Culture system, kit and method for cell reprogramming

Abstract

The present invention relates to the field of cells, and in particular to a culture system, kit and method for cell reprogramming. The culture system comprises a first culture system and a second culture system. In the present invention, by means of a chemical small molecule reprogramming method, senescence-associated epigenetic markers are erased from senescent somatic cells by means of a series of small molecule combinations, the senescent somatic cells are reversely induced into plastic intermediate-state cells akin to those in a salamander regeneration process, and such intermediate-state cells can be induced by specific chemical small molecules into more "youthful" mesenchymal stem cells, i.e., sub-pluripotent stem cells. The sub-pluripotent stem cells are characterized by a simplified and time-efficient preparation process, high cellular purity, and enhanced safety and stability; moreover, the preparation process does not require a prolonged pluripotent stem cell reprogramming phase, and thus has huge clinical application prospects.

Description

Culture system, kit and method for cell reprogramming TECHNICAL FIELD

[0001] The present application relates to the field of cells, in particular to a culture system, kit and method for reprogramming human somatic cells into sub-pluripotent stem cells. BACKGROUND

[0002] The essence of biological individual development is actually the process of establishing multiple cell lineages, in which cells face a series of fate regulation, and errors in cell fate regulation can lead to cells with abnormal state, function and type, which is an important reason for the formation of many major diseases. Establishing a method for precisely regulating cell fate is a basic approach to realize the regulation of individual development, physiology, metabolism and aging and other life activities as well as disease treatment. For a long time, cell fate regulation during development has been considered to be unidirectional and irreversible. However, after a long period of research by the laboratory of Deng Hongkui, Peking University, and other domestic and foreign laboratories, cell reprogramming technology has been established, which breaks this inherent cognition and opens a new era of stem cell and regenerative medicine research. In 2007, Japanese scientist Shinya Yamanaka reprogrammed human skin cells into induced pluripotent stem cells (iPSC or iPS) through four transcription factors OSKM, which has similar ability to embryonic stem cells. Through specific induction conditions, iPSCs can be differentiated into nearly 200 functional cells in the human body, and can be used for clinical treatment of many refractory / incurable diseases. However, since the transcription factors need to be inserted into the chromosome through a virus, it can damage the genomic stability and has a risk of tumorigenesis, which has great clinical risks.

[0003] It is well known that mesenchymal stem cells (MSCs) have attracted widespread attention from the public and academia due to their unique biological characteristics and potential therapeutic value. At present, the acquisition of MSCs can be generally divided into two categories. One is tissue-derived MSCs. Researchers have discovered and isolated mesenchymal stem cells from different human tissues, and MSCs from different tissues express tissue-specific genes. The therapeutic effects of MSCs from different tissues have obvious differences, and are also affected by many factors, including donor and tissue source, donor health status, cell heterogeneity, transplantation scheme, and reagents used for isolation, culture, cryopreservation and thawing, which greatly limit the therapeutic value of tissue-derived MSCs. The other is pluripotent stem cell-derived MSCs. The acquisition of this type of MSCs depends on pluripotent stem cell technology, and the preparation of iPSCs is affected by many factors, such as selection of somatic cells, selection and optimization of mediating factors and vectors, iPSC cell culture, iPSC screening and identification, reprogramming efficiency, preparation period, etc. Moreover, iPSCs have tumorigenicity and potential clinical application risks, which are not yet recognized by the public.

[0004] Professor Deng Hongkui's research group first used chemical small molecules to reprogram mouse fibroblasts into iPSCs. Through chemical small molecule regulation of cell fate, it provides an accurate, flexible and controllable means for cell fate regulation, and brings new possibilities for the treatment of major diseases and regenerative medicine. In 2022, Deng Hongkui's laboratory published again, claiming to use chemical small molecule reprogramming technology to reprogram human fibroblasts / adipose mesenchymal stem cells into iPSCs. International research groups have successively published papers claiming that important signaling pathways in cells can be regulated by chemical small molecules to reprogram skin cells into functional cells such as myocardial cells, neural progenitor cells, and liver cells.

[0005] Chemical reprogramming is fundamentally different from traditional reprogramming techniques: traditional transgenic reprogramming techniques such as induced pluripotent stem cell technology (iPS technology) drive direct changes in cell fate through overexpression of endogenous transcription factors, and the induction process is difficult to control; while chemical reprogramming uses exogenous chemical small molecules to simulate external signal stimulation to drive cell fate to change in a phased manner. Therefore, this method is highly controllable and is expected to achieve precise regulation of cell fate, reverse cell identity and functional state, and make reverse development possible. The method of chemical reprogramming opens up a new field for cells to go from mature to juvenile state, which is also considered a key principle of regeneration and rejuvenation. Cell reprogramming has proven that the developmental direction of cells is reversible, and the reprogramming process starts from somatic cells, which can be induced into "young" adult cells with specific functionality by chemical small molecules. During the process of reprogramming somatic cells to pluripotent state, aging-related epigenetic markers are erased, and pluripotent genome OCT4 and other genes are not activated, and the cells do not reach stem cell homeostasis and are not tumorigenic, but their unique plastic state can be used for clinical treatment of various diseases caused by cell loss, such as Parkinson's disease and diabetes, and has broad application space. In view of this, the present application provides a culture system, kit and method for reprogramming somatic cells into sub-pluripotent stem cells.

[0006] SUMMARY

[0007] The technical problem to be solved by the present application is to provide a culture system, kit and method for cell reprogramming. The purpose is to use a combination of chemical small molecules with specific components to reprogram human somatic cells into a plastic intermediate state cell similar to the regeneration process of newts, named SuperXell Stem Cell; this intermediate state cell is induced into a somatic sub-pluripotent stem cell (Aapollo Cell) by a combination of chemical small molecules, which is more young and highly uniform, stable in characteristics, and controllable in quality than mesenchymal stem cells (MSC).

[0008] The technical solution of the present application to solve the above technical problem is as follows:

[0009] In a first aspect, a culture system for cell reprogramming comprises a first culture system and a second culture system;

[0010] The first culture system comprises a GSK3β inhibitor, a TGFβR inhibitor, a RAR activator, a Smoothened receptor agonist, a SAH hydrolase inhibitor, a JAK1 / 2 inhibitor, a Menin-MLL interaction inhibitor, a C-jun N-terminal kinase inhibitor;

[0011] The second culture system comprises a TGFβR inhibitor, a RAR activator, a C-jun N-terminal kinase inhibitor, a SAH hydrolase inhibitor, a JAK1 / 2 inhibitor, a p38 MAPK inhibitor, a CREBBP / EP300 inhibitor, an AMP-activated protein kinase inhibitor, a Menin-MLL interaction inhibitor, a CK2 inhibitor.

