Method for differentiating pluripotent stem cells into desired cell type

Abstract

To provide a method of differentiating a pluripotent stem cell of mammalian origin into a desired cell type by predicting the direction of cell differentiation caused by induction of expression of a transcription factor.SOLUTION: The present invention provides a method for differentiating a pluripotent stem cell into a transformed cell, comprising introducing a transcription factor into a pluripotent stem cell of mammalian origin, wherein the transcription factor has a z-value of 3 or more for a specific cell type in the table of a human gene expression correlation matrix.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for differentiating pluripotent stem cells into a desired cell type and a differentiation inducer for use in said differentiation method. This application claims priority to Japanese Patent Application No. 2015-046318, which is incorporated herein by reference. [Background technology]

[0002] (Transcription factor network) Networks of transcription factors (TFs) determine cell identity and can be modified by overexpressing multiple transcription factors in combination. Selecting and identifying the combination of transcription factors that results in specific cell differentiation is difficult due to the large number of possible combinations of transcription factors (approximately 2,000). One of the goals of regenerative medicine is to generate differentiated cells of a desired type from pluripotent stem cells such as embryonic stem (ES) cells or induced pluripotent stem (iPS) cells {Non-Patent Document 1: Nature 292, 154-156 (1981); Non-Patent Document 2: Proc Natl Acad Sci USA 78, 7634-7638 (1981); Non-Patent Document 3: Science 282, 1145-1147 (1998); Non-Patent Document 4: Cell 126, 663-676 (2006)}. However, the vast number, diversity and complexity of transcription factor regulatory mechanisms pose a major challenge in finding the right combination of transcription factors.

[0003] To facilitate transcription factor network analysis, systems biology approaches have been applied for loss of function, i.e. knocking out or silencing, of transcription factors in mouse ES cells {Nat Genet 36, 543-544 (2004), Nature 442, 533-538 (2006), Cell 128, 9-13 (2007), Sci Rep 3, 1390 (2013)} followed by phenotypic or global transcriptomic analysis. However, since the alteration of cell identity information can be achieved to a great extent by forcibly inducing a combination of transcription factors, a gain-of-function approach, i.e., overexpression of transcription factors, is more preferable {Non-Patent Document 10: Cell 51, 987-1000 (1987); Non-Patent Document 4: Cell 126, 663-676 (2006); Non-Patent Document 11: Proc Natl Acad Sci USA 105, 6057-6062 (2008); Non-Patent Document 12: Nature 468, 521-526 (2010); Non-Patent Document 13: Nature 463, 1035-1041 (2010); Non-Patent Document 14: Nature 476, 224-227 (2011); Non-Patent Document 15: Cell Stem Cell 9, 205-218 (2011); Non-Patent Document 16: Nature 475, 390-393 (2011), Non-Patent Document 17: Nature 475, 386-389 (2011)}. Thus, the NIA Mouse ES Cell Bank {Non-Patent Document 18: Cell Stem Cell 5, 420-433 (2009), Non-Patent Document 19: Sci Rep 1, 167 (2011)} was established, in which 137 transcription factors, i.e., 7-10% of all transcription factors (1500-2000 transcription factors) encoded in the mouse genome {Non-Patent Document 20: Biochem Biophys Res Commun 322, 787-793 (2004)}, can be induced in a tetracycline-regulatable manner. Forty-eight hours after forced induction of each transcription factor, the global gene expression profile (i.e., transcriptome) was measured in each of these ES cell lines {Non-Patent Document 19: Sci Rep 1, 167 (2011)}. Meanwhile, expression profiles of all genes expressed in various cell types, tissues, and organs are available as public domain databases.One of these is the expression profile of various cell types by the Genomics Institute of the Novartis Research Foundation (GNF) {Non-Patent Document 21: Genome Biol 10, R130 (2009), Non-Patent Document 22: Proc Natl Acad Sci USA 99, 4465-4470 (2002)}. By comparing the transcription factor-induced gene expression profile obtained in the above experiment with the gene expression profile of GNF, a matrix showing the correlation of gene expression levels (gene expression correlation matrix) was created.

[0004] (Cell Differentiation) The differentiation state of a cell is thought to be determined by the set of specific transcription factors expressed in that cell and their expression levels. Transcription factors directly regulate gene expression and play an important role in forming cell-specific gene networks by binding to transcription-regulating regions such as promoters and enhancers, promoting or suppressing the process of transcribing genetic information from DNA into RNA. In recent years, there has been active research into the development of techniques for inducing differentiation of induced pluripotent stem cells (iPS cells) into any cell type, but problems such as low differentiation efficiency and the mixture of different cell lineages remain. Therefore, it is extremely important to identify transcription factors that can determine cell differentiation and control terminal differentiation.

[0005] (Mouse transcription factor network) In order to elucidate the structure of the mouse transcription factor network that determines the differentiation lineage of cells, the inventors have established the NIA mouse ES cell bank (cell lines for 137 transcription factor genes) in which the expression of mouse transcription factor genes can be freely induced {Non-Patent Document 18: Cell Stem Cell 5, 420-433 (2009), Non-Patent Document 19: Sci Rep 1, 167 (2011)}. In these cell lines, a single transcription factor can be rapidly and strongly forcibly expressed by removing doxycycline from the culture medium using the Tet-off system. The present inventors used these cell lines to comprehensively analyze the changes in the amount of transcripts 48 hours after gene expression induction using microarrays. By comparing the obtained gene expression profile with the gene expression patterns of each mouse organ and tissue (gene expression correlation matrix), it was possible to clearly observe which cell lineages tend to be determined by the changes in gene expression patterns caused by the induction of expression of a single transcription factor. This confirmed that the direction of cell differentiation caused by the induction of expression of a transcription factor can be predicted with a high degree of accuracy. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Nature 292, 154-156 (1981) [Non-Patent Document 2] Proc Natl Acad Sci USA 78, 7634-7638 (1981) [Non-Patent Document 3] Science 282, 1145-1147 (1998) [Non-Patent Document 4] Cell 126, 663-676 (2006) [Non-Patent Document 5] Nat Genet 36, 543-544 (2004) [Non-Patent Document 6] Nature 442, 533-538 (2006) [Non-Patent Document 7] Cell 128, 9-13 (2007) [Non-Patent Document 8] Sci Rep 3, 1390 (2013) [Non-Patent Document 9] Annu Rev Cell Dev Biol 26, 721-744 (2010) [Non-Patent Document 10] Cell 51, 987-1000 (1987) [Non-Patent Document 11] Proc Natl Acad Sci USA 105, 6057-6062 (2008) [Non-Patent Document 12] Nature 468, 521-526 (2010) [Non-Patent Document 13] Nature 463, 1035-1041 (2010) [Non-Patent Document 14] Nature 476, 224-227 (2011) [Non-Patent Document 15] Cell Stem Cell 9, 205-218 (2011) [Non-Patent Document 16] Nature 475, 390-393 (2011) [Non-Patent Document 17] Nature 475, 386-389 (2011) [Non-Patent Document 18] Cell Stem Cell 5, 420-433 (2009) [Non-Patent Document 19] Sci Rep 1, 167 (2011) [Non-Patent Document 20] Biochem Biophys Res Commun 322, 787-793 (2004) [Non-Patent Document 21] Genome Biol 10, R130 (2009) [Non-Patent Document 22] Proc Natl Acad Sci USA 99, 4465-4470 (2002) Summary of the Invention [Problem to be solved by the invention]

[0007] We have confirmed that the gene expression correlation matrix created using mouse ES cells developed and created by the inventors can predict with a high degree of accuracy the direction of cell differentiation caused by the induction of transcription factor expression. However, it is known that there are significant differences between human and mouse cell differentiation, even though they are both mammals. [Means for solving the problem]

[0008] The inventors have created a new human gene expression correlation matrix using human cells, and further confirmed that by introducing a transcription factor cocktail selected from the matrix into human pluripotent stem cells, they can differentiate into the desired cell type, thereby completing the present invention.

[0009] That is, the present invention comprises the following. 1. A method for differentiating pluripotent stem cells into neural cells, comprising the step of introducing any one or more of the following transcription factors (1) to (5) into mammalian-derived pluripotent stem cells: (1) Five transcription factors selected from NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2 (2) Four transcription factors selected from NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2 (3) Three transcription factors selected from NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2 (4) Two transcription factors selected from NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2 (5) A transcription factor selected from NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. 2. The differentiation method according to the preceding paragraph 1, wherein the nervous system cells include motor neurons. 3. The differentiation method according to the preceding paragraph 1 or 2, wherein the motor neurons are cells present in motor neurons. 4. A neural cell differentiation inducer capable of differentiating mammalian pluripotent stem cells, which contain one or more of the following transcription factors (1) to (5), into neural cells. (1) Five transcription factors selected from NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2 (2) Four transcription factors selected from NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2 (3) Three transcription factors selected from NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2 (4) Two transcription factors selected from NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2 (5) A transcription factor selected from NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. 5. The differentiation inducer according to the preceding item 4, wherein the motor nerve is a peripheral motor nerve. 6. The differentiation inducer according to the preceding item 4 or 5, wherein the motor neurons are cells present in motor neurons. 7. The differentiation inducer according to any one of the preceding aspects 4 to 6, wherein the transcription factor is an mRNA, a synthetic mRNA, a nucleic acid or a protein. 8. A method for differentiating pluripotent stem cells into hepatoblasts and / or hepatocytes, comprising the step of introducing any one or more of the following transcription factors (1) to (5) into mammalian-derived pluripotent stem cells: (1) a transcription factor selected from TGIF, TCF4, PITX2, SALL4, and MEIS1 (2) two transcription factors selected from TGIF, TCF4, PITX2, SALL4, and MEIS1; (3) three transcription factors selected from TGIF, TCF4, PITX2, SALL4, and MEIS1; (4) 4 transcription factors selected from TGIF, TCF4, PITX2, SALL4, and MEIS1 (5) Five transcription factors selected from TGIF, TCF4, PITX2, SALL4, and MEIS1 9. A hepatoblast and / or hepatocyte differentiation inducer capable of differentiating mammalian pluripotent stem cells, which contain any one or more of the following transcription factors (1) to (5), into hepatoblasts and / or hepatocytes: (1) a transcription factor selected from TGIF, TCF4, PITX2, SALL4, and MEIS1 (2) two transcription factors selected from TGIF, TCF4, PITX2, SALL4, and MEIS1; (3) three transcription factors selected from TGIF, TCF4, PITX2, SALL4, and MEIS1; (4) 4 transcription factors selected from TGIF, TCF4, PITX2, SALL4, and MEIS1 (5) Five transcription factors selected from TGIF, TCF4, PITX2, SALL4, and MEIS1 10. A method for differentiating pluripotent stem cells into hematopoietic stem cells and / or blood cells, comprising the step of introducing any one or more of the following transcription factors (1) to (7) into mammalian-derived pluripotent stem cells: (1) a transcription factor selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 (2) two transcription factors selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 (3) three transcription factors selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 (4) four transcription factors selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 (5) Five transcription factors selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 (6) Six transcription factors selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 (7) Seven transcription factors selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 11. A hematopoietic stem cell and / or blood cell differentiation inducer capable of differentiating mammalian pluripotent stem cells into hematopoietic stem cells and / or blood cells, comprising any one or more of the following transcription factors (1) to (7): (1) a transcription factor selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 (2) two transcription factors selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 (3) three transcription factors selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 (4) four transcription factors selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 (5) Five transcription factors selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 (6) Six transcription factors selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 (7) Seven transcription factors selected from CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, and SALL4 12. A method for differentiating pluripotent stem cells into chondrocytes, comprising the step of introducing the transcription factor SOX9 into mammalian-derived pluripotent stem cells. 13. A chondrocyte differentiation inducer capable of differentiating mammalian pluripotent stem cells containing the transcription factor SOX9 into chondrocytes. 14. A method for differentiating pluripotent stem cells into neural cells, comprising the step of introducing any one or more of the following transcription factors (1) to (11) into mammalian-derived pluripotent stem cells: (1) a transcription factor selected from NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB (2) NEUROG2 and one or more transcription factors selected from NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB (3) NEUROG2, NEUROG3, and one or more transcription factors selected from NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB. (4) NEUROG2, NEUROG3, NEUROG1, and one or more transcription factors selected from NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB. (5) NEUROG2, NEUROG3, NEUROG1, NEUROD1, and one or more transcription factors selected from NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB. (6) NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, and one or more transcription factors selected from HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB. (7) NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, and one or more transcription factors selected from ASCL1, PITX2, NEUROD2, PRDM1, and NFIB. (8) one or more transcription factors selected from NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, and PITX2, NEUROD2, PRDM1, and NFIB (9) one or more transcription factors selected from NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, and NEUROD2, PRDM1, and NFIB (10) One or more transcription factors selected from NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB. (11) Transcription factors NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1 and NFIB 15. A method for differentiating pluripotent stem cells into neural cells according to the preceding paragraph 14, further comprising introducing one or more transcription factors selected from TCF4, PDX1, SMAD7, SOX11, RNF2, MXI1 and YY1 into the human pluripotent stem cells. 16. The differentiation method according to the preceding paragraph 14 or 15, wherein the nervous system cells are cells present in the nervous system. 17. A neural cell differentiation inducer capable of differentiating mammalian pluripotent stem cells, which contains any one or more of the following transcription factors (1) to (11), into neural cells. (1) a transcription factor selected from NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB (2) NEUROG2 and one or more transcription factors selected from NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB (3) NEUROG2, NEUROG3, and one or more transcription factors selected from NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB. (4) NEUROG2, NEUROG3, NEUROG1, and one or more transcription factors selected from NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB. (5) NEUROG2, NEUROG3, NEUROG1, NEUROD1, and one or more transcription factors selected from NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB. (6) NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, and one or more transcription factors selected from HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB. (7) NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, and one or more transcription factors selected from ASCL1, PITX2, NEUROD2, PRDM1, and NFIB. (8) one or more transcription factors selected from NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, and PITX2, NEUROD2, PRDM1, and NFIB (9) one or more transcription factors selected from NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, and NEUROD2, PRDM1, and NFIB (10) One or more transcription factors selected from NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, and NFIB. (11) Transcription factors NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1 and NFIB 18. The nervous system cell differentiation inducer according to the preceding item 17, further comprising one or more transcription factors selected from TCF4, PDX1, SMAD7, SOX11, RNF2, MXI1 and YY1. 19. The nervous system cell differentiation inducer according to the preceding item 17 or 18, wherein the nervous system cells are cells present in the nervous system. Effect of the Invention

