Culture medium for dedifferentiation culture of somatic cells and limb bud cell in-vitro induction and multiplication culture method

Abstract

The invention provides a culture medium for dedifferentiation culture of somatic cells and a limb bud cell in-vitro induction and multiplication culture method, and belongs to the technical field of cell culture. A chemical induction dedifferentiation technology is developed on the basis of compounding of various cell signal channel inhibitors or activators, somatic cells are successfully induced to be converted into dedifferentiated cells with limb bud cell characteristics, and CiLB cells with osteogenic and chondrogenic differentiation potential are obtained through multiplication culture of a limb bud cell culture medium. The CiLB cell is expected to become a valuable cell resource for future clinical application, provides powerful cell support and solutions for a plurality of medical frontier fields such as bone injury repair, bone disease treatment, tissue engineering and the like, and leads the medical treatment technology to advance to a new height.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a culture medium for somatic cell dedifferentiation culture and an in vitro induction and amplification culture method for limb bud cells. Background Art

[0002] In nature, there is a class of animals with high regenerative ability. When they are injured, the cells in these organisms that were originally in a terminally differentiated state can quickly activate the dedifferentiation mechanism, a reverse development process, and transform themselves into progenitor cells with strong regenerative potential. These progenitor cells then form new tissues or organs through differentiation and development processes, thereby achieving regeneration, such as the regeneration of salamanders' broken limbs. In mammals, there is a group of cells with similar strong differentiation ability, called limb bud cells. Limb bud cells are produced by the lateral plate mesoderm (LPM) in the early stages of embryonic development and have the ability to differentiate into a variety of cell types that constitute the bones and soft tissues of the limbs. However, mammals, including humans, have relatively low somatic cells that show relatively low plasticity. Faced with the stimulation of external signals, they often find it difficult to respond quickly and initiate the dedifferentiation program, which in turn limits the ability of mammals to regenerate tissues or organs after damage. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a somatic cell dedifferentiation culture composition, which utilizes a variety of small molecule compounds to treat somatic cells, thereby realizing the transformation of somatic cells into cells with the molecular characteristics and differentiation potential of limb bud cells, thereby providing the possibility for the healing and regeneration of body injuries.

[0004] The invention provides a composition for somatic cell dedifferentiation culture, comprising 0-500 mg of vitamin C phosphate, 0-10 mmol of lithium chloride, 0-5 mmol of niacinamide, 0-2.2 g of bovine serum albumin, 0-500 mg of basic fibroblast growth factor and any combination of the following small molecule compounds: 0.5-15 μmol of WNT pathway activator, 5-50 μmol of transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 0.5-5 μmol of retinoic acid receptor agonist, 0.45-5 μmol of hedgehog signaling pathway activator, 0.3-10 μmol of vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 0.5-12 μmol of RhoA / ROCK pathway inhibitor, 0-5 μmol of telomere silencing interferon 1 inhibitor, 0-2.5 μmol of c-Jun amino-terminal kinase inhibitor, 0-2.2 μmol of monoamine oxidase inhibitor, 0-11 μmol of DNA methylation inhibitors, 0-1.1 μmol histone methyltransferase G9a inhibitors, 0-1.0 μmol histone methyltransferase EZH2 inhibitors, 0.1-5 μmol JAK protein inhibitors, 0-2.2 μmol P38 and B-RAF kinase inhibitors, 0-0.55 μmol adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitors, and 0-5 μmol p300 / CREB binding protein inhibitors;

[0005] The bovine serum albumin and basic fibroblast growth factor are not simultaneously 0;

[0006] The small molecule compounds are not 0 at the same time.

[0007] Preferably, it comprises a first composition and a second composition;

[0008] The first composition comprises 0-500 mg vitamin C phosphate, 0-10 mmol lithium chloride, 0-5 mmol niacinamide, 0.5-2.2 g bovine serum albumin and any combination of the following small molecule compounds: 0.5-15 μmol WNT pathway activator, 5-50 μmol transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 0.5-5 μmol retinoic acid receptor agonist, 0.45-5 μmol hedgehog signaling pathway activator, 0.3-10 μmol vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 0.5-12 μmol RhoA / ROCK pathway inhibitor, 0.5-5 μmol telomere silencing interferon 1 inhibitor, 0.01-1.0 μmol histone methyltransferase EZH2 inhibitor and 0.1-5 μmol JAK protein inhibitor;

[0009] The second composition comprises 0-500 mg vitamin C phosphate, 0-10 mmol lithium chloride, 0-5 mmol niacinamide, 10-500 mg basic fibroblast growth factor and any combination of the following small molecule compounds: 0.5-15 μmol WNT pathway activator, 5-50 μmol transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 0.5-5 μmol retinoic acid receptor agonist, 0.45-5 μmol hedgehog signaling pathway activator, 0.5-10 μmol vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 0.5-12 μmol RhoA / ROCK pathway inhibitor, 0.1-2.5 μmol c-Jun amino-terminal kinase inhibitor, 0.5-2.2 μmol monoamine oxidase inhibitor, 0.01-11 μmol DNA methylation inhibitor, 0.01-1.1 μmol histone methyltransferase G9a inhibitor, 0.1-5 μmol JAK protein inhibitor, 0.5-2.2 μmol P38 and B-RAF kinase inhibitors, 0.05-0.55 μmol adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitors and 0.1-5 μmol p300 / CREB binding protein inhibitors.

[0010] Preferably, the WNT pathway activator comprises at least one of the following: CHIR99021, SB-216763; CHIR99021 trihydrochloride, BIO-acetone oxime, GSK-3β inhibitor XII, GSK-3 inhibitor XV, TD114-2, TD114-3, IM12, CHIR98014 and SB-415286;

[0011] The transforming growth factor-β receptor I / activin-like kinase 5 inhibitor includes at least one of the following: 616452, A83-01, SB431542, SB 505124, GW 788388, doxorubicin and SB 525334;

[0012] The retinoic acid receptor agonist comprises at least one of the following: TTNPB, Ch55 and AM580;

[0013] The hedgehog signaling pathway activator comprises at least one of the following: SAG, purmorphamine, Hh-Ag1.5 and human SHH;

[0014] The vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor includes ABT-869;

[0015] The RhoA / ROCK pathway inhibitors include at least one of the following: Y27632, Tzv, fasudil, and thiazovivin;

[0016] The telomeric silencing interferon 1 inhibitor includes at least one of the following: EPZ004777, EPZ5676, EPZ5676 and SGC0946;

[0017] The c-Jun amino-terminal kinase inhibitor includes JNK-IN-8;

[0018] The monoamine oxidase inhibitors include tranylcypromine;

[0019] The DNA methylation inhibitor includes 5-azacytidine;

[0020] The histone methyltransferase G9a inhibitor includes UNC0224;

[0021] The histone methyltransferase EZH2 inhibitor includes DZNep;

[0022] The JAK protein inhibitor includes ruxolitinib;

[0023] The P38 and B-RAF kinase inhibitors include damanimod BIRB796;

[0024] The adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitors include dorsomorphin;

[0025] The p300 / CREB binding protein inhibitor includes SGC-CBP30.

[0026] The present invention provides a culture medium for somatic cell dedifferentiation culture, which uses KnockOut DMEM culture medium as a basic culture medium, and further comprises the following components: 8% to 12% serum replacement by volume, 8% to 12% fetal bovine serum by volume, 0.8% to 1.2% alanyl-glutamine by weight, 0.8% to 1.2% non-essential amino acids by weight, 0.8% to 1.2% penicillin and streptomycin, and the composition for somatic cell dedifferentiation culture described in the above technical solution;

[0027] The concentration of the composition is calculated in terms of vitamin C phosphate, and the final concentration of vitamin C phosphate in the culture medium is 45-55 μg / ml.

[0028] The present invention provides use of the somatic cell dedifferentiation culture composition or the culture medium in mammalian somatic cell dedifferentiation culture.

[0029] The present invention provides a method for dedifferentiation culture of mammalian somatic cells, comprising the following steps:

[0030] Using a culture medium containing the composition to carry out dedifferentiation induction culture on mammalian somatic cells, so as to obtain cells with erased somatic cell molecular characteristics and aging phenotype and increased differentiation potential;

[0031] Preferably, the dedifferentiation induction culture includes a first stage induction culture and a second stage induction culture;

[0032] The first stage of induction culture is to culture with a culture medium containing the first composition in the above technical solution to obtain epithelial cells, and when the confluence of the epithelial cells is above 80%, enter the second stage of induction culture;

[0033] The second stage of induction culture is carried out by using a culture medium containing the second composition in the above technical solution to obtain multiple cell colony cells.

[0034] The present invention provides a culture medium for culturing limb bud cells with somatic cells under in vitro conditions, comprising the culture medium for somatic cell dedifferentiation culture and the culture medium for limb bud cells;

[0035] The limb bud cell culture medium includes a KnockOut DMEM culture medium as a basic culture medium, and also includes the following components: 8% to 12% serum replacement, 0% to 15% fetal bovine serum, 0.9% to 1.1% alanyl-glutamine, 0.9% to 1.1% non-essential amino acids, 0.9% to 1.1% penicillin-streptomycin, 45 to 55 μg / ml vitamin C phosphate, 10 to 200 ng / ml basic fibroblast growth factor and the following small molecule compounds: WNT pathway activator 0.5 to 15 μM, transforming growth factor-β receptor I / activin-like kinase 5 inhibitor 2 to 20 μM, retinoic acid receptor agonist 0.5 to 5 μM, hedgehog signaling pathway activator 0.1 to 1 μM, RhoA / ROCK pathway inhibitor 2 to 20 μM, c-Jun amino-terminal kinase inhibitor 0.2 to 5 μM and p300 / CREB binding protein inhibitor 0.5 to 5 μM.

