Direct reprogramming of urine cells into neural stem cells using synthetic mRNA

Abstract

The present invention relates to a method for inducing dedifferentiation of neural stem cells from urine cells by introducing mRNA of dedifferentiation factors Oct4, Sox2, Klf4 and Glis1; and a composition for preventing or treating nerve damage diseases, the composition comprising neural stem cells induced by the method as an effective ingredient.

Description

Technical Field

[0001] The present invention relates to a method for directly reprogramming urine cells into neural stem cells and a pharmaceutical composition for treating nerve damage diseases, wherein the pharmaceutical composition comprises the reprogrammed neural stem cells prepared by the method. Background Art

[0002] Stem cells are cells that have unlimited potential for self-renewal or differentiation, which are related to specific cells and tissues required in the body. Stem cells are divided into three types: embryonic stem cells (ES cells) isolated from early embryos, embryonic germ cells (EG cells) isolated from embryonic primordial germ cells, and multipotent adult progenitor cells (MAPC cells) isolated from adult tissues.

[0003] Since stem cells have the potential to differentiate into cells with specific functions, they have become the subject of research for restoring the functions of various organs and as cell therapy agents. Recently, the application of stem cells has expanded to the fields of plastic surgery and cosmetology.

[0004] The role of adult stem cells in the body can be roughly summarized in two ways. First, the stem cells themselves differentiate into tissues and cells in our body and regenerate damaged tissues and cells; and second, stem cells continuously secrete growth factors and proteins (such as cytokines) during their lifespan to help neighboring cells grow and regenerate.

[0005] Direct reprogramming is a technology that allows somatic cells to be converted into other types of cells as needed. Since direct reprogramming does not experience a pluripotent stem cell state like the reprogramming of Professor Yamanaka in Japan, direct reprogramming can save time and show a high efficiency level, while solving the risks associated with tumorigenesis and the costs caused by further differentiation of pluripotent stem cells. However, the direct reprogramming method currently performed mainly uses mouse cells as a cell source, so the possibility of regeneration in the same way in human somatic cells is not high. In addition, skin cells (fibroblasts) are often used in human somatic cells, but in this case, an invasive method for collecting cell sources is required, and this brings pain and safety risks to the donor, and convenience deteriorates. In addition, clinical application is difficult, because the method of introducing the required reprogramming factor (gene) in the direct reprogramming process mainly uses viruses, and the gene integration system using viruses is not suitable for treatment, because the system can induce mutations due to indiscriminate insertion into the genome.

[0006] Therefore, the present inventors used easily collected cells derived from human urine as a cell source and used transfection of synthetic mRNA to introduce reprogramming factors into the cells to solve the above-mentioned problems, and confirmed the optimal culture conditions required for efficient and direct reprogramming of urine cells into neural stem cells, thereby completing the present invention.

[0007] Related technical literature

[0008] Korean Patent Application No. 10-2013-0087020 Summary of the invention

[0009] Technical issues

[0010] An object of the present invention is to provide a composition for directly reprogramming urine cells into neural stem cells.

[0011] Another object of the present invention is to provide a method for directly reprogramming urine cells into neural stem cells.

[0012] Still another object of the present invention is to provide a method for preventing or treating a nerve damage disease, the method comprising administering neural stem cells reprogrammed from urine cells as an active ingredient.

[0013] Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating a nerve damage disease, the pharmaceutical composition comprising neural stem cells reprogrammed from urine cells as an active ingredient.

[0014] Technical Solution

[0015] The present invention provides a composition for directly reprogramming urine cells into neural stem cells, the composition comprising:

[0016] (i) Oct4 protein or a nucleic acid encoding the Oct4 protein;

[0017] (ii) Sox2 protein or a nucleic acid encoding the Sox2 protein;

[0018] (iii) Klf4 protein or a nucleic acid encoding the Klf4 protein; and

[0019] (iv) Glis1 protein or a nucleic acid encoding the Glis1 protein.

[0020] As used herein, the term "reprogrammed neural stem cell" includes neural stem cells made by direct reprogramming for establishing undifferentiated stem cells with pluripotency similar to or identical to that of neural stem cells. The induced neural stem cells have the same or similar characteristics as neural stem cells, specifically can show similar cell morphology, have similar gene and protein expression patterns, and have pluripotency in vivo and in vitro. Therefore, the reprogrammed neural stem cells of the present invention can be neural stem cells that can differentiate into nerve cells (neurons), astrocytes, oligodendrocytes, GABAergic nerve cells, dopaminergic nerve cells, etc.

[0021] As used herein, the term "urine cell (UC)" refers to a somatic cell that can be easily and repeatedly obtained at any time without any inconvenience and pain.

[0022] The present inventors have found that when reprogramming factors Oct4, Sox2, Klf4, and Glis1 are expressed in urine cells, urine cells that have been differentiated into cell types are reprogrammed into neural stem cells having differentiation ability.

