Direct retrodifferentiation of urinary cells into neural stem cells using synthetic messenger RNA

Abstract

The present invention relates to a method for inducing reverse differentiation of neural stem cells from urine cells by introducing mRNA of the reverse differentiation factors Oct4, Sox2, Klf4, and Glis1, and to a composition for preventing or treating nerve damage diseases, which contains neural stem cells induced by said method as an active ingredient.

Description

[Technical field]

[0001] The present invention relates to a method for direct retrodifferentiation of urine cells into neural stem cells, and a pharmaceutical composition for treating nerve injury diseases, comprising the directly retrodifferentiated neural stem cells produced by said method. [Background technology]

[0002] Stem cells are cells that have unlimited self-renewal capacity and the ability to differentiate into specific cells and tissues required in the body. Stem cells are classified into three types: embryonic stem cells (ES cells) isolated from early embryos, embryonic germ cells (EG cells) isolated from primordial germ cells during the embryonic stage, and multipotent adult progenitor cells (MAPC cells).

[0003] Stem cells have the potential to differentiate into cells with specialized functions, and as such, they have been the subject of research into cell therapy to restore the functions of various organs. Recently, the scope of their use has been expanding to include plastic surgery and beauty treatments.

[0004] The roles of adult stem cells in the body can be broadly summarized into two. The first is that stem cells themselves differentiate into and regenerate damaged tissues and cells in our bodies. The second is that they continuously secrete proteins such as growth factors and cytokines throughout our lives to help the growth and regeneration of nearby cells.

[0005] Direct reprogramming is a technology that can convert somatic cells into other types of cells. Unlike the reverse differentiation reprogramming of Professor Yamanaka in Japan, it does not go through the state of pluripotent stem cells, so it is highly efficient in a short time, and it can solve the risk of tumor development and additional differentiation costs of pluripotent stem cells. However, the current direct reverse differentiation method mainly uses mouse cells as the cell source, so the probability of reproducing identical human somatic cells is low. In addition, skin cells (fibroblasts) are often used among human somatic cells, but in this case, an invasive method of cell source collection is required, which causes pain and safety risks for the donor and reduces convenience. In addition, the reverse differentiation factor (gene) introduction method required for direct reverse differentiation mainly uses viruses, making it difficult to use clinically, and in the case of a gene insertion system using viruses, it induces mutations due to indiscriminate insertion into the dielectric, making it unsuitable for treatment.

[0006] Therefore, the inventors solved the above problems by using human urine-derived cells, which are easy to collect, as a cell source and using transfection of synthetic mRNA to introduce reverse differentiation factors into cells, and confirmed the optimal culture conditions required for highly efficient direct reverse differentiation of urine cells into neural stem cells, thereby completing the present invention. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Korean Patent Publication No. 10-2013-0087020 Summary of the Invention [Problem to be solved by the invention]

[0008] It is an object of the present invention to provide a composition that induces retrodifferentiation of urine cells into neural stem cells.

[0009] Another object of the present invention is to provide a method for inducing retrodifferentiation of urine cells into neural stem cells.

[0010] A further object of the present invention is to provide a pharmaceutical composition for preventing or treating nerve damage diseases, which contains neural stem cells retrodifferentiated from urine cells as an active ingredient. [Means for solving the problem]

[0011] The present invention relates to (i) an Oct4 protein or a nucleic acid encoding an Oct4 protein; (ii) a Sox2 protein or a nucleic acid encoding a Sox2 protein; (iii) a Klf4 protein or a nucleic acid encoding a Klf4 protein; and (iv) A composition for inducing direct retrodifferentiation of urine cells into neural stem cells is provided, the composition comprising a Glis1 protein or a nucleic acid encoding a Glis1 protein.

[0012] In the present invention, the term "reverse-differentiated neural stem cells" includes cells prepared by using a reverse differentiation technique on differentiated cells to establish undifferentiated stem cells having similar or identical multi-potency to neural stem cells. Induced neural stem cells have the same or similar characteristics as neural stem cells, specifically, they show similar cell morphology, have similar gene and protein expression patterns, and can have multi-potency in and out of the body. Thus, the reverse-differentiated neural stem cells of the present invention may be capable of differentiating into nerve cells (neurons), astrocytes, oligodendrocytes, GABAergic neurons, dopaminergic neurons, etc.

