Method for converting human somatic cells into proliferable neural stem cells

Abstract

The present invention relates to a method for converting human fibroblasts into neural stem cells, and more particularly, to a method for converting human fibroblasts into neural stem cells by direct transdifferentiation using sendai virus, mRNA of stem cell-related factors, or a combination of miRNA and a small molecule compound, and to a use thereof. According to the present invention, high-quality neural stem cells can be induced in a short period of time from human fibroblasts by direct transdifferentiation, so that a sufficient amount of cells can be obtained for cell therapy without side effects of tumor formation, and thus the neural stem cells can be used as a cell therapeutic agent for brain diseases.

Description

Technical Field

[0001] The present invention relates to a method for converting human fibroblasts into neural stem cells, and more particularly, to a method for converting human fibroblasts into neural stem cells by direct transdifferentiation using a combination of Sendai virus or mRNA or miRNA and a small molecule compound, and its use. Background Art

[0002] Since the study of inducing human fibroblasts into induced pluripotent stem cells in 2007, the reprogramming of cells has been studied. In the past, human embryonic stem cell-derived neural stem cells used in stem cell research had ethical issues that may arise from the use of human embryos, immune rejection reactions, and tumor formation when undifferentiated embryonic stem cells are transplanted. Adult stem cells have problems such as difficulty in obtaining cells and limited differentiation ability. However, although induced pluripotent stem cells avoid ethical issues and have no immune rejection problems, there is a problem of teratoma formation when undifferentiated stem cells are transplanted. In addition, the recently published directly transdifferentiated neural stem cells have similar properties to induced pluripotent stem cells and embryonic stem cell-derived neural stem cells, but the virus system, which is the main method for forming neural stem cells, may form mutations due to random insertion of genes. When transplanted into the human body, plasmids, proteins, RNA, etc. are used to solve the problems caused by viruses, but when they are used, the differentiation efficiency of differentiation into neural stem cells is low and cancer genes are used, so new problems that have not been confirmed may arise.

[0003] As a solution to the problem of induced pluripotent stem cells, research on direct conversion of human fibroblasts into desired cells using direct transdifferentiation methods has been reported. Among them, with the purpose of treating intractable brain diseases, research on direct differentiation of fibroblasts into neurons has been actively carried out, and by introducing various combinations of neuron-related transcription factors into human fibroblasts, research on the formation of neurons has been successful. This shows that it can be used as a cell therapy for intractable brain diseases, but since neurons are already differentiated cells, it is difficult to obtain a sufficient amount of cells for cell therapy.

[0004] Due to the problems described above, methods for directly differentiating fibroblasts into neural stem cells are being studied recently. For example, methods for inducing neural stem cells from fibroblasts by introducing various transcription factors using a viral system are included. In the inventor's previous invention "Method for directly converting human fibroblasts into neural stem cells using small molecule compounds (PCT / KR2016 / 003819)", a method for inducing the process of directly differentiating fibroblasts into neural stem cells (direct transdifferentiation) using 8 small molecule compounds is proposed. For the direct transdifferentiation described above, it is further necessary to study a method that can proliferate a sufficient amount of neural stem cells for use as a therapeutic agent by further shortening the differentiation cycle and improving efficiency. Summary of the invention

[0005] Technical issues

[0006] Therefore, in the process of studying a method for preparing neural stem cells with shortened direct transdifferentiation cycle, high efficiency and high quality, the inventors completed the present invention by utilizing Sendai virus without inserted exogenous genes, one or more of mRNA or miRNA; and small molecule compounds to induce neural stem cells that can proliferate sufficient amounts of cells required for transplantation, have genetic stability (genomic DNA stability) and do not form tumors.

[0007] Therefore, an object of the present invention is to provide a direct transdifferentiation inducing composition for inducing fibroblasts to directly transform into neural stem cells, comprising one or more small molecule compounds selected from the group consisting of Thiazovivin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2'-deoxycytidine and DZNep, and one or more of Sendai virus, mRNA or miRNA.

[0008] Another object of the present invention is to provide a method for preparing neural stem cells, comprising the step of culturing somatic cells in a culture medium containing one or more small molecule compounds selected from the group consisting of Thiazovivin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2'-deoxycytidine and DZNep and one or more of Sendai virus, mRNA or miRNA.

[0009] Another object of the present invention is to provide a cell therapy agent for treating brain diseases, which comprises the neural stem cells prepared by the above preparation method.

[0010] Technical Solution

[0011] In order to achieve the above-mentioned purpose, the present invention provides a direct transdifferentiation inducing composition for inducing fibroblasts to directly transform into neural stem cells, which comprises one or more small molecule compounds selected from the group consisting of Thiazovivin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2'-deoxycytidine and DZNep, and one or more of Sendai virus, mRNA or miRNA.

[0012] In order to achieve another object of the present invention, the present invention provides a method for preparing neural stem cells, comprising the step of culturing human fibroblasts in a culture medium containing one or more small molecule compounds selected from the group consisting of Thiazovivin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2'-deoxycytidine and DZNep and one or more of Sendai virus, mRNA or miRNA.

[0013] In order to achieve another object of the present invention, the present invention provides a cell therapeutic agent for treating brain diseases, which comprises the neural stem cells prepared by the above preparation method.