[0023] The term "reprogramming factor" originated from reprogramming, a concept introduced by Yamanaka in 2006. All cells of an adult gradually differentiate in an undifferentiated state during normal development, and thus become cells with specialized functions. Among them, fertilized cells are omnipotent, and when fertilized cells develop into blastocysts as subsequent developmental steps, they can be divided into inner cell mass and outer cells, in which case, inner cell mass cells can produce embryonic cells and germ cells known as pluripotent. Embryonic stem cells show pluripotent specific gene expression aspects, and their representative examples include Oct4, Sox2, Nanog, Lin28, etc. Reprogramming can be said to be a technology that induces this specific gene expression in somatic cells to restore properties similar to undifferentiated cells (such as embryonic stem cells and adult stem cells). The inventors used Oct4, Sox2, Klf4 and Glis1 to reprogram urine cells into neural stem cells as part of various factors used in a series of studies related to reprogramming.

[0024] The Oct4, Sox2, Klf4 and Glis1 used in the composition of the present invention include all Oct4, Sox2, Klf4 and Glis1 derived from humans and animals (e.g., horses, sheep, pigs, goats, camels, antelopes and dogs), and preferably human Oct4, Sox2, Klf4 and Glis1. In addition, the Oct4, Sox2, Klf4 and Glis1 proteins of the present invention for reprogramming into neural stem cells may include proteins having their wild-type amino acid sequences, and may also include variants of Oct4, Sox2, Klf4 and Glis1 proteins.

[0025] In a specific exemplary embodiment, the gene sequences of Oct4, Sox2, Klf4 and Glis1, which were first used in reprogramming and published in Nature. 2011 Jun 8; 474(7350):225-9, were used.

[0026] Variants of Oct4, Sox2, Klf4 and Glis1 proteins refer to proteins that differ from the native amino acid sequences of Oct4, Sox2, Klf4 and Glis1 by deletion, insertion, non-conservative or conservative substitution or a combination thereof in at least one amino acid residue. Variants may be functional equivalents that exhibit the same biological activity as the native protein, or may be variants in which the physical and chemical properties of the protein are modified as desired. The variants are variants that have increased structural stability or increased physiological activity under certain physical or chemical conditions.

[0027] The nucleic acid comprising a nucleotide sequence encoding Oct4, Sox2, Klf4 and Glis1 proteins is a nucleic acid comprising a nucleotide sequence encoding wild-type Oct4, Sox2, Klf4 and Glis1 proteins or a variant form of Oct4, Sox2, Klf4 and Glis1 proteins as described above, which can be modified by substitution, deletion and insertion of one or more bases or a combination thereof, and can be naturally isolated or prepared using a chemical synthesis method.

[0028] The nucleic acid having the nucleotide sequence encoding Oct4, Sox2, Klf4 and Glis1 proteins can be single-stranded or double-stranded, and can be a DNA molecule (genomic, cDNA) or an RNA molecule. In a specific exemplary embodiment of the present invention, a synthetic messenger RNA (mRNA) is used as the nucleic acid encoding Oct4, Sox2, Klf4 and Glis1 proteins, and a synthetic mRNA expression vector into which the DNA expressing Oct4, Sox2, Klf4 and Glis1 is introduced can be used to prepare the synthetic messenger RNA.

[0029] The present invention provides a method for directly reprogramming urine cells into neural stem cells, the method comprising: introducing Oct4, Sox2, Klf4 and Glis1 proteins or nucleic acids encoding the proteins into urine cells.

[0030] More specifically, the method may include:

[0031] (a) isolating urine cells from urine and culturing the urine cells;

[0032] (b) introducing the composition of claim 1 or 3 into cultured urine cells;

[0033] (c) inducing direct reprogramming into neural stem cells by culturing the urine cells into which the composition has been introduced in a neural stem cell induction medium; and

[0034] (d) Selecting a neural stem cell line having characteristics similar to those of neural stem cells from cells that have been directly reprogrammed by a neural stem cell induction medium.

[0035] In step (a), urine cells may be cultured in a culture medium containing fetal bovine serum (FBS), basic fibroblast growth factor (bFGF) and epithelial growth factor (EGF).

[0036] As used herein, the term "culture medium" refers to a culture medium that can support cell growth and survival in vitro, and includes all typical culture mediums suitable for induction and cultivation of urine cells and reprogrammed neural stem cells in the art. According to the type of cell, culture medium and culture conditions can be selected. The culture medium for cultivation is preferably a cell culture basal medium (Cell culture minimum medium, CCMM), and generally includes a carbon source, a nitrogen source and trace element components. The example of this cell culture basal medium includes: Dulbecco's modified Eagle's Medium (Dulbecco's Modified Eagle's Medium, DMEM), Minimal Essential Medium (Minimal Essential Medium, MEM), Basal Medium Eagle (Basal Medium Eagle, BME), RPMI1640, F-10, F-12, α Minimal Essential Medium (αMinimalEssential Medium, αMEM), Glasgow's Minimal Essential Medium (Glasgow's Minimal EssentialMedium, GMEM), Iscove's Modified Dulbecco's Medium (Iscove's Modified Dulbecco's Medium, IMEM) etc., but are not limited thereto. Additionally, the culture medium may include antibiotics, such as penicillin, streptomycin or gentamicin.