[0013] In the present invention, the term "urine cells (UCs)" is known as somatic cells which can be easily and repeatedly obtained from urine at any time without any inconvenience or pain.

[0014] The present inventors discovered that when the de-differentiation factors Oct4, Sox2, Klf4 and Glis1 are expressed in urinary cells, the already differentiated cell type of urinary cells is de-differentiated into neural stem cells with differentiation potential.

[0015] The term "reverse differentiation factor" originated from the concept of reverse differentiation (reprogramming) introduced by Professor Yamanaka's team in 2006. All cells in an adult undergo normal developmental processes, gradually differentiating from an undifferentiated state and changing into cells with specialized functions. Among them, cells of a fertilized egg have totipotency, and when they progress to a blastocyst stage, they can be divided into an inner cell mass and outer cells. At this time, the inner cell mass cells can develop into embryonic cells and germ cells, which is called pluripotency. These embryonic stem cells show gene expression patterns specific to pluripotency, and representative examples include Oct4, Sox2, Nanog, and Lin28. Reverse differentiation can be said to be a technology that induces such specific gene expression in somatic cells and returns them to properties similar to undifferentiated cells such as embryonic stem cells and adult stem cells. Oct4, Sox2, Klf4 and Glis1 were used by the present inventors to induce retrodifferentiation of urinary cells into neural stem cells as part of various factors that have been used in a series of studies related to retrodifferentiation.

[0016] 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 such as horses, sheep, pigs, goats, camels, antelopes and dogs, and are preferably human Oct4, Sox2, Klf4 and Glis1. Furthermore, the Oct4, Sox2, Klf4 and Glis1 proteins of the present invention used for retrodifferentiation into neural stem cells may include not only proteins having the wild-type amino acid sequences but also mutants of Oct4, Sox2, Klf4 and Glis1 proteins.

[0017] In one specific embodiment, the gene sequences of Oct4, Sox2, Klf4, and Glis1, which were first used in the retrodifferentiation, were those disclosed in Nature. 2011 Jun 8; 474(7350):225-9.

[0018] Mutants of Oct4, Sox2, Klf4 and Glis1 proteins refer to proteins having sequences that differ from the native amino acid sequences of Oct4, Sox2, Klf4 and Glis1 by deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues, or a combination thereof. The mutants may be functional equivalents that exhibit the same biological activity as the native proteins, or may be mutants in which the physicochemical properties of the proteins have been altered, as necessary. Mutants have increased structural stability against physical and chemical environments, or increased physiological activity.

[0019] The nucleic acids consisting of nucleotide sequences encoding the Oct4, Sox2, Klf4 and Glis1 proteins are nucleic acids consisting of nucleotide sequences encoding wild-type or mutant forms of the Oct4, Sox2, Klf4 and Glis1 proteins as described above, in which one or more bases may be mutated by substitution, deletion, insertion or a combination thereof, and may be isolated from nature or produced using a chemical synthesis method.

[0020] The nucleic acid having a nucleotide sequence encoding the Oct4, Sox2, Klf4 and Glis1 proteins may be single-stranded or double-stranded, and may be a DNA molecule (genomic, cDNA) or an RNA molecule. In a specific embodiment of the present invention, the nucleic acid encoding the Oct4, Sox2, Klf4 and Glis1 proteins is a synthetic messenger RNA (mRNA), which may be produced using a synthetic mRNA expression vector into which DNA expressing Oct4, Sox2, Klf4 and Glis1 is introduced.

[0021] The present invention provides a method for directly inducing retrodifferentiation of urinary cells into neural stem cells comprising the step of introducing into urinary cells Oct4, Sox2, Klf4 and Glis1 proteins or nucleic acids encoding said proteins.

[0022] More specifically, (a) isolating and culturing urinary cells from urine; (b) introducing Oct4, Sox2, Klf4 and Glis1 proteins or nucleic acids encoding said proteins into said cultured urine cells; (c) culturing the urinary cells into which the composition has been introduced in a neural stem cell induction medium to directly induce reverse differentiation into neural stem cells; (d) selecting a neural stem cell line having the characteristics of neural stem cells from the cells directly induced to reversely differentiate into neural stem cells.

[0023] In the step (a), the urine cells may be cultured in a medium containing fetal bovine serum (FBS), basic fibroblast growth factor (bFGF), and epithelial growth factor (EGF).