[0036] The present invention provides a method for culturing chemically induced limb bud cells CiLB, comprising the following steps:

[0037] The multiple cell colony cells obtained by the dedifferentiation culture method of mammalian somatic cells are digested, and the obtained single cells are inoculated into the limb bud cell culture medium in the culture medium described in the above technical scheme for subculture to obtain chemically induced limb bud cells CiLB.

[0038] The present invention provides chemically induced limb bud cells CiLB obtained by the culture method described in the above technical solution.

[0039] The present invention provides the use of the chemically induced limb bud cells CiLB in preparing medical materials or medicines for repairing or regenerating damaged organs or tissues of mammals.

[0040] The invention provides a composition for somatic cell dedifferentiation culture, comprising 0-500 mg of vitamin C phosphate, 0-10 mmol of lithium chloride, 0-5 mmol of niacinamide, 0-2.2 g of bovine serum albumin, 0-500 mg of basic fibroblast growth factor and any combination of the following small molecule compounds: 0.5-15 μmol of WNT pathway activator, 5-50 μmol of transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 0.5-5 μmol of retinoic acid receptor agonist, 0.45-5 μmol of hedgehog signaling pathway activator, 0.3-10 μmol of vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 0.5-12 μmol of RhoA / ROCK pathway inhibitor, 0-5 μmol of telomere silencing interferon 1 inhibitor, 0-2.5 μmol of c-Jun amino-terminal kinase inhibitor, 0-2.2 μmol of monoamine oxidase inhibitor, 0-11 μmol of DNA methylation inhibitor, 0-1.1 μmol histone methyltransferase G9a inhibitor, 0-1.0 μmol histone methyltransferase EZH2 inhibitor, 0.1-5 μmol JAK protein inhibitor, 0-2.2 μmol P38 and B-RAF kinase inhibitor, 0-0.55 μmol adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitor and 0-5 μmol p300 / CREB binding protein inhibitor; the bovine serum albumin and basic fibroblast growth factor are not 0 at the same time; the small molecule compound is not 0 at the same time. The present invention compounds vitamin C phosphate, lithium chloride, niacinamide and bovine serum albumin with at least one of basic fibroblast growth factor and small molecule compounds that serve as inhibitors or activators in multiple signal pathways, which not only erases the original molecular characteristics of somatic cells, but also significantly reduces the aging-related secretory phenotype. It has been verified by the examples that the single cells cultured with the above-mentioned composition show a gene expression profile very similar to that of human embryonic limb bud cells at the transcriptome level, indicating that these single cells have a high degree of similarity in developmental potential. In the natural biological development process, limb bud cells undergo a delicate differentiation process and gradually evolve into chondrocytes and osteocytes. This series of changes eventually forms the appendage structure of the organism. It can be seen that the present invention deeply reveals the intrinsic mechanism of cell dedifferentiation and regeneration, provides an effective way to provide mammals with cells with high differentiation potential, and provides the possibility for the treatment of organ or limb regeneration. The results of in vitro differentiation experiments in the embodiments of the present invention show that compared with human cells, cultured single cells have significantly stronger bone and cartilage formation capabilities. More importantly, when these cells are transplanted into animals, they can not only spontaneously differentiate into cartilage and bone tissue, but also show strong damage repair capabilities, significantly promoting the healing and regeneration of damaged parts.

[0041] The present invention also provides a culture medium for culturing limb bud cells with somatic cells under in vitro conditions, comprising a culture medium comprising the composition described in the above technical solution and a limb bud cell culture medium. The culture medium comprising the composition can achieve the purpose of dedifferentiation of somatic cells, and the limb bud cell culture medium can achieve the amplification and culture of dedifferentiated cells, so that the human CiLB cells obtained by culture can be amplified stably for a long time. At the same time, they can also maintain the biological characteristics of limb bud cells, ensuring the high homogeneity of the cell population.

[0042] In addition, the present invention also uses the chemically induced limb bud cells CiLB obtained by the culture method described in the above technical scheme in the preparation of medical materials or drugs for the repair or regeneration of mammalian organ or tissue damage. The present invention successfully reverses the natural development trajectory of mature cells through chemically induced dedifferentiation technology, transforming them into CiLB cells with limb bud cell characteristics. This discovery not only profoundly reveals the intrinsic mechanism of cell dedifferentiation and regeneration, but also brings revolutionary treatment solutions to the field of clinical medicine. It is particularly noteworthy that the human CiLB cells prepared by the present invention that can be expanded on a large scale are expected to become a valuable cell resource for future clinical applications, providing strong cell support and solutions for multiple medical frontier fields such as bone injury repair, bone disease treatment and tissue engineering, and leading medical treatment technology to new heights. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of chemically induced somatic cell dedifferentiation and limb bud cell expansion culture;

[0044] Figure 2 The results of analyzing the expression of related genes at different time points of chemically induced dedifferentiation;

[0045] Figure 3 The results of gene ontology analysis of differentially expressed genes between human CiLB cells and parental somatic cells;

[0046] Figure 4 The bright field morphology of human CiLB cells and parental somatic cells (left) and the proliferation results of human CiLB cells under different conditions;

[0047] Figure 5 The results show the effects of single-reduction of small molecules in limb bud cell culture medium on the number of human CiLB cells and gene expression;

[0048] Figure 6 The results of qPCR (left) and immunofluorescence detection of the expression of limb bud cell-related genes in human CiLB cells;

[0049] Figure 7 The heat map shows the expression results of related genes in human CiLB cells and parental somatic cells;

[0050] Figure 8 The results of immunofluorescence detection of the expression of limb bud cell-related genes in human CiLB cells;

[0051] Fig. 9 Single cell clustering (left) and UPAM (right) show similarity results between human CiLB cells and different types of embryonic cells;

[0052] Fig.10 Single-cell RNA-Seq data analysis of highly expressed genes enriched in human CiLB cells relative to parental somatic cells;

[0053] Fig.11 The similarity results of human CiLB cells and different cell types of embryonic limb bud tissue were analyzed for single-cell RNA-Seq data;

[0054] Fig.12 Results of osteogenic (left) and adipogenic (right) analysis of human CiLB cells and parental cells;

[0055] Fig.13 Results of in vitro 3D analysis of human CiLB cells using Alcian blue staining (left) and immunohistochemistry (right);

[0056] Fig.14 Results of detection of Alcian blue (left) and chondrocyte-related gene expression for micromass chondrogenic differentiation;

[0057] Fig.15 Single-cell RNA-Seq pseudo-time analysis of molecular pathways in human CiLB cells differentiated in vitro for 7 days;

[0058] Fig.16 The results of spontaneous biochemical products of human CiLB cells were tested for subcutaneous and renal cyst transplantation in mice;

[0059] Fig.17 Repair test results for animal injuries;

[0060] Fig.18 Analysis of RNA-Seq data for the expression of genes related to aging and proliferation;

[0061] Fig.19 The test results of proliferation ability of human CiLB cells and Qindai cells;

[0062] Fig. 20 Results of gene expression analysis of human CiLB cells at different generations;

[0063] Fig.21 These are the safety test results for human CiLB cells. DETAILED DESCRIPTION

[0064] The invention provides a composition for somatic cell dedifferentiation culture, comprising 0-500 mg of vitamin C phosphate, 0-10 mmol of lithium chloride, 0-5 mmol of niacinamide, 0-2.2 g of bovine serum albumin, 0-500 mg of basic fibroblast growth factor and any combination of the following small molecule compounds: 0.5-15 μmol of WNT pathway activator, 5-50 μmol of transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 0.5-5 μmol of retinoic acid receptor agonist, 0.45-5 μmol of hedgehog signaling pathway activator, 0.3-10 μmol of vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 0.5-12 μmol of RhoA / ROCK pathway inhibitor, 0-5 μmol of telomere silencing interferon 1 inhibitor, 0-2.5 μmol of c-Jun amino-terminal kinase inhibitor, 0-2.2 μmol of monoamine oxidase inhibitor, 0-11 μmol of DNA methylation inhibitor, 0-1.1 μmol histone methyltransferase G9a inhibitor, 0-1.0 μmol histone methyltransferase EZH2 inhibitor, 0.1-5 μmol JAK protein inhibitor, 0-2.2 μmol P38 and B-RAF kinase inhibitor, 0-0.55 μmol adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitor and 0-5 μmol p300 / CREB binding protein inhibitor; the bovine serum albumin and basic fibroblast growth factor are not 0 at the same time; in the small molecule compound, the telomere silencing interferon 1 inhibitor and the c-Jun amino-terminal kinase inhibitor, monoamine oxidase inhibitor, DNA methylation inhibitor, P38 and B-RAF kinase inhibitor, adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitor, p300 / CREB binding protein inhibitor are not zero at the same time. At the same time, c-Jun amino-terminal kinase inhibitor, monoamine oxidase inhibitor, DNA methylation inhibitor, P38 and B-RAF kinase inhibitor, adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitor and p300 / CREB binding protein inhibitor are zero at the same time or not at the same time.