[0010] The method of the present invention for differentiating pluripotent stem cells into a desired cell type is capable of differentiating pluripotent stem cells into a desired cell type. [Brief description of the drawings]

[0011] [Figure 1] A human ES cell line expressing a specific transcription factor gene. [Diagram 2] Method for inducing desired cell type differentiation. [Figure 3-1] Human gene expression correlation matrix created in Example 1. [Figure 3-2] Human gene expression correlation matrix created in Example 1. [Figure 3-3] Human gene expression correlation matrix created in Example 1. [Diagram 3-4] Human gene expression correlation matrix created in Example 1. [Figure 4-1] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-2] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-3] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-4] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-5] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-6] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-7] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-8] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-9] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-10] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-11] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-12] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-13] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-14] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-15]Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-16] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-17] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-18] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-19] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Figure 4-20] Human gene expression correlation matrix (Z-scores entered) created in Example 1. [Diagram 5] Methods for inducing differentiation into desired cell types used in this example. [Figure 6] A: The process of differentiating pluripotent stem cells into neurons. B: The results showing that pluripotent stem cells were successfully differentiated into neurons. [Figure 7] Results showing that human embryonic stem cells can be differentiated into motor neurons. [Figure 8] Results showing that human induced pluripotent stem cells can be differentiated into motor neurons. [Figure 9] Results show that pluripotent stem cells can differentiate into hepatoblasts and hepatocytes. [Figure 10] Results show that pluripotent stem cells can differentiate into hematopoietic stem cells and blood cells. [Figure 11] Results showing that pluripotent stem cells were able to differentiate into chondrocytes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] (Method of Differentiating Pluripotent Stem Cells of the Present Invention into Desired Cell Types) The method of differentiating pluripotent stem cells of the present invention into a desired cell type (hereinafter, sometimes referred to as the "method of the present invention") is characterized in that a transcription factor or a transcription factor cocktail required for differentiation into the desired cell type is introduced into pluripotent stem cells in order to differentiate them into one or more cell types of tissues, organs, and cells (horizontal axis) listed in the human gene expression correlation matrix (correlation matrix) created in this Example 1. This will be explained below. In this specification, the term "gene" refers not only to double-stranded nucleic acid, but also to each of its constituent single-stranded, linear, and circular strands, such as the positive strand (or sense strand) or complementary strand (or antisense strand), and further includes DNA, RNA, mRNA, cDNA, etc., unless otherwise specified.

[0013] The method of the present invention comprises the steps of selecting at least one positive transcription factor from the matrix shown in FIG. 3 having a z-score of 3 or greater for a specific cell type; and introducing a nucleic acid, mRNA or protein encoding the at least one positive transcription factor, or all of these, into a pluripotent stem cell to form a transformed cell or a specific cell type. Furthermore, the steps required to differentiate a transformed cell into a specific cell type may further include not only the introduction of another transcription factor but also a specific treatment {e.g., culture under specific environment (culture conditions), etc.}. Furthermore, the method of the present invention may additionally or alternatively include the steps of selecting at least one negative transcription factor (coloured) from the matrix shown in Figure 3; and introducing a nucleic acid, mRNA or protein, or both, encoding said at least one negative transcription factor into pluripotent stem cells to form a transformed cell or a specific cell type. Furthermore, the method of the present invention may also reduce the expression of the transcription factor, or knock out the transcription factor gene. Any desired method may be used to reduce or knock out the expression of the transcription factor, including, for example, RNAi interference, targeted ribozyme, homologous recombination, site-directed mutagenesis, methylation, or any combination thereof.

[0014] (z value) The z value shown in FIG. 4 of the present invention can be determined, for example, by the following formula (1) described in the literature "Sci Rep 1, 167 (2011)".

[0015] z=(xset-xall) × √nset / SDall (1) Wherein, xset is the average expression change of a particular subset of genes, xall is the average expression change of all genes, nset is the size of the gene set, and SDall is the standard deviation of the expression change in all genes. xall can be the number of the most upregulated genes, the number of the most downregulated genes, or the sum of the most upregulated genes and the most downregulated genes. This sum can be equal to about 100, about 500, about 1,000, about 2,500, about 4,000, about 5,000, about 6,000, about 7,500, about 10,000, or a total number that is greater than or less than about 10, 25, 40, 50, 60, 75, 100, 250, 500, or 1,000 genes or more.

[0016] A desired number of positive and / or negative transcription factors can be selected from the matrix, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or more transcription factors can be selected for use. At least one positive transcription factor preferably has a z value of 3 or more for a particular cell type. However, other positive transcription factors can have z values ​​of 3 or more or less than 3. A positive transcription factor, or an average of a group of them, can have a z value of 0 or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more.

[0017] At least one negative transcription factor preferably has a z-score of -3 or less with respect to a particular cell type. Other negative transcription factors can have z-scores of -3 or less or greater. The average z-score of a negative transcription factor, or a group of negative transcription factors, can be less than 0, or less than -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -20, -25, -30, -40, -50.