[0024] The term "culture medium" as used in the present invention means a medium capable of supporting cell growth and survival in vitro, and includes all common media used in the art suitable for induction and culture of urine cells and dedifferentiated neural stem cells. The medium and culture conditions can be selected according to the type of cells. The medium used for culture is preferably a cell culture minimum medium (CCMM), which generally contains a carbon source, a nitrogen source, and trace element components. Examples of such cell culture minimum media include, but are not limited to, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (α Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), and IMEM (Iscove's Modified Dulbecco's Medium). The medium may also contain antibiotics such as penicillin, streptomycin, or gentamicin.

[0025] In a specific embodiment of the present invention, the cells isolated from urine can be cultured in a basic medium containing FBS, bFGF and EGF, and more specifically, can be cultured by adding bFGF and EGF to high glucose DMEM and REGM (Renal Epithelial Cell Growth Medium) medium containing FBS. More specifically, the high glucose DMEM and REGM medium may further contain L-glutamine and penicillin-streptomycin.

[0026] In the step (b), the method of introducing the retrodifferentiation factor into the urine cells can be any method commonly used in the art for providing a nucleic acid molecule or a protein to cells, without limitation, and preferably, the method of administering the retrodifferentiation factor to the culture medium of the differentiated cells or the method of directly injecting the retrodifferentiation factor into the differentiated cells can be used, and the retrodifferentiation factor used here can be in the form of a virus obtained from packaging cells transfected with a viral vector containing the gene of the factor, messenger RNA produced by in vitro transcription, or protein produced in various cell lines. In a specific embodiment of the present invention, the retrodifferentiation factor was introduced into the urine cells using messenger RNA produced by transcription of DNA encoding Oct4, Sox2, Klf4, and Glis1.

[0027] The method of directly injecting the transcribed messenger RNA into differentiated cells may be any method known in the art, including, but not limited to, microinjection, electroporation, particle bombardment, direct muscle injection, insulators, and transposons. Specifically, in the embodiments of the present invention, the messenger RNA of the retrodifferentiation factor was introduced into urine cells using electroporation.

[0028] In the step (c), the medium for inducing the urine cells into which the nucleic acids encoding Oct4, Sox2, Klf4 and Glis1 proteins have been introduced (neural stem cell induction medium) into neural stem cells may be a medium obtained by mixing DMEM / F12 medium and Neurobasal medium in a volume ratio of 1:1, and adding 1xN2, 1xB27, human LIF, TGF-beta inhibitor and GSK3-beta inhibitor to the medium mixture. More specifically, the medium may further include one or more of Shh agonist, adenylyl cyclase activator, histone deacetylase inhibitor and ascorbic acid 2-phosphate. In a specific embodiment of the present invention, it was confirmed that the neural stem cell conversion rate was the best in a medium to which all of Shh agonist, adenylyl cyclase activator, histone deacetylase inhibitor and ascorbic acid 2-phosphate were added.

[0029] Furthermore, when the culture of urinary cells in the medium is performed under hypoxic (O2 hypoxia) conditions, the induction efficiency can be increased.

[0030] The selection of the induced neural stem cells in step (d) is performed by collecting the neural stem cell colonies generated after performing step (C), and the selected neural stem cells can be cultured in a neural stem cell medium. The neural stem cell medium may be a medium in which DMEM / F12 medium and Neurobasal medium are mixed in a volume ratio of 1:1, to which 1xN2, 1xB27, human LIF, TGF-beta inhibitor and GSK3-beta inhibitor are added. Furthermore, the induced neural stem cells can be subcultured using 0.5 mM EDTA solution or Accutase solution.

[0031] The present invention also provides a pharmaceutical composition for preventing or treating nerve damage, comprising the neural stem cells induced by the above method as an active ingredient.

[0032] Neural stem cells are multipotent cells that can differentiate into nerve cells (neurons), astrocytes, oligodendrocytes, GABAergic neurons, dopaminergic neurons, etc., and can repair damaged or lost nerve cells, making it possible to treat diseases caused by damage or loss of such nerve cells without any restrictions.

