Method for direct transdifferentiation of somatic cells

Abstract

To provide a simple, reproducible, and highly efficient production method for direct transdifferentiation of somatic cells into other somatic cells in a short period of time.SOLUTION: A production method for direct transdifferentiation from the somatic cells into other somatic cells comprises: (a) introducing a GLIS family gene, a mutant GLIS family gene, or a gene product thereof into somatic cells; and (b) culturing the gene-introduced somatic cells in a medium containing a component that induces differentiation from the somatic cells or precursor cells of the somatic cells into other somatic cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing somatic cells by direct transdifferentiation of the somatic cells into other somatic cells. [Background technology]

[0002] Somatic cells such as adipocytes, neurons, cardiac myocytes, and hepatocytes are expected to be used as materials for regenerative medicine and for screening of diseases related to these cells. Therefore, there is a strong demand for the development of methods for preparing these somatic cells in large quantities in vitro.

[0003] Methods for producing these somatic cells using embryonic stem cells (hereinafter sometimes referred to as "ES cells") or induced pluripotent stem cells (hereinafter sometimes referred to as "iPS cells") have been proposed. However, these methods require the preparation of a culture environment, such as adding various inhibitors involved in development and differentiation to the cell culture medium, which is problematic in terms of complexity and the lack of reproducibility. Furthermore, these methods also produce cells other than the target somatic cells, which poses a problem in terms of efficiency. Furthermore, it takes at least 21 to 30 days to obtain the target somatic cells, making them difficult to produce in a short period of time. Furthermore, methods have been reported for inducing differentiation of these somatic cells, such as adipocytes, by culturing fibroblasts, mesenchymal stem cells, or precursor cells of these somatic cells in a medium containing components involved in transcription factors. However, these differentiation induction methods have low differentiation induction efficiency and are problematic in terms of efficiency, and therefore have not yet been adopted as a means for mass production of somatic cells.

[0004] GLIS family member GLIS1 (GLIS family zinc finger 1) is known to improve the efficiency of iPS cell establishment (see, for example, Patent Document 1). GLIS family member GLIS3 (GLIS family zinc finger 3) is known to be useful for inducing differentiation of human multipotent or pluripotent cells into functional pancreatic β cells that produce insulin (see, for example, Patent Document 2). Furthermore, the present inventors have discovered that somatic cells can be directly transdifferentiated into pancreatic secretory cells by introducing a GLIS family gene and a Neurogenin3 gene, or by introducing a GLIS family gene, a Neurogenin3 gene, and a Pdx1 gene into somatic cells, and have filed patent applications for this discovery (Patent Documents 3 and 4). However, it has not been known that GLIS family genes such as GLIS1 are solely involved in the direct conversion of somatic cells into somatic cells other than pancreatic secretory cells without going through stem cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2013-519371 [Patent Document 2] Special Publication No. 2009-533047 [Patent Document 3] International Publication No. 2016 / 002937 Brochure [Patent Document 4] International Publication No. 2017 / 073740 Brochure Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a production method for direct transdifferentiation of somatic cells into other somatic cells, which is simple, highly reproducible, highly efficient in production, and can be carried out in a short period of time. [Means for solving the problem]

[0007] The present inventors have therefore conducted extensive research into the effect of GLIS family genes alone on the transdifferentiation of somatic cells, and have found, quite unexpectedly, that the efficiency of transdifferentiation of somatic cells into other somatic cells is dramatically improved when somatic cells into which GLIS family genes have been introduced are cultured in a medium containing a differentiation-inducing component for those somatic cells. They have also found that the efficiency of transdifferentiation of somatic cells into other somatic cells is dramatically improved when somatic cells into which GLIS family genes and transcription factors have been introduced are cultured in a medium containing a growth factor for those somatic cells, and have thus completed the present invention.

[0008] That is, the present invention provides the following [1] to

[14] . [1] (a) introducing a GLIS family gene, a mutant GLIS family gene, or a gene product thereof into a somatic cell; (b) culturing the transfected somatic cells in a medium containing a component for inducing differentiation of the somatic cells or precursor cells of the somatic cells into other somatic cells; A method for producing somatic cells by direct transdifferentiation of the somatic cells into other somatic cells, comprising: [2] (c) introducing a GLIS family gene, a mutant GLIS family gene, or a gene product thereof and a transcription factor into a somatic cell; and (d) culturing the transfected somatic cells in a medium containing growth factors for other somatic cells; A method for producing somatic cells by direct transdifferentiation of the somatic cells into other somatic cells, comprising: [3] The method of [1] or [2], wherein the GLIS family gene is the GLIS1 gene. [4] The method according to any one of [1] to [3], wherein the mutant GLIS family gene is a gene encoding a protein in which some amino acid residues on the N-terminal side of the GLIS1 protein have been deleted. [5] The method of any one of [1] to [4], wherein the mutant GLIS family gene is a gene encoding a protein in which 100 to 360 amino acid residues on the N-terminal side of the GLIS1 protein are deleted. [6] The method according to any one of [1] to [5], wherein the somatic cells are fibroblasts or mesenchymal stem cells. [7] The method according to any one of [1] to [6], wherein the other somatic cells are selected from fat cells, nerve cells, cardiac muscle cells, liver cells, bone cells and blood cells. [8] Somatic cells produced by the production method described in any one of [1] to [7]. [9] The somatic cell according to [8], wherein the somatic cell is a cell selected from the group consisting of adipocytes, nerve cells, cardiac muscle cells, hepatocytes, bone cells and blood cells.

[10] A promoter for direct transdifferentiation of a somatic cell into another somatic cell, comprising a GLIS family gene, a mutant GLIS family gene, or a gene product thereof.

[11] A promoter for promoting direct differentiation of somatic cells into other somatic cells according to

[10] , wherein the somatic cells are fibroblasts or mesenchymal stem cells, and the other somatic cells are cells selected from adipocytes, nerve cells, cardiac muscle cells, hepatocytes, bone cells, and blood cells.

[12] A direct transdifferentiation agent for somatic cells into other somatic cells, comprising a GLIS family gene, a mutant GLIS family gene, or a gene product thereof, and a component for inducing differentiation of somatic cells into other somatic cells.

[13] A direct transdifferentiation agent for somatic cells into other somatic cells, comprising a GLIS family gene, a mutant GLIS family gene, or a gene product thereof, a transcription factor, and a growth factor for other somatic cells.

