WO2016167528A1 - Method for directly converting human fibroblasts into neural stem cells using small molecule compounds - Google Patents

Abstract

The present invention relates to a method for converting human fibroblasts into neural stem cells and, more specifically, to a method for directly converting human fibroblasts into neural stem cells using only a combination of small molecule compounds without an introduction of foreign genes; and a use thereof. The method according to the present invention for directly converting human fibroblasts into neural stem cells using only small molecule materials without gene introduction is useful for utilization in a cellular therapeutic agent of brain diseases, as it is possible to secure a sufficient amount of cells to be used in cell therapy through the induction of genetically stable neural stem cells, and to differentiate into various types of functional nerve cells, and tumors are not caused.

Description

Direct conversion of human fibroblasts into neural stem cells using small molecule compounds

The present invention relates to a method for converting human fibroblasts into neural stem cells, and more particularly, to a method for directly converting from human fibroblasts to neural stem cells using a combination of small molecule compounds without introducing an external gene and its use. .

Interest in cell reprogramming began to grow in 2007 when studies led to human fibroblasts induced into pluripotent stem cells. Human embryonic stem cells used in previous stem cell research have ethical problems due to the use of human embryos, immune rejection problems, and teratoma formation when undifferentiated embryonic stem cells are transplanted. In the case of stem cells, it is difficult to secure cells and has a problem in that they have differentiation ability. However, de-differentiated stem cells are avoided from ethical problems and do not have an immune rejection problem. However, when undifferentiated stem cells are transplanted, they may cause teratoma formation. Retrodifferentiated stem cells have similar properties to embryonic stem cells, but viral systems, a method commonly used to form pluripotent stem cells, can mutate by random insertion of genes. Plasmids, proteins, RNA, etc. are used to solve the problem of the virus, but new problems may occur due to low efficiency and use of oncogenes.

In order to solve the problem of the pluripotent stem cells, a study has been reported to directly convert human fibroblasts into desired cells using a direct cross-differentiation method. Among them, the direct differentiation research into fibroblasts using fibroblasts for the treatment of intractable brain disease has been actively conducted, introducing various combinations of neuronal cell-related transcription factors into human fibroblasts, Formed research was successful. This has been shown to be used as a cell therapy for intractable brain disease, but since the neurons are already differentiated cells, there is a difficulty in securing a sufficient amount of cells for use in cell therapy.

Due to these problems, methods for directly differentiating into neural stem cells using fibroblasts have recently been studied, and various transcription factors using a viral system are introduced to induce neural stem cells from fibroblasts. Only branched transcription factors were used to reach neural stem cells. Neural stem cells can self-renew and thus get as many cells as they want and can differentiate into neurons. In addition, neural stem cells derived from fibroblasts of each individual by direct cross-differentiation method are very useful cells for the treatment of refractory brain disease because there is no ethical problem, immune rejection and tumor formation after transplantation.

Cell therapy is a method of implanting and treating experimentally induced healthy cells to replace damaged cells and tissues in the human body. In the case of patients with a disease caused by genetic factors, skin cells can be used to directly convert to desired cells, and genetic manipulations can be used to replace normal genes before transplanting. However, direct cross-differentiation methods that have been studied previously are difficult to use as a practical cell therapy because they introduce a foreign gene.

Accordingly, the present inventors have made efforts to induce fibroblasts into neural stem cells using only small molecule compounds without introducing external genes, and thus, they are capable of proliferating in sufficient amounts for transplantation and are genetically stable neural stem cells without tumor formation. It was confirmed that the preparation can be completed the present invention.

Summary of the Invention

Disclosure of Invention An object of the present invention is to provide a method for producing neural stem cells by culturing human fibroblasts in a medium containing a small molecule compound, and a cell therapeutic agent for treating brain diseases containing the prepared neural stem cells as an active ingredient.