[0014] Hereinafter, the present invention will be described in detail.

[0015] The present invention is a method for preparing neural stem cells from fibroblasts by using Sendai virus, mRNA or miRNA and an optimal combination of small molecule compounds to induce neural stem cells from human fibroblasts.

[0016] In order to differentiate fibroblasts into neural stem cells without an additional gene introduction process, the present invention selects a direct transdifferentiation method, for which a culture solution in combination with a small molecule compound for differentiation is used, and the differentiation cycle can be shortened and the efficiency can be improved by further using Sendai virus, mRNA or miRNA to improve the transdifferentiation efficiency. As described in the present invention, various problems that occur when using embryonic stem cells can be overcome by transdifferentiation using small molecules and Sendai virus, mRNA or miRNA, and the side effects (tumor formation, etc.) of cell therapeutic agents using induced pluripotent stem cells can be reduced, so that it can be used as a cell therapeutic agent with improved safety.

[0017] In the present invention, "neural stem cells" are undifferentiated cells that have the ability to self-replicate and have the multipotency 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, through the stage of producing neural progenitor cells or glial progenitor cells of specific nervous system cells. The above-mentioned neural stem cells may be characterized by differentiation into one or more of the group consisting of astrocytes, oligodendrocytes, neurons, dopaminergic neurons, γ-aminobutyric acid neurons, motor neurons and cholinergic neurons, but are not limited thereto.

[0018] "Direct transdifferentiation" is one of the cell reprogramming technologies, which refers to the technology of directly reprogramming mature differentiated cells to restore them to stem cells. The present invention relates to a technology that can directly differentiate fibroblasts, which are human somatic cells, into neural stem cells by direct transdifferentiation. In particular, since it directly differentiates into neural cells without undergoing the process of dedifferentiation into induced pluripotent stem cells, it can overcome many shortcomings that occur during the differentiation process of induced pluripotent stem cells and improve cell differentiation efficiency.

[0019] As one embodiment of the present invention, the present invention relates to a method for preparing neural stem cells, so as to reprogram human fibroblasts and differentiate them into neural stem cells by direct transdifferentiation, the method comprising the step of culturing human fibroblasts in a culture medium containing one or more small molecule compounds of Thiazovivin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2'-deoxycytidine and DZNep and one or more of Sendai virus, mRNA or miRNA.

[0020] In the present invention, it is known that "Thiazovivin" blocks the Rho / ROCK signal that induces apoptosis of neural cells and neural stem cells and blocks the PTEN signal that inhibits the proliferation of neural stem cells, so it is expected to inhibit apoptosis of neural stem cells and increase self-regeneration and self-proliferation capabilities (Matthias Groszer, et al., Science 294: 2186, 2001). The above-mentioned Thiazovivin is a ROCK inhibitor (inhibitor of Rho-associated kinase (ROCK)), which is a substance that selectively inhibits Rho-associated kinase (ROCK). In addition to Thiazovivin, Y-27632 and the like can be used.

[0021] In the present invention, "2-propyl valeric acid (valproic acid, VPA)" is a histone deacetylase inhibitor, which refers to a substance that inhibits histone deacetylase, and is known to promote the expression of cell proliferation inhibitors and genes necessary for differentiation by placing chromatin in a high acetylation state, thereby inducing cell (cancer cell) differentiation and inhibiting angiogenesis, and fixing the cell cycle in the G1 state and inducing apoptosis of cancer cells, thereby showing a strong cell proliferation inhibition (cytostatic) anticancer activity. It is well known that histone deacetylase (HDAC) inhibits gene transcription through pRB / E2F, and the destruction of histone acetylation is related to the occurrence of various cancers, and HDAC is highly expressed under harsh environmental conditions such as hypoxia, low sugar, and cell carcinogenesis to inhibit the expression of cell proliferation inhibitors, thereby promoting cell proliferation, so HDAC is considered to be an important regulatory factor for cell carcinogenesis and differentiation regulation. In addition to VPA, trichostatin (TSA) or a derivative thereof can also be used as a histone deacetylase.

[0022] In the present invention, "purmorphamine" is a purine compound, which is known to be a substance involved in the Shh signaling system. The above-mentioned purmorphamine is not particularly limited as long as it can induce Shh signals, and various derivatives can be used. For example, commercially available 2-(1-naphthoxy)-6-(4-morpholinoanilino)-9-cyclohexylpurin) and the like can be used. The above-mentioned purmorphamine can be treated in a culture medium that is generally used to induce dedifferentiation into neural stem cell-like cells. As such, when purmorphamine, which is a Shh analog, is used for treatment, the advantage is that there is no need to introduce genes for preparing neural stem cells from human fibroblasts.

