the transplanted tissue. EM121-positive human cells were confirmed to be TH-positive dopamine neurons (Figure 48). This indicates that the transplanted ciNSC5 differentiated into dopamine neurons within the striatum.

[0218] Through this, we found that ciNSC5 not only significantly improves behavioral symptoms in a 6-OHDA-induced PD model, but also shows an effect of suppressing dopamine neuron death, demonstrating a more pronounced therapeutic effect than MSCs. In particular, since the transplanted neural progenitor cells ciNSC5 differentiated into dopamine neurons in the striatum, the transplantation site, it can be seen that ciNSC5 can exert its therapeutic effect by replacing dopamine neurons that have decreased due to PD when transplanted into nerve tissue.

[0219] Example 7. Confirmation of the therapeutic effect of ciNSC5 directly trans-fractionated by the compound of the present invention on chronic spinal cord injury. 7-1. Confirmation of the effect on improving motor function recovery. To confirm the effect of ciNSC5 on functional recovery in the chronic step after spinal cord injury, behavioral tests including the BBB open field test, grid walk test, and footprint analysis were performed after ciNSC5 implantation, and these were integrated to perform a functional recovery index (FRI) analysis. Specifically, to create a chronic spinal cord injury Rett model, adult Sprague-Dawley male and female Retts (230-250g, Sam:TacN(SD)BR, Samtako) were used before surgery. The weight of a Rett (Osan, Korea) was measured, anesthetized with chloral hydrate (500 mg / kg, intraperitoneal injection), and the back and neck were shaved. Laminectomy was performed at the T9-T10 level to expose the lower part of the spinal cord without damaging the papillary membrane. Next, the spinous processes of T8 and T11 were fixed with clamps to stabilize the vertebrae, and a moderate bruise injury (10 g × 25 mm) was inflicted on the dorsal surface of the exposed spinal cord to create a chronic spinal cord injury Rett model. After the injury, the muscles and skin were covered and the Rett was placed overnight in a temperature and humidity controlled chamber. Postoperatively, the Rett was subcutaneously injected with replacement fluid (5 ml, lactated ringer) and antibiotics (gentamicin, 5 mg / kg, intramuscular injection) were administered once daily for 5 days postoperatively. After the injury, the Retts were housed individually in cages to facilitate access to water and food. Weight and the weight of remaining food and water were checked and recorded for all animals. The bladder was passively emptied three times a day until reflex urination was established. Six weeks after spinal cord injury, Rett was anesthetized with chloral hydrate (500 mg / kg, ip) and given 10 μl of MSC or ciNSC5 (1 × 10) suspended in saline. 6Cells were injected into the lesion cavity. The control group received the same amount of saline solution at the corresponding time point. Behavioral analyses of the BBB open field test, grid walk test, and footprint analysis were performed weekly for 8 weeks post-transplant. The BBB open field test assessed post-bruising recovery in Rett's spinal cord by testing mobility function using the following open field mobility score (Table 5) according to the Basso, Beattie, and Bresnahan (BBB) ​​criteria, which use the BBB score to test functional recovery and mobility in chronic SCI studies, ranging from complete paralysis (score 0) to normal mobility (score 21). The grid walk test involved having animals walk on a horizontal runway of 1m long metal grid bars 30cm above the floor, and selecting 10 defined bar sectors for analysis. To prevent habituation to fixed bar intervals, the bars in such sectors were arranged irregularly (1-4cm intervals) and changed for each test section. The analysis was conducted by counting the number of errors in foot positioning. If an animal was unable to walk on its hind limbs, two errors were created per bar, resulting in a total of 20 errors. The percentage of foot missteps per step was then calculated. Footprint analysis involved dipping the animals' forelimbs and hindlimbs in non-toxic red and blue dyes, then having them walk across a narrow box (1m long and 7cm wide) to scan their footprints. The digitized images were then analyzed. In addition, the three behavioral tests were integrated to compare behavior at the time of cell transplantation and 8 weeks post-transplantation. The 8-week mobility function recovery delta value was calculated to quantify the steps of motor function recovery and derive the Functional Recovery Index (FRI). The standard points given for each behavioral experiment are shown in Table 6 below.

