Method for inducing somatic cells into pluripotent stem cells and pluripotent stem cells

Abstract

The invention relates to the technical field of cell culture, and particularly discloses a method for inducing somatic cells into pluripotent stem cells and the pluripotent stem cells. Comprising the following steps: sequentially carrying out induction culture on somatic cells by adopting a culture medium I, a culture medium II, a culture medium III and a culture medium IV to obtain the pluripotent stem cells, the I-IV culture medium adopts a DMEM high-glucose culture medium as a basic culture medium; each of the culture medium I and the culture medium II comprises the following components: a retinoic acid receptor stimulant, a glycogen kinase inhibitor, a TGF (Transforming Growth Factor) beta inhibitor, a selective inhibitor of cytoskeleton rearrangement related coiled-coil protein kinase, a receptor tyrosine kinase inhibitor, an SMO (Sequence Matrix Oxidase) stimulant, a Janus kinase 1 inhibitor, a Janus kinase 2 inhibitor and a Menin-MLL interaction inhibitor. The method for inducing the somatic cells into the pluripotent stem cells has the advantages of being low in cost, easy to operate, high in transformation efficiency and the like, and the human somatic cells can be efficiently and stably induced and recompiled into the pluripotent stem cells.

Description

Technical Field

[0001] The present application relates to the field of cell culture technology, and in particular to a method for inducing somatic cells into pluripotent stem cells and pluripotent stem cells. Background Art

[0002] During the development of organisms, maintenance of homeostasis, and disease progression, the dynamic changes in cell identity are closely dependent on complex external signals from the microenvironment. These signals not only shape the initial identity of cells, but also continuously regulate their maintenance and transformation. In the plant kingdom and some invertebrates, stimuli from the external environment can directly stimulate the ability of cells to differentiate and regenerate, showing amazing plasticity. However, for mammalian cells, especially highly differentiated human somatic cells, it is particularly difficult to rely solely on external perturbations to awaken their plasticity potential.

[0003] In recent years, in order to overcome this problem, scientists have used small molecule compounds as chemical perturbation tools to successfully induce mouse somatic cells into pluripotent stem cells. This discovery not only verifies the potential of chemicals in cell fate reprogramming, but also reveals the feasibility of precisely manipulating cell fate by regulating multiple cell signaling pathways and chromatin states. Despite this, there are still many challenges to achieving efficient and stable reprogramming of human somatic cells into pluripotent cells.

[0004] Therefore, it is necessary to continue to develop a new small molecule composition for inducing reprogramming of human somatic cells into pluripotent stem cells, so as to achieve efficient and stable conversion of human somatic cells into pluripotent stem cells. Summary of the invention

[0005] In order to improve the efficiency and stability of the transformation of human somatic cells into pluripotent cells, the present application provides a method for inducing somatic cells into pluripotent stem cells and pluripotent stem cells.

[0006] In a first aspect, the present application provides a method for inducing somatic cells into pluripotent stem cells, using the following technical solution: A method for inducing somatic cells into pluripotent stem cells, comprising the following steps: Inducing and culturing the somatic cells in sequence using medium I, medium II, medium III, and medium IV to obtain pluripotent stem cells; The culture media I to IV all use DMEM high-glucose culture media as the basic culture media; The medium I and medium II both include the following components: retinoic acid receptor agonist, glycogen kinase inhibitor, TGFβ inhibitor, selective inhibitor of cytoskeleton rearrangement-related coiled-coil protein kinase, receptor tyrosine kinase inhibitor, SMO agonist, Janus kinase 1 and Janus kinase 2 inhibitor, Menin-MLL interaction inhibitor; The retinoic acid receptor agonist is BMS961, The glycogenokinase inhibitor is CHIR 98014, The TGFβ inhibitor is SB-431542, The selective inhibitor of the cytoskeletal rearrangement-related coiled-coil protein kinase is Y27632, The receptor tyrosine kinase inhibitor is RG3635, The SMO agonist is Cyclopamine, The Janus kinase 1 and Janus kinase 2 inhibitor is Baricitinib, The Menin-MLL interaction inhibitor is MI503.