In order to achieve the above object, the present invention comprises the steps of culturing human fibroblasts in a medium comprising thiazovivin, Valproic acid, Purmorphamine, A8301, SB431542 and CHIR99021 It provides a method for producing a neural stem cell comprising.

The present invention also provides a cell therapy for the treatment of brain diseases containing neural stem cells produced by the above method.

1 shows the results of confirming the morphological changes of the cells according to the addition of 13 small molecule materials.

Figure 2 shows the results of confirming the gene expression according to the addition of 13 small molecule materials.

Figure 3 is a result confirming the morphological changes of the cells according to the use of four, six, eight small molecule compounds.

4 is a result of confirming the morphological confirmation of the induced neural stem cells, the expression of marker genes and proteins.

5 is a result of confirming the expression of marker genes and proteins in the early and late stages of induced neural stem cells.

6 is a result of confirming the chromosome through the chromosome analysis of the induced neural stem cells.

Figure 7 shows the results of analyzing the cell proliferation rate of the induced neural stem cells.

FIG. 8 shows the results of confirming epigenetic genetic changes of induced neural stem cells using bisulfite PCR analysis.

9 is a result confirming the differentiation of induced neural stem cells into trigeminal nervous system cells.

10 is a result of confirming the differentiation of the induced neural stem cells into various types of neurons.

Figure 11 shows the results of analyzing the brain of the mouse transplanted induced neural stem cells.

Detailed description of the invention and preferred Embodiment

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, the small molecule material was used to construct an optimal combination of small molecule materials for inducing human fibroblasts into neural stem cells, and the function of each of the various small molecule materials was confirmed. This induces human neural stem cells with a culture medium consisting of a combination of small molecule materials, secures genetic stability without chromosomal abnormalities, and establishes optimized culture conditions for prolonged proliferation and maintenance of induced neural stem cells. Thereafter, the basic characteristics of human neural stem cells and the differentiation ability of trigeminal nervous system cells and various kinds of neurons were confirmed, and induced human neural stem cells were confirmed to differentiate into trigeminal neural cells without tumor formation after transplantation. In addition, as some small molecule substances regulate the expression of endoderm and mesoderm-related genes, the inducibility of endoderm and mesoderm-related cells was confirmed.

Accordingly, the present invention includes the step of culturing human fibroblasts in a medium comprising thiazovivin, Valproic acid, Purmorphamine, A8301, SB431542 and CHIR99021. It relates to a method for producing neural stem cells.

In the present invention, "Thiazovivin (N-benzyl-2- (pyrimidin-4-ylamino) thiazole-4-carboxamide)" prevents Rho / ROCK signal inducing apoptosis of neurons and neural stem cells and neural stem cells. It is known to block PTEN signaling, which inhibits the proliferation of cells, and is expected to inhibit neural stem cell apoptosis, increase self-renewal and self-proliferation (Matthias Groszer, et al., Science 294: 2186, 2001). . The thiazovivin is a substance that selectively inhibits ROCK (Rho-associated kinase) as an inhibitor of Rho-associated kinase (ROCK), and Y-27632 may be used in addition to thiazovivin. . Treating the thiazobibin in the medium to be included in an effective concentration, the effective concentration may be affected depending on factors well known in the art, such as the type and culture method of the medium.

In the present invention, "VPA (valproic acid, 2-propylpentanoic acid)" refers to a substance that inhibits histone deacetylase as a histone deacetylase inhibitor, and a high acetylation state of chromatin. Promote expression of cell proliferation inhibitors and genes essential for differentiation, induce differentiation of cells (cancer cells), inhibit angiogenesis, and fix cell cycle to G1 status to apoptosis of cancer cells It is known to exhibit strong cytostatic anti-cancer activity by inducing apoptosis. Histone deacetylase (HDAC) inhibits gene transcription via pRB / E2F, and the destruction of histone acetylation is associated with a number of cancer developments, and HDAC is associated with hypoxia, mortgage ( Iii) HDAC is known to be recognized as an important regulator of cell carcinogenesis and differentiation by inhibiting the expression of cell proliferation factor and expressing it under severe environmental conditions such as cell carcinogenesis. In particular, the VPA is known to cause inositol reduction, inhibit GSK-3β, activate the ERK pathway, and stimulate PPAR activity.