[0018] (Transcription Factors) The form of the transcription factor used in the method of the present invention is not particularly limited, and examples thereof include nucleic acids, synthetic mRNAs, proteins, and the like. Furthermore, the vector for introducing the transcription factor into the pluripotent stem cell is not particularly limited, and may be, for example, a viral vector, such as a Sendai virus vector. In addition, nanoparticle capsules, liposomes, exosomes, etc. containing synthetic mRNAs or proteins may also be used to introduce the transcription factor into the pluripotent stem cell. In addition, examples of transcription factors as used herein include the following: DLX3(distal-less homeobox 3)、NEUROG3(neurogenin 3)、NEUROG2(neurogenin 2)、NEUROG1(neurogenin 1)、ASCL1(achaete-scute family bHLH transcription factor 1)、NEUROD1(neurogenic differentiation 1)、YY1(YY1 transcription factor)、SOX11(SRY (sex determining region Y)-box 11)、GLIS2(GLIS family zinc finger 2)、PDX1(pancreatic and duodenal homeobox 1)、E2F6(E2F transcription factor 6)、SOX2(SRY (sex determining region Y)-box 2)、CDX2(caudal type homeobox 2)、DLX4(distal-less homeobox 4)、NANOG(Nanog homeobox)、MXI1(MAX interactor 1, dimerization protein)、RNF2(ring finger protein 2)、NEUROD2(neurogenic differentiation 2)、ASCL2(achaete-scute family bHLH transcription factor 2)、SREBF2(sterol regulatory element binding transcription factor 2)、SOX15(SRY (sex determining region Y)-box 15)、FOXA2(forkhead box A2)、FOXA1(forkhead box A1)、TBX3(T-box 3)、ARNT2(aryl-hydrocarbon receptor nuclear translocator 2)、PITX2(paired-like homeodomain 2)、PRDM1(PR domain containing 1, with ZNF domain)、TCF4(transcription factor4)、NFIB(nuclear factor I / B)、ZNF281(zinc finger protein 281)、TBX2(T-box 2)、NR2F2(nuclear receptor subfamily 2, group F, member 2)、NFIC{nuclear factor I / C (CCAAT-binding transcription factor)}、NRF1(nuclear respiratory factor 1)、HOXA2(homeobox A2)、TBX5(T-box 5)、ZIC1(Zic family member 1)、HEY1(hes-related family bHLH transcription factor with YRPW motif 1)、CTCF{CCCTC-binding factor (zinc finger protein)}、HES1(hes family bHLH transcription factor 1)、TFAP2C{transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma)}、MYOD1(myogenic differentiation 1)、SALL4(spalt-like transcription factor 4)、TP73(tumor protein p73)、TFE3(transcription factor binding to IGHM enhancer 3)、FOXP1(forkhead box P1)、FOS(FBJ murine osteosarcoma viral oncogene homolog)、IRF4(interferon regulatory factor 4)、GATA3(GATA binding protein 3)、JUNB(jun B proto-oncogene)、ESX1(ESX homeobox 1)、TGIF1(TGFB-induced factor homeobox 1)、MAB21L3(mab-21-like 3)、DLX6(distal-less homeobox 6)、IRF5(interferon regulatory factor5)、HSF1(heat shock transcription factor 1)、JUN(jun proto-oncogene)、FOSL1(FOS-like antigen 1)、CTCFL{CCCTC-binding factor (zinc finger protein)-like}、FOSL2(FOS-like antigen 2)、FOXG1(forkhead box G1)、THAP11(THAP domain containing 11)、CUX1(cut-like homeobox 1)、ESRRB(estrogen-related receptor beta)、HNF4A(hepatocyte nuclear factor 4, alpha)、HNF1A(HNF1 homeobox A)NKX2-5(NK2 homeobox 5)、KLF9(Kruppel-like factor 9)、TFAP4{transcription factor AP-4 (activating enhancer binding protein 4)}、ERG(v-ets avian erythroblastosis virus E26 oncogene homolog)、KLF3(Kruppel-like factor 3)、MKRN1(makorin ring finger protein 1)、OLIG2(oligodendrocyte lineage transcription factor 2)、ELF5{E74-like factor 5 (ets domain transcription factor)}、HOXA9(homeobox A9)、NKX2-1(NK2 homeobox 1)、GRHL2{grainyhead-like 2 (Drosophila)}、USF2(upstream transcription factor 2, c-fos interacting)、KLF4{Kruppel-like factor 4 (gut)}、ELF1{E74-like factor 1 (ets domain transcription factor)}、CEBPB{CCAAT / enhancer binding protein (C / EBP),beta}、ETS1(v-ets avian erythroblastosis virus E26 oncogene homolog 1)、ETS2(v-ets avian erythroblastosis virus E26 oncogene homolog 2)、SPI1(Spi-1 proto-oncogene)、IRF1(interferon regulatory factor 1)、IRF2(interferon regulatory factor 2)、DMRT1(doublesex and mab-3 related transcription factor 1)、GLI1(GLI family zinc finger 1)、SPIC{Spi-C transcription factor (Spi-1 / PU.1 related)}、RUNX3(runt-related transcription factor 3)、GATA2(GATA binding protein 2)、MEF2C(myocyte enhancer factor 2C)、FOXL2(forkhead box L2)、FBXO15(F-box protein 15)、HHEX(hematopoietically expressed homeobox)、SMAD7(SMAD family member 7)、MEIS2(Meis homeobox 2)、ARID3A{AT rich interactive domain 3A (BRIGHT-like)}、WRNIP1(Werner helicase interacting protein 1)、PPARG(peroxisome proliferator-activated receptor gamma)、PTF1A(pancreas specific transcription factor, 1a)、RFX2{regulatory factor X, 2 (influences HLA class II expression)}、EOMES(eomesodermin)、TFCP2L1(transcription factor CP2-like 1)、ZNF274(zinc finger protein 274)、EGR1(earlygrowth response 1)、LHX2(LIM homeobox 2)、TFAP2A{transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha)}、OTX1(orthodenticle homeobox 1)、OVOL2(ovo-like zinc finger 2)、E2F4(E2F transcription factor 4, p107 / p130-binding)、RUVBL2(RuvB-like AAA ATPase 2)、SMARCA4(SWI / SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4)、GTF2F1(general transcription factor IIF, polypeptide 1, 74kDa)、GBX2(gastrulation brain homeobox 2)、ID1(inhibitor of DNA binding 1, dominant negative helix-loop-helix protein)、PLXNB3(plexin B3)、MYC(v-myc avian myelocytomatosis viral oncogene homolog)、ATF2(activating transcription factor 2)、CDYL2(chromodomain protein, Y-like 2)、ZBTB45(zinc finger and BTB domain containing 45)、RSPO1(R-spondin 1)、STAT5A(signal transducer and activator of transcription 5A)、LMO1{LIM domain only 1 (rhombotin 1)}、SMARCB1(SWI / SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1)、GADD45A(growth arrestand DNA-damage-inducible, alpha)、SETDB1(SET domain, bifurcated 1)、SRSF6(serine / arginine-rich splicing factor 6)、ZFAND3(zinc finger, AN1-type domain 3)、IRF3(interferon regulatory factor 3)、KAT8{K(lysine) acetyltransferase 8}、ZSCAN4(zinc finger and SCAN domain containing 4)、CRY1{cryptochrome 1 (photolyase-like)}、SIN3A(SIN3 transcription regulator family member A)、LMO2{LIM domain only 2 (rhombotin-like 1)}、NFYB(nuclear transcription factor Y, beta)、L3MBTL2{l(3)mbt-like 2 (Drosophila)}、TP53(tumor protein p53)、RHOXF2(Rhox homeobox family, member 2)、RFX5{regulatory factor X, 5 (influences HLA class II)、EGFLAM(EGF-like, fibronectin type III and laminin G domains)、NELFE(negative elongation factor complex member E)、XRCC4(X-ray repair complementing defective repair in Chinese hamster cells 4)、ZFP57(ZFP57 zinc finger protein)、SAP30(Sin3A-associated protein, 30kDa)、Emerald (A virant form of green fluorescence protein [GFP])、BCL6(B-cell CLL / lymphoma 6)、RXRA(retinoid X receptor, alpha)、STAT3{signaltransducer and activator of transcription 3 (acute-phase response factor)}、ELL2(elongation factor, RNA polymerase II, 2)、TRPV2(transient receptor potential cation channel, subfamily V, member 2)、HOXC9(homeobox C9)、RARA(retinoic acid receptor, alpha)、ZNF263(zinc finger protein 263)、SMAD5(SMAD family member 5)、SUB1{SUB1 homolog (S. cerevisiae)}、SUZ12(SUZ12 polycomb repressive complex 2 subunit)、JAG1(jagged 1)、ATF3(activating transcription factor 3)、ATF1(activating transcription factor 1)、FLI1(Fli-1 proto-oncogene, ETS transcription factor)、ETV5(ets variant 5)、KDM5A{lysine (K)-specific demethylase 5A}、NELFA(negative elongation factor complex member A)、TCF23(transcription factor 23)、ZNF646(zinc finger protein 646)、SIX5(SIX homeobox 5)、MYBL2(v-myb avian myeloblastosis viral oncogene homolog-like 2)、PAX6(paired box 6)、SMAD2(SMAD family member 2)、SOX9{SRY (sex determining region Y)-box 9}、STRA13(stimulated by retinoic acid 13)、TBX6(T-box 6)、SMAD1(SMAD family member 1)、FOXH1(forkhead boxH1), OTX2 (orthodenticle homeobox 2), TGIF (TGFB induced factor homeobox 1), MEIS1 (Meis homeobox 1).

[0019] (pluripotent stem cells) The pluripotent stem cells used in the method of the present invention are derived from mammals, and are preferably derived from humans, for example, human ES cells, human iPS cells, or any combination thereof, including, but not limited to, tissue stem cells derived from tissues or organs, skin fibroblasts, and any type of cells derived from tissues or organs.

[0020] (ES cell line with transcription factor gene introduced) In the steps of the method of the present invention, the method for introducing a transcription factor gene into a pluripotent stem cell can be a method known per se and is not particularly limited, but preferably, an expression cassette sandwiched between PiggyBac transposase recognition sequences (PB sequences) developed by Woltjen et al. (Reference: Nature 458:766-770, 2009.) can be used as a mechanism for actively incorporating the gene to be introduced into the genome of human ES cells. By introducing a drug selection cassette into the expression cassette, it is possible to efficiently establish a genetically modified human ES cell line (see FIG. 1).

[0021] (Methods for inducing desired cell types) In the steps of the method of the present invention, the method of inducing the cells to the desired cell type can be a method known per se and is not particularly limited. Preferably, however, a gene expression method using synthetic mRNA developed by Warren, Rossi et al. (Reference: Cell Stem Cell 7:618-630, 2010) is used as a footprint-free forced gene expression method without gene integration into the host genome, and a method of efficiently introducing synthetic mRNA of a transcription factor gene into human pluripotent stem cells to induce differentiation (Reference: Figure 2) is used.

[0022] (How to use the human gene expression correlation matrix) The present inventors have already created a mouse gene expression correlation matrix and confirmed that the direction of cell differentiation caused by the induction of transcription factor expression can be predicted with a high degree of accuracy. However, it is known that the cell differentiation of humans and mice is significantly different, even though they are both mammals. Furthermore, the present inventors have now newly created a human gene expression correlation matrix using a combination of human pluripotent stem cells and human transcription factors. By comparing this human gene expression correlation matrix, which is disclosed for the first time in the present invention, with a previously reported mouse gene expression correlation matrix, it has been confirmed that the transcription factors and combinations of transcription factors required for the differentiation of a desired cell type are significantly different between mice and humans than expected, and that human cells differentiate faster than mouse cells. In addition, the human gene expression correlation matrix of the present invention (see Figures 3 and 4) also includes desired organs, tissues, and cells that are not included in the mouse gene expression correlation matrix.

[0023] In the gene expression correlation matrix developed and created by the present inventors, transcription factors showing Z values ​​above a certain value (cutoff value, for example, 7 or more) and / or transcription factors with Z values ​​in the top three or more can be selected and introduced into mammalian pluripotent stem cells, allowing induction into a desired cell type. For example, the following are given as examples. In order to differentiate into skeletal muscle as the desired cell type, we selected Myod1, Mef2c, and Esx1 from among the transcription factors with a Z value of 11 or more in the gene expression correlation matrix, and introduced them individually into mouse ES cells. We confirmed that they differentiated into skeletal muscle. In order to differentiate into hepatocytes as the desired cell type, we selected Hnf4a, Foxa1, Gata2, and Gata3 from among the transcription factors with a Z value of 10 or more in the gene expression correlation matrix, and introduced each of them alone into mouse ES cells, and confirmed that they were able to differentiate into hepatocytes. In order to differentiate into blood cells as the desired cell type, we selected Sfpi1, Elf1, Elf5, Myc, Irf2, and Ets1 from transcription factors with a Z value of 15 or more in the gene expression correlation matrix, and introduced each of them alone into mouse ES cells, and confirmed that they were able to differentiate into blood cells. In order to differentiate into neurons as the desired cell type, we selected Ascl1, Smad7, Nr2f1, Sox11, Dmrt1, Sox9, Foxg1 and Sox2 from among the transcription factors with a Z value of 12 or more in the gene expression correlation matrix, and introduced each of them alone into mouse ES cells, and confirmed that they could be differentiated into neurons.

[0024] In this example, it was confirmed that when the transcription factors NEUROD1, NEUROD2, NEUROG2 and NEUROG3, which have a Z value of 8 or more in the gene expression correlation matrix, were introduced into human embryonic stem cells, they were able to differentiate into neurons. In this example, NEUROD1 (sequence number 1 for the base sequence and sequence number 2 for the protein sequence), NEUROD2 (sequence number 3 for the base sequence and sequence number 4 for the protein sequence), NEUROG1 (sequence number 5 for the base sequence and sequence number 6 for the protein sequence), NEUROG2 (sequence number 7 for the base sequence and sequence number 8 for the protein sequence) and NEUROG3 (sequence number 9 for the base sequence and sequence number 10 for the protein sequence) were introduced into human embryonic stem cells, and it was confirmed that they could be differentiated into motile cells. In this example, it was confirmed that when one or more of TGIF (homeobox protein TGIF1 isoforms a to e may be used, but the base sequence is SEQ ID NO: 11 and the protein sequence is SEQ ID NO: 12), TCF4 (transcription factor 4 isoforms a to n may be used, but the base sequence is SEQ ID NO: 13 and the protein sequence is SEQ ID NO: 14), PITX2 (pituitary homeobox 2 isoforms a to c may be used, but the base sequence is SEQ ID NO: 15 and the protein sequence is SEQ ID NO: 16), SALL4 (sal-like protein 4 isoforms 1 to 2 may be used, but the base sequence is SEQ ID NO: 17 and the protein sequence is SEQ ID NO: 18), and MEIS1 (the base sequence is SEQ ID NO: 19 and the protein sequence is SEQ ID NO: 20) were introduced into human embryonic stem cells, and differentiation into hepatocytes (hepatoblasts) was possible. Note that these are completely different from the transcription factors (Hnf4a, Foxa1) used in differentiation into hepatocytes from mouse ES cells. In this example, when one or more of CDYL2 (the base sequence is SEQ ID NO: 21 and the protein sequence is SEQ ID NO: 22), ETS2 (any of transcriptional regulator ERG isoforms 1 to 7 may be used, but the base sequence is SEQ ID NO: 23 and the protein sequence is SEQ ID NO: 24), SPI1 (any of transcription factor PU.1 isoforms 1 to 2 may be used, but the base sequence is SEQ ID NO: 25 and the protein sequence is SEQ ID NO: 26), OVOL2 (any of transcription factor Ovo-like 2 isoforms 1 to 2 may be used, but the base sequence is SEQ ID NO: 27 and the protein sequence is SEQ ID NO: 28), CDX2 (the base sequence is SEQ ID NO: 29 and the protein sequence is SEQ ID NO: 30), and CEBPB (any of CCAAT / enhancer-binding protein beta isoforms a to c may be used, but the base sequence is SEQ ID NO: 31 and the protein sequence is SEQ ID NO: 32) were introduced into human embryonic stem cells, and it was confirmed that they could be differentiated into blood cells (or hematopoietic stem cells). In this example, it was confirmed that when SOX9 (the base sequence is SEQ ID NO: 33 and the protein sequence is SEQ ID NO: 34) was introduced into human embryonic stem cells, they were able to differentiate into chondrocytes.