[0007] The present application provides a method for inducing somatic cells into pluripotent stem cells, which uses culture medium I to IV to induce and culture human somatic cells in sequence, and by regulating multiple key signal pathways and chromatin modification processes in the cells, the original fate restrictions of the cells are broken, and their efficient and stable transformation to a pluripotent state is promoted. Among them, the use of culture medium I for the first stage of culture can transform somatic cells into single-layer epithelial-like cells; the use of culture medium II for the second stage of culture can produce plastic state cells with a regenerative program; the use of culture medium III for the third stage of culture can establish an initial pluripotency network, form extraembryonic endoderm characteristics, and obtain extraembryonic endoderm characteristic-like cells; the use of culture medium IV for the fourth stage of culture can completely establish a pluripotency network and obtain primary pluripotent stem cells. In summary, the method for inducing somatic cells into pluripotent stem cells provided by the present application has the advantages of low cost, simple operation, high conversion efficiency, good stability, etc., and provides new ideas and tools for the field of cell fate reprogramming, thereby promoting the development of fields such as regenerative medicine, disease model construction and drug screening.

[0008] Optionally, the medium I further comprises the following components: FBS, GlutaMAX TM , NEAA, penicillin-streptomycin, 2-mercaptoethanol, VC, niacinamide, histone methyltransferase inhibitors, S-adenosylhomocysteine ​​hydrolase inhibitors; The histone methyltransferase inhibitor is EPZ004777, and the S-adenosyl homocysteine ​​hydrolase inhibitor is 3-deazaadenine A.

[0009] Optionally, the medium II further comprises the following components: FBS, GlutaMAX TM , NEAA, penicillin-streptomycin, 2-mercaptoethanol, VC, NAM, bFGF, c-Jun kinase inhibitors, DNA methyltransferase inhibitors, monoamine oxidase inhibitors, EHMT2 inhibitors, BMP receptor / AMPK inhibitors, CREB binding protein and CREB binding protein bromodomain inhibitors; The c-Jun kinase inhibitor is AS601245, The DNA methyltransferase inhibitor is Vidaza, The monoamine oxidase inhibitor is tranylcypromine, The EHMT2 inhibitor is UNC0224, The BMP receptor / AMPK inhibitor is Dorsomorphin, and the CREB binding protein and CREB binding protein bromodomain inhibitor is SGC-CBP30.

[0010] Optionally, the III medium further comprises the following components: N2 supplement, B27 supplement, GlutaMAXTM, NEAA, penicillin-streptomycin, 2-mercaptoethanol, AlbuMAX TM -II, HRG, glycogen kinase inhibitors, TGFβ inhibitors, selective inhibitors of cytoskeletal rearrangement-associated coiled-coil protein kinases, histone deacetylase inhibitors, mitogen-activated protein kinase inhibitors, histone methyltransferase inhibitors, S-adenosylhomocysteine ​​hydrolase inhibitors, monoamine oxidase inhibitors.

[0011] Optionally, the IV culture medium includes DMEM high-glucose medium and the following components: N2 supplement, B27 supplement, GlutaMAXTM, NEAA, penicillin-streptomycin, 2-mercaptoethanol, HRG, glycogen kinase inhibitor, selective inhibitor of cytoskeletal rearrangement-associated coiled-coil protein kinase, mitogen-activated protein kinase inhibitor, Wnt inhibitor, B-Raf inhibitor, and histone deacetylase inhibitor.

[0012] Optionally, the histone deacetylase inhibitor is valproic acid; the Wnt inhibitor is Wnt-C59, and the B-Raf inhibitor is PLX4032.

[0013] Optionally, the mitogen-activated protein kinase inhibitor is selected from Mirdametinib, MEK162 and LY2228820.

[0014] In some embodiments, the mitogen-activated protein kinase inhibitor can be a mixture of Mirdametinib and MEK162, a mixture of Mirdametinib and LY2228820, or a mixture of MEK162 and LY2228820.

[0015] In a specific embodiment, the mitogen-activated protein kinase inhibitor may also be Mirdametinib, MEK162 or LY2228820.

[0016] Optionally, the mitogen-activated protein kinase inhibitor is a mixture of Mirdametinib and LY2228820 in a molar ratio of 1:(0.25-0.45).

[0017] In the present application, the mitogen-activated protein kinase inhibitor can block or reduce the activity of mitogen-activated protein kinase, thereby affecting the signal transduction process in which it participates, and then regulating the proliferation, differentiation, apoptosis and other life activities of the regulation cells. Through experimental exploration, it was found that the present application further uses a mixture of Mirdametinib and LY2228820 as a mitogen-activated protein kinase inhibitor, and the prepared culture medium can make the conversion efficiency of human somatic cells to pluripotent stem cells higher, and the expression level of the specific antibody OCT4 of the obtained pluripotent stem cells can reach more than 90%, the expression level of the specific antibody SOX2 can reach more than 32%, and the expression level of the specific antibody NANOG can reach more than 94%.