[0023] In the present invention, "A-8301" is a TGF-β type I receptor inhibitor, which refers to a substance that interferes with the normal signal transduction process of TGF-β type I by interacting with the TGF-β type I receptor (TojoM et al., Cancer Sci. 96: 791-800, 2005). It is well known that TGF-β type I (transforming growth factor-β type I) is a multifunctional peptide that plays various roles in cell proliferation, differentiation and various types of cells. This multifunctionality plays a core role in the growth and differentiation of various tissues such as adipocyte formation, muscle cell formation, osteoblast formation, epithelial cell differentiation, and inhibits the proliferation of neural stem cells. In addition to the above-mentioned TGF-β type I receptor inhibitor A-8301, all TGF-β type I receptor inhibitors including SB432542 can also be used. The above-mentioned low molecular weight substance TGF-β type I receptor inhibitor A-8301 can be commercially available or prepared, and the proliferation of neural stem cells can be promoted by treating with the above-mentioned inhibitor.

[0024] In the present invention, "SB431542" is an activin receptor-like kinase-5 (ALK5) inhibitor, which plays a role in improving chromosome stability by inducing rapid dedifferentiation.

[0025] In the present invention, "CHIR99021" is a glycogen synthase kinase (GSK) inhibitor, which is a substance targeting GSK1 / 2, which is an upstream molecule of GSK1 / 2 involved in the GSK signal transduction process. The above-mentioned CHIR99021 is represented by aminopyrimidine, and all GSK inhibitors can be used except CHIR99021.

[0026] In the present invention, "5-Aza-2'-deoxycytidine" is a cytidine analog called decitabine, which acts as a DNA demethylating agent. DNA demethylating agents participate in the DNA replication process and regulate gene expression by removing methyl groups (-CH3) present on DNA. 5-azacytidine can be used as a cytidine analog such as decitabine.

[0027] In the present invention, "3-Deazaneplanocin A (DZNep)" is an inhibitor of S-adenosyl homocysteine ​​hydrolase and histone methyltransferase EZH2, which induces apoptosis by activating suppressed tumor suppressor genes and inhibiting the expression of EZH2, and blocks the EZH2 activity and trimethylation of lysine 27 of histone H3 in vitro. It plays a role in inducing apoptosis of cancer cells, inhibiting S-adenosyl homocysteine ​​(SAH) hydrolase, reducing overall DNA methylation, and enhancing Oct 4 expression in chemically induced pluripotent stem cells (CiPSCs).

[0028] In the present invention, the above-mentioned small molecules can be included in the culture medium at an effective concentration, and the effective concentration can be adjusted and included according to factors known in the art such as the type of culture medium and the culture method, so as to differentiate fibroblasts into neural stem cells.

[0029] The Sendai virus contained in the culture medium for direct transdifferentiation of the present invention is used to reprogram fibroblasts as somatic cells, and because it contains Yamanaka factors, it has the characteristics of being able to be induced into cells with pluripotency. In one embodiment of the present invention, a CytoTune-ips 2.0 Sendai reprogramming kit is used, which contains Sendai virus, including Oct4, Sox2, Klf4, and C-myc. In the present invention, reprogramming for transdifferentiation into neural stem cells is performed by treating human fibroblasts with the above-mentioned CytoTune-ips 2.0 Sendai reprogramming kit.

[0030] Furthermore, in the present invention, the culture medium for differentiation of neural stem cells contains mRNA and a small molecule compound for expression of OCT4 and / or SOX4, and thus it is confirmed that the transdifferentiation efficiency is higher even compared to the case of inducing direct transdifferentiation. It is well known that OCT4 is a transcription factor required for maintaining the pluripotency of stem cells, and SOX2 is a representative marker of neural stem cells and one of the factors required for converting somatic cells into induced pluripotent stem cells. In one embodiment of the present invention, human fetal fibroblasts are transfected with OCT4 mRNA and SOX2 mRNA to regulate gene expression, and then an experiment is conducted to compare the results of inducing direct transdifferentiation into neural stem cells using an existing iNSC culture medium with the results of inducing direct transdifferentiation in the culture medium of the present invention.

[0031] Furthermore, direct transdifferentiation can be induced by miRNA302 / 367, which inhibits the expression of NR2F2 gene, which inhibits OCT4, a gene that maintains the pluripotency of stem cells. miRNA302 / 367 is an important cluster that can induce the expression of OCT4 and induce direct transdifferentiation into stem cells by inhibiting NR2F2. Therefore, in one embodiment of the present invention, human Huntington skin fibroblasts were transfected with the miRNA302 / 307 cluster to regulate genes, and then the differentiation efficiency of inducing direct transdifferentiation into neural stem cells was compared with that of inducing direct transdifferentiation in the culture medium of the present invention.

[0032] Furthermore, in the present invention, the culture medium includes all culture mediums commonly used for culturing neural stem cells. The culture medium used for culturing generally contains a carbon source, a nitrogen source and trace element components. The above culture medium may include DMEM / F12, N2, B27, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), but is not limited thereto.

[0033] The medium used to culture the induced stem cells of the present invention can use a basal medium known in the art without limitation. The basal medium can be prepared by artificial synthesis, or a commercially available medium can be used. Examples of commercially available culture media include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, α-Minimalessential Medium (α-MEM), Glasgow's Minimal Essential Medium (G-MEM), and Isocove's Modified Dulbecco's Medium, etc., but are not limited thereto.