[0012] Further, the first culture system further comprises at least one of a sirtuins inhibitor, a BMP4, a DOT1L histone methyltransferase inhibitor, an AKT Kinase Inhibitor, a histone methyltransferase SETD2 inhibitor;

[0013] The second culture system further comprises at least one of a bFGF, a Smoothened receptor agonist, a DOT1L histone methyltransferase inhibitor, an adenosine kinase inhibitor, a DNA / RNA methyltransferase inhibitor, an AKT Kinase Inhibitor.

[0014] By adding the above components, the cell growth rate and reprogramming efficiency are higher, and the preparation period is greatly shortened.

[0015] Further, a third culture system is further included, and the third culture system comprises at least one of a TGF-beeta / Smad signaling pathway activator and a ROCK inhibitor.

[0016] Further, the GSK3β inhibitor comprises at least one of LiCl, CHIR99021, Laduviglusib (CHIR-99021) HCl, SB216763, TWS119, BIO, LY2090314, CHIR-98014; the sirtuins inhibitor comprises Nicotinamide, Sirtinol, Selisistat, SIRT-IN-3, Nicotinamide-d4, 4'-Bromo-resveratrol, Nicotinamide- 15N， 13at least one of C3; the TGFpR inhibitor comprises at least one of E-616452, SB-431542, LY2109761, GW788388, SB 525334, Galunisertib; the RAR activator comprises at least one of TTNPB, AM580, Adapalene, Etretinate, Tamibarotene; the Smoothened receptor agonist comprises at least one of SAG, SAG HC1, Sonic Hedgehog, LY2940680, Purmorphamine; the DOT1L histone methyltransferase inhibitor comprises at least one of EPZ-5676, EPZ-6438, 3-deazaneplanocin A HC1, GSK126, EPZ004777, BIX-01294, GSK343, UNC1999, SGC0946; the JAK1 / 2 inhibitor comprises at least one of Ruxolitinib, AZD-1480, Fedratinib, WP1066, Tofacitinib; the Menin-MLL interaction inhibitor comprises at least one of VTP50469, MI-503, MI-463; the C-jun N-terminal kinase inhibitor comprises at least one of JNKIN8, SP600125, Tanzisertib, JNK-IN-7, JNK Inhibitor VIII; the histone methyltransferase SETD2 inhibitor comprises at least one of SETD2-IN-1 TFA, EPZ-719, EZM0414; the p38 MAPK inhibitor comprises at least one of BIRB 796, SB 202190, Adezmapimod, Ralimetinib dimesylate, VX-702; the CREBBP / EP300 inhibitor comprises at least one of SGC-CBP30, Curcumin, C646, ICG-001, A-485; the AMP-activated protein kinase inhibitor comprises at least one of Dorsormorphin, Dorsomorphin dihydrochloride, WZ4003, HTH-01-015; the adenosine kinase inhibitor comprises at least one of 5-Iodotubercidin, AK-IN-1, GP3269, ABT-702 dihydrochloride, GP3269; the DNA / RNA methyltransferase inhibitor comprises at least one of 5-Azacytidine, Decitabine, RG108, Zebularine, SGI-1027;The CK2 inhibitor includes at least one of CX-4945, LY294002, Silmitasertib sodium salt, (E / Z)-GO289; the TGF-beeta / Smad signaling pathway activator includes at least one of BMP4, Kartogenin, lsoxazole 9, L-Quebrachitol, SJ000291942; the ROCK inhibitor includes Y27632.

[0017] Further, the use amount of each component in the first culture system is as follows: 0.1-20 mM GSK3β inhibitor, 1-20 μM TGFβR inhibitor, 1-20 μM RAR activator, 0.25-10 μM Smoothened receptor agonist, 0.05-10 μM SAH hydrolase inhibitor, 0.5-20 μM JAK1 / 2 inhibitor, 0.25-10 μM Menin-MLL interaction inhibitor, 0.2-10 μM C-jun N-terminal kinase inhibitor;

[0018] The use amount of each component in the second culture system is as follows: 1-15 μM TGFβR inhibitor, 1-5 μM RAR activator, 0.2-5 μM C-jun N-terminal kinase inhibitor, 0.05-1 μM SAH hydrolase inhibitor, 0.5-20 μM JAK1 / 2 inhibitor, 1-20 μM p38 MAPK inhibitor, 1-10 μM CREBBP / EP300 inhibitor, 0.25-10 μM AMP-activated protein kinase inhibitor, 0.25-10 μM Menin-MLL interaction inhibitor, 0.5-20 μM CK2 inhibitor.

[0019] Further, the use amount of each component in the first culture system is as follows: 0.1-20 mM GSK3β inhibitor, 1-100 ng / mL BMP4, 1-20 μM TGFβR inhibitor, 1-20 μM RAR activator, 0.25-10 μM Smoothened receptor agonist, 1-30 μM DOT1L histone methyltransferase inhibitor, 0.05-10 μM SAH hydrolase inhibitor, 0.5-20 μM JAK1 / 2 inhibitor, 0.25-10 μM Menin-MLL interaction inhibitor, 0.2-10 μM AKT Kinase Inhibitor, 0.2-10 μM C-jun N-terminal kinase inhibitor, and 0.1-10 μM histone methyltransferase SETD2 inhibitor;

[0020] The amount of each component in the second culture system is as follows: 10-300 ng / ml bFGF, 1-15 μM TGFβR inhibitor, 1-5 μM RAR activator, 0.25-10 μM Smoothened receptor agonist, 0.2-5 μM C-jun N-terminal kinase inhibitor, 1-10 μM DOT1L histone methyltransferase inhibitor, 0.05-1 μM SAH hydrolase inhibitor, 0.5-20 μM JAK1 / 2 inhibitor, 1-20 μM p38 MAPK inhibitor, 1-10 μM CREBBP / EP300 inhibitor, 0.25-10 μM AMP-activated protein kinase inhibitor, 0.25-10 μM Menin-MLL interaction inhibitor, 0.25-10 μM adenosine kinase inhibitor, 1-30 μM DNA / RNA methyltransferase inhibitor, 0.2-10 μM AKT Kinase Inhibitor, and 0.5-20 μM CK2 inhibitor;

[0021] The amount of each component in the third culture system is as follows: 1-100 ng / mL BMP4 and 1-20 μΜ Y27632.

[0022] Further, the first culture system, or the second culture system, or the third culture system further comprises a basal medium, and the basal medium comprises Knockout DMEM, 2% B27 supplement, 10% KSR, 10% FBS, 1% GlutaMAX, 1% NEAA, 1% Penicillin Streptomycin, 50 mg / ml Vitamin C; wherein % is the volume percentage.

[0023] In a second aspect, a kit for cell reprogramming comprises the first culture system and the second culture system, or comprises the first culture system to the third culture system.

[0024] In a third aspect, a method for cell reprogramming comprises the following steps: using the first culture system and the second culture system to sequentially culture adult cells to obtain super-repair stem cells (SuperXell Stem Cell).