[0025] As described above, by selecting transcription factors exhibiting a Z value of 6 or more (or 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more) and / or the top three (or 4, 5, 6, 7, 8, 9, 10) transcription factors in descending order of Z value in the human gene expression correlation matrix of the present invention, and introducing these transcription factors alone or in combination with two or more transcription factors into human pluripotent stem cells, it is possible to induce the cells to become of the desired cell type. Specific examples of the method of the present invention are described below.

[0026] (Method of differentiation into neural cells) The method for differentiating the neural cells of the present invention (particularly cells present in the fetal brain, cerebellar peduncle, cerebellum, whole brain, thalamus, hypothalamus, prefrontal cortex, occipital lobe, amygdala, caudate nucleus, cingulate cortex, medulla oblongata, globus pallidus, subthalamic nucleus, parietal lobe, temporal lobe, and pons) is as follows. Fetal brain: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-1 (Fetal_brain) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Cerebellum peduncles: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-1 (Cerebellum_Peduncles) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Cerebellum: A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-1 (Cerebellum) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Whole brain: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-2 (Whole_Brain) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Brain_Thalamus: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-2 (Brain_Thalamus) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Hypothalamus: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-2 (Hypothalamus) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Prefrontal cortex: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-2 (Prefrontal_Cortex) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Occipital lobe: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-3 (Occipital_Lobe) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. A single transcription factor or two or more transcription factors selected from the transcription factors listed in Brain_Amygdala (Figure 4-3) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Caudate nucleus: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-3 (Cauda_tenucleus) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Cingulate cortex: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-3 (Cingulate_Cortex) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Medulla oblongata: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-4 (Medulla_Oblongata) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Globus pallidus: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-4 (Globus_pallidus) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Subthalamic nucleus: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-4 (Subthalamic_nucleus) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Parietal lobe: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-4 (Parietal_Lobe) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Temporal lobe: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-5 (Temporal_Lobe) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Pons: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-5 (Pons) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0027] {A method for differentiating into cells in the pituitary gland (especially cells present in the pituitary gland)} The method for differentiation of the pituitary gland of the present invention is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-1 (Pituitary) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0028] {Method of differentiation into olfactory nerves (especially cells present in the olfactory nerves)} The method for differentiating olfactory nerves (particularly, olfactory bulbs) of the present invention is as follows. Olfactory bulb: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-5 (Olfactory_Bulb) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0029] {A method for differentiating into spinal nerves (especially cells present in spinal nerves)} The method for differentiating spinal nerves (particularly spinal cord) of the present invention is as follows. Spinal cord: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-5 (Spinal_cord) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0030] {Method of differentiation into skeletal muscle (especially cells present in skeletal muscle)} The method for differentiating skeletal muscles (particularly, psoas muscle and tongue) of the present invention is as follows. Psoas muscle: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-6 (Skeletal_Muscle_Psoas) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Tongue: A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-6 (Tongue) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0031] {Method of differentiation into skin (especially cells present in the skin)} The method for differentiation of skin according to the present invention is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-6 (Skin) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0032] {Method of differentiation into ganglia (especially cells present in ganglia)} The method for differentiating ganglia (particularly dorsal root ganglia, superior cervical ganglia, atrioventricular node, trigeminal ganglion, and ciliary ganglion) of the present invention is as follows. Dorsal root ganglion: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-7 (Dorsal_root_ganglion) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Superior cervical ganglion: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-7 (Superior_Cervical_Ganglion) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Atrioventricular node: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-7 (Atrioventricular_node) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Trigeminal Ganglion: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-8 (Trigeminal_Ganglion) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Ciliary ganglion: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-8 (Ciliary_ganglion) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0033] {A method for differentiating into ovarian tissue (especially cells present in the ovarian tissue)} The ovarian differentiation method of the present invention is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-8 (Ovary) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0034] {A method for differentiating into adrenal gland cells (especially cells present in the adrenal gland)} The method for differentiation of the adrenal gland (particularly, the adrenal cortex or adrenal gland) of the present invention is as follows. Adrenal cortex: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-9 (Adrenal_Cortex) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Adrenal gland: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-9 (Adrenal_gland) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0035] {A method for differentiation into the appendix (specifically into cells present in the appendix)} The method for differentiation of the appendix of the present invention is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-7 (Appendix) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0036] {Method of differentiation into kidney cells (especially cells present in the kidney)} The kidney differentiation method of the present invention is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-10 (Kidney) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0037] {Method of differentiation into liver cells (especially liver-resident cells)} The method for liver (particularly liver, fetal liver) differentiation of the present invention is as follows. Liver: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-10 (Liver) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Fetal liver: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-12 (Fetal_liver) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0038] {A method for differentiating into salivary glands (especially cells present in salivary glands)} The method for differentiation of salivary glands of the present invention is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-12 (salivary_gland) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0039] {Method for differentiating into pancreatic islets (especially cells present in pancreatic islets)} The method for differentiating into the pancreatic islets (particularly, pancreatic islet cells) of the present invention is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-12 (Islet_Cell) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0040] {A method for differentiating into pancreatic cells (especially cells present in the pancreas)} The method for differentiating into pancreas of the present invention is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-13 (Pancreas) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0041] {Method of differentiation into prostate (especially cells present in the prostate)} The method for differentiation into prostate gland of the present invention is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-11 (Prostate) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0042] {A method for differentiating into thyroid gland (especially cells present in the thyroid gland)} The method for differentiation into the thyroid gland (particularly, fetal thyroid gland) of the present invention is as follows. Thyroid: A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-11 (Thyroid) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Fetal thyroid: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-11 (Fetal_Thyroid) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0043] (Method of differentiation into fat cells) The method for differentiating into the adipocytes (particularly, cultured adipocytes) of the present invention is as follows. Cultured adipocytes: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-11 (Cultured_adipocyte) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0044] {Method of differentiation into uterus (especially cells present in uterus)} The method for differentiating into the uterus (particularly, uterus and uterine body) of the present invention is as follows. Uterus: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-12 (Uterus) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Uterus Corpus: A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-8 (Uterus_Corpus) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0045] (Method of differentiation into blood cells) The method for differentiation into blood cells of the present invention (particularly whole blood, bone marrow, mononuclear cells, lymph nodes, tonsils, thymus, natural killer cells, dendritic cells, B cells, B lymphoblasts, T cells (PB_CD8, PB_CD4), and early red blood cells) is as follows. Whole blood: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-14 (Whole_blood (JJV)) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Bone marrow: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-14 (Myeloid (BM_CD33)) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Monocytes: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-14 (Monocytes (PB_CD14)) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Lymph node: A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-15 (Lymphnode) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Tonsil: A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-15 (Tonsil) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Thymus: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-15 (Thymus) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Natural killer cells: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-16 (Natural_Killer_Cells (PB_CD56)) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Dendritic cells: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-16 (Dendritic_Cells (PB_BDCA4)) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. B cells: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-16 (B_Cells (PB_CD19)) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. B lymphoblasts: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-17 (B_lymphoblasts (721)) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. T cells (PB_CD8): A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-16 (T_Cells (PB_CD8)) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. T cells (PB_CD4): A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-17 (T_Cells (PB_CD4)) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Early erythroid: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-17 (Early_Erythroid (BM_CD71)) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Lymphoma-derived Raji cells: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-17 (Lymphoma_Raji) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Chronic myelogenous leukemia cells: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-18 (Leukemia_chronic_myelogenous (k562)) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Burkitt's lymphoma Daudi cell line: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-18 (Lymphoma_burkitts_Daudi) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Promyelocytic leukemia cells: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-19 (Leukemia_promyelocytic (hl60)) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Lymphoblastic leukemia cells: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-19 (Leukemia_lymphoblastic (molt4)) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0046] {Method of differentiation into myeloid cells (cells present in bone marrow)} The method for myeloid differentiation of the present invention is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-15 (Bone_marrow) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0047] (Method of differentiation into hematopoietic stem cells) The method of the present invention for differentiating into hematopoietic stem cells is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-18 (Bone_marrow (CD34)) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0048] (Method for differentiating into vascular endothelial cells) The method for differentiating into vascular endothelial cells of the present invention is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-18 (Endothelial_cells (BM_CD105)) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0049] {A method for differentiating into testis (especially cells present in the testis)} The method for differentiating into the testis of the present invention (particularly into testis, testicular Leydig cells, testicular germ cells, testicular seminiferous tubules, and testicular interstitial cells) is as follows. Testis: A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-19 (Testis) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Testis Leydig cells: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-19 (Testis_Leydig_Cell) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Testis germ cells: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-20 (Testis_Germ_Cell) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Testis seminiferous tubule: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-20 (Testis_Seminiferous_Tubule) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Testicular interstitial cells: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-20 (Testis_Intersitial) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0050] {Method of differentiation into cardiac cells (especially cardiac cells)} The method for differentiation into cardiac (particularly cardiac, cardiomyocytes) is as follows. Heart: A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-6 (Heart) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Cardiomyocytes: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-13 (Cardiac_Myocytes) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0051] {Method of differentiation into placenta (especially cells present in the placenta)} The method for differentiation into placenta of the present invention is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-9 (Placenta) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0052] {A method for differentiating into smooth muscle cells (especially cells present in smooth muscle cells)} The method for differentiating into smooth muscle cells of the present invention is as follows. A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-13 (Smooth_Muscle) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0053] {Method of differentiation into lung cells (especially cells present in the lung)} The method for differentiation into lung (particularly lung, bronchial epithelial cells, fetal lung, trachea) of the present invention is as follows. Lung: A single transcription factor or two or more transcription factors selected from the transcription factors shown in Figure 4-9 (Lung) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors shown in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Bronchial epithelial cells: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-14 (Bronchial_epithelial_cells (HBEC)) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Fetal lung: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-10 (Fetal_lung) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells. Trachea: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-10 (Trachea) are introduced into human pluripotent stem cells. Furthermore, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0054] (Method of Differentiating into Colorectal Adenocarcinoma Cells) The method for differentiating into colorectal adenocarcinoma cells of the present invention is as follows. Colorectal adenocarcinoma cells: A single transcription factor or two or more transcription factors selected from the transcription factors listed in Figure 4-13 (Colorectal_Adenocarcinoma) are introduced into human pluripotent stem cells. In addition, if necessary, one or more transcription factors listed in other gene expression correlation matrices are simultaneously introduced into human pluripotent stem cells.