[0033] Specifically, the disease caused by damage to nerve cells may be selected from the group consisting of Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotriophic lateral sclerosis, ischemic stroke, demyelinating disease, multiple sclerosis, epilepsy, degenerative neurological diseases, and spinal cord injury. Effect of the Invention

[0034] The present invention is a technology that utilizes human urine-derived cells as a cell source, maximizing the convenience of securing a cell source, and increasing the possibility of clinical use through a non-integration direct reverse differentiation method using synthetic mRNA. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 is a schematic diagram of the T7-VEE-OKS-iG vector for producing synthetic mRNA of OCT4, KLF4, SOX2 and GLIS1 genes. [Diagram 2] FIG. 2 shows the results of FACS analysis to confirm whether or not synthetic mRNA of OCT4, KLF4, SOX2 and GLIS1 genes linked with GFP (fluorescent gene) was introduced into urine cells. [Diagram 3] FIG. 3 is a schematic diagram of the entire process of obtaining induced neural stem cell colonies using the direct reverse differentiation technique from urine cells. [Figure 4]Figure 4 shows the results of mRNA level analysis by RT-PCR using neural stem cells derived from H9 embryonic stem cells as a positive control to confirm whether the induced neural stem cells express neural stem cell marker genes such as SOX1, SOX2, PAX6, and PLZF. [Diagram 5] FIG. 5 shows the results of immunoassay to confirm whether the induced neural stem cells express neural stem cell marker genes such as SOX1, NESTIN, SSEA1, SOX2, PAX6, and PLZF at the protein level. [Figure 6] FIG. 6 shows the results of confirming whether the induced neural stem cells have the differentiation ability through Ki67 immunostaining. [Figure 7] Figure 7 shows the results of quantitative analysis of mRNA by qRT-PCR to confirm whether neural stem cells induced from H9 embryonic stem cells express neural stem cell marker genes such as SOX1, SOX2, PLZF, and PAX6. [Figure 8] Figure 8 shows the results of RT-PCR mRNA level analysis to confirm whether the induced neural stem cells express marker genes specific to the forebrain, midbrain, hindbrain, and spinal cord in developmental biology. [Figure 9a] Figures 9 and 10 show the results of total RNA sequencing to confirm whether the induced neural stem cells show similar mRNA and long non-coding RNA (lnc-RNA) expression patterns at the global gene expression level as neural stem cells derived from H9 embryonic stem cells rather than from the original urine-derived cells. [Figure 9b]Figures 9 and 10 show the results of total RNA sequencing to confirm whether the induced neural stem cells show similar mRNA and long non-coding RNA (lnc-RNA) expression patterns at the global gene expression level as neural stem cells derived from H9 embryonic stem cells rather than from the original urine-derived cells. [Figure 9c] Figures 9 and 10 show the results of total RNA sequencing to confirm whether the induced neural stem cells show similar mRNA and long non-coding RNA (lnc-RNA) expression patterns at the global gene expression level as neural stem cells derived from H9 embryonic stem cells rather than from the original urine-derived cells. [Figure 9d] Figures 9 and 10 show the results of total RNA sequencing to confirm whether the induced neural stem cells show similar mRNA and long non-coding RNA (lnc-RNA) expression patterns at the global gene expression level as neural stem cells derived from H9 embryonic stem cells rather than from the original urine-derived cells. [Figure 10a] Figures 9 and 10 show the results of total RNA sequencing to confirm whether the induced neural stem cells show similar mRNA and long non-coding RNA (lnc-RNA) expression patterns at the global gene expression level as neural stem cells derived from H9 embryonic stem cells rather than from the original urine-derived cells. [Figure 10b]Figures 9 and 10 show the results of total RNA sequencing to confirm whether the induced neural stem cells show similar mRNA and long non-coding RNA (lnc-RNA) expression patterns at the global gene expression level as neural stem cells derived from H9 embryonic stem cells rather than from the original urine-derived cells. [Figure 10c] Figures 9 and 10 show the results of total RNA sequencing to confirm whether the induced neural stem cells show similar mRNA and long non-coding RNA (lnc-RNA) expression patterns at the global gene expression level as neural stem cells derived from H9 embryonic stem cells rather than from the original urine-derived cells. [Figure 10d] Figures 9 and 10 show the results of total RNA sequencing to confirm whether the induced neural stem cells show similar mRNA and long non-coding RNA (lnc-RNA) expression patterns at the global gene expression level as neural stem cells derived from H9 embryonic stem cells rather than from the original urine-derived cells. [Figure 11] Figure 11 shows the results of RT-PCR of the VEE gene and genomic DNA PCR analysis to determine whether the synthetic mRNA introduced into the induced neural stem cells remains in the host and is integrated into the host matrix. [Figure 12] FIG. 12 shows the results of STR analysis to confirm whether the induced neural stem cells were derived from human urine cells. [Figure 13] FIG. 13 shows the results of karyotype analysis to confirm whether the induced neural stem cells retain normal chromosomes. [Figure 14]FIG. 14 shows the results of confirming the expression of neuronal gene markers TUJ1 and MAP2 by immunological screening in order to verify the differentiation ability of the induced neural stem cells into neurons. [Figure 15] FIG. 15 shows the results of immunostaining confirming that the induced neural stem cells can differentiate into GABA neurons, motor neurons, and dopamine neurons. [Figure 16] FIG. 16 shows the results of confirming, by immunological screening, the expression of S100beta and GFAP, which are markers for verifying the differentiation ability of induced neural stem cells into astrocytes, which are neuroglial cells. [Figure 17] FIG. 17 shows the results of confirming, by immunodetection, the expression of PDGFR, OLIG2, and O4, which are markers for verifying the differentiation ability of induced neural stem cells into oligodendrocytes, which are neuroglial cells. [Figure 18] FIG. 18 shows 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. [Figure 19] FIG. 19 shows the results of confirming the expression of pluripotency stage marker genes in H9 embryonic stem cells and induced neural stem cells. [Figure 20] FIG. 20 shows the results of comparing the conversion rate when urine-derived cells are induced to neural stem cells under hypoxic conditions (5% hypoxia) with basic conditions (21% O 2 ) as a control group. [Figure 21]21 shows the results of confirming that the induction efficiency of neural stem cells is increased when the medium further contains one or more of Shh agonist, adenylyl cyclase activator, histone deacetylase inhibitor, and ascorbic acid 2-phosphate in a medium in which DMEM / F12 medium and Neurobasal medium are mixed at a volume ratio of 1:1 and the mixture is supplemented with 1xN2, 1xB27, human LIF, TGF-beta inhibitor, and GSK3-beta inhibitor. It was confirmed that the neural stem cell conversion rate was the best in the medium supplemented with all of Shh agonist, adenylyl cyclase activator, histone deacetylase inhibitor, and ascorbic acid 2-phosphate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The present invention will be described in more detail with reference to the following examples. However, the following examples are provided for easier understanding of the present invention, and the present invention is not limited to the following examples.