[0220] [Table 6]

[0221] [Table 7]

[0222] The results showed that both the ciNSC5 transplant group and the MSC transplant group significantly increased the BBB mobility score after spinal cord injury (Figures 49A and D). Furthermore, the grid error rate (foot slips per step) for all ciNSC5 and MSC transplants was even lower in the saline-treated group, and the rate of foot slips in the ciNSC5 transplant group was significantly reduced compared to the MSC transplant group in both male and female reds (Figures 49B and E). At 14 weeks post-injury (8 weeks post-transplant), footprints in both ciNSC5-transplant reds and MSC-transplant reds showed fairly consistent forelimb-hindlimb harmony and very little toe drag. In contrast, footprints in the saline-treated group showed inconsistent forelimb-hindlimb harmony and significant drag, with subsequent ink tracks on both hind limbs. Additionally, toe drag was significantly reduced in the ciNSC5 transplant group compared to the MSC transplant group (Figures 49C and F). Furthermore, functional recovery index (FRI) analysis revealed that the FRI scores of ciNSC5 and MSCs increased significantly compared to the saline transplantation group, and in particular, the FRI score of the ciNSC5 transplantation group improved even more remarkably than that of the MSC transplantation group (Figures 50A, B, D, and E). No abnormal weight changes were observed with ciNSC5 and MSC transplantation during the period in which the behavioral experiment was conducted (Figures 50C and F).

[0223] 7-2. Confirmation of the effect of reducing lesion volume and myelin loss. Eight weeks after MSC or ciNSC5 transplantation, animals were anesthetized with chloral hydrate (500 mg / kg) and perfused via cardiac puncture with 0.1 M PBS (pH 7.4) and then with 4% paraformaldehyde in 0.1 M PBS (pH 7.4). A spinal cord section (1.5 cm) concentrated at the lesion site was incised and post-fixed by impregnation in the same fixative for 6 hours, and then placed in 30% sucrose in 0.1 M PBS (pH 7.4). The segment was placed on OCT to prepare frozen sections, and longitudinal or transverse sections were cut to 10 or 16 μm. To confirm the lesion volume using serial longitudinal sections (10 μm) through the dorsal-ventral axis of the spinal cord, sections were stained with cresyl violet acetate every 50 μm and observed under a light microscope. Lesion areas were determined using MetaMorph software (Molecular devices) with a low-magnification lens (1.25X), and the area at each longitudinal level was determined. The total lesion volume was inferred by the numerical average of the continuous areas. In addition, continuous transverse cryosections (16 μm thick) were placed on glass slides to confirm myelin loss after injury. Selected slides were incubated overnight at 60°C with 0.1% Luxol fast blue (Solvent Blue 38; Sigma) acidified with 95% ethanol. Differentiation was performed with 0.05% lithium carbonate.

[0224] To confirm tissue loss after spinal cord injury, serial longitudinal sections were stained with Cresyl violet and lesion volume was measured. At 8 weeks post-transplant, the total lesion volume was significantly reduced in both the ciNSC5 and MSC-treated groups compared to the saline-infusion group, and the lesion volume was significantly further reduced in the ciNSC5 transplant group compared to the MSC transplant group (Figures 51A and B). Furthermore, myelin loss after injury was confirmed by Luxol-fast blue staining. Both the ciNSC5 and MSC-treated groups showed significantly suppressed myelin loss compared to the saline-infusion group, and the ciNSC5 transplant group showed significantly further reduced myelin loss compared to the MSC transplant group (Figures 51C and D).