[0037] In a specific example of the present invention, a culture medium can be obtained by culturing cells separated from urine in a basal culture medium comprising FBS, bFGF and EGF, specifically, a culture medium can be obtained by adding bFGF and EGF to a high glucose DMEM and a renal epithelial cell growth medium (REGM) comprising FBS, and culturing cells separated from urine in the culture medium. More specifically, in the present invention, the high glucose DMEM and REGM culture mediums can additionally include L-glutamine and penicillin-streptomycin.

[0038] In step (b), as a method for introducing reprogramming factors into urine cells, methods for providing nucleic acids or proteins into cells commonly used in the art can be used without limitation, and preferably a method for applying reprogramming factors to a culture solution of differentiated cells or a method for directly injecting reprogramming factors into differentiated cells can be used, and the reprogramming factors used in this case can be used in the form of viruses obtained from packaging cells transfected with viral vectors into which genes of the corresponding factors have been inserted, messenger RNA produced by in vitro transcription, proteins produced in various cell lines, etc. In a specific exemplary embodiment of the present invention, when introducing reprogramming factors into urine cells, messenger RNA produced by transcription of DNA encoding Oct4, Sox2, Klf4, and Glis1 is used.

[0039] For the method of directly injecting the synthetic messenger RNA into the differentiated cells, any method known in the art can be selected and used, and a method can be appropriately selected and applied from the methods using microinjection, electroporation, particle bombardment, direct intramuscular injection, insulators and transposons. Specifically, in an exemplary embodiment of the present invention, the messenger RNA of the reprogramming factor is introduced into the urine cell using electroporation.

[0040] In step (c), the culture medium (neural stem cell induction medium) for inducing urine cells into neural stem cells into which nucleic acids encoding Oct4, Sox2, Klf4 and Glis1 proteins have been introduced can be a culture medium obtained by mixing DME / MF12 culture medium and neural basal culture medium in a volume ratio of 1:1, and adding 1×N2, 1×B27, human LIF, TGF-β inhibitor and GSK3-β inhibitor to the culture medium mixture. More specifically, the culture medium can further include one or more of the following: Shh agonist, adenylate cyclase activator, histone deacetylase inhibitor and ascorbic acid 2-phosphate. In a specific exemplary embodiment of the present invention, it was confirmed that the neural stem cell conversion rate was optimal in a culture medium supplemented with all of the above Shh agonists, adenylate cyclase activators, histone deacetylase inhibitors and ascorbic acid 2-phosphate.

[0041] Furthermore, the induction efficiency can be improved when urine cells are cultured in culture medium under hypoxic (O2 deficient) conditions.

[0042] In step (d), the induced neural stem cells are selected to collect neural stem cell colonies generated after step (c), and the selected neural stem cells can be cultured in a neural stem cell culture medium. The neural stem cell culture medium can be a culture medium obtained by adding 1×N2, 1×B27, human LIF, TGF-β inhibitor and GSK3-β inhibitor to a culture medium (in which DMEM / F12 culture medium and neural basal culture medium are mixed in a volume ratio of 1:1). In addition, the induced neural stem cells can be subcultured using a 0.5mM EDTA solution or an Accutase solution.

[0043] In addition, the present invention provides a pharmaceutical composition for preventing or treating a nerve damage disease, the pharmaceutical composition comprising the neural stem cells induced by the method as an active ingredient.

[0044] Furthermore, the present invention provides a method for preventing or treating a nerve damage disease, which comprises administering directly reprogrammed neural stem cells prepared by the method as an active ingredient.

[0045] Neural stem cells are multipotent cells that can differentiate into nerve cells (neurons), astrocytes, oligodendrocytes, GABAergic nerve cells, dopaminergic nerve cells, etc., and can repair or restore damaged or lost nerve cells, so it is possible to treat diseases caused by damage or disappearance of nerve cells without limitation.

[0046] Specifically, the disease caused by nerve cell damage can be selected from the group consisting of Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, ischemic encephalopathy (stroke), demyelinating disease, multiple sclerosis, epilepsy, degenerative nerve disease and spinal cord injury.

