Composition for preventing or treating nervous system diseases using human fibroblast-derived neural stem cells

Abstract

The present invention relates to: a composition of induced neural stem cells (ciNSCs) derived through direct cross-differentiation from somatic cells such as human fibroblasts, in which the composition is for preventing or treating nervous system diseases such as Huntington's disease, stroke, Alzheimer's disease, epilepsy, and multiple sclerosis; and uses of the induced neural stem cells for the treatment of nervous system diseases.

Description

Composition for preventing or treating nervous system diseases using human fibroblast-derived neural stem cells

[0001] The present invention relates to a composition for preventing or treating neurological diseases such as Huntington's disease, stroke, Alzheimer's disease, epilepsy, and multiple sclerosis using neural stem cells (ciNSCs) derived from human fibroblasts.

[0002] Research on cellular reprogramming began in 2007 with the development of human fibroblasts into induced pluripotent stem cells (iPSCs). Previous stem cell research, using human embryonic stem cell-derived neural stem cells, has been plagued by ethical concerns stemming from the use of human embryos, concerns about immune rejection, and the potential for tumor formation when undifferentiated embryonic stem cells are transplanted. Adult stem cells, on the other hand, are difficult to obtain and have limited differentiation potential. While iPSCs avoid ethical concerns and are immune-free, transplantation of undifferentiated stem cells can lead to teratoma formation. Furthermore, recently announced directly cross-differentiated neural stem cells possess similar properties to iPSCs and embryonic stem-derived neural stem cells. However, the viral system, the primary method used to generate neural stem cells, carries the risk of mutation due to random insertion of genes. In addition, plasmids, proteins, RNA, etc. are being used to solve the problem of viruses when transplanted into the human body, but their efficiency is low and new, unconfirmed problems may arise because they use oncogenes.

[0003] Accordingly, active research has been conducted since then, successfully introducing various combinations of neural cell-related transcription factors into human fibroblasts and inducing neural cell formation. This has demonstrated potential as a cell therapy for intractable brain diseases. However, because these cells are already differentiated into neurons, securing sufficient numbers of cells for cell therapy remains challenging. Due to this issue, recent research has focused on methods for directly differentiating fibroblasts into neural stem cells.

[0004] The central nervous system (CNS) receives stimuli and signals from both inside and outside the body, transmits them to other organs, and induces responses, regulating and controlling the body's activities accordingly. The CNS consists of the brain and spinal cord, and the peripheral nervous system, which connects it to the rest of the body. The nervous system is composed of basic units called neurons, which are arranged in networks that transmit electrical and chemical messages to the brain. Diseases of this nervous system can be fatal and are often incurable.

[0005] Huntington's disease is a degenerative genetic disease of the nervous system. It causes chorea, which causes involuntary movement of the limbs, dementia, mental illness such as depression, and death within 10 years of onset. Huntington's disease is caused by the excessive increase of the CAG repeat sequence located in the 16.3 section of the autosomal chromosome (chromosome 4), which creates a mutant huntingtin protein, resulting in the loss of GABAergic medium spiny neurons in the putamen. Current pharmacological treatments for Huntington's disease have not shown a clear therapeutic effect.

[0006] A stroke occurs when a blood vessel supplying blood to the brain becomes blocked or bursts, damaging the brain and causing neurological abnormalities such as paralysis, speech impairment, and loss of consciousness. Ischemic stroke accounts for more than 80% of all strokes, and occurs when a blood vessel supplying blood to the brain is blocked due to a blood clot or high blood pressure, causing brain cells to die. Hemorrhagic stroke occurs when a blood vessel supplying the brain bursts, causing bleeding and resulting brain damage. The mortality rate is approximately 50% within 30 days of the stroke, and half of these deaths occur within the first two days. When a cerebral hemorrhage occurs, brain tissue around the hemorrhage is destroyed, and the hematoma formed by the hemorrhage increases pressure in the existing brain, causing secondary problems.

[0007] Alzheimer's disease is a degenerative neurological disease in which abnormal proteins, such as amyloid beta and tau, accumulate in the brain, causing the gradual death of brain cells. Alzheimer's disease is the most common cause of dementia, accounting for 50-60% of all dementia cases. It is caused by brain abnormalities that lead to the gradual decline of memory.

[0008] Neuromyelitis optica and multiple sclerosis (MS) are inflammatory demyelinating diseases of the central nervous system, presenting with similar clinical manifestations of recurrent attacks of the brain, optic nerves, or spinal cord. Previously, NMO was considered a subtype of MS. However, the discovery of a disease-specific antibody (AQP4) in the serum of NMO patients in 2004 expanded the definition of the disease to include NMO. Furthermore, widespread inflammation and neuronal cell death occur throughout the nervous system, causing lesions in the spinal cord, optic nerves, and brain, resulting in various symptoms such as vision loss and paralysis of the lower extremities.

[0009] Epilepsy is a chronic, recurrent brain disease characterized by temporary paralysis of brain functions, such as loss of consciousness, seizures, and behavioral changes, caused by temporary abnormalities in brain neurons that lead to excessive excitement. The causative factors of epilepsy change with age, and it can also occur in conjunction with various brain diseases and genetics. Various brain diseases, including congenital diseases, various types of brain damage, brain inflammation, brain tumors, cerebrovascular diseases (cerebral hemorrhage, cerebral infarction), and degenerative diseases, can cause epileptic seizures. However, in more than half of epilepsy cases, the cause is unknown, and various drug treatments are currently being administered concurrently.

[0010] In a previous study, the inventors of the present invention developed neural stem cells directly converted from human fibroblasts using a small molecule compound (PCT / KR2016 / 003819), and administered the neural stem cells to various animal models of diseases, confirming their effective disease treatment effects and completing the present invention.

[0011] Accordingly, an object of the present invention is to provide a composition for preventing or treating a neurological disease, comprising induced neural stem cells directly converted from fibroblasts using a direct cross-differentiation inducing composition comprising at least one small molecule compound selected from the group consisting of Thiazovivin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2′-deoxycytidine and DZNep; or at least one of Sendai virus, OCT4 and / or SOX4 expression mRNA and miRNA 302 / 367.