[0065] In the present invention, the composition preferably includes a first composition and a second composition. The first composition preferably includes 0-500 mg vitamin C phosphate, 0-10 mmol lithium chloride, 0-5 mmol niacinamide, 0.5-2.2 g bovine serum albumin and any combination of the following small molecule compounds: 0.5-15 μmol WNT pathway activator, 5-50 μmol transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 0.5-5 μmol retinoic acid receptor agonist, 0.45-5 μmol hedgehog signaling pathway activator, 0.3-10 μmol vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 0.5-12 μmol RhoA / ROCK pathway inhibitor, 0.5-5 μmol telomere silencing interferon 1 inhibitor, 0.01-1.0 μmol histone methyltransferase EZH2 inhibitor and 0.1-5 μmol JAK protein inhibitors; more preferably, small molecule compounds including 45-55 mg vitamin C phosphate, 4.5-5.5 mmol lithium chloride, 0.9-1.1 mmol niacinamide, 1.8-2.2 g bovine serum albumin and any combination of the following: 9-14 μmol WNT pathway activator, 9-11 μmol transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 1.8-2.2 μmol retinoic acid receptor agonist, 0.45-0.55 μmol hedgehog signaling pathway activator, 0.9-1.1 μmol vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 1.8-2.2 μmol RhoA / ROCK pathway inhibitor, 1.8-2.2 μmol telomeric silencing disruptor 1 inhibitor, 0.018-0.022 μmol histone methyltransferase DZNep and 0.9-1.1 μmol JAK protein inhibitor; further preferably including 50 mg vitamin C phosphate, 5 mmol lithium chloride, 1 mmol niacinamide, 2 g bovine serum albumin and any combination of the following small molecule compounds: 10 μmol WNT pathway activator, 10 μmol transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 2 μmol retinoic acid receptor agonist, 0.5 μmol hedgehog signaling pathway activator, 1 μmol vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 2 μmol RhoA / ROCK pathway inhibitor, 2 μmol telomeric silencing disruptor 1 inhibitor, 0.02 μmol histone methyltransferase EZH2 inhibitor and 1 μmol JAK protein inhibitor.

[0066] In the present invention, the second composition comprises 0-500 mg vitamin C phosphate, 0-10 mmol lithium chloride, 0-5 mmol niacinamide, 10-500 mg basic fibroblast growth factor and any combination of the following small molecule compounds: 0.5-15 μmol WNT pathway activator, 5-50 μmol transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 0.5-5 μmol retinoic acid receptor agonist, 0.45-5 μmol hedgehog signaling pathway activator, 0.5-10 μmol vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 0.5-12 μmol RhoA / ROCK pathway inhibitor, 0.1-2.5 μmol c-Jun amino-terminal kinase inhibitor, 0.5-2.2 μmol monoamine oxidase inhibitor, 0.01-11 μmol DNA methylation inhibitor, 0.01-1.1 μmol histone methyltransferase G9a inhibitor, 0.1-5 μmol The invention also comprises the following small molecule compounds: a JAK protein inhibitor, 0.5-2.2 μmol P38 and B-RAF kinase inhibitor, 0.05-0.55 μmol adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitor, and 0.1-5 μmol p300 / CREB binding protein inhibitor, and more preferably comprises 45-55 mg vitamin C phosphate, 4.5-5.5 mmol lithium chloride, 0.9-1.1 mmol niacinamide, 90-110 mg basic fibroblast growth factor, and any combination of the following small molecule compounds: 9-14 μmol WNT pathway activator, 9-11 μmol transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 1.8-2.2 μmol retinoic acid receptor agonist, 0.45-0.55 μmol hedgehog signaling pathway activator, 0.9-1.1 μmol vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 9-12 μmol RhoA / ROCK pathway inhibitor, 0.9-1.1 μmol c-Jun amino-terminal kinase inhibitor, 1.8-2.2 μmol monoamine oxidase inhibitor, 9-11 μmol DNA methylation inhibitor, 0.018-1.1 μmol histone methyltransferase G9a inhibitor, 0.9-1.1 μmol JAK protein inhibitor, 1.8-2.2 μmol P38 and B-RAF kinase inhibitor, 0.45-0.55 μmol adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitor and 1.8-2.2μmol p300 / CREB binding protein inhibitor; further preferably including 50mg vitamin C phosphate, 5mmol lithium chloride, 1mmol niacinamide, 100μg basic fibroblast growth factor and any combination of the following small molecule compounds: 10μmol WNT pathway activator, 10μmol transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 2μmol retinoic acid receptor agonist, 0.5μmol hedgehog signaling pathway activator, 1μmol vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 10μmol RhoA / ROCK pathway inhibitor, 101μmol c-Jun amino-terminal kinase inhibitor, 2μmol monoamine oxidase inhibitor, 10μmol DNA methylation inhibitor, 1μmol histone methyltransferase G9a inhibitor, 1μmol JAK protein inhibitor, 2μmol P38 and B-RAF kinase inhibitor, 0.5μmol adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitor and 2μmol p300 / CREB binding protein inhibitor. .

[0067] In the present invention, the WNT pathway activator preferably includes at least one of the following: CHIR99021, SB-216763; CHIR 99021 trihydrochloride, BIO-acetone oxime, GSK-3β inhibitor XII, GSK-3 inhibitor XV, TD114-2, TD114-3, IM12, CHIR98014 and SB-415286; more preferably CHIR99021. The transforming growth factor-β receptor I / activin-like kinase 5 inhibitor preferably includes at least one of the following: 616452, A83-01, SB431542, SB 505124, GW 788388, doxorubicin and SB 525334; more preferably 646452. The retinoic acid receptor agonist preferably includes at least one of the following: TTNPB, Ch55 and AM580; more preferably TTNPB. The hedgehog signaling pathway activator includes at least one of the following: SAG, purmorphamine, Hh-Ag1.5 and human SHH; more preferably SAG. The vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor preferably includes ABT-869. The RhoA / ROCK pathway inhibitor preferably includes at least one of the following: Y27632, Tzv, fasudil and thiazovivin; more preferably Y27632. The telomeric silencing interferon 1 inhibitor preferably includes at least one of the following: EPZ004777, EPZ5676, EPZ5676 and SGC0946; more preferably EPZ004777. The c-Jun amino-terminal kinase inhibitor preferably includes JNK-IN-8. The monoamine oxidase inhibitor preferably includes tranylcypromine. The DNA methylation inhibitor preferably includes 5-azacytidine. The histone methyltransferase G9a inhibitor preferably includes UNC0224. The histone methyltransferase EZH2 inhibitor preferably includes DZNep. The JAK protein inhibitor preferably includes ruxolitinib. The P38 and B-RAF kinase inhibitors preferably include damanimide BIRB796. The adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitors preferably include dorsomorphin. The p300 / CREB binding protein inhibitor preferably includes SGC-CBP30. The present invention does not impose any special restrictions on the sources of the components in the composition, and they can be obtained by commercial purchase channels known in the art.

[0068] The invention provides a culture medium for somatic cell dedifferentiation culture, which uses KnockOut DMEM culture medium as a basic culture medium and further comprises the following components: 8% to 12% by volume of serum replacement, 0% to 15% by volume of fetal bovine serum, 0.8% to 1.2% by volume of alanyl-glutamine, 0.8% to 1.2% by volume of non-essential amino acids, 0.8% to 1.2% by volume of penicillin and streptomycin, and the composition for somatic cell dedifferentiation culture described in the above technical scheme; the concentration of the composition is calculated in terms of vitamin C phosphate, and the final concentration of the vitamin C phosphate in the culture medium is 0 to 500 mg / L.

[0069] In the present invention, the culture medium for somatic cell dedifferentiation culture is based on KnockOut DMEM culture medium, preferably including the following components: 10% serum replacement by volume, 10% fetal bovine serum by volume, 1% alanyl-glutamine by volume, 1% non-essential amino acids by volume, 1% penicillin and streptomycin by volume, and the composition for somatic cell dedifferentiation culture described in the above technical solution; the concentration of the composition is calculated as vitamin C phosphate, and the final concentration of vitamin C phosphate in the culture medium is 50 μg / ml. The WNT pathway activator preferably includes at least one of the following: CHIR99021, SB-216763; CHIR 99021 trihydrochloride, BIO-acetone oxime, GSK-3β inhibitor XII, GSK-3 inhibitor XV, TD114-2, TD114-3, IM12, CHIR98014 and SB-415286; more preferably CHIR99021. The transforming growth factor-β receptor I / activin-like kinase 5 inhibitor preferably includes at least one of the following: 616452, A83-01, SB431542, SB 505124, GW 788388, doxorubicin and SB 525334; more preferably 646452. The retinoic acid receptor agonist preferably includes at least one of the following: TTNPB, Ch55 and AM580; more preferably TTNPB. The hedgehog signaling pathway activator includes at least one of the following: SAG, purmorphamine, Hh-Ag1.5 and human SHH; more preferably SAG. The RhoA / ROCK pathway inhibitor preferably includes at least one of the following: Y27632, Tzv, fasudil and thiazovivin; more preferably Y27632. The c-Jun amino-terminal kinase inhibitor preferably includes JNK-IN-8. The p300 / CREB binding protein inhibitor preferably includes SGC-CBP30.

[0070] The present invention does not impose any particular limitation on the preparation method of the culture medium, and any preparation method known in the art may be used. The culture medium is prepared by chemically induced dedifferentiation technology (see Figure 1 ), which can erase the molecular characteristics and aging phenotype of parental cells. In the present embodiment, the RNA-Seq results show that the chemically induced dedifferentiated cells not only erase the properties and aging characteristics of the original cells, but also reactivate the molecular characteristics of limb bud cells.

[0071] The present invention provides use of the somatic cell dedifferentiation culture composition or the culture medium in mammalian somatic cell dedifferentiation culture.

[0072] In the present invention, the mammal preferably includes primates, more preferably humans. The mammalian somatic cells include at least one of the following: fibroblasts, blood cells, epithelial cells and endothelial cells. The fibroblasts preferably include at least one of the following: fat fibroblasts, skin fibroblasts, adipose-derived mesenchymal stromal cells and umbilical cord mesenchymal stem cells. In an embodiment of the present invention, primary human adult adipose-derived mesenchymal stromal cells (hADSCs) derived from human adipose tissue and primary human adult skin fibroblasts (hASFs) derived from human skin tissue are used respectively.