[0047] Beneficial effects

[0048] The present invention is a technology that utilizes cells derived from human urine as a cell source to maximize the convenience of obtaining the cell source and enhances clinical feasibility through a non-integrating direct reprogramming method using synthetic mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is a schematic diagram of the T7-VEE-OKS-iG vector used to prepare synthetic mRNAs of OCT4, KLF4, SOX2 and GLIS1 genes;

[0050] Figure 2 The results of confirming whether the synthesized mRNAs of OCT4, KLF4, SOX2, and GLIS1 genes combined with GFP (fluorescent gene) were introduced into urine cells by FACS analysis are shown;

[0051] Figure 3 The whole process of obtaining neural stem cell colonies derived from urine cells using direct reprogramming technology is shown;

[0052] Figure 4 The figure shows the results of confirming whether the induced neural stem cells express neural stem cell marker genes (such as SOX1, SOX2, PAX6, and PLZF) by mRNA level analysis using RT-PCR using neural stem cells derived from H9 embryonic stem cells as a positive control;

[0053] Figure 5 The results of confirming whether the induced neural stem cells express neural stem cell marker genes (such as SOX1, SOX2, PAX6, and PLZF) at the protein level by immunoassay are shown;

[0054] Figure 6 The results of confirming whether the induced neural stem cells have differentiation ability by immunostaining of Ki67 are shown;

[0055] Figure 7 The figure shows the results of confirming whether the induced neural stem cells express neural stem cell marker genes (such as SOX1, SOX2, PAX6, and PLZF) by mRNA quantitative analysis using qRT-PCR compared with neural stem cells derived from H9 embryonic stem cells;

[0056] Figure 8 The results of confirming whether the induced neural stem cells express developmental forebrain, midbrain, hindbrain and spinal cord specific marker genes by mRNA level analysis using RT-PCR are shown;

[0057] Figures 9 and 10 show the results of confirming whether the induced neural stem cells exhibited mRNA and long noncoding RNA (Inc-RNA) expression patterns that were more similar to those of neural stem cells derived from H9 embryonic stem cells than to the original urine-derived cells by performing total RNA sequencing at the global gene expression level;

[0058] Fig.11 The results of confirming whether the synthesized mRNA introduced into neural stem cells is retained in the host and whether the synthesized mRNA is integrated into the host genome by RT-PCR of the VEE gene and genomic DNA PCR analysis are shown;

[0059] Fig.12 The results of confirming whether the induced neural stem cells are derived from human urine cells by STR analysis are shown;

[0060] Fig.13The results of confirming whether the induced neural stem cells retain normal chromosomes by karyotype analysis are shown;

[0061] Fig.14 The results of confirming the expression of neural cell gene markers TUJ1 and MAP2 by immunoscreening to verify the ability of induced neural stem cells to differentiate into neural cells are shown;

[0062] Fig.15 The results of confirming by immunostaining that the induced neural stem cells can differentiate into GABA neurons, motor neurons, and dopamine neurons are shown;

[0063] Fig.16 The results of confirming the expression of S100β and GFAP, which are markers for verifying the ability of induced neural stem cells to differentiate into astrocytes (astrocytes are glial cells), by immunoscreening are shown;

[0064] Fig.17 The results of confirming the expression of PDGFR, OLIG2 and O4, which are markers for verifying the ability of induced neural stem cells to differentiate into oligodendrocytes (oligodendrocytes are glial cells), by immunoscreening are shown;

[0065] Fig.18 The results of confirming the relative mRNA expression levels of OCT4, REX4, and NANOG by date during the process of inducing neural stem cells from urine cells are shown;

[0066] Fig.19 The results of confirming whether marker genes of pluripotent H9 embryonic stem cells and induced neural stem cells are expressed are shown;

[0067] Fig. 20 The comparison results of the conversion rate when urine-derived cells were induced into neural stem cells under low oxygen conditions (5% hypoxia) using alkaline conditions (21% O2) as a control are shown;

[0068] Fig.21The results show that when one or more of Shh agonist, adenylate cyclase activator, histone deacetylase inhibitor and ascorbic acid 2-phosphate are further included in the culture medium, the induction efficiency of neural stem cells is improved, and the culture medium is obtained by mixing DMEMF12 medium and neural basal medium at a volume ratio of 1:1, and adding 1×N2, 1×B27, human LIF, TGF-β inhibitor and GSK3-β inhibitor to the culture medium mixture. It is confirmed that the neural stem cell conversion rate is optimal in the culture medium supplemented with all of the above Shh agonist, adenylate cyclase activator, histone deacetylase inhibitor and ascorbic acid 2-phosphate. DETAILED DESCRIPTION

[0069] The present invention will be described in more detail below with reference to Examples. However, the following Examples are provided only to make it easier to understand the present invention, and the present invention is not limited to the following Examples.

[0070] Example 1: Isolation of urine cells from urine

[0071] Based on the technology developed by Sutherland and Bain in the UK in 1972, the details are as follows. First, the urine donated by the donor was centrifuged at 1000g for 10 minutes. After removing the supernatant, the precipitate remaining in the lower layer was diluted with 20ml of PBS solution containing 1% penicillin / streptomycin / amphotericin B antibiotics. Then the diluted PBS+precipitation solution was centrifuged at 1000g for 10 minutes. After removing the supernatant again, the precipitate remaining in the lower layer was diluted with 1ml basal culture medium (based on DMEMF12 containing 1% penicillin / streptomycin / amphotericin B antibiotics, 1% L-glutamine and 10% FBS) and inoculated into a 12-well cell culture dish coated with gelatin. Next, after culturing the cells for 3 days by adding 1 ml of the basal medium, the medium was replaced with a growth medium (a medium containing 1% penicillin / streptomycin antibiotics, 1% L-glutamine, 5% FBS, 10 ng / ml bFGF and 10 g / ml EGF based on a medium obtained by mixing DMEM and REGM at 1:1) to culture the cells.