[0012] In order to achieve the above object, the present invention provides a composition for preventing or treating a nervous system disease, which comprises induced neural stem cells directly converted from fibroblasts using a direct cross-differentiation inducing composition comprising at least one small molecule compound selected from the group consisting of Thiazovin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2′-deoxycytidine, and DZNep; or at least one of Sendai virus, OCT4 and / or SOX4 expression mRNA, and miRNA 302 / 367.

[0013] Hereinafter, the present invention will be described in detail.

[0014] The present invention relates to a composition for preventing or treating a neurological disease, comprising neural stem cells directly converted from fibroblasts using a direct cross-differentiation-inducing composition comprising at least one small molecule compound selected from the group consisting of Thiazovivin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2′-deoxycytidine, and DZNep; or at least one of Sendai virus, OCT4 and / or SOX4 expression mRNA, and miRNA 302 / 367.

[0015] The neural stem cells of the present invention are transformed by directly inducing cross-differentiation of fibroblasts in a culture medium containing at least one of a combination of small molecule compounds, Sendai virus, OCT4 and / or SOX4 expression mRNA, and miRNA 302 / 367, without separate gene introduction or manipulation.

[0016] In the present invention, the term "direct cross-differentiation" refers to one of the cell reprogramming technologies, which directly reprograms already mature differentiated cells to return them to stem cells. The present invention relates to a technology that can directly differentiate human somatic cells, for example, one or more somatic cells among fibroblasts, fibroblast-derived neural stem cells, or neural progenitor cells, into neural stem cells by reprogramming them through direct cross-differentiation, and this can overcome numerous disadvantages that appear during the differentiation of induced pluripotent stem cells and increase the efficiency of cell differentiation, since it directly differentiates them into neural cells without going through a dedifferentiation process into induced pluripotent stem cells.

[0017] "Neural stem cells" are undifferentiated cells that have the ability to self-renew and have the ability to differentiate into neurons and / or glia, such as astrocytes, oligodendrocytes and / or Schwann cells. Neural stem cells differentiate into neural cells, such as neurons or glia, after passing through the stage of neural progenitor cells or glial progenitor cells that produce specific nervous system cells. The neural stem cells can then differentiate into any one or more selected from the group consisting of astrocytes, oligodendrocytes, neurons, dopaminergic neurons, GABA neurons, motor neurons and cholinergic neurons.

[0018] In the present invention, the small molecule compound included in the culture medium used for direct cross-differentiation of the neural stem cells may be a compound selected from Thiazovin, Valproic acid (VPA), Purmorphamine, A8301 (TGF-β type I receptor inhibitor), SB43154 (ALK5 (Activin Receptor-Like Kinase-5) inhibitor), CHIR99021 (GSK (glycogen synthase kinase) inhibitor), 5-Aza-2'-deoxycytidine (decitabine), and DZNep (3-Deazaneplanocin A), and the culture medium may include a combination of these. Each of the small molecules is included in the medium at an effective concentration for differentiation of fibroblasts into neural stem cells, and the effective concentration is adjusted according to factors known in the art, such as the type of medium and the culture method. Can be.

[0019] Additionally, the culture medium of the present invention may contain Sendai virus. The Sendai virus is intended for reprogramming somatic cells, such as fibroblasts, and has the characteristic of being able to induce cells with multipotency by including Yamanaka factors.

[0020] The mRNA that can be included in the culture medium of the present invention is for the expression of OCT4 and / or SOX4. OCT4 is a transcription factor necessary for maintaining the pluripotency of stem cells, and SOX2 is a representative marker of neural stem cells and a factor necessary for converting somatic cells into induced pluripotent stem cells. By transfecting fetal human fibroblasts with OCT4 mRNA and SOX2 mRNA, gene expression can be regulated, and then direct cross-differentiation into neural stem cells can be induced using iNSC media.

[0021] In addition, the miRNA included in the culture solution of the present invention is miRNA302 / 367, which plays a role in suppressing the expression of the NR2F2 gene, which suppresses OCT4, a gene that maintains the pluripotency of stem cells.

[0022] In addition to the above components, the culture medium of the present invention may include all media commonly used for culturing neural stem cells. The media used for culturing generally include carbon sources, nitrogen sources, and trace element components, and are not limited thereto, but may include DMEM / F12, N2, B27, bFGF (basic fibroblast growth factor), and EGF (epidermal growth factor).

[0023] Neural stem cells directly differentiated using the above-described culture medium may have preventive or therapeutic effects on nervous system diseases. Therefore, the above-described neural stem cells may be included in a pharmaceutical composition for the prevention or treatment of nervous system diseases.

[0024] The above neurological disease may be one or more diseases selected from the group consisting of, but not limited to, Huntington's disease, Alzheimer's disease, stroke, epilepsy, multiple sclerosis, amyotrophic axial sclerosis (Lou Gehrig's disease), meningitis, cerebral palsy, encephalitis, and ischemic cerebrovascular disease.

[0025] In the present invention, the term “prevention” means any action that can suppress or delay the onset of the neurological disease by administering the pharmaceutical composition according to the present invention.

[0026] The term “treatment” as used herein refers to any action that improves or benefits the symptoms of the neurological disease by administering the pharmaceutical composition according to the present invention.

[0027] As used herein, the term "administration" refers to providing a given substance to a subject by any suitable method. The pharmaceutical composition of the present invention may be administered parenterally via any conventional route as long as it can reach the target tissue. Furthermore, the pharmaceutical composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells.

[0028] The pharmaceutical composition of the present invention may be a "cell therapy agent", and in this specification, the "cell therapy agent" refers to a medicine used for the purposes of treatment, diagnosis, and prevention by cells and tissues isolated from humans, cultured, and specially manufactured, and means a medicine used for the purposes of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological characteristics of cells by other methods to restore the function of cell tissues.