In addition to the histone deacetylase inhibitor (HDAC inhibitor) VPA (valproic acid, 2-propylpentanoic acid), trachostatin (TSA) or a derivative thereof may be used, and the derivative may be variously pharmaceutically acceptable. Inorganic or organic salts. If the treatment concentration is too low, it will be difficult to see the effect. If the concentration is too high, it will be toxic, so it is necessary to check the appropriate concentration according to the cell type.

In the present invention, "purmorphamine" is a purine compound, and is known to be involved in the Shh signaling system. The permopamine is not particularly limited as long as it can induce the Shh signal, and various derivatives may be used. For example, 2- (1-Naphthoxy) -6- (4-morpholinoanilino) -9-cyclohexylpurin) can be purchased commercially. The permophamine may be treated with a medium commonly used to induce differentiation into neural stem cell-like cells. This treatment of Shh analogs, permophamine, there is an advantage that does not need to introduce a gene to produce neural stem cells from human fibroblasts. The permophamine is included in the medium at an effective concentration. Effective concentrations of permopamine may be affected by factors well known in the art, such as media type and culture method.

In the present invention, "A-8301" is a TGF-β type I receptor inhibitor that binds to the TGF-β type I receptor and interferes with the normal signaling process of TGF-β type I. (Tojo M et al., Cancer Sci. 96: 791-800, 2005). TGF-β type I (Transforming growth factor-β type I) is a multifunctional peptide that has various functions on cell proliferation, differentiation and various cell types. It is known to play a pivotal role in the growth and differentiation of various tissues such as cell differentiation and to inhibit the proliferation of neural stem cells. In addition to the TGF-β type I receptor inhibitor A-8301, any TGF-β type I receptor inhibitor including SB432542 may be used, and the low molecular weight TGF-β type I receptor inhibitor A-8301 may be purchased or used on the market. Can be used to promote neural stem cell proliferation by the inhibitor treatment. The TGF-β type I receptor inhibitor A-8301 is treated in the medium to be included in an effective concentration. Effective concentrations may be affected by factors well known in the art, such as media type and culture method.

In the present invention, "SB431542" serves to improve chromosome stability by inducing rapid reverse differentiation with an ALK5 (Activin Receptor-Like Kinase-5) inhibitor.

In the present invention, "CHIR99021" is a GSK (glycogen synthase kinase) inhibitor and is a substance targeting GSK1 / 2, an upstream molecule of GSK1 / 2 involved in GSK signaling. The CHIR99021 is represented as aminopyrimidine, and all GSK inhibitors may be used in addition to CHIR99021.

In the present invention, it is preferable to further include DZNep (Deazaneplanocin A) or 5-AZA, but is not limited thereto. In addition, it is preferable to further include at least one small molecule compound selected from the group consisting of PD0325901, Ascorbic acid, PS48, Forskolin and Tranylcypromine, but It is not limited.

In the present invention, "5-Azacitidine (5-AZA)" means 4-amino-1-β-D-ribofuranosyl-1,3,5-triazine-2 (1H) -one It is named (4-amino-1-β-D-ribofuranosyl-1,3,5-triazin-2 (1H) -one) and is a compound known to have a DNA demethylation action. 5-azacytidine is also known as an anti-neoplastic drug that is active against leukemia, lymphoma and various solid tumors. The 5-azacytidine can be obtained by synthesizing by a general chemical synthesis method known in the art, or can be purchased commercially available (eg Sigma-Aldrich (St Louis, Mo, USA)).

In the present invention, PD0325901 is one of the inhibitors of MEK / ERK signaling pathway, and ascorbic acid is one of the water-soluble vitamins and has strong antioxidant properties.

