CN118048296B - Culture system, kit and method for reprogramming cells - Google Patents
Culture system, kit and method for reprogramming cells Download PDFInfo
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- CN118048296B CN118048296B CN202410451919.6A CN202410451919A CN118048296B CN 118048296 B CN118048296 B CN 118048296B CN 202410451919 A CN202410451919 A CN 202410451919A CN 118048296 B CN118048296 B CN 118048296B
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Abstract
The invention relates to a culture system, a kit and a method for reprogramming cells, and relates to the field of cells, which comprises a first culture system and a second culture system. According to the invention, by adopting a chemical micromolecule reprogramming method, the aged adult cells are subjected to aging-related epigenetic mark erasure through a series of micromolecule combinations, the aged adult cells are reversely induced into the plastic intermediate-state cells similar to the salamander in the regeneration process, and the intermediate-state cells can be induced into more 'young' mesenchymal stem cells, namely the sub-pluripotent stem cells through specific chemical micromolecules.
Description
Technical Field
The invention relates to the field of cells, in particular to a culture system, a kit and a method for reprogramming human adult cells into pluripotent stem cells.
Background
The essence of biological ontogenesis is in fact the process of establishment of multiple cell lineages, in which cells are subjected to a series of fate regulation, the error of which can lead to the production of cells with abnormal states, functions, types, which are also important causes of the formation of numerous serious diseases. The method for establishing the accurate regulation and control of the cell fate is a basic way for realizing the regulation and control of vital activities such as individual development, physiology, metabolism, aging and the like and the treatment of diseases. Cell fate regulation during development has long been considered unidirectional and irreversible, however, long-time researches in the laboratory of the Beijing university Deng Hongkui teacher and other laboratories in China and abroad have found that a cell reprogramming technology is established, the inherent cognition is broken, and a new era of research on stem cells and regenerative medicine is opened. In 2007, the mountain of Japanese scientist is extended to reprogram human skin cells into induced pluripotent stem cells (iPSC or iPS) through four transcription factors OSKM, and the induced pluripotent stem cells are similar to the capacity of embryonic stem cells, and can differentiate the iPSC into near 200 functional cells of human body through specific induction conditions, so that the human skin cells can be used for clinically treating various refractory/non-refractory diseases. However, because transcription factors need to be inserted into chromosomes through virus mediation, genome stability can be destroyed, the risk of tumorigenesis is caused, and the clinical hidden trouble is great.
It is well known that Mesenchymal Stem Cells (MSCs) have attracted public and academic attention due to their unique biological properties, as well as potential therapeutic value. At present, the obtaining modes of MSCs are mainly divided into two major categories, one category is MSCs from tissue, researchers have found and separated mesenchymal stem cells from different human tissues, and MSCs of different tissues express tissue-specific genes. The therapeutic effects of mesenchymal stem cells of different tissue sources have obvious variability and are also affected by a variety of factors, including donor and tissue sources, health status of the donor, cell heterogeneity, transplantation protocol and reagents for isolation, culture, cryopreservation and thawing, so that the therapeutic value of MSCs of tissue sources is greatly limited. The other type is pluripotent stem cell derived MSCs, the acquisition of which depends on pluripotent stem cell technology, while the preparation of iPSC is affected by various factors, such as somatic cell selection, mediating factors and mediating vector selection and optimization, iPSC cell culture, iPSC screening and identification, reprogramming efficiency, preparation cycle and the like, and iPSC has tumorigenic effect, has potential clinical application risk and is not accepted by the masses.
The proposal group of the north large Deng Hongkui teaches that the fibroblast of the mouse is reprogrammed to the iPSC by utilizing the chemical small molecule combination for the first time, and the cell fate is regulated and controlled by the chemical small molecule, so that an accurate, flexible and controllable means is provided for regulating and controlling the cell fate, and a new possibility is brought for treating serious diseases and regenerative medicine. In 2022, deng Hongkui laboratory re-transmits the report that human fibroblasts/adipose mesenchymal stem cells are reprogrammed to ipscs by using a chemical small molecule reprogramming technology, and several international subject groups published papers in succession, namely that skin cells can be reprogrammed to human functional cells such as myocardial cells, neural progenitor cells, liver cells and the like by regulating important signal pathways of cells through chemical small molecules.
Chemical reprogramming is essentially different from traditional reprogramming techniques: traditional transgenic reprogramming technologies such as induced pluripotent stem cell technology (iPS technology) drive direct transformation of cell fate through over-expression of endogenous transcription factors of cells, and the induction process is difficult to control; and chemical reprogramming is to simulate the stimulation of external signals by using an exogenous chemical small molecule to drive the cell fate to change in a staged manner. Therefore, the method has strong controllability, is hopeful to realize accurate regulation and control of cell fate, reversion of cell identity and functional state, and makes reverse development possible. The method of chemical reprogramming opens up a completely new area for cells from a mature state to a naive state, which is also considered a key principle for regeneration and rejuvenation. Cell reprogramming has demonstrated that the direction of development of cells is reversible, the reprogramming process starts from somatic cells, and the cells can be induced into 'young' somatic cells with specific functions through chemical small molecules, during the reprogramming process of somatic cells to a pluripotent state, the epigenetic markers related to aging can be erased, meanwhile, the cells do not activate the pluripotent genome OCT4 and the like, do not reach the steady state of stem cells, do not have tumorigenicity, but the unique plastic state can be used for clinically treating various diseases caused by cell loss, such as parkinson and diabetes, and has wide application space. In view of this, the present invention provides a culture system, kit and method for reprogramming of adult cells into pluripotent stem cells.
Disclosure of Invention
The invention aims to provide a culture system, a kit and a method for reprogramming cells. The aim is to reprogram human adult cells into a plastic intermediate cell similar to the salamander regeneration process by using chemical small molecule combinations with determined components, and the cell is named as super-reparative stem cell (SuperXell Stem Cell); the intermediate state cells are induced into adult sub-pluripotent stem cells (AapolloCells) through chemical small molecule combination conditions, which are younger than Mesenchymal Stem Cells (MSC), are highly uniform, have stable characteristics and are controllable in quality.
