Method for regulating degree of differentiation of cells

Abstract

The present invention relates to a method for regulating the degree of differentiation of cells, comprising a step for forcibly expressing an MYC family gene and a BMI1 gene in a cell selected from the group consisting of (i) mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, and lymphocytes having an arbitrary degree of differentiation, and (ii) megakaryocytes and megayoung granulocytes having a high degree of differentiation.

Description

Technical Field

[0001] The present invention broadly relates to methods for regulating the degree of cell differentiation, etc. Background Art

[0002] Mesoderm-derived tissues such as bones, blood vessels, and cardiomyocytes are derived from mesenchymal stem cells through various lines of precursor cells. Mesenchymal stem cells are somatic stem cells that give rise to mesoderm-derived tissues such as osteoblasts, adipocytes, and chondrocytes. Mesenchymal stem cells are found in tissues such as bone marrow, fat, and dental pulp. Furthermore, peripheral blood cells differentiate from hematopoietic stem cells through various lines of hematopoietic precursor cells. Myeloid common precursor cells are hematopoietic precursor cells that give rise to platelets, red blood cells, and white blood cells other than lymphocytes (neutrophils, macrophages, basophils, dendritic cells, etc.). Normally, they are found in the bone marrow and differentiate as needed in response to trauma, infection, and other events to provide mature blood cells. The resulting neutrophils, macrophages, basophils, and dendritic cells are key players in innate immunity, helping to defend against various pathogens, eliminate tumors and degenerated endogenous cells, and address allergic reactions, acute, and chronic inflammation.

[0003] These cells hold promise for screening drugs for inflammation and allergies, and as a starting point for cell-based therapies to eliminate foreign substances in the body. Conventional methods employ in vitro differentiation induction from umbilical cord blood, bone marrow, or human ES / iPS cells, but these methods are cumbersome and difficult to prepare large quantities of cells.

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent literature 1: Jie Z, Zhang Y, Wang C, Shen B, Guan X, Ren Z, et al. Large-scale ex vivo generation of human neutrophils from cord blood CD34+ cells. PLoS One. 2017; 12(3).

[0007] Non-patent Document 2: Caux C, Vanbervliet B, Massacrier C, Dezutter-Dambuyant C, DeSaint-Vis B, Jacquet C, et al. CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNFα. J Exp Med. 1996;184(2):695-706.

[0008] Non-patent Document 3: Lachmann N, Ackermann M, Frenzel E, Liebhaber S, Brennig S, Happle C, et al. Large-scale hematopoietic differentiation of human induced pluripotent stem cells provides granulocytes or macrophages for cell replacement therapies. Stem Cell Reports. 2015;

[0009] Non-patent Document 4: Hiramoto T, Ebihara Y, Mizoguchi Y, Nakamura K, Yamaguchi K, Ueno K, et al. Wnt3a stimulates maturation of impaired neutrophils developed from severe congenital neutropenia patient-derived pluripotent stem cells. Proc Natl Acad Sci U S A. 2013;110(8):3023-8.

[0010] Non-Patent Document 5: Sweeney CL, Teng R, Wang H, Merling RK, Lee J, Choi U, et al. Molecular Analysis of Neutrophil Differentiation from Human Induced Pluripotent Stem Cells Delineates the Kinetics of Key Regulators of Hematopoiesis. Stem Cells. 2016;34(6):1513-26.

[0011] Non-Patent Document 6: Takata K, Kozaki T, Lee CZW, Thion MS, Otsuka M, Lim S, et al. Induced-Pluripotent-Stem-Cell-Derived Primitive Macrophages Provide a Platform for Modeling Tissue-Resident Macrophage Differentiation and Function. Immunity. 2017;47(1):183-198.e6.

[0012] Non-Patent Document 7: Cao X, Yakala GK, van den Hil FE, Cochrane A, Mummery CL, Orlova V V. Differentiation and Functional Comparison of Monocytes and Macrophages from hiPSCs with Peripheral Blood Derivatives. Stem Cell Reports. 2019;12(6):1282-97.

[0013] Non-Patent Document 8: Ackermann M, Kempf H, Hetzel M, Hesse C, Hashtchin AR, Brinkert K, et al. Bioreactor-based mass production of human iPSC-derived macrophages enables immunotherapies against bacterial airway infections. Nat Commun. 2018;9(1).

[0014] Non-patent document 9: Combination of immortalization and inducibledeath strategies to generate a human mesenchymal stromal cell line with controlled survival. Bourgine P, Le Magnen C, Pigeot S, Geurts J, Scherberich A, Martin I. StemCell Res. 2014Mar; 12(2):584-98 Summary of the Invention

[0015] Problems to be solved by the invention

[0016] Although the method using hematopoietic progenitor cells derived from umbilical cord blood and bone marrow blood can obtain neutrophils, macrophages, etc. by adding growth factors (see non-patent documents 1 and 2), the proliferation of umbilical cord blood and bone marrow blood itself is limited, and there are large differences between batches, making it difficult to prepare cells with stable quality in large quantities.

[0017] In the case of methods using human ES / iPS cells, although ES / iPS cells themselves can proliferate almost indefinitely, the differentiation induction method is complicated and the induction efficiency into blood cells is poor, making it difficult to supply them in large quantities at a clinical application level (see Non-Patent Documents 3 to 7). For example, in the case of reported differentiation of macrophages from human iPS cells, in a 250 mL culture system, 10 8 From the perspectives of drug screening and cell therapy, the number of cells in existing technologies is clearly insufficient, and there is a need to develop more efficient induction methods.

[0018] Some immortalized myeloid cell lines have been established through culture at low rates from leukemia patients. However, these cells stably express leukemia mutations and are unable to differentiate normally, making them incapable of drug screening using mature cells. Furthermore, they carry the risk of cancerous transformation, making them unsuitable as a starting point for cell therapy.

[0019] As an immortalized mesenchymal stem cell line, there is a cell line created by forcibly expressing hTERT in human bone marrow-derived mesenchymal stromal cells (Non-Patent Document 9). However, this requires bone marrow puncture and presents invasiveness issues. This paper does not describe rejuvenating MSCs at any stage of differentiation (converting them to a more undifferentiated state) and does not demonstrate the ability to rejuvenate progenitor cells.

[0020] In view of this situation, the purpose of the present invention is to provide a novel method for regulating cell differentiation in order to establish a stable production system for cells such as mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, and lymphocytes, neutrophils, macrophages, basophils, and dendritic cells.

[0021] Solutions for solving problems

[0022] The present inventors have conducted repeated studies to solve the above-mentioned technical problems and have discovered that by forcibly expressing MYC family genes and BMI1 genes in mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, and lymphocytes with any degree of differentiation, as well as in megakaryocytes and granulocytes with a high degree of differentiation, the degree of cell differentiation can be regulated, thereby completing the present invention.

[0023] That is, the present invention includes the following inventions. [1]

[0025] A method for regulating cell differentiation degree, comprising the following steps:

[0026] MYC family genes and BMI1 gene are forcibly expressed in cells selected from the group consisting of (i) mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, and lymphocytes with any degree of differentiation, and (ii) megakaryocytes and myelocytes with a high degree of differentiation. [2]

[0028] The method according to [1], wherein the cells are mesenchymal stem cells with any degree of differentiation. [3]

[0030] The method according to [1] or [2], wherein the cells are mesenchymal stem cells that do not express the cell surface marker CD90. [4]

[0032] The method according to any one of [1] to [3], wherein the cells are mesenchymal stem cells expressing the cell surface marker CD90. [5]

[0034] The method according to any one of [1] to [4], wherein the cells are vascular endothelial cells having any degree of differentiation. [6]

[0036] The method according to any one of [1] to [5], wherein the cells are smooth muscle cells having any degree of differentiation. [7]

[0038] The method according to any one of [1] to [6], wherein the cells are vascular smooth muscle cells having any degree of differentiation. [8]

[0040] The method according to any one of [1] to [7], wherein the cells are vascular smooth muscle cells expressing a cell surface marker of CD140b, KDR, or CD34. [9]

[0042] The method according to any one of [1] to [8], wherein the cells are neural crest cells having any degree of differentiation.

[10]

[0044] The method according to any one of [1] to [9], wherein the cells are lymphocytes having any degree of differentiation.

[11]

[0046] The method according to any one of [1] to

[10] , wherein the cells are megakaryocytes expressing cell surface markers CD41 and CD42b.

[12]

[0048] The method according to any one of [1] to

[11] , wherein the cells are myelocytes expressing cell surface markers CD14 and CD11b.

[13]

[0050] The method according to any one of [1] to

[12] , further comprising the step of inhibiting the expression of MYC family genes and BMI1 gene, or inhibiting the function of their expression products.

[14]

[0052] The method according to any one of [1] to

[13] , further comprising the step of forcibly expressing the BCL-XL gene.

[15]

[0054] The method according to any one of [1] to

[14] , further comprising the step of inhibiting the expression of the BCL-XL gene or inhibiting the function of its expression product.

[16]

[0056] The method according to any one of [1] to

[15] , further comprising the step of inhibiting the expression of at least one of the CDKN1A gene and the P53 gene, or inhibiting the function of the expression product thereof.

[17]

[0058] A method for producing precursor cells of mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, or myelocytes, comprising the step of culturing cells obtained by the method of any one of [1] to

[16] .

[18]

[0060] A method for producing differentiated cells of mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, or myelocytes, comprising the step of differentiating cells obtained by the method of any one of [1] to

[16] .

[19]

[0062] A cell obtained by the method described in any one of [1] to

[18] .

[20]

[0064] A pharmaceutical composition comprising cells obtained by the method of any one of [1] to

[18] . [twenty one]

[0066] A method for treating or preventing a disease, comprising the step of administering the cell of

[19] or the pharmaceutical composition of

[20] to a patient in need thereof. [twenty two]

[0068] A cell differentiation regulator for cells selected from the group consisting of (i) mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, and lymphocytes with any degree of differentiation, and (ii) megakaryocytes and myelocytes with a high degree of differentiation, comprising as an active ingredient a molecule that forces expression of a MYC family gene and a BMI1 gene. [twenty three]

[0070] A method for producing macrophages, comprising the following steps:

[0071] 1) A process for forcibly expressing MYC family genes and BMI1 genes in highly differentiated myelocytes;

[0072] 2) a step of culturing the cells obtained in step 1 to allow them to proliferate;

[0073] 3) A step of promoting differentiation and maturation into macrophages by inhibiting forced expression of MYC family genes and BMI1 genes in the cells obtained in step 2 and further culturing the cells under macrophage differentiation conditions. [twenty four]

[0075] The method according to

[23] , wherein step 1 further comprises the step of forcibly expressing the BCL-XL gene in myelocytes with a high degree of differentiation.

[25]

[0077] The method according to

[23] or

[24] , wherein step 3 further comprises a step of inhibiting forced expression of the BCL-XL gene in the cells obtained in step 2.

[26]

[0079] The method according to any one of

[23] to

[25] , wherein step 1 further comprises the step of inhibiting the expression of the CDKN1A gene and / or the p53 gene, or inhibiting the function of their expression products, in highly differentiated myelocytes.

[27]

[0081] A method for producing mesenchymal stem cells having proliferation ability, comprising the step of forcibly expressing a MYC family gene and a BMI1 gene in mesenchymal stem cells having any degree of differentiation.

[28]

[0083] The production method according to

[27] , wherein the mesenchymal stem cells having any degree of differentiation are mesenchymal stem cells that do not express the cell surface marker CD90.

[29]

[0085] The production method according to

[27] or

[28] , wherein the mesenchymal stem cells having any degree of differentiation are mesenchymal stem cells expressing the cell surface marker CD90.

[30]

[0087] The production method according to any one of

[27] to

[29] , wherein the mesenchymal stem cells having proliferation ability express the cell surface marker CD90.

[31]

[0089] A method for producing vascular endothelial cells with proliferation ability, comprising the step of forcibly expressing MYC family genes and BMI1 genes in vascular endothelial cells with any degree of differentiation.

[32]

[0091] A method for producing smooth muscle cells with proliferation ability, comprising the step of forcibly expressing MYC family genes and BMI1 genes in smooth muscle cells with any degree of differentiation.

[33]

[0093] The production method according to

[32] , wherein the smooth muscle cells having any degree of differentiation are vascular smooth muscle cells.

[34]

[0095] The production method according to

[33] , wherein the vascular smooth muscle cells are vascular smooth muscle cells expressing cell surface markers such as CD140b, KDR or CD34.

[35]

[0097] A method for producing neural crest cells with proliferation ability, comprising the step of forcibly expressing MYC family genes and BMI1 genes in neural crest cells with any degree of differentiation.

[36]

[0099] A method for producing lymphocytes having proliferation ability, comprising the step of forcibly expressing MYC family genes and BMI1 genes in lymphocytes having any degree of differentiation.

[37]

[0101] A method for producing megakaryocytes having proliferation ability, comprising the step of forcibly expressing a MYC family gene and a BMI1 gene in megakaryocytes expressing the cell surface markers CD41 and CD42b.

[38]

[0103] A method for producing macrophages having proliferation ability, comprising the step of forcibly expressing a MYC family gene and a BMI1 gene in macrophages expressing the cell surface markers CD14 and CD11b.

[39]

[0105] A method for promoting the differentiation of iPS cells, ES cells, hematopoietic stem cells or hematopoietic precursor cells into lymphocytes or lymphocyte precursor cells, comprising the step of co-culturing non-senescent vascular endothelial cells with iPS cells, ES cells, hematopoietic stem cells or hematopoietic precursor cells.

[40]

[0107] The method according to

[39] , wherein the lymphocytes express cell surface markers of CD45 and CD56, or express cell surface markers of CD19 and CD45.

[41]

[0109] The method according to

[39] or

[40] , wherein the lymphocytes are natural killer cells or B cells.

[42]

[0111] The method according to any one of

[39] to

[41] , wherein the hematopoietic stem cells or hematopoietic progenitor cells are derived from umbilical cord blood or iPS cells.

[43]

[0113] The method according to any one of

[39] to

[42] , wherein the non-senescent vascular endothelial cells express Notch ligand.

[44]

[0115] The method according to

[43] , wherein the Notch ligand is DLL4 and / or Jagged 1.

[0116] Effects of the Invention

[0117] According to the present invention, the degree of cell differentiation can be regulated by forcibly expressing MYC family genes and BMI1 genes in mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, and lymphocytes of any degree of differentiation, as well as megakaryocytes and myelocytes of high degree of differentiation.

[0118] According to the present invention, immortalized cell lines of mesenchymal, megakaryocyte, or myelocyte lineages can be efficiently induced from various iPS cells. Therefore, by using iPS cells transfected with receptors targeting foreign-specific antigens, iPS cells genetically modified to enhance cytotoxicity toward target cells, HLA-null iPS cells to suppress immune rejection, and iPS cells for genetic diseases, these cells can be more physiologically targeted, and advantages in both drug screening and cell therapy are expected. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 The results of evaluating the cell proliferation of mesenchymal stem cells are shown.

[0120] Figure 2 Shown are the results of flow cytometry analysis of mesenchymal stem cells.

[0121] Figure 3 The graph shows the results of flow cytometric analysis when mesenchymal stem cells were separated into CD90- cells and CD90+ cells and cultured to force MB expression.

[0122] Figure 4 The results are shown, comparing the cell proliferation of mesenchymal stem cells under Dox-on and Dox-off conditions.

[0123] Figure 5 The results of flow cytometric analysis of mesenchymal stem cells under Dox on and Dox off conditions are shown.

[0124] Figure 6 The results of induction of terminal differentiation of mesenchymal stem cells are shown.

[0125] Figure 7 The results of evaluating the cell proliferation of vascular endothelial cells are shown.

[0126] Figure 8 Shown are the results of flow cytometric analysis of vascular endothelial cells.

[0127] Figure 9 The results of morphological observation of vascular endothelial cells are shown.

[0128] Figure 10 Shown are the results of immunostaining of vascular endothelial cells.

[0129] Figure 11 The results of vascular endothelial cell marker expression are shown.

[0130] Figure 12 The results of evaluating the cell proliferation of vascular smooth muscle cells are shown.

[0131] Figure 13 Shown are the results of immunostaining of vascular smooth muscle cells.

[0132] Figure 14 Schematic diagram showing the experiment of precursor cell reprogramming of vascular smooth muscle cells.

[0133] Figure 15 Show Figure 14 Results of morphological observation of vascular smooth muscle cells in the experiment.