[0072] Example 2: Introduction of reprogramming factors into urine cells using electroporation

[0073] The method of introducing the synthesized mRNA into the urine-derived cells cultured in Example 1 by electroporation is as follows. The synthesized mRNA was synthesized by a typical in vitro transcription kit (RiboMAX◎ Large-Scale RNA Production System, Promega), and the DNA that became the backbone of the mRNA to be synthesized was T7-VEE-OKS-iG (Steven Dowdy, Addgene), which includes genes of OCT4, KLF4, SOX2, and GLIS1 ( Figure 1 ). As the sequences of OCT4, KLF4, SOX2 and GLIS1 genes, those disclosed in Nature. 2011Jun 8; 474(7350): 225-9 were used, and for the corresponding gene sequences and mRNA application methods, reference was made to Cell Stem Cell. 2013Aug1; 13(2): 246-54 and U.S. Patent No. 9,862,930B2.

[0074] 1×10 cells pretreated with 0.2 ug / ml B18R protein were electroporated 3 times at 1600 V and 10 ms for 1 hour with 0.5 μg of synthesized mRNA. 6 The urine-derived cells were treated to introduce the synthesized mRNA. The introduced urine-derived cells were cultured in a growth medium containing 0.2 ug / ml of B18R protein for 2 days.

[0075] The inventors visualized, determined and verified the efficiency of introduction by electroporation by simultaneously introducing synthetic mRNA including GFP fluorescent protein, and confirmed the efficiency of human urine-derived cells expressing GPF fluorescent protein by FACS analysis. It was confirmed by verification that the efficiency of human urine-derived introduction of synthetic mRNA by electroporation reached 72.2% about 48 hours after treatment, which was determined to be sufficient for the next step of neural stem cell induction experiment ( Figure 2 ).

[0076] Example 3: Induction of reprogrammed neural stem cells from urine

[0077] Urine-derived cells into which mRNA had been introduced were seeded onto Matrigel-coated TMAfter being placed in a cell culture dish, the cells were cultured for 7 to 10 days in a neural stem cell induction medium based on a neural stem cell induction medium (a medium obtained by mixing DMEM / F12 medium and Neurobasal (Gibco) medium at 1:1 and adding 1×N2 (Gibco), 1×B27 (Gibco), 10 ng / ml human LIF, 2 uM SB431542 (TGF-β inhibitor) and 3 uM CHIR99021 (GSK3-β inhibitor) to the medium mixture) supplemented with 0.5 uM purmorphamine (Shh agonist), 10 uM Forskolin (adenylate cyclase activator), 100 uM sodium butyrate (histone deacetylase inhibitor) and 64 ug / ml ascorbic acid 2-phosphate. When the induction is completed, the development of neural stem cell colonies can be confirmed ( Figure 3 ).

[0078] In addition, when one or more of Shh agonist, adenylate cyclase activator, histone deacetylase inhibitor and ascorbic acid 2-phosphate were further included in the medium further supplemented with 1×N2, 1×B27, human LIF, TGF-β inhibitor and GSK3-β inhibitor, the effect on the induction efficiency of neural stem cells was confirmed. It was confirmed that in the medium supplemented with all of Shh agonist, adenylate cyclase activator, histone deacetylase inhibitor and ascorbic acid 2-phosphate, the neural stem cell conversion rate was optimal ( Fig.21 ).

[0079] The induced neural stem cell colonies are collected and cultured by placing the neural stem cell culture medium in a cell culture dish coated with Matrigel. The induced neural stem cells can then be subcultured using a 0.5 mM EDTA solution or an Accutase solution, and then experiments to verify various properties of the induced neural stem cells are performed in the following examples.

[0080] Example 4: Analysis of Molecular Biological Characteristics of Induced Neural Stem Cells

[0081] Experiments were conducted to verify the molecular biological characteristics of the neural stem cells derived from the present invention.

[0082] <4-1>

[0083] Using H9 embryonic stem cell-derived neural stem cells as a positive control, we confirmed that the induced neural stem cells expressed neural stem cell marker genes such as SOX1, SOX2, PAX6, and PLZF by mRNA level analysis using RT-PCR. Figure 4The primer sequences used in this experiment are as follows: SOX1 (forward-ACACTTGAAGCCCAGATGG; SEQ ID NO: 1, reverse-ATAGGCTCACTTTTGGACGG; SEQ ID NO: 2), SOX2 (forward-TCAGGAGTTGTCAAGGCAGAGA; SEQ ID NO: 3, reverse-CCGCCGCCGATGATTGTTATTA; SEQ ID NO: 4), PAX6 (forward-GGCTCAAATGCGACTTCAG; SEQ ID NO: 5, reverse-CCCTTCGATTAGAAAACCATACC; SEQ ID NO: 6), PLZF (forward-TATACAGCCACGCTGCAAGCCA; SEQ ID NO: 7, reverse-TGGTCTCCAGCATCTTCAGGCA; SEQ ID NO: 8).