[0034] In the present invention, the culture period of the fibroblasts used for direct transdifferentiation is preferably 10 to 20 days, and it can be confirmed that the fibroblasts differentiate into neural stem cells within 15 days. In one embodiment of the present invention, neural stem cells began to be observed after 13 days of culture, and it can be confirmed that neural stem cell colonies were formed between 15 and 19 days. In addition, the expression of Sox1, Sox2, MSH1, and Nestin, which are neural stem cell markers, was confirmed by immunostaining to differentiate into neural stem cells.

[0035] In the present invention, the neural stem cells are characterized in that they can maintain chromosome stability and can maintain an undifferentiated state for more than 10 passages, but are not limited thereto.

[0036] As another embodiment of the present invention, the present invention relates to a cell therapeutic agent comprising neural stem cells cultured by the above method.

[0037] The above-mentioned cell therapy agents are cells and tissues prepared by separation, cultivation and recombination from the human body. They are drugs used for treatment, diagnosis and prevention purposes. They refer to cells that are used for the purpose of diagnosis, prevention or treatment of diseases through genetic modification or change of biological characteristics.

[0038] The cell therapy agent of the present invention may be a pharmaceutical composition for preventing or treating brain diseases.

[0039] In the present specification, the term "prevention" refers to all actions that can inhibit or delay the occurrence of vascular calcification by administering the above-mentioned pharmaceutical composition of the present invention.

[0040] In the present specification, the term "treatment" refers to all actions that can improve or beneficially change the symptoms by administering the above-mentioned pharmaceutical composition of the present invention.

[0041] The above-mentioned pharmaceutical composition of the present invention can be prepared into the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, and can further include carriers or excipients required for the above-mentioned preparations. Pharmaceutically acceptable carriers, excipients and diluents that can be further included in the above-mentioned active ingredients include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate and mineral oil, etc. When preparing, it is prepared by using diluents or excipients such as commonly used fillers, extenders, adhesives, wetting agents, disintegrants, surfactants, etc.

[0042] For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., which are prepared by mixing one or more excipients such as flour, starch, calcium carbonate, sucrose or lactose, gelatin, etc. in the above-mentioned extracts or compounds. In addition, lubricants such as magnesium stearate and talcum powder can also be used. Liquid preparations for oral administration include suspensions, internal liquids, emulsions, syrups, etc., and in addition to simple diluents such as commonly used water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. can also be included.

[0043] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used as non-aqueous solvents and suspensions. Semi-synthetic fatty acid esters (witepsol), polyethylene glycol, Tween 61, cocoa butter, lauric acid glyceride, glyceryl gelatin, etc. can be used as bases for suppositories.

[0044] The pharmaceutical composition of the present invention can be administered orally or parenterally (intravenous injection, subcutaneous injection, intraperitoneal administration or topical administration) according to the desired method, and the dosage varies according to the patient's condition and weight, the severity of the disease, the drug form, the route of administration and the time, and the appropriate form can be selected by a person of ordinary skill in the art. In particular, since the pharmaceutical composition of the present invention contains a cell therapy agent, it can be administered by any device that can move the cell therapy agent to the target cells.

[0045] The above-mentioned pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease in a reasonable amount applicable to medical treatment, and its standard may depend on the patient's disease, severity, drug activity, sensitivity to the drug, administration time, route of administration, excretion rate, treatment cycle, ingredients used in combination, and other matters. The above-mentioned pharmaceutical composition of the present invention may be administered as a single therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with existing therapeutic agents. Taking all of the above factors into account, the dosage may be determined at a level that can minimize side effects, and this may be determined by a person of ordinary skill in the art at an easy level.

[0046] Specifically, the dosage of the above-mentioned pharmaceutical composition can be varied according to the patient's age, weight, severity, gender, etc. For example, 1.0×10 4 to 1.0×10 10cells / kg body weight, preferably 1.0×10 5 to 1.0×10 9 The dosage of the active ingredient may be administered once or in multiple doses at the amount of 10 cells / kg body weight. However, the actual dosage of the active ingredient should be determined by taking into account various relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and gender, and the above dosage can be set differently as needed, but is not limited to the above-specified dosage.

[0047] Effects of the Invention

[0048] The present invention relates to a method for converting human fibroblasts into neural stem cells, and more specifically, to a method for converting human fibroblasts into neural stem cells by direct transdifferentiation using a combination of Sendai virus, mRNA or miRNA and a small molecule compound, and its use. When human fibroblasts are directly converted into neural stem cells using a combination of Sendai virus, mRNA or miRNA and a small molecule compound, high-quality neural stem cells can be induced due to the absence of gene insertion, and a sufficient amount of cells can be obtained for cell therapy. Since there is no side effect of tumors, it has an ideal effect as a cell therapy agent for brain diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 A schematic diagram of an experiment in which human fetal fibroblasts were made to express Oct4, Sox2, Klf4, C-myc (or L-myc) genes without additional insertions using cytotune-ips 2.0 Sendai virus and differentiated into neural stem cells using small molecule compounds.

[0050] Figure 2 The results show the results of observing the process of inducing differentiation of human fibroblasts into neural stem cells when the cells were cultured for 19 days containing Sendai virus (SeV) and 8 small molecule compounds (8SMs).

[0051] Figure 3 The results of experiments on various combinations of culture solutions containing small molecule compounds and Sendai virus for inducing direct transdifferentiation of human fetal fibroblasts into neural stem cells are shown.