[0134] Figure 16 Show Figure 14 Results of the experiment evaluating the cell proliferation of vascular smooth muscle cells.

[0135] Figure 17-1 Show Figure 14 Results of flow cytometric analysis of vascular smooth muscle cells (CD140b) in the experiments.

[0136] Figure 17-2 Show Figure 14 Results of flow cytometric analysis of vascular smooth muscle cells (KDR) in the experiments.

[0137] Figure 17-3 Show Figure 14 Results of flow cytometric analysis of vascular smooth muscle cells (CD34) in the experiments.

[0138] Figure 18 Show Figure 14 Results of immunostaining of vascular smooth muscle cells in the experiments.

[0139] Figure 19 The results of morphological observation of neural crest cells are shown.

[0140] Figure 20 The results of evaluating the cell proliferation of neural crest cells are shown.

[0141] Figure 21 Shown are the results of flow cytometric analysis of neural crest cells (CD271).

[0142] Figure 22 Shown are the results of flow cytometric analysis of neural crest cells (CD271).

[0143] Figure 23 The results of evaluating the effect of fibronectin coating on neural crest cells are shown.

[0144] Figure 24 The results of evaluating the cell proliferation of CD45+ / CD56+ cells are shown.

[0145] Figure 25 Shown are the results of flow cytometric analysis of CD45+ / CD56+ cells.

[0146] Figure 26 The results of evaluating the cell proliferation of CD45+ / CD19+ cells are shown.

[0147] Figure 27 Shown are the results of flow cytometric analysis of CD45+ / CD19+ cells.

[0148] Figure 28 The results of evaluation of cell proliferation of megakaryocytes and granulocytes (macrophages) and the results of flow cytometry analysis are shown.

[0149] Figure 29 The results of PCA (Principal Component Analysis) are shown for non-senescent vascular endothelial cells set to dox on, non-senescent vascular endothelial cells set to dox off, human iPS cells, human aortic endothelial cells (HAEC), and human umbilical cord blood endothelial cells (HUVEC).

[0150] Figure 30 The results of DLL4 and Jagged 1 expression analysis in dox-on non-senescent vascular endothelial cells, dox-off non-senescent vascular endothelial cells, human iPS cells, human aortic endothelial cells (HAEC), and human umbilical cord blood endothelial cells (HUVEC) are shown.

[0151] Figure 31 The graph shows the results of flow cytometric analysis of CD45+ / CD56+ cells when non-senescent vascular endothelial cells were co-cultured with CD34-positive umbilical cord blood cells.

[0152] Figure 32 The results of evaluating the cell proliferation of CD45+ / CD56+ cells when non-senescent vascular endothelial cells were co-cultured with CD34-positive umbilical cord blood cells are shown.

[0153] Figure 33The results of flow cytometric analysis of CD45+ / CD19+ cells when human hematopoietic stem cells derived from iPS cells were co-cultured with non-senescent vascular endothelial cells or genetically recombinant DLL4 are shown.

[0154] Figure 34 The results of flow cytometric analysis of CD45+ / CD56+ cells and CD45+ / CD19+ cells when non-senescent vascular endothelial cells were co-cultured with iPS cell-derived human hematopoietic stem cells after IL-3 administration are shown. DETAILED DESCRIPTION

[0155] The method for regulating the degree of cell differentiation of this embodiment includes the following steps: changing the expression levels of MYC family genes and BMI1 genes in mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes with a high degree of differentiation, and myelocytes with any degree of differentiation (for example, forcibly expressing MYC family genes and BMI1 genes or inhibiting their expression). By forcibly expressing MYC family genes and BMI1 genes in mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes with a high degree of differentiation, and myelocytes with any degree of differentiation, cells (mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, and myelocytes) with a lower degree of differentiation than before forced expression can be obtained. By forcibly expressing MYC family genes and BMI1 genes in mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, and myelocytes with a high degree of differentiation, it is possible to obtain cells (mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, and myelocytes) with a low degree of differentiation compared to before forced expression. Cells with a low degree of differentiation may have proliferation capacity. In addition, by suppressing the expression of MYC family genes and BMI1 genes in cells with a low degree of differentiation obtained by forced expression of MYC family genes and BMI1 genes, it is possible to obtain cells (mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, and myelocytes) with a high degree of differentiation compared to before the expression inhibition.

[0156] When referring to "degree of differentiation" or "degree of cell differentiation", it refers to the level of differentiation or degree of differentiation or maturity of the cells.

[0157] The degree of cell differentiation can also be determined based on the expression level or degree of cell surface markers. For example, the degree of differentiation can be determined by detecting the expression level of cell surface markers expressed by cells.

[0158] The degree of cell differentiation can be determined relatively. For example, if, during the differentiation process of a cell, there are cells with a certain degree of differentiation and cells with a different degree of differentiation, the degree of differentiation of the two cells can be compared to determine whether the cell has a relatively low degree of differentiation or a relatively high degree of differentiation. When determining the degree of cell differentiation relatively, the determination of the degree of differentiation can also be made based on the expression level or degree of expression of cell surface markers.

[0159] In this embodiment, cells "having any degree of differentiation" or "having a high degree of differentiation" can be produced by inducing pluripotent stem cells such as iPS cells and ES cells, or can be produced by the method of regulating the degree of cell differentiation of the present invention.

[0160] In this embodiment, the regulation of cell differentiation degree can be an increase in cell proliferation (property), a decrease in cell proliferation, or both an increase and a decrease in cell proliferation. That is, the decrease in cell proliferation can be a regulation to achieve a high degree of cell differentiation, and the increase in cell proliferation can be a regulation to achieve a low degree of cell differentiation.

[0161] 1-1. Mesenchymal Stem Cells (MSCs)

[0162] "Mesenchymal stem cells" refer to stem cells that have the ability to differentiate into cells belonging to the mesenchyme. Mesenchymal stem cells may include adipose stromal cells and adipose stem cells.

[0163] Mesenchymal stem cells can be obtained by differentiation and induction from pluripotent stem cells such as iPS cells and ES cells using methods well known to those skilled in the art. For example, according to a report by Fukuta et al. (Plos One, 2014, 9(12):e112291), neural crest cells (NCCs) can be induced by culturing iPS cells on a matrigel-coated culture dish in the presence of SB-431542 and CHIR99021, and the NCCs can be expanded by culturing the NCCs on a fibronectin-coated culture dish in a medium containing EGF, FGF2, and SB-431542. MSCs can then be induced by culturing the NCCs on a fibronectin-coated culture dish in a medium containing 10% FBS and FGF2.

[0164] The induction of mesenchymal stem cells can be confirmed by subjecting the cultured cells to flow cytometry analysis to detect the appearance of cells having a mesenchymal stem cell-specific cell surface marker expression profile described below, or by subjecting the cells to a colony formation assay to confirm their ability to differentiate into mesenchymal stem cells.

[0165] Mesenchymal stem cells can differentiate into vascular endothelial cells, osteoblasts, adipocytes, chondrocytes, fibroblasts, myoblasts, bone marrow stromal cells, tendon cells, hepatocytes, bile duct epithelial cells, glial cells, neurons, cardiomyocytes, smooth muscle cells, lymphatic endothelial cells, etc.

[0166] In this embodiment, the "precursor cells of mesenchymal stem cells" can be precursor cells of cells selected from the group consisting of bone vascular endothelial cells, osteoblasts, adipocytes, chondrocytes, fibroblasts, myoblasts, bone marrow stromal cells, tendon cells, hepatocytes, bile duct epithelial cells, glial cells, neurons, cardiomyocytes, smooth muscle cells, and lymphatic endothelial cells.

[0167] In this embodiment, the "differentiated cells of mesenchymal stem cells" can be cells selected from the group consisting of vascular endothelial cells, osteoblasts, adipocytes, chondrocytes, fibroblasts, myoblasts, bone marrow stromal cells, tendon cells, hepatocytes, bile duct epithelial cells, glial cells, neurons, cardiomyocytes, smooth muscle cells, and lymphatic endothelial cells.

[0168] Mesenchymal stem cells can be characterized, for example, by the expression of cell surface markers CD90, CD73, and CD105.

[0169] Mesenchymal stem cells can be characterized by their degree of differentiation through the expression spectrum of cell surface markers in flow cytometric analysis. For example, as the degree of differentiation of mesenchymal stem cells increases, the expression of the cell surface marker CD90 decreases. That is, mesenchymal stem cells with a high degree of differentiation may not express the cell surface marker CD90, but express the cell surface markers CD73 and CD105. In one embodiment, mesenchymal stem cells with a high degree of differentiation are mesenchymal stem cells that are CD90-, CD90- / CD73+, or CD90- / CD105+ in flow cytometric analysis. If mesenchymal stem cells are repeatedly passaged from pluripotent stem cells such as iPS cells, differentiation gradually advances and stem cell properties (proliferative properties) decrease. If passaged for more than 10 generations, mesenchymal stem cells with a high degree of differentiation (CD90- / CD73+ or CD90- / CD105+) can reach about 90% of viable cells. In addition, if the degree of differentiation of mesenchymal stem cells decreases, the expression of the cell surface marker CD90 increases. That is, mesenchymal stem cells with a low degree of differentiation may express the cell surface marker CD90 and the cell surface markers CD73 and CD105. In one embodiment, mesenchymal stem cells with a low degree of differentiation are mesenchymal stem cells that are CD90+, CD90+ / CD73+, or CD90+ / CD105+ as determined by flow cytometry. In another embodiment, mesenchymal stem cells with a low degree of differentiation may be mesenchymal stem cells in a fraction in which no more than 10% of viable cells are CD90- / CD73+ or CD90- / CD105+ as determined by flow cytometry.

[0170] 1-2. Vascular endothelial cells

[0171] "Vascular endothelial cells" refer to cells that constitute the vascular endothelium or cells that can differentiate into such cells. Among vascular endothelial cells, fetal cells that have the ability to differentiate into blood cells are called hematopoietic endothelial cells.

[0172] As the hematopoietic mechanism in a living body, in addition to the hematopoietic mechanism that supplies blood cells derived from stem cells, a hematopoietic mechanism that supplies blood cells through the development of vascular endothelial cells is also known.

[0173] The vascular endothelial cells may be VE-cadherin (VE-cad)-positive, CD41-positive, and CXCR4-positive cells. For example, the vascular endothelial cells may be derived from pluripotent stem cells such as ES cells or iPS cells, and may be induced from ES cells or iPS cells through a process of inducing a reticular structure.

[0174] Cell culture conditions suitable for preparing reticular structures from human pluripotent stem cells, such as human ES cells and human iPS cells, vary depending on the pluripotent stem cells used. For example, IMDM supplemented with FBS to a final concentration of 15% can be used, or serum-free medium supplemented with growth factors and supplements as appropriate. Furthermore, to efficiently form reticular structures, VEGF can be added at a concentration of 0-100 ng / ml, more preferably 20 ng / ml. The culture environment varies depending on the type of ES cells or iPS cells used; for example, conditions such as 5% CO2 and 36-38°C, preferably 37°C, can be used. The culture time required for reticular structure formation varies depending on the type of pluripotent stem cells and the induction conditions. Generally, after seeding pluripotent stem cells onto feeder cells (e.g., 10T1 / 2 cells), cell aggregates containing hemogenic endothelial cells form approximately 7 days later, and reticular structures containing vascular endothelial progenitor cells and hematopoietic progenitor cells form approximately 14-16 days later.

[0175] The formed reticular structures become follicular structures, within which endothelial progenitor cells and hematopoietic progenitor cells exist in a concentrated state. The hematopoietic endothelial cells contained in the cell mass and the endothelial progenitor cells and hematopoietic progenitor cells present within the reticular structures can be separated by physical means, such as passing them through a sterile sieve-like device (e.g., a cell strainer). Subsequently, CD34+ endothelial progenitor cells can be isolated using a cell sorter and subsequently cultured in the presence of VEGF to obtain endothelial cells.

[0176] The induction of vascular endothelial cells can be confirmed by subjecting the cultured cells to flow cytometry analysis and detecting the appearance of cells having a vascular endothelial cell-specific cell surface marker expression profile described below, or by subjecting them to a colony formation assay and confirming their ability to differentiate into vascular endothelial cells.

[0177] Vascular endothelial cells can be characterized by, for example, the expression of cell surface markers such as VE-cad, CD31 (PECAM1), CD105, and CD146.

[0178] The degree of differentiation of vascular endothelial cells can be characterized by the expression profile of cell surface markers in flow cytometric analysis. For example, as the degree of differentiation of vascular endothelial cells increases, the expression of cell surface markers such as VE-cad, CD31, CD105, and CD146 increases. That is, vascular endothelial cells with a high degree of differentiation can express cell surface markers such as VE-cad, CD31, CD105, and CD146. In one embodiment, vascular endothelial cells with a high degree of differentiation can be VE-cad+ / CD31+ vascular endothelial cells in flow cytometric analysis, and can further express one or two cell surface markers selected from the group consisting of CD105 and CD146, preferably VE-cad+ / CD31+ / CD105+ / CD146+. In addition, when the degree of differentiation of vascular endothelial cells decreases, the expression of cell surface markers such as VE-cad, CD31, CD105, and CD146 decreases. That is, poorly differentiated vascular endothelial cells may not express one, two, three, or four cell surface markers selected from the group consisting of VE-cad, CD31, CD105, and CD146. In one embodiment, poorly differentiated vascular endothelial cells may be VE-cad+ / CD31+ vascular endothelial cells as determined by flow cytometry, and may not express one or two cell surface markers selected from the group consisting of CD105 and CD146.

[0179] 1-3. Smooth muscle cells

[0180] "Smooth muscle cells" refer to cells that constitute smooth muscle or cells that can differentiate into such cells (e.g., smooth muscle precursor cells, smooth muscle stem cells, etc.). In addition, "smooth muscle cells" may be, for example, vascular smooth muscle cells, digestive tract smooth muscle cells, bladder smooth muscle cells, uterine smooth muscle cells, etc.

[0181] Among smooth muscle cells, vascular smooth muscle cells can be differentiated and induced by pluripotent stem cells such as iPS cells and ES cells of mammals such as humans, and induced by ES cells or iPS cells through the process of inducing a network-like structure. By using a cell sorter to separate the CD34- / VEGFR (KDR) + vascular smooth muscle precursor cells contained in the network-like structure, and then continuing to culture, vascular smooth muscle cells can be obtained. Regarding the fact that vascular smooth muscle cells have been obtained, the cultured cells can be subjected to flow cytometry analysis and detection of the appearance of cells having a vascular smooth muscle cell-specific cell surface marker expression profile described later, or subjected to a colony formation test and confirmation of the ability to differentiate into vascular endothelial cells.

[0182] Smooth muscle cells can be derived from mesenchymal stem cells, neural crest cells, bone marrow cells, etc. Smooth muscle cells are known to include differentiated smooth muscle cells and dedifferentiated smooth muscle cells. Differentiated smooth muscle cells and dedifferentiated smooth muscle cells can be confirmed by gene expression profiling well known to those skilled in the art.

[0183] In this embodiment, the "smooth muscle cell precursor cells" may be the aforementioned mesenchymal stem cell precursor cells. In this embodiment, the "smooth muscle cell differentiated cells" may be differentiated smooth muscle cells or dedifferentiated smooth muscle cells.

[0184] Smooth muscle cells can be characterized, for example, by the expression of markers for VEGFR (KDR), Calponin, and αSMA.

[0185] The degree of differentiation of smooth muscle cells can be characterized by the expression profile of the above-mentioned markers. For example, as the degree of differentiation of vascular smooth muscle cells increases, the expression of markers such as calponin and αSMA increases. That is, vascular smooth muscle cells with a high degree of differentiation can express markers such as calponin and αSMA. In one embodiment, vascular smooth muscle cells with a high degree of differentiation can be vascular smooth muscle cells that are calponin+ / αSMA+ in flow cytometry analysis, and can also express markers for calponin and / or αSMA.

[0186] In addition to the above markers, smooth muscle cells can also be characterized by expressing cell surface markers well known to those skilled in the art. In the case of smooth muscle cells, for example, vascular smooth muscle cells can be characterized by expressing cell surface markers such as CD140b, KDR, and CD34.