[0025] The adult cells described above include adult human fibroblasts, tissue-derived mesenchymal stem cells, and other human-derived adult cells.

[0026] Further, the method further comprises the following step: using the third culture system to culture the super-repair stem cells to obtain sub-pluripotent stem cells.

[0027] Further, the method comprises the following specific steps:

[0028] (1) forming epithelioid cells: inducing and culturing adult cells into epithelioid cells by using a first culture system;

[0029] (2) forming super-repair stem cells: inducing and culturing the epithelioid cells into super-repair stem cells by using a second culture system;

[0030] (3) forming sub-pluripotent stem cells: inducing and culturing the super-repair stem cells into sub-pluripotent stem cells by using a third culture system.

[0031] The present application has the following advantages: the present application reprograms the aged adult cells by using a series of small molecule combinations (the first culture system to the third culture system) to erase the aging-related epigenetic markers, and reversely induces the aged adult cells into plastic intermediate cells similar to the plastic intermediate cells in the newt regeneration process. The intermediate cells are more "young" mesenchymal stem cells, i.e. sub-pluripotent stem cells, which have stronger proliferation ability, differentiation potential and factor secretion ability, and are safe and non-tumorigenic after being transplanted into the body. The sub-pluripotent stem cells have the characteristics of relatively consistent genetic background, low cost, high purity, stability and safety, and the preparation process does not need to go through the long pluripotent stem cell reprogramming process, and has great clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0032] Fig. 1 is a cell morphology diagram of each stage of hADSC reprogrammed sub-pluripotent stem cells according to the embodiment 1 of the present application;

[0033] Fig. 2 is a cell morphology diagram of each stage of hADSC reprogrammed sub-pluripotent stem cells according to the embodiment 2 of the present application;

[0034] Fig. 3 is a cell morphology diagram of each stage of hASFs reprogrammed sub-pluripotent stem cells according to the embodiment 3 of the present application;

[0035] Fig. 4 is a cell morphology diagram of each stage of hASFs reprogrammed sub-pluripotent stem cells according to the embodiment 4 of the present application;

[0036] Fig. 5 is a cell morphology diagram of each stage of UCMSC reprogrammed sub-pluripotent stem cells according to the embodiment 5 of the present application;

[0037] Fig. 6 is a cell morphology diagram of each stage of UCMSC reprogrammed sub-pluripotent stem cells according to the embodiment 6 of the present application;

[0038] Fig. 7 is a flow cytometry result diagram of hADSC reprogrammed sub-pluripotent stem cells according to the embodiment 1 of the present application;

[0039] Figure 8 is a flow cytometry result of UCMSC reprogrammed sub-pluripotent stem cells of Example 3 of the present application;

[0040] Figure 9 is a flow cytometry result of UCMSC reprogrammed sub-pluripotent stem cells of Example 5 of the present application. DETAILED DESCRIPTION

[0041] The principles and features of the present application are described below, and the examples are only used to explain the present application and not to limit the scope of the present application. If a specific technique or condition is not specified in the examples, it is performed according to the technique or condition described in the literature in the art or according to the product manual. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be commercially available through a regular channel.

[0042] The method of the present application is performed under conditions suitable for producing induced mesenchymal stem cells, including, for example, the composition and concentration of the culture medium, the culture temperature, the culture time, and other conditions. Based on the sufficient teachings of the prior art and in combination with the exemplary description of the present application, those skilled in the art can easily determine the above-mentioned culture conditions without undue experimentation. The key is to select the desired inhibited or activated cell signaling pathways and determine the order of the acting cell signaling pathways. In addition, the concentration of small molecule compounds or combinations thereof and other conditions can also be adaptively adjusted based on the scope provided by the present application.

[0043] And since there is still room for improvement for those skilled in the art in the regulation of small molecules and proteins involved in the above-mentioned same target or signaling pathway, there is a possibility that the small molecules provided by the present patent do not completely cover the range of small molecules for the corresponding pathway.

[0044] The sources of materials and reagents used in the following examples are shown in Table 1:

[0045] The present example provides a culture system for cell reprogramming, which includes a first culture system and a second culture system;

[0046] The first culture system includes a GSK3β inhibitor, a TGFβR inhibitor, a RAR activator, a Smoothened receptor agonist, a SAH hydrolase inhibitor, a JAK1 / 2 inhibitor, a Menin-MLL interaction inhibitor, and a C-jun N-terminal kinase inhibitor.

[0047] The second culture system includes a TGFβR inhibitor, a RAR activator, a C-jun N-terminal kinase inhibitor, a SAH hydrolase inhibitor, a JAK1 / 2 inhibitor, a p38 MAPK inhibitor, a CREBBP / EP300 inhibitor, an AMP-activated protein kinase inhibitor, a Menin-MLL interaction inhibitor, and a CK2 inhibitor.

[0048] In some embodiments, the first culture system further comprises at least one of a sirtuins inhibitor, BMP4, a DOT1L histone methyltransferase inhibitor, an AKT Kinase Inhibitor, a histone methyltransferase SETD2 inhibitor.

[0049] In some embodiments, the second culture system further comprises at least one of bFGF, a Smoothened receptor agonist, a DOT1L histone methyltransferase inhibitor, an adenosine kinase inhibitor, a DNA / RNA methyltransferase inhibitor, an AKT Kinase Inhibitor.

[0050] In some embodiments, the method further comprises a third culture system, wherein the third culture system comprises at least one of a TGF-beeta / Smad signaling pathway activator and a ROCK inhibitor.