[0037] Example 1: Isolation of urinary cells from urine The method is based on a technique developed by Sutherland and Bain in the UK in 1972, and is specifically as follows: First, urine provided by a donor was centrifuged at 1000g for 10 minutes. After removing the supernatant, the pellet remaining at the bottom was diluted with 20ml of PBS solution containing 1% Penicillin / Streptomycin / Amphotericin B antibiotics. Next, the diluted PBS + pellet solution was centrifuged at 1000g for 10 minutes. After removing the supernatant again, the pellet remaining at the bottom was diluted with 1ml of basic medium (medium based on DMEM / F12 containing 1% Penicillin / Streptomycin / Amphotericin B antibiotics, 1% L-glutamine, and 10% FBS) and seeded on a gelatin-coated 12-well cell culture dish. After that, 1 ml of basic medium was added and the cells were cultured for 3 days, after which the medium was changed to growth medium (a medium based on a 1:1 mixture of DMEM and REGM, containing 1% Penicillin / Streptomycin antibiotics, 1% L-glutamine, 5% FBS, 10 ng / ml bFGF, and 10 g / ml EGF) and cultured.

[0038] Example 2: Introduction of retrodifferentiation factors into urinary cells using electroporation The method of introducing synthetic mRNA into the urine-derived cells cultured in Example 1 by electroporation is as follows. Synthetic mRNA is synthesized using a conventional in vitro transcription kit (RiboMAX, Large Scale RNA Production Systems, Promega), and the DNA that serves as the backbone of the synthesized mRNA is T7-VEE-OKS-iG (Steven Dowdy, Addgene), which contains the genes OCT4, KLF4, SOX2, and GLIS1 (Figure 1). The sequences of the OCT4, KLF4, SOX2, and GLIS1 genes are those disclosed in Nature. 2011 Jun 8; 474 (7350): 225-9. The gene sequences and the mRNA application method were as follows: Cell Stem cell. 2013 Aug 1; 13 (2): 246-54 and US Patent US 9,862,930 B2.