[0018] In some embodiments, the molar ratio of Mirdametinib to LY2228820 can be 1: (0.25-0.30), (0.25-0.35), (0.25-0.40), (0.25-0.45), (0.30-0.35), (0.30-0.40), (0.30-0.45), (0.35-0.40), (0.35-0.45) or (0.40-0.45).

[0019] In a specific embodiment, the molar ratio of Mirdametinib to LY2228820 can also be 1:0.25, 1:0.30, 1:0.35, 1:0.40 or 1:0.45.

[0020] Optionally, the culture conditions of the medium I are: 37° C., 5% O 2 , 5% CO 2 , induction culture for 8-10 days, and the culture medium is changed every 3-4 days; The culture conditions of the medium II, medium III and medium IV are: 37°C, 5% CO2, and the culture medium is changed every 3-4 days; the medium II is induced for 10-12 days, the medium III is induced for 12-16 days, and the medium IV is induced for 12-14 days.

[0021] Optionally, the somatic cells are human somatic cells separated from human donor tissues, including but not limited to human adipose mesenchymal stem cells and human skin fibroblasts.

[0022] In a second aspect, the present application provides a method for inducing somatic cells into pluripotent stem cells to obtain pluripotent stem cells.

[0023] In the present application, the pluripotent stem cells obtained by the above method can be expanded for more than 20 generations, for example, up to 25, 30, 35, 40, 41, 42 generations, and can proliferate with a doubling time similar to that of human mesenchymal stem cells.

[0024] In summary, this application has the following beneficial effects: 1. The present application provides a method for inducing somatic cells into pluripotent stem cells. The method uses four new culture media to induce and culture human somatic cells, so that they can be induced to be reprogrammed into pluripotent stem cells efficiently and stably. This method provides new ideas and tools in the field of cell fate reprogramming, thereby promoting the development of regenerative medicine, disease model construction, and drug screening.

[0025] 2. The small molecules used in the culture medium of the present application are domestically produced, so the preparation cost of pluripotent stem cells is relatively low and can be widely promoted and used.

[0026] 3. The present application further uses a mixture of Mirdametinib and LY2228820 in a molar ratio of 1: (0.25-0.45) as a mitogen-activated protein kinase inhibitor, which can increase the expression levels of the specific antibodies OCT4, SOX2 and NANOG in the obtained pluripotent stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a cell morphology diagram of human adipose-derived mesenchymal stem cells (hADMSC) before induction in Example 1; Figure 2 is a cell morphology diagram of epithelial cells obtained in the first stage of Example 1; Figure 3 is a cell morphology diagram of cells in a plastic state obtained in the second stage of Example 1; Figure 4 This is a cell morphology diagram of the extraembryonic endoderm-like cells obtained in the third stage of Example 1; Figure 5 This is a cell morphology diagram of the pluripotent stem cells obtained in the fourth stage in Example 1. DETAILED DESCRIPTION