[0051] In some embodiments, the GSK3β inhibitor comprises at least one of LiCl, CHIR99021, Laduviglusib (CHIR-99021) HC1, SB216763, TWS119, BIO, LY2090314, CHIR-98014; the sirtuins inhibitor comprises at least one of Nicotinamide, Sirtinol, Selisistat, SIRT-IN-3, Nicotinamide-d4, 4'-Bromo-resveratrol, Nicotinamide- 15 N, 13at least one of C3; the TGFpR inhibitor comprises at least one of E-616452, SB-431542, LY2109761, GW788388, SB 525334, Galunisertib; the RAR activator comprises at least one of TTNPB, AM580, Adapalene, Etretinate, Tamibarotene; the Smoothened receptor agonist comprises at least one of SAG (Smoothened Agonist), SAG HC1, Sonic Hedgehog, LY2940680, Purmorphamine; the DOT1L histone methyltransferase inhibitor comprises at least one of EP-Z5676, EPZ-6438, 3-deazaneplanocin A HC1, GSK126, EPZ004777, BIX-01294, GSK343, UNC1999, SGC0946; the JAK1 / 2 inhibitor comprises at least one of Ruxolitinib, AZD-1480, Fedratinib, WP1066, Tofacitinib; the Menin-MLL interaction inhibitor comprises at least one of VTP50469, MI-503, MI-463; the C-jun N-terminal kinase inhibitor comprises at least one of JNKIN8, SP600125, Tanzisertib, JNK-IN-7, JNK Inhibitor VIII; the histone methyltransferase SETD2 inhibitor comprises at least one of SETD2-IN-1 TFA, EPZ-719, EZM0414; the p38 MAPK inhibitor comprises at least one of BIRB 796, SB 202190, Adezmapimod, Ralimetinib dimesylate, VX-702; the CREBBP / EP300 inhibitor comprises at least one of SGC-CBP30, Curcumin, C646, ICG-001, A-485; the AMP-activated protein kinase inhibitor comprises at least one of Dorsormorphin, Dorsomorphin dihydrochloride, WZ4003, HTH-01-015; the adenosine kinase inhibitor comprises at least one of 5-Iodotubercidin, AK-IN-1, GP3269, ABT-702 dihydrochloride, GP3269;The DNA / RNA methyltransferase inhibitor includes at least one of 5-Azacytidine, Decitabine, RG108, Zebularine, SGI-1027; the CK2 inhibitor includes at least one of CX-4945, LY294002, Silmitasertib sodium salt, (E / Z)-GO289; the TGF-beeta / Smad signaling pathway activator includes at least one of BMP4, Kartogenin, lsoxazole 9, L-Quebrachitol, SJ000291942; the ROCK inhibitor includes Y27632.

[0052] Preferably, in the first culture system, the components are used in the following amounts: 0.1-20 mM GSK3β inhibitor, 1-20 μM TGFβR inhibitor, 1-20 μM RAR activator, 0.25-10 μM Smoothened receptor agonist, 0.05-10 μM SAH hydrolase inhibitor, 0.5-20 μM JAK1 / 2 inhibitor, 0.25-10 μM Menin-MLL interaction inhibitor, 0.2-10 μM C-jun N-terminal kinase inhibitor.

[0053] In the second culture system, the components are used in the following amounts: 1-15 μM TGFβR inhibitor, 1-5 μM RAR activator, 0.2-5 μM C-jun N-terminal kinase inhibitor, 0.05-1 μM SAH hydrolase inhibitor, 0.5-20 μM JAK1 / 2 inhibitor, 1-20 μM p38 MAPK inhibitor, 1-10 μM CREBBP / EP300 inhibitor, 0.25-10 μM AMP-activated protein kinase inhibitor, 0.25-10 μM Menin-MLL interaction inhibitor, 0.5-20 μM CK2 inhibitor.

[0054] Preferably, the first culture system comprises the following components: 0.1-20 mM GSK3 inhibitor, 1-100 ng / mL BMP4, 1-20 mM TGF receptor inhibitor, 1-20 mM RAR agonist, 0.25-10 mM Smoothened receptor agonist, 1-30 mM DOT1L histone methyltransferase inhibitor, 0.05-10 mM SAH hydrolase inhibitor, 0.5-20 mM JAK1 / 2 inhibitor, 0.25-10 mM Menin-MLL interaction inhibitor, 0.2-10 mM AKT Kinase Inhibitor, 0.2-10 mM C-jun N-terminal Kinase inhibitor, and 0.1-10 mM histone methyltransferase SETD2 inhibitor.

[0055] Preferably, the second culture system comprises the following components: 10-300 ng / mL bFGF, 1-15 mM TGF receptor inhibitor, 1-5 mM RAR agonist, 0.25-10 mM Smoothened receptor agonist, 0.2-5 mM C-jun N-terminal Kinase inhibitor, 1-10 mM DOT1L histone methyltransferase inhibitor, 0.05-1 mM SAH hydrolase inhibitor, 0.5-20 mM JAK1 / 2 inhibitor, 1-20 mM p38 MAPK inhibitor, 1-10 mM CREBBP / EP300 inhibitor, 0.25-10 mM AMP-activated protein kinase inhibitor, 0.25-10 mM Menin-MLL interaction inhibitor, 0.25-10 mM adenosine kinase inhibitor, 1-30 mM DNA / RNA methyltransferase inhibitor, 0.2-10 mM AKT Kinase Inhibitor, and 0.5-20 mM CK2 inhibitor.

[0056] Preferably, the third culture system comprises the following components: 1-100 ng / mL BMP4 and 1-20 mM Y27632.

[0057] Preferably, the base medium in the first culture system, or the second culture system, or the third culture system comprises: Knockout DMEM + 2% B27 supplement + 10% KSR + 10% FBS + 1% GlutaMAX + 1% NEAA + 1% Penicillin Streptomycin + 50 mg / mL Vitamin C; wherein % is the volume percentage.

[0058] The embodiment also provides a kit for cell reprogramming, comprising the first culture system and the second culture system, or comprising the first culture system to the third culture system. The adult cells mentioned above include adult skin fibroblasts, tissue-derived mesenchymal stem cells and other human-derived adult cells.

[0059] The embodiment also provides a method for cell reprogramming, comprising the following steps: sequentially culturing adult cells by using the first culture system and the second culture system to obtain super-repair stem cells.

[0060] The preferred embodiment further comprises the following step: culturing the obtained super-repair stem cells by using the third culture system to obtain sub-pluripotent stem cells.

[0061] The preferred embodiment comprises the following specific steps:

[0062] (1) Formation of epithelial-like cells: inducing and culturing adult cells into epithelial-like cells by using the first culture system;

[0063] (2) Formation of super-repair stem cells: inducing and culturing the obtained epithelial-like cells into super-repair stem cells by using the second culture system;

[0064] (3) Formation of sub-pluripotent stem cells: inducing and culturing the obtained super-repair stem cells into sub-pluripotent stem cells by using the third culture system.

[0065] In summary, the present application uses chemical small molecules to reprogram somatic cells into a state similar to the regeneration process of animals: chemical small molecules stimulate somatic cells in response to exogenous damage signals, and then dedifferentiate to produce plastic intermediate cells that mediate tissue regeneration. The intermediate cells are defined as super-repair stem cells. By using specific culture conditions, the cells at this stage are further induced into sub-pluripotent stem cells (which are superior to stable cells of mesenchymal stem cells) by chemical small molecules, providing a safer, simpler and autologous source of clinical treatment, and having a wider application space and clinical value.

[0066] The specific culture medium used in the embodiment is as follows:

[0067] 1) Culture medium: high-sugar DMEM + 15% FBS or Mesenchymal Stem Cell Growth Medium 2.

[0068] 2) First culture system:

[0069] First culture system A:

[0070] Knockout DMEM + 2% B27 supplement + 10% KSR + 10% FBS + 1% GlutaMAX + 1% NEAA + 1% Penicillin-Streptomycin + 50 mg / ml Vitamin C + 5 mM LiCl + 1 mM Nicotinamide + 20 ng / ml BMP4 + CHIR99021 (5 mM) + 616452 (10 mM) + TTNPB (2 mM) + SAG (0.5 mM) + EPZ5676 (2 mM) + DZNep (0.05 mM) + Ruxolitinib (1 mM) + VTP50469 (0.5 mM) + AKT Kinase Inhibitor (1 mM) + JNKIN8 (0.2 mM) + SETD2-IN-1 (0.2 mM).