[0039] The synthesized mRNA was transfected into 1x10^6 urine-derived cells pretreated with 0.2ug / ml B18R protein for 1 hour by electroporation at 1600V, 10ms, 3 times. The transfected urine-derived cells were cultured for 2 days in growth medium containing 0.2ug / ml B18R protein.

[0040] In order to visualize and evaluate the efficiency of electroporation, the present inventors simultaneously introduced synthetic mRNA containing the GFP fluorescent gene and examined the efficiency of human urine-derived cells expressing GFP fluorescent protein through FACS analysis. Through this examination, it was confirmed that the efficiency of electroporation-based synthetic mRNA introduction into human urine-derived cells reached 72.2% at about 48 hours after treatment, and it was determined that this was sufficient for the next step, the neural stem cell induction experiment (Figure 2).

[0041] Example 3: Induction of urine-derived dedifferentiated neural stem cells Matrigel TMAfter seeding the urine-derived cells with mRNA on a cell culture dish coated with 1xN2 (Gibco), 1xB27 (Gibco), 10ng / ml human LIF, 2uM SB431542 (TGF-beta inhibitor) and 3uM CHIR99021 (GSK3-beta inhibitor)), the cells were cultured for 7-10 days in neural stem cell induction medium supplemented with 0.5uM purmorphamine (Shh agonist), 10uM folskolin (adenylyl cyclase activator), 100uM sodium butyrate (histone deacetylase inhibitor) and 64ug / ml ascorbic acid 2-phosphate. At the completion of induction, the generation of neural stem cell colonies was confirmed (Figure 3).

[0042] In addition, we investigated the effect of adding one or more of Shh agonist, adenylyl cyclase activator, histone deacetylase inhibitor, and ascorbic acid 2-phosphate to the medium supplemented with 1xN2, 1xB27, human LIF, TGF-beta inhibitor, and GSK3-beta inhibitor on the induction efficiency of neural stem cells. We confirmed that the medium supplemented with all of Shh agonist, adenylyl cyclase activator, histone deacetylase inhibitor, and ascorbic acid 2-phosphate had the highest neural stem cell conversion rate (Figure 21).

[0043] The induced neural stem cell colonies were harvested and cultured in neural stem cell medium on Matrigel-coated cell culture dishes. The induced neural stem cells were then subcultured using 0.5 mM EDTA solution or Accutase solution, and experiments were carried out to verify various characteristics of the induced neural stem cells in the following examples.

[0044] Example 4: Molecular biological characterization of induced neural stem cells An experiment was carried out to verify the molecular biological properties of the neural stem cells derived from the present invention.

[0045] <4-1> Using H9 embryonic stem cell-derived neural stem cells as a positive control, we confirmed through RT-PCR mRNA level analysis that the induced neural stem cells expressed neural stem cell marker genes such as SOX1, SOX2, PAX6, and PLZF (Figure 4). The 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; sequence number 6), PLZF (forward-TATACAGCCACGCTGCAAGCCA; sequence number 7, reverse-TGGTCTCCAGCATCTTCAGGCA; sequence number 8).

[0046] <4-2> Protein level analysis using immunostaining confirmed that the induced neural stem cells expressed neural stem cell marker genes such as SOX1, NESTIN, SSEA1, SOX2, PAX6, and PLZF (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), and PLZF (AF2944, R&D Systems).

[0047] <4-3> The induced neural stem cells were confirmed to have the ability to divide through immunostaining for Ki67 (Figure 6). The antibodies used in this experiment were as follows: Ki67 (Ab9260, Millipore)

[0048] <4-4> Quantitative analysis of mRNA by qRT-PCR confirmed that the induced neural stem cells from H9 embryonic stem cells expressed neural stem cell marker genes such as SOX1, SOX2, PLZF, and PAX6 (Figure 7).

[0049] <4-5> Analysis of mRNA levels by RT-PCR confirmed that the induced neural stem cells expressed marker genes specific to 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), 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).