[0029] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, and may additionally include carriers or excipients necessary for the formulation. Pharmaceutically acceptable carriers, excipients, and diluents that may be additionally included in the active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, and mineral oil. When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0030] For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the extract or compound with at least one excipient, such as cotton, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.

[0031] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0032] The pharmaceutical composition of the present invention may be administered, but is not limited to, parenterally, particularly intravenously, or by direct transplantation or transport into a desired tissue site, thereby regenerating or functionally restoring a damaged nervous system. For example, the neural stem cells of the present invention are directly transplanted into the damaged nerve site. Transplantation can be performed using a single-cell suspension or aggregates of a certain cell density.

[0033] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to a reasonable amount applicable to medical treatment and an amount sufficient to treat a disease. The standard for this amount may be determined based on the patient's disease, severity, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concomitantly administered ingredients, and other factors. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with conventional therapeutic agents. The dosage may be determined at a level that minimizes side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0034] Specifically, the dosage of the pharmaceutical composition may vary depending on the patient's age, weight, severity, sex, etc., and for example, 1.0Х10^2 to 1.0×10^10 cells / kg body weight, preferably 1.0×10^3 to 1.0Х10^6 cells / kg body weight, may be administered once or in several divided doses. However, the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the administration route, the patient's weight, age, and sex, and the dosage may be set differently as necessary and is not limited by the dosage described above.

[0035] The present invention relates to a composition for preventing or treating neurological diseases such as Huntington's disease, stroke, Alzheimer's disease, epilepsy, and multiple sclerosis using neural stem cells (ciNSCs) derived from human fibroblasts, wherein the neural stem cells, when transplanted into an animal model that has actually induced a neurological disease, have a high therapeutic effect by alleviating the disease and various symptoms caused by the disease.

[0036] Figure 1 illustrates the process of direct transformation of neural stem cells using patient-derived skin cells in a Huntington's disease animal model using quinolinic acid, a toxic substance.

[0037] Figure 2 shows the results of observing the motor recovery effect when a neural stem cell treatment was injected into an animal model in which Huntington's disease was induced by a toxic substance.

[0038] Figure 3 shows the results of a dose test on an animal model in which Huntington's disease was induced by a toxic substance.

[0039] Figure 4 shows the results of confirming the exercise recovery effect on an animal model in which Huntington's disease was induced through transformation by inserting a mutant HTT gene.

[0040] Figure 5 shows the results of measuring the recovery of motor function and body weight change in an animal model in which Huntington's disease was induced through transformation by inserting a mutant HTT gene.

[0041] Figure 6 shows the results of brain tissue analysis of an animal model in which Huntington's disease was induced through transformation by inserting a mutant HTT gene, confirming changes in the amount of neurotransmitter secretion.

[0042] Figure 7 shows the results of a toxicity test of neural stem cells on an animal model in which Huntington's disease was induced through transformation by inserting a mutant HTT gene.

[0043] Figure 8 shows the recovery of motor ability due to administration of the neural stem cells of the present invention to the MCAO (Middle Cerebral Artery Occlusion) stroke model.

[0044] Figure 9 shows the effect of neural stem cell administration on endogenous nerve regeneration in the MCAO (Middle Cerebral Artery Occlusion) stroke model.

[0045] Figure 10 shows the results of an experiment to confirm whether the cerebral infarction area recovered when neural stem cells were administered to the MCAO (Middle Cerebral Artery Occlusion) stroke model.

[0046] Figure 11 shows the results of confirming the effects of neural stem cells administered to the MCAO (Middle Cerebral Artery Occlusion) stroke model on cell death and inflammatory response.

[0047] Figure 12 shows the results of confirming the expression of cytokines at the mRNA level to investigate changes in the inflammatory response within the tissue when neural stem cells are administered to the MCAO (Middle Cerebral Artery Occlusion) stroke model.

[0048] Figure 13 is an overall experimental design diagram for confirming the effect of dementia treatment when neural stem cells are administered to Alzheimer's disease model mice (5xFAD).

[0049] Figure 14 shows the results of immunohistochemistry analysis performed on neural stem cells administered to Alzheimer's disease model mice (5xFAD).

[0050] Figure 15 shows the results of comparing the number of beta-amyloid deposits, a dementia-causing substance, in the cerebral cortex when neural stem cells were administered to Alzheimer's disease model mice (5xFAD) with the control group.

[0051] Figure 16 shows the results of comparing the number of beta-amyloid deposits, a dementia-causing substance, in the hippocampus of the brain when neural stem cells were administered to Alzheimer's disease model mice (5xFAD) with the control group.

[0052] Figures 17 and 18 show the results of a Western blot experiment performed on BACE1 and amyloid precursor protein (APP), proteins extracted from the cerebral cortex and hippocampus of 5xFAD mice and normal mice (Wild Type: WT).

[0053] Figure 19 shows the results of confirming whether the normal distribution of astrocytes and microglial cells, which are increased by the toxicity of beta-amyloid in 5xFAD mice, is restored by the injection of neural stem cells.

[0054] Figures 20 and 21 show the results of a water maze experiment (Morris Water Maze) to determine whether spatial learning ability and cognitive ability are improved when neural stem cells are administered to 5xFAD mice.

[0055] Figure 22 shows the results of a Novel Object Recognition experiment conducted to determine whether concentration is improved when neural stem cells are administered to 5xFAD mice.

[0056] Figure 23 shows the results of a T-maze experiment to confirm the improvement in spatial cognitive ability when neural stem cells are administered to 5xFAD mice.

[0057] Figure 24 shows the results of an open field test to confirm spontaneous motility evaluation when neural stem cells are administered to 5xFAD mice.

[0058] Figure 25 shows the changes in cytokine and chemokine secretion due to neural stem cell administration in 5xFAD mice, as confirmed in terms of mRNA expression levels.

[0059] Figure 26 shows the results of an experiment to analyze the amount of inflammatory (immune) and neurotrophic factors in terms of RNA by neural stem cell administration in 5xFAD mice.