[0071] First culture system B:

[0072] Knockout DMEM + 2% B27 supplement + 10% KSR + 10% FBS + 1% GlutaMAX + 1% NEAA + 1% Penicillin-Streptomycin + 50 mg / ml Vitamin C + 5 mM LiCl + CHIR99021 (5 mM) + 616452 (10 mM) + TTNPB (2 mM) + SAG (0.5 mM) + Ruxolitinib (1 mM) + VTP50469 (0.5 mM) + JNKIN8 (0.2 mM).

[0073] First culture system C:

[0074] Knockout DMEM + 2% B27 supplement + 10% KSR + 10% FBS + 1% GlutaMAX + 1% NEAA + 1% Penicillin-Streptomycin + 50 mg / ml Vitamin C + 5 mM LiCl + CHIR99021 (5 mM) + 616452 (10 mM) + TTNPB (2 mM) + Ruxolitinib (1 mM).

[0075] 3) Second culture system:

[0076] Second culture system A:

[0077] Knockout DMEM + 2% B27 supplement + 10% KSR + 10% FBS + 1% GlutaMAX + 1% NEAA + 1% Penicillin Streptomycin + 50 mg / ml Vitamin C + JNK IN8 (0.5 mM) + Ruxolitinib (1 mM) + BIRB796 (2 mM) + SGC-CBP30 (2 mM) + Dorsormorphin (0.5 mM) + VTP50469 (0.5 mM) + 5-Iodotubercidin (0.5 mM) + 5-Azacytidine (2 mM) + AKT Kinase Inhibitor (0.2 mM) + CX-4945 (1 mM).

[0078] Second culture system B:

[0079] Knockout DMEM + 2% B27 supplement + 10% KSR + 10% FBS + 1% GlutaMAX + 1% NEAA + 1% Penicillin Streptomycin + 50 mg / ml Vitamin C + JNK IN8 (0.5 mM) + Ruxolitinib (1 mM) + BIRB796 (2 mM) + SGC-CBP30 (2 mM) + Dorsormorphin (0.5 mM) + VTP50469 (0.5 mM) + CX-4945 (1 mM).

[0080] Third culture system: medium required for differentiation of pluripotent stem cells:

[0081] First two days: third culture system:

[0082] Knockout DMEM + 2% B27 supplement + 10% KSR + 10% FBS + 1% GlutaMAX + 1% NEAA + 1% Penicillin Streptomycin + 50 mg / ml Vitamin C + 20 ng / ml BMP4 + 10 mM Y27632.

[0083] Medium change after two days: commercial MSC serum-free medium (purchased from Becton Dickinson).

[0084] Embodiments of the present application will be described in detail below with reference to specific examples.

[0085] Example 1: Induction culture of hADSCs (adipose-derived mesenchymal stem cells)

[0086] 1. Obtaining of hADSCs

[0087] 1.1 Isolation and obtaining of hADSCs (adipose-derived mesenchymal stem cells)

[0088] The isolation and obtaining of hADSCs comprises the following steps:

[0089] (1) The obtained 2-4 cm 3 tissue is washed with PBS containing 2% penicillin-streptomycin for 2-3 times;

[0090] (2) The tissue is placed in a 100 mm culture dish and cut into 1-2 mm 3 pieces with scissors, and then 5-10 ml of 2 mg / ml collagenase IV is added and placed in a 37 °C incubator for 1 h;

[0091] (3) After 1 h of digestion and dissociation, the culture dish is taken out, 10-20 ml of DMEM medium containing 15% FBS is added, and then the suspension is blown several times with a pipette;

[0092] (4) The suspension is collected into a 50 ml centrifuge tube containing 30 ml (the suspension can be divided into 2-3 centrifuge tubes), and the cells are released by blowing or gently shaking for 1-2 min;

[0093] (5) The suspension is placed in a centrifuge at 400 g for 5 min, and after centrifugation, the centrifuge tube is removed to remove the supernatant, and then the medium (Mesenchymal Stem Cell Growth Medium 2) is added to resuspend the precipitate;

[0094] (6) The suspension is inoculated into a 100 mm culture dish and placed in a 37 °C carbon dioxide (5%) incubator for 18-24 h;

[0095] (7) The cells are taken out of the incubator to remove the supernatant, and fresh medium (Mesenchymal Stem Cell Growth Medium 2) is added;

[0096] (8) After 3-5 days of cell culture, the confluence reaches 85%-90%, and the subsequent experiment is prepared.

[0097] 1.2 Culture of purchased hADSCs cells

[0098] The culture of purchased hADSCs cells comprises the following steps:

[0099] (1) The cells are cultured in Mesenchymal Stem Cell Growth Medium 2 and placed in a 37 °C carbon dioxide (5%) incubator, and the medium is changed every 2-3 days;

[0100] (2) When the cell fusion reaches 85%~90%, prepare for the following experiment.

[0101] 2. Reprogramming of hADSCs to sub-pluripotent stem cells

[0102] Reprogramming of hADSCs to sub-pluripotent stem cells includes the following steps:

[0103] (1) Digestion and inoculation of ADSCs cells

[0104] a) Take the hADSCs cells out of the incubator, and observe the cells under a microscope. As shown in FIG. 1, the cell fusion degree is 85%~90%, and the cells are digested;

[0105] b) Place the cells in a clean bench, discard the supernatant, wash the cells with PBS, discard the PBS, add 0.25% trypsin, and place the cells in a 37° incubator (5% CO2) for 3 minutes. Then, add DMEM high-sugar medium containing 15% FBS to stop the digestion;

[0106] c) Collect the cell suspension into a 15ml centrifuge tube, and transfer it to a centrifuge for 3 minutes at 300g;

[0107] d) Discard the supernatant, resuspend the cells with 5ml DMEM high-sugar medium containing 15% FBS, and aspirate a small amount of cells for AO / PI counting;

[0108] e) Inoculate the cells according to 1x10^4 cells per well of a 12-well plate, and use DMEM high-sugar medium containing 15% FBS as the culture medium. After 24h of culture, replace the first culture system.