[0187] Vascular smooth muscle cells can be characterized by their degree of differentiation through cell surface marker expression profiles in flow cytometry analysis. For example, as the degree of differentiation of vascular smooth muscle cells increases, the expression of cell surface markers such as CD140b, KDR, and CD34 increases. That is, vascular smooth muscle cells with a high degree of differentiation can express cell surface markers such as CD140b, KDR, and CD34. In one embodiment, vascular smooth muscle cells with a high degree of differentiation can further express 1, 2, or 3 cell surface markers selected from the group consisting of CD140b, KDR, and CD34 in flow cytometry analysis. In addition, when the degree of differentiation of vascular smooth muscle cells decreases, the expression of cell surface markers such as CD140b, KDR, and CD34 decreases. That is, vascular smooth muscle cells with a low degree of differentiation may not express 1, 2, or 3 cell surface markers in the group consisting of CD140b, KDR, and CD34. In one embodiment, the vascular smooth muscle cells with a low degree of differentiation may be vascular smooth muscle cells that do not express one, two, or three cell surface markers selected from the group consisting of CD140b, KDR, and CD34 in flow cytometry analysis.

[0188] 1-4. Neural crest cells

[0189] Neural crest cells are migratory stem cells that originate from a population of cells called the neural crest that arises temporarily during development.

[0190] Neural crest cells can be obtained by differentiation and induction from pluripotent stem cells such as iPS cells and ES cells using methods well known to those skilled in the art. For example, according to the report of Fukuta et al. (Plos One, 2014, 9(12):e112291), iPS cells can be cultured on a matrigel-coated culture dish in the presence of an ALK5 inhibitor such as SB-431542 and a GSK3 inhibitor such as CHIR99021 to induce neural crest cells (NCC). In addition, in the culture of neural crest cells, in order to promote cell fixation and growth, the culture dish, wells, etc. can be coated with fibronectin. In the culture of neural crest cells, in order to promote cell growth, growth factors such as EGF and FGF2 can be added to the culture medium. In one embodiment, neural crest cells are cultured in a culture medium containing an ALK5 inhibitor such as SB-431542, EGF, and FGF2.

[0191] The induction of neural crest cells can be confirmed by subjecting the cultured cells to flow cytometry analysis to detect the appearance of cells having a neural crest cell-specific cell surface marker expression profile described below, or by subjecting them to a colony formation assay to confirm their ability to differentiate into neural crest cells.

[0192] Neural crest cells can differentiate into neurons, osteoblasts, chondrocytes, myoblasts, Schwann cells, glial cells, teeth, skeletal muscle cells, smooth muscle cells, melanocytes, etc.

[0193] In this embodiment, the "neural crest cell precursor cells" may be precursor cells of cells selected from the group consisting of neurons, osteoblasts, chondrocytes, myoblasts, Schwann cells, glial cells, teeth, skeletal muscle cells, smooth muscle cells, and melanocytes.

[0194] Neural crest cells can be characterized, for example, by expressing cell surface markers such as CD271.

[0195] The degree of differentiation of neural crest cells can be characterized by the expression profile of cell surface markers in flow cytometry analysis. For example, as the degree of differentiation of neural crest cells increases, the expression of cell surface markers such as CD271 decreases. That is, neural crest cells with a high degree of differentiation may be neural crest cells that do not express cell surface markers such as CD271. In addition, when the degree of differentiation of neural crest cells decreases, the expression of cell surface markers such as CD271 increases. That is, neural crest cells with a low degree of differentiation may be neural crest cells that express cell surface markers such as CD271, or may be neural crest cells that express cell surface markers such as CD271 more than neural crest cells with a high degree of differentiation.

[0196] Whether or not fibronectin is used to coat the culture dish or well during culture does not affect the proliferation of neural crest cells or the expression of cell surface markers such as CD271.

[0197] 1-5. Lymphocytes

[0198] Lymphocytes are a type of white blood cell and can be divided into natural killer cells, B cells, and T cells.

[0199] Lymphocytes can be obtained by differentiation induction from pluripotent stem cells such as iPS cells and ES cells, hematopoietic stem cells, and hematopoietic precursor cells using methods well known to those skilled in the art. When inducing lymphocyte differentiation, co-culturing the pluripotent stem cells, hematopoietic stem cells, and hematopoietic precursor cells with non-senescent vascular endothelial cells as feeder cells, or coating the pluripotent stem cells, hematopoietic stem cells, and hematopoietic precursor cells with Notch ligands such as DLL1, DLL4, and Jagged 1, can more efficiently induce lymphocyte differentiation. Furthermore, lymphocyte differentiation induction is preferably performed in the presence of cytokines such as IL7, IL15, IL2, and IL7.

[0200] Therefore, this embodiment also provides: a method for promoting the differentiation of pluripotent stem cells such as iPS cells and ES cells, hematopoietic stem cells or hematopoietic precursor cells into lymphocytes or lymphocyte precursor cells, which includes the process of co-culturing non-senescent vascular endothelial cells with pluripotent stem cells such as iPS cells and ES cells, hematopoietic stem cells or hematopoietic precursor cells.

[0201] In the method for regulating the degree of cell differentiation of the present invention, the immortalized vascular endothelial cells can be vascular endothelial cells with proliferation ability obtained by forcibly expressing MYC family genes (e.g., c-Myc gene) and BMI1 gene (and optionally BCL-X1 gene) in vascular endothelial cells. The vascular endothelial cells with proliferation ability can be cells with a low degree of differentiation compared to before forced expression. Mature vascular endothelial cells induced by stopping the forced expression of MYC family genes (e.g., c-Myc gene) and BMI1 gene (and optionally BCL-X1 gene) in the vascular endothelial cells with proliferation ability are also included in the immortalized vascular endothelial cells.

[0202] It is known that Notch signals, which are transmitted through the interaction between Notch and Notch ligands (Delta-like 4 (hereinafter referred to as DLL4), Jagged 1, etc.), are essential for cell differentiation. The non-senescent vascular endothelial cells used in the above-mentioned co-culture can express Notch ligands, such as DLL4 and / or Jagged 1. By causing non-senescent vascular endothelial cells to express DLL4 and / or Jagged 1, which are essential for Notch signaling, differentiation of pluripotent stem cells such as iPS cells and ES cells, hematopoietic stem cells, or hematopoietic precursor cells into lymphocytes or lymphocyte precursor cells can be promoted. By causing non-senescent vascular endothelial cells to express DLL4 and / or Jagged 1, differentiation into a wide range of cells can be promoted. Therefore, the method of this embodiment can be a method for promoting the differentiation of pluripotent stem cells such as iPS cells and ES cells, hematopoietic stem cells, or hematopoietic precursor cells into any cell type, comprising the step of co-culturing non-senescent vascular endothelial cells with pluripotent stem cells such as iPS cells and ES cells, hematopoietic stem cells, or hematopoietic precursor cells.

[0203] Pluripotent stem cells such as iPS cells and ES cells, hematopoietic stem cells, or hematopoietic progenitor cells used in co-culture can be pre-integrated with expression cassettes for desired genes (e.g., MYC family genes and the BMI1 gene, and optionally, the BCL-XL gene). This allows for forced expression of these genes while inducing differentiation of the iPS cells, ES cells, hematopoietic stem cells, or hematopoietic progenitor cells into target cells. In this case, differentiation can be induced into non-senescent target cells.

[0204] The hematopoietic stem cells or hematopoietic progenitor cells used in the co-culture may be collected from an organism, for example, derived from umbilical cord blood, bone marrow, or peripheral blood, or may be induced from pluripotent stem cells such as iPS cells.

[0205] Since the reticular structures induced from pluripotent stem cells such as iPS cells contain hematopoietic progenitor cells, the reticular structures can be used as hematopoietic stem cells or hematopoietic progenitor cells for co-culture.

[0206] The non-senescent vascular endothelial cells used in the co-culture may be cells whose differentiation degree has been regulated by the method for regulating the degree of cell differentiation of this embodiment, thereby preventing them from senescence. Furthermore, the non-senescent vascular endothelial cells used in the co-culture may be cells that have been treated with antibiotics such as mitomycin C or irradiation with radiation such as gamma rays to prevent proliferation, or may be untreated cells.

[0207] The conditions for co-culture can be appropriately determined by those skilled in the art based on the state of pluripotent stem cells such as iPS cells and ES cells, hematopoietic stem cells or hematopoietic precursor cells, and non-senescent vascular endothelial cells. For example, the culture temperature can be set to about 35°C to about 42°C, about 36°C to about 40°C, or about 37°C to about 39°C, the carbon dioxide concentration can be set to, for example, 5% CO2, and the oxygen concentration can be set to, for example, 20% O2. It can be a static culture or a shaking culture. The shaking speed during shaking culture is not particularly limited and can be set to, for example, 10rpm to 200rpm, 30rpm to 150rpm, etc. The culture medium can be an Iscove modified Dulbecco's medium (IMDM) culture medium containing serum, insulin, transferrin, serine, monothioglycerol, ascorbic acid, and TPO. In this case, the IMDM culture medium can further contain SCF and can further contain heparin. Phorbol esters (e.g., phorbol-12-myristate-13-acetate; PMA) can be added. In addition, the culture medium can contain serum or plasma, or can be serum-free. In the case of using serum, preferably human serum.As required, culture medium can also contain more than one material such as albumin, insulin, transferrins, iron complexes, selenium, fatty acid, trace element, 2-mercaptoethanol, thioglycerol, monothioglycerol (MTG), lipid, amino acid (such as L-glutamine), ascorbic acid, heparin, non-essential amino acid, vitamin, somatomedin, low molecular weight compound, antibiotic, antioxidant, pyruvic acid, buffer, inorganic salts, cytokine.As cytokine, can illustrate such as vascular endothelial growth factor (VEGF), thrombopoietin (TPO), various TPO sample action substances, stem cell factor (SCF), erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL3), ITS (insulin-transferrins, iron complexes-selenite) supplement, ADAM (A Disintegrin And Metalloprotease, disintegrin metalloproteinase) inhibitor etc.

[0208] In one embodiment, a combination of Flt3L, SCF, IL-7, and IL-15 is added as a cytokine. This combination may also include IL-3. In one embodiment, non-senescent vascular endothelial cells are co-cultured with pluripotent stem cells such as iPS cells and ES cells, hematopoietic stem cells, or hematopoietic precursor cells in a culture medium containing IL-3, Flt3L, SCF, IL-7, and IL-15, and then co-cultured in a culture medium containing Flt3L, SCF, IL-7, and IL-15. The culture medium used for the continued culture may not contain IL-3.

[0209] In addition, the order of adding non-senescent vascular endothelial cells and pluripotent stem cells such as iPS cells and ES cells, hematopoietic stem cells or hematopoietic precursor cells to the culture medium can be arbitrary. Both non-senescent vascular endothelial cells can be added first, and pluripotent stem cells such as iPS cells and ES cells, hematopoietic stem cells or hematopoietic precursor cells can be added first. Co-culture can be performed by adding pluripotent stem cells such as iPS cells and ES cells, hematopoietic stem cells or hematopoietic precursor cells to non-senescent vascular endothelial cells cultured in the culture medium, or by adding non-senescent vascular endothelial cells to pluripotent stem cells such as iPS cells and ES cells, hematopoietic stem cells or hematopoietic precursor cells cultured in the culture medium. Co-culture can be a two-dimensional culture using a multi-well plate or the like, or a three-dimensional culture in an environment close to that in vivo.

[0210] Co-culturing with non-senescent vascular endothelial cells can more efficiently induce differentiation into target cells compared to co-culturing without non-senescent vascular endothelial cells or co-culturing with mutant DLL4. Specifically, co-culturing with non-senescent vascular endothelial cells can induce differentiation into a greater number and / or a higher ratio of target cells.

[0211] The induction of lymphocytes or lymphocyte precursor cells can be ascertained by subjecting the cultured cells to flow cytometry analysis and detecting the appearance of cells having a lymphocyte-specific cell surface marker expression profile described below, or by subjecting them to a colony formation assay and confirming their lymphocyte differentiation ability.

[0212] Lymphocytes can be derived from hematopoietic stem cells, lymphocyte precursor cells, etc.

[0213] In the present embodiment, the "lymphocyte precursor cell" may be a precursor cell of a cell selected from the group consisting of natural killer cells, B cells, and T cells.

[0214] Lymphocytes can be characterized, for example, by the expression of cell surface markers such as CD45, CD56, and CD19. CD45 is a cell surface marker expressed by all blood cells except red blood cells and platelets, CD56 is a cell surface marker specific for natural killer cells, and CD19 is a cell surface marker specific for B cells. For example, natural killer cells are characterized by the expression of CD45 and CD56, while B cells are characterized by the expression of CD45 and CD19.

[0215] Lymphocyte can characterize its degree of differentiation by cell surface marker expression spectrum in flow cytometry analysis.For example, when the degree of differentiation of lymphocyte improves, the expression of cell surface markers such as CD45, CD56, CD19 reduces.That is, when the degree of differentiation of lymphocyte improves, in flow cytometry analysis, the expression of cell surface markers of less than CD45 and CD56 can be confirmed, the expression of cell surface markers of less than CD45 and CD19 can be confirmed, and the expression of cell surface markers of less than CD45, CD56 and CD19 can be confirmed.In addition, when the degree of differentiation of lymphocyte reduces, the expression of cell surface markers such as CD45, CD56, CD19 increases.That is, when the degree of differentiation of lymphocyte reduces, the number of cells expressing 1,2 or 3 kinds of cell surface markers selected from the group consisting of CD45, CD56 and CD19 increases.

[0216] 1-6. Megakaryocytes

[0217] "Megakaryocytes" refer to cells that can produce platelets and are sometimes also called erythroid precursor cells (Megacaryocyte-erythrocyte progenitor, MEP, megakaryocyte-erythroid progenitor). Megakaryocytes can be CD41a-positive, CD41b-positive, and CD42b-positive cells.

[0218] Megakaryocytes can be obtained by differentiation and induction from pluripotent stem cells such as iPS cells and ES cells using methods well known to those skilled in the art. For example, hematopoietic progenitor cells contained in a reticular structure derived from pluripotent stem cells such as human ES cells and iPS cells can be isolated using a cell sorter and cultured under megakaryocyte-inducing conditions (on feeder cells in the presence of TPO, SCF, and heparin) to obtain megakaryocytes (International Publication No. 2008 / 041370, Takayama Blood 2008, 111(11): 5298-5306). Alternatively, hematopoietic progenitor cells contained in a reticular structure induced from pluripotent stem cells such as human ES cells and iPS cells are forced to express MYC and BMI1 and cultured under megakaryocyte-inducing conditions (on feeder cells in the presence of TPO, SCF, and heparin) to obtain immortalized megakaryocytes. Mature megakaryocytes can be obtained by stopping the expression of MYC and BMI1 in these immortalized megakaryocytes (International Publication No. 2011 / 034073).

[0219] The induction of megakaryocytes can be confirmed by subjecting the cells to a colony formation assay and confirming their ability to differentiate into megakaryocytes.

[0220] Megakaryocytes are a type of blood cell and can be derived from bone marrow cells (hematopoietic stem cells) and the like. Hematopoietic stem cells can differentiate into hematopoietic precursor cells, which can then differentiate into blood cells such as erythrocytes, granulocytes, monocytes, macrophages, megakaryocytes, and platelets. Granulocytes include myelocytes, promyelocytes, metamyelocytes, granulocytes, band-shaped granulocytes, and segmented granulocytes (neutrophils, eosinophils, basophils, etc.).

[0221] In this embodiment, the "megakaryocyte precursor cell" may be a megakaryocyte-erythroid progenitor cell (MEP).

[0222] In this embodiment, the "megakaryocyte differentiated cells" may be megakaryocytes, platelets, or the like.

[0223] Megakaryocytes can be characterized by expression of cell surface markers such as CD41 and CD42b in flow cytometric analysis.

[0224] The degree of differentiation of megakaryocytes can be characterized by their cell surface marker expression profile in flow cytometric analysis. For example, as the degree of differentiation of megakaryocytes increases, the expression of cell surface markers CD41 and CD42b increases. That is, megakaryocytes with a high degree of differentiation can express cell surface markers CD41 and CD42b. Megakaryocytes with a high degree of differentiation can be CD41+ / CD42b+ megakaryocytes in flow cytometric analysis. In addition, when the degree of differentiation of megakaryocytes decreases, the expression of the cell surface marker CD42b decreases. That is, megakaryocytes with a low degree of differentiation are CD41+ and may not express the cell surface marker CD42b. Megakaryocytes with a low degree of differentiation can be CD41+ / CD42b- megakaryocytes.

[0225] 1-7. Myelocytes

[0226] "Myelocytes" refer to cells that give rise to eosinophils, monocytes, neutrophils, and basophils.