The technical scheme for solving the technical problems is as follows:
In a first aspect, a culture system for reprogramming cells includes a first culture system and a second culture system;
The first culture system comprises a GSK3 beta inhibitor, a TGF beta R 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;
The second culture system comprises a TGF-beta R 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.
Further, the first culture system further comprises at least one of sirtuins Inhibitor, BMP4, DOT1L histone methyltransferase Inhibitor, AKT KINASE Inhibitor and histone methyltransferase SETD2 Inhibitor;
the second culture system further comprises at least one of bFGF, smoothened receptor agonist, DOT1L histone methyltransferase Inhibitor, adenosine kinase Inhibitor, DNA/RNA methyltransferase Inhibitor, AKT KINASE Inhibitor.
By adding the components, the cell growth speed and reprogramming efficiency are higher, and the preparation period is greatly shortened.
Further, a third culture system is also included, the third culture system including at least one of a TGF-beeta/Smad signaling pathway activator and a ROCK inhibitor.
Further, the gsk3β inhibitor comprises at least one of LiCl, CHIR99021, laduviglusib (CHIR-99021) HCl, SB216763, TWS119, BIO, LY2090314, CHIR-98014; the sirtuins inhibitors include at least one of Nicotinamide、Sirtinol、Selisistat、SIRT-IN-3、Nicotinamide-d4、4'-Bromo-resveratrol、Nicotinamide-15N,13C3; the TGF-beta R inhibitor comprises at least one of E-616452, SB-431542, LY2109761, GW788388, SB525334, 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 HCl, sonic Hedgehog, LY2940680 and Purmorphamine; the DOT1L histone methyltransferase inhibitor comprises at least one of EPZ-5676, EPZ-6438, 3-deazaneplanocin A HCl, GSK126, EPZ004777, BIX-0194, GSK343, UNC1999 and SGC 0946; The JAK1/2 inhibitor comprises at least one of Ruxolitinib, AZD-1480, fedratinib, WP1066 and 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 JNKIN, SP600125, tanzisertib, JNK-IN-7, JNK Inhibitor VIII; the histone methyltransferase SETD2 inhibitor comprises at least one of SETD2-IN-1TFA, EPZ-719 and EZM 0414; The p38 MAPK inhibitor comprises at least one of BIRB796, SB202190, adezmapimod, ralimetinib dimesylate and VX-702; the CREBBP/EP300 inhibitor comprises at least one of SGC-CBP30, curcumin, C646, ICG-001 and 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 and GP 3269; the DNA/RNA methyltransferase inhibitor comprises at least one of 5-Azacytidine, decitabine, RG108, zebularine, SGI-1027; the CK2 inhibitor comprises at least one of CX-4945, LY294002, silmitasertib sodium salt, (E/Z) -GO 289; The TGF-beeta/Smad signaling pathway activator comprises at least one of BMP4, kartogenin, lsoxazole 9, L-Quebrachitol, SJ 000291942; the ROCK inhibitor includes Y27632.
Further, the amounts of the components in the first culture system are as follows: 0.1-20 mM G beta inhibitor, 1-20 mu M TGF beta R inhibitor, 1-20 mu M RAR activator, 0.25-10 mu M Smoothened receptor agonist, 0.05-10 mu M SAH hydrolase inhibitor, 0.5-20 mu M JAK1/2 inhibitor, 0.25-10 mu M Menin-MLL interaction inhibitor, 0.2-10 mu M C-jun N-terminal kinase inhibitor;
The second culture system comprises the following components in percentage by weight: 1-15 mu M TGF beta R inhibitor, 1-5 mu M RAR activator, 0.2-5 mu M C-jun N-terminal kinase inhibitor, 0.05-1 mu M SAH hydrase inhibitor, 0.5-20 mu M JAK1/2 inhibitor, 1-20 mu M p MAPK inhibitor, 1-10 mu M CREBBP/EP300 inhibitor, 0.25-10 mu M AMP activated protein kinase inhibitor, 0.25-10 mu M menu-MLL interaction inhibitor, 0.5-20 mu M CK2 inhibitor.
Further, the amounts of the components in the first culture system are as follows: 0.1-20 mM G < beta > -3 Inhibitor, 1-100 ng/mL BMP4, 1-20 mu M TGF < beta > -R Inhibitor, 1-20 mu M RAR activator, 0.25-10 mu M Smoothened receptor agonist, 1-30 mu M DOT1L histone methyltransferase Inhibitor, 0.05-10 mu M SAH hydrolase Inhibitor, 0.5-20 mu M JAK1/2 Inhibitor, 0.25-10 mu M Menin-MLL interaction Inhibitor, 0.2-10 mu MAKT KINASE Inhibitor, 0.2-10 mu M C-jun N-terminal kinase Inhibitor, and 0.1-10 mu M histone methyltransferase SETD2 Inhibitor;
The second culture system comprises the following components in percentage by weight: 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 μ M C-jun N-terminal kinase Inhibitor, 1-10 μM DOT1L histone methyltransferase Inhibitor, 0.05-1 μM SAH hydrase Inhibitor, 0.5-20 μM JAK1/2 Inhibitor, 1-20 μ M p38 MAPK Inhibitor, 1-10 μM CREBBP/EP300 Inhibitor, 0.25-10 μM AMP activated protein kinase Inhibitor, 0.25-10 μM menu-MLL interaction Inhibitor, 0.25-10 μM adenosine kinase Inhibitor, 1-30 μM DNA/RNA methyltransferase Inhibitor, 0.2-10 μ MAKT KINASE Inhibitor and 0.5-20 μM2 Inhibitor;
The third culture system comprises the following components in percentage by weight: 1-100 ng/mLBMP < 4 > and 1-20 μm Y27632.
Further, the first culture system, or the second culture system, or the third culture system, further comprises a basal medium comprising KnockoutDMEM, 2% B27supplement, 10% ksr, 10% FBS, 1% GlutaMAX, 1% NEAA, 1% PenicillinStreptomycin, 50mg/ml vitamin C; wherein% is the volume percent.
In a second aspect, a kit for reprogramming cells comprises the first culture system and the second culture system, or comprises the first culture system to the third culture system.