[0050] <4-6> Through total RNA sequencing, we confirmed that the induced neural stem cells showed mRNA and lncRNA expression patterns similar to those of neural stem cells derived from H9 embryonic stem cells rather than those of the original urine-derived cells at the global gene expression level (Figures 9 and 10).

[0051] <4-7> Through RT-PCR and genomic DNA PCR analysis of the VEE gene, it was confirmed that the synthetic mRNA introduced into the induced neural stem cells no longer remained and was not integrated into the host matrix (Figure 11). The primer sequences used in this experiment are as follows: VEE (forward-ACGAAGGGCAAGTCGCTGTT; SEQ ID NO: 25, reverse-TTTCGTCGGCCCAGTTGGTA; SEQ ID NO: 26).

[0052] <4-8> STR analysis confirmed that the human urine-derived cells and the induced neural stem cells were derived from the same individual (Figure 12).

[0053] <4-9> Karyotype analysis confirmed that the induced neural stem cells retained normal chromosomes (Figure 13).

[0054] Example 5: Differentiation potential analysis of induced neural stem cells <5-1> Analysis of differentiation ability into neural cells When the induced neural stem cells were differentiated into neurons, the expression of the neural cell marker genes TUJ1 and MAP2 was confirmed by immunostaining (Figure 14). The neural cell differentiation method is as follows: neural stem cells are cultured in a neural cell differentiation medium containing DMEM / F12 medium supplemented with 1xN2, 1xB27, 300ng / ml cAMP, 0.2mM vitamin C, 10ng / ml BDNF, and 10ng / ml GDNF for 14 days, and then analyzed. The antibodies used in this experiment are as follows: Tuj1 (801202, BioLegend), MAP2 (AB5622, Millipore).

[0055] In addition, the induced neural stem cells were able to differentiate into GABAergic neurons, motor neurons, and dopamine neurons by immunohistochemistry, staining for GABA, HB9, and TH, respectively (Figure 15). The antibodies used in this experiment were as follows: GABA (A2052, Sigma), HB9 (81.5C10, DSHB), and TH (AB152, Millipore).

[0056] <5-2> Analysis of differentiation ability into glial cells Through immunostaining of S100beta and GFAP, it was confirmed that the induced neural stem cells could differentiate into astrocytes, a type of neuroglial cell (Figure 16). The method for differentiation of astrocytes is as follows: DMEM / F12 medium containing 1xN2 and 1xB27, supplemented with 2.5% FBS or 20ng / ml CNTF and 10ng / ml BMP4, was used for culturing for 14 days or more, and then analyzed. The antibodies used in this experiment were as follows: S100beta (S2535, Sigma), GFAP (SAB4501162, Sigma).

[0057] Through immunostaining of PDGFR, OLIG2, and O4, it was confirmed that the induced neural stem cells could differentiate into oligodendrocytes, a type of neuroglial cell (Figure 17). The differentiation method for oligodendrocytes is as follows: After culturing for more than 5 days in DMEM / F12 medium supplemented with 1xN2, 1xB27, 25ug / ml insulin, 1uM Shh agonist, and 100nM retinoic acid, the cells were then cultured for more than 7 days in DMEM / F12 medium supplemented with 1xN2, 1xB27, 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, and then analyzed. Antibodies used in this study were as follows: PDGFR (SC338, Santacruz), OLIG2 (AB9610, Millipore), O4 (MAB345, Millipore).

[0058] Example 6: Verification and analysis of neural stem cell induction method without going through the pluripotency stage The combination of the retrodifferentiation factors OCT4, SOX2, KLF4, and GLIS1 genes used in the present invention is known to be capable of establishing induced pluripotent stem cells under certain conditions (US Pat. No. 09,862,930).

[0059] However, it was verified that the neural stem cells induced in the present invention do not pass through a pluripotent stage for the following reasons.

[0060] First, the present invention uses a neural stem cell induction medium that does not contain bFGF, which is known to be essential for establishing human induced pluripotent stem cells, and B18R protein, which is necessary for the expression of endogenous gene mRNA. Therefore, if one follows the research results reported to date, it would be impossible to establish human induced pluripotent stem cells.

[0061] Second, the time period in which the induced neural stem cells in the present invention are formed is approximately 8 days after the induction process according to the protocol (Figure 3), whereas known human induced pluripotent stem cells are formed after more than 3 weeks after the induction process, which is too short a time for them to have passed through the pluripotency stage.