[0052] Figure 4 The qRT-PCR results of stem cell markers of neural stem cells directly transdifferentiated from human fetal fibroblasts are shown.

[0053] Figure 5 The figure shows an immunofluorescence staining image of a neural stem cell marker for confirming neural stem cells directly transdifferentiated from human fetal fibroblasts.

[0054] Figure 6 The figures show the results of subculture to confirm the genetic stability of the induced neural stem cells and confirmation of chromosome stability at the second and tenth cultures.

[0055] Figure 7 The figure shows the culture results (colonies) of neural stem cells confirmed to have been directly transdifferentiated from adult human fibroblasts.

[0056] Figure 8 The results of immunofluorescence staining of stem cell markers for confirming that neural stem cells were directly transdifferentiated from adult human fibroblasts are induced.

[0057] Fig. 9 The qRT-PCR results of neural stem cell markers of neural stem cells directly transdifferentiated from adult human fibroblasts are shown.

[0058] Fig.10 This figure shows the results of confirming the culture of neural stem cells (colonies) that were directly transdifferentiated from human fibroblasts of Huntington's disease patients.

[0059] Fig.11 The results of immunofluorescence staining of stem cell markers for confirming that neural stem cells were directly transdifferentiated from human fibroblasts of Huntington's disease patients.

[0060] Fig.12 The qRT-PCR results of neural stem cell markers of neural stem cells directly transdifferentiated from human fibroblasts from Huntington's disease patients are shown.

[0061] Fig.13 The graphs compare the neural stem cell conversion efficiencies confirmed in human fibroblasts (fetal fibroblasts, adult fibroblasts, and human fibroblasts from Huntington's disease patients).

[0062] FIG. 14 shows the results of differentiation into GABAergic neural cells to confirm the differentiation ability of the neural stem cells induced in the present invention.

[0063] FIG. 15 is a diagram showing the results of immunostaining of differentiated GABAergic neurons, in which expression of Tuj1, a neuron initial marker, and GABA, a GABAergic neuron marker, was confirmed.

[0064] Fig.16 The graph shows the results of inducing direct transdifferentiation into neural stem cells by injecting 375 ng of OCT4 mRNA and 125 ng of SOX2 mRNA.

[0065] Fig.17This is a diagram showing the confirmation of whether stem cell genes are expressed in neural stem cells induced to differentiate by transduction with OCT4 and SOX2 mRNA.

[0066] Fig.18 The diagram shows the culture process and results of confirming the differentiation-induced neural stem cells by transfection with miRNA302 / 367.

[0067] Fig.19 This is a diagram showing whether stem cell genes are expressed in neural stem cells induced to differentiate by transfection with miRNA302 / 367. DETAILED DESCRIPTION

[0068] As one embodiment of the present invention, the present invention relates to a direct transdifferentiation induction composition for inducing fibroblasts to directly transform into neural stem cells, which comprises one or more small molecule compounds selected from the group consisting of Thiazovivin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2'-deoxycytidine and DZNep, and Sendai virus.

[0069] As another embodiment of the present invention, the present invention relates to a method for preparing neural stem cells, comprising the step of culturing human fibroblasts in a culture medium containing one or more small molecule compounds selected from the group consisting of Thiazovivin, Valproic acid, Purmorphamine, A8301, SB43154, CHIR99021, 5-Aza-2'-deoxycytidine and DZNep, and Sendai virus.

[0070] As another embodiment of the present invention, the present invention relates to a cell therapeutic agent, which comprises neural stem cells prepared by a method for preparing neural stem cells, wherein the method for preparing the neural stem cells comprises the step of culturing human fibroblasts in the above-mentioned direct transdifferentiation induction composition; or in a culture medium containing the above-mentioned composition.

[0071] As another embodiment of the present invention, the present invention relates to a pharmaceutical composition for treating brain diseases selected from the group consisting of cerebral apoplexy, cerebral hemorrhage, cerebral infarction, Alzheimer's disease, dementia, Huntington's disease, Parkinson's disease, multiple sclerosis, multiple system neuropathy, epilepsy, Pick's disease and Creutzfeldt-Jakob disease, which comprises the above-mentioned cell therapy agent.

[0072] Implementation

[0073] Hereinafter, the present invention will be described in detail by way of examples to specifically illustrate the present invention. However, the embodiments according to the present invention may be modified into various other forms, and the scope of the present invention is not to be construed as being limited to the embodiments described in detail below. The embodiments of the present invention are provided to more fully illustrate the present invention to those of ordinary skill in the art.

[0074] Example 1. Inducing direct transdifferentiation of human fetal fibroblasts into neural stem cells

[0075] To induce human fibroblasts to transform into neural stem cells, 1×10 5 Human fetal fibroblasts, and then 8 small molecule compounds related to reprogramming (Thiazovivin, Valproic acid, Purmorphamine, A8301, SB431542, CHIR99021, 5-Aza-2'-deoxycytidine and DZNep) and Sendai virus (OSKM, cytotune-ips2.0 Sendai reprogramming kit) are added to the neurobasal medium composed of DMEM / F12, N2 supplement, B27 supplement, bFGF and EGF and the prepared cells are cultured. The properties of each of the above 8 small molecule compounds are as described in the existing patent (PCT / KR2016 / 003819).