In a third aspect, a method for reprogramming a cell, comprising the steps of: and culturing the adult cells sequentially by adopting the first culture system and the second culture system to obtain the super-reparative stem cells (SuperXell Stem Cell).
The above-mentioned adult cells include adult cells derived from human body such as adult fibroblasts and tissue-derived mesenchymal stem cells.
Further, the method also comprises the following steps: and culturing the obtained super-repairable stem cells by adopting the third culture system to obtain the sub-pluripotent stem cells.
Further, the method comprises the following specific steps:
(1) Formation of epithelial-like cells: inducing and culturing the adult cells into epithelial-like cells by adopting a first culture system;
(2) Formation of super-reparative stem cells: performing induction culture on the obtained skin-like cells by adopting a second culture system until the cells are super-reparative stem cells;
(3) And (3) forming the sub-pluripotent stem cells, and performing induction culture on the obtained super-reparative stem cells by adopting a third culture system until the sub-pluripotent stem cells are obtained.
The beneficial effects of the invention are as follows: according to the invention, by a chemical micromolecule reprogramming method, aging adult cells are subjected to aging-related epigenetic mark erasure through a series of micromolecule combinations (a first culture system to a third culture system), the aging adult cells are reversely induced into plastic intermediate cells similar to the salamander regeneration process, the intermediate cells are more 'young' mesenchymal stem cells, namely sub-pluripotent stem cells, induced by chemical micromolecules, the cells have stronger proliferation capacity, differentiation potential and factor secretion capacity, the cells are safe and not tumorigenic after being transplanted into a body, the sub-pluripotent stem cells are relatively consistent with the genetic background of the body, and the characteristics of low cost, high purity, stability and safety are realized, and the preparation process does not need to be subjected to a long-time pluripotent stem cell reprogramming process, so that the method has a huge clinical application prospect.
Drawings
FIG. 1 is a cell morphology diagram of the various stages of hADSC reprogramming sub-pluripotent stem cells of example 1 of the present invention;
FIG. 2 is a cell morphology diagram of the various stages of hADSC reprogramming sub-pluripotent stem cells of example 2 of the present invention;
FIG. 3 is a cell morphology diagram of the stages of hASFs reprogrammed pluripotent stem cells of example 3 of the invention;
FIG. 4 is a cell morphology diagram of the stages of hASFs reprogrammed pluripotent stem cells of example 4 of the invention;
FIG. 5 is a cell morphology diagram of UCMSC reprogramming sub-pluripotent stem cells at each stage of example 5 of the present invention;
FIG. 6 is a cell morphology diagram of UCMSC reprogramming sub-pluripotent stem cells at each stage of example 6 of the present invention;
FIG. 7 is a graph of the results of hADSC reprogramming sub-pluripotent stem cells of example 1 of the present invention;
FIG. 8 is a graph of results of UCMSC reprogramming of a sub-pluripotent stem cell stream according to example 3 of the present invention;
FIG. 9 is a graph of results of UCMSC reprogramming of a sub-pluripotent stem cell stream according to example 5 of the present invention.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
The method of the present invention is performed under conditions suitable for the production of induced mesenchymal stem cells, including, for example, the composition, concentration, culture temperature, culture time and other conditions of the culture solution. Based on the full teachings of the prior art and in combination with the exemplary illustrations of the present invention, one skilled in the art can readily determine the above-described culture conditions without undue experimentation. The key is to select the desired inhibited or activated cell signaling pathway and to determine the order in which the cell signaling pathways are affected. In addition, the concentration of the small molecule compound or combination thereof and other conditions may be adaptively adjusted based on the scope provided by the present invention.
And because the man skilled in the art still has room for improvement to regulate and control small molecules and proteins and the like participating in the same target or signal path, the small molecules provided by the patent cannot completely cover the small molecule range of the corresponding path.
The materials and sources of reagents in the following examples are detailed in table 1:
the present embodiment provides a culture system for cell reprogramming comprising a first culture system and a second culture system;
The first culture system comprises a GSK3 beta inhibitor, a TGF beta R 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;
The second culture system comprises a TGF-beta R 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.
Preferably, the first culture system further comprises at least one of sirtuins Inhibitor, BMP4, DOT1L histone methyltransferase Inhibitor, AKT KINASE Inhibitor, histone methyltransferase SETD2 Inhibitor;
the second culture system further comprises at least one of bFGF, smoothened receptor agonist, DOT1L histone methyltransferase Inhibitor, adenosine kinase Inhibitor, DNA/RNA methyltransferase Inhibitor, AKT KINASE Inhibitor.
The preferred embodiment further comprises a third culture system comprising at least one of a TGF-beeta/Smad signaling pathway activator and a ROCK inhibitor.
Preferably, the GSK3 beta inhibitor comprises at least one of LiCl, CHIR99021, laduviglusib (CHIR-99021) HCl, SB216763, TWS119, BIO, LY2090314, CHIR-98014; the sirtuins inhibitors include at least one of Nicotinamide、Sirtinol、Selisistat、SIRT-IN-3、Nicotinamide-d4、4'-Bromo-resveratrol、Nicotinamide-15N,13C3; the TGF-beta R inhibitor comprises at least one of E-616452, SB-431542, LY2109761, GW788388, SB525334, 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 HCl, sonic Hedgehog, LY2940680 and Purmorphamine; the DOT1L histone methyltransferase inhibitor comprises at least one of EP-Z5676, EPZ-6438, 3-deazaneplanocin A HCl, GSK126, EPZ004777, BIX-0194, GSK343, UNC1999 and SGC 0946; The JAK1/2 inhibitor comprises at least one of Ruxolitinib, AZD-1480, fedratinib, WP1066 and 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 JNKIN, SP600125, tanzisertib, JNK-IN-7, JNK Inhibitor VIII; the histone methyltransferase SETD2 inhibitor comprises at least one of SETD2-IN-1TFA, EPZ-719 and EZM 0414; The p38 MAPK inhibitor comprises at least one of BIRB796, SB202190, adezmapimod, ralimetinib dimesylate and VX-702; the CREBBP/EP300 inhibitor comprises at least one of SGC-CBP30, curcumin, C646, ICG-001 and 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 and GP 3269; the DNA/RNA methyltransferase inhibitor comprises at least one of 5-Azacytidine, decitabine, RG108, zebularine, SGI-1027; the CK2 inhibitor comprises at least one of CX-4945, LY294002, silmitasertib sodium salt, (E/Z) -GO 289; The TGF-beeta/Smad signaling pathway activator comprises at least one of BMP4, kartogenin, lsoxazole 9, L-Quebrachitol, SJ 000291942; the ROCK inhibitor includes Y27632.