[14] A direct differentiation transduction agent for somatic cells into other somatic cells according to

[13] , wherein the somatic cells are fibroblasts or mesenchymal stem cells, and the other somatic cells are cells selected from adipocytes, nerve cells, cardiac muscle cells, hepatocytes, bone cells, and blood cells. [Effects of the Invention]

[0009] According to the present invention, direct transdifferentiation of somatic cells into other somatic cells can be achieved simply, reproducibly, efficiently, and in a short period of time, thereby enabling the supply of somatic cells as materials for regenerative medicine and for research into various diseases. [Brief explanation of the drawings]

[0010] [Figure 1] The time course of neuronal differentiation from 1 week to 3 weeks after the start of differentiation induction is shown by immunostaining with a neuronal marker (Tuj-1). GFP indicates the control. K1#14 shows the results of culturing in a medium containing differentiation-inducing components after GLIS1 gene transduction. K2#9 (4-OH(-)) shows the results of culturing in a medium containing differentiation-inducing components after mutant GLIS1 gene transduction. K2(Ert)#9 (4-OH(+)) shows the results of culturing in a medium containing differentiation-inducing components and an estrogen receptor antagonist after mutant GLIS1 gene transduction. [Figure 2] This shows the efficiency of neuronal induction after two weeks of culture. GFP indicates the control. K2#14 indicates the results of culturing in a medium containing a differentiation-inducing component after introduction of the GLIS1 gene. K2#9 (4-OH(-)) indicates the results of culturing in a medium containing a differentiation-inducing component after introduction of the mutant GLIS1 gene. K2(Ert)#9 (4-OH(+)) indicates the results of culturing in a medium containing a differentiation-inducing component and an estrogen receptor antagonist after introduction of the mutant GLIS1 gene. [Figure 3] This shows an image of Lipi Dye staining of adipocytes after two weeks of culture. The green and light blue (light blue) areas are the areas of oil droplets stained with Lipi Dye within the adipocytes. GFP indicates the control. K1#14 shows the results of culturing in a medium containing differentiation-inducing components after introduction of the GLIS1 gene. K2#9 (4-OH(-)) shows the results of culturing in a medium containing differentiation-inducing components after introduction of the mutant GLIS1 gene. K2(Ert)#9 (4-OH(+)) shows the results of culturing in a medium containing differentiation-inducing components and an estrogen receptor antagonist after introduction of the mutant GLIS1 gene. [Figure 4] This shows the induction efficiency of adipocytes after two weeks of culture. GFP indicates the control. K1#14 indicates the results of culturing in a medium containing a differentiation-inducing component after introduction of the GLIS1 gene. K2#9 (4-OH(-)) indicates the results of culturing in a medium containing a differentiation-inducing component after introduction of the mutant GLIS1 gene. K2(Ert)#9 (4-OH(+)) indicates the results of culturing in a medium containing a differentiation-inducing component and an estrogen receptor antagonist after introduction of the mutant GLIS1 gene. [Figure 5] This shows the efficiency of neuronal induction after two weeks of culture. The left panel shows the change in the expression level of the neuronal marker (TUBB3). The right panel shows the change in the expression level of the neuronal differentiation-inducing component (Brn2). [Figure 6] This shows the efficiency of adipogenesis after two weeks of culture. The left panel shows the change in the expression level of an adipocyte marker (FABP4). The right panel shows the change in the expression level of an adipocyte differentiation-inducing component (PPARγ). [Figure 7] This shows the efficiency of osteocyte induction after two weeks of culture. The left panel shows the change in the expression level of the early osteocyte marker (ALP). The right panel shows the change in the expression level of the osteocyte differentiation inducer (BGLAP). [Figure 8] This shows the efficiency of cardiomyocyte induction after 10 days of culture. The left panel shows the change in the expression level of an immature cardiomyocyte marker (Sall1). The right panel shows the change in the expression level of a cardiomyocyte transcription factor (Tbx5). [Figure 9] This shows the efficiency of cardiomyocyte induction after two weeks of culture. The left panel shows the change in the expression level of the cardiomyocyte marker (cTnT). The right panel shows the change in the expression level of the cardiomyocyte transcription factor (Tbx5). [Figure 10] This shows the efficiency of hepatocyte induction after two weeks of culture. The left panel shows changes in the expression level of a hepatocyte marker (MAOA). The right panel shows changes in the expression level of a hepatocyte transcription factor (GATA4). [Figure 11] This shows the efficiency of astrocyte induction after two weeks of culture. The left panel shows the change in the expression level of the astrocyte marker (GFAP). The right panel shows the change in the expression level of the astrocyte transcription factor (NFIA). DETAILED DESCRIPTION OF THE INVENTION

[0011] A first aspect of the method of the present invention for direct transdifferentiation of a somatic cell into another somatic cell is characterized by comprising the following steps (a) and (b): (a) introducing a GLIS family gene, a mutated GLIS family gene, or a gene product thereof into a somatic cell; (b) culturing the transfected somatic cells in a medium containing a component for inducing differentiation of the somatic cells or precursor cells of the somatic cells into other somatic cells;

[0012] The GLIS family gene used in step (a) is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include GLIS1, GLIS2, and GLIS3. These may be used alone or in combination of two or more. Among the GLIS family members, GLIS1 and GLIS3 are preferred, with GLIS1 being more preferred, in terms of their excellent effect of improving the efficiency of direct transdifferentiation of somatic cells into other somatic cells. The origin of the GLIS family gene is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include human and mouse. Sequence information of the GLIS family genes can be obtained from publicly known databases. For example, at NCBI, the following sequences are available under the accession numbers NM_147193 (human GLIS1), NM_147221 (mouse GLIS1), NM_032575 (human GLIS2), NM_031184 (mouse GLIS2), NM_152629 (human GLIS3), NM_175459, and NM_172636 (mouse GLIS3).

[0013] The mutant GLIS family gene used in step (a) is a mutant of the GLIS family gene, preferably a mutant of the GLIS1 gene, more preferably a gene encoding a protein in which some amino acid residues on the N-terminus of the GLIS1 protein have been deleted, and even more preferably a gene encoding a protein in which 100 to 360 amino acid residues on the N-terminus of the GLIS1 protein have been deleted. Specific examples include genes having 85% or more sequence identity with the nucleotide sequence represented by either SEQ ID NO: 1 or 2. The base sequence shown in SEQ ID NO: 1 is the sequence of a gene encoding a protein in which 360 amino acid residues are deleted from the N-terminus of mouse GLIS1 protein. The base sequence shown in SEQ ID NO: 2 is the sequence of a gene encoding a protein in which 190 amino acid residues are deleted from the N-terminus of human GLIS1 protein. The sequence identity with the base sequence represented by either of SEQ ID NOs: 1 and 2 is not particularly limited as long as it is 85% or more and can be selected appropriately depending on the purpose, but 90% or more is preferred, 95% or more is more preferred, 98% or more is even more preferred, and 99% or more is particularly preferred. The method for determining the sequence identity is not particularly limited, and any known method can be appropriately selected. For example, the sequence identity can be determined using the BLAST algorithm by Karlin and Altschul (Karlin, S. & Altschul, SF (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, Karlin, S. & Altschul, SF, Proc. Natl. Acad. Sci. USA 90:5873).