In the present invention, phospholine acts to directly activate the catalytic subunit of dadenylate cyclase to increase the concentration of intracellular cAMP, and tranylcipromine is monoamine oxidation, an enzyme that normally degrades norepinephrine at the nerve endings. It acts to inhibit enzyme (MAO).

In the present invention, the culture medium includes all media commonly used for culturing neural stem cells. The medium used for culturing generally contains a carbon source, a nitrogen source and a trace element component. Although not limited thereto, the medium is preferably composed of DMEM / F12, N2, B27, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF).

The medium for inducing stem cell culture of the present invention can be used without limitation a basal medium known in the art. The basal medium may be prepared by artificially synthesizing, or a commercially prepared medium may be used. Examples of commercially prepared media include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, α-MEM (α-Minimal). essential medium), G-MEM (Glasgow's Minimal Essential Medium), and Isocove's Modified Dulbecco's Medium, but are not limited thereto. In a specific embodiment of the present invention was cultured in DMEM medium.

In the present invention, the culture period is preferably 10 to 15 days, but is not limited thereto.

In the present invention, the step of subcultured cells and suspended culture to form a sphere; And it is preferable to further include a step of the suspension culture after the attached spear formed culture, but is not limited thereto. Preferably, the suspension culture and the adhesion culture are cultured for 7 to 10 days, respectively, and the step of floating culture again after attaching the formed spheres is preferably repeated 2 to 4 times, but is not limited thereto.

In the present invention, "nerve stem cells" have a self-replicating ability and neurons and / or glia, for example, astrocytes, oligodendrocytes and / or Schwann cells ( It is an undifferentiated cell having multi-differentiation ability to differentiate into Schwann cell). Neural stem cells are differentiated into neural cells, such as neurons or glia, through the steps of neural precursor cells or glia precursor cells that produce specific nervous system cells.

In the present invention, the neural stem cells may be characterized by expressing nestin, sox1 or musashi1, astrocytes, oligodendrocytes, neurons (neurons), dopamine neurons, gaba neurons , Motor neuron and choline neurons may be characterized in that the differentiation into any one or more selected from the group consisting of, but is not limited thereto.

In the present invention, the neural stem cells may be characterized by maintaining chromosome stability, and may be characterized by maintaining an undifferentiated state of 10 passages or more, but is not limited thereto.

Most methods of inducing human fibroblasts into neural stem cells using a direct conversion method are performed by introducing external genes related to neural stem cell formation. At this time, the external genes are introduced through various methods, and the most commonly used method is using a lentiviral or retroviral vector system. The introduction of genes using viruses causes genetic instability due to random integration of foreign genes, which may lead to cancer in future clinical applications. For this reason, methods for using small molecules without gradually injecting external genes have been proposed. However, despite the recent progress in research on inducing direct conversion using various small molecule compounds, at least one gene has been used, and without the introduction of genes, it is still unable to convert from human somatic cells to desired neural stem cells.

However, the technology of the present invention is a method of ensuring genetic stability of inducing neural stem cells from human fibroblasts without introducing an external gene, and is designed to solve a method of inducing genetic defects using existing genes.

In the present invention, human fibroblasts are directly converted to neural stem cells using only a combination of small molecule materials. Thus, by overcoming many of the problems of the existing technology, for example, ethical and immune rejection problems associated with embryonic stem cells, tumorigenesis problems of embryonic stem cells and dedifferentiated stem cells, It is very likely to be used as a cell therapy. In addition, the production of neural stem cells directly using somatic cells of brain disease patients can be used as a new cell model for studying the pathogenesis of brain diseases due to damage or death of nerve cells. Neural stem cells produced in this way are very fast and economical compared to dedifferentiated stem cells. De-differentiated stem cells are more than double the time and cost required to produce dedifferentiated stem cells and to differentiate them back into neurons.