[0073] The present invention provides a method for dedifferentiation culture of mammalian somatic cells, comprising the following steps:

[0074] Mammalian somatic cells are cultured in a culture medium containing the composition for dedifferentiation induction, thereby obtaining cells with the molecular characteristics and aging phenotype of somatic cells erased and differentiation potential increased.

[0075] The limitations on the types of mammals and somatic cells in the present invention are the same as those in the above technical solution and will not be elaborated here.

[0076] In the present invention, the mammalian somatic cell expansion method is preferably that the mammalian somatic cells are inoculated into mesenchymal stem cell growth medium 2 (PromoCell, C-28009), cultured at 37°C with 21% O2 and 5% CO2, and when the cell confluence reaches 80%, digested into single cells, inoculated into fresh culture medium for culture, and the culture medium is replaced every 2 days.

[0077] In the present invention, before dedifferentiation induction culture, the mammalian somatic cells are preferably inoculated in DMEM culture medium containing 15% fetal bovine serum by volume and cultured for 1 day, and then dedifferentiation induction culture is performed the next day. The inoculation density of the mammalian somatic cells is preferably (1-5)×10 4 cells / well.

[0078] In the present invention, the dedifferentiation induction culture preferably includes a first stage induction culture and a second stage induction culture. The first stage induction culture is preferably cultured with a culture medium containing the above-mentioned first composition to obtain epithelial cells, and when the confluence value of the epithelial cells is above 80%, the second stage induction culture is entered. Epithelial cells are produced in the culture for 4 to 6 days, and the confluence of the cells reaches 80% within 8 to 20 days of culture. hADSCs are cultured for 8 to 12 days to reach a cell confluence of 80%. hASFs are cultured for 10-20 days to reach a cell confluence of 80%. The second stage induction culture is cultured with a culture medium containing the composition to obtain multiple cell colony cells. The time for the second stage induction culture preferably includes 18 to 22 days, and most preferably 20 days.

[0079] In the present invention, after the second stage of induction culture, the obtained cells gradually lost their proliferation ability after three passages. It is speculated that the epigenetic regulatory small molecules that erase the original identity of the cells may hinder the capture and proliferation ability of this group of cells.

[0080] In order to obtain a large number of dedifferentiated cells, the present invention provides a culture medium for culturing limb bud cells with somatic cells under in vitro conditions, including the culture medium for dedifferentiation of somatic cells and the culture medium for limb bud cells;

[0081] The limb bud cell culture medium includes a KnockOut DMEM culture medium as a basic culture medium, and also includes the following components: 8% to 12% serum replacement, 0% to 15% fetal bovine serum, 0.9% to 1.1% alanyl-glutamine, 0.9% to 1.1% non-essential amino acids, 0.9% to 1.1% penicillin-streptomycin, 0 to 500 μg / ml vitamin C phosphate, 10 to 200 ng / ml basic fibroblast growth factor and the following small molecule compounds: WNT pathway activator 4.5 to 5.5 μM, transforming growth factor-β receptor I / activin-like kinase 5 inhibitor 8 to 12 μM, retinoic acid receptor agonist 1.8 to 2.2 μM, hedgehog signaling pathway activator 0.45 to 0.55 μM, RhoA / ROCK pathway inhibitor 8 to 12 μM, c-Jun amino-terminal kinase inhibitor 1 μM and p300 / CREB binding protein inhibitor 1.8 to 2.2 μM.

[0082] In the present invention, the limb bud cell culture medium comprises a KnockOut DMEM culture medium as a basal culture medium, preferably comprising the following components: 10% serum replacement, 10% fetal bovine serum, 1% alanyl-glutamine, 1% non-essential amino acids, 1% penicillin-streptomycin, 50 μg / ml vitamin C phosphate, 40 ng / ml basic fibroblast growth factor and the following small molecule compounds: WNT pathway activator 5 μM, transforming growth factor-β receptor I / activin-like kinase 5 inhibitor 10 μM, retinoic acid receptor agonist 2 μM, hedgehog signaling pathway activator 0.5 μM, RhoA / ROCK pathway inhibitor 10 μM, c-Jun amino-terminal kinase inhibitor 1 μM and p300 / CREB binding protein inhibitor 2 μM. The limb bud cell culture medium can be used for long-term, homogeneous expansion of limb bud cells.

[0083] In an embodiment of the present invention, a new culture medium, i.e., limb bud cell culture medium, is prepared by re-formulating the epigenetic regulatory small molecules in the second-stage induction solution, which can be used for dedifferentiated cell proliferation culture. At the same time, through the single small molecule compound subtraction experiment, the key small molecules in the limb bud cell culture medium are determined. The absence of any small molecule compound in the limb bud cell culture medium will reduce the total number of human CiLB cells, which indicates that the subtraction of any small molecule will also impair the expression of dedifferentiation-related genes, indicating that the combination of small molecule compounds used to regulate the corresponding signal pathway is essential for maintaining limb bud cells.

[0084] The present invention provides a method for culturing chemically induced limb bud cells CiLB, comprising the following steps:

[0085] The multiple cell colony cells obtained by the dedifferentiation culture method of mammalian somatic cells described in the above technical solution are digested, and the obtained single cells are inoculated into the limb bud cell culture medium in the culture medium described in the above technical solution for subculture, so as to obtain chemically induced limb bud cells CiLB.

[0086] In the present invention, the digestion solvent is preferably an ethylenediaminetetraacetic acid (EDTA) solution containing 0.25% by mass of trypsin. The digestion time is preferably 8 to 12 minutes, more preferably 10 minutes. The volume ratio of the single cell obtained by digestion to the limb bud cell culture medium is preferably 1: (1 to 1.5), more preferably 1: 1.12. The conditions for the subculture are preferably: culture gas 21% O2, 5% CO2, and temperature 37°C. The subculture is preferably replaced with fresh culture medium every two days. When the cell confluence reaches 80% during the subculture, a new round of subculture is carried out. After three subcultures, the unreprogrammed somatic cells disappear, and uniform chemically induced limb bud cells CiLB are obtained.

[0087] In the present invention, the chemically induced limb bud cells CiLB cells have the molecular characteristics of limb bud cells and the potential for osteogenic and chondrogenic differentiation and the ability to repair damage in vivo. The chemically induced limb bud cells CiLB cells are a cell population with increased differentiation potential, having the following characteristics: epithelial-like cells positive for the pluripotency marker Lin28A; and compared with the somatic cells, the expression level of epithelial cell surface markers is increased, and the expression level of somatic cell markers is decreased. The epithelial cell surface markers preferably include at least one of the following: KRT8, KRT18 and KRT19; the somatic cell markers preferably include at least one of the following: VIM, SNAI2, COL5A1, PRKX1, COL6A2, ZEB1, MMP1, TWIST1 and COL1A1.

[0088] The chemically induced limb bud cells CiLB cells prepared by the present invention are purified limb bud cells, and the purity is more than 50%, more preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the pluripotent stem cell population according to the selected limb bud cell markers. The limb bud cell markers preferably include at least one of the following: SALL4, LIN28A, HOXB9, NMYC, PRRX1, MSX1, MSX2, PITX1, HOXA5, HOXA6, HES6, COL2A1, PBX1, HEY1, ISL1, MEIS1, MEIS2.

[0089] In the embodiment of the present invention, q-PCR and immunofluorescence were used to detect the expression of key genes in the chemically induced limb bud cells CiLB cells. The results showed that dedifferentiation-related genes such as LIN28A and SALL4 were highly expressed in human CiLB cells. Compared with somatic cells, the expression levels of genes such as THY1, COL5A1 and COL1A1 in human CiLB cells were significantly reduced. Gene ontology analysis showed that in human CiLB cells, the original characteristics of somatic cells were erased, and the characteristics of mesenchymal development and limb development were enriched.

[0090] In the examples of the present invention, since aging is an intrinsic obstacle to cell dedifferentiation, in order to evaluate the aging of human CiLB cells, the changes in the expression of aging-related genes in human CiLB cells and their parental cells were detected. The results showed that the main aging marker CDKN1A (P21 protein) was significantly downregulated in human CiLB cells, while other aging-related genes, including MMP1, SERPINE1, BCL2L1 and STING1, were also significantly reduced in human CiLB cells. In addition, nuclear laminin B1 (LMNB1) is a structural component of the cell nucleus, and its expression level decreases during aging, while related markers (such as MKI76, CCNB1 and BIRC5) are upregulated in human CiLB cells. The senescence-associated secretory phenotype (SASP) is a key feature of aging, which is related to the high expression of pro-inflammatory and anti-inflammatory related factors, and the results of the examples of the present invention show that the expression level of the SASP gene in human CiLB cells is significantly reduced compared with the parental cells. It can be seen that the chemically induced limb bud cells CiLB cells show lower cell aging characteristics.

[0091] In the present invention, the chemically induced limb bud cells CiLB cells have long-term proliferation ability. In the present embodiment, after 120 hours of culture, human CiLB cells increased by more than 10 times, and more than 90% of the cells were Ki-67 positive, which was consistent with the expression results of highly expressed proliferation-related genes.

[0092] The present invention also verifies whether the chemically induced limb bud cells CiLB cells can effectively maintain their limb bud cell characteristics after long-term passage. The expression of limb bud cell genes in human CiLB cells of three different generations, early, middle and late, was detected respectively. The results showed that even after 32 passages, the expression levels of LIN28A and HOXB9 genes remained consistent in early and middle generations of human CiLB cells, and somatic cell-related genes were still not expressed or expressed at a low level; aging-related genes remained unexpressed or expressed at a low level in long-term passaged human CiLB cells. Human CiLB cells maintain their differentiation ability even after long-term passage. High-resolution G-banding analysis confirmed the normal diploid karyotype, indicating the stability of the genome after more than 30 passages. And the transplantation experiment of immunodeficient mice showed that the transplantation of human CiLB cells does not cause tumor risk.