[0227] Myelocytes can be obtained from hematopoietic stem cells in the bone marrow during maturation to produce white blood cells such as neutrophils, eosinophils, basophils, monocytes, and lymphocytes, as well as red blood cells and platelets, which are cellular components of the blood. Myelocytes can be derived by differentiation induction from pluripotent stem cells such as iPS cells and ES cells using methods well known to those skilled in the art.

[0228] The induction of myelocytes can be confirmed by subjecting the cultured cells to flow cytometry analysis and detecting the appearance of cells having a myelocyte-specific cell surface marker expression profile described below, or by subjecting the cells to a colony formation assay and confirming their ability to differentiate into myelocytes.

[0229] Myelocytes are a type of blood cell that can be derived from bone marrow cells (hematopoietic stem cells). Hematopoietic stem cells can differentiate into hematopoietic precursor cells, which can then differentiate into blood cells such as erythrocytes, granulocytes, monocytes, macrophages, megakaryocytes, and platelets. Examples of granulocytes include myelocytes, promyelocytes, metamyelocytes, myeloblasts, band-shaped granulocytes, and segmented granulocytes (neutrophils, eosinophils, basophils, etc.).

[0230] In this embodiment, the "myelocyte precursor cells" may be granulocyte-macrophage progenitor cells (GMP).

[0231] In this embodiment, the "myelocyte-differentiated cells" may be myelocytes, segmented granulocytes (neutrophils, eosinophils, basophils, etc.), monocytes, macrophages, and the like.

[0232] Myelocytes can be characterized in flow cytometric analysis by the expression of cell surface markers such as CD11b, CD43, CD16, and CD14.

[0233] The degree of differentiation of myelocytes can be characterized by the aforementioned cell surface marker expression profiles in flow cytometric analysis. For example, as the degree of differentiation of macrophages increases, the expression of CD11b and CD14 cell surface markers increases. That is, highly differentiated macrophages may express CD11b and CD14 cell surface markers. Highly differentiated macrophages may be CD11b+ / CD14+ macrophages in flow cytometric analysis. Furthermore, as the degree of differentiation of macrophages decreases, the expression of CD11b+ cell surface markers decreases. That is, poorly differentiated macrophages may be CD14+ and may not express CD11b cell surface markers. Poorly differentiated macrophages may be CD11b- / CD14+ macrophages.

[0234] 2. Extraction process

[0235] The method for regulating the degree of cell differentiation may further include a step of extracting (isolating or purifying) cells of a desired specific degree of differentiation.

[0236] Extraction of cells with a specific degree of differentiation can be performed before or after forced expression of the MYC family genes and the BMI1 gene.

[0237] In one embodiment, the extraction step can be performed before forced expression of the MYC family gene and the BMI1 gene, and forced expression of the MYC family gene and the BMI1 gene can be achieved in the extracted cells with a specific degree of differentiation (eg, highly differentiated cells).

[0238] In another way, a cell group comprising cells of a desired specific degree of differentiation (e.g., cells of low degree of differentiation) forcibly expressing MYC family genes and BMI1 genes is prepared, and then cells of a desired specific degree of differentiation (e.g., cells of low degree of differentiation) are extracted from the cell group. MYC family genes and BMI1 genes can be forced to be expressed in the cells of the extracted specific degree of differentiation. By extracting target cells, applying the method of the present invention to the extracted cells, or extracting target cells from a cell group to which the method of the present invention is applied and continuing to cultivate the cells, the target cell species can be efficiently proliferated. From the viewpoint of efficiently improving the proliferative properties of the extracted cell species, the extracted cells are preferably set to a single cell of only mesenchymal stem cells, only vascular endothelial cells, only smooth muscle cells, etc., or can be set to a cell group formed by a mixture of two or more of these cells.

[0239] Examples of cells to be extracted include mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, and myelocytes. Target cells can be extracted using antibodies against cell surface markers specifically expressed (or not expressed) by flow cytometry analysis, panning, magnetic beads, and other methods well known to those skilled in the art. Mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, and myelocytes can be extracted by isolating cells that meet the above-mentioned cell surface marker expression profiles.

[0240] The target cells can be isolated so that the proportion of the target cells in the cell population after the extraction operation reaches, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more (e.g., 100%). Single target cells can also be isolated.

[0241] 3. BCL-XL gene

[0242] The method of regulating cell differentiation of the present invention may further include the step of forcibly expressing the BCL-XL gene in mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, highly differentiated megakaryocytes, and myelocytes of any degree of differentiation. By expressing the BCL-XL gene in addition to the MYC family genes and the BMI1 gene, it is expected that regulation of cell differentiation to a low degree of differentiation can be further promoted.

[0243] The aforementioned forced expression time of the BCL-XL gene can be appropriately determined by those skilled in the art.

[0244] The BCL-XL gene is a gene that has the function of inhibiting cell apoptosis.

[0245] Forced expression of the MYC family gene, the BMI1 gene, and / or the BCL-XL gene can be performed simultaneously or sequentially.

[0246] For example, forced expression of MYC family genes and BMI1 followed by forced expression of BCL-XL can produce cells with a low degree of differentiation. Alternatively, forced expression of MYC family genes, BMI1 genes, and BCL-XL genes simultaneously can produce cells with a low degree of differentiation. To maintain a low degree of differentiation, it is preferable to maintain forced expression of BCL-XL in addition to forced expression of MYC family genes and BMI1 genes during culture.

[0247] When forcibly expressing genes such as MYC family genes, BMI1 genes, and BCL-XL genes in cells, any method well known to those skilled in the art can be used. For example, genes can be introduced into cells and expressed using gene delivery systems based on viral vectors such as lentivirus, retrovirus, and Sendai virus, plasmid vectors, and episomal vectors. Alternatively, a method can be used to integrate the target gene into the genome of the cell in a non-viral manner using a transposon to establish a stable expression cell line, and then remove the excess introduced gene by transposonase (e.g., the PiggyBac Transposon system).

[0248] The expression vector (e.g., viral vector) of the desired gene (e.g., MYC family gene and BMI1 gene, optionally and BCL-XL gene) can be transfected into the target cell, or the target cell can be induced by a pluripotent stem cell (e.g., ES cell, iPS cell) pre-integrated with an expression cassette of the desired gene (e.g., MYC family gene and BMI1 gene, optionally and BCL-XL gene) and forced to express the gene at this stage. Alternatively, the gene can be forced to express in a pluripotent stem cell (e.g., ES cell, iPS cell) pre-integrated with an expression cassette of the desired gene (e.g., MYC family gene and BMI1 gene, optionally and BCL-XL gene) while the pluripotent stem cell is induced to differentiate into the target cell. When gene expression is performed using a gene introduction vector, the gene can be operably connected downstream of a suitable promoter and inserted into a gene introduction vector, introduced into the cell, and the target gene is expressed.

[0249] This promoter can be said to be an exogenous promoter. In this specification, the "endogenous" promoter of a gene refers to a promoter that is connected to the gene in the genome under natural conditions, and the "exogenous" promoter of a gene refers to a promoter configured in a manner that guides the transcription of the gene by a promoter that is operably connected near the gene by genetic manipulation (i.e., molecular biology techniques). Here, being connected in an "operably" manner refers to connecting the promoter and the target gene in a manner that controls the target gene cis-wise and achieves the desired expression of the target gene. An exogenous promoter can be a constitutive promoter or a regulated promoter. As a constitutive promoter, for example, CMV promoter, EF1 promoter, ubiquitin promoter, etc. can be enumerated. A regulated promoter refers to a promoter that can be induced or can be derepressed, and refers to a promoter that can be combined with any one of a repressor or an inducer, and has a DNA sequence that works with the promoter. When the promoter is induced or derepressed, it is in an "open state", and when the promoter is not induced or derepressed, the promoter is in a "closed state". As examples of regulated promoters, drug-responsive promoters such as tetracycline-responsive promoters, steroid-responsive promoters, and metallothionein promoters can be cited. A tetracycline-responsive promoter refers to a known regulated promoter that is reversibly controlled by the presence or absence of tetracycline or its derivatives (e.g., doxycycline (Dox)). A tetracycline-responsive promoter is a promoter that has a tetracycline response element (TRE) configured internally, and is activated (i.e., induces the expression of a target protein) by the binding of a reverse tetracycline-controlled transactivator (rtTA) protein or a tetracycline-controlled transactivator (tTA) to TRE. The rtTA protein binds to the TRE in the presence of Dox, while the tTA protein binds to the TRE in the absence of Dox, inducing the expression of a target gene that is functionally connected to the promoter downstream of the TRE sequence. When a tetracycline-responsive promoter is used, by culturing cells into which the gene functionally linked to the tetracycline-responsive promoter and the rtTA or tTA protein are introduced in the presence of Dox, the expression of the gene can be induced or inhibited in a Dox-dependent manner. The exogenous promoter is preferably a regulated promoter. By using a regulated promoter, the inducible expression of the target gene can also be controlled, for example, by adding a drug. Such a drug-based gene expression system can be easily selected by a person skilled in the art to achieve the desired expression control of MYC family genes, BMI1 genes, BCL-XL genes, etc. In order to perform such expression, commercially available kits, etc. can be used. In addition, the MYC family genes, BMI1 genes, and BCL-XL genes as target genes for expression control can be inserted into different vectors or into the same vector.

[0250] While MYC family genes, BMI1, and BCL-XL promote the regulation of cell differentiation to a low degree, they inhibit the terminal differentiation of differentiated cells (e.g., adipocytes, osteoblasts, chondrocytes, macrophages, dendritic cells, neutrophils, and erythrocytes). Therefore, the expression of these genes can be inhibited before entering the terminal differentiation process. By inhibiting the expression of these genes in cells, it is easier to induce functional, more mature differentiated cells (e.g., adipocytes, osteoblasts, chondrocytes, vascular endothelial cells, vascular smooth muscle cells, megakaryocytes, platelets, macrophages, dendritic cells, neutrophils, erythrocytes, and megakaryocytes).

[0251] Inhibition of intracellular expression of MYC family genes, BMI1 genes, BCL-XL genes, and the like can also be achieved, for example, by removing the drug, etc., to reverse the induction of expression induced by the aforementioned drug-inducible expression system using a regulated promoter. Alternatively, the expression of these genes can be inhibitorily controlled by removing the introduced MYC family genes, BMI1 genes, BCL-XL genes, and the like using the Cre / lox system. Commercially available kits and the like can be used as appropriate to suppressively regulate the expression of MYC family genes, BMI1 genes, BCL-XL genes, and the like.

[0252] In order to achieve the forced expression of each of the above genes and its removal (inhibition), a commercially available drug-responsive gene expression induction system such as Tet-on (registered trademark) or Tet-off (registered trademark) system can be used. In this case, the culture medium can be made to contain a corresponding drug, such as tetracycline or doxycycline, in the process of forced expression, and forced expression is suppressed by removing these from the culture medium. When using a drug (such as doxycycline) responsive gene expression induction system, the drug inductivity can cause cells (mesenchymal stem cells with any degree of differentiation, etc.) to proliferate stably for a long time, and only by removing the drug from the culture medium at any opportunity, desired differentiated cells can be prepared in large quantities while suppressing proliferation. Alternatively, a temperature-sensitive gene expression control system or a light-sensitive gene expression control system can be used.

[0253] The forced expression of gene and the release (suppression) of forced expression can be carried out by International Publication No. 2011 / 034073 (above) and U.S. Patent Application Publication No. 2012 / 0238023, International Publication No. 2012 / 157586 (above) and U.S. Patent Application Publication No. 2014 / 0127815, International Publication No. 2014 / 123242 and U.S. Patent Application Publication No. 2016 / 0002599 or Nakamura S et al, Cell Stem Cell.14, 535-548, the method described in 2014, other known methods or methods based thereon. In addition, it can be carried out by International Publication No. 2015 / 046229, International Publication No. 2016 / 125364 or International Publication No. 2020 / 045651, other known methods or methods based thereon.

[0254] 4.CDKN1A gene

[0255] The method of regulating cell differentiation degree of the present invention may include the step of inhibiting the expression of at least one of the CDKN1A gene and the p53 gene, or inhibiting the function of their expression products, in mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, and lymphocytes of any degree of differentiation, as well as megakaryocytes and myelocytes of high degree of differentiation.

[0256] Here, the term "expression" is used to encompass both transcription and translation. Reference to inhibiting expression includes inhibition at both the transcriptional and translational levels. The methods of the present invention are expected to further improve regulation of cell differentiation (towards a lower degree of differentiation) by inhibiting expression of the CDKN1A gene and / or p53 gene or inhibiting the function of their expression products.

[0257] The CDKN1A (cyclin-dependent kinase inhibitor 1A) gene encodes the cell cycle inhibitor p21 and is also well-known as a downstream gene of the tumor suppressor gene p53. Activated p53 protein acts as a transcription factor, increasing the expression of genes downstream of p53. Therefore, as used herein, "inhibiting the expression of a gene or the function of its expression product" can be achieved by directly inhibiting the expression of the target gene or the function of its expression product (e.g., p21 in the case of the CDKN1A gene), or by controlling the expression of genes upstream of the target gene or the function of their expression products. However, in this specification, inhibition of the expression of the CDKN1A gene or inhibition of the function of its expression product does not include the p53 gene, which is an upstream gene of the CDKN1A gene, or other tumor suppressor genes upstream of the p53 gene, such as the INK4A gene and the ARF gene.

[0258] It is preferred to inhibit not only the expression of the CDKN1A gene but also the expression of the p53 gene, or to inhibit the function of their expression products.

[0259] The inhibition of the expression of each of the above genes or the function of their expression products can be carried out by known methods, for example, by introducing various molecules such as siRNA, shRNA, antisense nucleic acids (referred to as "expression inhibitory nucleic acids") that can specifically inhibit the expression of each gene or expression vectors that can express these expression inhibitory nucleic acids into cells. Alternatively, other techniques can be used, such as knocking down genes using genome editing technology. For example, when knocking down a gene using the CRISPR-Cas system, a fusion protein of an inactivated Cas and repressor domain such as a guide RNA targeting the gene and dCas is used.

[0260] siRNA is typically a double-stranded oligoRNA, consisting of an RNA having a sequence complementary to the nucleotide sequence of the target gene's mRNA or a partial sequence thereof, and its complementary strand. When used in mammalian cells, the length of siRNA is generally about 19 to 30 bases, preferably about 21 to 25 bases. The nucleotide sequence of these RNAs can be appropriately designed by those skilled in the art based on the sequence information of the gene to be inhibited. shRNA can also be used instead of siRNA.

[0261] Antisense nucleic acid refers to a nucleic acid that contains a nucleotide sequence that can specifically hybridize with the target mRNA under the physiological conditions of the cell expressing the target mRNA (mature mRNA or initial transcription product), and can inhibit the translation of the polypeptide encoded by the target mRNA when hybridized. Antisense nucleic acid is usually a single-stranded nucleic acid with a length of 10 to 100 bases, preferably 15 to 30 bases. The type of antisense nucleic acid can be DNA or RNA, or it can be a chimera of DNA and RNA. The nucleotide sequence of the antisense nucleic acid can be appropriately designed by those skilled in the art based on the sequence information of the gene whose expression is to be inhibited.

[0262] In addition to the above techniques, compounds known to inhibit gene expression can also be used. For example, compounds that inhibit CDKN1A gene expression include p21 inhibitors such as UC2288, butyrolactone I, LLW10, sorafenib, and scutellarin. Furthermore, p53 inhibitors include pifithrin α, Nutlin-3, ReACp53, and RG7388.

[0263] Alternatively, in order to inhibit the expression of a gene or the function of its expression product, the target gene can also be knocked out using known techniques. Knockout of a gene refers to that all or part of a gene is destroyed or mutated and does not play its original function. For a gene, one allele on the genome can be destroyed or mutated in a way that does not play a function. In addition, multiple alleles can also be destroyed or mutated. Knockout can be carried out by known methods, for example: a method of knocking out by introducing a DNA construct made in a way that genetic recombination occurs with the target gene into a cell; a method of knocking out by introducing a base insertion, deletion, or substitution using genome editing technologies such as TALEN and CRISPR-Cas systems.

[0264] In addition, compounds that inhibit the transcription of each gene and the transcription products, or inhibitors of the binding between the produced protein and the target protein (p53 binding inhibition: pifitarin α, Nutlin-3, ReACp53, RG7388, etc.; p21 binding inhibition: UC2288, butyrolactone I, LLW10, sorafenib, scutellariae toxin, etc.) can also be used.

[0265] The expression of a gene or the function of its expression product can be inhibited by the above-mentioned method.