In this embodiment, the following components are preferably used in the first culture system: 0.1-20 mM G beta inhibitor, 1-20 mu M TGF beta R inhibitor, 1-20 mu M RAR activator, 0.25-10 mu M Smoothened receptor agonist, 0.05-10 mu M SAH hydrolase inhibitor, 0.5-20 mu M JAK1/2 inhibitor, 0.25-10 mu M Menin-MLL interaction inhibitor, 0.2-10 mu M C-jun N-terminal kinase inhibitor;
The second culture system comprises the following components in percentage by weight: 1-15 mu M TGF beta R inhibitor, 1-5 mu M RAR activator, 0.2-5 mu M C-jun N-terminal kinase inhibitor, 0.05-1 mu M SAH hydrase inhibitor, 0.5-20 mu M JAK1/2 inhibitor, 1-20 mu M p MAPK inhibitor, 1-10 mu M CREBBP/EP300 inhibitor, 0.25-10 mu M AMP activated protein kinase inhibitor, 0.25-10 mu M menu-MLL interaction inhibitor, 0.5-20 mu M CK2 inhibitor.
In this embodiment, the following components are preferably used in the first culture system: 0.1-20 mM G < beta > -3 Inhibitor, 1-100 ng/mL BMP4, 1-20 mu M TGF < beta > -R Inhibitor, 1-20 mu M RAR activator, 0.25-10 mu M Smoothened receptor agonist, 1-30 mu M DOT1L histone methyltransferase Inhibitor, 0.05-10 mu M SAH hydrolase Inhibitor, 0.5-20 mu M JAK1/2 Inhibitor, 0.25-10 mu M Menin-MLL interaction Inhibitor, 0.2-10 mu MAKT KINASE Inhibitor, 0.2-10 mu M C-jun N-terminal kinase Inhibitor, and 0.1-10 mu M histone methyltransferase SETD2 Inhibitor;
The second culture system comprises the following components in percentage by weight: 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 μ M C-jun N-terminal kinase Inhibitor, 1-10 μM DOT1L histone methyltransferase Inhibitor, 0.05-1 μM SAH hydrase Inhibitor, 0.5-20 μM JAK1/2 Inhibitor, 1-20 μ M p38 MAPK Inhibitor, 1-10 μM CREBBP/EP300 Inhibitor, 0.25-10 μM AMP activated protein kinase Inhibitor, 0.25-10 μM menu-MLL interaction Inhibitor, 0.25-10 μM adenosine kinase Inhibitor, 1-30 μM DNA/RNA methyltransferase Inhibitor, 0.2-10 μ MAKT KINASE Inhibitor and 0.5-20 μM2 Inhibitor;
The third culture system comprises the following components in percentage by weight: 1-100 ng/mLBMP < 4 > and 1-20 μm Y27632.
In this embodiment, preferably, the basal medium in the first culture system, the second culture system, or the third culture system includes: knockoutDMEM +2% B27supplement+10% KSR+10% FBS+1% Glutamax+1% NEAA+1% Penicillium strepavidin+50 mg/ml vitamin C; wherein% is the volume percent.
The present embodiment also provides a kit for reprogramming cells, comprising the first culture system and the second culture system, or comprising the first culture system to the third culture system. The above-mentioned adult cells include adult skin fibroblasts, adult-derived adult cells such as tissue-derived mesenchymal stem cells.
The present embodiment also provides a method for reprogramming a cell, comprising the steps of: and culturing the adult cells sequentially by adopting the first culture system and the second culture system to obtain the super-reparative stem cells (SuperXell Stem Cell).
The preferred embodiment further comprises the following steps: and culturing the obtained super-repairable stem cells by adopting the third culture system to obtain the sub-pluripotent stem cells.
The preferred steps of this embodiment include the following specific steps:
(1) Formation of epithelial-like cells: inducing and culturing the adult cells into epithelial-like cells by adopting a first culture system;
(2) Formation of super-reparative stem cells: performing induction culture on the obtained skin-like cells by adopting a second culture system until the cells are super-reparative stem cells;
(3) And (3) forming the sub-pluripotent stem cells, and performing induction culture on the obtained super-reparative stem cells by adopting a third culture system until the sub-pluripotent stem cells are obtained.
In summary, the present invention uses chemical small molecule reprogramming of somatic cells for a similar animal regeneration process: the chemical small molecules enable somatic cells to respond to the stimulation of exogenous injury signals, then the somatic cells are dedifferentiated, and plastic intermediate cells mediating tissue regeneration are generated, the intermediate cells are defined as super-repair stem cells, the cells at the stage are further induced into sub-pluripotent stem cells (steady state cells superior to mesenchymal stem cells) through the chemical small molecules under specific culture conditions, safer, simpler and more convenient clinical treatment means from autologous sources are provided, and the application space and the clinical value are wider.
The specific culture medium used in this example, the culture medium and the culture system used were as follows:
1) Culture medium: high glucose dmem+15% fbs or MESENCHYMAL STEM CELL Growth Medium 2.
2) First culture system:
first culture system a:
KnockoutDMEM +2% B27support+10% KSR+10% FBS+1% Glutamax+1% NEAA+1% Penicillin-Streptomycin +50mg/ml vitamin C+5mM LiCl+1mM Nicotinamide
+20ng/mLBMP4+CHIR99021(5μM)+616452(10μM)+TTNPB(2μM)+SAG(0.5μM)+EPZ5676(2μM)+DZNep(0.05μM)+Ruxolitinib(1μM)+VTP50469(0.5μM)+AKT Kinase Inhibitor(1μM)+JNKIN8 (0.2μM)+SETD2-IN-1(0.2μM).