[0225] 7-3. Confirmation of the effect of reducing gliotic wound formation. To confirm the effect of ciNSC5 on glial wound formation, it was evaluated by double immunohistochemistry against GFAP and CSPG. Since CSPG, secreted from wounds densely formed within the lesion site after spinal cord injury, is considered a chemical impediment to axonal regeneration and functional recovery, the presence or absence of CSPG was confirmed by staining with CS-56. Specifically, frozen sections were analyzed by immunohistochemistry using antibodies against GFAP (DAKO, Santa Clara, CA, USA) and CS-56 (CSPG, Millipore, Billerica, MA). For double labeling, FITC or cy3-conjugated secondary antibodies were used (Jackson ImmunoResearch, West Grove, PA), and the nuclei were labeled with DAPI (Molecular Probes, Eugene, OR). In immunohistochemical control studies, the primary antibody was replaced with non-immunochemical and control antibodies, and pre-adsorbed with an excess (10 μg / ml) of each antigen to remove the primary antibody. Fluorescence intensity above titer was quantified and averaged using MetaMorph software (Molecular devices, Sunnyvale, CA). Titer values ​​were at least three background levels, and the background was quantified and averaged against the primary antibody dropout control.

[0226] When the presence or absence of CSPG was confirmed by staining with CS-56, the lesion sites in the spinal cord of the saline-treated group were richly filled with CS-56 and GFAP-positive staining signals, whereas Both CSPG and GFAP intensities were significantly reduced in the MSC and ciNSC5 transplantation groups compared to the saline-treated group (Figure 52A-C). Western blot showed G The results of the FAP level examination also showed that, similar to the immunohistochemical staining results, the GFAP level was significantly reduced in the ciNSC5 transplanted group compared not only to the saline group but also to the MSC transplanted group (Figure 52D). Through this, we confirmed that ciNSC5 inhibits gliotic scar formation.

[0227] 7-4. Confirmation of the effect on improving axonal regeneration To confirm the effect of ciNSC5 on corticospinal tract axon growth, leading axonal tracking was performed using BDA, and fluorogold (FG) was injected into the lesion site via the tail portion at 14 weeks post-injury to determine the scale of the extra descending axon pathway leading to the injury site. Selected FG-labeled neurons in the supraspinal nuclei were counted. Specifically, 12 rets (3 from the saline infusion group, 5 from the MSC transplantation group, and 5 from the ciNSC5 transplantation group) were anesthetized with chloral hydrate (500 mg / kg) at 8 weeks post-MSC or ciNSC5 transplantation, and bone on the motor cortex was removed. 10% fluorescently labeled biotinylated dextranamine (BDA; Molecular Probes, Eugene, OR) was injected into the motor cortex at eight locations in both directions (four on each side). Two weeks after BDA injection, the retto was perfused with 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4). Spinal cord was collected and longitudinally sectioned to a thickness of 20 mm on a cryomicrotome, and axonal regeneration was quantified under a 20x fluorescence microscope. Each section was prepared on a grid with lines drawn at 0, 500, 1000, 1500, 2000, 2500, and 3000 μm intervals, and the number of axons was counted by angular distance from the door edge of the lesion cavity. BDA-positive axons were counted in four selected tissues at 100 μm intervals, including the corticospinal tract. The total number of axons in each animal was averaged per group. The total number of axons present in four sections per animal was counted and averaged.Furthermore, to determine the extent to which extra descending axons contribute to lateral lumbar vertebral widening through retrograde tracking using Fluorogold (FG; Invitrogen, Grand Island, NY, USA), 4% FG was injected into the YJ101 spinal cord fraction four weeks after injury, at four locations on each plane (0.5 μl per injection) according to the following stereotactic coordinates: (1) 0.5 mm medial-lateral (ML), 0.5 mm dorsal-ventral (DV); (2) 1.0 mM ML, 0.5 mm DV; (3) 0.5 mm ML, 2.0 mM DV; and (4) 1.0 mM ML, 2.0 mM DV. The dye was allowed to disperse for at least two minutes after each injection, and histological analysis was performed one week later by sacrificing the animals. The extent of bulk injection, including white matter, was confirmed by sequentially cutting the cross-section of 5-mm spinal cord sections containing the injection site, and it was confirmed that the dye did not diffuse beyond the injected spinal cord section. Cases that did not meet these criteria were excluded from further studies. Transverse brain sections (40 μm thick) containing the sensorimotor cortex were cut on cryostat, and the third section of each section was placed on a glass slide. Ten sections were analyzed per lett, and the number of labeled neurons was counted per lett and averaged across the entire lett. In addition, the expression pattern of GAP43, a known axonal regeneration factor, was confirmed by Western blotting.