[0266] Inhibition of expression of the CDKN1A gene and / or the p53 gene is preferably performed by introducing into cells an expression vector that expresses an inhibitory nucleic acid targeting each gene. Forced expression of an inhibitory nucleic acid targeting genes such as the CDKN1A gene and the p53 gene within cells can be achieved using any method known to those skilled in the art. For example, nucleic acid encoding the inhibitory nucleic acid can be introduced into cells using a gene delivery system based on viral vectors such as lentiviruses and retroviruses, or non-viral vectors such as plasmid vectors or episomal vectors, leading to expression. Alternatively, a method using a transposon to non-virally integrate the nucleic acid encoding the inhibitory nucleic acid into the cellular genome to establish a cell line stably expressing the inhibitory nucleic acid, followed by removal of excess introduced nucleic acid using a transposonase (e.g., the PiggyBac Transposon system), is also preferred. The target cell can be transfected with an expression vector (e.g., a viral vector) containing an expression inhibitory nucleic acid for a desired gene (e.g., CDKN1A gene, p53 gene), or the target cell can be induced by a pluripotent stem cell (e.g., ES cell, iPS cell) pre-integrated with an expression cassette for an expression inhibitory nucleic acid for a desired gene (e.g., CDKN1A gene, p53 gene), and the siRNA, shRNA, or antisense nucleic acid can be forced to be expressed at this stage. Alternatively, the pluripotent stem cell (e.g., ES cell, iPS cell) pre-integrated with an expression cassette for an expression inhibitory nucleic acid for a desired gene (e.g., CDKN1A gene, p53 gene) can be induced to differentiate into the target cell. When an expression vector is used to express an expression inhibitory nucleic acid in a cell, a nucleic acid (e.g., DNA) encoding the expression inhibitory nucleic acid can be operably linked downstream of a suitable promoter, inserted into the expression vector, and introduced into the cell to express the target expression inhibitory nucleic acid. The promoter can be an exogenous promoter. The exogenous promoter can be a constitutive promoter or a regulated promoter, preferably a constitutive promoter. Examples of constitutive promoters include Pol III promoters such as the U6 promoter, H1 promoter, tRNA promoter, retroviral LTR promoter, adenovirus VA1 promoter, 5SrRNA promoter, 7SK RNA promoter, and 7SL RNA promoter when expressing relatively small RNAs such as siRNA and shRNA. The nucleic acid encoding the nucleic acid that inhibits expression of the CDKN1A gene and the nucleic acid encoding the nucleic acid that inhibits expression of the p53 gene can be inserted into separate expression vectors or into the same expression vector.

[0267] In this embodiment, the expression of the CDKN1A gene or the p53 gene, or the function of its expression product, can be suppressed simultaneously with the forced expression of any one of the MYC family genes, the BMI1 gene, or the BCL-XL gene, preferably simultaneously with the forced expression of the BCL-XL gene, or after, for example, confirming that cell proliferation has decreased. As an example, the cell proliferation rate at a certain moment can be compared with its most recent cell proliferation rate (for example, the proliferation of cells is confirmed every week, and the cell proliferation rate of a certain week is compared with the proliferation rate of the week before), and it is confirmed that the proliferation rate reaches a state of less than 1 / 2. Decreased cell proliferation is not intended to be limited, and observation is performed immediately after about 30 days, about 40 days, about 50 days, about 60 days, about 70 days, about 80 days, or about 90 days after the forced expression of the MYC family genes and the BMI1 gene. In one embodiment, a MYC family gene (e.g., c-Myc gene) and a BMI1 gene are forcibly expressed in a subject cell, and concurrently, the expression of the CDKN1A gene and / or the p53 gene is suppressed, or the function of their expression products is inhibited. In one embodiment, a MYC family gene (e.g., c-Myc gene), a BMI1 gene, and a BCL-XL gene are forcibly expressed in a subject cell, and concurrently, the expression of the CDKN1A gene and / or the p53 gene is suppressed, or the function of their expression products is inhibited.

[0268] 5. Homolog

[0269] As used herein, each gene, such as the MYC family gene, BMI1 gene, BCL-XL gene, CDKN1A gene, and p53 gene, refers to a gene encoded by its known nucleic acid sequence, such as a cDNA sequence. Each gene also includes homologs identified based on homology to known nucleic acid sequences. A "homolog" refers to a gene whose cDNA sequence is substantially identical to the nucleic acid sequence of the gene.

[0270] Among MYC family genes, a homolog of the c-MYC gene refers to a gene whose cDNA sequence is substantially identical to, for example, the nucleic acid sequence set forth in SEQ ID NO: 1. A cDNA consisting of a sequence substantially identical to the nucleic acid sequence set forth in SEQ ID NO: 1 refers to a DNA consisting of a sequence having about 60% or greater, preferably about 70% or greater, more preferably about 80% or greater, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, still more preferably about 90% or greater, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, and most preferably about 99% or greater identity to the DNA consisting of the sequence set forth in SEQ ID NO: 1, or a DNA that can hybridize under stringent conditions with a DNA or RNA consisting of a complementary sequence to the nucleic acid sequence set forth in SEQ ID NO: 1, and wherein the protein encoded by such DNA inhibits the cell cycle. Alternatively, a cDNA consisting of a sequence substantially identical to the nucleic acid sequence set forth in SEQ ID NO: 1 refers to a DNA comprising a sequence in which one or more bases, for example, 1 to 10, preferably several, for example, 1 to 5, 1 to 4, 1 to 3, or 1 to 2, are deleted, substituted, or added to the sequence set forth in SEQ ID NO: 1, and wherein the protein encoded by such DNA inhibits the cell cycle. The c-MYC gene may be a gene encoding c-MYC fused to a destabilization domain (DD). The destabilization domain can be purchased and used from ProteoTuner or Clontech.

[0271] The sequence shown in sequence number 1 is the following sequence.

[0272]

[0273] A homolog of the BMI1 gene refers to a gene whose cDNA sequence is substantially identical to, for example, the nucleic acid sequence set forth in SEQ ID NO: 2. A cDNA consisting of a sequence substantially identical to the nucleic acid sequence set forth in SEQ ID NO: 2 refers to a DNA having a sequence identity of about 60% or greater, preferably about 70% or greater, more preferably about 80% or greater, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, still more preferably about 90% or greater, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, and most preferably about 99% or greater, to the DNA consisting of the sequence set forth in SEQ ID NO: 2, or a DNA that can hybridize under stringent conditions with a DNA or RNA consisting of a sequence complementary to the nucleic acid sequence set forth in SEQ ID NO: 2, and wherein the protein encoded by such DNA inhibits the cell cycle. Alternatively, the cDNA composed of a sequence substantially identical to the nucleic acid sequence shown in sequence number 2 refers to a DNA composed of a sequence in which one or more bases, for example, 1 to 10, preferably several, for example, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases are deleted, substituted, or added to the sequence shown in sequence number 2, and the protein encoded by these DNAs inhibits the cell cycle.

[0274] The sequence shown in sequence number 2 is the following sequence.

[0275]

[0276] A homolog of the BCL-XL gene refers to a gene whose cDNA sequence is substantially identical to, for example, the nucleic acid sequence set forth in SEQ ID NO: 3. A cDNA consisting of a sequence substantially identical to the nucleic acid sequence set forth in SEQ ID NO: 3 refers to a DNA having a sequence identity of about 60% or greater, preferably about 70% or greater, more preferably about 80% or greater, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, still more preferably about 90% or greater, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, and most preferably about 99% or greater, to the DNA consisting of the sequence set forth in SEQ ID NO: 3, or a DNA that can hybridize under stringent conditions with a DNA or RNA consisting of a complementary sequence to the nucleic acid sequence set forth in SEQ ID NO: 3, and wherein the protein encoded by such a DNA inhibits the cell cycle. Alternatively, the cDNA composed of a sequence substantially identical to the nucleic acid sequence shown in sequence number 3 refers to a DNA composed of a sequence in which one or more bases, for example, 1 to 10, preferably several, for example, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases are deleted, substituted, or added to the sequence shown in sequence number 3, and the protein encoded by these DNAs inhibits the cell cycle.

[0277] The sequence shown in sequence number 3 is the following sequence.

[0278]

[0279] A homolog of the CDKN1A gene refers to a gene whose cDNA sequence is substantially identical to, for example, the nucleic acid sequence set forth in SEQ ID NO: 4. A cDNA consisting of a sequence substantially identical to the nucleic acid sequence set forth in SEQ ID NO: 4 refers to a DNA having a sequence identity of about 60% or greater, preferably about 70% or greater, more preferably about 80% or greater, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, still more preferably about 90% or greater, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, and most preferably about 99% or greater, to the DNA consisting of the sequence set forth in SEQ ID NO: 4, or a DNA that can hybridize under stringent conditions with a DNA or RNA consisting of a sequence complementary to the nucleic acid sequence set forth in SEQ ID NO: 4, and wherein the protein encoded by such DNA inhibits the cell cycle. Alternatively, the cDNA composed of a sequence substantially identical to the nucleic acid sequence shown in sequence number 4 refers to a DNA composed of a sequence in which one or more bases, for example, 1 to 10, preferably several, for example, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases are deleted, substituted, or added to the sequence shown in sequence number 4, and the protein encoded by these DNAs inhibits the cell cycle.

[0280] The sequence shown in sequence number 4 is the following sequence.

[0281]

[0282] The p53 gene refers to a gene whose cDNA sequence is substantially identical to the nucleic acid sequence set forth in SEQ ID NO: 5. A cDNA consisting of a sequence substantially identical to the nucleic acid sequence set forth in SEQ ID NO: 5 refers to a DNA consisting of a sequence having an identity of about 60% or more, preferably about 70% or more, more preferably about 80% or more, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, still more preferably 90% or more, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, and most preferably about 99% or more to the DNA consisting of the sequence set forth in SEQ ID NO: 5, or a DNA that can hybridize under stringent conditions with a DNA consisting of a sequence complementary to the nucleic acid sequence set forth in SEQ ID NO: 5, and wherein the protein encoded by the DNA is a cancer suppressor. Alternatively, the cDNA composed of a sequence substantially identical to the nucleic acid sequence shown in sequence number 5 refers to a DNA composed of a sequence in which one or more bases, for example, 1 to 10, preferably several, for example, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases are deleted, substituted, or added to the sequence shown in sequence number 5, and the protein encoded by these DNAs inhibits cancer.

[0283] The sequence shown in sequence number 5 is the following sequence.

[0284]

[0285] Here, stringent conditions refer to hybridization conditions that can be easily determined by those skilled in the art and are generally empirical experimental conditions that depend on the base length of the nucleic acid, the washing temperature, and the salt concentration. Generally, longer bases require higher annealing temperatures, while shorter bases require lower temperatures. Hybrid formation generally depends on the ability of complementary strands to reanneal in an environment slightly below their melting points.

[0286] Specifically, low stringency conditions include, for example, washing in a 0.1×SSC, 0.1% SDS solution at 37°C to 42°C during the filter washing step after hybridization. High stringency conditions include, for example, washing in a 5×SSC, 0.1% SDS solution at 65°C during the washing step. By further increasing the stringency of the conditions, polynucleotides with high homology can be obtained.

[0287] 6. Cultivation process

[0288] The method of producing precursor cells of mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, or myelocytes according to this embodiment includes a step (culturing step) of culturing cells obtained by the method of regulating the degree of cell differentiation according to this embodiment.

[0289] "Precursor cells" refer to cells in the process of differentiating from cells with proliferation ability to differentiated cells, which are the final differentiated form.

[0290] The culture conditions of the cells can be appropriately determined by those skilled in the art according to the type of cells and their state. For example, the culture temperature can be set to about 35°C to about 42°C, about 36°C to about 40°C, or about 37°C to about 39°C, the carbon dioxide concentration can be set to, for example, 5% CO2, and the oxygen concentration can be set to, for example, 20% O2. The culture can be static or shaking. The shaking speed during shaking culture is not particularly limited and can be set to, for example, 10 rpm to 200 rpm, 30 rpm to 150 rpm, etc.

[0291] The culture medium can be Iscove's modified Dulbecco's modifier (IMDM) medium containing serum, insulin, transferrin, serine, monothioglycerol, ascorbic acid, and TPO. In this case, the IMDM culture medium can also contain SCF and, further, heparin. Furthermore, phorbol esters (e.g., phorbol-12-myristate-13-acetate; PMA) can be added.

[0292] The cell culture process can be carried out in the presence or absence of feeder cells. In this specification, "feeder cells" refer to cells co-cultured with target cells in order to adjust the environment required for the culture of target cells to be proliferated or differentiated. Feeder cells can be derived from the same species or from a different species as long as they can recognize target cells. Feeder cells can be cells that have been treated with antibiotics or gamma rays to prevent proliferation, or they can be untreated cells.

[0293] Culture medium can contain serum or blood plasma, or, also can be serum-free.When using serum, preferably human serum.As required, culture medium can also contain more than one material in for example albumin, insulin, transferrins, selenium, fatty acid, trace element, 2-mercaptoethanol, thioglycerol, monothioglycerol (MTG), lipid, amino acid (for example L-glutamine), ascorbic acid, heparin, non-essential amino acid, vitamin, somatomedin, low molecular compound, antibiotic, antioxidant, pyruvic acid, buffer, inorganic salts, cytokine etc.As cytokine, can illustrate for example vascular endothelial growth factor (VEGF), thrombopoietin (TPO), various TPO sample action substances, stem cell factor (SCF), erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL3), ITS (insulin-transferrins-selenite) supplement, ADAM (ADisintegrin And Metalloprotease, disintegrin metalloproteinase) inhibitor etc.

[0294] 7. Differentiation process

[0295] The method of producing differentiated cells of mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, or myelocytes according to this embodiment includes a step of differentiating cells obtained by the method of regulating cell differentiation degree according to the embodiment described above (differentiation step).

[0296] "Differentiated cells" refer to cells in the process of differentiation from cells with proliferation capacity, or cells in their final differentiated form.

[0297] The differentiation step can be carried out by culturing cells under culture conditions appropriate to the type of cells to be differentiated, and reference can be made to the description of the above-mentioned culturing step.

[0298] 8. Cells

[0299] The present invention provides mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, or myelocytes, or precursor cells or differentiated cells thereof (hereinafter referred to as cells of the present invention), which have a MYC family gene operably linked to a first exogenous promoter and a BMI1 gene operably linked to a second exogenous promoter.

[0300] MYC family gene is preferably c-MYC. The first exogenous promoter and the second exogenous promoter can independently be constitutive promoters or regulated promoters, preferably regulated promoters. The regulated promoter is preferably a drug-responsive promoter, more preferably a tetracycline-responsive promoter. The types of the first exogenous promoter and the second exogenous promoter can be the same or different, preferably promoters of the same type. By using promoters of the same type, the MYC family gene and the BMI1 gene can be expressed synchronously, and synchronous expression can be suppressed. The first exogenous promoter and the second exogenous promoter are preferably the same regulated promoter (e.g., drug-responsive promoter), more preferably both are tetracycline-responsive promoters. The first exogenous promoter and the second exogenous promoter can be independently and operably connected to the MYC family gene and the BMI1 gene, or can be operably connected to the MYC family gene and the BMI1 gene on one exogenous promoter. In this case, by linking the MYC family gene and the BMI1 gene via a spacer sequence such as an IRES, bicistronic expression can be achieved under the control of a single exogenous promoter. The MYC family gene operably linked to the first exogenous promoter and the BMI1 gene operably linked to the second exogenous promoter can be integrated into the genome of the cells of the present invention or can be present in an expression vector introduced into the cells of the present invention. Preferably, the MYC family gene operably linked to the first exogenous promoter and the BMI1 gene operably linked to the second exogenous promoter are integrated into the genome of the cells of the present invention.

[0301] When a tetracycline-responsive promoter is used as the first exogenous promoter and / or the second exogenous promoter, tetracycline-dependent expression control can be achieved. Therefore, the cells of the present invention preferably further comprise an rtTA gene or a tTA gene operably linked to a third exogenous promoter. The third exogenous promoter may be a constitutive promoter or a regulated promoter, preferably a constitutive promoter. The rtTA gene or tTA gene operably linked to the third exogenous promoter may be integrated into the genome of the cells of the present invention or may be present in an expression vector introduced into the cells of the present invention. Preferably, the rtTA gene or tTA gene operably linked to the third exogenous promoter is integrated into the genome of the cells of the present invention.