In addition, when comparing adult neural stem cells with cross-differentiated neural stem cells according to the present invention, cross-differentiated neural stem cells do not have ethical problems because they do not need to secure neural stem cells from the fetus or the adult brain, and adult stem cells have self-renewal ability. Although limited, cross-differentiated neural stem cells have the advantage that they can produce as many cells as desired because there is no limit to self-renewal capacity. In addition, cross-differentiated neural stem cells can be produced using the patient's own cells, there is no immune rejection reaction, patient-specific cell modeling for disease research, patient-specific toxicity evaluation of the developed drug and new drug development Can be. In particular, since the cross-differentiated neural stem cells of the present invention do not use an external gene, there is no possibility of tumorigenesis caused by indiscriminate insertion of an external gene, and thus, the cross-differentiated neural stem cells of the present invention will be highly applicable to cell therapy.

Accordingly, the present invention relates to a cell therapy agent for treating brain diseases containing neural stem cells produced by a method comprising culturing human fibroblasts in a medium containing a small molecule compound in another aspect.

In the present invention, the brain disease may be characterized by stroke, stroke, cerebral hemorrhage, cerebral infarction, Alzheimer's disease, dementia, Huntington's disease, Parkinson's disease, multiple sclerosis, multiple nerve atrophy, epilepsy, peak disease and Creutzfeldt-Jakob disease. It is not limited to this.

The present invention can be used as a clinically applicable neural stem cells for the treatment of brain diseases, and can be used as a brain disease cell model that can be directly used in pathogenesis research when producing diseased neuronal stem cells derived from patients with hereditary cerebral disease. By replacing the mutated gene with a normal gene, it can be used as a cell therapy for the treatment of patients with hereditary brain diseases.

As used herein, the term "cellular therapeutic agent" refers to a medicinal product (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared through isolation, culture, and special manipulation from humans. Or through a series of actions such as proliferating and screening living autologous, allogeneic, or heterologous cells in vitro or otherwise altering the biological properties of a cell to restore tissue function. Means the drug used for the purpose.

In the present invention, the term "treatment" means any action that improves or benefits the condition of the disease by administration of the cell therapy agent.

The route of administration of the cell therapy composition of the present invention may be administered via any general route as long as it can reach the desired tissue. Parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration may be, but is not limited thereto.

The composition may be formulated in a suitable form with a pharmaceutical carrier generally used for cell therapy. 'Pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not cause an allergic or similar reaction, such as gastrointestinal disorders, dizziness or the like, when administered to a human. Pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, carriers for parenteral administration such as aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to those described in the following references (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The composition may also be administered by any device in which the cell therapy agent can migrate to the target cell.

The cell therapy composition of the present invention may include a therapeutically effective amount of cell therapy for the treatment of a disease.

The term therapeutically effective amount means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinician. This includes amounts that induce alleviation of the symptoms of the disease or disorder being treated.

It will be apparent to those skilled in the art that the cell therapy agent included in the composition of the present invention will vary depending on the desired effect. Therefore, the optimal cell therapy content can be readily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of other components contained in the composition, the type of formulation, and the age, weight, general health, sex and diet of the patient. It can be adjusted according to various factors including the time of administration, the route of administration and the rate of secretion of the composition, the duration of treatment, and the drugs used simultaneously. In consideration of all the above factors, it is important to include an amount that can achieve the maximum effect in a minimum amount without side effects. For example, the daily dose of stem cells of the present invention is 1.0 × 10 ⁴ to 1.0 × 10 ¹⁰ cells / kg body weight, preferably 1.0 × 10 ⁵ to 1.0 × 10 ⁹ cells / kg body weight divided into one or several times May be administered. However, it should be understood that the actual dosage of the active ingredient should be determined in light of several 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 therefore the dosage may It does not limit the scope of the present invention in terms of aspects.