[0076] Differentiation was induced in the above medium for 19 days, with the medium replaced every 2 to 3 days. Figure 2 The process of differentiation of fibroblasts into neural stem cells was confirmed as shown. Figure 2 As shown, cells with small neural stem cell shapes were observed on day 13 after culture, neural stem cell colonies were confirmed on day 15, and sufficient colonies were confirmed on day 19 to obtain single neural stem cell colonies.

[0077] Previous studies have shown that when Sendai virus is not added, it takes about 60 days for fibroblasts to directly transdifferentiate into neural stem cells, but this time is significantly shortened when Sendai virus is co-treated.

[0078] Example 2. Comparison of direct transdifferentiation efficiency of small molecule compounds and Sendai virus

[0079] Experiments were conducted using the combinations shown in Table 1 below to compare the direct transdifferentiation efficiency of the culture solution composition comprising Sendai virus and small molecule compounds with the previous research results using only small molecule compounds according to the present invention.

[0080] Table 1

[0081]

[0082] The results of inducing direct transdifferentiation of human fetal fibroblasts for 15 days under the culture medium containing the small molecule compound of the above 7 conditions are as follows: Figure 3 As shown, neural stem cell-shaped colonies were not observed under other conditions, but under the culture medium conditions shown in 7), neural stem cell-shaped cells began to appear from the 13th day, and small neural stem cell colonies could be confirmed on the 15th day.

[0083] Example 3. Confirmation of marker expression of directly transdifferentiated neural stem cells

[0084] The expression of neural stem cell markers was confirmed by qRT-PCR to confirm whether human neural stem cells induced by Sendai virus and 8 small molecule compounds showed the characteristics of general neural stem cells.

[0085] First, human fetal fibroblasts were treated with Sendai virus and then cultured in a neurobasal medium consisting of DMEM F12, N2 supplement, B27 supplement, bFGF and EGF for 19 days to induce neural stem cells (SeV iNSC only). After human fibroblasts were treated with Sendai virus, 8 small molecule compounds, including thiazovivin, valproic acid, and valproic acid, were added to the neurobasal medium consisting of DMEM F12, N2 supplement, B27 supplement, bFGF and EGF. acid), Purmorphamine, A8301, SB431542, CHIR99021, 5-Aza-2'-deoxycytidine and DZNep and cultured for 19 days to induce neural stem cells (SeV+8SMs iNSC).

[0086] Furthermore, without the process of treating with Sendai virus, human fibroblasts were cultured for 60 days in a culture medium composed of DMEM F12, N2 supplement, B27 supplement, bFGF and EGF, to which 8 small molecule compounds Thiazovivin, Valproic acid, Purmorphamine, A8301, SB431542, CHIR99021, 5-Aza-2'-deoxycytidine and DZNep were added to induce neural stem cells (8SMs iNSC only (8SMsiNSC only)).

[0087] For each neural stem cell induced by the above method (SeV iNSC only / SeV+8SMs iNSC / 8SMsiNSC only), the expression levels of Sox2, Nestin, and MSH1, which are neural stem cell markers, were confirmed by qRT-PCR. Figure 4 As shown, it can be confirmed that the neural stem cell markers in the induced neural stem cells increased by about 200 to 600 times compared with the neonatal fibroblasts as the control group. In addition, compared with SeV iNSC alone, SeV+8SMs iNSC and 8SMs iNSC alone showed more than 3 times higher expression levels of Sox2 and MSH1. However, with regard to the time of inducing differentiation, it was confirmed that SeV+8SMs iNSC could obtain a large number of neural stem cells after 19 days (Example 1), which, as described in the previous invention (PCT / KR2016 / 003819), was about 40 days faster than when only 8 small molecule compounds were used.

[0088] Furthermore, as described above, the results of immunostaining (immunocytochemistry (ICC)) of neural stem cell markers induced by human fetal fibroblasts using Sox1, Sox2, MSH1, and nestin as neural stem cell markers showed that neural stem cell markers were confirmed in all the screened neural stem cells, and it was confirmed that the differentiated cells were neural stem cells. Figure 5 ).

[0089] Example 4. Confirmation of chromosome stability by subculture

[0090] Subculture was performed to increase the stem cell properties of neural stem cells induced from human fetal fibroblasts. In order to confirm genetic stability, a total of 10 subcultures were performed, and the chromosome stability of neural stem cells in the second and tenth subcultures was confirmed. Figure 6 As shown, it was confirmed that all of them had normal chromosome stability.

[0091] Example 5. Induction of neural stem cells using adult fibroblasts

[0092] In the above Examples 1 to 4, fetal fibroblasts were used to induce neural stem cells. However, in order to confirm whether adult fibroblasts can also induce neural stem cells, adult fibroblasts were used to induce neural stem cells in the same manner as in the above Examples 1 to 4 and their characteristics were confirmed.

[0093] Results, such as Figure 7 As shown, as a result of inducing differentiation for 19 days and observing in the same manner as in Example 1, the shape of neural stem cells was observed from the 13th day, and colonies of neural stem cells were confirmed on the 19th day.