[0302] When the cells of the present invention are cultured under conditions in which mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, or myelocytes can proliferate, MYC family genes (e.g., c-Myc gene) and BMI1 genes are expressed in vitro in an amount that promotes the proliferation of these cells. The amount of MYC family genes (e.g., c-Myc) and BMI1 genes that promotes the proliferation of these cells in vitro means that the proliferation rate of these cells expressing this amount of MYC family genes and BMI1 genes is significantly increased compared to mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, or myelocytes prepared in the same manner as these cells except that the MYC family genes and BMI1 genes are not expressed.

[0303] From the perspective of enhancing proliferation capacity, the cells of the present invention may further comprise a BCL-XL gene operably linked to a fourth exogenous promoter. When the cells are cultured under conditions in which the fourth exogenous promoter operates, the BCL-XL gene is expressed, and it is expected that the proliferation of the cells of the present invention will be further promoted. The fourth exogenous promoter can independently be a constitutive promoter or a regulated promoter, preferably a regulated promoter. The regulated promoter is preferably a drug-responsive promoter, more preferably a tetracycline-responsive promoter. The type of the fourth exogenous promoter may be the same or different from the first exogenous promoter and / or the second exogenous promoter, and preferably the first, second, and fourth exogenous promoters are of the same type. By using promoters of the same type, the MYC family genes, the BMI1 gene, and the BCL-XL gene can be expressed synchronously, or their expression can be suppressed. The first, second, and fourth exogenous promoters are preferably the same regulated promoter (e.g., a drug-responsive promoter), and more preferably, all are tetracycline-responsive promoters. The fourth exogenous promoter can be operably linked to the BCL-XL gene independently of the first and second exogenous promoters. A MYC family gene and the BCL-XL gene can be operably linked to the first exogenous promoter, a BMI1 gene and the BCL-XL gene can be operably linked to the second exogenous promoter, or a MYC family gene, a BMI1 gene, and a BCL-XL gene can be operably linked to a single exogenous promoter. By linking multiple genes via a spacer sequence such as a RES, bicistronic expression can be achieved under the control of a single exogenous promoter. The BCL-XL gene operably linked to the fourth exogenous promoter can be integrated into the genome of the cells of the present invention or present in an expression vector introduced into the cells of the present invention. Preferably, the BCL-XL gene operably linked to the fourth exogenous promoter is integrated into the genome of the cells of the present invention.

[0304] From the perspective of enhancing proliferation capacity, the cells of the present invention may further comprise a nucleic acid encoding a nucleic acid that inhibits expression of the CDKN1A gene (e.g., siRNA, shRNA, antisense nucleic acid) operably linked to a fifth exogenous promoter and / or a nucleic acid encoding a nucleic acid that inhibits expression of the p53 gene operably linked to a sixth exogenous promoter. When the cells are cultured under conditions in which the fifth and / or sixth exogenous promoters operate, expression of the nucleic acid that inhibits expression of the CDKN1A gene and / or the nucleic acid that inhibits expression of the p53 gene is expected to further promote proliferation of the cells of the present invention. The fifth and sixth exogenous promoters can independently be constitutive or regulated, preferably constitutive. A constitutive promoter is preferably a Pol III-based promoter such as the H1 promoter. The type of the fifth and sixth exogenous promoters may be the same or different. The nucleic acid encoding the nucleic acid that inhibits the expression of the CDKN1A gene operably linked to the fifth exogenous promoter and the nucleic acid encoding the nucleic acid that inhibits the expression of the p53 gene operably linked to the sixth exogenous promoter can be integrated into the genome of the cell of the present invention or can be present in an expression vector introduced into the cell of the present invention. Preferably, they are integrated into the genome of the cell of the present invention.

[0305] In one embodiment, the cell of the present invention has:

[0306] A MYC family gene operably linked to a first exogenous promoter,

[0307] a BMI1 gene operably linked to a second exogenous promoter, and

[0308] Mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, or myelocytes, or precursor cells thereof, expressing the BCL-XL gene operably linked to a fourth exogenous promoter. When a tetracycline-responsive promoter is used as the first exogenous promoter and / or the second exogenous promoter, the cells of the present invention may further have an rtTA gene or a tTA gene operably linked to a third exogenous promoter.

[0309] In one embodiment, the cell of the present invention has:

[0310] A MYC family gene operably linked to a first exogenous promoter,

[0311] a BMI1 gene operably linked to a second exogenous promoter,

[0312] A nucleic acid encoding a nucleic acid that inhibits expression of the CDKN1A gene and is operably linked to a fifth exogenous promoter, and

[0313] Mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, or myelocytes, or precursor cells thereof, containing a nucleic acid encoding a nucleic acid that inhibits the expression of the p53 gene operably linked to a sixth exogenous promoter. When a tetracycline-responsive promoter is used as the first exogenous promoter and / or the second exogenous promoter, the cells of the present invention may further contain an rtTA gene or a tTA gene operably linked to a third exogenous promoter.

[0314] In one embodiment, the cell of the present invention has:

[0315] A MYC family gene operably linked to a first exogenous promoter,

[0316] a BMI1 gene operably linked to a second exogenous promoter,

[0317] BCL-XL gene operably linked to a fourth exogenous promoter

[0318] A nucleic acid encoding a nucleic acid that inhibits expression of the CDKN1A gene and is operably linked to a fifth exogenous promoter, and

[0319] Mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, or myelocytes, or precursor cells thereof, containing a nucleic acid encoding a nucleic acid that inhibits the expression of the p53 gene operably linked to a sixth exogenous promoter. When a tetracycline-responsive promoter is used as the first exogenous promoter and / or the second exogenous promoter, the cells of the present invention may further contain an rtTA gene or a tTA gene operably linked to a third exogenous promoter.

[0320] The cells of the present invention can be obtained by the above-mentioned method of the present invention for regulating the degree of differentiation of mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells or lymphocytes with any degree of differentiation, or megakaryocytes or myelocytes with a high degree of differentiation, or the method for producing precursor cells of mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes or myelocytes.

[0321] In addition, the present invention provides a cell population comprising the cells of the present invention described above (referred to as the cell population of the present invention). The cell population is rich in the cells of the present invention described above, and the proportion of the cells of the present invention contained in the entire cell population is, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more (e.g., 100%). Such a cell population rich in the cells of the present invention can be obtained by extracting target cells (mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes or myelocytes, or precursor cells thereof) having a specific degree of differentiation (e.g., high degree of differentiation, low degree of differentiation) from a cell population to which the method of the present invention is applied. In a preferred embodiment, the cell population of the present invention is rich in cells of the present invention having a specific degree of differentiation (e.g., high degree of differentiation, low degree of differentiation). In one embodiment, the proportion of the cells of the present invention (the cells are mesenchymal stem cells) contained in the entire cell population is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more (e.g., 100%). In one embodiment, the proportion of the cells of the present invention (the cells are vascular endothelial cells) contained in the entire cell population is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more (e.g., 100%). In one embodiment, the proportion of the cells of the present invention (the cells are smooth muscle cells) contained in the entire cell population is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more (e.g., 100%). In one embodiment, the proportion of the cells of the present invention (the cells are neural crest cells) contained in the entire cell population is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more (e.g., 100%). In one embodiment, the proportion of the cells of the present invention (the cells are lymphocytes) contained in the entire cell population is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more (e.g., 100%). In one embodiment, the proportion of the cells of the present invention (the cells are megakaryocytes) contained in the entire cell population is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more (e.g., 100%).In one embodiment, the proportion of the cells of the present invention (the cells are myelocytes) contained in the entire cell population is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more (e.g., 100%). Such a cell population rich in cells of the present invention at a specific differentiation stage (e.g., highly differentiated or poorly differentiated) can be obtained by isolating and extracting cells at the desired differentiation stage from a cell population to which the above-described method of the present invention has been applied using an antibody against a cell surface marker specifically expressed by cells at that differentiation stage, using a cell sorter or the like.

[0322] The cells and cell populations of the present invention can be obtained by the above-mentioned method of the present invention for regulating the degree of differentiation of mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells or lymphocytes having any degree of differentiation, or megakaryocytes or myelocytes having a high degree of differentiation, or the method for producing precursor cells of mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes or myelocytes.

[0323] 9. Cell Preparation

[0324] In addition, the present invention provides a cell preparation comprising the cell population of the present invention described above (referred to as the cell preparation of the present invention). The cell preparation of the present invention can be prepared by suspending the cell population of the present invention described above in an appropriate physiological aqueous solution (e.g., physiological saline, isotonic solution containing glucose or other auxiliary agents, liquid culture medium). The physiological aqueous solution can be compounded with a buffer (e.g., phosphate buffer, sodium acetate buffer), a soothing agent (e.g., lidocaine hydrochloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), a preservative (e.g., sodium benzoate, benzalkonium chloride, etc.), an antioxidant (e.g., ascorbic acid, sodium edetate, etc.), etc. The cell concentration in the cell preparation is, for example, 1.0×10 1 ~1.0×10 12 The cell population of the present invention is suspended at 10 cells / mL.

[0325] Depending on the type of cells or cell populations of the present invention contained in the cell preparation, a combination of cytokines suitable for cell proliferation can be added to the cell preparation. For example, in the case of a cell preparation containing mesenchymal stem cells, bFGF can be added to the cell preparation. In the case of a cell preparation containing vascular endothelial cells, VEGF can be added to the cell preparation. In the case of a cell preparation containing smooth muscle cells, VEGF can be added to the cell preparation. In the case of a cell preparation containing neural crest cells, ALK5 inhibitors such as SB-431542 and GSK3 inhibitors such as CHIR99021 can be added to the cell preparation. Alternatively, in the case of a cell preparation containing neural crest cells, ALK5 inhibitors such as SB-431542, EGF, and FGF2 can be added to the cell preparation. In the case of a cell preparation containing lymphocytes, IL-3 can be added to the cell preparation. In the case of a cell preparation containing megakaryocytes, TPO and SCF can be added to the cell preparation.

[0326] 10. Cell differentiation regulator

[0327] The cell differentiation degree regulator of this embodiment contains as an active ingredient a molecule that forces the expression of MYC family genes and BMI1 gene, optionally contains as an active ingredient a molecule that forces the expression of BCL-XL gene, or contains as an active ingredient a molecule that inhibits the expression of CDKN1A gene or p53 gene or inhibits the function of their expression products.

[0328] 11. Pharmaceutical Compositions

[0329] The pharmaceutical composition of this embodiment comprises cells obtained by the method of regulating cell differentiation degree or the method of producing precursor cells or differentiated cells of mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, or myelocytes according to the present invention.

[0330] 12. Treatment or prevention methods

[0331] The treatment or prevention method of this embodiment includes the step of administering cells or a pharmaceutical composition containing the cells to a subject in need thereof, wherein the cells are cells obtained by the method of regulating cell differentiation degree of the present invention, the method of producing precursor cells or differentiated cells of mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes or myelocytes.

[0332] The present invention will be described in detail below based on the following examples, but the present invention is not limited to these examples. Those skilled in the art can modify the present invention in various forms without departing from the scope of the present invention, and such modifications are included in the scope of the present invention.

[0333] Example

[0334] Example 1-1: Mesenchymal stem cells

[0335] According to a report by Fukuta et al. (Plos One, 2014, 9(12):e112291), neural crest cells (NCCs) were induced from iPS cells by culturing them on Matrigel-coated culture dishes in the presence of SB-431542 and CHIR99021 for 7 days. NCCs were then expanded in fibronectin C-coated culture dishes by switching to a medium containing EGF, FGF2, and SB-431542. Furthermore, MSCs were induced in fibronectin C-coated culture dishes by switching to a medium containing 10% FBS and FGF2.

[0336] MSCs at passage 9, one month after induction, were infected with any of the viral vectors shown below.

[0337] Control (no infection or empty viral vector)

[0338] c-MYC only

[0339] c-MYC+BMI1(MB)

[0340] c-MYC+BMI1+BCLXL (MBX)

[0341] c-MYC+BMI1+BCLXL+shp53

[0342] ·c-MYC+BMI1+BCLXL+shp53+shp21

[0343] shp53 (p53KD)

[0344] shp21(p21KD)

[0345] The basic culture medium of iPS-MSCs used alfa MEM+10% FCS+5 ng / ml bFGF.

[0346] The forced expression vectors for MYC, BMI1, and BCLXL used a doxycycline-induced expression system (dox-on system). Immediately after infection, 1 μg / ml of doxycycline was added to the culture medium and the number of viable cells was counted. The results are shown in Figure 1 .like Figure 1As shown, the control (uninfected or empty viral vector) gradually stopped proliferation. In the case of p53KD, although transient proliferation was obtained, proliferation also gradually stopped. In addition, in the case of forced expression of c-MYC alone and in the case of p21KD, proliferation stopped earlier than in the control condition. On the other hand, in the case of combinations containing MB (c-MYC+BMI1, c-MYC+BMI1+BCLXL, c-MYC+BMI1+BCLXL+shp53, c-MYC+BMI1+BCLXL+shp53+shp21), exponential proliferation was confirmed even after more than 60 days of infection.

[0347] In addition, the cells were stained with CD90-FITC, CD73-PE, and CD105-APC, which are representative markers of mesenchymal stem cells, and analyzed by flow cytometry. Figure 2 . It is quite interesting that the proportion of CD90+ / CD73+ / CD105+ cell populations decreased in the control group, but increased in the MB and MBX groups. CD90+ cell populations and CD90- cell populations were observed in the control group. CD90 is a representative marker of mesenchymal stem cells. As mesenchymal stem cells enter terminal differentiation and lose their stemness, they also lose CD90 expression. These results indicate that in the control group, mesenchymal stem cells (CD90-) gradually lost their stemness with passage and entered terminal differentiation, while in the MB and MBX groups, relatively undifferentiated mesenchymal stem cells (CD90+) maintained their stemness.

[0348] In addition, a cell population containing 9th generation differentiated cells (CD90-) was stained with CD90 antibody, separated into CD90+ and CD90- fractions, purified, and uninfected as a control. After MB infection, doxycycline 1 μg / mL was added to both groups and cultured. Flow cytometry analysis was performed on days 14 and 21 of infection. The results are shown in Figure 3 and Figure 5 .

[0349] FACS analysis of CD105, CD90, and CD73 on day 21 of infection revealed that 13% of the CD90+ purified control group remained CD90+, with the majority becoming CD90- differentiated cells. Furthermore, the CD90- purified group comprised a majority of CD90- differentiated cells. The CD90+ fraction generated both CD90+ and CD90- fractions, while the CD90- fraction did not generate a CD90+ fraction, indicating that the CD90+ fraction is located higher in the hierarchy. On the other hand, in the MB group, the majority of the cell population expressed CD90+ in both the CD90+ and CD90- purified groups. By forced expression of c-MYC+BMI1, both the CD90+ fraction and the CD90- fraction produced CD90+CD73+CD105+ fractions, indicating that through c-MYC+BMI1, mesenchymal stem cells (CD90-) entering terminal differentiation were converted into relatively undifferentiated mesenchymal stem cells (CD90+) (mesenchymal stem cells rejuvenated into precursor cells, and precursor cells were reprogrammed).

[0350] Furthermore, it was confirmed that when MB or MBX expression was shut down by Dox off, the proliferation rate slowed down (about 1 / 2 to 1 / 3 of that of Dox on), and CD90 expression was gradually lost (gradual differentiation) ( Figure 4 The introduction of MBX rejuvenated MSC, returning it to near generation 0.

[0351] Example 1-2: Induction of differentiation into chondrocytes, osteoblasts, and adipocytes

[0352] Based on Example 1-1, iPS-derived MSCs were forced to express MB or MBX, and the proliferation-capable MSCs (non-aging MSCs) thus established were differentiated into adipocytes, osteoblasts, and chondrocytes, and the amounts of each series were compared with those of MSCs derived from human bone marrow. Adipocyte differentiation was performed using the StemPro (trademark) Adipogenesis Differentiation Kit gibco (cat: A1007001) and confirmed by staining with HCS (High-content screening) LipidTOX (trademark) green neutral lipid stain (Invitrogen (Molecular Probes) Cat: H34475). Osteoblast differentiation was performed using the StemPro (trademark) Adipogenesis Differentiation Kit gibco (cat: A1007001) and confirmed by staining with Alizarin Red S staining quantitative assay Scien Cell Research Laboratories #8678. Chondrocyte differentiation was performed using StemPro (trademark) Osteogenesis Differentiation Kit Gibco (cat: A1007201) and confirmed by staining with Alcian blue staining solution pH = 2.5 (Sigma) cat: A3157. Figure 6 .