In addition, the composition comprising the cell therapy of the present invention as an active ingredient in the treatment method of the present invention is rectal, intravenous (intravenous therapy, iv), intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular Or via the intradermal route.

The present invention provides a method of treatment comprising administering to a mammal a therapeutically effective amount of said cell therapy composition of the invention. The term mammal, as used herein, refers to a mammal that is the subject of treatment, observation or experiment, preferably human.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Function Verification and Combination of Small Molecule Compounds

1-1: Confirmation of function of small molecule compound

In order to induce human fibroblasts into neural stem cells, 13 kinds of various small molecule compounds related to reprogramming were selectively selected and used (Table 1).

To confirm the function of each small molecule substance and to find the optimal combination, 1 × 10 ⁵ human fibroblasts were prepared in a 60 mm dish and the next day 13 small molecule substances were placed in a neurobasal medium consisting of DMEM / F12, N2, B27, bFGF and EGF. (PD0325901, SB431542, Thiazovivin, Ascorbic acid, PS48, CHIR99021, Deazaneplanocin A, Valproic acid, Forskolin, Tranylcypromine, A8301, 5-AZA, Purmorphamine) were added one by one. After changing the culture solution every 2 to 3 days to confirm the morphological changes of the cells (Fig. 1). Subcultures were performed every 5 days, and some cells were obtained to confirm gene expression patterns at this stage. Fibroblast markers (Thy1), neural stem cell markers (nestin, sox1, sox2, pax6), MET or EMT markers (snail. AFP, GATA4), gene expression patterns of markers related to maintaining chromosomal stability (Zsacn4) were confirmed using quantitative PCR (FIG. 2).

As a result, it was confirmed that eight small molecule substances (Thiazovivin, Valproic acid, Purmorphamine, A8301, SB431542, CHIR99021, Deazaneplanocin A, 5-AZA) significantly increased the expression of neural stem cell marker genes.

1-2: Combination of small molecule compounds

For the most efficient neural stem cell production conditions, the fibroblasts were transformed into neural stem cells using the culture medium under various conditions except one small molecule material from the conditions in which the eight small molecule materials identified in Example 1-1 were added all at once. It was. 1 × 10 ⁵ human fibroblasts were prepared in a 60 mm dish, and the morphological changes of the cells were confirmed by changing the culture medium under various conditions every 2 to 3 days from the following day. In addition, changes in expression patterns of various genes were observed through the cells obtained during subculture.

As a result, eight (Thiazovivin, Valproic acid, Purmorphamine, A8301, SB431542, CHIR99021, Deazaneplanocin A, 5-) increased the gene expression of neural stem cell markers (Thiazovivin, Valproic acid, Purmorphamine, A8301, SB431542, CHIR99021). A combination of small molecule materials of AZA) was selected. It was confirmed that human fibroblasts can be induced into neural stem cells using only 6-8 small molecule materials. In addition, despite the same human fibroblasts, it was confirmed that the timing of the discovery of cells having a morphology similar to that of neural stem cells depends on the kind of the source from which the cells are derived.

Example 2: Induction and Cultivation of Neural Stem Cells from Human Fibroblasts

As the combination of small molecule materials needed to convert human fibroblasts into neural stem cells was determined, it was intended to establish conditions for stably inducing and culturing. In addition, in order to confirm the efficiency induced by neural stem cells, a comparison was made using a culture medium to which at least four small molecule compounds were added. First, 1 × 10 ⁵ fibroblasts were prepared, and then 4 species (Thiazovivin, Valproic acid, Purmorphamine, A8301), 6 species (Thiazovivin, Valproic acid, Three kinds of culture medium containing Purmorphamine, A8301, SB431542, CHIR99021) and 8 small molecules (Thiazovivin, Valproic acid, Purmorphamine, A8301, SB431542, CHIR99021, Deazaneplanocin A, 5-AZA) were treated.