[0109] (2) Reprogramming of hADSCs to epithelial-like cells

[0110] a) Take the cells cultured for 24h out of the incubator, and observe the cells under a microscope (take a photo for record), as shown in FIG. 1:

[0111] b) Place the cells in a clean bench, discard the supernatant (DMEM medium containing 15% FBS), add 1ml of the first culture system A to each well of a 12-well plate, and then place the cells in a low-oxygen (5% O 2， 5% CO2) 37° incubator. Replace the fresh culture medium every 3~4 days;

[0112] c) The epithelial-like cells can be observed generally 4~6 days after induction. Continue to induce until the cell fusion degree reaches about 100% (hADSCs generally 8~10 days), and replace the second culture system A;

[0113] (3) Reprogramming of plasticity intermediate cells

[0114] a) The cells with a fusion degree of about 100% are placed in a clean bench, the supernatant is discarded, 1 ml of the second culture system A is added to the hole of the 12-hole plate, and then the cells are placed in a 37 °C incubator (5% CO2). The cells are replaced with fresh culture medium every 3-4 days;

[0115] b) The multi-layer cell clones generally appear in the second culture system A for 4-6 days. After 8-10 days of continuous induction, the cells are digested and enter the plasticity intermediate state cell differentiation.

[0116] (4) Reprogramming of sub-pluripotent stem cells

[0117] The plasticity intermediate state cells are taken out of the incubator, the cell morphology is observed under a microscope and photographed, as shown in Figure 1, then the cells are placed in a clean bench, the supernatant is aspirated, washed once with PBS, then 300 μL of Accutase digestion enzyme is added, and the cells are digested in a 37 °C incubator for 3 min. Then the digestion is stopped by adding Knockout DMEM medium, the cells are collected into a 15 mL centrifuge tube, centrifuged in a centrifuge at 300 g for 3 min, the supernatant is discarded, and the cell pellet is resuspended with 2 mL of the third culture system. A small amount of cells is aspirated for AO / PI counting: 2 x 10 5 The cells are inoculated, then the cells are placed in the incubator for two days, then the supernatant is aspirated, and a commercial MSC culture medium (purchased from Zhuhai Baisuo Cell Science and Technology Co., Ltd.) is added. The cells are cultured to a density of about 80%, then digested, passaged, and detected.

[0118] 3. Detection

[0119] Detection results: The obtained sub-pluripotent stem cells are subjected to cell morphology and surface marker (antibodies used: APC-CD105, FITC-CD90, PE-CD73, APC-CD79a, FITC-CD45, PE-CD34, FITC-CD14) flow detection. The results are shown in Figures 1 and 7. As shown in Figure 1, the cell morphology is observed at each stage of reprogramming. Under the action of small molecules, the cell morphology gradually changes. As shown in Figure 7, flow detection identifies that the positive rate of CD105, CD73 and CD90 is ≥ 90%, and CD45, CD34, CD14, CD79a and HLA-DR are negative, and the positive rate is ≤ 5%.

[0120] Example 2: Induction culture of hADSCs

[0121] Compared with Example 1, except that in step (2) the hADSCs cells are reprogrammed to epithelioid cells, the first culture system B is used and the plasticity intermediate state cells are reprogrammed using the second culture system B, the rest are the same.

[0122] Detection results: the obtained sub-pluripotent stem cells were subjected to cell morphology detection, and the results are shown in Figure 2. As shown in Figure 2, the morphology of the cells at each stage of reprogramming was observed, and the morphology of the cells gradually changed under the action of the chemical small molecules. Surface markers (antibodies used: APC-CD105, FITC-CD90, PE-CD73, APC-CD79a, FITC-CD45, PE-CD34, FITC-CD14) were detected by flow cytometry. The flow cytometry results showed that the positive rates of CD105, CD73 and CD90 were ≥90%, and CD45, CD34, CD14, CD79a and HLA-DR were negative, with a positive rate of ≤5%.

[0123] Example 3: Reprogramming of hASFs (adult skin dermal fibroblasts)

[0124] 1. Obtaining hASFs

[0125] 1.1 Isolation and obtaining of hASFs

[0126] The isolation and obtaining of hASFs include the following steps:

[0127] (1) The 0.5-1 cm tissue was washed with PBS containing 2% penicillin-streptomycin for 2-3 times; 2

[0128] (2) The tissue block was cut into small pieces of 0.5-1 mm 2

[0129] (3) The tissue block was placed in a 100 mm cell culture dish, and 1 drop of DMEM medium containing 15% FBS was added to each piece of tissue;

[0130] (4) The culture dish was incubated in a 37°C carbon dioxide (5%) incubator for 4-12 hours (to prevent the tissue block from drying out);

[0131] (5) Then, 3-5 ml of c was gently added to the culture dish (to ensure that the tissue block does not detach from the culture dish);

[0132] (6) The culture medium (Mesenchymal Stem Cell Growth Medium 2) was replaced every 2-3 days;

[0133] (7) Generally, the fibroblasts in the tissue began to grow within 4-7 days;

[0134] (8) The cell confluence reached 85%-90% within 10-14 days, and the subsequent experiments were ready.

[0135] 1.2 Culture of purchased hASFs cells ​​

[0136] Culture of purchased hASFs cells, including the following steps:

[0137] (1) The cells were cultured in Mesenchymal Stem Cell Growth Medium 2, placed in a 37 ° carbon dioxide (5%) incubator, and replaced every 2-3 days;

[0138] (2) When the cells were fused to 85%-90%, the subsequent experiments were prepared.

[0139] 4. Reprogramming of hASFs cells to sub-pluripotent stem cells

[0140] Reprogramming of hASFs cells to sub-pluripotent stem cells, including the following steps:

[0141] (1) Digestion and inoculation of hASFs cells

[0142] a. Take the hASFs cells out of the incubator, observe the cells under a microscope, as shown in Figure 3, the cell fusion degree is 85%-90%, and the cells are digested;

[0143] b. Place the cells in a clean bench, discard the supernatant, wash the cells with PBS, discard the PBS, add 0.25% trypsin, and place the cells in a 37 ° incubator (5% CO2) for 3 minutes, then add DMEM high glucose medium containing 15% FBS to stop digestion;

[0144] c. Collect the cell suspension into a 15 ml centrifuge tube and transfer it to a centrifuge, 300g, 3 minutes;

[0145] d. Discard the supernatant, add 5 ml of DMEM high glucose medium containing 15% FBS to resuspend the cells, and aspirate a small amount of cells for AO / PI counting;

[0146] e. According to 1x10 4 Cell inoculation, using medium: DMEM high glucose medium containing 15% FBS, replacing the first culture system A after 24h culture.

[0147] (2) Reprogramming of hASFs cells to epithelial-like cells

[0148] a. Take the cells cultured for 24h out of the incubator, observe the cells under a microscope (take pictures), as shown in Figure 3;

[0149] b. Place the cells in a clean bench, discard the supernatant (15% FBS DMEM medium), add 1 ml of the first culture system A to the wells of a 12-well plate, then place the cells in a low oxygen (5% O 2，5% CO2) 37°C incubator, cells were replaced with fresh medium every 3-4 days;

[0150] c. Epithelial-like cells were observed generally in 9-16 days of induction, and the second culture system A was replaced when the cells reached 100% confluence.