[0084] <4-2>

[0085] We also confirmed that the induced neural stem cells expressed neural stem cell marker genes such as SOX1, NESTIN, SSEA1, SOX2, PAX6, and PLZF by protein level analysis using immunostaining ( Figure 5 The antibodies used in this experiment are as follows: SOX1 (AF3369, R&D Systems), NESTIN (MAB5326, Millipore), SSEA1 (MAB4301, Millipore), SOX2 (SC-20088, Santacruz), PAX6 (PAX6, DSHB), PLZF (AF2944, R&D Systems).

[0086] <4-3>

[0087] The induced neural stem cells had the ability to divide by immunostaining of Ki67. Figure 6 ). The antibodies used in this experiment are as follows: Ki67 (Ab9260, Millipore).

[0088] <4-4>

[0089] We also confirmed by quantitative analysis of mRNA using qRT-PCR that the induced neural stem cells expressed neural stem cell marker genes such as SOX1, SOX2, PLZF, and PAX6 compared with neural stem cells derived from H9 embryonic stem cells ( Figure 7 ).

[0090] <4-5>

[0091] RT-PCR was also used to confirm that the induced neural stem cells expressed marker genes specific to the developmental forebrain, midbrain, hindbrain, and spinal cord ( Figure 8 ). The primer sequences used in this experiment are as follows: EMX1 (forward -CAGGCCCAGGTAGTTCAATGGG; SEQ ID NO: 9, reverse -GCTCAGCCTTAAGCCCTGTCTC; SEQ ID NO: 10), FOXG1 (forward -ACCCGTCAATGACTTCGCAGAG; SEQ ID NO: 11, reverse -AGGGTTGGAAGAAGACCCCTGA; SEQ ID NO: 12), OTX1 (forward -CTCAAACAACCCCCATACGGCA; SEQ ID NO: 13, reverse -GGTAGCGAGTCTTGGCGAAGAG; SEQ ID NO: 14), OTX2 (forward -TTCAGGGCTGTGTGAATTGTGTGA; SEQ ID NO: 15, reverse -CAGAGGTGGAGTTCAAGGTTGCAT; SEQ ID NO: 16), EN1 (forward -GAGTTCCAGGCAAACCGCTACA; SEQ ID NO: 17, reverse -GTTGTACAGTCCCTGGGCCATG; SEQ ID NO: 18), NO: 18), GBX2 (forward -CGGTTAGCAGCCACCTTTCCAT; SEQ ID NO: 19, reverse -GACGTGCTTCACATGGCTCAGA; SEQ ID NO: 20), HOXB2 (forward -GAGACCCAGGAGCCAAAAGC; SEQ ID NO: 21, reverse -GAAGGAGACGTGGCGGATTG; SEQ ID NO: 22), HOXB4 (forward -CTGAGGGCCAGAATGACTGCTC; SEQ ID NO: 23, reverse -CAGAACTCAACTGGCCCCTCAC; SEQ ID NO: 24).

[0092] <4-6>

[0093] Total RNA sequencing confirmed that induced neural stem cells exhibited mRNA and lnc-RNA expression patterns more similar to those of neural stem cells derived from H9 embryonic stem cells than to the original urine-derived cells at the global gene expression level ( FIGS. 9 and 10 ).

[0094] <4-7>

[0095] RT-PCR and genomic DNA PCR analysis of the VEE gene confirmed that the synthesized mRNA introduced into neural stem cells was no longer retained in the host and was not integrated into the host genome ( Fig.11 The primer sequences used in this experiment are as follows: VEE (forward-ACGAAGGGCAAGTCGCTGTT; SEQ ID NO: 25, reverse-TTTCGTCGGCCCAGTTGGTA; SEQ ID NO: 26).

[0096] <4-8>

[0097] STR analysis confirmed that human urine cells and induced neural stem cells were derived from the same individual ( Fig.12 ).

[0098] <4-9>

[0099] Karyotype analysis confirmed that the induced neural stem cells retained normal chromosomes ( Fig.13 ).

[0100] Example 5: Analysis of differentiation ability of induced neural stem cells

[0101] <5-1> Analysis of the ability to differentiate into neural cells

[0102] Immunostaining confirmed that when induced neural stem cells differentiated into neurons, they expressed the neural cell marker genes TUJ1 and MAP2 ( Fig.14 ). The neural cell differentiation method is as follows. Neural stem cells were analyzed after 14 days of culture in a neural cell differentiation medium obtained by adding 1×N2, 1×B27, 300ng / ml cAMP, 0.2mM vitamin C, 10ng / ml BDNF and 10ng / ml GDNF to DMEM / F12 medium. The antibodies used in this experiment are as follows. Tuj1 (801202, BioLegend), MAP2 (AB5622, Millipore).