As a result, about 3 days after the induction, the morphology of the cells began to change slowly. In the culture condition of four small molecule compounds added about 7-10 days, the cell morphology did not change as much as that of fibroblasts. However, in the culture condition in which six and eight small molecule compounds were added, cells similar to neural stem cells were formed.

Subsequently, passage was incubated in suspension using petri-dish for the purpose of increasing the properties of stem cells. After 7-10 days of incubation by the suspension method, most cells form small spheres under the culture conditions of 6 and 8 small molecule compounds. On the other hand, it was confirmed that spheres were not formed properly under the conditions in which the four small molecule compounds were added. The small spheres thus formed are attached to a PLO / FN-coated dish and incubated for 7-10 days. It was confirmed that the morphology of the cells at this stage is more similar to that of neural stem cells (FIG. 3). When the suspension culture and the adhesion culture were repeated two to four times, it was confirmed that the morphology of the cells changed more clearly in the culture conditions in which six and eight small molecule compounds were added. However, it was confirmed that the morphology of the cells did not change in the culture medium to which the four small molecule compounds were added, despite the continuous culture. The neural stem cells induced through the culture medium containing 6 or 8 small molecule compounds maintained the shape of the neural stem cells even when cultured for a long time with continuous passage.

Example 3 Characterization of Human Neural Stem Cells Induced by Small Molecule Compounds

It was confirmed that human neural stem cells induced and maintained with six small molecule compounds in DMEM / F12 + N2B27 exhibited general characteristics of neural stem cells. First, PCR and immunocytochemistry (ICC) were performed to determine whether neural stem cells express markers of neural stem cells. As a result, it was confirmed that neural stem cells express nestin through immunocytochemistry (ICC) staining, and PCR expression of the neural stem cell markers nestin, sox1, and musashi1 is similar to neural stem cells derived from human embryonic stem cells. It was confirmed that the degree (Fig. 4).

In order to check whether the properties of neural stem cells are maintained during long-term culture, the morphology of the cells was observed at each of the early and late stages of induced neural stem cells. In addition, the expression of nestin through immunocytochemistry (ICC) staining and gene expression of the nestin, sox1, and musashi1 neural stem cell markers using PCR were confirmed (FIG. 5). In addition, chromosomal analysis confirmed the presence or absence of chromosomal abnormalities in the early and late stages of the induced neural stem cells (Fig. 6), compared with the growth rate of the cells during the proliferation and maintenance of neural stem cells derived from human embryonic stem cells ( 7). As a result, the induced neural stem cells were found to be able to continue culturing without chromosomal abnormalities while maintaining the shape and properties of neural stem cells even during long-term culture.

Subsequently, bisulfite PCR showed methylation / acetylation of the promoter region of nestin, a neuronal stem cell marker, to confirm epigenetic genetic changes. It was confirmed that the characteristics of fibroblasts were completely changed to the characteristics of neural stem cells (Fig. 8).

Example 4: In vitro differentiation of induced human neural stem cells

Examples 1 to 3 confirmed that inducing neural stem cells from human fibroblasts using small molecule materials was successfully performed. More specifically, in order to confirm the characteristics and availability of neural stem cells, differentiation ability in vitro was confirmed. First, they differentiated into trigeminal neurons (astrocyte), oligodendrocyte (neuron) and neurons (neuron).

As a result, it was confirmed that neural stem cells induced by the small molecule compound were successfully differentiated into trigeminal neural cells (FIG. 9).

Then, as a result of confirming that the differentiation of various types of neurons, it was confirmed that the differentiation into dopamine neurons, Gaba neurons, motor neurons, choline neurons and glutamate neurons (Fig. 10). Through this, it was confirmed that the human neural stem cells induced by the present invention can be differentiated into various neurons.

Induction, proliferation, and differentiation into various types of functional neurons using human fibroblasts show that human fibroblasts are highly useful as cell therapeutics for the treatment of brain diseases.