First culture system B:
KnockoutDMEM +2% B27support+10% KSR+10% FBS+1% Glutamax+1% NEAA+1% Penicillin-Streptomycin +50mg/ml vitamin C+5mM LiCl+CHIR99021 (5. Mu.M) +
616452(10μM)+TTNPB(2μM)+SAG(0.5μM)+Ruxolitinib(1μM)+VTP50469(0.5μM)+JNKIN8 (0.2μM)。
First culture system C:
KnockoutDMEM +2% B27support+10% KSR+10% FBS+1% GlutataMAX+1% NEAA+1% Penicillin-Streptomycin +50mg/ml vitamin C+5mM LiCl+CHIR99021 (5. Mu.M) +616452 (10. Mu.M) + TTNPB (2. Mu.M) +Ruxolitinib (1. Mu.M).
3) Second culture system:
second culture system a:
KnockoutDMEM +2% B27support+10% KSR+10% FBS+1% Glutamax+1% NEAA+1% Penicillium strepavidin+50 mg/ml vitamin C++JNKIN8(0.5μM)+EPZ5676(2μM) +DZNep(0.2μM)+Ruxolitinib(1μM)+BIRB796(2μM)+SGC-CBP30(2μM)+Dorsormorphin(0.5μM)+VTP50469(0.5μM)+5-Iodotubercidin(0.5μM)+5-Azacytidine(2μM)+AKT Kinase Inhibitor(0.2μM)+CX-4945(1μM).
Second culture system B:
KnockoutDMEM +2% B27support+10% KSR+10% FBS+1% Glutamax+1% NEAA+1% Penicillium strepavidin+50 mg/ml vitamin C++JNKIN8(0.5μM) +Ruxolitinib(1μM)+BIRB796(2μM)+SGC-CBP30(2μM)+Dorsormorphin(0.5μM)+VTP50469(0.5μM)+CX-4945(1μM).
Third culture system: culture medium required for differentiation of pluripotent stem cells:
The first two days: third culture system:
KnockoutDMEM +2% B27support+10% KSR+10% FBS+1% Glutamax+1% NEAA+1% Penicillium Streptomycin+50mg/ml vitamin C+20ng/ml BMP4+10 μΜ Y27632.
After two days the medium was changed: commercial MSC serum-free medium (purchased from bessel organisms).
Embodiments of the present invention will be described in detail below with reference to specific examples.
Example 1: hADSCs (adipose-derived mesenchymal stem cells) induced culture
1. HADSCs acquisition of hADSCs
1.1 HADSCs (adipose-derived mesenchymal stem cells) isolation
HADSCs, comprising the steps of:
(1) Washing the obtained tissue of 2-4 cm 3 with PBS containing 2% penicillin-streptomycin for 2-3 times;
(2) Placing the tissue in a 100mm culture dish, cutting to 1-2 mm 3 with scissors, adding 5-10ml collagenase IV 2 mg/ml, and placing in a 37-degree incubator for digestion for 1h;
(3) After 1h of digestion and dissociation, the petri dish is taken out, 10-20ml of DMEM medium containing 15% FBS is added, and then a pipetting gun is used for blowing for a plurality of times;
(4) Collecting the suspension into 50ml centrifuge tubes filled with 30ml (the suspension can be divided into 2-3 centrifuge tubes), blowing or slightly vibrating for 1-2 minutes, and releasing cells;
(5) Putting the suspension into a centrifuge, centrifuging for 5 minutes, taking out a centrifuge tube after centrifuging to remove supernatant, and then adding a culture Medium (MESENCHYMAL STEM CELL Growth Medium 2) to resuspend and precipitate;
(6) Inoculating the suspension into a culture dish of 100 mm, and placing the culture dish into a 37-DEG carbon dioxide (5%) incubator for culturing for 18-24 hours;
(7) Removing the cell supernatant from the incubator and replacing fresh Medium (MESENCHYMAL STEM CELL Growth Medium 2);
(8) After the cells are cultured for 3-5 days, the fusion degree reaches 85% -90%, and the subsequent experiments are prepared.
1.2 Cultivation of hADSCs cells purchased
Culture of purchased hADSCs cells comprising the steps of:
(1) Culturing the cells in MESENCHYMAL STEM CELL Growth Medium 2, and placing the cells in a 37-DEG carbon dioxide (5%) incubator, wherein the cells are replaced every 2-3 days;
(2) And after the cells are fused to 85% -90%, preparing for a subsequent experiment.
2. HADSCs reprogramming of cells to pluripotent sub-stem cells
HADSCs reprogramming of cells to sub-pluripotent stem cells comprising the steps of:
(1) Digestive seeding of ADSCs cells
A) Taking hADSCs cells out of the incubator, observing the cells under a microscope, and digesting the cells with the cell fusion degree of 85% -90% as shown in fig. 1;
b) Placing the cells in a super clean bench, discarding the supernatant, washing the cells with PBS, discarding the PBS, adding 0.25% pancreatin, placing the cells in a 37 DEG incubator (5% CO 2), digesting for 3 minutes, then adding DMEM high sugar medium containing 15% FBS, and stopping the digestion;
c) The cell suspension was collected in a 15ml centrifuge tube, transferred to a centrifuge, 300g,3 minutes;
d) The supernatant was discarded, 5ml of DMEM high-sugar medium containing 15% FBS was added to resuspend the cells, a few cells were aspirated, and AO/PI counting was performed;
e) Inoculating cells according to 1x10 x 4 cells per well of a 12-well plate, using a medium of: DMEM high sugar medium containing 15% fbs was cultured for 24 hours and then the first culture system was replaced.
(2) HADSCs reprogramming of cells to epithelial-like cells
A) Cells after 24h of incubation were removed from the incubator and observed under a microscope (photograph recording), as shown in fig. 1:
b) Placing cells in an ultra-clean bench, discarding supernatant (DMEM medium of 15% FBS), adding 1ml of first culture system A into a 12-pore plate hole, placing the cells in a low-oxygen (5% O 2,5%CO2) 37-degree incubator, and replacing the cells with fresh medium for 3-4 days;
c) The epithelial-like cells are generally observed in 4-6 days of induction, the induction is continued until the cell fusion degree reaches about 100% (hADSCs is generally 8-10 days), and the second culture system A is replaced;
(3) Reprogramming plastic intermediate cells
A) Placing cells with the fusion degree reaching about 100% in an ultra-clean bench, discarding the supernatant, adding 1ml of second culture system A into a 12-pore plate hole, placing the cells in a 37-degree incubator (5% CO 2), and replacing fresh culture medium for every 3-4 days;
b) The multi-layer cell cloning generally occurs in 4-6 days of the second culture system A, and after the induction is continued for 8-10 days, the cells are digested and enter into the plastic intermediate cell differentiation.