[0055] (Differentiation inducer for use in a method for differentiating pluripotent stem cells into a desired cell type) The differentiation inducer used in the method of the present invention for differentiating pluripotent stem cells into a desired cell type (hereinafter, sometimes referred to as the "differentiation inducer of the present invention") is a composition containing at least a transcription factor necessary for the method of the present invention. In detail, the differentiation inducer of the present invention includes transcription factors in the form of nucleic acids, synthetic mRNAs, proteins, and viral vectors carrying them, nanoparticle capsules carrying them, liposomes carrying them, or exosomes carrying them.

[0056] Examples of the transcription factors that are proteins contained in the differentiation inducer of the present invention include the following. (1) A transcription factor, or a protected, glycosylated, acylated, or acetylated derivative of the transcription factor. (2) A transcription factor that has 90% (or 92%, 94%, 96%, 98%, or 99%) or more homology with a transcription factor and has a transcription factor-specific action that is substantially the same as that of the transcription factor. (3) A transcription factor according to any one of the above, which comprises 100 to 10, 50 to 30, 40 to 20, 10 to 5, or 5 to 1 amino acids substituted, deleted, inserted and / or added, and has an action specific to a transcription factor that is substantially identical to that of the transcription factor. Examples of transcription factors in the mRNA, synthetic mRNA, and nucleic acid contained in the differentiation inducer of the present invention include the following. (4) A gene encoding a polypeptide consisting of one or more of the above transcription factors. (5) A gene encoding a polypeptide in which 1 to 20 (or 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 3) amino acids have been substituted, deleted, inserted and / or added in the amino acid sequence of any one or more of the above transcription factors and which has a transcription factor-specific action that is substantially identical to that of the transcription factor. (6) A gene encoding a polypeptide having 90% (or 92%, 94%, 96%, 98%, or 99%) or more homology to the amino acid sequence of any one or more of the above transcription factors and having a transcription factor-specific function that is substantially identical to that of the transcription factor. (7) A gene having a homology of 90% (or 92%, 94%, 96%, 98%, or 99%) or more to the base sequence of any one or more of the above transcription factors and encoding a polypeptide having a transcription factor-specific function that is substantially identical to that of the transcription factor. The transcription factor having a mutation may be one that exists in nature, or may be one obtained by introducing a mutation into a naturally occurring gene. Means for introducing a mutation are known per se, and for example, site-directed mutagenesis, homologous recombination, primer extension, polymerase chain reaction (hereinafter abbreviated as PCR), and the like can be used alone or in appropriate combination. For example, the method described in the textbooks (Sambrook et al., eds., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, 1989; Muramatsu Masami, ed., Laboratory Manual of Genetic Engineering, Maruzen Co., Ltd., 1988) or a modified version of the method can be used, and Ulmer's technique (Ulmer, KM, Science, 1983, vol. 219, pp. 666-671) can also be used. In the case of peptides, from the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the peptide upon introduction of mutations, for example, mutual substitutions between homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) can be easily assumed.

[0057] The differentiation inducers of the present invention are listed below. Neuronal differentiation inducers (especially fetal brain differentiation inducers, cerebellar peduncle differentiation inducers, cerebellum differentiation inducers, whole brain differentiation inducers, thalamus differentiation inducers, hypothalamus differentiation inducers, prefrontal cortex differentiation inducers, occipital lobe differentiation inducers, amygdala differentiation inducers, caudate nucleus differentiation inducers, cingulate cortex differentiation inducers, medulla oblongata differentiation inducers, pallidum differentiation inducers, subthalamic nucleus differentiation inducers, parietal lobe differentiation inducers, temporal lobe differentiation inducers, pons differentiation inducers) Motor neuron differentiation inducer Motor neurons are classified into two types: central and peripheral. Central motor neurons are nerves that run from the brain to the spinal cord, while peripheral motor neurons are nerves that run from the spinal cord to the muscles. Pituitary differentiation inducer Olfactory nerve differentiation inducers (especially olfactory bulb differentiation inducers) Spinal cord neuronal differentiation inducers (especially spinal cord differentiation inducers) ○Skeletal muscle differentiation inducers (especially psoas muscle differentiation inducers and tongue differentiation inducers), Skin differentiation inducer Ganglion differentiation inducers (especially dorsal root ganglion differentiation inducers, superior cervical ganglion differentiation inducers, atrioventricular node differentiation inducers, trigeminal ganglion differentiation inducers, ciliary ganglion differentiation inducers) Ovarian differentiation inducer Adrenal differentiation inducers (especially adrenal cortex differentiation inducers, adrenal differentiation inducers) Appendiceal differentiation inducer Renal differentiation inducer ○Liver differentiation inducers (especially liver differentiation inducers, fetal liver differentiation inducers) Salivary gland differentiation inducer ○ Pancreatic islet differentiation inducers (especially pancreatic islet cell differentiation inducers), Pancreatic differentiation inducer Prostate differentiation inducer Thymic differentiation inducers (especially thyroid differentiation inducers and fetal thyroid differentiation inducers) Adipocyte differentiation inducers (especially cultured adipocyte differentiation inducers) ○ Uterine differentiation inducers (especially uterine differentiation inducers, uterine body partialization inducers) Blood cell differentiation inducers (especially whole blood differentiation inducers, bone marrow differentiation inducers, mononuclear cell differentiation inducers, lymph node differentiation inducers, tonsil differentiation inducers, thymus differentiation inducers, natural killer cell differentiation inducers, dendritic cell differentiation inducers, B cell differentiation inducers, B lymphoblast differentiation inducers, T cell (PB_CD8, PB_CD4) differentiation inducers, early red blood cell differentiation inducers) Bone marrow differentiation inducer Blood cell (or hematopoietic stem cell) differentiation inducer Vascular endothelial cell differentiation inducer ○ Testicular differentiation inducers (especially testicular differentiation inducers, testicular Leydig cell differentiation inducers, testicular germ cell differentiation inducers, testicular seminiferous tubule differentiation inducers, testicular interstitial cell differentiation inducers) Cardiac differentiation inducers (especially cardiac differentiation inducers, cardiomyocyte differentiation inducers), Placental differentiation inducer Smooth muscle differentiation inducer, Pulmonary differentiation inducers (especially pulmonary differentiation inducers, bronchial epithelial cell differentiation inducers, fetal lung differentiation inducers, tracheal differentiation inducers) Cartilage differentiation inducer

[0058] The present invention will be specifically described with reference to the following examples, but the present invention is not limited thereto. All of the examples were approved by the Ethics Committee of Keio University School of Medicine. EXAMPLES

[0059] (Creating a human gene expression correlation matrix) In this example, a human gene expression correlation matrix (see Figs. 3 and 4) was created. The details are as follows.

[0060] With reference to the literature "Cell Stem Cell 5, 420-433 (2009)" and "Sci Rep 1, 167 (2011)", gene expression profiles of human ES cells were obtained by RNA sequencing (RNA-seq) under forced expression (DOX+) or non-forced expression (DOX-) for 48 hours for 175 transcription factors one by one. Furthermore, the sequenced RNA fragments of 50-bp length were aligned with the human genome sequence by computer analysis. The RNA fragments were then identified to match mRNA / ncRNA from Ensembl and RefSeq (gene coordinates of transcripts were downloaded from UCSC database, genome version hg19, hgdownload.soe.ucsc.edu / goldenPath / hg19 on August 5, 2014). Ensembl data was used primarily, and uncovered transcripts were supplemented with RefSeq. Matches to genomes were counted if the number of mismatches was 2 or less and the number of hits in the genome was 10 or less. Fragments were weighted by 1 / n, where n is the number of hits in the genome. Fragments were identified as transcription factors if they matched in orientation, matched within 5-bp of gene and intron boundaries, and the total matched length was 90% or less of the read length. Gene expression changes induced by forced expression of each transcription factor were normalized by subtracting the log-transformed expression value of the DOX+ sample (one replicate per clone) from the log-transformed expression value of the corresponding DOX- sample and adding the median log-transformed expression value of all DOX- samples. The relationship between gene expression changes induced by overexpression of transcription factors and tissue-specific gene expression was evaluated based on the correlation between the GNF database (see Non-Patent Document 22) and the results of RNA-seq. Correlations were analyzed between gene expression response to transcription factor transduction and median-subtracted log-transformed gene expression values ​​in various tissues in the human GNF database ver. 2. Correlation analysis was performed using 9,980 genes that showed significant values ​​in both data sets. Criteria for significance in the GNF database were a false positive rate (FDR) of 0.05 or less and a 2-fold change or greater. Correlation matrices were calculated using ExAtlas sorting using hierarchical clustering, lgsun.grc.nia.nih.gov / exatlas.

[0061] The results of creating a human gene expression correlation matrix are shown in Figures 3 and 4. Figure 3 shows a correlation matrix between "changes in gene expression induced by forced expression of specific transcription factors (horizontal axis)" and "tissue-specific gene expression from the GNF database (vertical axis)." Each cell in this matrix shows the significance (z-value) of the correlation between "changes in gene expression induced by forced expression of specific transcription factors (measured as log-ratio, horizontal axis)" and "tissue-specific gene expression from the GNF database (vertical axis)." Figure 4 shows the top 30 Z-scores, expressed as actual numbers, for each cell, tissue, and organ listed in Figure 3. EXAMPLES

[0062] (Methods of Differentiation into Desired Cell Types) By using the human gene expression correlation matrix (FIGS. 3 and 4) obtained in this Example 1, pluripotent stem cells can be differentiated into the cells, tissues, and organs shown in FIG. 3. As a differentiation method, a molecular biology technique known per se can be used. For example, a method using synthetic mRNA, nanoparticle-encapsulated synthetic mRNAs, or Sendai virus vectors can be used. These methods are footprint-free and can introduce transcription factor mRNA and proteins into pluripotent stem cells. Details are as follows.

[0063] (Synthesis of mRNA encoding the gene for the transcription factor) Modified mRNA was synthesized by referring to the method described in the literature "Warren et al., Cell Stem Cell, 2010 Nov 5;7(5):618-30". More specifically, mRNA was synthesized by in vitro transcription using a mixture of modified dNTPs {(dNTPs: 3-0-Me-m7G(5')ppp(5')G ARCA cap analog, 5-methylcytidine triphosphate, and pseudouridine triphosphate)} of template DNAs encoding the genes of the transcription factors shown in Figure 3. To express mammalian (especially human) transcription factors, we used a Sendai virus vector capable of expressing the human transcription factors shown in Figure 3. In particular, mutants of Sendai virus vectors, such as those lacking F protein, are non-infectious and easy to manipulate (see Inoue et al., J. Virol. 77: 23238-3246, 2003).

[0064] (Method of Differentiating Pluripotent Stem Cells into Desired Cell Types) A single transcription factor or a cocktail of two or more transcription factors was prepared with reference to the Z value shown in FIG. 4. The form of the transcription factor is not particularly limited, and may be any of synthetic mRNAs, Sendai virus vectors incorporating a transcription factor (or multiple transcription factors), and nanoparticle capsules containing synthetic mRNAs. Note that the synthetic mRNA may carry gene sequences of multiple transcription factors on the same gene. The method for introducing these single or a cocktail of two or more transcription factors into cells is not particularly limited, and transfection using lipofectamine, viral infection, and the like can be used. Furthermore, the type of cells to be introduced (particularly, mammalian cells, more preferably human cells) is not particularly limited, and includes pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), tissue stem cells derived from tissues or organs, skin fibroblasts, and any type of cell derived from tissues or organs.