Example 5: In vivo differentiation of induced human neural stem cells

In Examples 3 and 4 it was confirmed in vitro that the neural stem cells derived from human fibroblasts can be successfully differentiated into trigeminal nerve cells using small molecule materials.

In order to observe the in vivo differentiation capacity and tumor formation of neural stem cells derived from human fibroblasts using small molecule materials, human neural stem cells induced in the brain of mouse (Oriental Bio, Balb / c) were transplanted. It was. Then, the mice transplanted with human neural stem cells induced for 3 to 7 months were continuously observed.

As a result, the brain of the mouse transplanted with the neural stem cells induced by the small molecule compound showed no tumor formation externally for 3 to 7 months (FIG. 11A), and brain tissue was confirmed by H & E (Haematoxylin and Eosin) staining. It was also confirmed that no tumor formation was observed (FIG. 11B). In addition, it was confirmed that the induced neural stem cells were successfully transplanted through the expression of neural stem cell markers (FIG. 11C), and the induced neural stem cells 3 to 7 months after transplantation expressed human cell-specific markers and neuron-specific markers. It was confirmed that the transplanted cells were differentiated into neurons (FIG. 11D). This indicates that human neural stem cells induced by small molecule compounds can be differentiated into neurons without transplantation after transplantation.

These induced neural stem cells can be used as an optimal model cell to study the development of neurons and the pathogenesis of brain disease, and as a cell therapy for the treatment of brain diseases in humans due to the advantage of not generating tumors. The probability is very high.

The method of directly converting human fibroblasts into neural stem cells using only small molecule materials without introducing the genes according to the present invention secures a sufficient amount of cells and various kinds of functional neurons for use in cell therapy through induction of genetically stable neural stem cells. Because it can be differentiated and does not generate tumors, it is useful for brain disease cell therapy.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

1. A method for producing neural stem cells, comprising culturing human fibroblasts in a medium comprising thiazovivin, Valproic acid, Permorphamine, A8301, SB431542 and CHIR99021.
2. The method of claim 1, further comprising DZNep (Deazaneplanocin A) or 5-AZA.
3. The method of claim 2, further comprising at least one small molecule compound selected from the group consisting of PD0325901, Ascorbic acid, PS48, Forskolin, and Tranylcypromine. Method for producing neural stem cells.
4. The method of claim 1, wherein the medium is DMEM / F12 containing N2, B27, bFGF, and EGF.
5. The method of claim 1, wherein the culturing is carried out for 10 to 15 days.
6. The method of claim 1, further comprising: subcultured with the cells, followed by suspension culture to form a sphere; And culturing the attached spheres and then culturing the suspension again.
7. 7. The method of claim 6, wherein the suspension culture and the adhesion culture are cultured for 7 to 10 days, respectively.
8. According to claim 6, The method of producing neural stem cells, characterized in that the step of culturing the suspension attached again after the culture attached to the formed spheres.
9. The method of claim 1, wherein the neural stem cells express nestin, sox1, or musashi1.
10. According to claim 1, wherein the neural stem cells (astrocyte), oligodendrocyte (oligodendrocyte), neurons (neuron), dopamine neurons, gaba neurons, motor neurons and choline neurons are selected from the group consisting of Method for producing a neural stem cell, characterized in that it is differentiated into any one or more.
11. The method of producing neural stem cells according to claim 1, wherein the chromosome stability is maintained.
12. The method of claim 1, wherein the neural stem cells are maintained in an undifferentiated state for 10 passages or more.
13. Cell therapy for the treatment of brain diseases containing neural stem cells produced by the method of any one of claims 1 to 12.
14. The method of claim 13, wherein the brain disease is selected from the group consisting of stroke, stroke, cerebral hemorrhage, cerebral infarction, Alzheimer's disease, dementia, Huntington's disease, Parkinson's disease, multiple sclerosis, multiple neurotrophic, epilepsy, peak disease and Creutzfeldt-Jakob disease Cell therapy, characterized in that.

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