[0093] In view of the differentiation potential of the chemically induced limb bud cells CiLB, the present invention provides the use of the chemically induced limb bud cells CiLB obtained by the culture method described in the above technical scheme in the preparation of medical materials or drugs for repairing or regenerating damaged organs or tissues in mammals.

[0094] In the present embodiment, since limb bud cells are the progenitor cells of chondrocytes and osteocytes, and eventually develop into limbs or appendages, the chemically induced limb bud cells CiLB were tested for their osteogenic, adipogenic and chondrogenic differentiation abilities, and the results showed that human CiLB cells have the ability to differentiate into osteoblasts and chondrocytes, but no longer have the ability of parent cells to generate fat. This can be confirmed by single-cell sequencing results.

[0095] In the embodiment of the present invention, in order to evaluate the spontaneous differentiation potential of the chemically induced limb bud cells CiLB in animals, experiments such as renal capsule transplantation and subcutaneous transplantation were carried out respectively. The results showed that the chemically induced limb bud cells CiLB had in vivo differentiation potential, and Alcian blue staining in the subcutaneous tissue of immunodeficient mice showed that oval chondrocyte-like cells were present in the graft; at the same time, immunohistochemical staining after renal capsule transplantation showed that oval chondrocyte-like cells in the graft expressed SOX9 and COL2A1. Movat five-color staining showed that cartilage-like cells labeled with Alcian blue and bone-like cell clusters labeled with saffron yellow were present in the same graft, indicating that human CiLB cells have the potential to spontaneously form cartilage and bone in vivo. The results of the rabbit model femoral condyle defect repair experiment showed that after transplanting human CiLB cells, the damaged site was filled with newly formed bones, indicating that they have the ability to promote bone damage repair. In summary, the human CiLB cells prepared by the present invention have strong bone and cartilage differentiation ability.

[0096] The following is a detailed description of a somatic cell dedifferentiation culture medium and a limb bud cell in vitro induction and expansion culture method provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0097] Example 1

[0098] Dedifferentiation and expansion culture methods of human cells

[0099] The embodiments of the present invention respectively take primary human adult adipose-derived mesenchymal stromal cells (hADSCs) derived from human adipose tissue and primary human adult skin fibroblasts (hASFs) derived from adult skin tissue as examples to illustrate the dedifferentiation culture and expansion culture methods.

[0100] 1. Pre-culture of human cells: hADSCs (human adipose-derived mesenchymal stem cells) and hASFs were inoculated in mesenchymal stem cell growth medium 2 (PromoCell, C-28009) and cultured at 37°C in 21% O2 and 5% CO2. When the confluence of somatic cells reached 80%, 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco) was used for digestion. 1.5 x 10 cells were inoculated into a 100 mm culture dish. 6hADSCs or hASFs were cultured per dish and fresh culture medium was replaced the next day; and fresh culture medium was replaced every 2 days thereafter.

[0101] 2. Preparation of Chemically Induced Dedifferentiation Medium

[0102] Medium for inducing dedifferentiation in the first stage (induction medium in the second stage): Basic medium KnockOut DMEM (ThermoFisher, 10829018) also includes the following components: 10% serum replacement (KSR), 10% fetal bovine serum (FBS), 1% alanyl-glutamine (GlutaMax), 1% non-essential amino acids (NEAA), 1% penicillin-streptomycin, 50 μg / ml vitamin C phosphate (Vc2p), 5 mM lithium chloride (LiCl), 1 mM niacinamide (NAM), 2 mg / ml bovine serum albumin (AlbuMax-II) and the following molar concentrations of small molecule compounds: 10 μM WNT pathway activator CHIR99021), 10 μM transforming growth factor-β receptor I / activin-like kinase 5 inhibitor 616452, 2 μM retinoic acid receptor agonist TTNPB, 0.5 μM hedgehog signaling pathway activator SAG, 1 μM vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor linifanib / ABT-869, 2 μM RhoA / ROCK pathway inhibitor Y27632, 2μM telomeric silencing disruptor 1 inhibitor EPZ004777, 0.02μM histone methyltransferase DZNep and 1μM JAK protein inhibitor Ruxolitinib.

[0103] The medium for inducing dedifferentiation in the second stage (the second stage induction solution) is the basic medium KnockOut DMEM, which also includes the following components: 10% serum replacement (KSR), 10% fetal bovine serum (FBS), 1% alanine glutamine (GlutaMax), 1% non-essential amino acids (NEAA), 1% penicillin-streptomycin, 50 μg / ml vitamin C phosphate (Vc2p), 5 mM lithium chloride (LiCl), 1 mM niacinamide (NAM), 100 ng / ml basic fibroblast growth factor (bFGF) and the following molar concentrations of small molecule compounds: 12 μM WNT pathway activator CHIR99021, 10 μM transforming growth factor-β receptor I / activin-like kinase 5 inhibitor 616452, 2 μM retinoic acid receptor agonist TTNPB, 0.5 μM hedgehog signaling pathway activator SAG, 1 μM vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor linifanib / ABT-869, 10 μM RhoA / ROCK pathway inhibitor Y27632, 1μM c-Jun amino-terminal kinase inhibitor JNK-IN-8, 2μM monoamine oxidase inhibitor Tranylcypromine, 10μM DNA methylation inhibitor 5-azacytidine, 1μM histone methyltransferase G9a inhibitor UNC0224, 1μM JAK protein inhibitor Ruxolitinib, 2μM P38 and B-RAF kinase inhibitor damanimide BIRB796, 0.5μM adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitor dorsomorphin and 2μM p300 / CREB binding protein inhibitor SGC-CBP30.

[0104] Limb bud cell culture medium:

[0105] 3. Chemically induced dedifferentiation culture method

[0106] hADSCs or hASFs were plated at 1 × 10 per well. 4 The cells were seeded at a density of 100 cells / mL in a 12-well plate and cultured in DMEM containing 15% fetal bovine serum. The next day, the medium was replaced with the first stage dedifferentiation induction medium.

[0107] In the first stage of induction: hADSCs will produce a layer of epithelial cells within 4-6 days and reach 80% confluence within 8-12 days, and hASFs will reach 80% confluence after 10-20 days of culture. Then the second stage of induction of dedifferentiation medium is replaced for culture.

[0108] Second stage induction: After 8 to 12 days of treatment, multilayered cell colonies appear and continue to grow. The second stage of induction lasts about 20 days.

[0109] The cells obtained after culture were digested and inoculated into fresh medium for inducing dedifferentiation in the second stage for culture, and the number of cells was determined. The above method was repeated for three times.

[0110] 4. Preparation of Limb Bud Cell Culture Medium

[0111] The culture medium is based on KnockOut DMEM and also includes the following components: 10% serum replacement (KSR), 10% fetal bovine serum (FBS), 1% alanine glutamine (GlutaMax), 1% non-essential amino acids (NEAA), 1% penicillin-streptomycin, 50 μg / ml vitamin C phosphate (Vc2p), 40 ng / ml basic fibroblast growth factor (bFGF), and small molecule compounds including the following molar concentrations: 5 μM WNT pathway activator CHIR99021, 10 μM transforming growth factor-β receptor I / activin-like kinase 5 inhibitor 616452, 2 μM retinoic acid receptor agonist TTNPB, 0.5 μM hedgehog signaling pathway activator SAG, 10 μM RhoA / ROCK pathway inhibitor Y27632, 1 μM c-Jun amino-terminal kinase inhibitor JNK-IN-8 and 2 μM p300 / CREB binding protein inhibitor SGC-CBP30.

[0112] 5. Cultivation of Human CiLB Cells

[0113] The cells after the second stage induction were treated with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) for about 10 minutes, and the dissociated cells were inoculated in limb bud cell culture medium at a ratio of 1:12, incubated at 37°C with 21% O2 and 5% CO2, and the culture medium was replaced every two days. The number of cells was measured after the culture. When the cells reached about 85% confluence (about 3-7 days), they were passaged. Within three passages, the unreprogrammed somatic cells disappeared and uniform human CiLB cells were generated. Human CiLB cells were replaced with fresh culture medium every two days and passaged every 4-6 days. The cells were digested into single cells using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) and passaged at a ratio of 1:20 to 1:30. Human CiLB cells can be passaged continuously for more than 30 times.

[0114] Example 2

[0115] Adipogenic and osteogenic induction of human CiLB cells and their parental cells

[0116] 1. Adipogenic induction: Adipogenic differentiation kit (Oricell, HUXMD-90031) was used. Human CiLB cells were cultured at 2×10 4 Cells / cm 2The cells were seeded at a density of 100 μg / mL in gelatin-coated culture plates. When the cell confluence reached 100%, the limb bud cell culture medium was replaced with adipogenic differentiation medium A. After 3 days, adipogenic differentiation medium A was replaced with adipogenic differentiation medium B and cultured for 1 day. Adipogenic differentiation medium A and B were then used alternately every day until lipid droplets appeared at about 16 days. Adipogenic cells were quantitatively analyzed by Oil Red O staining.

[0117] 2. Osteogenic induction: using MSCgo TM Human CiLB cells were cultured at 3×10 4 cells / cm 2 When the cell confluence reached 80% to 90%, the limb bud cell expansion medium was replaced with osteogenic differentiation medium. The osteogenic differentiation medium was replaced every 3 days. After 15 days, the osteoblasts were quantitatively analyzed by Alizarin Red S staining.