[0103] In addition, it was confirmed that the induced neural stem cells could differentiate into GABA neurons, motor neurons and dopamine neurons by immunostaining for GABA, HB8 and TH, respectively ( Fig.15 The antibodies used in this experiment are as follows: GABA (A2052, Sigma), HB9 (81.5C10, DSHB), TH (AB152, Millipore).

[0104] <5-2> Analysis of the ability to differentiate into glial cells

[0105] By immunostaining, S100β and GFAP staining confirmed that the induced neural stem cells can differentiate into astrocytes (astrocytes are a type of glial cells) ( Fig.16 ). The astrocyte differentiation method is as follows. The induced neural stem cells were cultured in an astrocyte differentiation medium for 14 days or longer and then analyzed. The astrocyte differentiation medium was supplemented with 2.5% FBS or 20 ng / ml CNTF and 10 ng / ml BMP4 in DMEM / F12 medium containing 1×N2 and 1×B27. The antibodies used in this experiment are as follows: S100β (S2535, Sigma), GFAP (SAB4501162, Sigma).

[0106] By immunostaining, PDGFR, OLIG2 and O4 staining confirmed that the induced neural stem cells could differentiate into oligodendrocytes (oligodendrocytes are a type of glial cells) ( Fig.17 ). The oligodendrocyte differentiation method is as follows. After the induced neural stem cells are cultured in a medium obtained by adding 1×N2, 1×B27, 25ug / ml insulin, 1uM Shh agonist and 100nM retinoic acid to DMEM / F12 medium for 5 days or more, the cells are cultured in a medium obtained by adding 1×N2, 1×B27, 25ug / ml insulin, 100ng / ml biotin, 60ng / ml T3, 10ng / ml PDGF-AA, 10ng / ml IGF-1, 5ng / ml HGF, 10ng / ml NT-3 and 1uM cAMP to DMEM / F12 medium for 7 days or more, and then analyzed. The antibodies used in this experiment are as follows: PDGFR (SC338, Santa Cruz), OLIG2 (AB9610, Millipore), O4 (MAB345, Millipore).

[0107] Example 6: Validation and analysis of a method for inducing neural stem cells without undergoing a pluripotent stage

[0108] It is known that the combination of the reprogramming factors OCT4, SOX2, KLF4, and GLIS1 genes used in the present invention can establish induced pluripotent stem cells under specific conditions (US Pat. No. 09862930).

[0109] However, it was verified that the neural stem cells induced in the present invention did not pass through the pluripotent stage for the following reasons.

[0110] First, a neural stem cell induction medium was used that did not contain bFGF, which is essential for establishing induced human pluripotent stem cells, and B18R protein, which is required for expressing mRNA of endogenous genes, and thus it was impossible to establish induced human pluripotent stem cells based on the research results reported so far.

[0111] Second, the time of the neural stem cells derived from the present invention is according to the scheme ( Figure 3 ) are formed about 8 days after the induction process of , and it is known that human pluripotent stem cells induced after the induction process require 3 weeks or more, so the time to go through the pluripotent stage is too short.

[0112] Third, when observed by date-based qRT-PCR analysis, the expression of pluripotent stage marker genes OCT4, REX1, and NANOG remained low from day 0 to day 12 ( Fig.18 The primer sequences used in this experiment are as follows: OCT4 (forward -GACAGGGGGAGGGGAGGAGCTAGG; SEQ ID NO: 27, reverse -CTTCCCTCCAACCAGTTGCCCCAAAC; SEQ ID NO: 28), REX1 (forward -CTGAAGAAACGGGCAAAGAC; SEQ ID NO: 29, reverse -GAACATTCAAGGGAGCTTGC; SEQ ID NO: 30), NANOG (forward -CAGCCCTGATTCTTCCACCAGTCCC; SEQ ID NO: 31, reverse -GGAAGGTTCCCAGTCGGGTTCACC; SEQ ID NO: 32).

[0113] Fourth, it was confirmed that when induced neural stem cells were verified by immunostaining, unlike H9 embryonic stem cells, the expression of OCT4 and NANOG was not detected ( Fig.19 The antibodies used in this experiment are as follows: OCT4 (SC5279, Santa Cruz), NANOG (AF1997, R&D Systems).

[0114] Based on the above points, it was confirmed that the method of the present invention can immediately induce urine cells into neural stem cells without going through the pluripotent stem cell stage.

[0115] Example 7: Experiment on improving the efficiency of neural stem cell induction

[0116] Furthermore, the culture conditions and the like that can improve the efficiency of transformation from urine cells to neural stem cells were confirmed.