[0060] Figure 27 is a result comparing the aging and oligodendrocyte production by neural stem cell administration in 5xFAD mice in terms of RNA.

[0061] Figure 28 shows the results of RNA sequencing performed on hemispheres extracted from the neural stem cell administration group, HBSS administration group, and WT control group.

[0062] Figure 29 shows the results of DAVID analysis performed to predict major gene functions by statistically analyzing the correlation of genes based on various databases in 5xFAD mice, and confirms the effect of suppressing hyperphosphorylation reaction by neural stem cell administration.

[0063] Figure 30 shows the results of a heat map analysis of 13 genes related to nerve regeneration, comparing the neural stem cell administration group and the control group.

[0064] Figure 31 shows the results of DAVID analysis for analyzing the correlation of 13 genes related to nerve regeneration.

[0065] Figure 32 shows the results of confirming whether recovery of motor ability, reduction of inflammation in the central nervous system, and reduction of demyelination were achieved by administering neural stem cells to an EAE (experimental autoimmune encephalomyelitis) model.

[0066] Figure 33 shows the results of analyzing the changes in reactive astrocytes due to a decrease in immune response caused by neural stem cell administration in the AE mouse model.

[0067] Figure 34 shows the results of analyzing changes in microglia due to a decrease in immune response caused by neural stem cell administration in an EAE mouse model.

[0068] Figure 35 shows the results of measuring the intensity of immune response and the degree of oligodendrocyte loss in the spinal cord through various staining and mRNA expression analyses in an EAE mouse model.

[0069] Figure 36 shows the results confirming that fewer reactive astrocytes and microglia were observed in the brain of the neural stem cell administration group, and more myelin sheaths were preserved.

[0070] Figures 37 and 38 illustrate a schematic diagram and experimental process for analyzing whether epilepsy is improved through several animal behavioral experiments after inducing an epilepsy model and injecting iNSC cells. Figure 39 illustrates a product used to induce an epilepsy model.

[0071] Figure 40 shows the results of a T-maze experiment to evaluate spatial cognition when neural stem cells are injected into an epilepsy model.

[0072] Figure 41 shows the results of a Novel Objection Recognition experiment conducted to evaluate attention concentration when neural stem cells were injected into an epilepsy model.

[0073] Figures 42 and 43 show the results of confirming the regeneration effect of cells lost by paracrine effect when neural stem cells were transplanted in an epilepsy model.

[0074] Figure 44 shows the results of confirming that astrogliosis, which is an abnormal increase in the number of astrocytes due to cell loss caused by epilepsy, was reduced to a normal number through cell injection.

[0075] Figure 45 shows the results of comparing the number of microglia cells measured to show that cell loss due to epilepsy causes an increase in immune cells.

[0076] Figure 46 shows the results of RT-qPCR for analyzing changes in cytokine secretion after administering neural stem cells to an epilepsy model.

[0077] Hereinafter, examples will be described in detail to specifically explain this specification. However, the embodiments according to this specification may be modified in various different forms, and the scope of this specification is not limited to the embodiments described below. The embodiments of this specification are provided to more fully explain this specification to those of average skill in the art.

[0078]

[0079] <Experimental Example 1> Direct cross-differentiation of neural stem cells from fibroblasts

[0080] To induce human fibroblasts into neural stem cells, 1x10 human fetal fibroblasts 5 Dogs were prepared in 60 mm dishes, and eight small molecule compounds related to reprogramming (Thiazovivin, Valproic acid, Purmorphamine, A8301, SB431542, CHIR99021, 5-Aza-2′-deoxycytidine, and DZNep) and Sendai virus (OSKM, cytotune-ips 2.0 sendai reprogramming kit) were added to neurobasal medium consisting of DMEM / F12, N2 supplement, B27 supplement, bFGF, and EGF, and the prepared cells were cultured.

[0081] Differentiation was induced in the above medium for 19 days, and the culture medium was changed every 2-3 days thereafter to confirm the process of fibroblasts differentiating into neural stem cells, as shown in Fig. 2. As shown in Fig. 2, small neural stem cell-like cells were observed on the 13th day after culture, colonies of neural stem cells were confirmed on the 15th day, and colonies sufficient to secure a single colony of neural stem cells were confirmed on the 19th day.

[0082]

[0083] <Example 1> Effect of neural stem cell administration on Huntington's disease model

[0084] 1-1. Effect of neural stem cell administration in a quinolic acid-induced Huntington's disease model

[0085] A Huntington's disease animal model was created in mice. First, a toxic substance, quinolinic acid, was injected directly into the brain to induce neuronal cell death. Quinolinic acid activates NMDA receptors in neurons, inducing neuronal cell death through oxidation similar to that seen in Huntington's disease. This process is illustrated in Figure 1. After transplanting the neural stem cell treatment into the lesioned area at a density of 1x10^6 cells / head, the Rotarod, Balanced Beam, and Apomorphin-induced Rotation tests were performed. As a result, recovery of motor function was observed starting two weeks after cell transplantation (Figure 2).

[0086] In addition, a dose test was conducted to analyze the appropriate dosage. 1x10^3 cells, 1x10^4 cells, and 1x10^5 cells were directly administered into the brain for each group, and an apomorphine-induced rotation test, a rotarod test, and a grip strength test were performed, respectively. From about 6 weeks after cell transplantation, the group administered 1x10^4 cells or more showed the efficacy of motor function recovery by neural stem cell administration, and the group with the best motor function recovery efficacy was confirmed to be the 1x10^5 cell administration group (Fig. 3).

[0087]

[0088] 1-2. Effects of neural stem cell injection in a Huntington's disease model transformed with a mutant Huntington's gene.

[0089] A Huntington's disease animal model was created by transgenic mice with the mutant HTT gene that causes Huntington's disease (Trangenic mice R6 / 2 mice).