[0151] (3) Reprogramming of plasticity intermediate cells

[0152] a. Cells reaching 100% confluence were placed in a clean bench, the supernatant was discarded, 1 ml of the second culture system A was added to each well of a 12-well plate, and then the cells were placed in a 37°C incubator (5% CO2), and the cells were replaced with fresh medium every 3-4 days;

[0153] b. Multilayer cell clones were generally observed in 4-6 days of induction in the second culture system A, and after 8-10 days of continuous induction, the cells were digested and induced to differentiate into plasticity intermediate cells.

[0154] (4) Reprogramming of sub-pluripotent stem cells

[0155] The induced plasticity intermediate cells were taken out of the incubator, the cell morphology was observed under a microscope and photographed, as shown in Figure 3, then the cells were placed in a clean bench, the supernatant was aspirated, washed once with PBS, then 300 μL of Accutase enzyme was added, digested for 3 min in a 37°C incubator, then the digestion was stopped by adding Knockout DMEM medium, the cells were collected into a 15 mL centrifuge tube, centrifuged at 300 g for 3 min, the supernatant was discarded, and the cell pellet was resuspended with 2 mL of the third culture system, a small amount of cells was aspirated for AO / PI counting: 2 x 10 5 The cells were inoculated, then the cells were placed in the incubator for two days, then the supernatant was aspirated, and a commercial MSC medium (purchased from Zhuhai Baisuo Cell Science and Technology Co., Ltd.) was added, and the cells were cultured to a density of about 80%, then the cells were digested and passaged, and detected.

[0156] 3. Detection

[0157] Detection results: the obtained sub-pluripotent stem cells were subjected to cell morphology detection, surface marker (antibodies used: APC-CD105, FITC-CD90, PE-CD73, APC-CD79a, FITC-CD45, PE-CD34, FITC-CD14) flow detection. The results are shown in Figures 3 and 8. As shown in Figure 3, the cell morphology at each stage of reprogramming was observed, and under the action of chemical small molecules, the cell morphology gradually changed. As shown in Figure 8, the flow detection results of mesenchymal cell surface markers: negative expression of CD14, CD45, CD79a, CD34 and HLA-DR were all lower than 1%, and positive expression of CD90, CD73 and CD105 was higher than 90%.

[0158] Example 4: hASFs reprogramming

[0159] Compared with Example 3, except that in step (2) hADSCs cells were reprogrammed to epithelial-like cells, the first culture system B was used and step (3) reprogrammed plasticity intermediate cells, the second culture system B was used, the rest were the same.

[0160] Detection results: the obtained sub-pluripotent stem cells were subjected to cell morphology detection, surface marker (antibodies used: APC-CD105, FITC-CD90, PE-CD73, APC-CD79a, FITC-CD45, PE-CD34, FITC-CD14) flow detection. The results are shown in Figures 3 and 8. As shown in Figure 3, the cell morphology at each stage of reprogramming was observed, and under the action of chemical small molecules, the cell morphology gradually changed. As shown in Figure 8, the flow detection results of mesenchymal cell surface markers: negative expression of CD14, CD45, CD79a, CD34 and HLA-DR were all lower than 1%, and positive expression of CD90, CD73 and CD105 was higher than 90%.

[0161] Example 5: UCMSC reprogramming to sub-pluripotent stem cells

[0162] 1. Culture of purchased UCMSCs cells

[0163] Culture of purchased UCMSCs cells includes the following steps:

[0164] (1). The cells were cultured in Mesenchymal Stem Cell Growth Medium 2 and placed in a 37°C carbon dioxide (5%) incubator, and replaced every 2-3 days;

[0165] (2) When the cells were fused to 85%-90%, the subsequent experiments were prepared.

[0166] 2. UCMSC cell reprogramming to sub-pluripotent stem cells

[0167] UCMSC cell reprogramming to sub-pluripotent stem cells, including the following steps:

[0168] (1) UCMSC cell digestion inoculation

[0169] a. Take the UCMSC cells out of the incubator, and observe the cells under a microscope. As shown in FIG. 5, the cell confluence is 85% to 90%. Digest the cells;

[0170] b. Place the cells in a clean bench, discard the supernatant, wash the cells with PBS, discard the PBS, add 0.25% trypsin, and place the cells in a 37°C incubator (5% CO2) for 3 minutes. Then, add DMEM high glucose medium containing 15% FBS to stop the digestion;

[0171] c. Collect the cell suspension into a 15ml centrifuge tube and transfer it to a centrifuge at 300g for 3 minutes;

[0172] d. Discard the supernatant, resuspend the cells with 5ml DMEM high glucose medium containing 15% FBS, and aspirate a small amount of cells for AO / PI counting;

[0173] e. Seed the cells at 1x10^4 cells per well of a 12-well plate, and use DMEM high glucose medium containing 15% FBS as the culture medium. After 24 hours of culture, replace the first culture system A.

[0174] (2) UCMSC cell induction to epithelial-like cells

[0175] a. Take the cells cultured for 24 hours out of the incubator, and observe the cells under a microscope (take a photo for record), as shown in FIG. 5:

[0176] b. Place the cells in a clean bench, discard the supernatant (DMEM medium containing 15% FBS), add 1ml of the first culture system A to each well of a 12-well plate, and then place the cells in a low-oxygen (5% O 2， 5% CO2) 37°C incubator. Replace the fresh culture medium every 3 to 4 days;

[0177] c. Epithelial-like cells can be observed generally 4 to 6 days after induction. Continue to induce until the cell confluence reaches about 100% (hADSCs generally 8 to 10 days), and replace the second culture system;

[0178] (3) Reprogramming plasticity intermediate cells

[0179] a. Place the cells with a confluence of about 100% in a clean bench, discard the supernatant, add 1ml of the second culture system to each well of a 12-well plate, and then place the cells in a 37°C incubator (5% CO2). Replace the fresh culture medium every 3 to 4 days;

[0180] b. Multilayer cell clones generally appear in the second culture system for 4-6 days, continue to induce for 8-10 days, then digest the cells, and enter the plasticity intermediate cell differentiation.

[0181] (4) Reprogramming of sub-pluripotent stem cells

[0182] Take the plasticity intermediate cells out of the incubator, observe the cell morphology under a microscope and take pictures, as shown in Figure 5, then place the cells in a clean bench, aspirate the supernatant, wash once with PBS, then add 300 μL of Accutase enzyme, digest for 3 min in a 37°C incubator, then add Knockout DMEM medium to stop digestion, collect the cells into a 15 mL centrifuge tube, centrifuge in a centrifuge at 300g for 3 min, discard the supernatant, resuspend the cell pellet with 2 mL of the third culture system, aspirate a small amount of cells for AO / PI counting: 2x10 5 Inoculate the cells, then place the cells in the incubator for two days, then aspirate the supernatant, add commercial MSC culture medium (purchased from Zhuhai Baisuo Cell Science and Technology Co., Ltd.), and culture the cells to a density of about 80%, then digest and passage and detect.