(4) Reprogramming of pluripotent stem cells
Taking out the plastic intermediate cells from the incubator, observing the cell morphology under a microscope and photographing, as shown in fig. 1, placing the cells in an ultra clean bench, sucking off the supernatant, washing once with PBS, then adding 300 μl of Accutase digestive enzyme, stopping digestion in a 37 ° incubator for 3min, then adding Knockout DMEM medium to stop digestion, collecting the cells into a 15mL centrifuge tube, centrifuging in the centrifuge for 300g,3min, discarding the supernatant, re-suspending the cell pellet with a third culture system of 2mL, sucking out a few cells, and performing AO/PI count: cells were inoculated according to 2x10 5 per well of a 6-well plate, then placed in an incubator for two days, after which the supernatant was aspirated, and commercial MSC medium (purchased from the pearl oyster cell science and technology company) was added, and after culturing the cells to a density of about 80%, digestion passaging and detection were performed.
3. Detection of
Detection result: the obtained sub-pluripotent stem cells were subjected to cell morphology and surface markers (antibodies adopted, APC-CD105, FITC-CD90, PE-CD73, APC-CD79a, FITC-CD45, PE-CD34, FITC-CD 14) were subjected to flow detection. The results are shown in FIGS. 1 and 7. As can be seen from fig. 1, the cell morphology of each reprogramming stage is gradually changed under the action of small chemical molecules; as can be seen from fig. 7, the flow detection identifies: the positive rate of CD105, CD73 and CD90 is more than or equal to 90 percent; while CD45, CD34, CD14, CD79a and HLA-DR are negative, and the positive rate is less than or equal to 5%.
Example 2: hADSCs Induction culture
In comparison with example 1, the procedure was the same as in example 1 except that the cells were reprogrammed to the epithelial-like cells in step (2) hADSCs, the first culture system B was used and the plastic intermediate cells were reprogrammed in step (3), the second culture system B was used.
Detection result: performing cell morphology detection on the obtained sub-pluripotent stem cells, and the result is shown in figure 2; as can be seen from FIG. 2, the cell morphology was gradually changed by the action of small chemical molecules, as observed by reprogramming the cell morphology at each stage. Surface markers (antibodies adopted APC-CD105, FITC-CD90, PE-CD73, APC-CD79a, FITC-CD45, PE-CD34, FITC-CD 14) were detected in a flow format; the stream detection result is: the positive rate of CD105, CD73 and CD90 is more than or equal to 90 percent; while CD45, CD34, CD14, CD79a and HLA-DR are negative, and the positive rate is less than or equal to 5%.
Example 3: hASFs reprogramming (adult dermal fibroblasts)
1. HASFs to obtain
1.1 HASFs separation to obtain
HASFs, comprising the steps of:
(1) Washing 0.5-1 cm 2 tissues with PBS containing 2% penicillin-streptomycin for 2-3 times;
(2) Shearing the tissue blocks into small blocks of 0.5-1 mm 2 by using scissors;
(3) Placing the tissue blocks in a 100 mm cell culture dish, and dripping 1 drop of DMEM medium containing 15% FBS into each piece of tissue;
(4) The dishes were placed in a 37℃carbon dioxide (5%) incubator for 4-12 hours (preventing drying of the tissue pieces);
(5) Afterwards, c of 3-5 ml is gently added to the culture dish (to ensure that the tissue mass does not leave the dish);
(6) Medium (MESENCHYMAL STEM CELL Growth Medium 2) was changed every 2-3 days;
(7) Fibroblast growth in tissue typically begins within 4-7 days;
(8) The cell fusion degree reaches 85% -90% within 10-14 days, and the subsequent experiment is prepared.
1.2 Cultivation of hASFs cells purchased
Culture of purchased hASFs cells comprising the steps of:
(1) Culturing the cells in MESENCHYMAL STEM CELL Growth Medium 2, and placing the cells in a 37-DEG carbon dioxide (5%) incubator, wherein the cells are replaced every 2-3 days;
(2) And after the cells are fused to 85% -90%, preparing for a subsequent experiment.
4. HASFs reprogramming of cells to pluripotent sub-stem cells
HASFs reprogramming of cells to sub-pluripotent stem cells comprising the steps of:
(1) Digestion and seeding of hASFs cells
A. taking hASFs cells out of the incubator, observing the cells under a microscope, and digesting the cells with the cell fusion degree of 85% -90% as shown in fig. 3;
b. Placing the cells in a super clean bench, discarding the supernatant, washing the cells with PBS, discarding the PBS, adding 0.25% pancreatin, placing the cells in a 37 DEG incubator (5% CO 2), digesting for 3 minutes, then adding DMEM high sugar medium containing 15% FBS, and stopping the digestion;
c. the cell suspension was collected in a 15ml centrifuge tube, transferred to a centrifuge, 300g,3 minutes;
d. The supernatant was discarded, 5ml of DMEM high-sugar medium containing 15% FBS was added to resuspend the cells, a few cells were aspirated, and AO/PI counting was performed;
e. cells were seeded at 1x10 4 cells per well of a 12 well plate using the following media: DMEM high sugar medium containing 15% fbs was cultured for 24 hours and then the first culture system a was replaced.