[0028] The present application provides a method for inducing somatic cells into pluripotent stem cells, comprising the following steps: (1) Inducing and culturing human somatic cells in medium No. 1 at 37° C., 5% O 2 , 5% CO 2 for 8-10 days, changing the culture medium every 3-4 days to obtain epithelial-like cells; The medium No. 1 uses DMEM high-glucose medium as the basic medium and includes the following components: FBS, GlutaMAX TM , NEAA, penicillin-streptomycin, 2-mercaptoethanol, VC, niacinamide, histone methyltransferase inhibitor (EPZ004777), S-adenosylhomocysteine ​​hydrolase inhibitor (3-deazaadenine A), retinoic acid receptor agonist (BMS961), glycogen kinase inhibitor (CHIR 98014), TGFβ inhibitor (SB-431542), selective inhibitor of cytoskeletal rearrangement-associated coiled-coil protein kinase (Y27632), receptor tyrosine kinase inhibitor (RG3635), SMO agonist (Cyclopamine), Janus kinase 1 and Janus kinase 2 inhibitor (Baricitinib), Menin-MLL interaction inhibitor (MI503); (2) replacing the epithelial culture cells obtained in step (1) with medium II, inducing and culturing them at 37° C., 5% CO 2 for 10-12 days, changing the culture medium every 3-4 days, and obtaining cells in a plastic state; The medium II uses DMEM high-glucose medium as the basic medium and includes the following components: FBS, GlutaMAX TM, NEAA, penicillin-streptomycin, 2-mercaptoethanol, VC, NAM, bFGF, c-Jun kinase inhibitor (AS601245), DNA methyltransferase inhibitor (Vidaza), monoamine oxidase inhibitor (tranylcypromine), EHMT2 inhibitor (UNC0224), BMP receptor / AMPK inhibitor (Dorsomorphin), CREB binding protein and CREB binding protein bromodomain inhibitor (SGC-CBP30), retinoic acid receptor agonist (BMS961), glycogen kinase inhibitor (CHIR 98014), TGFβ inhibitor (SB-431542), selective inhibitor of cytoskeletal rearrangement-associated coiled-coil protein kinase (Y27632), receptor tyrosine kinase inhibitor (RG3635), SMO agonist (Cyclopamine), Janus kinase 1 and Janus kinase 2 inhibitor (Baricitinib), Menin-MLL interaction inhibitor (MI503); (3) replacing the plastic state cells obtained in step (2) with medium III, inducing and culturing at 37° C., 5% CO 2 for 12-16 days, changing the culture medium every 3-4 days, and obtaining extraembryonic endoderm-like cells; The culture medium III uses DMEM high-glucose culture medium as the basic culture medium and includes the following components: N2 supplement, B27 supplement, GlutaMAXTM, NEAA, penicillin-streptomycin, 2-mercaptoethanol, AlbuMAX TM -II, HRG, glycogen kinase inhibitor (CHIR 98014), TGFβ inhibitor (SB-431542), selective inhibitor of cytoskeletal rearrangement-associated coiled-coil protein kinase (Y27632), histone deacetylase inhibitor (valproic acid), mitogen-activated protein kinase inhibitor, histone methyltransferase inhibitor (EPZ004777), S-adenosylhomocysteine ​​hydrolase inhibitor (3-deazaadenine A), monoamine oxidase inhibitor (tranylcypromine).

[0029] (4) replacing the extraembryonic endoderm-like cells obtained in step (3) with medium IV, inducing and culturing them at 37° C., 5% CO 2 for 12-14 days, and replacing the culture medium every 3-4 days to obtain pluripotent stem cells; The IV culture medium uses DMEM high-glucose culture medium as the basic culture medium and includes the following components: N2 supplement, B27 supplement, GlutaMAXTM, NEAA, penicillin-streptomycin, 2-mercaptoethanol, HRG, glycogen kinase inhibitor (CHIR98014), selective inhibitor of cytoskeletal rearrangement-associated coiled-coil protein kinase (Y27632), mitogen-activated protein kinase inhibitor, Wnt inhibitor (Wnt-C59), B-Raf inhibitor (PLX4032), and histone deacetylase inhibitor (valproic acid).

[0030] In the steps (3) and (4), the mitogen-activated protein kinase inhibitor is selected from Mirdametinib, MEK162 and LY2228820; further, the mitogen-activated protein kinase inhibitor is a mixture of Mirdametinib and LY2228820 in a molar ratio of 1:(0.25-0.45).

[0031] In the present application, human adipose-derived mesenchymal stem cells (hADMSC) and human skin fibroblasts (hASF) were from the cell bank of Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine; Mirdametinib and MEK162 were purchased from MCE; LY2228820 was purchased from Shanghai Zeye Biotechnology Co., Ltd.; the raw materials, reagents, solvents, etc. used in this application can all be obtained commercially.

[0032] The present application is further described in detail below in combination with embodiments, performance testing and accompanying drawings.