[0228] BDA anterograde axon tracing revealed very little regeneration in the saline-transplant and MSC-transplant groups, while the ciNSC5-transplant group showed vigorous CST axon regeneration through and beyond the lesion cavity (Figure 53). Axon counting showed that no axons were observed at 1000 μm from the lesion cavity starting point in the saline-transplant and MSC-transplant groups, but axons were observed at 2000 μm in the ciNSC5-transplant group. Furthermore, Fluorogold retrograde axon tracing showed that the number of FG-labeled neurons in RN, PnC, LVe, MdV, and MdD was significantly higher in the ciNSC5-transplant group compared to the saline-treated control group, while in the MSC-transplant group, FG-labeled neurons increased only in LVe, with no effect in other sites (Figure 54). In addition, Western blot analysis revealed that intraspinal GAP43 expression in grouplets transplanted with ciNSC5 was significantly increased compared to the saline transplantation group or the MSC transplantation group (Figure 55).

[0229] 7-5. Confirmation of differentiation into mature nerve cells Six weeks after transplanting ciNSC5 into a chronic spinal cord injury model, immunohistochemical staining was performed using a human-specific STEM121 antibody.

[0230] As a result, STEM121-positive cells were observed near the transplantation site, indicating that the transplanted ciNSC5s remained in the tissue. To confirm the cell type, double immunostaining was performed, showing that the transplanted ciNSC5s were double-stained with the neuronal cell markers NeuN, MAP2, and Tuj1 (Figure 56). This suggests that the transplanted ciNSC5s differentiated into mature neurons in the spinal cord tissue.

Claims

1. Compared to adult stem cells, (1) Increased expression of Tuj1, TBR2, MASH1, GAP-43, or p75; (2) Increased expression of Tuj1 protein, CD325 (N-cadherin) protein, p75 protein, GAP-43 protein, CD54 protein, CD309 protein, CD56 (NCAM) protein, PSA-NCAM protein, CD29 protein, MASH1 protein, or TBR2 protein; (3) Decreased expression of CD44, CD73, or CD105; (4) Increased secretion of PlGF, NGF, BDNF, VEGFA, MMP1, MMP2, MMP7, TIMP2, SHH, Notch1, TNFSF12 or IL-16; and (5) Oligomerization of CtBPs (C-terminal binding proteins) was induced. cell.

2. The cells according to claim 1, wherein, compared to adult stem cells, the expression of Tuj1 protein is increased by 6 times or more, CD325 (N-cadherin) protein by 7 times or more, p75 protein by 4 times or more, GAP43 protein by 4 times or more, CD54 protein by 2 times or more, CD309 protein by 3 times or more, CD56 (NCAM) protein by 3.5 times or more, PSA-NCAM protein by 4 times or more, CD29 protein by 4.5 times or more, MASH1 protein by 4 times or more, or TBR2 protein by 3 times or more.

3. The cells according to claim 1, wherein the expression of CD44 protein is reduced by 50-90%, the expression of CD73 protein is reduced by 30-70%, or the expression of CD105 protein is reduced by 50-90% compared to adult stem cells.

4. The cells according to claim 1, wherein, compared to adult stem cells, the secretion of PlGF is increased by 1.5 to 4 times, the secretion of NGF is increased by 1.5 to 6 times, the secretion of BDNF is increased by 1.5 to 3 times, or the secretion of VEGFA is increased by 1.5 to 5 times.

5. The cells according to claim 1, wherein the secretion of MMP1 is increased by 1.5 to 3.5 times or the secretion of MMP2 is increased by 2 to 6 times compared to adult stem cells.

6. The cells according to claim 1, wherein the secretion of TNFSF12 is increased by 1.5 to 4 times compared to adult stem cells.