[0065] The outline of the steps of the method for differentiating into a desired cell type in this embodiment is shown in Figure 5. By introducing a transcription factor (or a transcription factor cocktail) selected from the human gene expression correlation matrix in this embodiment 1 into a cell by the method described in this embodiment 2, the cell can be differentiated into a desired cell type as shown below. nerve cells (particularly fetal brain, cerebellar peduncle, cerebellum, whole brain, thalamus, hypothalamus, prefrontal cortex, occipital lobe, amygdala, caudate nucleus, cingulate cortex, medulla oblongata, globus pallidus, subthalamic nucleus, parietal lobe, temporal lobe, pons), pituitary gland, olfactory nerve (particularly olfactory bulb), spinal nerve (particularly spinal cord), skeletal muscle (particularly psoas muscle, tongue), skin, ganglia (particularly dorsal root ganglion, superior cervical ganglion, atrioventricular node, trigeminal ganglion, ciliary ganglion), ovary, adrenal gland (particularly adrenal cortex, adrenal gland), appendix, kidney, liver (particularly liver, fetal liver), salivary gland, pancreatic islet (particularly pancreatic islet cell), pancreas, prostate, thymus (particularly thyroid, fetal thyroid gland), adipocytes (particularly cultured adipocytes), uterus (particularly uterus, uterine body), blood cells (particularly whole blood, bone marrow, mononuclear cells, lymph nodes, tonsils, thymus, natural killer cells, dendritic cells, B cells, B lymphoblasts, T cells (PB_CD8, PB_CD4), early red blood cells), bone marrow, hematopoietic stem cells, vascular endothelial cells, testis (particularly testis, testicular Leydig cells, testicular germ cells, testicular seminiferous tubules, testicular interstitial cells), heart (particularly heart, cardiomyocytes), placenta, smooth muscle, and lung (particularly lung, bronchial epithelial cells, fetal lung, trachea), motor neurons, hepatoblasts, hepatocytes, chondrocytes. EXAMPLES

[0066] (Differentiation into neural cells) In this example, it was confirmed that a transcription factor cocktail selected from the human gene expression correlation matrix of this example 1 can be introduced into human embryonic stem cells by the method described in this example 2, and the cells can be differentiated into neural cells. The details are as follows.

[0067] (Method of differentiation into neural cells) With reference to the transcription factors and their Z values ​​shown in Figures 3 and 4, synthetic mRNAs of a cocktail of four transcription factors (NEUROD1, NEUROD2, NEUROG2, and NEUROG3) with a Z value of 12 or more were introduced (transfected) into human embryonic stem cells using RNAiMAX transfection reagent. The transfection was performed twice on the first day and twice on the second day (see Figure 6A). On the third day, the medium was replaced with a standard neuronal differentiation medium to obtain cells on the seventh day of culture.

[0068] (Confirmation of differentiation into neural cells) The differentiation state of the cells was confirmed using a marker for mature neurons (beta-3-tubulin, Bill) (see Figure 6B). NEUROD1 alone increased cells stained with Bill, but no clear neuronal morphology was observed. In contrast, the cocktail of four transcription factors not only increased the number of Bill-stained cells but also the number of cells with mature neuronal morphology. From the above, it was confirmed that pluripotent stem cells can be differentiated into neurons by introducing a cocktail of transcription factors involved in differentiation into neurons, selected from the human gene expression correlation matrix of this Example 1, into the pluripotent stem cells. EXAMPLES

[0069] (Differentiation into motor neurons) In this example, it was confirmed that a transcription factor cocktail selected from the human gene expression correlation matrix of this example 1 can be added (introduced) to human embryonic stem cells or human induced pluripotent stem cells by the method described in this example 2, and can be differentiated into motor neurons. The details are as follows.

[0070] With reference to the transcription factors and their Z values ​​shown in Figures 3 and 4, synthetic mRNAs of a cocktail of five transcription factors (NEUROD1, NEUROD2, NEUROG1, NEUROG2, and NEUROG3) were introduced (transfected) into human embryonic stem cells using RNAiMAX transfection reagent. The transfection was performed twice on the first day, and then the cells were cultured in a standard neuronal differentiation medium for six days to obtain cells on the seventh day of culture.

[0071] The cells were immunostained with neuronal markers to confirm that they had differentiated into neurons (see Figure 7). Furthermore, electrophysiological analysis confirmed that these neurons were motor neurons based on the expression of markers such as choline-acetyl transferase (ChAT), ISL1, and HB9 (see Figure 7). Culture conditions for differentiating human embryonic stem cells into neural cells are already known. However, in conventional methods, it usually takes several weeks to more than a month for neural cell markers to be expressed. However, in this example, it was confirmed that rapid and highly efficient differentiation was achieved by adding five types of synthetic mRNA to human embryonic stem cells. Furthermore, it was confirmed that it was also possible to transfect three types of transcription factors in the first transfection and two types of transcription factors in the second transfection.

[0072] For human induced pluripotent stem cells, the same conditions were used as for human embryonic stem cells. The results for human induced pluripotent stem cells confirmed that they differentiated rapidly and efficiently, similar to the results for human embryonic stem cells (see Figure 8). In this example, it was confirmed for the first time that pluripotent stem cells can be differentiated into motor neurons (motor neuron cells, particularly peripheral motor neuron cells). EXAMPLES

[0073] (Hepatocyte differentiation) In this example, it was confirmed that human embryonic stem cells can be differentiated into hepatocytes and hepatoblasts by forcibly expressing transcription factors selected from the human gene expression correlation matrix of this example 1 using the method described in this example 1. Details are as follows.

[0074] Each transcription factor (TGIF, TCF4, PITX2, SALL4, or MEIS1) was selected with reference to the transcription factors and their Z values ​​shown in Figures 3 and 4. These transcription factors were introduced as doxycycline-inducible transgenes into human embryonic stem cells, and these transcription factors were forcibly expressed in the human embryonic stem cells by adding doxycycline. Forced expression was performed for 24 hours on the first day, and the cells were then cultured in a known hepatocyte differentiation medium for 8 days to obtain cells on the 9th day of culture.

[0075] The expression of albumin, a marker for hepatocytes, confirmed that the cells had differentiated into hepatocytes and hepatoblasts (see Figure 9). In the figure, "TFIF-, TCF4-, PITX2-, SALL4-, MEIS1-" refers to the control group in which no transcription factors were added. Culture conditions for differentiating human embryonic stem cells into hepatocytes are already known. For example, Hay DC, et al. (2008), Stem Cells 26:894-902 can be mentioned. In addition, Kajiwara et al. (2012) {Proc Natl Acad Sci US A. 109:12538-43} et al. reported that by adding various cell growth and differentiation factors to the medium, the expression of albumin, a marker of liver cells, began on the 17th day. However, in this example, under the same culture conditions, the expression of albumin could be confirmed on the 9th day of culture. In other words, the culture time could be shortened by about half. In addition, in the method described in the literature, the addition of HGF, a hepatocyte-specific differentiation factor, is necessary from the 10th day onwards, but this was not necessary in this example. Considering the above, the differentiation into hepatocytes of the present invention has a remarkable effect of rapid differentiation and a unique effect of eliminating the need for the addition of HGF, which is a differentiation factor specific to hepatocytes, compared with conventional methods. Furthermore, in this example, differentiation was achieved by introducing one copy of each of the five transcription factors, but a method in which five, four, three, or two transcription factors are transfected simultaneously or separately is also possible. EXAMPLES

[0076] (Hematopoietic stem cells / differentiation into blood cells) In this example, it was confirmed that human embryonic stem cells can be differentiated into hematopoietic stem cells / blood cells by forcibly expressing the transcription factors selected from the human gene expression correlation matrix in this example 1 using the method described in this example 1. The details are as follows.

[0077] Each transcription factor (CDYL2, ETS2, SPI1, OVOL2, CDX2, CEBPB, or SALL4) was selected with reference to the transcription factors and their Z values ​​shown in Figures 3 and 4. These transcription factors were introduced as transgenes induced by doxycycline into human embryonic stem cells, and these transcription factors were forcibly expressed in the human embryonic stem cells by adding doxycycline. Forced expression was carried out for 48 hours on days 3 to 5 of culture, and the cells were cultured in a known hematopoietic progenitor cell medium to obtain cells on the 5th day of culture.

[0078] Culture conditions for differentiating human ES cells into hematopoietic progenitor cells are already known. For example, they are disclosed in the literature Wang et al. (2012) Cell Res. 22:194-207. The literature reports that by adding various cell growth and differentiation factors to the culture medium, expression of CD43, a marker for hematopoietic progenitor cells, begins on the 17th day. However, in this example, it was found that the speed and rate of differentiation into hematopoietic progenitor cells was significantly increased by forcibly expressing (inducing with DOX) a transcription factor under known hematopoietic progenitor cell conditions. Differentiation was possible using either a single transcription factor or a combination of transcription factors. More specifically, in this example, expression of CD43, a marker for hematopoietic progenitor cells, was confirmed on the fifth day of culture (see FIG. 10). Furthermore, on the fifth day, almost no expression of CD43 was observed in the control group (conventional technology) in which no transcription factor was added. As a result, the method of the present invention has approximately four times the differentiation induction ability (differentiation rate) compared to the conventional method. EXAMPLES

[0079] (Differentiation into chondrocytes) In this example, it was confirmed that human embryonic stem cells can be differentiated into chondrocytes by forcibly expressing a transcription factor selected from the human gene expression correlation matrix of this example 1 using the method described in this example 1. The details are as follows.

[0080] SOX9 was selected with reference to the transcription factors and their Z values ​​shown in Figures 3 and 4. SOX9 was introduced as a transgene induced by doxycycline into human embryonic stem cells, and SOX9 was forcibly expressed in the human embryonic stem cells by adding doxycycline. Forced expression was performed for 24 hours on the first day, and the cells were then cultured in a known human embryonic stem cell culture medium for two days to obtain cells on the third day of culture.

[0081] Culture conditions for differentiating human embryonic stem cells into chondrocytes are already known. For example, they are disclosed in Oldershaw et al. (2010). Nat Biotechnol. 28:1187-94. In this document, Type II Collagen-positive chondrocytes are produced 14 days after differentiation induction using a complex method in which various cell growth and differentiation factors are added to the medium in stages. However, in this example, it was confirmed that by forcibly expressing SOX9, Type II Collagen-positive chondrocytes could be created two days later (i.e., the third day after differentiation induction) (see "SOX9+for 1 day" in Figure 11). In this example, the differentiation-promoting medium used in the literature was not used, but a normal embryonic stem cell culture medium was used. In the control without SOX9 (see "SOX9-" in Figure 11), the expression of Type II Collagen could not be confirmed. As described above, the differentiation method of the present invention does not require the use of a special medium and has about four times the differentiation induction ability (differentiation rate) compared to conventional methods. EXAMPLES