[0353] The results showed that the differentiation ability of all lines of MSCs was equivalent to that of human bone marrow-derived MSCs, indicating that the MSCs with proliferation ability have the same differentiation ability as MSCs in vivo.

[0354] Example 2: Vascular endothelial cells

[0355] The cells were co-cultured with 10T1 / 2 cells using the method of Takayama Blood 2008, 111(11): 5298-5306; Takayama JEM 2010, 207(13): 2817-2830. Vascular progenitor cells were induced from human iPS cells in the presence of 20 ng / ml VEGF. On day 10-11, CD34+ vascular endothelial progenitor cells were sorted using FACS AriaIII and infected with Dox-onc-MYC / Dox-on BMI1 / Dox-on BCL-XL lentiviral vectors. The cells were further cultured in the presence of 1 μg / ml doxycycline and 100 ng / ml VEGF, and the cell number was counted. Compared with the control (uninfected with virus), cell proliferation was significantly increased in the dox-on group ( Figure 7) In addition, immunostaining of the cell surface marker VE-cadherin was performed in the dox-on state and in the mature state after dox-off for 7 days.

[0356] As a result, the expression of VE-cadherin in doxed cells was relatively increased, and it was confirmed that the cells differentiated into vascular endothelial cells ( Figures 8-10 ).

[0357] Furthermore, qPCR was used to evaluate the gene expression of vascular endothelial cell differentiation markers ENG (CD105), MCAM (CD146), and PECAM1 in human iPS, imVEC-dox on, imVEC-dox off, HAEC (human aortic endothelial cells), and HUVEC (human unbilical vein endothelial cells).

[0358] As a result, it was confirmed that cells obtained by dox-offing human imVECs expressed differentiation markers of vascular endothelial cells ( Figure 11 ).

[0359] Example 3: Vascular smooth muscle cells

[0360] The cells were co-cultured with 10T1 / 2 cells using the method of Takayama Blood 2008, 111(11): 5298-5306; Takayama JEM 2010, 207(13): 2817-2830. Vascular progenitor cells were induced from human iPS cells in the presence of 20 ng / ml VEGF. On day 10-11, CD34- / VEGFR<KDR>+ vascular smooth muscle progenitor cells were sorted using FACS AriaIII and infected with Dox-on c-MYC / Dox-on BMI1 / Dox-on BCL-XL lentiviral vectors. The cells were further cultured in the presence of 1 μg / ml doxycycline, and the cell number was counted. Compared with the control (uninfected with virus), cell proliferation was significantly increased in the dox-on group ( Figure 12 ).

[0361] Furthermore, immunostaining for calponin and αSMA, smooth muscle differentiation markers, was performed on cells cultured on day 26 (dox-on precursor cell stage) and cells doxed for one week from day 22 (day 29).

[0362] As a result, it was confirmed that the expression of vascular smooth muscle differentiation markers in dox-off cells was enhanced ( Figure 13 ).

[0363] MBX-introduced non-senescent vascular smooth muscle cells were dox-off (MBX-off) and incubated with PDGFR2-APC antibody on day 3. The cells were analyzed using FACS Canto II and sorted into PDGFR2+ undifferentiated vascular smooth muscle cells and PDGFR2-differentiated vascular smooth muscle cells. Figure 14 The cells were then divided again into a group receiving 1 μg / ml doxycycline and a group remaining on dox-free. Morphological observation, cell count, and FACS-based cell surface marker analysis (CD140b, KDR, and CD34) were performed. This resulted in a total of four groups being tested.

[0364] I) PDGFR2+ undifferentiated vascular smooth muscle cells+dox on

[0365] II) PDGFR2+ undifferentiated vascular smooth muscle cells dox off

[0366] III) PDGFR2-differentiated vascular smooth muscle cells + dox on

[0367] IV) PDGFR2-differentiated vascular smooth muscle cells doxed

[0368] The morphological observation results of groups I) to IV) 5 days after sorting are shown in Figure 15 The cell count results are shown in Figure 16 The results of cell surface marker expression confirmation are shown in Figures 17-1 to 17-3 .according to Figure 16 and Figures 17-1 to 17-3The results showed that compared to the dox-off conditions (Group I) PDGFR2+ undifferentiated vascular smooth muscle cells (dox-on) and Group III) PDGFR2-differentiated vascular smooth muscle cells (dox-on), the expression of surface markers CD140b, KDR, and CD34 was suppressed to a lower level in the dox-on group (Group II) PDGFR2+ undifferentiated vascular smooth muscle cells (dox-off) and Group IV PDGFR2-differentiated vascular smooth muscle cells (dox-off), respectively), and the cell proliferation rate increased, indicating that the cells returned to the state of undifferentiated vascular smooth muscle cells. This result suggests that forced expression of MBX in PDGFR2-differentiated vascular smooth muscle cells reverts to proliferative undifferentiated vascular smooth muscle cells (vascular smooth muscle cell precursor cells, PDGFR2+) (reprogramming of precursor cells). Conversely, it was shown that when MBX expression is stopped in MBX-expressing undifferentiated vascular smooth muscle cells (vascular smooth muscle cell precursor cells, PDGFR2+), they differentiate into mature vascular smooth muscle cells (PDGFR2-).

[0369] Furthermore, PDGFR2+ undifferentiated vascular smooth muscle cells and PDGFR2- differentiated vascular smooth muscle cells cultured under dox-on conditions for 28 days after sorting and dox-off for one week from day 29 were immunostained for smooth muscle differentiation markers, calponin and αSMA.

[0370] As a result, it was confirmed that the expression of smooth muscle differentiation markers was enhanced in both PDGFR2+ undifferentiated vascular smooth muscle cells and PDGFR2-differentiated vascular smooth muscle cells ( Figure 18 The expression of smooth muscle differentiation markers was also enhanced in PDGFR2-differentiated vascular smooth muscle cells forced to express MBX (via precursor cell reprogramming), indicating that good differentiation capacity was maintained even after precursor cell reprogramming.

[0371] Example 4: Neural Crest Cells

[0372] According to the report of Fukuta et al. (Plos One, 2014, 9(12):e112291), iPS cells were cultured on matrigel-coated culture dishes in the presence of SB-431542 and CHIR99021 for 7 days to induce neural crest cells (NCC). The culture medium was then changed to NCC medium containing CDMi medium (CDMi medium prepared based on IMDM medium supplemented with F-12w, BSA, CD concentrated lipids, apo-transferrin, 1-thioglycerol, P / S, and insulin) supplemented with EGF, FGF2, and SB-431542, and NCC was expanded on fibronectin-coated culture dishes.

[0373] The cultured NCCs were infected with c-MYC+BMI1(MB) lentiviral vector or c-MYC+BMI1+BCLXL(MBX) lentiviral vector and cultured in the presence of 1 μg / ml doxycycline for morphological observation, cell number counting and FACS-based surface marker (CD271) confirmation ( Figures 19-23 ).

[0374] The morphological observation results of NCCs in the control group (no infection or empty viral vector), the group infected with c-MYC+BMI1(MB) lentiviral vector, or the group infected with c-MYC+BMI1+BCLXL(MBX) lentiviral vector 10 days after sorting are shown in FIG. Figure 19 In addition, if Figure 20 As shown, cell proliferation in the control group gradually stopped, while NCC infected with the c-MYC+BMI1(MB) lentiviral vector or c-MYC+BMI1+BCLXL(MBX) lentiviral vector showed exponential cell proliferation.

[0375] In addition, the expression of CD271, a cell surface marker of neural crest cells, was analyzed by flow cytometry. The results are shown in Figures F and G. As neural crest cells enter terminal differentiation and lose their stem cell properties, they also lose CD271 expression. In the control group, CD271 expression decreased with increasing passage number ( Figure 21 ), while the expression of CD271 in MB and MBX groups was restored ( Figure 22 These results indicate that the control group gradually lost stem cell properties and entered terminal differentiation with passage, while the MB and MBX groups maintained stem cell properties and remained undifferentiated NCC.

[0376] Furthermore, it was confirmed whether coating the culture dish with fibronectin had little effect on the cell number and CD271 expression in the MB and MBX groups ( Figure 23 ).

[0377] Example 5: Lymphocytes

[0378] 0.1% gelatin was added to the wells of a 6-well plate at 1 ml / well and incubated at 37°C for 30 minutes. The gelatin was removed and non-senescent vascular endothelial cells were plated at 2-3 × 10 ＾5Cells / well were seeded with basic culture medium (IMDM + 15% FBS + 10μg / ml human insulin, 5μg / ml transferrin + 5ng / ml sodium selenite + 2mM L-glutamine + 0.45mM α-monomercaptoglycerol + 50μl / ml ascorbic acid + 20ng / ml VEGF). The immortalized vascular endothelial cells used in this experiment were mature vascular endothelial cells whose proliferation was stopped by irradiation, obtained by culturing the vascular endothelial cells whose proliferation was improved by introducing MBX under dox off for 7 days according to Example 2. The cells were cultured at 37°C and CO2 5.0%. The culture medium was removed the next day, and human hematopoietic stem cells derived from iPS were cultured at 1×10 ＾5 iPS cell-derived hematopoietic progenitor cells were seeded at 10 cells / well in minimally soluble medium (IMDM + 15% FBS + 10 μg / ml human insulin + 5.5 μg / ml transferrin + 5 ng / ml sodium selenite + 2 mM L-glutamine + 0.45 mM α-monomercaptoglycerol + 50 μl / ml ascorbic acid + 50 ng / ml human Flt3L + 50 ng / ml human SCF + 20 ng / ml IL-7 + 20 ng / ml IL-15). On the same day, a doxycycline-inducible c-MYC + BMI1 + BCLXL (MBX) viral vector was added, followed by 1 μg / ml doxycycline. Regarding cytokine concentrations, hFlt3L 50 ng / ml + hSCF 50 ng / ml + IL-7 20 ng / ml + IL-15 20 ng / ml was used as the base concentration. In the IL-3 supplementation group, IL-3 was administered at 20 ng / ml for the first two weeks only. All cells were cultured at 37°C and 5.0% CO2. Thereafter, the culture medium was replaced halfway every 2 to 3 days. Furthermore, CD45+ / CD56+ cells and CD45+ / CD19+ cells were stained with CD45-BV421, CD56-FITC, and CD19-PC7, and FACS CantoII was used to analyze them. Figures 24-27 ). CD45-BV421 and CD56-FITC are cell surface markers of natural killer cells, and CD45-BV421 and CD19-PC7 are cell surface markers of B cells.

[0379] like Figure 24 As shown, the total cell number and CD45+ / CD56+ cell number in the MBX(+)+IL-3 group increased exponentially over 2 months, whereas the MBX(-) group (uninfected or with an empty viral vector) gradually stopped increasing from culture day 50. Due to the lack of CD45+ / CD56+ cell proliferation in the MBX(+) group, culture was terminated on day 38.

[0380] In addition, if Figure 25As shown, the ratio of CD45+ / CD56+ cell populations in the MBX(-) group was 89.9% on the 30th day of culture and decreased to 32.9% on the 64th day, while that in the MBX(+)+IL-3 group was 55.9% on the 30th day and 69.2% on the 66th day. Even after 66 days, the cell population was confirmed at a high ratio of more than 50%.

[0381] Therefore, the MBX(+)+IL-3 group was able to obtain CD45+ / CD56+ cells with high proliferation capacity, successfully producing non-senescent natural killer cells efficiently and in large quantities.

[0382] In addition, if Figure 26 and 27 As shown, a CD45+ / CD19+ cell population also emerged. The number of CD45+ / CD19+ cells increased exponentially over two months in the MBX(+)+IL-3 group, whereas the increase in the MBX(-) group (uninfected or with an empty viral vector) gradually stopped after culture day 50. Due to the lack of CD45+ / CD19+ cell proliferation in the MBX(+) group, culture was discontinued on day 38.

[0383] Therefore, the MBX(+)+IL-3 group was able to obtain CD45+ / CD19+ cells with high proliferation capacity, successfully producing non-senescent B cells efficiently and in large quantities.

[0384] Based on the above results, non-senescent lymphocytes such as non-senescent natural killer cells and non-senescent B cells can be obtained by forcibly expressing c-MYC, BMI1, and BCLXL (MBX) and culturing with the addition of IL-3.

[0385] Example 6: Myelocytes and Megakaryocytes

[0386] The experiment was conducted using megakaryocytes and myelocytes induced from human iPS cells whose proliferation had been enhanced by the introduction of MBX. IMDM+ was used as the basal medium. Megakaryocytes were cultured with 50 ng / mL SCF and 50 ng / mL TPO, while myelocytes were cultured with 50 ng / mL M-CSF, 50 ng / mL GM-CSF, 25 ng / mL G-CSF, 25 ng / mL IL-3, 25 ng / mL SCF, and 5 ng / mL TPO.

[0387] Megakaryocytes

[0388] On day 3 of the dox-off (MBX-off) phase, CD41+CD42b- undifferentiated megakaryocytes and CD41+CD42b+ differentiated megakaryocytes were sorted and purified using an AriaIII FACS system. The cells were then divided again into a group receiving 1 μg / ml doxycycline and a group remaining dox-off. Cell counts and surface marker analysis were performed using FACS. The following four groups were tested in total.

[0389] I)CD41+CD42b- undifferentiated megakaryocytes+dox on

[0390] II) CD41+CD42b- undifferentiated megakaryocytes dox off

[0391] III)CD41+CD42b+differentiated megakaryocytes+dox on

[0392] IV) CD41+CD42b+ differentiated megakaryocytes dox off

[0393] The results are shown in Figure 28 In the doxycycline-treated groups (Group I) (CD41+CD42b- undifferentiated megakaryocytes + dox-on) and Group III (CD41+CD42b+ differentiated megakaryocytes + dox-on), cells proliferated exponentially, and all cells returned to the CD41+CD42b- undifferentiated megakaryocyte state. On the other hand, in the dox-off groups (Group II) (CD41+CD42b- undifferentiated megakaryocytes + dox-off) and Group IV (CD41+CD42b+ differentiated megakaryocytes + dox-off), cell proliferation ceased, resulting in CD41+CD42b+ differentiated megakaryocytes. These results suggest that forced expression of MBX in differentiated megakaryocytes (CD41+CD42b+) reverts them to proliferative, undifferentiated megakaryocytes (megakaryocyte precursor cells, CD41+CD42b-) (reprogramming of precursor cells). On the other hand, if MBX expression is stopped in undifferentiated megakaryocytes (megakaryocyte precursor cells, CD41+CD42b-) that express MBX, they lose their proliferative ability and differentiate into mature megakaryocytes (CD41+CD42b+).

[0394] Myelocytes

[0395] On the third day of dox-off (MBX-off) treatment, CD14+CD11b- undifferentiated macrophages and CD14+CD11b+ differentiated macrophages were sorted and purified using AriaIII FACS. The cells were then divided again into a group receiving 1 μg / ml doxycycline and a group remaining dox-off. Cell counts and surface marker analysis were performed using FACS. The following four groups were tested in total.

[0396] I)CD14+CD11b-undifferentiated macrophages+dox on

[0397] II) CD14+CD11b- undifferentiated macrophages doxed

[0398] III)CD14+CD11b+differentiated macrophages+dox on

[0399] IV) CD14+CD11b+ differentiated macrophages dox off

[0400] The results are shown in Figure 28 In both groups (Group I) CD14+CD11b- undifferentiated macrophages + doxon and Group III) CD14+CD11b+ differentiated macrophages + doxon), cells in the doxycycline+ group proliferated exponentially, while all cells returned to the CD14+CD11b- undifferentiated macrophage state.

[0401] On the other hand, in the dox-off groups (Group II) (CD14+CD11b- undifferentiated macrophages) and (Group IV) (CD14+CD11b+ differentiated macrophages), cell proliferation ceased, resulting in the formation of CD14+CD11b+ differentiated macrophages. This result suggests that forced expression of MBX in differentiated macrophages (CD14+CD11b+) reverts them to proliferative undifferentiated macrophages (CD14+CD11b-) (reprogramming of precursor cells). Conversely, the results suggest that inactivating MBX expression in MBX-expressing undifferentiated macrophages (CD14+CD11b-) leads to a loss of proliferative capacity and differentiation into mature macrophages (CD14+CD11b+).

[0402] Example 7-1: Co-culture with non-senescent vascular endothelial cells

[0403] It is known that the notch signal, which is transmitted through the interaction between Notch and Notch ligands (Delta-like 4 (hereinafter referred to as DLL4), Jagged 1, etc.), is essential for cell differentiation.