(2) HASFs reprogramming of cells to epithelial-like cells
A. The cells after 24h of culture were taken out of the incubator and observed under a microscope (photograph recording), as shown in fig. 3;
b. placing cells in an ultra-clean bench, discarding supernatant (DMEM medium of 15% FBS), adding 1ml of first culture system A into a 12-pore plate hole, placing the cells in a low-oxygen (5% O 2,5%CO2) 37-degree incubator, and replacing the cells with fresh medium for 3-4 days;
c. the epithelial-like cells are generally observed in 9-16 days of induction, the induction is continued until the cell fusion degree reaches about 100%, and the second culture system A is replaced;
(3) Reprogramming plastic intermediate cells
A. placing cells with the fusion degree reaching about 100% in an ultra-clean bench, discarding the supernatant, adding 1ml of second culture system A into a 12-pore plate hole, placing the cells in a 37-degree incubator (5% CO 2), and replacing fresh culture medium for every 3-4 days;
b. the multi-layer cell cloning generally occurs in 4-6 days induced by the second culture system A, and after the induction is continued for 8-10 days, the cells are digested and enter into the induction of plastic intermediate cell differentiation;
(4) Reprogramming of pluripotent stem cells
Taking out the induced plasticity intermediate cells from the incubator, observing the cell morphology under a microscope and photographing, as shown in fig. 3, then placing the cells in an ultra clean bench, sucking off the supernatant, washing once with PBS, then adding 300 μl of Accutase digestive enzyme, digesting 3min in a 37 ° incubator, then adding KnockoutDMEM medium to stop digestion, collecting the cells into a 15mL centrifuge tube, centrifuging 300g in a centrifuge for 3min, discarding the supernatant, resuspending the cell pellet with a third culture system of 2mL, sucking out a few cells, and performing AO/PI count: cells were inoculated according to 2x10 5 per well of a 6-well plate, then placed in an incubator for two days, after which the supernatant was aspirated, and commercial MSC medium (purchased from the pearl oyster cell science and technology company) was added, and after culturing the cells to a density of about 80%, digestion passaging and detection were performed.
3. Detection of
Detection result: the obtained sub-pluripotent stem cells were subjected to cell morphology and surface markers (antibodies adopted, APC-CD105, FITC-CD90, PE-CD73, APC-CD79a, FITC-CD45, PE-CD34, FITC-CD 14) were subjected to flow detection. The results are shown in FIGS. 3 and 8. As can be seen from fig. 3, the cell morphology was gradually changed by the action of the small chemical molecules when the cell morphology was observed at each stage of reprogramming. As can be seen from fig. 8, the mesenchymal cell surface marker was detected by flow assay: negative expression of CD14, CD45, CD79a, CD34 and HLA-DR were all less than 1%, positive expression of CD90, CD73, CD105 was more than 90%.
Example 4: hASFs reprogramming
In comparison with example 3, the procedure was the same except that the cells in step (2) hADSCs were reprogrammed to the epithelial-like cells, the first culture system B was used and the step (3) was used to reprogram the plastic intermediate cells, the second culture system B was used.
Detection result: performing cell morphology detection on the obtained sub-pluripotent stem cells, and the result is shown in fig. 4; from FIG. 4, it is clear that the cell morphology changes gradually under the action of small chemical molecules, as observed by reprogramming the cell morphology at each stage. Surface markers (antibodies adopted APC-CD105, FITC-CD90, PE-CD73, APC-CD79a, FITC-CD45, PE-CD34, FITC-CD 14) were detected in a flow format; stream detection result: negative expression of CD14, CD45, CD79a, CD34 and HLA-DR were all less than 1%, positive expression of CD90, CD73, CD105 was more than 90%.
Example 5: UCMSC reprogramming to sub-pluripotent stem cells
1. Culture of purchased UCMSCs cells
Culture of purchased UCMSCs cells, comprising the steps of:
(1) Culturing the cells in MESENCHYMAL STEM CELL Growth Medium 2, and placing the cells in a 37-DEG carbon dioxide (5%) incubator, and changing every 2-3 days;
(2) And after the cells are fused to 85% -90%, preparing for a subsequent experiment.
2. Reprogramming UCMSC cells to sub-pluripotent stem cells
The reprogramming of UCMSC cells to sub-pluripotent stem cells comprises the steps of:
(1) Digestion and seeding of UCMSC cells
A. Taking UCMSC cells out of the incubator, observing the cells under a microscope, and digesting the cells with the cell fusion degree of 85% -90% as shown in fig. 5;
b. Placing the cells in a super clean bench, discarding the supernatant, washing the cells with PBS, discarding the PBS, adding 0.25% pancreatin, placing the cells in a 37 DEG incubator (5% CO 2), digesting for 3 minutes, then adding DMEM high sugar medium containing 15% FBS, and stopping the digestion;
c. the cell suspension was collected in a 15ml centrifuge tube, transferred to a centrifuge, 300g,3 minutes;
d. The supernatant was discarded, 5ml of DMEM high-sugar medium containing 15% FBS was added to resuspend the cells, a few cells were aspirated, and AO/PI counting was performed;
e. Inoculating cells according to 1x10 x 4 cells per well of a 12-well plate, using a medium of: DMEM high sugar medium containing 15% fbs was cultured for 24 hours and then the first culture system a was replaced.
(2) Induction of UCMSC cells into epithelial-like cells
A. Cells after 24h of incubation were removed from the incubator and observed under a microscope (photograph recording), as shown in fig. 5:
b. placing cells in an ultra-clean bench, discarding supernatant (DMEM medium of 15% FBS), adding 1ml of first culture system A into a 12-pore plate hole, placing the cells in a low-oxygen (5% O 2,5%CO2) 37-degree incubator, and replacing the cells with fresh medium for 3-4 days;
c. The epithelial-like cells are generally observed in 4-6 days of induction, the induction is continued until the cell fusion degree reaches about 100% (hADSCs is generally 8-10 days), and the second culture system is replaced;
(3) Reprogramming plastic intermediate cells
A. placing cells with the fusion degree reaching about 100% in an ultra-clean bench, discarding the supernatant, adding 1ml of a second culture system into a 12-pore plate hole, placing the cells in a 37-degree incubator (5% CO 2), and replacing fresh culture medium for every 3-4 days;
b. the multi-layer cell cloning generally occurs in 4-6 days of the second culture system, and after the continuous induction is carried out for 8-10 days, the cells are digested and enter into the plastic intermediate cell differentiation.