[0082] (Neuronal differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling them to differentiate into neural cells. The method for differentiating the neural cells of the present invention (particularly cells present in the fetal brain, cerebellar peduncle, cerebellum, whole brain, thalamus, hypothalamus, prefrontal cortex, occipital lobe, amygdala, caudate nucleus, cingulate cortex, medulla oblongata, globus pallidus, subthalamic nucleus, parietal lobe, temporal lobe, and pons) is as follows. Fetal brain: NEUROG2, NEUROG1, NEUROG3, SOX11, MXI1, PDX1, NEUROD2, HOXA2, NEUROD1 are introduced into human pluripotent stem cells. Cerebellar peduncle: NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, ASCL1, HOXA2, PITX2, NEUROD2 introduced into human pluripotent stem cells. Cerebellum: NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, HOXA2, ASCL1, PITX2, NEUROD2, PRDM1, NFIB are introduced into human pluripotent stem cells. Whole brain: NEUROG2, NEUROG3, NEUROG1, NEUROD1, NRF1, and HOXA2 are introduced into human pluripotent stem cells. Brain thalamus: NEUROG2, NEUROG3, HOXA2, NEUROG1, and ASCL1 are introduced into human pluripotent stem cells. Hypothalamus: NEUROG2, SOX2, NEUROG1, NEUROG3, HOXA2, and SOX11 are introduced into human pluripotent stem cells. Prefrontal cortex: NEUROG2, NEUROG3, NEUROG1, SOX11, MXI1, SOX2, HOXA2, PDX1, ASCL1, NEUROD1, and NEUROD2 were introduced into human pluripotent stem cells. Occipital cortex: NEUROG2, NEUROG1, NEUROG3, HOXA2, SOX2, SOX11, PDX1, NEUROD1, ASCL1, MXI1 are introduced into human pluripotent stem cells. Brain amygdala: NEUROG2, NEUROG1, NEUROG3, HOXA2, SOX2, PDX1, SOX11, MXI1, NEUROD1, and ASCL1 are introduced into human pluripotent stem cells. Caudate nucleus: NEUROG2, HOXA2, NEUROG1, NEUROG3, SOX2, NRF1, ASCL1, and PDX1 are introduced into human pluripotent stem cells. Cingulate cortex: NEUROG2, NEUROG3, NEUROG1, HOXA2, ASCL1, SOX11, PDX1, NRF1, NEUROD1, PRDM1 are introduced into human pluripotent stem cells. Medulla oblongata: NEUROG2, HOXA2, NEUROG1, NEUROG3, PDX1, SOX11, SOX2, NRF1, ASCL1, NR2F2, and GLIS2 are introduced into human pluripotent stem cells. Pale blue cells: Introducing NEUROG2, HOXA2, NEUROG1, NEUROG3, NRF1, ASCL1, SOX11, PDX1 into human pluripotent stem cells. Subthalamic nucleus: NEUROG2, HOXA2, NEUROG1, NEUROG3, NRF1, ASCL1, SOX11, and PDX1 are introduced into human pluripotent stem cells. Parietal cortex: NEUROG2, HOXA2, NEUROG3, NEUROG1, NRF1, SOX11, ASCL1, PDX1, SOX2 are introduced into human pluripotent stem cells. Temporal lobe: NEUROG2, HOXA2, NRF1, NEUROG1, and NEUROG3 are introduced into human pluripotent stem cells. Pontine: HOXA2, NRF1, NEUROG2, NEUROG1, NEUROG3, CTCF, NR2F2, HES1, NFIC, PDX1, SOX11, ERG introduced into human pluripotent stem cells. EXAMPLES

[0083] (Pituitary Gland Differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells by the method described in this Example 2, thereby enabling differentiation into cells present in the pituitary gland, specifically cells present in the pituitary gland. The method for differentiation of the pituitary gland of the present invention is as follows. SOX2, NANOG, ASCL1, DLX4, DLX3, and CDX2 are introduced into human pluripotent stem cells. EXAMPLES

[0084] (Differentiation of olfactory nerves) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling differentiation into olfactory nerves, particularly cells present in the olfactory nerves. The method for differentiating olfactory nerves (particularly, olfactory bulbs) of the present invention is as follows. Olfactory bulb: NFIB, TBX2, TBX3, SOX2, NFIC, HES1, JUNB, FOS, and FOXA2 are introduced into human pluripotent stem cells. EXAMPLES

[0085] (Differentiation of spinal nerves) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells by the method described in this Example 2, thereby enabling them to differentiate into spinal nerves, particularly cells present in spinal nerves. The method for differentiating spinal nerves (particularly spinal cord) of the present invention is as follows. Spinal cord: NFIB, SOX2, HOXA2, TBX3, and E2F6 are introduced into human pluripotent stem cells. EXAMPLES

[0086] (Skeletal muscle differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling them to differentiate into skeletal muscle, particularly cells present in skeletal muscle. The method for differentiating skeletal muscles (particularly, psoas muscle and tongue) of the present invention is as follows. Psoas: MYOD1, NRF1, SALL4, ZIC1, KLF9, ZNF281, CTCF, HES1, HOXA2, TBX5, TP73, ERG, MAB21L3, PRDM1, NFIC, CTCFL, FOXP1, HEY1, PITX2 into human pluripotent stem cells. Tongue: MYOD1, TP73, HES1, JUNB, KLF4, SALL4, ZIC1, ESX1, ZNF281, TBX5, NRF1, HEY1, TFAP2C, FOS, FOXP1, TFE3, CTCF, FOSL1, GRHL2, TBX2, NFIB, PITX2, KLF9, IRF4 into human pluripotent stem cells. EXAMPLES

[0087] (Skin Differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling them to differentiate into skin, and in particular into cells present in the skin. The method for differentiation of skin according to the present invention is as follows. Introducing HES1, CTCF, TP73, JUNB, HEY1, ZIC1, TBX5, NFIC, TFAP2C, ESX1, NRF1, HOXA2, ELF1, NR2F2, KLF9, GRHL2, IRF4, ERG, FOS, TBX2, SALL4, and KLF4 into human pluripotent stem cells. EXAMPLES

[0088] (Ganglionic differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells by the method described in this Example 2, thereby enabling them to differentiate into ganglia, specifically cells present in ganglia. The method for differentiating ganglia (particularly dorsal root ganglia, superior cervical ganglia, atrioventricular node, trigeminal ganglion, and ciliary ganglion) of the present invention is as follows. Dorsal root ganglion: HES1, HOXA2, CTCF, TBX2, NR2F2, NRF1, HEY1, NFIC, TBX3, JUNB, TBX5, E2F6, GLIS2, ZIC1, ERG, KLF9 are introduced into human pluripotent stem cells. Superior cervical ganglion: HES1, CTCF, NRF1, HOXA2, HEY1, NFIC, NR2F2, TBX5, KLF9, ZIC1, ERG, FLI1, TBX2, JUNB, ELF1, GLIS2, TBX3, TFAP4, IRF4, PDX1 to introduce into human pluripotent stem cells. Atrioventricular node: HES1, CTCF, NRF1, HEY1, HOXA2, NFIC, and NR2F2, ZIC1, TBX5, KLF9, TBX2, ERG, JUNB, TFAP2C, ELF1, TP73, TFAP4, ESX1, E2F6, IRF4, FLI1, SALL4, TBX3, ARNT2 are introduced into human pluripotent stem cells. Trigeminal ganglion: HES1, CTCF, HOXA2, HEY1, NRF1, NR2F2, NFIC, TBX5, JUNB, ZIC1, TBX2, KLF9, ERG, ELF1, TBX3, E2F6, ESX1, ARNT2, GLIS2, TP73, IRF4 introduced into human pluripotent stem cells. Ciliary ganglion: HES1, CTCF, HEY1, NFIC, JUNB, HOXA2, NRF1, NR2F2, TBX2, ZIC1, TBX5, NFIB, ARNT2, ESX1, IRF4, ERG, TBX3, TFAP2C, ELF1, FOS, TP73, HSF1, KLF9, GLIS2, E2F6, PITX2, ZNF281, FOSL1, IRF1, FOXP1, GATA3 into human pluripotent stem cells. EXAMPLES

[0089] (Ovarian Differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling them to differentiate into ovaries, specifically cells present in the ovaries. The ovarian differentiation method of the present invention is as follows. HES1, HEY1, CTCF, NR2F2, NFIC, TBX2, TBX3, NRF1, ZIC1, HOXA2, TBX5, JUNB, and ERG are introduced into human pluripotent stem cells. EXAMPLES

[0090] (Adrenal Differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby differentiating them into cells present in the adrenal gland, specifically cells present in the adrenal gland. The method for differentiation of the adrenal gland (particularly, the adrenal cortex or adrenal gland) of the present invention is as follows. Adrenal cortex: SALL4, HES1, HEY1, ZIC1, FOXP1, ESX1, PITX2, NRF1, TP73, JUNB, DLX6, TGIF1 are introduced into human pluripotent stem cells. Adrenal gland: TGIF1, SALL4, TFE3, NFIB, ZIC1, and DLX6 are introduced into human pluripotent stem cells. EXAMPLES

[0091] (Differentiation of the appendix) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling them to differentiate into cells present in the appendix, specifically cells present in the appendix. The method for differentiation of the appendix of the present invention is as follows. HES1, CTCF, NR2F2, HEY1, HOXA2, NFIC, TBX2, NRF1, JUNB, TBX5, ZIC1, ERG, GLIS2, KLF9, ELF1, TBX3, IRF4, ARNT2, E2F6, IRF1, HSF1, SOX2, TFAP2C, TFAP4, FLI1, PDX1, RUNX3, MYOD1, HNF1A, NFIB, ESX1, TP73 are introduced into human pluripotent stem cells. EXAMPLES

[0092] (Kidney Differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling them to differentiate into kidney cells, specifically cells present in the kidney. The kidney differentiation method of the present invention is as follows. Introducing HNF4A, SALL4, TGIF1, HNF1A, ZIC1, NFIB, TFE3, TP73, TFAP2C, NRF1, SMAD7, and MAB21L3 into human pluripotent stem cells. EXAMPLES

[0093] (Liver Differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells by the method described in this Example 2, thereby enabling them to differentiate into liver cells, particularly cells present in the liver. The method for liver (particularly liver, fetal liver) differentiation of the present invention is as follows. Liver: SALL4, TGIF1, MAB21L3, ZIC1, EGFLAM, PITX2, HNF4A, NRF1, ZNF281, CTCFL, TP73, TFE3, DLX6, TCF4 introduced into human pluripotent stem cells. Fetal liver: Introducing SIX5, HNF4A, SIN3A, ID1, and HNF1A into human pluripotent stem cells. EXAMPLES

[0094] (Salivary gland differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling them to differentiate into salivary glands, specifically cells present in the salivary glands. The method for differentiation of salivary glands of the present invention is as follows. Introducing HES1, HEY1, ELF1, CTCF, and FLI1 into human pluripotent stem cells. EXAMPLES

[0095] (Islet differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells by the method described in this Example 2, thereby enabling them to differentiate into pancreatic islets, specifically cells present in pancreatic islets. The method for differentiating into the pancreatic islets (particularly, pancreatic islet cells) of the present invention is as follows. Introducing ASCL1, CEBPB, HES1, JUNB, and TFE3 into human pluripotent stem cells. EXAMPLES

[0096] (Pancreatic Differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling the cells to be differentiated into the pancreas, specifically into cells present in the pancreas. The method for differentiating into pancreas of the present invention is as follows. Introducing HNF4A, ELF1, ZIC1, SALL4, and JUNB into human pluripotent stem cells. EXAMPLES

[0097] (Prostate Differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling them to differentiate into prostate cells, particularly cells present in the prostate. The method for differentiation into prostate gland of the present invention is as follows. NFIB, TP73, FOS, IRF5, ESRRB, TFAP2C, GRHL2, HHEX, HOXA9, DLX6, ESX1, TGIF1, SALL4, CEBPB, and JUNB are introduced into human pluripotent stem cells. EXAMPLES

[0098] (Thyroid Differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells by the method described in this Example 2, thereby enabling differentiation into the thymus, specifically into cells present in the thymus. The method for differentiating into thyroid gland (particularly thyroid gland, fetal thyroid gland) of the present invention is as follows. Thyroid: MYOD1, NFIB, HHEX, ASCL2, and PPARG are introduced into human pluripotent stem cells. Fetal thyroid: NFIB, MYOD1, HHEX, TGIF1, and TFAP2C are introduced into human pluripotent stem cells. EXAMPLES