[0183] 3. Detection

[0184] The results of the detection: the obtained sub-pluripotent stem cells were subjected to cell morphology and surface marker (antibodies used: APC-CD105, FITC-CD90, PE-CD73, APC-CD79a, FITC-CD45, PE-CD34, FITC-CD14) flow detection. The results are shown in Figures 5 and 9. As shown in Figure 5, the cell morphology was observed at each stage of reprogramming, and under the action of small molecules, the cell morphology gradually changed; as shown in Figure 9, the cell surface markers of the reprogrammed CiMSCs were identified as: the positive rate of CD105, CD73 and CD90 was ≥90%; while CD45, CD34, CD14, CD79a and HLA-DR were negative, and the positive rate was ≤5%.

[0185] Example 6: Reprogramming of UCMSCs to sub-pluripotent stem cells

[0186] Compared with Example 5, except that in step (2) hADSCs cells are reprogrammed to epithelioid cells, the first culture system B and step (3) reprogramming of plasticity intermediate cells are used, and the second culture system B is different, the rest are the same.

[0187] The results of the detection: the obtained sub-pluripotent stem cells were detected for cell morphology, and the results are shown in Figure 6. As shown in Figure 6, the cell morphology of each stage of reprogramming was observed, and under the action of the chemical small molecules, the cell morphology gradually changed. Surface markers (antibodies used: APC-CD105, FITC-CD90, PE-CD73, APC-CD79a, FITC-CD45, PE-CD34, FITC-CD14) were detected by flow cytometry. The flow cytometry results showed that the positive rates of CD105, CD73 and CD90 were ≥90%, and the positive rates of CD45, CD34, CD14, CD79a and HLA-DR were ≤5%.

[0188] Comparative Example 1

[0189] Comparative Example 1 is the same as Example 1, except that in step (2), the hADSCs cells were reprogrammed to epithelial-like cells using the first culture system C.

[0190] The detection results: sub-pluripotent stem cells could not be obtained.

[0191] Comparative Example 2

[0192] Comparative Example 2 is the same as Example 3, except that in step (2), the hADSCs cells were reprogrammed to epithelial-like cells using the first culture system C.

[0193] The detection results: sub-pluripotent stem cells could not be obtained.

[0194] Comparative Example 3

[0195] Comparative Example 3 is the same as Example 5, except that in step (2), the hADSCs cells were reprogrammed to epithelial-like cells using the first culture system C.

[0196] The detection results: sub-pluripotent stem cells could not be obtained.

[0197] Although the embodiments of the present application have been shown and described above, it should be understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A medium for cell reprogramming, characterized by, It comprises a first culture system, a second culture system and a third culture system; The first culture system consists of the following components in the indicated amounts: KnockOut DMEM, 2% B27-supplement, 10% KSR, 10% FBS, 1% GlutaMAX, 1% NEAA, 1% Penicillin-Streptomycin, 50 mg / ml Vitamin C, 5 mM LiCl, 5 mM CHIR99021, 10 mM 616452, 2 mM TTNPB, 0.5 mM SAG, 1 mM Ruxolitinib, 0.5 mM VTP50469, 0.2 mM JNKIN8; The second culture system consists of the following components in the indicated amounts: KnockOut DMEM, 2% B27-supplement, 10% KSR, 10% FBS, 1% GlutaMAX, 1% NEAA, 1% Penicillin-Streptomycin, 50 mg / ml Vitamin C, 0.5 mM JNKIN8, 1 mM Ruxolitinib, 2 mM BIRB796, 2 mM SGC-CBP30, 0.5 mM Dorsormorphin, 0.5 mM VTP50469, 1 mM CX-4945; The third culture system consists of the following components in the indicated amounts: KnockOut DMEM, 2% B27-supplement, 10% KSR, 10% FBS, 1% GlutaMAX, 1% NEAA, 1% Penicillin-Streptomycin, 50 mg / ml Vitamin C, 20 ng / ml BMP4, 10 mM Y27632.

2. A medium for cell reprogramming, characterized in that, It comprises a first culture system, a second culture system and a third culture system; The first culture system consists of the following components in the indicated amounts: KnockOut DMEM, 2% B27-supplement, 10% KSR, 10% FBS, 1% GlutaMAX, 1% NEAA, 1% Penicillin-Streptomycin, 50 mg / ml Vitamin C, 5 mM LiCl, 1 mM Nicotinamide, 20 ng / mL BMP4, 5 mM CHIR99021, 10 mM 616452, 2 mM TTNPB, 0.5 mM SAG, 2 mM EPZ5676, 0.05 mM DZNep, 1 mM Ruxolitinib, 0.5 mM VTP50469, 1 mM AKT Kinase Inhibitor, 0.2 mM JNKIN8, 0.2 mM SETD2-IN-1; The second culture system consists of the following components in the indicated amounts: KnockOut DMEM, 2% B27-supplement, 10% KSR, 10% FBS, 1% GlutaMAX, 1% NEAA, 1% Penicillin-Streptomycin, 50 mg / ml Vitamin C, 0.5 μΜ JNKIN8, 2 μΜ EPZ5676, 0.2 μΜ DZNep, 1 μΜ Ruxolitinib, 2 μΜ BIRB796, 2 μΜ SGC-CBP30, 0.5 μΜ Dorsormorphin, 0.5 μΜ VTP50469, 0.5 μΜ 5-Iodotubercidin, 2 μΜ 5-Azacytidine, 0.2 μΜ AKT Kinase Inhibitor, 1 μΜ CX-4945; The third culture system consists of the following components in the indicated amounts: KnockOut DMEM, 2% B27-supplement, 10% KSR, 10% FBS, 1% GlutaMAX, 1% NEAA, 1% Penicillin-Streptomycin, 50 mg / ml Vitamin C, 20 ng / ml BMP4, 10 μΜ Y27632. A culture medium for cell reprogramming comprising one or more of the following components: KnockOut DMEM, 2% B27-supplement, 10% KSR, 10% FBS, 1% GlutaMAX, 1% NEAA, 1% Penicillin-Streptomycin, 50 mg / ml Vitamin C, 20 ng / ml BMP4, 10 μΜ Y27632.

3. A kit for cell reprogramming, characterized in that, A method for obtaining mesenchymal stem cells comprising the step of culturing adult cells in a culture medium for cell reprogramming comprising one or more of the following components: KnockOut DMEM, 2% B27-supplement, 10% KSR, 10% FBS, 1% GlutaMAX, 1% NEAA, 1% Penicillin-Streptomycin, 50 mg / ml Vitamin C, 20 ng / ml BMP4, 10 μΜ Y27632.

4. A method for cell reprogramming, characterized in that, ​

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