[0094] Furthermore, the results of immunostaining of the induced neural stem cells showed the expression of Sox1, Sox2, MSH1 and nestin ( Figure 8 ) and the results of qRT-PCR observation showed that the expression levels of Sox2, nestin, and MSH1 were higher than those of adult fibroblasts ( Fig. 9 ), thus confirming that neural stem cells can be successfully induced using adult fibroblasts.

[0095] Example 6. Induction of neural stem cells using fibroblasts from Huntington's disease patients

[0096] In order to confirm whether neural stem cells can be induced by direct transdifferentiation using fibroblasts from actual brain disease patients, neural stem cells were induced by the same method as in Examples 1 to 4 above and their characteristics were confirmed.

[0097] Results, such as Fig.10 As shown, as a result of inducing differentiation for 19 days and observing in the same manner as in Example 1, the shape of neural stem cells was observed from the 13th day, and the colony of neural stem cells was confirmed on the 19th day.

[0098] Furthermore, the results of immunostaining of the induced neural stem cells showed that Sox1, Sox2, MSH1 and nestin ( Fig.11 ) and the results of qRT-PCR observation showed that the expression levels of Sox2, nestin, and MSH1 were higher than those of adult fibroblasts ( Fig.12 ), thus confirming that neural stem cells can be successfully induced using adult fibroblasts.

[0099] As a result, it was confirmed that neural stem cells were successfully induced from all human fetal fibroblasts, adult fibroblasts, and fibroblasts of Huntington's disease patients by using the culture medium containing Sendai virus and 8 small molecule compounds of the present invention, and the results of comparing the conversion efficiency of each fibroblast were as follows: Fig.13 As shown, human fetal fibroblasts showed a transformation efficiency of about 1.2%, and fibroblasts from adults and Huntington's disease patients showed a transformation efficiency of about 0.4%.

[0100] Example 7. Confirmation of differentiation ability of induced neural stem cells

[0101] In order to confirm the differentiation ability of the neural stem cells induced by the above examples, they were induced to differentiate into GABAergic neurons in vitro. GABAergic neurons secrete GABA in the brain. GABA (γ-aminobutyric acid, GABA) is a non-protein amino acid distributed in nature and is known to be a neurotransmitter present in the brain or spinal cord of mammals.

[0102] On the first day (-Day 1), 5×10 4Cells / well were seeded in a 24-well plate coated with poly-L-ornithine / fibronectin (PLO / FN), and the media was a 1:1 mixture of DMEM F12 and Neurobasal media, supplemented with 0.5X N2 supplement, 0.5X B27 supplement, 1X MEM-NEAA, 100ug / ml penicillin and streptomycin, 10uM valpronic acid, 0.65uM purmorphamine, 2uM SB431542, 0.1uM retinoic acid, and 100ug / ml penicillin and streptomycin. The cells were cultured in a medium containing 1uM CHIR99021, 200nM dosormorphin, 100nM LDN193189, and 200uM ascorbic acid for 0 to 7 days, and then cultured in a medium containing a 1:1 mixture of DMEM F12 and Neurobasal media supplemented with 0.5X N2 supplement, 0.5X B27 supplement, 1X MEM-NEAA, 100ug / ml penicillin and streptomycin, and 200uM ascorbic acid until day 21 of differentiation, and differentiation into neuron-shaped neural cells (GABA neurons) was confirmed (Figure 14).

[0103] Furthermore, the results of cell immunostaining using the differentiated GABAergic neurons confirmed the expression of Tuji, an initial marker of neurons, and GABA, a marker of GABAergic neurons (Figure 15). That is, it was confirmed that the neural stem cells produced by direct transdifferentiation induced by the culture medium containing Sendai virus and 8 small molecule compounds of the present invention can successfully differentiate into neurons.

[0104] Example 8. Induction of neural stem cells by gene transfer using nRNA

[0105] When human fetal fibroblasts are used to induce direct transdifferentiation into neural stem cells, in order to confirm the influence of OCT4, a transcription factor used to maintain the pluripotency of stem cells, and SOX2, a factor required for converting somatic cells into induced pluripotent cells, human fetal fibroblasts are directly transfected with OCT4 mRNA and SOX2 mRNA, which are stem cell genes, to introduce the genes, and then the iNSC media of the present invention is used to induce direct transdifferentiation into neural stem cells.

[0106] Human fetal fibroblasts were cultured at 5×10 4 Cells were seeded in 24-well plates and cultured overnight in DMEM + 10% FBS medium. The next day, Lipofectamine messengerMAX reagent and OptiMEM medium (25ul) that met each condition were vortexed and incubated at room temperature for 10 minutes (mixture 1). The required amount of OCT4 mRNA and SOX2 mRNA (mixture 2) was diluted in Opti-MEM medium (50ul), and mixture 1 and mixture 2 were mixed and incubated at room temperature for 5 minutes. Then, each 24-well plate was placed for transfection for 6 hours or 24 hours. Then, it was replaced with iNSC medium and direct transdifferentiation was induced.

[0107] DMEM F12, N2 supplement, B27 supplement, Thiazovivin, Valproic acid, Purmorphamine, A8301, SB431542 and CHIR99021 were added to the above iNSCs and cultured for further 14 days. Fig.16 As shown, the cells decreased in size until day 3 after transfection and then began to elongate, but did not change in shape to neural stem cells even after day 14.