[0090] The neural stem cells of Experimental Example 1 were transplanted into the animal model described above, and the recovery of motor function was confirmed (Fig. 4). Using the model, the degree of body contraction and motor function abnormalities were confirmed through a clasping test, and the recovery of function was confirmed 4 weeks after neural stem cell transplantation. In addition, the recovery of motor function was confirmed through the rotarod test and grip strength test, and it was confirmed that the group transplanted with neural stem cells showed reduced weight loss and extended lifespan compared to the control group (Fig. 5).

[0091] In addition, brain tissues of the neural stem cell-administered and control groups of the Trangenic mice R6 / 2 were analyzed to investigate the expression rates of GDNF, BDNF, and GABA genes, which are neuroprotective substances or their transmitter genes. As a result, their expression was increased, and ELISA analysis confirmed that the amount of GABA secretion was increased. In addition, tissue staining was performed to confirm the survival of the transplanted neural stem cells and their normal differentiation into neural cells. (Fig. 6)

[0092] 1-3. Toxicity assessment

[0093] Toxicity, tumorigenicity, and distribution tests were conducted using immunocompromised scid mice after neural stem cell transplantation. As shown in Fig. 7, the results of the toxicity and tumorigenicity tests demonstrated that the administration of the neural stem cells of the present invention was safe. In addition, the distribution test demonstrated that the administered neural stem cells existed only within the brain, the site of administration, and did not migrate to other organs (Fig. 7).

[0094]

[0095] <Example 2> Effect of neural stem cell administration on a stroke model

[0096] 2-1. Effect of neural stem cell administration on motor ability recovery in a stroke model

[0097] Using the MCAO (Middle Cerebral Artery Occlusion) stroke model, neural stem cells from Experimental Example 1 were administered intravenously or intrathecally for 4 weeks, and the recovery of motor ability was observed. The mNSS test is an experiment that measures the neurological behavioral index of experimental animals and comprehensively evaluates and scores motor ability, sensory function, and balance ability. It was confirmed that the scores were improved by administration of the neural stem cells of the present invention. In addition, the results of the rotarod test also confirmed that motor ability was further improved by administration of neural stem cells. (Fig. 8)

[0098] 2-2. Endogenous nerve regeneration effect when neural stem cells are administered in a stroke model

[0099] In the MCAO (Middle Cerebral Artery Occlusion) stroke model, the intravenous and intrathecal administration of neural stem cells, as described in 2-1 above, was confirmed to have an endogenous nerve regeneration effect. For the model, BrdU, a marker for proliferating cells, and DCX, an immature neuron marker, were used to stain the medial ventricle of the brain, confirming that endogenous nerve regeneration was promoted in both the intravenous and intrathecal administration groups. (Figure 9)

[0100] 2-3. Effects on recovery and improvement of cell death in the cerebral infarction area in a stroke model

[0101] Mice were sacrificed 28 days after MCAO stroke modeling, and the damaged area of ​​the cerebral infarction was measured. Results showed that infarct volume was significantly reduced in both the intravenous and intrathecal groups when neural stem cells were transplanted compared to the control group. Quantitative area calculations confirmed a significant reduction in the size of the infarct area. These results demonstrate that the cerebral infarction lesion caused by ischemic stroke was restored by neural stem cell transplantation. (Figure 10)

[0102] We also observed changes in cell death in the lesion site following neural stem cell administration. The number of cells undergoing apoptosis was determined using TUNEL staining. As a result, both intravenous and intrathecal neural stem cell administration groups demonstrated a statistically significant decrease in TUNEL-positive cells. Therefore, we demonstrated that neural stem cell transplantation improves cell death in the rat brain, and such changes could have a significant impact on stroke recovery. (Figure 11)

[0103] To investigate changes in the inflammatory response within tissues, cytokine secretion was assessed at the mRNA level. Changes in mRNA levels of pro-inflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6, as well as anti-inflammatory cytokines such as TGF-beta, IL-4, IL-10, and IL-13 were observed in brain tissue. In the intravenous administration group, pro-inflammatory cytokines decreased and anti-inflammatory cytokines increased, suggesting a reduced inflammatory response. (Figure 12)

[0104]

[0105] <Example 3> Effect of neural stem cell administration on Alzheimer's disease model

[0106] 3-1. Effect of neural stem cell administration on the production of dementia-causing substances in an Alzheimer's disease model

[0107] The efficacy of neural stem cells transplanted into 5xFAD, a mouse model of Alzheimer's disease (AD), was investigated. 5xFAD mice begin to produce beta-amyloid, a causative agent of dementia, starting at 3 months of age. At 6 months, amyloid accumulates throughout the brain, leading to neuronal loss and cognitive decline. Therefore, neural stem cell transplantation was performed at this time. 1*10^6 cells were transplanted into each hippocampus of the mice. One month after transplantation, animal behavioral tests, including the Morris water maze, T-maze, open field test, and novel object recognition, were performed to determine whether behavioral and cognitive functions improved. Ten months later, the 5xFAD brains were removed to determine whether beta-amyloid, a causative agent of dementia, was reduced and whether immune suppression and neuroregeneration were achieved through neural stem cell transplantation. (Figure 13)

[0108] To confirm whether neural stem cells transplanted into the hippocampus of 5xFAD mouse brains had successfully engrafted, immunohistochemistry (IHC) experiments were performed. Using Hochest antibody, which stains mouse cell nuclei, and hNU antibody, which stains human neural stem cells, we confirmed that neural stem cells had engrafted into the hippocampus of 5xFAD brains, as shown in Figure 14.

[0109] In addition, an experiment was conducted to confirm the number of beta-amyloid deposits in the mouse brain. Figure 15 shows the results of an experiment comparing the number of beta-amyloid deposits, a dementia-causing substance, in the cerebral cortex of the mouse brain, and Figure 16 shows the results of an experiment comparing the group transplanted with neural stem cells (iNSCs) and the group transplanted with HBSS, in the hippocampus. IHC experiments were conducted using the Hochest antibody, which stains the nucleus of cells, and the 6E10 antibody, which stains beta-amyloid. As a result of analyzing the ratio of plaques formed by beta-amyloid clumping in the cerebral cortex, it was confirmed that the number of plaques was significantly reduced in the iNSC group compared to the HBSS group in both the cerebral cortex and hippocampus, and the number of plaques per area was also significantly reduced in the iNSC group.