[0118] 3. Chondrogenic Induction of Human CiLB Cells and Their Parental Cells

[0119] High-density micromass culture system: Human CiLB cells or hADSCs were digested with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) and cultured at 1×10 7 Resuspend at a density of 10 cells / mL. Drop 20 μL of the cell suspension into each well of a 12-well plate. Incubate the cells at 37°C and 5% CO2 for about 1.5 h to form cell aggregates, and then gently add culture medium for 1 day of culture. Afterwards, replace the culture medium with chondrogenic induction medium: chondrogenic induction basal medium includes high-glucose DMEM containing 1% fetal bovine serum (FBS), 1% insulin-transferrin-selenium (ITS), 1% L-glutamine, 1% sodium pyruvate, 1% non-essential amino acids (NEAA), 1% penicillin-streptomycin, and 50 μg / ml ascorbic acid-2-phosphate (Vc2p). To prepare a chondrogenic induction medium containing chondrogenic factors, cytokines including 30 ng / ml bone morphogenetic protein 4 (BMP4), 10 ng / ml growth differentiation factor 5 (GDF5), 10 ng / ml transforming growth factor β1 (TGFβ1) and 10 ng / ml basic fibroblast growth factor 2 (FGF2) were added to the chondrogenic induction basal medium. During the induction period, the medium was replaced every 3 days. After 7 and 14 days, the cell aggregates were analyzed.

[0120] Aggregate culture system: 1×10 7Individual CiLB cells were resuspended in 1 mL of limb bud cell culture medium and then transferred to a 24-well ultra-low adhesion plate. The next day, the chondrogenic induction medium containing chondrogenic factors was replaced with fresh medium every 3 days. Cell clumps were collected on day 53.

[0121] 4. RNA Sequencing of Human CiLB Cells

[0122] Total RNA was extracted using Direct-zol RNA Mini Prep Kit (Zymo Research, R2053). RNA sequencing library was constructed using NEB Next Ultra RNA Library Prep Kit. The 2×150 bp paired-end library generated by fragmentation and random primers was sequenced using the Illumina HiSeq X Ten system.

[0123] 5. Single-cell RNA sequencing (scRNA-seq)

[0124] The cells were collected and cultured at 1 × 10 6 The density of cells was resuspended in phosphate buffered saline (PBS) containing 0.04% bovine serum albumin (BSA). Using Single cell 3'Library and Gel BeadKitV3.1 (10×Genomics, 1000075) and Chromium Single Cell B Chip Kit (10x Genomics, 1000074), a cell suspension containing 300-600 viable cells per microliter was loaded onto the Chromium single cell controller (10x Genomics) to generate single cell gel beads in emulsions (GEMs) according to the manufacturer's instructions. On the S1000TM Touch Thermal Cycler (Bio Rad), the captured cells were lysed and the released RNA was barcoded by reverse transcription in a single GEM. These steps were performed at 53°C for 45 minutes, followed by 5 minutes at 85°C and maintained at 4°C. Single-cell RNA sequencing libraries were constructed using the Single Cell 3'Library and Gel Bead Kit V3.1 (10x Genomics) and sequenced using an Illumina Novaseq6000 sequencer. The sequencing depth of each cell was at least 1 × 10 5 Reads were paired-end 150 bp (PE150).

[0125] 6. Reverse transcription (RT)-quantitative PCR (qPCR)

[0126] Total RNA was extracted using Direct-zol RNA MiniPrep Kit (Zymo Research, R2053) and Tguide S32 Blood / Cell / Tissue RNA Kit (TIANGEN, DP661). ENA was reversed into cDNA using TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, AT311-03). qPCR was performed on the CFX Connect Real-Time System using KAPA SYBR FAST qPCRKit Master Mix (KAPABiosystems, KM4101) (Bio-Rad). GAPDH was used as the internal reference gene and the ΔΔCt technique was used to analyze the data. The specific steps are as follows:

[0127] 1. RT-qPCR system see Table 1, primers see Table 2.

[0128] Table 1 RT-qPCR system

[0129] Element Volume / μL 2×KAPA SYBR FAST qPCR Master Mix 10 Forward primer 0.4 Reverse primer 0.4 cDNA 1 <![CDATA[ddH2O]]> 8.2

[0130] 2. Reverse transcription-quantitative PCR program settings:

[0131]

[0132] Melting curve program: 65°C to 95°C, heating rate 0.5°C, lasting 5 seconds.

[0133] Table 2 Primer sequences

[0134]

[0135]

[0136] 7. Transplantation experiment of human CiLB cells

[0137] 7.1. Renal capsule transplantation

[0138] Human CiLB cells were resuspended in Matrigel (Corning, CB40234A), and 50 μl of the cell suspension (1×10 8 Cells / ml) were injected under the renal capsule of 8-12 week old immunodeficient mice. Six weeks later, the grafts were surgically removed for examination.

[0139] 7.2. Subcutaneous transplantation

[0140] Human CiLB cells were resuspended in Matrigel (Corning, CB40234A), and 50 μl of the cell suspension (1×10 8 The researchers injected 100 cells / ml of 50 μg of 5 μg of 1 ...

[0141] 7.3. Repair of femoral condyle defect in rabbit model

[0142] New Zealand white rabbits (male, 12 weeks old) were anesthetized, depilated and disinfected. The lateral femoral condyle was exposed through a 2 cm long incision, and a 6 mm wide and 8 mm deep defect was made using a trephine. 100 μl of cell suspension (1×10 8 Cells / ml) were filled into sponges (Medtronic, 400402) and transplanted into the defect site. The incision was then sutured. After 4 weeks, the animals were euthanized, the femurs were removed and micro-CT scans were performed using Skyscan1276 (Bruker, Germany) equipment to detect the femoral condyle defect.

[0143] 8. Flow Cytometry

[0144] Cells were dissociated with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) at 37°C for 3 min and filtered through a 40 μm cell strainer to ensure single-cell suspension. Fluorescence activated cell sorting (FACS) analysis was performed according to the manufacturer's instructions of the BD Cytofix / Cytoperm fixation / permeabilization kit. Flow cytometry was performed using the CytoFlEX S system (Beckman Coulter).

[0145] 9. Immunostaining

[0146] Immunofluorescence: After cells were fixed with 4% paraformaldehyde (DingGuo) for 30 min, they were permeabilized and blocked with phosphate buffered saline (PBS) containing 0.1% Triton X-100 (Sigma-Aldrich) and 2% donkey serum (Jackson Immuno Research) at 37°C for 1 h. The primary antibody was incubated overnight at 4°C in the same buffer at appropriate dilutions. After washing three times with PBS, the cells were incubated with secondary antibodies in PBS containing 2% donkey serum at 4°C overnight. Afterwards, the cells were washed three times with PBS and DNA was stained with DAPI solution (Roche Life Science). Detailed information on the antibodies is shown in Table 3.

[0147] Table 3 Antibody information

[0148]

[0149]

[0150] 10. Immunohistochemistry: Serial tissue sections of renal capsule transplants and cartilage pellet sections were examined by hematoxylin-eosin (HE), Alcian blue, Movat's pentachrome, and primary antibody staining. Serial tissue sections of subcutaneous transplants were examined by HE and Alcian blue. Serial tissue sections of femoral condylar defects were examined by Safranin O-fast green staining and human nuclear antigen (Sci-tech). Primary antibody descriptions are shown in the Key Resources table.

[0151] 11. Chromosome Karyotype Analysis

[0152] High-resolution G-banding (400G-500G) karyotype studies were performed at Beijing Jiaen Hospital and analyzed using CytoVision (Leica). At least 20 metaphases were examined for each analysis. The number of chromosomes and the presence of chromosome structural abnormalities were examined.

[0153] 12. Teratoma formation

[0154] For teratoma development, harvest approximately 3 × 10 6 Individual CiLB cells were treated with trypsin-ethylenediaminetetraacetic acid (EDTA), resuspended in Matrigel, and injected subcutaneously into 2- to 3-month-old male immunodeficient NPG mice (Vitalstar Biotechnology, Beijing). Teratomas were analyzed 8 weeks after transplantation. All animal studies were authorized by the Institutional Animal Care and Use Committee of Peking University and were performed in accordance with the Peking University Animal Care Guidelines.

[0155] 12.RNA-seq analysis

[0156] The raw sequencing data were quality controlled using FastQC. Then, the raw reads were trimmed using “ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:7:1:true LEADING:3TRAILING:3SLIDINGWINDOW:4:15MINLEN:36” to remove the detected adapters. Next, the processed reads were mapped to the human reference genome hg19 using STAR. The read counts of the genes were calculated using the program featureCounts of the Subread package. The gene count matrix was normalized for visualization using the R package DESeq2.

[0157] 13. Single-cell RNA-seq analysis

[0158] Single-cell RNA-seq reads from all samples were mapped to the human reference genome hg19 using Cellranger v3.1.0. After quantifying the expression of transcripts in each barcode using Cellranger, the output filtered matrix was loaded into the R environment and preprocessed using Seurat.

[0159] 14. Single-cell RNA sequencing data preprocessing

[0160] First, we performed quality control, and cells that met one of the following conditions were removed: (1) total UMI counts were less than 1000 or greater than 50000; (2) the number of detected genes did not exceed 500; (3) the percentage of mitochondrial genes was greater than 20%. Then, we checked the doublet score using the computeDoubletDensity function in the R package scDblFinder and removed cells with significantly high doublet scores. Next, we followed the standard Seurat process for normalization, feature selection, scaling, PCA dimensionality reduction, clustering, and UMAP dimensionality reduction. Seurat's FindMarkers function was used to calculate differentially expressed genes between samples. GO analysis was performed using the R package clusterProfiler.