[0099] (adipocyte differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby differentiating them into adipocytes. The method for differentiating into the adipocytes (particularly, cultured adipocytes) of the present invention is as follows. Cultured adipocytes: Introducing JUN, NFIB, FOSL1, FOS, JUNB, and SREBF2 into human pluripotent stem cells. EXAMPLES

[0100] (uterine differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling them to differentiate into cells present in the uterus, specifically cells present in the uterus. The method for differentiating into the uterus (particularly, uterus and uterine body) of the present invention is as follows. Uterine: NFIB, JUN, FOSL1, SOX2, RUNX3, NFIC, JUNB, IRF5, PTF1A, HSF1, TBX2, TBX3, FOS, MEF2C, ARNT2, GATA2 to introduce into human pluripotent stem cells. Uterine corpus: HES1, JUNB, CTCF, HEY1, FOS, ZIC1, HOXA2, NFIC, FOSL1, NRF1, TBX5, ARNT2, NFIB, TFAP2C, ESX1, TBX2, TBX3, NR2F2, TP73, IRF4, THAP11, ELF1, JUN, ERG, HSF1, KLF9 are introduced into human pluripotent stem cells. EXAMPLES

[0101] (Blood cell differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby differentiating them into blood cells. The method for differentiation into blood cells of the present invention (particularly whole blood, bone marrow, mononuclear cells, lymph nodes, tonsils, thymus, natural killer cells, dendritic cells, B cells, B lymphoblasts, T cells (PB_CD8, PB_CD4), and early red blood cells) is as follows. Whole blood: CEBPB, SPI1, ETS1, ELF1, IRF1, ETS2, IRF2, DMRT1, KLF4 are transduced into human pluripotent stem cells. Bone marrow: SALL4, CEBPB, ESX1, ELF1, ZIC1, ZNF281, ETS1, KLF4, FOXP1, NRF1, SPI1 are introduced into human pluripotent stem cells.Bone marrow: CEBPB, SPI1, ETS1, ELF1, CDYL2, IRF1, GADD45A are introduced into human pluripotent stem cells. Mononuclear cells: SPI1, CEBPB, ETS1, ELF1, IRF1, CDYL2, GADD45A are introduced into human pluripotent stem cells. Lymph node: IRF1, IRF2, ELF1, ETS1, SPI1, ETS2, IRF4, RUNX3 are introduced into human pluripotent stem cells. Tonsil: ELF1, SPI1, IRF1, IRF2, ESX1, IRF4, KLF4, SALL4, ETS1 introduced into human pluripotent stem cells. Thymus: SALL4, ESX1, ETS1, SPI1, and ETS2 are introduced into human pluripotent stem cells. Natural killer cells: Introducing ETS1, CDYL2, GADD45A, IRF1, and IRF2 into human pluripotent stem cells. Dendritic cells: CDYL2, SPI1, GADD45A, ETS1, and MYC are introduced into human pluripotent stem cells. B cells: CDYL2, MYC, ATF2, IRF2, and GBX2 are introduced into human pluripotent stem cells. B lymphoblasts: MYC, CDYL2, GADD45A, GBX2, ATF2, RUVBL2, PLXNB3, L3MBTL2, E2F4, SMARCA4, ID1, ZSCAN4 are introduced into human pluripotent stem cells. T cells (PB_CD8): CDYL2, MYC, GBX2, ETS1, IRF2 are introduced into human pluripotent stem cells. T cells (PB_CD4): CDYL2, MYC, GBX2, ETS1, and ATF2 are introduced into human pluripotent stem cells. Early erythroid: Introducing CDYL2, E2F4, GADD45A, and ZSCAN4 into human pluripotent stem cells. EXAMPLES

[0102] (Bone marrow differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling them to differentiate into bone marrow, specifically into cells present in bone marrow. The method for myeloid differentiation of the present invention is as follows. Introducing SALL4, CEBPB, ESX1, ELF1, ZIC1, ZNF281, ETS1, KLF4, FOXP1, NRF1, and SPI1 into human pluripotent stem cells. EXAMPLES

[0103] (Differentiation of hematopoietic stem cells) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby differentiating the cells into hematopoietic stem cells. The method of the present invention for differentiating into hematopoietic stem cells is as follows. Introducing MYC, GBX2, CDYL2, GADD45A, ATF2, ID1, ZSCAN4, SMARCA4, E2F4, and RUVBL2 into human pluripotent stem cells. EXAMPLES

[0104] (Vascular endothelial cell differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby differentiating the cells into vascular endothelial cells. The method for differentiating into vascular endothelial cells of the present invention is as follows. Introducing MYC, RUVBL2, GBX2, CDYL2, GADD45A, ATF2, ID1, and E2F4 into human pluripotent stem cells. EXAMPLES

[0105] (Testicular Differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling them to differentiate into testis, specifically into cells present in the testis. The method for differentiating into the testis of the present invention (particularly into testis, testicular Leydig cells, testicular germ cells, testicular seminiferous tubules, and testicular interstitial cells) is as follows. Testis: SALL4, MYBL2, RFX2, TGIF1, and CTCFL are introduced into human pluripotent stem cells. Testicular Leydig cells: MYBL2, NR2F2, KLF9, GLIS2, and SIX5 are introduced into human pluripotent stem cells. Testicular germ cells: MYBL2, L3MBTL2, E2F4, KDM5A, and DMRT1 are introduced into human pluripotent stem cells. Testicular seminiferous tubules: MYBL2, E2F4, KLF9, YY1, and NEUROD1 are introduced into human pluripotent stem cells. Testicular interstitial cells: MYBL2, E2F4, NR2F2, KLF9, and GTF2F1 are introduced into human pluripotent stem cells. EXAMPLES

[0106] (Cardiac Differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells by the method described in this Example 2, thereby enabling them to differentiate into cells that reside in the heart, specifically cells that reside in the heart. The method for differentiation into cardiac (particularly cardiac, cardiomyocytes) is as follows. Heart: SALL4, TGIF1, PITX2, ZNF281, NRF1, ZIC1, TP73, FOXP1, CTCFL, NFIB, TFE3, EGFLAM, DLX6, TFAP2C, MYOD1, ESX1, PRDM1, MAB21L3, FOS, TCF4, JUNB, SMAD7, KLF4, ARID3A, TBX5, HOXA9, HES1, FOXG1, FOSL2, USF2, ERG, ARNT2 into human pluripotent stem cells. Cardiomyocytes: FOSL1, JUN, FOS, FOSL2, JUNB, HSF1, CUX1, IRF5, ESX1, ETS2 are introduced into human pluripotent stem cells. EXAMPLES

[0107] (Placenta differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells by the method described in this Example 2, thereby enabling them to differentiate into placenta, specifically into cells present in the placenta. The method for differentiation into placenta of the present invention is as follows. TFAP2C, ESX1, FOS, JUNB, TP73, IRF5, GATA3, TFE3, CEBPB, FOSL1, DLX6, JUN, FOXP1, ESRRB, NFIB, ETS2, HES1, ELF1, ZIC1, SALL4, TFAP2A, HSF1, HEY1, HHEX, TGIF1, THAP11, ETS1, ARNT2, IRF4, CUX1, GRHL2, HOXA9, TBX2, TBX5, ELF5 into human pluripotent stem cells. EXAMPLES

[0108] (Smooth muscle differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells by the method described in this Example 2, thereby enabling them to differentiate into smooth muscle, particularly cells present in smooth muscle. The method for differentiating into smooth muscle cells of the present invention is as follows. Introducing JUN, FOSL1, FOS, GADD45A, FOSL2, HSF1, JUNB, CUX1, IRF5, GATA3, and ETS2 into human pluripotent stem cells. EXAMPLES

[0109] (Lung Differentiation) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling them to differentiate into lung cells, particularly cells present in the lung. The method for differentiation into lung (particularly lung, bronchial epithelial cells, fetal lung, trachea) of the present invention is as follows. Lung: SALL4, TGIF1, FOS, TP73, NFIB, TFAP2C, ESX1, DLX6, PITX2, TFE3, JUNB, FOXP1ZNF281, CEBPB, ZIC1, IRF5, CTCFL, HOXA9, FOSL1, TCF4, GATA3, ETS2, EGFLAM, ARID3A, KLF4, FOSL2, HHEX, ETS1, ELF1, ESRRB, IRF4, NRF1, HES1, GRHL2, FOXG1, ELF5, PRDM1, RFX2, JUN, HNF4A, TFAP2A, ERG, ARNT2, HEY1 into human pluripotent stem cells. Bronchial epithelial cells: GADD45A, JUN, FOSL1, MYC, CUX1, IRF5, ESRRB, FOS, L3MBTL2, TRPV2, FOSL2 are introduced into human pluripotent stem cells. Fetal lung: CEBPB, GATA3, ESX1, NFIB, JUNB, IRF5, JUN, ETS2, HSF1, ESRRB, FOSL1, TGIF1, TBX2, TFAP2C, FOS, HNF4A into human pluripotent stem cells. Trachea: JUNB, HES1, TP73, TFAP2C, ESX1, CEBPB, GATA3, ELF1, FOXA2, FOS, IRF5, HEY1, NFIB, IRF4, ZIC1, FOXA1, NFIC, TBX5, CTCF, ESRRB, E2F6, FOSL1, HSF1, IRF1 into human pluripotent stem cells.

[0110] (General remarks) In this example, a transcription factor cocktail selected from the human gene expression correlation matrix of this Example 1 can be introduced into human embryonic stem cells using the method described in this Example 2, thereby enabling differentiation into a desired cell type.

[0111] 1. A method for differentiating human-derived pluripotent stem cells into hematopoietic stem cells and / or blood cells, comprising the step of introducing a CDYL2 transcription factor into the pluripotent stem cells. 2. A method for differentiating human-derived pluripotent stem cells into hematopoietic stem cells and / or blood cells, comprising the step of introducing an ETS2 transcription factor into the pluripotent stem cells. 3. A method for differentiating human-derived pluripotent stem cells into hematopoietic stem cells and / or blood cells, comprising the step of introducing only SPI1 as a transcription factor into the pluripotent stem cells. 4. A method for differentiating human-derived pluripotent stem cells into hematopoietic stem cells and / or blood cells, comprising the step of introducing a CEBPB transcription factor into the pluripotent stem cells. 5. A method for differentiating human-derived pluripotent stem cells into hematopoietic stem cells and / or blood cells, comprising the step of introducing a SALL4 transcription factor into the pluripotent stem cells. 6. A hematopoietic stem cell and / or blood cell differentiation inducer capable of differentiating human-derived pluripotent stem cells into hematopoietic stem cells, comprising one or more transcription factors of CDYL2, ETS2, SPI1, CEBPB and SALL4. 7. The differentiation method according to any one of the preceding items 1 to 6, wherein the transcription factor is mRNA, synthetic mRNA, a nucleic acid or a protein. 8. The differentiation inducer according to the preceding item 6, wherein the transcription factor is an mRNA, a synthetic mRNA, a nucleic acid or a protein. [Industrial Applicability]

[0112] The present invention can provide a method for differentiating a novel pluripotent stem cell into a desired cell type.

Claims

[Claim 1] A method for differentiating a pluripotent stem cell into a transformed cell, comprising: introducing a transcription factor into a mammalian pluripotent stem cell; wherein the transcription factor has a z-score of 3 or greater for a particular cell type in the table of human gene expression correlation matrices as defined herein; method.

Images