[0110] In addition, proteins from the cerebral cortex and hippocampus were extracted and Western blotting was performed. The protein amount was analyzed between the stem cell administration group, HBSS administration group, and control group using BACE1 (Beta-site Amyloid precursor protein Cleaving Enzyme1) antibody that blocks the production of beta-amyloid protein known to be the cause of Alzheimer's disease. As a result, there was no significant difference in the amount of BACE1 in the cerebral cortex of the neural stem cell administration group, but in the hippocampus, it was confirmed that there was a significant difference in the amount of BACE1 between the stem cell administration group and the HBSS administration group. (Figure 17) In addition, the expression levels of Amyloid Precursor Protein (APP), which is the parent protein of beta-amyloid, C-terminal fragment β (CTF β), which is the terminal fragment of APP cleaved by β enzyme, which is the cause of beta-amyloid formation, and C-terminal fragment α (CTF α), which is the terminal fragment of APP cleaved by α enzyme, which is generally produced, were confirmed. As a result, there was no significant change in the total amount of APP, but it was confirmed that CTF β, which is the terminal fragment of beta-amyloid formation, was significantly reduced in the neural stem cell administration group compared to the HBSS administration group. (Figure 18)

[0111] Additionally, we conducted IHC experiments to determine whether the increased astrocyte and microglia populations due to beta-amyloid toxicity were normalized by the injected neural stem cells. The distribution patterns in the cortex and hippocampus of 5xFAD mice and HBSS-treated mice were examined, and a significant decrease in the astrocyte and microglia distribution was observed in the neural stem cell-treated group compared to the HBSS-treated group. (Figure 19)

[0112]

[0113] 3-2. Behavioral Analysis and Cognitive Effects of Neural Stem Cell Administration in Alzheimer's Disease Models

[0114] To determine the effects of neural stem cell administration on behavioral analysis and cognitive function in 5xFAD mice, we conducted the Morris Water Maze experiment, which assesses spatial learning and cognitive function among animal behavior analysis experiments. One month after neural stem cell transplantation, we analyzed how quickly 5xFAD and control mice arrived at the platform within 1 minute for 5 days. The results showed that the neural stem cell-treated group showed improved cognitive function compared to the HBSS-treated group. Furthermore, three months after transplantation, the neural stem cell-treated group showed improved cognitive function compared to the HBSS-treated group and returned to normal as much as the control group. (Fig. 20)

[0115] Additionally, on the sixth and final day of the experiment, the platform was removed and the mice were tested for one minute to determine which zone they remained in the longest. If cognitive function improved, they would remain in Zone 3, where the platform was located, for a longer period. Analysis results showed that, at one and three months after transplantation, the neural stem cell-treated group remained in Zone 3 longer than the HBSS-treated group. Morris Water Maze results showed that the neural stem cell-treated group showed better cognitive function in Zone 3 than the HBSS-treated group, and that the results were similar to those of the WT group. (Figure 21)

[0116] Additionally, a Novel Object Recognition experiment was conducted to assess attentional capacity. The experiment lasted three days. On the first day, habituation was conducted for five minutes. On the second day, the subjects explored identical objects for ten minutes. On the final day, only one of the two identical objects was switched with a different object and the subjects were allowed to explore for ten minutes. The duration of exploration of the different object was recorded. The total time spent exploring all objects was similar across all experimental groups. However, the discrimination ratio values ​​in both the neural stem cell-treated and control groups were positive and higher than zero. A positive discrimination ratio value indicates that the subjects explored the different object longer, while a negative value indicates that they failed to recognize the different object and instead explored the same object longer. The results of the experiment confirmed that the group that received neural stem cells exhibited improved attentional capacity.

[0117] Additionally, a T-maze experiment was conducted to assess spatial cognition. Food was placed on either side of the maze, and the mice were allowed to explore either side for 60 seconds. When placed back at the starting point, the mice were observed to determine which direction they would explore. Mice have a natural instinct to explore new areas, so they will try to navigate in the opposite direction from where they first explored. However, Alzheimer's disease mice, due to memory loss, are more likely to return to the same area. The results confirmed that the rate of exploring new areas was significantly higher in the neural stem cell-treated group than in the HBSS-treated group, demonstrating improved spatial cognition in the neural stem cell-treated group (Figure 23).

[0118] We also conducted an Open Field Test experiment to assess spontaneous mobility. Dementia mice showed less movement and inactivity, while increased mobility was observed in both the neural stem cell-treated and control groups. The distance traveled and the distance traveled from the edge to the center were significantly increased in the neural stem cell-treated group compared to the HBSS-treated group. (Figure 24)

[0119]

[0120] 3-3. Effects of Neural Stem Cell Administration on Cytokine Secretion and Neurotrophic Factor Expression in Alzheimer's Disease Models

[0121] To analyze the amount of cytokines and chemokines in terms of RNA, RT-qPCR experiments were conducted. In the hippocampus transplanted with neural stem cells, we observed a significant decrease in IL1β, CCL2, and CXCL10 gene expression, while in the cerebral cortex, a significant decrease in CCL2 gene expression was observed. (Figure 25)

[0122] Additionally, RT-qPCR experiments were conducted to analyze the amount of immune (inflammatory) factors and neurotrophic factors from the RNA perspective. Immune-related genes such as IBA-1 and GFAP were significantly expressed in both the cerebral cortex and hippocampus. Furthermore, neurotrophic factor genes secreted by neural stem cells, such as GAP43, TUJ1, and MAP2, were found to increase in the hippocampus transplanted with neural stem cells compared to the HBSS-treated group. (Figure 26)

[0123] We also conducted RT-qPCR experiments to analyze anti-aging and oligodendrogenesis levels from an RNA perspective. COL1A1 significantly decreased in the cortex and hippocampus, while expression levels of oligodendrocyte-related genes (FGF17, OLIG2, PDGFRa, NG2) significantly increased in the hippocampus. (Figure 27)

[0124] 3-4. RNA sequencing of Alzheimer's disease models

[0125] RNA-sequencing experiments were performed on hemispheres extracted from the neural stem cell-administered group, HBSS-administered group, and control group of the Alzheimer's disease model. A total of 23,183 genes were analyzed, and to identify significant genes, the following conditions were applied: Fold change = 1.5, Normalized Data = 2.0, and P-value = 0.5. As a result, 737 genes were identified, but no significant differences in expression levels were found between the neural stem cell-administered group and the HBSS-administered group.