(4) Reprogramming of pluripotent stem cells
Taking out the plastic intermediate cells from the incubator, observing the cell morphology under a microscope and photographing, as shown in fig. 5, then placing the cells in an ultra clean bench, sucking off the supernatant, washing once with PBS, then adding 300 μl of Accutase digestive enzyme, stopping digestion in a 37 ° incubator for 3min, then adding Knockout DMEM medium to stop digestion, collecting the cells into a 15mL centrifuge tube, centrifuging 300g in the centrifuge for 3min, discarding the supernatant, re-suspending the cell pellet with a third culture system of 2mL, sucking out a small amount of cells, and performing AO/PI count: cells were inoculated according to 2x10 5 per well of a 6-well plate, then placed in an incubator for two days, after which the supernatant was aspirated, and commercial MSC medium (purchased from the pearl oyster cell science and technology company) was added, and after culturing the cells to a density of about 80%, digestion passaging and detection were performed.
3. Detection of
Results of the detection: the obtained sub-pluripotent stem cells were subjected to cell morphology and surface markers (antibodies adopted, APC-CD105, FITC-CD90, PE-CD73, APC-CD79a, FITC-CD45, PE-CD34, FITC-CD 14) were subjected to flow detection. The results are shown in FIGS. 5 and 9. As can be seen from fig. 5, the cell morphology of each reprogramming stage is gradually changed under the action of small chemical molecules; as can be seen from fig. 9, the reprogrammed cell surface markers CiMSC were identified as: the positive rate of CD105, CD73 and CD90 is more than or equal to 90 percent; while CD45, CD34, CD14, CD79a and HLA-DR are negative, and the positive rate is less than or equal to 5%.
Example 6: UCMSC reprogramming to sub-pluripotent stem cells
In comparison with example 5, the procedure was the same as in example 5 except that the cells were reprogrammed to the epithelial-like cells in step (2) hADSCs, the first culture system B was used and the plastic intermediate cells were reprogrammed in step (3) using the second culture system B.
Results of the detection: the obtained sub-pluripotent stem cells were subjected to cell morphology, and the results are shown in fig. 6; as can be seen from fig. 6, the cell morphology was gradually changed by the action of small chemical molecules, as observed by reprogramming the cell morphology at each stage. Surface markers (antibodies adopted APC-CD105, FITC-CD90, PE-CD73, APC-CD79a, FITC-CD45, PE-CD34, FITC-CD 14) were detected in a flow format; stream detection result: the positive rate of CD105, CD73 and CD90 is more than or equal to 90 percent; while CD45, CD34, CD14, CD79a and HLA-DR are negative, and the positive rate is less than or equal to 5%.
Comparative example 1
In comparison to example 1, the procedure was the same except that the cells were reprogrammed to epithelial-like cells in step (2) hADSCs, using the first culture system C.
Detection result: sub-pluripotent stem cells cannot be obtained.
Comparative example 2
In comparison to example 3, the procedure was the same except that the cells were reprogrammed to epithelial-like cells in step (2) hADSCs, using the first culture system C.
Detection result: sub-pluripotent stem cells cannot be obtained.
Comparative example 3
In comparison to example 5, the procedure was the same except that the reprogramming of cells to epithelial-like cells in step (2) hADSCs was performed using the first culture system C.
Detection result: sub-pluripotent stem cells cannot be obtained.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (4)
1. A culture medium for reprogramming of cells, comprising a first culture system, a second culture system, and a third culture system;
The first culture system comprises ：KnockOut DMEM、2% B27-supplement、10% KSR、10% FBS、1% GlutaMAX、1% NEAA、1% Penicillin-Streptomycin、50mg/ml vitamins with the following dosage components C、5mM LiCl、5μM CHIR99021、10μM 616452、2μM TTNPB、0.5μM SAG、1μM Ruxolitinib、0.5μM VTP50469、0.2μM JNKIN8;
The second culture system comprises ：KnockOut DMEM、2% B27-supplement、10% KSR、10% FBS、1% GlutaMAX、1% NEAA、1% PenicillinStreptomycin、50mg/ml vitamins with the following dosage components C、0.5μM JNKIN8、1μM Ruxolitinib、2μM BIRB796、2μM SGC-CBP30、0.5μM Dorsormorphin、0.5μM VTP50469、1μM CX-4945;
The third culture system consists of ：KnockOut DMEM、2% B27-supplement、10% KSR、10% FBS、1% GlutaMAX、1% NEAA、1% PenicillinStreptomycin、50mg/ml vitamin C, 20ng/ml BMP4 and 10 mu m Y27632.
2. A culture medium for reprogramming of cells, comprising a first culture system, a second culture system, and a third culture system;
The first culture system comprises ：KnockOut DMEM、2% B27supplement、10% KSR、10% FBS、1% GlutaMAX、1% NEAA、1% Penicillin-Streptomycin、50mg/ml vitamins with the following dosage components C、5mM LiCl、1mM Nicotinamide、20ng/mL BMP4、5μM CHIR99021、10μM 616452、2μM TTNPB、0.5μM SAG、2μM EPZ5676、0.05μM DZNep、1μM Ruxolitinib、0.5μM VTP50469、1μM AKT Kinase Inhibitor、0.2μM JNKIN8、0.2μM SETD2-IN-1;
The second culture system comprises ：KnockOut DMEM、2% B27-supplement、10% KSR、10% FBS、1% GlutaMAX、1% NEAA、1% Penicillin-Streptomycin、50mg/ml vitamins with the following dosage components C、0.5μM JNKIN8、2μM EPZ5676、0.2μM DZNep、1μM Ruxolitinib、2μM BIRB796、2μM SGC-CBP30、0.5μM Dorsormorphin、0.5μM VTP50469、0.5μM 5-Iodotubercidin、2μM 5-Azacytidine、0.2μM AKT Kinase Inhibitor、1μM CX-4945;
The third culture system consists of ：KnockOut DMEM、2% B27-supplement、10% KSR、10% FBS、1% GlutaMAX、1% NEAA、1% PenicillinStreptomycin、50mg/ml vitamin C, 20ng/ml BMP4 and 10 mu m Y27632.
3. A kit for cell reprogramming comprising a medium for cell reprogramming as claimed in any one of claims 1 to 2.
4. A method for reprogramming a cell, comprising the steps of: culturing the adult cells with a medium for reprogramming cells according to any one of claims 1 to 2 to obtain mesenchymal stem cells.
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