[0014] A gene product refers to mRNA transcribed from a gene and a protein translated from the mRNA. Examples of gene products used in the present invention include mRNA transcribed from the GLIS family gene, a protein translated from the mRNA, mRNA transcribed from the mutant GLIS family gene, and a protein translated from the mRNA.

[0015] The sequence of the GLIS family gene, mutant GLIS family gene, or gene product thereof may consist of only the portion of the sequence of each gene that is translated into a protein, or may include a portion other than the portion that is translated into a protein.

[0016] In step (a), the cell into which the gene or gene product is introduced is a somatic cell. The somatic cell is not particularly limited and can be appropriately selected depending on the purpose. The somatic cell may be an undifferentiated precursor cell or a terminally differentiated mature cell. The somatic cell may be derived from an ES cell or an iPS cell, but is preferably a mature cell derived from a somatic cell or a precursor cell of the target somatic cell. Specific examples of the somatic cells include adipose tissue-derived stromal (stem) cells, mesenchymal stem cells, fibroblasts, etc. Among these, fibroblasts and mesenchymal stem cells are more preferred. The species of the individual from which the somatic cells are collected is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include humans and mice. The individual from which the somatic cells are collected is not particularly limited and can be selected appropriately depending on the purpose, but when the other somatic cells of interest are to be used for regenerative medicine purposes, the individual itself or another individual with the same or substantially the same MHC type is preferred from the viewpoint of rejection. Here, "substantially the same MHC type" means that the MHC types are identical to the extent that the transplanted cells can engraft when other somatic cells derived from the somatic cells are transplanted into the individual, using an immunosuppressant or the like. The time from which the somatic cells are collected from the individual is not particularly limited and can be selected appropriately depending on the purpose.

[0017] The culture conditions for the somatic cells are not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include a culture temperature of about 37°C and a CO2 concentration of about 2% to 5%, etc. The medium used for culturing the somatic cells is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include minimum essential medium (hereinafter sometimes referred to as "MEM"), Dulbecco's modified Eagle's medium (hereinafter sometimes referred to as "DMEM"), RPMI1640 medium, 199 medium, F12 medium, etc., each containing 5% by mass to 20% by mass of serum.

[0018] The method for introducing each of the genes or their gene products into somatic cells in step (a) is not particularly limited and can be appropriately selected depending on the purpose. Examples include a method using a vector, a method using synthetic mRNA (messenger RNA), and a method using a recombinant protein.

[0019] The vector is not particularly limited and can be appropriately selected depending on the purpose. Examples of the vector include viral vectors and non-viral vectors. Specific examples of the viral vector include retroviral vectors, lentiviral vectors, etc. Specific examples of the non-viral vector include plasmid vectors, episomal vectors, etc.

[0020] The method for introducing the vector into the somatic cells is not particularly limited, and a known method can be appropriately selected depending on the purpose. For example, when using a retroviral vector, the method described in International Publication No. 2007 / 69666, Cell, 126, 663-676 (2006), Cell, 131, 861-872 (2007), etc. can be used. When using a lentiviral vector, the method described in Science, 318, 1917-1920 (2007), etc. can be used. When using a plasmid vector, the method described in Science, 322, 949-953 (2008), etc. can be used. When using an episomal vector, the method described in Science, 324: 797-801 (2009), Biochemical and Biophysical Research Communications, 426: 141-147 (2012), etc. can be used.

[0021] When the viral vector is used, viral particles obtained using packaging cells may be used. The packaging cells are cells into which a gene encoding a viral structural protein has been introduced, and when a recombinant viral vector incorporating a gene of interest is introduced into the cells, recombinant viral particles incorporating the gene of interest are produced. The packaging cells are not particularly limited and can be selected appropriately depending on the purpose. Examples include packaging cells based on human kidney-derived HEK293 cells or mouse fibroblast-derived NIH3T3 cells; packaging cells Platinum-E (hereinafter, sometimes referred to as "Plat-E cells") that express the viral structural proteins gag-pol and env under the control of MoMuLV (Moloney Murine Leukemia Virus) LTR (long terminal repeats), which enable long-term production of high-titer virus; PLAT-A cells designed to express an amphotropic virus-derived envelope glycoprotein; and PLAT-GP cells designed to express a vesicular stomatitis virus-derived envelope glycoprotein.

[0022] The method for introducing the viral vector into the packaging cells is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include lipofection, electroporation, calcium phosphate method, etc. The method for infecting the somatic cells with the obtained viral particles is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include polybrene method.

[0023] The vector may contain a marker gene for confirming the introduction of each of the genes. The marker gene refers to a gene that enables cell sorting or selection by introducing the marker gene into cells. Specific examples of the marker gene include drug resistance genes, fluorescent protein genes, luciferase genes, and chromogenic enzyme genes. These may be used alone or in combination of two or more. Specific examples of the drug resistance gene include a neomycin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene, a zeocin resistance gene, and a hygromycin resistance gene. Specific examples of the fluorescent protein gene include the green fluorescent protein (GFP) gene, the yellow fluorescent protein (YFP) gene, and the red fluorescent protein (RFP) gene. A specific example of the luciferase gene is a luciferase gene. Specific examples of the chromogenic enzyme gene include the β-galactosidase gene, the β-glucuronidase gene, and the alkaline phosphatase gene.

[0024] The method for introducing the mRNA into the somatic cells is not particularly limited, and any known method can be appropriately selected and used. The method for introducing the recombinant protein into the somatic cells is not particularly limited, and any known method can be appropriately selected and used.

[0025] The number of times each of the genes or gene products thereof is introduced into somatic cells is not particularly limited and can be appropriately selected depending on the purpose, and may be once or twice or more.

[0026] The timing of introduction of each of the genes or their gene products into somatic cells is not particularly limited and can be selected appropriately depending on the purpose. All of the genes or their gene products may be introduced at the same time or at different times.

[0027] The amount of each gene or its gene product introduced into somatic cells is not particularly limited and can be selected appropriately depending on the purpose. All genes or their gene products may be introduced in equal amounts, or in different amounts.

[0028] The gene or its gene product may be the same gene or its gene product, and may be an embodiment in which only the gene is used, only the gene product is used, or both are used. There are no particular limitations on the combination of different genes or their gene products, and these can be selected appropriately depending on the purpose. The same genes or their gene products may be used, or different genes or their gene products may be used. In the gene or gene product introduction step, substances other than the gene or its gene product may be introduced, as long as the effects of the present invention are not impaired.