[0126] From the 737 genes, we selected genes whose expression was identical between the neural stem cell-administered and control groups, but opposite in the HBSS-administered group. We derived 119 genes and created a heatmap for these genes (Figure 28).

[0127] For the above genes, we conducted a DAVID analysis based on various databases to statistically analyze gene correlations and predict key functions of the 119 genes. The following gene functions were identified, with a P value of 0.05 or less. The neural stem cell-injected groups showed increased cell activity and transcription (RNA splicing, mRNA processing) activity compared to the HBSS-treated group, and hyperphosphorylation, a cause of dementia, was suppressed. (Figure 29)

[0128] Additionally, the results of creating a heat map of 13 genes related to neurogeneration show that the gene expression levels between the neural stem cell administration group and the control group are similar (Figure 30).

[0129] To analyze the correlation between the 13 genes associated with neuroregeneration, DAVID was used. The results showed that genes related to nervous system development and neurodevelopment were most frequently associated. Cell injection demonstrated a memory-enhancing effect by regenerating damaged neurons (Figure 31).

[0130] <Example 4> Effect of neural stem cell administration on EAE (multiple sclerosis) model

[0131] We created an EAE (experimental autoimmune encephalomyelitis, multiple sclerosis) model and confirmed recovery of motor ability, reduction of inflammation in the central nervous system, and reduction of demyelination following 8 weeks of neural stem cell administration.

[0132] EAE clinical scoring quantifies the severity of the disease by assigning scores to each stage of symptoms based on the gradual progression of motor loss and paralysis in EAE model animals. The group administered neural stem cells demonstrated improvements in motor function, with the effects becoming most pronounced at week 4. (Figure 32)

[0133] Furthermore, to confirm changes in reactive astrocytes due to reduced immune response following neural stem cell administration in an EAE mouse model, the spinal cord of the EAE mouse model was stained with GFAP, a marker of reactive astrocytes. As a result, fewer reactive astrocytes were observed in the neural stem cell-administered group, and GFAP mRNA expression in the spinal cord was also reduced (Figure 33).

[0134] Additionally, we conducted experiments to analyze changes in microglia. Microglia play a role as immune cells in the central nervous system and, when an immune response is induced, are characterized by proliferation and thick swelling. We stained the spinal cord of an EAE mouse model with the microglia marker Iba1. Fewer microglia were observed in the neural stem cell-administered group, and a decrease in Iba1 mRNA expression in the spinal cord was also observed. (Figure 34)

[0135] In an EAE mouse model, we conducted experiments to determine the intensity of immunoreactivity and the extent of oligodendrocyte loss within the spinal cord using various staining and mRNA expression analyses. H&E staining revealed a lower level of immunoreactivity in the neural stem cell-treated group, while Luxol fast blue (LFB) staining confirmed that myelin sheaths were more preserved in the neural stem cell-treated group. (Figure 35)

[0136] In addition, Fig. 36 shows the results showing that fewer reactive astrocytes and microglia were observed in the brain of the neural stem cell-administered group, and more myelin sheaths were preserved. When the EAE mouse model brain was analyzed using MBP, a myelin sheath marker, and Iba1, a microglia marker, it was confirmed that a lower number of microglia were observed in the neural stem cell-administered group, and more myelin sheaths were present.

[0137] <Example 5> Effect of neural stem cell administration on epilepsy model

[0138] 5-1. Creation of an epilepsy mouse model and experimental design

[0139] Figure 37 is the overall experimental plan for inducing an epilepsy model, injecting neural stem cells, and analyzing whether epilepsy is improved through animal behavioral experiments.

[0140] Epilepsy models were induced via intraperitoneal injection of pilocarpine, and epilepsy scores were assessed using the Racine scale. Cell transplantation was performed at a dose of 1*10^6 cells into the hippocampus. Animal behavior was assessed using the T-maze and novel object recognition tests to assess cognitive function. Subsequent tissue analysis was performed to determine whether epilepsy improved. (Figure 38)

[0141] Figure 39 shows the products used to induce an epilepsy model. N-methylscopolamine was injected 30 minutes before pilocarpine to minimize peripheral side effects, and pilocarpine was injected to initiate epilepsy induction. After epilepsy induction, behavioral analysis was performed to determine the epilepsy score based on the Racine scale.

[0142]

[0143] 5-2. Improvement effect of neural induction cell administration in a mouse model of epilepsy

[0144] The neural stem cells of the present invention were administered to a mouse model of epilepsy, and a T-maze experiment was conducted to assess spatial cognition. As a result, compared to the control group, the epilepsy-induced group showed a loss of short-term memory and cognitive function. However, the group transplanted with neural stem cells showed an improvement in short-term memory, comparable to the control group. (Figure 40)

[0145] We also conducted a Novel Object Recognition experiment to assess concentration. In the epilepsy-induced model, the ability to explore novel objects was significantly reduced compared to the control group. However, in the group transplanted with neural stem cells, the ability to explore novel objects was significantly improved compared to the control group due to cell transplantation. (Figure 41)