[0108] For human fetal fibroblasts induced to differentiate under the above-mentioned SOX2+OCT+8SMs conditions, strongly expressed RNA was confirmed by qRT-PCR on day 7 and day 14, and the expression levels were compared.

[0109] Human fetal fibroblasts (NEOhDF D0) were used as a control group, and it was confirmed that the expression level of SOX2 increased more than 100 times under the conditions of NEOhDF D7 SOX2+OCT4 and NEOhDF D14 SOX2+OCT4. Fig.17 ).

[0110] Example 9. Inducing neural stem cells using miRNA

[0111] The experiment of inducing direct transdifferentiation into neural stem cells was conducted by inhibiting the NR2F2 gene, which inhibits the OCT4 gene, through the expression of miRNA302 / 367. Human Huntington Dermal Fibroblasts were transfected with miRNA302 / 307 cluster to regulate the gene, and then induced direct transdifferentiation into neural stem cells using existing iNSC culture medium and confirmed the induction efficiency. Human Huntington Dermal Fibroblasts were cultured at 2×10 5 The cells were seeded in a 6-well plate, and 5ul of Lipofectamin RNAiMAX and 5nM of miRNA302 / 307 were added, and then incubated at room temperature for 5 minutes. Then, it was mixed with Opti-MEM medium (1ml) and transfected for 5 hours. Then, it was replaced with DMEM+ medium (media) and cultured for 1 day (T1), 2 days (T2), and 3 days (T3), and replaced with a medium containing 8SMs and cultured for 16 days to observe whether differentiation was induced. The results of observing the cells after subculture on the 7th day showed that the cell shape was small and became round, but the cell amount decreased and did not proliferate. ( Fig.18 ).

[0112] In addition, in order to confirm whether the neural stem cell genes of the cultured cells are expressed, RT-PCR experiments were performed using OCT4, SOX1, SOX2, nestin, and Musashi-1 on the 7th and 14th days of expression. As a result, it was confirmed that the expression of stem cell-related markers was the highest on the third day compared with the control group (contorl). It was confirmed that the miRNA302 / 367 cluster contributes to direct transdifferentiation into stem cells ( Fig.19 ).

[0113] So far, the present invention has been studied with the preferred embodiments of the present invention as the center. It can be understood by those skilled in the art that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a restrictive point of view. The scope of the present invention should be interpreted as indicated in the claims rather than the above description, and all differences within the scope equivalent thereto are included in the present invention.

Claims

1. A direct transdifferentiation induction composition for inducing fibroblasts to directly transform into neural stem cells, characterized in that: The invention comprises one or more small molecule compounds selected from the group consisting of Thiazovivin, valproic acid, purmorphamine, A8301, SB43154, CHIR99021, 5-aza-2'-deoxycytidine and DZNep, and Sendai virus.

2. The composition according to claim 1, characterized in that The above composition further comprises a culture medium for cell culture.

3. The composition according to claim 1, characterized in that The above-mentioned Sendai virus contains the Yamanaka factor.

4. The composition according to claim 1, characterized in that The above-mentioned fibroblasts are derived from humans.

5. The composition according to claim 1, characterized in that The neural stem cells differentiate into one or more selected from the group consisting of astrocytes, oligodendrocytes, neurons, dopaminergic neurons, γ-aminobutyric acid neurons, motor neurons, and cholinergic neurons.

6. A method for preparing neural stem cells, characterized in that: The method comprises the step of culturing human fibroblasts in a culture medium containing one or more small molecule compounds selected from the group consisting of Thiazovivin, valproic acid, purmorphamine, A8301, SB43154, CHIR99021, 5-aza-2'-deoxycytidine and DZNep and Sendai virus.

7. The method for preparing neural stem cells according to claim 6, characterized in that: The above-mentioned Sendai virus contains the Yamanaka factor.

8. The method for preparing neural stem cells according to claim 6, characterized in that: The culture medium is DMEM / F12 containing N2, B27, basic fibroblast growth factor and epidermal growth factor.

9. The method for preparing neural stem cells according to claim 6, characterized in that: The above-mentioned culture is carried out for 10 to 20 days.

10. The method for preparing neural stem cells according to claim 6, characterized in that: The neural stem cells differentiate into one or more selected from the group consisting of astrocytes, oligodendrocytes, neurons, dopaminergic neurons, γ-aminobutyric acid neurons, motor neurons, and cholinergic neurons.

11. A cell therapy agent, characterized in that: Comprising the culture solution composition according to any one of claims 1 to 5; or neural stem cells prepared by the preparation method according to any one of claims 6 to 10.

12. A pharmaceutical composition for treating brain diseases, characterized in that: Comprising the cell therapeutic agent according to claim 11.

13. The pharmaceutical composition according to claim 12, characterized in that The above brain disease is selected from the group consisting of cerebral apoplexy, stroke, cerebral hemorrhage, cerebral infarction, Alzheimer's disease, dementia, Huntington's disease, Parkinson's disease, multiple sclerosis, multiple system neuropathy, epilepsy, Pick's disease and Creutzfeldt-Jakob disease.

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