[0117] Normal oxygen conditions (21% O2) were used as a control, and the induction was performed by adjusting the oxygen concentration. Specifically, 1×10 5 After the neural stem cell induction method was performed on urine-derived cells, the number of colonies positive for the neural stem cell marker genes SOX1 and PLZF was counted by immunostaining, and it was confirmed that the transformation efficiency was increased by about 2 times under hypoxic conditions ( Fig. 20 ). Sequence Listing <110> Star Technology <120> Direct reprogramming of urine cells into neural stem cells using synthetic mRNA <130> G21U13C0007P / CN <150> KR1020180101208 <151> 2018-08-28 <160> 32 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SOX1 forward <400> 1 acacttgaag cccagatgg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOX1 reverse <400> 2 ataggctcac ttttggacgg 20 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> SOX2 forward <400> 3 tcaggagttg tcaaggcaga ga 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SOX2 reverse <400> 4 ccgccgccga tgattgttat ta 22 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PAX6 forward <400> 5 ggctcaaatg cgacttcag 19 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PAX6 reverse <400> 6 cccttcgatt agaaaaccat acc 23 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PLZF forward <400> 7 tatacagcca cgctgcaagc ca 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PLZF reverse <400> 8 tggtctccag catcttcagg ca 22 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> EMX1 forward <400> 9 caggcccagg tagttcaatg gg 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> EMX1 reverse <400> 10 gctcagcctt aagccctgtc tc 22 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> FOXG1 forward <400> 11 acccgtcaat gacttcgcag ag 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> FOXG1 reverse <400> 12 agggttggaa gaagacccct ga 22 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OTX1 forward <400> 13 ctcaaacaac ccccatacgg ca 22 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse OTX1 <400> 14 ggtagcgagt cttggcgaag ag 22 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OTX2 forward <400> 15 ttcagggctg tgtgaattgt gtga 24 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse OTX2 <400> 16 cagaggtgga gttcaaggtt gcat 24 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> EN1 forward <400> 17 gagttccagg caaaccgcta ca 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> EN1 reverse <400> 18 gttgtacagt ccctgggcca tg 22 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GBX2 forward <400> 19 cggttagcag ccacctttcc at 22 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GBX2 reverse <400> 20 gacgtgcttc acatggctca ga 22 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HOXB2 forward <400> 21 gagacccagg agccaaaagc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HOXB2 reverse <400> 22 gaaggagacg tggcggattg 20 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HOXB4 forward <400> 23 ctgagggcca gaatgactgc tc 22 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HOXB4 reverse <400> 24 cagaactcaa ctggcccctc ac 22 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VEE forward <400> 25 acgaagggca agtcgctgtt 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VEE reverse <400> 26 tttcgtcggc ccagttggta 20 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OCT4 forward <400> 27 gacaggggga ggggaggagc tagg 24 <210> 28 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> OCT4 reverse <400> 28 cttccctcca accagttgcc ccaaac 26 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> REX1 forward <400> 29 ctgaagaaac gggcaaagac 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> REX1 reverse <400> 30 gaacattcaa gggagcttgc 20 <210> 31 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NANOG forward <400> 31 cagccctgat tcttccacca gtccc 25 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse NANOG <400> 32 ggaaggttcc cagtcgggtt cacc 24

Claims

1. A method for directly reprogramming urine cells into neural stem cells, the method comprising: (a) isolating urine cells from urine and culturing the urine cells; (b) introducing into the cultured urine cells a composition comprising: (i) Oct4 protein or a nucleic acid encoding the Oct4 protein; (ii) Sox2 protein or a nucleic acid encoding the Sox2 protein; (iii) Klf4 protein or a nucleic acid encoding the Klf4 protein; and (iv) Glis1 protein or a nucleic acid encoding the Glis1 protein; (c) inducing direct reprogramming into neural stem cells by culturing the urine cells into which the composition has been introduced in a neural stem cell induction medium, wherein the neural stem cell induction medium is obtained by mixing DMEM / F12 medium and neural basal medium in a volume ratio of 1:1 and adding 1×N2, 1×B27, human LIF, TGF-β inhibitor and GSK3-β inhibitor to the medium mixture; and (d) Selecting a neural stem cell line having characteristics similar to those of neural stem cells from cells that have been directly reprogrammed by a neural stem cell induction medium, said method not being a method for treating a disease.

2. The method according to claim 1, wherein: In step (b), the nucleic acid is a synthetic mRNA, and the introduction of the synthetic mRNA encoding the Oct4, Sox2, Klf4 and Glis1 uses electroporation to introduce the synthetic mRNA.

3. The method according to claim 1, wherein: The neural stem cell induction medium further comprises one or more of the following: a Shh agonist, an adenylate cyclase activator, a histone deacetylase inhibitor and ascorbic acid 2-phosphate.

4. The method according to claim 3, wherein: Cultivation in the neural stem cell induction medium was performed under O 2 -deficient conditions.

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