[0146] Figures 42 to 45 show the results of confirming the regenerative effect of cells lost due to epilepsy. In a mouse model of epilepsy, we examined whether the paracrine effect of transplanting neural stem cells regenerated lost cells. As a result, we confirmed using the TUJ1 antibody that, compared to the group administered neural stem cells, the cells differentiated into immature early neurons. This was also confirmed through quantitative analysis. (Figure 42)

[0147] In addition, we conducted an experiment to confirm that cell loss caused by epilepsy was regenerated by neural stem cell transplantation (Fig. 43). When comparing the CA3 and DG regions with the control group, excluding the CA1 region of the hippocampus, we confirmed that there was no difference in the number of cells. In addition, we conducted an experiment to determine whether astrogliosis, which is an abnormal increase in the number of astrocytes caused by cell loss due to epilepsy, would be reduced to a normal number by cell injection. We confirmed that the number of astrocytes increased when epilepsy was induced, but significantly decreased in the group administered neural stem cells. (Fig. 44) In addition, we measured the number of microglia to confirm whether immune cells increased due to epilepsy. When comparing the epilepsy group with the control group, there was a large difference in microglia, but the difference was not significant in the group injected with neural stem cells.

[0148] Additionally, RT-qPCR experiments were conducted to analyze the amount of cytokine secretion from the RNA perspective during epilepsy induction. Analysis of mRNA changes in pro-inflammatory IL-1β revealed a decrease in the iNSC group compared to the HBSS group, while an increase in anti-inflammatory IL-6 was observed.

[0149]

[0150] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than restrictive. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0151] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating a neurological disease, comprising induced neural stem cells (iNSCs) directly converted from somatic cells using a direct cross-differentiation inducing composition comprising a small molecule compound selected from the group consisting of Thiazovin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2′-deoxycytidine, and DZNep; or one or more of Sendai virus, OCT4 and / or SOX4 expressing mRNA, and miRNA 302 / 367.

[0152] In one embodiment, the neurological disease is at least one disease selected from the group consisting of Huntington's disease, Alzheimer's disease, stroke, epilepsy, multiple sclerosis, amyotrophic axial sclerosis (Lou Gehrig's disease), meningitis, meningitis, cerebral palsy, encephalitis, and ischemic cerebrovascular disease, and more preferably, Huntington's disease, Alzheimer's disease, stroke, epilepsy, or multiple sclerosis.

[0153] In another embodiment, the somatic cells may be selected from fibroblasts, fibroblast-derived neural stem cells, or neural progenitor cells.

[0154] In another embodiment, the pharmaceutical composition may be a cell therapy agent, and the pharmaceutical composition may be administered intravenously or by direct transplantation or transport to a desired tissue site.

[0155] In another embodiment, the pharmaceutical composition may be administered once a day or in several divided doses in an amount of 1.0Х10^2 to 1.0×10^10 cells / kg.

[0156] In another aspect, the present invention relates to a method for preventing or treating a nervous system disease, comprising the steps of treating a somatic cell with a direct cross-differentiation-inducing composition comprising a small molecule compound selected from the group consisting of Thiazovin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2′-deoxycytidine, and DZNep and Sendai virus, OCT4 and / or SOX4 expression mRNA, and miRNA 302 / 367, thereby directly converting induced neural stem cells (iNSCs); and administering the directly converted induced neural stem cells (iNSCs) to a subject.

[0157] In another aspect, the present invention relates to the use of induced neural stem cells (iNSCs) directly converted by treating somatic cells with a direct cross-differentiation inducing composition comprising a small molecule compound selected from the group consisting of Thiazovin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2′-deoxycytidine and DZNep and Sendai virus, OCT4 and / or SOX4 expressing mRNA and miRNA 302 / 367, for the prevention or treatment of neurological diseases.

Claims

1. A small molecule compound selected from the group consisting of Thiazovivin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2′-deoxycytidine and DZNep; or A pharmaceutical composition for preventing or treating a neurological disease, comprising induced neural stem cells (iNSCs) directly converted from somatic cells using a direct cross-differentiation inducing composition comprising Sendai virus, OCT4 and / or SOX4 expressing mRNA and miRNA 302 / 367.

2. In paragraph 1, A composition for preventing or treating a nervous system disease, wherein the nervous system disease is at least one disease selected from the group consisting of Huntington's disease, Alzheimer's disease, stroke, epilepsy, multiple sclerosis, amyotrophic axial sclerosis (Lou Gehrig's disease), meningitis, cerebral palsy, encephalitis, and ischemic cerebrovascular disease.

3. In paragraph 2, A composition for preventing or treating a nervous system disease, wherein the nervous system disease is selected from Huntington's disease, Alzheimer's disease, stroke, epilepsy or multiple sclerosis.

4. In paragraph 1, A composition characterized in that the somatic cells are selected from fibroblasts, fibroblast-derived neural stem cells, or neural progenitor cells.

5. In paragraph 1, A composition characterized in that the above pharmaceutical composition is a cell therapy agent.

6. In paragraph 1, A composition characterized in that the pharmaceutical composition is administered intravenously or by direct transplantation or transport to a desired tissue site.

7. In paragraph 1, A composition characterized in that the above pharmaceutical composition is administered once a day or in several divided doses in an amount of 1.0Х10^2 to 1.0×10^10 cells / kg.

8. A step of directly converting induced neural stem cells (iNSCs) by treating a composition for inducing direct cross-differentiation comprising a small molecule compound selected from the group consisting of Thiazovivin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2′-deoxycytidine, and DZNep and Sendai virus, OCT4 and / or SOX4 expressing mRNA and miRNA 302 / 367 to somatic cells; A method for preventing or treating a nervous system disease by administering the directly converted induced neural stem cells (iNSCs) to a subject.

9. Use of induced neural stem cells (iNSCs) directly converted by treating somatic cells with a composition for inducing direct cross-differentiation comprising a small molecule compound selected from the group consisting of Thiazovivin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2′-deoxycytidine, and DZNep and Sendai virus, OCT4 and / or SOX4 expressing mRNA and miRNA 302 / 367 for the prevention or treatment of neurological diseases.

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