7. The cell according to claim 1, wherein the CtBPs include CtBP1 or CtBP2.

8. The cells according to claim 1, wherein the expression of HES1, Sox2, or OCT4 is increased compared to adult stem cells.

9. The cells according to claim 1, which do not express MAP2, NSE, NeunN, doublecortin, NeuroD1 (Neurogenic Difference 1), Nestin, Mussashi 1, or GFAP.

10. The cells according to claim 1, wherein the expression of genes that induce neurogenesis, genes that secrete inducible neurotrophic factors, genes related to neurogenesis, neurons marker genes, neurons receptor genes, and channels related genes is increased compared to adult stem cells.

11. The cells according to claim 1, wherein the expression of enzymes for nerve glioscar degradation is increased compared to adult stem cells.

12. The cell according to claim 1, which is capable of differentiating into a dopaminergic neuron, a neuron, or a mature neuron.

13. The cells according to claim 1, wherein adipogenesis is not induced.

14. The cells according to claim 1, which are directly differentiated from adult stem cells into neural progenitor cells.

15. The cell according to claim 1, wherein the adult stem cell is a mesenchymal stem cell.

16. The cell according to claim 15, wherein the mesenchymal stem cell is umbilical cord blood-derived mesenchymal stem cell (UCB-MSC), umbilical cord-derived mesenchymal stem cell (UC-MSC), adipose-derived mesenchymal stem cell (AD-MSC), or bone marrow-derived mesenchymal stem cell (BM-MSC).

17. A pharmaceutical composition for the prevention or treatment of nerve injury diseases, comprising the cells, stem cell culture, or suspension culture described in claim 1 as an active ingredient.

18. The pharmaceutical composition for the prevention or treatment of a nerve injury disorder according to claim 17, wherein the nerve injury disorder is a central or peripheral nervous system injury or a neurodegenerative disease.

19. The pharmaceutical composition for the prevention or treatment of a nerve injury disease according to claim 18, wherein the injury to the central or peripheral nervous system is spinal cord injury (SCI), traumatic brain injury (TBI), peripheral nerve injury, stroke, or brain cancer.

20. The pharmaceutical composition for the prevention or treatment of a nerve injury disease according to claim 19, wherein the spinal cord injury is induced by trauma or inflammation.

21. The pharmaceutical composition for the prevention or treatment of a nerve injury disease according to claim 19, wherein the spinal cord injury is induced by one or more selected from the group consisting of acute transverse myelitis, acute seed-sowing myelitis, spinal cord disease, non-Hodgkin lymphoma, hydrocephalus, hereditary ataxia, neurosyphilis, Minamata disease, Lou Gehrig's disease, and multiple sclerosis.

22. The pharmaceutical composition for the prevention or treatment of nerve damage disease according to claim 18, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), multiple sclerosis (MS), or multiple system atrophy.

23. A pharmaceutical composition for the prevention or treatment of nerve injury disease according to claim 17, for suppressing the death of motor nerve cells in the spinal cord.

24. A pharmaceutical composition for the prevention or treatment of nerve injury diseases according to claim 17, for suppressing demyelination of the central nervous system (CNS).

25. A pharmaceutical composition for the prevention or treatment of nerve injury disease according to claim 17, for inhibiting the penetration of mononuclear cells or macrophages into the spinal white matter.

26. A pharmaceutical composition for the prevention or treatment of nerve injury disease according to claim 17, for suppressing the activation of microglia or astrocytic cells.

27. A pharmaceutical composition for the prevention or treatment of nerve injury disease according to claim 17, for suppressing the loss of dopamine neurons.

28. A pharmaceutical composition for the prevention or treatment of nerve injury disease according to claim 17, for reducing myelin loss.

29. A pharmaceutical composition for the prevention or treatment of nerve injury disease according to claim 17, for reducing the formation of gliotic scars.

30. A pharmaceutical composition for the prevention or treatment of nerve injury disease according to claim 17, for increasing axons.

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