[0062] Third, the expression of the pluripotency stage marker genes OCT4, REX1, and NANOG, as seen through date-specific qRT-PCR analysis, maintained low expression 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).

[0063] Fourth, when the induced neural stem cells were examined by immunostaining, it was confirmed that, unlike H9 embryonic stem cells, the expression of OCT4 and NANOG was not detected (Figure 19). The antibodies used in this experiment were as follows: OCT4 (SC5279, Santacruz), NANOG (AF1997, R&D systems).

[0064] Based on the above evidence, it was confirmed that the method of the present invention can directly induce urine cells into neural stem cells without passing through the stage of pluripotent stem cells.

[0065] Example 7: Experiment to increase induction efficiency of neural stem cells We further confirmed the culture conditions that can improve the conversion efficiency of urine cells to neural stem cells.

[0066] Normal oxygen conditions (21% O2) were used as the control group, and induction was performed by adjusting the oxygen concentration. Specifically, 1x10^5 urine-derived cells were subjected to neural stem cell induction under each basic condition (21% O2) and hypoxic condition (5% hypoxia), and then the number of positive colonies for neural stem cell marker genes SOX1 and PLZF were counted using immunostaining, confirming that the conversion efficiency increased by about 2-fold under hypoxic conditions (Figure 20).

Claims

1. (i) a nucleic acid encoding an Oct4 protein; (ii) a nucleic acid encoding a Sox2 protein; and (iii) a nucleic acid encoding a Klf4 protein; and (iv) a nucleic acid encoding a Glis1 protein; and (v) a neural stem cell induction medium obtained by mixing a DMEM / F12 medium and a Neurobasal medium in a volume ratio of 1:1, the neural stem cell induction medium comprising N2, B27, human LIF, a TGF-beta inhibitor, a GSK3-beta inhibitor, an Shh agonist, an adenylyl cyclase activator, a histone deacetylase inhibitor and ascorbic acid 2-phosphate; 13. A composition for inducing direct retrodifferentiation of urine cells into neural stem cells, comprising:

2. The composition for inducing direct retrodifferentiation of urinary cells into neural stem cells according to claim 1 , wherein the urinary cells are exfoliated somatic cells contained in urine.

3. The composition for inducing direct retrodifferentiation of urinary cells to neural stem cells according to claim 1 , wherein the nucleic acids encoding the Oct4, Sox2, Klf4 and Glis1 proteins are synthetic mRNAs.

4. (a) isolating and culturing urinary cells from urine; (b) introducing into said cultured urine cells a composition according to claim 1 or 3; (c) culturing the urine cells transfected with the composition in a neural stem cell induction medium for 7-10 days to directly induce retrodifferentiation into neural stem cells; (d) selecting a neural stem cell line having neural stem cell-like properties from the cells directly induced to undergo reverse differentiation using the neural stem cell induction medium; Including, In the step (c), the neural stem cell induction medium is obtained by mixing DMEM / F12 medium and Neurobasal medium in a volume ratio of 1:1, and adding N2, B27, human LIF, TGF-beta inhibitor, GSK3-beta inhibitor, Shh agonist, adenylyl cyclase activator, histone deacetylase inhibitor, and ascorbic acid 2-phosphate to the medium mixture. A method for inducing direct retrodifferentiation of urine cells into neural stem cells.

5. 5. The method for inducing direct reverse differentiation of urine cells into neural stem cells according to claim 4, wherein in step (b), the synthetic mRNAs encoding Oct4, Sox2, Klf4 and Glis1 proteins are introduced using electroporation.

6. The culture in the neural stem cell induction medium is in a hypoxic (O ２ The method for inducing direct retrodifferentiation of urinary cells into neural stem cells according to claim 4, wherein the method is carried out under conditions of hypoxia.

7. A pharmaceutical composition for preventing or treating a nerve cell injury disease, comprising as an active ingredient directly reverse-differentiated neural stem cells produced by the method according to any one of claims 4 to 6, wherein the directly reverse-differentiated neural stem cells express genes encoding EN1 and OTX2 proteins.

8. The pharmaceutical composition according to claim 7, wherein the nerve cell damaging disease is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, ischemic stroke, demyelinating disease, multiple sclerosis, epilepsy, degenerative neurological disease, and spinal cord injury.

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