[0029] Next, step (b) will be described. Step (b) is a step of culturing the transfected somatic cells in a medium containing a component that induces differentiation of the somatic cells or precursor cells of the somatic cells into other somatic cells (also referred to as target somatic cells).

[0030] The combination of source somatic cells and target somatic cells in step (b) is not particularly limited, but the source somatic cells are preferably the above-mentioned fibroblasts and mesenchymal stem cells, and the mesenchymal stem cells are preferably bone marrow-derived stem cells. Examples of target somatic cells include fat cells, nerve cells, cardiac muscle cells, liver cells, bone cells, and blood cells. As blood cells, white blood cells and red blood cells are particularly preferred.

[0031] The components contained in the medium used in step (b) are components that induce differentiation of the somatic cells or precursor cells of the somatic cells into other somatic cells (also referred to as target somatic cells). Such differentiation-inducing components can be components known for each target somatic cell type.

[0032] Components known to induce differentiation of fibroblasts or mesenchymal stem cells into adipocytes include 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and insulin, and these can be used alone or in combination. Of these components, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and insulin are particularly preferred.

[0033] Components known to induce differentiation of fibroblasts or mesenchymal stem cells into neurons include EGF (epidermal growth factor) and FGF-2 (fibroblast growth factor-2), and these can be used alone or in combination. Of these components, EGF and FGF-2 are particularly preferred.

[0034] Components known to induce differentiation of fibroblasts or mesenchymal stem cells into cardiomyocytes include VEGF and Wnt / β-catenin inhibitors (e.g., IWP2), and these can be used alone or in combination of two or more. Of these components, it is particularly preferable to use VEGF, Wnt / β-catenin inhibitors (e.g., IWP2).

[0035] Oncostatin M (OsM), DEX, hepatocyte growth factor (HGF), and other components are known to induce differentiation of fibroblasts or mesenchymal stem cells into hepatocytes, and these can be used alone or in combination. Of these components, OsM, DEX, and HGF are particularly preferred.

[0036] Components that induce differentiation of fibroblasts or mesenchymal stem cells into blood cells vary depending on the blood cells of interest, but known examples include BMP4, VEGF, FGF1, bFGF, SCF, FIt3-L, TPO, GM-CSF, IL-2, IL-4, IL-15, G-CSF, IL-3, IL-6, IL-7, TNF-α, EPO, and IGF-II, and one or more of these can be used in combination. Components that induce differentiation of fibroblasts or mesenchymal stem cells into bone cells include Runx2, Runx3, Dlx5, ATF4, Osx, Smad1, Wnt, Fgf, Hedgehog, Msx2, Twist, AP-1, Tnc, Ncam1, and Pth1h, and these can be used alone or in combination of two or more.

[0037] In addition to the differentiation-inducing components for target somatic cells, it is also preferable to add an estrogen receptor antagonist to the culture medium as a differentiation-inducing component in order to improve the direct transdifferentiation efficiency of the present invention. Examples of estrogen receptor antagonists include tamoxifen, fulvestrant, and mepitiostane. The addition of this estrogen receptor antagonist is particularly preferable when the transgene is a mutant GLIS1 gene.

[0038] The content of the differentiation-inducing components in the medium is not particularly limited as long as it is a known amount for each component, but is preferably 0.001 μM to 50 μM for each component in a normal medium.

[0039] The basal medium to which the differentiation-inducing components can be added is not particularly limited and can be selected appropriately depending on the purpose. Examples include minimum essential medium (hereinafter sometimes referred to as "MEM") containing 5% to 20% by mass of serum, Dulbecco's modified Eagle's medium (hereinafter sometimes referred to as "DMEM"), RPMI1640 medium, 199 medium, F12 medium, etc. Furthermore, some media containing the differentiation-inducing components are already commercially available, and such commercially available media can also be used.

[0040] The culture conditions for step (b) are not particularly limited and can be appropriately selected depending on the purpose. For example, the culture temperature is about 37°C, and the CO2 concentration is about 2% to 5%.

[0041] Whether or not target somatic cells have been obtained by the culture in step (b) can be confirmed by detecting known markers for each target somatic cell. The method for confirming the expression of proteins among these markers is not particularly limited, and any known method can be appropriately selected, for example, immunostaining. Furthermore, the method for confirming gene expression is not particularly limited, and any known method can be appropriately selected, for example, quantitative PCR.

[0042] A second embodiment of the method of the present invention for direct transdifferentiation of a somatic cell into another somatic cell is characterized by comprising the following steps (c) and (d): (c) introducing a GLIS family gene, a mutant GLIS family gene, or a gene product thereof and a transcription factor into a somatic cell; (d) culturing the transgenic somatic cells in a medium containing growth factors for other somatic cells;

[0043] Step (c) of the second embodiment is the same as step (a) above, except that a transcription factor is introduced into the somatic cells in addition to the GLIS family gene and the like. The transcription factors used are preferably those known to be involved in the differentiation induction of target somatic cells. Examples of such transcription factors include Tbx4, GATA4, Mef2c, and Hand2 for cardiomyocytes. Examples of such factors include HNF4a, FOXA3, HNF1a, GATA4, TCF-1, SALL4, TGIF1, MAB21L3, ZIC1, EGFLAM, PITX2, NRF1, ZNF281, CTCFL, TP73, TFE3, DLX6, and TCF4 for hepatocytes. Examples of such factors include NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2 for neurons. Examples of such factors include Nfia, Nfib, and Sox9 for astrocytes. The method for introducing these transcription factors into somatic cells is the same as the method for introducing the GLIS gene.

[0044] The growth factors used in step (d) of the second embodiment are growth factors for other somatic cells, and known growth factors can be used. For example, growth factors for cardiomyocytes include FGF, VEGF, BMP, EGF, Nrg, TGF, PGF, PDGF, etc. Growth factors for hepatocytes include HGF, EGF, FGF, IGF, etc. Growth factors for neurons and glial cells include NGF, EGF, BDNF, NT, HGF, GDNF, FGF, LIF, HIF, PDGF, M-CSF, IGF, VEGF, BMP, etc. Growth factors for bone cells include M-CSF, BMP, TGFβ, RANKL, FGF, etc. These growth factors can be used alone or in combination of two or more. The culture in step (d) of the second embodiment can be carried out in the same manner as in step (b) of the first embodiment.

[0045] According to the method of the present invention for producing somatic cells by direct transdifferentiation from somatic cells to other somatic cells, other somatic cells can be produced directly from somatic cells by transdifferentiation, which is advantageous in that the desired somatic cells can be produced without going through iPS cells, which have the risk of tumorigenesis. The term "direct transdifferentiation" refers to the direct conversion of a somatic cell into another somatic cell without passing through a stem cell.