[0161] 15. Integration with public datasets

[0162] To better compare our human limb buds with single-cell sequencing data of human long bones (GSE143753 and E-MTAB-8813) and single-cell sequencing data of early organogenesis in human embryos (GSE157329), we performed batch effect correction and integration analysis using the R package Harmony. Specifically, to ensure that the mapped genomes were consistent, we first remapped our data to the human reference genome hg38 using Cellranger v3.1.0 and performed the same preprocessing as described above. Then, different batches were set according to the samples to remove the genomic background of different cell lines. The batches set above will be used as parameters of the RunHarmony function. UMAP dimensionality reduction was performed using the first 30 adjusted principal components. Hierarchical clustering was performed using the function BuildClusterTree, which constructed a phylogenetic tree connecting the "average" cells of each identity class.

[0163] 16. Cell differentiation trajectory analysis

[0164] We performed trajectory inference and pseudo-time analysis on our samples using Monocle 2, a tool that performs pseudo-time sorting of cells based on transcriptome similarity. Specifically, we used human CiLB cells and cells after 7 days of differentiation as input. Pseudo-time trajectories were constructed using the top 1000 differentially expressed genes. Branch-specific gene expression patterns were isolated using the Monocle2 BEAM statistical test. GO analysis of branch-specific genes was performed using the enrichGO function of the R package clusterProfiler. Heatmaps and gene expression pattern plots were drawn using the correlation function of Monocle2.

[0165] result

[0166] In the early stages of chemical reprogramming, a population of cells with high plasticity appears transiently. The generation of this population of cells requires two chemically induced dedifferentiation steps, including the first and second stages (see Figure 1 In the first stage, the original identity of the somatic cells is erased, laying the foundation for further dedifferentiation; when the cells are switched to the induction solution in the second stage, they respond quickly and activate a series of genes in the early embryonic development process. This technology is called chemically induced dedifferentiation (CID). In the present invention, the molecular characteristics of chemically induced dedifferentiated cells are most obvious at the end of the second stage ( Figure 2 ). RNA-Seq data showed that these chemically induced dedifferentiated cells not only erased the properties and aging characteristics of the original cells, but also reactivated the molecular characteristics of limb bud cells ( Figure 3 ), which are called human chemically induced limb bud cells (human chemically induced limb bud cells, hereinafter referred to as "human CiLB cells").

[0167] In order to determine whether human CiLB cells can be expanded and cultured, the second stage induction dedifferentiation medium was first tested. Although the second stage induction dedifferentiation medium is essential for inducing cell dedifferentiation, human CiPS cells cannot be maintained under this condition and gradually lose their proliferation ability after three passages ( Figure 4 ). It is speculated that epigenetic regulatory small molecules that erase the original identity of cells may hinder the capture and expansion of this group of cells. Therefore, the epigenetic regulatory small molecules in the second stage induction solution, including monoamine oxidase inhibitor Tranylcypromine, DNA methylation inhibitor 5-azacytidine and histone methyltransferase G9a inhibitor UNC0224, were formulated into a new culture medium called limb bud cell culture medium. In the limb bud cell culture medium, human CiPS cells can continue to proliferate ( Figure 4 ).

[0168] Then, a small molecule single reduction experiment (based on the limb bud cell culture medium, each treatment medium lacks a small molecule compound, see Figure 5 ) in order to identify the key small molecules in the limb bud cell culture medium. The results showed that removing any small molecule would reduce the total number of human CiLB cells ( Figure 5 Middle left), especially the Rho / ROCK signaling pathway inhibitor Y27632, the WNT pathway activator CHIR99021, and the transforming growth factor-β receptor I / activin-like kinase 5 inhibitor 616452. In addition, the reduction of any small molecule alone also impaired the expression of dedifferentiation-related genes ( Figure 5 Middle right), indicating that the combined use of small molecules to regulate corresponding signaling pathways is essential for maintaining limb bud cells.

[0169] To further verify the properties of human CiLB cells, we first used q-PCR and immunofluorescence to detect the expression of key genes in limb bud cells and parental cells. The results showed that dedifferentiation-related genes such as LIN28A and SALL4 were highly expressed in human CiLB cells ( Figure 6 , where 1013, 06918, 0325, etc. represent different donor numbers). Gene ontology analysis showed that somatic genes such as THY1, COL5A1, and COL1A1 were highly expressed in parental somatic cells, but their expression levels were significantly reduced in human CiLB cells ( Figure 7 ). Gene ontology analysis showed that in human CiLB cells, the original characteristics of somatic cells were erased, and the characteristics of mesenchymal development and limb development were enriched ( Figure 3 In addition, the present invention demonstrates that human CiLB cells can be prepared from human adipose-derived mesenchymal stromal cells (hADSCs) and adult human skin dermal fibroblasts (hASFs) obtained from different donors ( Figure 5 , Figure 7 and Figure 8 ).

[0170] To further analyze the similarities between human CiLB cells and limb bud cells at the molecular level, single-cell transcriptome sequencing was performed. These data were integrated with a human embryonic dataset of Carnegie stages 12-16 (4-6 weeks of gestation) (including all embryonic tissues and organs). Unified manifold approximation and projection (UMAP) and cluster analysis showed that human CiLB cells are highly similar to human embryonic limb bud cells ( Fig. 9 ). Given that gene ontology analysis also showed that highly expressed genes in human CiLB cells were enriched in embryonic limb development characteristics ( Fig.10), and integrated the sequencing data with the sequencing data of three limb bud cell subpopulations (named limb bud 1, limb bud 2, and limb bud 3) of human embryos that developed to the fifth week, as well as the differentiated cell types of the eighth week. The analysis results showed that human CiLB cells were highly similar to embryonic limb bud cells at the single-cell transcriptome level ( Fig.11 Human CiLB cells expressed genes related to limb bud development, such as MSX1, HOXA9, and MEIS2 ( Fig.11 ), which is more similar to limb bud 1 in the embryonic limb bud at 30 days after fertilization.

[0171] Limb bud cells are the progenitors of chondrocytes and osteocytes, which eventually develop into limbs or appendages. In order to further study the bone and cartilage generation ability of human CiLB cells, the bone, cartilage and fat directional differentiation ability of human CiLB cells was first tested in vitro. The results showed that compared with the parental cells hADSCs and hASFs, the osteogenic ability of human CiLB cells was significantly improved ( Fig.12 ). In contrast, dedifferentiated human CiLB cells no longer have the ability to produce fat ( Fig.12 ).

[0172] Then, human CiLB cells were 3D differentiated under chondrogenic conditions. Alcian blue staining and immunohistochemistry showed that the differentiation products of human CiLB cells secreted cartilage-specific acidic mucopolysaccharides and upregulated the expression of chondrocyte-related genes ( Fig.13 In addition, micromass chondrogenic differentiation results showed that human CiLB cells were able to generate cartilage nodules, showing clear chondrocyte morphology ( Fig.14 ). SOX9, a key transcription factor for chondrogenesis, and COL2A1, a key marker for hyaline cartilage, are highly expressed in cells differentiated from human CiLB cells ( Fig.14 ). Single-cell sequencing technology was used to analyze the changes in gene expression of human CiLB cells after 7 days of in vitro differentiation. As differentiation progressed, the molecular characteristics of bone and cartilage production gradually increased ( Fig.15 ).

[0173] To evaluate the spontaneous differentiation potential of human CiLB cells in vivo, human CiLB cells were seeded onto collagen sponges and transplanted into the subcutaneous tissue of immunodeficient mice. Alcian blue staining showed the presence of oval chondrocyte-like cells in the grafts ( Fig.16 ). In addition, human CiLB cells were transplanted under the renal capsule of immunodeficient mice. After 6 weeks, the grafts were fixed and sectioned. Immunohistochemical staining showed that oval chondrocytes in the grafts expressed SOX9 and COL2A1 ( Fig.16 Further staining with Movat's five-color method showed that in the same graft, there were both cartilage-like cells labeled with Alcian blue and osteoid cells labeled with saffron yellow ( Fig.16 ), indicating that human CiLB cells have the potential to spontaneously form cartilage and bone in vivo. To further test the function of human CiLB cells, human CiLB cells were transplanted into a rabbit femoral condyle defect model. The results showed that after transplantation of human CiLB cells, the damaged area was filled with newly formed bone ( Fig.17 ), indicating that they have the ability to promote bone repair. These results together indicate that human CiLB cells have a strong osteo- and chondrogenic differentiation capacity.

[0174] Since senescence is an intrinsic barrier to cell dedifferentiation, changes in senescence-related gene expression in human CiLB cells and their parental cells were analyzed. Notably, the major senescence marker CDKN1A (P21 protein) was significantly downregulated in human CiLB cells ( Fig.18 ). The expression levels of other aging-related genes, including MMP1, SERPINE1, BCL2L1, and STING1, were also significantly reduced in human CiLB cells ( Fig.18 Interestingly, nuclear lamin B1 (LMNB1), a structural component of the cell nucleus, is expressed at reduced levels during aging but is upregulated in human CiLB cells that highly express cell cycle-related markers such as MKI76, CCNB1, and BIRC5 ( Fig.18 ). The senescence-associated secretory phenotype (SASP) is a key feature of aging and is associated with high expression of pro-inflammatory and anti-inflammatory factors. We further examined the expression of SASP-related genes and found that the expression levels of SASP genes in human CiLB cells were significantly reduced compared with parental cells ( Fig.18 ). These results indicate that chemically induced dedifferentiation significantly reduces cellular senescence characteristics.

[0175] Then, the present invention also detected the long-term proliferation ability of human CiLB cells. Although the parental cells gradually lost their proliferation ability, human CiLB cells always maintained a stable proliferation ability ( Fig.19 We observed that after 120 h of culture, the number of human CiLB cells increased more than 10-fold ( Fig.19 Flow cytometry analysis showed that more than 90% of the cells were Ki-67 positive ( Fig.19 ), confirming their proliferation ability, which is consistent with the results that human CiLB cells highly express proliferation-related genes ( Fig.18 ).