[0404] Furthermore, PCA (principal component analysis) was performed on non-senescent vascular endothelial cells set to dox on, non-senescent vascular endothelial cells set to dox off, human iPS cells, human aortic endothelial cells (HAEC), and human umbilical cord blood endothelial cells (HUVEC). The results showed that the main components of non-senescent vascular endothelial cells set to dox off were similar to those of human aortic endothelial cells (HAEC) and human umbilical cord blood endothelial cells (HUVEC). Figure 29 ). It was also found that the expression of DLL4 and Jagged 1 was stronger in non-senescent vascular endothelial cells set to dox off than in non-senescent vascular endothelial cells set to dox on, human iPS cells, human aortic endothelial cells (HAEC), and human umbilical cord blood endothelial cells (HUVEC). Figure 30 ).

[0405] Therefore, the effects of non-senescent vascular endothelial cells on the differentiation of hematopoietic stem cells or hematopoietic progenitor cells into lymphocytes were investigated.

[0406] Example 7-2: Co-culture of non-senescent vascular endothelial cells and CD34-positive umbilical cord blood cells

[0407] The immortalized vascular endothelial cells used in this experiment were mature vascular endothelial cells whose proliferation was enhanced by the introduction of MBX according to Example 2 and cultured under dox-off for 7 days, and whose proliferation was stopped by irradiation.

[0408] 0.1% gelatin was added to the wells of a 6-well plate at 1 ml / well and incubated at 37°C for 30 minutes. The gelatin was removed and non-senescent vascular endothelial cells were plated at 2-3 × 10 ＾5 Cells / well were seeded in basic culture medium and cultured at 37°C and 5.0% CO2. The next day, the culture medium was removed and CD34-positive umbilical cord blood cells were plated at 5×10 ＾3 10 cells / well were seeded with a basic medium for hematopoietic progenitor cells derived from CD34-positive umbilical cord blood cells (X-VIVO10 + 10% BSA). In addition, CD34-positive umbilical cord blood cells were also seeded at 5×10 ＾3 Cells / well were seeded in a basic medium for CD34-positive umbilical cord blood cell-derived hematopoietic progenitor cells. Regarding cytokine conditions, four types of cytokines, 1 to 4, were prepared and all were cultured at 37°C and 5.0% CO2. Thereafter, half of the medium was replaced every 2-3 days and culture was continued. On the 24th day of culture, the cells were stained with natural killer cell surface markers CD45-BV421 and CD56-FITC, and the CD45+ / CD56+ cells were analyzed using FACSCanto II ( Figures 31-32 The conditions for cytokines 1 to 4 are shown below.

[0409] Cytokine 1:

[0410] X-VIVO10+10% BSA+50ng / ml human Flt3+50ng / ml human SCF+20ng / ml IL-7+20ng / ml IL-15

[0411] Cytokine 2:

[0412] X-VIVO10+10% BSA+50ng / ml human Flt3+50ng / ml human SCF+20ng / ml IL-7+20ng / ml IL-15+20ng / ml IL-2+50ng / ml TPO

[0413] Cytokine 3:

[0414] X-VIVO10+10% BSA+50ng / ml human Flt3+50ng / ml human SCF+20ng / ml IL-7+20ng / ml IL-15+20ng / ml IL-2+50ng / ml TPO+10pg / ml GM-CSF+250pg / ml G-CSF+50pg / ml IL-6

[0415] Cytokine 4:

[0416] On days 0-9: X-VIVO10 + 10% BSA + 50 ng / ml human Flt3 + 50 ng / ml human SCF + 20 ng / ml IL-7 + 50 ng / ml TPO + 10 pg / ml GM-CSF + 250 pg / ml G-CSF + 50 pg / ml IL-6

[0417] On days 9-14: X-VIVO10 + 10% BSA + 50 ng / ml human Flt3 + 50 ng / ml human SCF + 20 ng / ml IL-7 + 20 ng / ml IL-15 + 10 pg / ml GM-CSF + 250 pg / ml G-CSF + 50 pg / ml IL-6

[0418] Day 14-;

[0419] like Figure 31As shown, CD45+ / CD56+ cell populations appeared under all conditions of cytokines 1 to 4. In addition, the proportion of CD45+ / CD56+ cell populations was higher when co-cultured with non-senescent vascular endothelial cells (imEC) than when not co-cultured with non-senescent vascular endothelial cells (feeder-free).

[0420] In addition, if Figure 32 As shown, even when focusing on the absolute number of cells, when comparing cells cultured under the same cytokine conditions (e.g., im EC cytokine3 versus feeder-free cytkine2), the number of CD45+ / CD56+ cells is higher when co-cultured with non-senescent vascular endothelial cells.

[0421] The above results indicate that co-culturing with non-senescent vascular endothelial cells can promote the differentiation of hematopoietic stem cells or hematopoietic progenitor cells into natural killer cells. Furthermore, it is also shown that differentiation into lymphocyte progenitor cells, an intermediate before differentiation into natural killer cells, can be promoted.

[0422] Example 7-3: Non-senescent vascular endothelial cells or genetically recombinant DLL4 and human hematopoietic stem cells derived from iPS cells Co-culture of cells

[0423] The immortalized vascular endothelial cells used in this experiment were mature vascular endothelial cells whose proliferation was enhanced by the introduction of MBX according to Example 2 and cultured under dox-off for 7 days, and whose proliferation was stopped by irradiation.

[0424] 0.1% gelatin was added to the wells of a 6-well plate at 1 ml / well and incubated at 37°C for 30 minutes. The gelatin was removed and non-senescent vascular endothelial cells were plated at 2-3 × 10 ＾5 Cells / well were seeded in basic medium (IMDM + 15% FBS + 10 μg / ml human insulin + 5.5 μg / ml transferrin + 5 ng / ml sodium selenite + 2 mM L-glutamine + 0.45 mM α-monomercaptoglycerol + 50 μl / ml ascorbic acid + 20 ng / ml VEGF) and cultured at 37°C and 5.0% CO2. In addition, recombinant DLL4 was seeded in another well and refrigerated at 4°C. The next day, the medium was removed and iPS-derived human hematopoietic stem cells were cultured at 8.4 × 10 ＾4Cells / well were seeded in the basic medium for iPS cell-derived hematopoietic progenitor cells (IMDM+15% FBS+10μg / ml human insulin+5.5μg / ml transferrin+5ng / ml sodium selenite+2mM L-glutamine+0.45mMα-monomercaptoglycerol+50μl / ml ascorbic acid+10ng / ml human Flt3L+50ng / ml human SCF+5ng / ml IL-7+30ng / ml TPO). Doxycycline-inducible c-MYC+BMI1+BCLXL(MBX) viral vector was added, and doxycycline was added at 1μg / ml. All were cultured at 37°C and CO2 5.0%. Thereafter, the culture medium was replaced every 2 to 3 days. The cells were then stained with the cell surface markers CD45-BV421 and CD19-PC7 of B cells, and the CD45+ / CD19+ cells ( Figure 33 ).

[0425] like Figure 33 As shown, the ratio of CD45+ / CD19+ cells and the absolute number of cells were greater when co-cultured with non-senescent endothelial cells (imECs) than when co-cultured with recombinant DLL4. This suggests that non-senescent endothelial cells may also express high levels of other Notch ligands, such as Jagged 1, in addition to DLL4.

[0426] These results demonstrate that co-culturing with non-senescent vascular endothelial cells can further promote the differentiation of hematopoietic stem cells or hematopoietic progenitor cells into lymphoid lineage cells such as B cells, compared to co-culturing with recombinant DLL4. Furthermore, it was shown that co-culturing with non-senescent vascular endothelial cells can further promote the differentiation of hematopoietic stem cells or hematopoietic progenitor cells into lymphoid lineage cells such as B cells. Furthermore, it was shown that co-culturing with non-senescent vascular endothelial cells can further promote the differentiation of hematopoietic stem cells or hematopoietic progenitor cells into lymphoid progenitor cells, which are intermediates before differentiation into B cells.

[0427] Example 7-4: Co-expression of IL-3-administered non-senescent vascular endothelial cells and human hematopoietic stem cells derived from iPS cells nourish

[0428] The immortalized vascular endothelial cells used in this experiment were mature vascular endothelial cells whose proliferation was enhanced by the introduction of MBX according to Example 2 and cultured under dox-off for 7 days, and whose proliferation was stopped by irradiation.

[0429] 0.1% gelatin was added to the wells of a 6-well plate at 1 ml / well and incubated at 37°C for 30 minutes. The gelatin was removed and non-senescent vascular endothelial cells were plated at 2-3 × 10 ＾5Cells / well were seeded in basic medium (IMDM + 15% FBS + 10 μg / ml human insulin + 5.5 μg / ml transferrin + 5 ng / ml sodium selenite + 2 mM L-glutamine + 0.45 mM α-monomercaptoglycerol + 50 μl / ml ascorbic acid + 20 ng / ml VEGF) and cultured at 37°C and 5.0% CO2. The next day, the medium was removed and iPS-derived human hematopoietic stem cells were plated at 8.4 × 10 ＾4 Cells were seeded per well in a basal medium for iPS cell-derived hematopoietic progenitor cells (IMDM + 15% FBS + 10 μg / ml human insulin + 5.5 μg / ml transferrin + 5 ng / ml sodium selenite + 2 mM L-glutamine + 0.45 mM α-monomercaptoglycerol + 50 μl / ml ascorbic acid + 50 ng / ml human Flt3L + 50 ng / ml human SCF + 20 ng / ml IL-7 + 20 ng / ml IL-15). A doxycycline-inducible c-MYC + BMI1 + BCLXL (MBX) viral vector was added, followed by 1 μg / ml doxycycline, and the cells were cultured at 37°C and 5.0% CO2. IL-3 was administered at 20 ng / ml for only the first week. Thereafter, the culture medium was replaced by half every 2-3 days. Afterwards, the cells were stained with CD45-BV421, CD56-FITC, and CD19-PC7, and CD45+ / CD56+ cells and CD45+ / CD19+ cells were analyzed using FACSCanto II. Figure 34 CD45-BV421 and CD56-FITC are cell surface markers for natural killer cells, and CD45-BV421 and CD19-PC7 are cell surface markers for B cells. The compositions of the culture media used in the culture are shown below.

[0430] like Figure 34 As shown, the proportions of CD45+ / CD56+ cell populations and CD45+ / CD19+ cell populations were higher when co-cultured with non-senescent vascular endothelial cells (im EC) than when not co-cultured with non-senescent vascular endothelial cells (feeder free).

[0431] The above results indicate that co-culturing with non-senescent vascular endothelial cells can promote the differentiation of hematopoietic stem cells or hematopoietic progenitor cells into natural killer cells or B cells. Furthermore, it is shown that differentiation into lymphocyte progenitor cells, which are intermediates before differentiation into natural killer cells or B cells, can be promoted.

Claims

1. A method for regulating cell differentiation degree, comprising the following steps: MYC family genes and BMI1 gene are forcibly expressed in cells selected from the group consisting of (i) mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, and lymphocytes with any degree of differentiation, and (ii) megakaryocytes and myelocytes with a high degree of differentiation.

2. The method according to claim 1, wherein The cells are mesenchymal stem cells with any degree of differentiation.

3. The method according to claim 1, wherein The cells are mesenchymal stem cells that do not express the cell surface marker CD90.

4. The method according to claim 1, wherein The cells are mesenchymal stem cells expressing the cell surface marker CD90.

5. The method according to claim 1, wherein The cells are vascular endothelial cells with any degree of differentiation.

6. The method according to claim 1, wherein The cells are smooth muscle cells of any degree of differentiation.

7. The method according to claim 1, wherein The cells are vascular smooth muscle cells with any degree of differentiation.

8. The method according to claim 1, wherein The cells are vascular smooth muscle cells expressing cell surface markers of CD140b, KDR or CD34.

9. The method according to claim 1, wherein The cells are neural crest cells with any degree of differentiation.

10. The method according to claim 1, wherein The cells are lymphocytes of any degree of differentiation.

11. The method according to claim 1, wherein The cells are megakaryocytes expressing the cell surface markers CD41 and CD42b.

12. The method according to claim 1, wherein The cells are myelocytes expressing cell surface markers CD14 and CD11b.

13. The method according to claim 1, wherein The method further includes the step of inhibiting the expression of MYC family genes and BMI1 gene, or inhibiting the function of their expression products.

14. The method according to claim 1, wherein The method also includes a step of forcibly expressing the BCL-XL gene.

15. The method according to claim 1, wherein The method also includes the step of inhibiting the expression of the BCL-XL gene or the function of its expression product.

16. The method according to claim 1, wherein The method further includes the step of inhibiting the expression of at least one of the CDKN1A gene and the P53 gene, or inhibiting the function of the expression product thereof.

17. A method for producing precursor cells of mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes, or myelocytes, comprising the step of culturing the cells obtained by the method according to claim 1.

18. A method for producing differentiated cells of mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells, lymphocytes, megakaryocytes or myelocytes, comprising the step of differentiating the cells obtained by the method according to claim 1.

19. A cell obtained by the method according to any one of claims 1 to 18.

20. A pharmaceutical composition comprising cells obtained by the method of any one of claims 1 to 18.

21. A method for treating or preventing a disease, comprising the step of administering cells obtained by the method according to any one of claims 1 to 18 to a patient in need thereof.

22. A cell differentiation regulator for cells selected from the group consisting of (i) mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, neural crest cells and lymphocytes with any degree of differentiation, and (ii) megakaryocytes and myelocytes with a high degree of differentiation, comprising as an active ingredient a molecule that forces the expression of a MYC family gene and a BMI1 gene.

23. A method for producing mesenchymal stem cells having proliferation ability, comprising the step of forcibly expressing a MYC family gene and a BMI1 gene in mesenchymal stem cells having any degree of differentiation.

24. The manufacturing method according to claim 23, wherein: Mesenchymal stem cells with any degree of differentiation are mesenchymal stem cells that do not express the cell surface marker CD90.

25. The manufacturing method according to claim 23, wherein: Mesenchymal stem cells having any degree of differentiation are mesenchymal stem cells expressing the cell surface marker CD90.

26. The production method according to any one of claims 23 to 25, wherein Mesenchymal stem cells with proliferation capacity express the cell surface marker CD90.

27. A method for producing vascular endothelial cells having proliferation ability, comprising the step of forcibly expressing a MYC family gene and a BMI1 gene in vascular endothelial cells having any degree of differentiation.

28. A method for producing smooth muscle cells having proliferation ability, comprising the step of forcibly expressing a MYC family gene and a BMI1 gene in smooth muscle cells having any degree of differentiation.

29. The manufacturing method according to claim 28, wherein: Smooth muscle cells of any degree of differentiation are vascular smooth muscle cells.

30. The manufacturing method according to claim 29, wherein: Vascular smooth muscle cells are vascular smooth muscle cells that express cell surface markers such as CD140b, KDR, or CD34.

31. A method for producing neural crest cells with proliferation ability, comprising the step of forcibly expressing MYC family genes and BMI1 gene in neural crest cells with any degree of differentiation.

32. A method for producing lymphocytes having proliferation ability, comprising the step of forcibly expressing MYC family genes and BMI1 gene in lymphocytes having any degree of differentiation.

33. A method for producing megakaryocytes having proliferation ability, comprising the step of forcibly expressing a MYC family gene and a BMI1 gene in megakaryocytes expressing the cell surface markers CD41 and CD42b.

34. A method for producing macrophages having proliferation ability, comprising the step of forcibly expressing a MYC family gene and a BMI1 gene in macrophages expressing the cell surface markers CD14 and CD11b.

35. A method for promoting the differentiation of iPS cells, ES cells, hematopoietic stem cells or hematopoietic precursor cells into lymphocytes or lymphocyte precursor cells, comprising the step of co-culturing non-senescent vascular endothelial cells with iPS cells, ES cells, hematopoietic stem cells or hematopoietic precursor cells.

36. The method according to claim 35, wherein Lymphocytes express cell surface markers of CD45 and CD56, or cell surface markers of CD19 and CD45.

37. The method according to claim 35 or 36, wherein Lymphocytes are natural killer cells or B cells.

38. The method according to claim 35 or 36, wherein Hematopoietic stem cells or hematopoietic precursor cells are derived from umbilical cord blood or iPS cells.

39. The method according to claim 35 or 36, wherein Non-senescent vascular endothelial cells express Notch ligands.

40. The method of claim 39, wherein Notch ligands are DLL4 and / or Jagged 1.

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