[0046] Furthermore, the production method of the present invention by direct transdifferentiation of somatic cells into other somatic cells is a simple and easily reproducible method in which a gene or its gene product is introduced into a somatic cell and the gene-introduced cell is cultured in a medium containing a growth factor or a differentiation-inducing component, and yet it can efficiently produce the desired somatic cells in a short period of time.

[0047] Another embodiment of the present invention includes an agent for promoting direct transdifferentiation of somatic cells into other somatic cells, which comprises a GLIS family gene, a mutant GLIS family gene, or a gene product thereof. In other words, as mentioned above, it was completely unknown that GLIS family genes, mutant GLIS family genes, or their gene products alone have the function of promoting direct transdifferentiation of somatic cells into other somatic cells. As the GLIS family gene, mutant GLIS family gene, or gene product thereof, the same as those described above are used, and the same as those described above are preferred.

[0048] The direct transdifferentiation promoter of the present invention is particularly useful when the somatic cells are fibroblasts or mesenchymal stem cells and the other somatic cells are cells selected from adipocytes, nerve cells, cardiomyocytes, hepatocytes, osteocytes, and blood cells. The method of using the direct transdifferentiation promoter of the present invention is also the same as described above.

[0049] Since the direct transdifferentiation agent of the present invention is used in combination with a component for inducing differentiation of somatic cells or somatic cell precursor cells into other somatic cells as described above, examples of the direct transdifferentiation agent for somatic cells into other somatic cells include a direct transdifferentiation agent for somatic cells into other somatic cells that contains a GLIS family gene, a mutant GLIS family gene, or a gene product thereof, and a component for inducing differentiation of somatic cells into other somatic cells. Further examples include a direct transdifferentiation agent for somatic cells into other somatic cells that contains a GLIS family gene, a mutant GLIS family gene, or a gene product thereof, a transcription factor, and a growth factor for other somatic cells. Here, the GLIS family gene, mutant GLIS family gene, or gene product thereof is the same as that described above, and is preferably the same as that described above.

[0050] The direct transdifferentiation promoter of the present invention is particularly useful when the somatic cells are fibroblasts or mesenchymal stem cells and the other somatic cells are cells selected from adipocytes, nerve cells, cardiomyocytes, hepatocytes, osteocytes, and blood cells. The method of using the direct transdifferentiation promoter of the present invention is also the same as described above.

[0051] The direct transdifferentiation agent of the present invention may be configured such that each gene or its gene product is separated into individual containers, collected in a single container, or collected in containers in any number of containers. This direct transdifferentiation agent can be suitably used as a kit for producing somatic cells. This kit for producing somatic cells contains at least the direct transdifferentiation agent and may further contain other components as necessary. [Example]

[0052] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples in any way.

[0053] Example 1 To verify the effect of GLIS family genes on the efficiency of differentiation into various cell types, we introduced the GLIS1 gene into human bone marrow-derived mesenchymal stem cells (MSCs) using a lentiviral vector and conducted differentiation induction experiments into various cell types. (1) To produce lentivirus, HEK293FT cells (Thermo Fisher Scientific) were cultured in 10 mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin. 2 × 10 cells were cultured in a 60 mm culture dish (TPP). 6 Cells were seeded at a cell count of 1000 and cultured in 4 mL of antibiotic-free medium. Sixteen hours after seeding, each plasmid carrying the GLIS1 gene was introduced using Lipofectamine 2000 (Thermo Fisher). 24 hours after gene introduction, the medium was replaced with 3 mL of antibiotic-containing medium, and the medium supernatant was collected 48 hours later. When collecting the virus solution, it was filtered through a 0.45 mm pore size filter (Whatman) and then Polybrene solution was added to a final concentration of 8 μg / mL.

[0054] (2) MSCs (human bone marrow-derived mesenchymal stem cells, Lonza) were cultured in 10 mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin, and 1 × 10 cells were plated in a 24-well culture plate (TPP) at 1 × 10 cells / well 24 hours before lentivirus infection.4 Cells were seeded at a cell count of 100 cells / well. 24 hours after seeding, the culture supernatant was completely removed, and 300 μL of the aforementioned lentivirus solution was added. The cells were then cultured in a 37°C, 5% CO2 incubator. 24 hours after the addition of the virus solution, the medium was replaced with 300 μL of differentiation induction medium specific to each cell type (PromoCell). After replacement, the medium was changed every 2-3 days. 7, 14, and 21 days after the start of differentiation induction, samples were fixed, immunocytochemically analyzed, and observed under a fluorescent microscope.

[0055] (3) The differentiation-inducing medium used was a medium containing components (EGF and FGF-2) that induce differentiation of mesenchymal stem cells into neurons. Neurons were detected based on the criteria of Tuj-1 neural marker-positive cells, the extension of axon-like processes, and the significantly reduced size of the nuclei compared to MSCs. The results are shown in Figures 1 and 2.

[0056] Example 2 To examine the effect of mutant GLIS family genes on the efficiency of differentiation into various cell types, we introduced the mutant GLIS1 gene into human bone marrow-derived mesenchymal stem cells (MSCs) using a lentiviral vector and conducted differentiation induction experiments into various cell types. (1) To produce lentivirus, HEK293FT cells (Thermo Fisher Scientific) were cultured in 10 mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin. 2 × 10 cells were cultured in a 60 mm culture dish (TPP). 6 Cells were seeded at a cell count of 1000 and cultured in 4 mL of antibiotic-free medium. Sixteen hours after seeding, each plasmid carrying the mutant GLIS1 gene (SEQ ID NO: 2) was introduced using Lipofectamine 2000 (Thermo Fisher). 24 hours after gene introduction, the medium was replaced with 3 mL of antibiotic-containing medium, and the medium supernatant was collected 48 hours later. When collecting the virus solution, it was filtered using a 0.45 mm pore size filter (Whatman) and then Polybrene solution was added to a final concentration of 8 μg / mL.

[0057] (2) MSCs (human bone marrow-derived mesenchymal stem cells, Lonza) were cultured in 10 mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin, and 1 × 10 cells were plated in a 24-well culture plate (TPP) at 1 × 10 cells / well 24 hours before lentivirus infection. 4 Cells were seeded at 100 cells / well. 24 hours after seeding, the culture supernatant was completely removed, and 300 μL of the aforementioned lentivirus solution was added. The cells were cultured in a 37°C, 5% CO2 incubator. 24 hours after virus addition, the medium was replaced with 300 μL of cell type-specific differentiation medium (Mesenchymal Stem Cell Adipogenic Differentiation Medium 2 (Ready-to-use) or Mesenchymal Stem Cell Neurogenic Differentiation Medium (Ready-to-use), C-28016, PromoCell). After replacement, the medium was changed every 2–3 days. 7, 14, and 21 days after the start of differentiation induction, samples were fixed and subjected to immunocytochemistry and observed under a fluorescent microscope.