[0176] In order to comprehensively evaluate whether human CiLB cells can effectively maintain their limb bud cell characteristics after long-term passage in limb bud cell culture medium, the expression of limb bud cell genes in human CiLB cells of three different passages, early, middle and late, was deeply analyzed ( Fig. 20The results showed that even after 32 passages, the expression levels of these genes remained consistent in early and late passages of human CiLB cells ( Fig. 20 In long-term passaged human CiLB cells, somatic cell-related genes are still not expressed or expressed at low levels ( Fig. 20 Flow cytometry analysis showed that human CiLB cells maintained homogeneity after long-term passaging, with more than 90% of cells expressing LIN28A and HOXB9 ( Fig. 20 ). Aging-related genes remain unexpressed or expressed at low levels in long-term cultured human CiLB cells ( Fig. 20 In addition, human CiLB cells retain their differentiation capacity even after long-term passage ( Fig.21 ). High-resolution G-banding analysis confirmed a normal diploid karyotype ( Fig.21 ), indicating the stability of the genome after more than 30 passages. In addition, human CiLB cells were transplanted subcutaneously into immunodeficient mice and no tumors developed within 8 weeks ( Fig.21 These results indicate that human CiLB cells can be passaged for a long time, maintaining genomic stability while maintaining a homogenous cell population and differentiation potential. This is critical for large-scale expansion for further clinical application.

[0177] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A somatic cell dedifferentiation culture composition, characterized in that: The invention comprises 0-500 mg vitamin C phosphate, 0-10 mmol lithium chloride, 0-5 mmol niacinamide, 0-2.2 g bovine serum albumin, 0-500 mg basic fibroblast growth factor and any combination of the following small molecule compounds: 0.5-15 μmol WNT pathway activator, 5-50 μmol transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 0.5-5 μmol retinoic acid receptor agonist, 0.45-5 μmol hedgehog signaling pathway activator, 0.3-10 μmol vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 0.5-12 μmol RhoA / ROCK pathway inhibitor, 0-5 μmol telomere silencing interferon 1 inhibitor, 0-2.5 μmol c-Jun amino-terminal kinase inhibitor, 0-2.2 μmol monoamine oxidase inhibitor, 0-11 μmol DNA methylation inhibitors, 0-1.1 μmol histone methyltransferase G9a inhibitors, 0-1.0 μmol histone methyltransferase EZH2 inhibitors, 0.1-5 μmol JAK protein inhibitors, 0-2.2 μmol P38 and B-RAF kinase inhibitors, 0-0.55 μmol adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitors, and 0-5 μmol p300 / CREB binding protein inhibitors; The bovine serum albumin and basic fibroblast growth factor are not simultaneously 0; The small molecule compounds are not 0 at the same time.

2. The somatic cell dedifferentiation culture composition according to claim 1, characterized in that: comprising a first composition and a second composition; The first composition comprises 0-500 mg vitamin C phosphate, 0-10 mmol lithium chloride, 0-5 mmol niacinamide, 0.5-2.2 g bovine serum albumin and any combination of the following small molecule compounds: 0.5-15 μmol WNT pathway activator, 5-50 μmol transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 0.5-5 μmol retinoic acid receptor agonist, 0.45-5 μmol hedgehog signaling pathway activator, 0.3-10 μmol vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 0.5-12 μmol RhoA / ROCK pathway inhibitor, 0.5-5 μmol telomere silencing interferon 1 inhibitor, 0.01-1.0 μmol histone methyltransferase EZH2 inhibitor and 0.1-5 μmol JAK protein inhibitor; The second composition comprises 0-500 mg vitamin C phosphate, 0-10 mmol lithium chloride, 0-5 mmol niacinamide, 10-500 mg basic fibroblast growth factor and any combination of the following small molecule compounds: 0.5-15 μmol WNT pathway activator, 5-50 μmol transforming growth factor-β receptor I / activin-like kinase 5 inhibitor, 0.5-5 μmol retinoic acid receptor agonist, 0.45-5 μmol hedgehog signaling pathway activator, 0.5-10 μmol vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor, 0.5-12 μmol RhoA / ROCK pathway inhibitor, 0.1-2.5 μmol c-Jun amino-terminal kinase inhibitor, 0.5-2.2 μmol monoamine oxidase inhibitor, 0.01-11 μmol DNA methylation inhibitor, 0.01-1.1 μmol histone methyltransferase G9a inhibitor, 0.1-5 μmol JAK protein inhibitor, 0.5-2.2 μmol P38 and B-RAF kinase inhibitors, 0.05-0.55 μmol adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitors and 0.1-5 μmol p300 / CREB binding protein inhibitors.

3. The somatic cell dedifferentiation culture composition according to claim 1 or 2, characterized in that: The WNT pathway activator comprises at least one of the following: CHIR99021, SB-216763; CHIR 99021 trihydrochloride, BIO-acetone oxime, GSK-3β inhibitor XII, GSK-3 inhibitor XV, TD114-2, TD114-3, IM12, CHIR98014 and SB-415286; The transforming growth factor-β receptor I / activin-like kinase 5 inhibitor includes at least one of the following: 616452, A83-01, SB431542, SB 505124, GW 788388, doxorubicin and SB 525334; The retinoic acid receptor agonist comprises at least one of the following: TTNPB, Ch55 and AM580; The hedgehog signaling pathway activator comprises at least one of the following: SAG, purmorphamine, Hh-Ag1.5 and human SHH; The vascular endothelial growth factor receptor / platelet-derived growth factor receptor inhibitor includes ABT-869; The RhoA / ROCK pathway inhibitors include at least one of the following: Y27632, Tzv, fasudil, and thiazovivin; The telomeric silencing interferon 1 inhibitor includes at least one of the following: EPZ004777, EPZ5676, EPZ5676 and SGC0946; The c-Jun amino-terminal kinase inhibitor includes JNK-IN-8; The monoamine oxidase inhibitors include tranylcypromine; The DNA methylation inhibitor includes 5-azacytidine; The histone methyltransferase G9a inhibitor includes UNC0224; The histone methyltransferase DZNep includes UNC0224; The JAK protein inhibitor includes ruxolitinib; The P38 and B-RAF kinase inhibitors include damanimod BIRB796; The adenosine monophosphate-dependent protein kinase and bone morphogenetic protein inhibitors include dorsomorphin; The p300 / CREB binding protein inhibitor includes SGC-CBP30.

4. A culture medium for somatic cell dedifferentiation culture, characterized in that: A KnockOut DMEM medium is used as a basic medium, and further comprises the following components: 8% to 12% serum replacement by volume, 8% to 12% fetal bovine serum by volume, 0.8% to 1.2% alanyl-glutamine by weight, 0.8% to 1.2% non-essential amino acids by weight, 0.8% to 1.2% penicillin and streptomycin by weight, and the composition for somatic cell dedifferentiation culture according to any one of claims 1 to 3; The concentration of the composition is calculated in terms of vitamin C phosphate, and the final concentration of vitamin C phosphate in the culture medium is 0 to 500 mg / L.

5. Use of the composition for somatic cell dedifferentiation culture according to any one of claims 1 to 3 or the culture medium according to claim 4 in mammalian somatic cell dedifferentiation culture.

6. A method for dedifferentiation culture of mammalian somatic cells, characterized in that: The following steps are involved: Dedifferentiation-inducing and culturing mammalian somatic cells using a culture medium containing the composition of any one of claims 1 to 3 to obtain cells with erased somatic cell molecular characteristics and aging phenotypes and increased differentiation potential; Preferably, the dedifferentiation induction culture includes a first stage induction culture and a second stage induction culture; The first stage of induction culture is to culture using a culture medium containing the first composition of claim 2 to obtain epithelial cells, and when the confluence of the epithelial cells is above 80%, enter the second stage of induction culture; The second stage of induction culture is carried out using a culture medium comprising the second composition according to claim 2 to obtain multiple cell colony cells.

7. A culture medium for culturing limb bud cells from somatic cells under in vitro conditions, characterized in that: Comprising the somatic cell dedifferentiation culture medium and the limb bud cell culture medium as claimed in claim 4; The limb bud cell culture medium includes a KnockOut DMEM culture medium as a basic culture medium, and also includes the following components: 8% to 12% serum replacement, 0% to 15% fetal bovine serum, 0.9% to 1.1% alanyl-glutamine, 0.9% to 1.1% non-essential amino acids, 0.9% to 1.1% penicillin-streptomycin, 45 to 55 μg / ml vitamin C phosphate, 10 to 200 ng / ml basic fibroblast growth factor and the following small molecule compounds: WNT pathway activator 0.5 to 15 μM, transforming growth factor-β receptor I / activin-like kinase 5 inhibitor 2 to 20 μM, retinoic acid receptor agonist 0.5 to 5 μM, hedgehog signaling pathway activator 0.1 to 1 μM, RhoA / ROCK pathway inhibitor 2 to 20 μM, c-Jun amino-terminal kinase inhibitor 0.2 to 5 μM and p300 / CREB binding protein inhibitor 0.5 to 5 μM.

8. A method for culturing chemically induced limb bud cells CiLB, comprising the following steps: The multiple cell colony cells obtained by the dedifferentiation culture method of mammalian somatic cells according to claim 6 are digested, and the obtained single cells are inoculated into the limb bud cell culture medium in the culture medium according to claim 7 for subculture, thereby obtaining chemically induced limb bud cells CiLB.

9. The chemically induced limb bud cells CiLB obtained by the culture method of claim 8.

10. Use of the chemically induced limb bud cells CiLB according to claim 9 in preparing medical materials or drugs for repairing or regenerating damaged organs or tissues in mammals.

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