[0058] (3) The differentiation-inducing medium used was a medium containing components for inducing differentiation of mesenchymal stem cells into neurons (Mesenchymal Stem Cell Neurogenic Differentiation Medium (Ready-to-use), C-28015, PromoCell). Neurons were detected based on the criteria of Tuj-1 neural marker-positive cells, the extension of axon-like processes, and the significantly reduced size of the nuclei compared to MSCs. The results are shown in Figures 1 and 2.

[0059] Example 3 The effect of adding an estrogen receptor antagonist was examined. (1) To produce lentivirus, HEK293FT cells (Thermo Fisher Scientific) were cultured in 10 mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin. 2 × 10 cells were cultured in a 60 mm culture dish (TPP). 6Cells were seeded at a cell count of 1000 and cultured in 4 mL of antibiotic-free medium. 16 hours after seeding, plasmids carrying the GLIS1 gene or mutant GLIS1 gene (SEQ ID NO: 2) were introduced using Lipofectamine 2000 (Thermo Fisher). 24 hours after gene introduction, the medium was replaced with 3 mL of antibiotic-containing medium, and the medium supernatant was collected 48 hours later. When collecting the virus solution, the solution was filtered through a 0.45 mm pore size filter (Whatman) and then Polybrene solution was added to a final concentration of 8 μg / mL.

[0060] (2) MSCs (mesenchymal stem cells, Lonza) were cultured in 10 mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin, and 1 × 10 cells were plated in a 24-well culture plate (TPP) at 1 × 10 cells per well 24 hours before lentivirus infection. 4 Cells were seeded at a cell count of 100 cells / well. 24 hours after seeding, the culture supernatant was completely removed, and 300 μL of the aforementioned lentivirus solution was added. The cells were then cultured in a 37°C, 5% CO2 incubator. 24 hours after the addition of the virus solution, the medium was replaced with 300 μL of an estrogen receptor antagonist (tamoxifen) and a differentiation-inducing medium specific to each cell type (PromoCell). After the replacement, the medium was changed every 2-3 days. 7, 14, and 21 days after the start of differentiation induction, samples were fixed, immunocytochemically analyzed, and observed under a fluorescent microscope.

[0061] (3) The differentiation-inducing medium used was a medium containing components for inducing differentiation of mesenchymal stem cells into neurons (Mesenchymal Stem Cell Neurogenic Differentiation Medium (Ready-to-use), C-28015, PromoCell). Neurons were detected based on the criteria of cells positive for the Tuj-1 neural marker, the extension of axon-like processes, and the significantly reduced size of the nuclei compared to MSCs. The results are shown in Figures 1 and 2.

[0062] Example 4 (Direct transdifferentiation of fibroblasts into adipocytes) MSCs (human bone marrow-derived mesenchymal stem cells, Lonza) were cultured in 10 mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin, and 1 × 10 cells were plated in a 24-well culture plate (TPP) at 1 × 10 cells / well 24 hours before lentivirus infection. 4 Cells were seeded at a cell count of 100 cells / well. 24 hours after seeding, the culture supernatant was completely removed, and 300 μL of the aforementioned lentivirus solution was added. The cells were then cultured in a 37°C, 5% CO2 incubator. 24 hours after the addition of the virus solution, the medium was replaced with 300 μL of a cell type-specific differentiation induction medium (Mesenchymal Stem Cell Adipogenic Differentiation Medium 2 (Ready-to-use), C-28016, PromoCell). After replacement, the medium was changed every 2–3 days. 7, 14, and 21 days after the start of differentiation induction, samples were fixed, immunocytochemically analyzed, and observed under a fluorescent microscope. The results are shown in Figures 3 and 4.

[0063] Example 5 Direct transdifferentiation of human bone marrow-derived mesenchymal stem cells (MSCs) into neurons, adipocytes, and bone cells (confirmed by quantitative PCR) To examine the effects of the full-length and N-terminal truncated GLIS1 genes on the differentiation efficiency into various cell types, we introduced each GLIS1 gene into human bone marrow-derived mesenchymal stem cells (MSCs) using a lentiviral vector and conducted differentiation induction experiments into various cell types. To generate lentivirus, HEK293FT cells were cultured in 10 mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin. 2 × 10 cells were added to a 60 mm culture dish (TPP). 6Cells were seeded at a cell count of 1000 and cultured in 4 mL of antibiotic-free medium. 16 hours after seeding, plasmids carrying the full-length or N-terminally truncated GLIS1 gene were introduced using Lipofectamine 2000 (ThermoFisher). 24 hours after gene introduction, the medium was replaced with 7 mL of antibiotic-containing medium, and the medium supernatant was collected 48 hours later. For virus collection, the solution was filtered through a 0.45 mm pore size filter (Whatman) and then supplemented with Polybrene solution at a final concentration of 8 μg / mL. MSCs (Lonza) were cultured in 10 mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin, and 1 × 10 cells were plated in a 24-well culture plate (TPP) at 1 × 10 cells / well 24 hours before lentivirus infection. 4 Cells were seeded at a cell count of 100 cells / well. 24 hours after seeding, the culture supernatant was completely removed, and 300 μL of the aforementioned lentivirus solution was added. The cells were then cultured in a 37°C, 5% CO2 incubator. 24 hours after the addition of the virus solution, the medium was replaced with 300 μL of differentiation induction medium (PromoCell) specific to each cell type, with subsequent medium changes every 2-3 days. Cells were harvested 7, 14, and 21 days after the start of differentiation induction, and changes in gene expression of markers specific to each cell type were examined using quantitative PCR.

[0064] The primers (differentiation-inducing components and target somatic cell markers) used in the quantitative PCR in the above test are shown in Table 1.

[0065] [Table 1]

[0066] The direct transdifferentiation effect of GLIS1 into neurons is shown in Figure 5. The GLIS1 gene was introduced into human bone marrow-derived MSCs, and then the cells were cultured for two weeks in a neuronal differentiation-inducing medium (AscI1, Brn2, Myt1I). After this, the expression levels of the neuronal marker (TUBB3) and the differentiation-inducing component (Brn2) were significantly increased. The effect of direct transdifferentiation into adipocytes using GLIS1 is shown in Figure 6. The GLIS1 gene was introduced into human bone marrow-derived MSCs, and then the cells were cultured for two weeks in adipocyte differentiation-inducing medium (PPARγ). After this, the expression levels of the adipocyte marker (FABP4) and differentiation-inducing component (PPARγ) were significantly increased. The effect of direct transdifferentiation into osteocytes using GLIS1 is shown in Figure 7. The GLIS1 gene was introduced into human bone marrow-derived MSCs, and then the cells were cultured for two weeks in osteocyte differentiation-inducing medium (ALP). After this, the expression levels of the osteocyte marker (BGLAP) and differentiation-inducing component (ALP) were significantly increased.

[0067] Example 6 (Method for direct transdifferentiation of fibroblasts into cardiomyocytes by introducing the GLIS1 gene and transcription factors) To examine the effects of the full-length and N-terminal truncated GLIS1 genes on the differentiation efficiency into various cells, we introduced cardiomyocyte transcription factors and the full-length or N-terminal truncated GLIS1 gene into mouse embryonic fibroblasts (MEFs) using lentiviral and retroviral vectors, and then performed experiments to induce differentiation into cardiomyocytes. To generate each gene transfer virus, HEK293FT or Plat-E cells cultured in 10 mL of DMEM supplemented with 10% FBS and 1% penicillin / streptomycin were seeded at 2 x 10 cells per 100 mm culture dish (TPP) and cultured in 7 mL of antibiotic-free medium. Sixteen hours after seeding, plasmids carrying cardiomyocyte transcription factors and full-length or N-terminally truncated GLIS1 genes were transfected using Lipofectamine 2000 (ThermoFisher). 24 hours after transfection, the medium was replaced with 7 mL of antibiotic-containing medium, and the medium supernatant was harvested 48 hours later. For virus collection, the supernatant was collected after centrifugation at 400 x g for 10 min and filtered through a 45 mm pore size filter (Whatman) before adding Polybrene solution to a final concentration of 8 μg / mL. MEFs were cultured in 10 mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin, and seeded at 5 x 104 cells / well onto fibronectin-coated 24-well culture plates (TPP) 24 hours before virus infection. After 24 hours of seeding, the culture supernatant was completely removed, and 500 μL of each virus solution was added. The cells were then cultured in a 37°C, 5% CO2 incubator. 24 hours after virus addition, the medium was replaced with 500 μL of cardiomyocyte differentiation-inducing medium, with subsequent medium changes every 2–3 days. Cells were harvested 0, 1, 4, 7, 14, 21, and 28 days after the start of differentiation induction and observed over time under a microscope. Changes in gene expression of specific markers for each cell type were examined using quantitative PCR.

[0068] The plasmids used in Examples 6 to 8 are shown in Table 2.

[0069] [Table 2]

[0070] The primers (transcription factors and target somatic cell markers) used for quantitative PCR in Examples 6 to 8 are shown in Table 3.

[0071] [Table 3]

[0072] The growth factor-containing media used in Example 6 are as follows: Base medium: StemPro-34 SF medium (Gibco, 10639-011) Additive reagents and cytokines: Gluta MAX (10 μL / mL, Gibco, 35050-061) Ascorbic acid (50μg / mL, Sigma Aldrich, A-4544) Recombinant human VEGF 165 (5ng / mL, Biolegend) Recombinant human FGF basic 146 aa(10ng / mL, Biolegend) Recombinant human FGF 10 (50ng / mL, Biolegend)

[0073] The effect of direct transdifferentiation into cardiomyocytes using GLIS1 and cardiomyocyte transcription factors (Tbx5, Mef2c, GATA4) is shown in Figure 8. Mouse embryonic fibroblasts (P2-3) were transfected with the GLIS1 gene and cardiomyocyte transcription factors (Tbx5, Mef2c, GATA4) and then cultured in a medium containing cardiomyocyte growth factor for 10 days. After this, expression of the transcription factor (Tbx5) was confirmed, and the expression level of the cardiomyocyte marker (Sall1) was significantly increased. The effect of direct transdifferentiation into cardiomyocytes using GLIS1 and cardiomyocyte transcription factors (Tbx5, Mef2c, GATA4, and Hand2) is shown in Figure 9. Mouse embryonic fibroblasts (P2-3) were transfected with the GLIS1 gene and cardiomyocyte transcription factors (Tbx5, Mef2c, GATA4, and Hand2), and then cultured in a medium containing cardiomyocyte growth factor for two weeks. After this, expression of the cardiomyocyte transcription factor (Tbx5) was confirmed, and the expression level of the cardiomyocyte marker (cTnT) was significantly increased.

[0074] Example 7 (Method for direct transdifferentiation of fibroblasts into hepatocytes by introducing the GLIS1 gene and transcription factors) To examine the effects of full-length and N-terminal truncated GLIS1 on the differentiation efficiency into various cells, we introduced hepatocyte transcription factors and full-length or N-terminal truncated GLIS1 genes into mouse embryonic fibroblasts (MEFs) using lentiviral and retroviral vectors, and then performed hepatocyte differentiation experiments. To prepare each gene transfer virus, HEK293FT cells or Plat-E cells were cultured in 10 mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin. 2 × 10 cells were added to a 100 mm culture dish (TPP). 6Cells were seeded at 1000 x g and cultured in 7 mL of antibiotic-free medium. Sixteen hours after seeding, plasmids carrying a hepatocyte transcription factor (GATA4) or full-length or N-terminally truncated GLIS1 gene were transfected using Lipofectamine 2000 (ThermoFisher). 24 hours after transfection, the medium was replaced with 7 mL of antibiotic-containing medium, and the medium supernatant was collected 48 hours later. For virus collection, the supernatant was collected after centrifugation at 400 x g for 10 minutes and filtered through a 45 mm pore size filter (Whatman), followed by the addition of Polybrene solution to a final concentration of 8 μg / mL. MEFs were cultured in 10 mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin and seeded at 4.5 × 104 cells / well onto gelatin-coated 24-well culture plates (TPP) 24 hours before virus infection. After 24 hours of seeding, the culture supernatant was completely removed, and 500 μL of each virus solution was added. The cells were then cultured in a 37°C, 5% CO2 incubator. 24 hours after the addition of the virus solution, the medium was replaced with 500 μL of hepatocyte medium (1). On day 7 of differentiation induction, the cells were replated onto collagen I-coated 12-well culture plates and cultured in hepatocyte medium (2). Medium was replaced every 2–3 days during differentiation induction. Cells were harvested at days 0, 1, 4, 7, 14, 21, and 28 after the start of differentiation induction and observed over time under a microscope. Changes in gene expression of specific markers for each cell type were examined using quantitative PCR.

[0075] Hepatocyte medium (1) (differentiation induction 1-7 days) Base medium: DMEM / F12 medium (Gibco) Additives and cytokines: FBS (8%, Gibco) Penicillin / Streptomycin(1%,NacalaiTesque) GlutaMAX (10μL / mL, Gibco, 35050-061) Nicotinamide (10mM, Sigma Aldrich) Insulin (1 μg / mL, Wako) β-mercaptoethanol (50 μM, Nacalai Tesque) Dexamethasone (0.1uM, Sigma-Aldrich) Hepatocyte medium (2) (after 7 days of differentiation induction) Recombinant human HGF (20ng / mL, Biolegend) Recombinant human EGF (20ng / mL, Biolegend)

[0076] The effect of direct transdifferentiation into hepatocytes using GLIS1 (full-length and N-terminal truncated GLIS1 genes) and the hepatocyte transcription factor (GATA4) is shown in Figure 10. Mouse embryonic fibroblasts (P2-3) were transfected with the GLIS1 gene and the hepatocyte transcription factor (GATA4), and then cultured in a medium containing hepatocyte growth factor for 2 weeks. Expression of the hepatocyte transcription factor (GATA4) was confirmed, and the expression level of the hepatocyte marker (MAOA) was significantly increased.

[0077] Example 8 (Direct transdifferentiation of fibroblasts into astrocytes by introducing the GLIS1 gene and transcription factors) To examine the effects of the full-length and N-terminal truncated GLIS1 genes on the differentiation efficiency into various cell types, we introduced astrocyte transcription factors and the full-length or N-terminal truncated GLIS1 genes into mouse embryonic fibroblasts (MEFs) using retroviral vectors, and then performed astrocyte differentiation induction experiments. To prepare each gene transfer virus, Plat-E cells were cultured in 10 mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin. 2 × 10 cells were added to a 100 mm culture dish (TPP). 6Cells were seeded at 1000 x g and cultured in 7 mL of antibiotic-free medium. Sixteen hours after seeding, plasmids carrying the full-length or N-terminal-deleted GLIS1 gene and astrocyte transcription factor (NFIA) were transfected using Lipofectamine 2000 (Thermo Fisher). 24 hours after transfection, the medium was replaced with 7 mL of antibiotic-containing medium, and the medium supernatant was collected 48 hours later. For virus collection, the supernatant was collected after centrifugation at 400 x g for 10 minutes and filtered through a 45 mm pore size filter (Whatman), followed by the addition of Polybrene solution to a final concentration of 8 μg / mL. MEFs were cultured in x mL of DMEM medium containing 10% FBS and 1% penicillin / streptomycin, and 5 × 10 cells were plated onto a gelatin-coated 24-well culture plate (TPP) 24 hours before virus infection. 4 Cells were seeded at a cell count of 100 cells / well. 24 hours after seeding, the culture supernatant was completely removed, and 500 μL of each virus solution was added. The cells were then cultured in a 37°C, 5% CO2 incubator. 24 hours after the addition of the virus solution, the medium was replaced with 500 μL of astrocyte medium, with subsequent medium changes every 2-3 days. Cells were harvested 0, 1, 4, 7, 14, and 21 days after the start of differentiation induction and observed microscopically over time. Changes in gene expression of markers specific to each cell type were examined using quantitative PCR.

[0078] Astrocyte medium Basic medium: DMEM (Sigma) Additives and cytokines: FBS (10%, Gibco) Penicillin / Streptomycin 1%, Nacalai Tesque) β-mercaptoethanol (100 μM, Nacalai Tesque)

[0079] The effect of direct transdifferentiation into astrocytes using GLIS1 (full-length and N-terminal truncated GLIS1 genes) and astrocyte transcription factor (NFIA) is shown in Figure 11. Mouse embryonic fibroblasts (P2-3) were transfected with the GLIS1 gene and astrocyte transcription factor (NFIA), and then cultured in astrocyte growth factor-containing medium for 2 weeks. Expression of astrocyte transcription factor (NFIA) was confirmed, and the expression level of the astrocyte marker (GFAP) was significantly increased.

Claims

1. (a) introducing the GLIS1 gene, a gene encoding a protein in which 100 to 360 amino acid residues on the N-terminal side of the GLIS1 gene are deleted, or a gene product thereof, into somatic cells selected from fibroblasts and mesenchymal stem cells; (b) culturing the transfected somatic cells in a medium containing a component for inducing differentiation of the somatic cells or precursor cells of the somatic cells into other somatic cells; A method for producing somatic cells by direct transdifferentiation from the somatic cells to other somatic cells, comprising: the other somatic cells are cells selected from adipocytes (excluding brown adipocytes), nerve cells, cardiac muscle cells, hepatocytes, and blood cells; When the other somatic cells are adipocytes (excluding brown adipocytes), the differentiation-inducing component is one or more selected from 3-isobutyl-1-methylxanthine, dexamethasone, and insulin; when the other somatic cells are nerve cells, the differentiation-inducing component is one or two selected from EGF and FGF-2; when the other somatic cells are cardiomyocytes, the differentiation-inducing component is one or two or more selected from VEGF and a Wnt / β-catenin inhibitor; and when the other somatic cells are hepatocytes. In the case where the other somatic cells are blood cells, the differentiation-inducing component is one or more selected from oncostatin M, DEX, and HGF, and in the case where the other somatic cells are blood cells, the differentiation-inducing component is one or more selected from BMP4, VEGF, FGF1, bFGF, SCF, Flt3-L, TPO, GM-CSF, IL-2, IL-4, IL-15, G-CSF, IL-3, IL-6, IL-7, TNF-α, EPO, and IGF-II.

2. (c) introducing the GLIS1 gene, a gene encoding a protein in which 100 to 360 amino acid residues on the N-terminal side of the GLIS1 gene have been deleted, or a gene product thereof and a transcription factor into somatic cells selected from fibroblasts and mesenchymal stem cells; (d) a method for producing a somatic cell by direct transdifferentiation into another somatic cell, the method comprising the step of culturing the transgenic somatic cell in a medium containing a growth factor of the other somatic cell, the other somatic cells are selected from cardiomyocytes, hepatocytes, and nerve cells or glial cells; When the other somatic cells are cardiomyocytes, the transcription factor is selected from Tbx4, GATA4, Mef2c, and Hand2; when the other somatic cells are hepatocytes, the transcription factor is selected from HNF4a, FOXA3, HNF1a, GATA4, TCF-1, SALL4, TGIF1, MAB21L3, ZIC1, EGFLAM, PITX2, NRF1, ZNF281, CTCFL, TP73, TFE3, DLX6, and TCF4; when the other somatic cells are nerve cells or glial cells, the transcription factor is selected from NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2; When the other somatic cells are cardiomyocytes, the growth factor is one or more selected from FGF, VEGF, BMP, EGF, Nrg, TGF, PGF, and PDGF; when the other somatic cells are hepatocytes, the growth factor is one or more selected from HGF, EGF, FGF, and IGF; and when the other somatic cells are nerve cells or glial cells, the growth factor is one or more selected from NGF, EGF, BDNF, NT, HGF, GDNF, FGF, LIF, HIF, PDGF, M-CSF, IGF, VEGF, and BMP.

Images