[0033] Example 1 Embodiment 1 provides a method for inducing somatic cells into pluripotent stem cells, comprising the following steps: (1) Phase I: Human adipose-derived mesenchymal stem cells (hADMSCs) were cultured in medium I at 37°C, 5% O2, 5% CO2 for 8-10 days, with the culture medium changed every 3-4 days to obtain epithelial-like cells; The medium No. 1 uses DMEM high-glucose medium as the basic medium and includes the following components: 15% FBS, 1% GlutaMAX TM, 1% NEAA, 1% penicillin-streptomycin, 0.055mM 2-mercaptoethanol, 50μg / mL VC, 1mM nicotinamide, 0.2μM histone methyltransferase inhibitor (EPZ004777), 0.01μM S-adenosylhomocysteine ​​hydrolase inhibitor (3-deazaadenine A), 5μM retinoic acid receptor agonist (BMS961), 10μM glycogen kinase inhibitor (CHIR98014), 10μM TGFβ inhibitor (SB-431542), 2μM selective inhibitor of cytoskeletal rearrangement-associated coiled-coil protein kinase (Y27632), 0.2μM receptor tyrosine kinase inhibitor (RG3635, purchased from Selleck), 0.5μM SMO agonist (Cyclopamine), 0.5μM Janus kinase 1 and Janus kinase 2 inhibitor (Baricitinib), 0.5 μM Menin-MLL interaction inhibitor (MI503); (2) The second stage: the epithelial culture cells obtained in step (1) are replaced with medium II, and induced and cultured at 37° C., 5% CO 2 for 10-12 days, with the culture medium replaced every 3-4 days to obtain cells in a plastic state; The medium II uses DMEM high-glucose medium as the basic medium and includes the following components: 15% FBS, 1% GlutaMAX TM , 1% NEAA, 1% penicillin-streptomycin, 0.055mM 2-mercaptoethanol, 50μg / mL VC, 1mM NAM, 40ng / mL bFGF, 0.5μM c-Jun kinase inhibitor (AS601245), 1μM DNA methyltransferase inhibitor (Vidaza, purchased from Selleck), 10μM monoamine oxidase inhibitor (tranylcypromine), 1μM EHMT2 inhibitor (UNC0224), 0.5μM BMP receptor / AMPK inhibitor (Dorsomorphin), 2μM CREB binding protein and CREB binding protein bromodomain inhibitor (SGC-CBP30), 5μM retinoic acid receptor agonist (BMS961), 10μM glycogen kinase inhibitor (CHIR 98014), 10μM TGFβ inhibitor (SB-431542), 2μM selective inhibitor of cytoskeletal rearrangement-associated coiled-coil protein kinase (Y27632), 0.2μM receptor tyrosine kinase inhibitor (RG3635), 0.5μM SMO agonist (Cyclopamine), 0.5μM Janus kinase 1 and Janus kinase 2 inhibitor (Baricitinib), 0.5μM Menin-MLL interaction inhibitor (MI503); (3) The third stage: the plastic state cells obtained in step (2) are replaced with medium III, and induced and cultured at 37° C., 5% CO 2 for 12-16 days, and the culture medium is changed every 3-4 days to obtain extraembryonic endoderm-like cells; The III medium uses DMEM high glucose medium as the basic medium and includes the following components: 1% N2 supplement, 2% B27 supplement, 1% GlutaMAXTM, 1% NEAA, 1% penicillin-streptomycin, 0.055mM 2-mercaptoethanol, 5mg / mL AlbuMAX TM -II, 20ng / mL HRG, 10μM glycogen kinase inhibitor (CHIR 98014), 10μM TGFβ inhibitor (SB-431542), 10μM selective inhibitor of cytoskeletal rearrangement-associated coiled-coil protein kinase (Y27632), 0.1μM histone deacetylase inhibitor (valproic acid), 0.1μM mitogen-activated protein kinase inhibitor (Mirdametinib), histone methyltransferase inhibitor (EPZ004777), 0.01μM S-adenosylhomocysteine ​​hydrolase inhibitor (3-deazaadenine A), 10μM monoamine oxidase inhibitor (tranylcypromine).

[0034] (4) The fourth stage: the extraembryonic endoderm-like cells obtained in step (3) are replaced with medium IV, and induced and cultured at 37° C., 5% CO 2 for 12-14 days, with the culture medium replaced every 3-4 days to obtain primary pluripotent stem cells; The IV culture medium uses DMEM high-glucose culture medium as the basal culture medium and includes the following components: 1% N2 supplement, 2% B27 supplement, 1% GlutaMAXTM, 1% NEAA, 1% penicillin-streptomycin, 0.055mM 2-mercaptoethanol, 20ng / mL HRG, 10μM glycogen kinase inhibitor (CHIR 98014), 10μM selective inhibitor of cytoskeletal rearrangement-associated coiled-coil protein kinase (Y27632), 0.1μM mitogen-activated protein kinase inhibitor (Mirdametinib), 2μM Wnt inhibitor (Wnt-C59), 0.5μM B-Raf inhibitor (PLX4032), and 0.1μM histone deacetylase inhibitor (valproic acid).

[0035] Embodiment 2-10 Example 2-10 provides a method for inducing somatic cells into pluripotent stem cells.

[0036] The difference between the above embodiment and embodiment 1 is that in steps (3) and (4), the types and ratios of mitogen-activated protein kinase inhibitors are specifically shown in Table 1 below.

[0037] Table 1 Types and ratios of mitogen-activated protein kinase inhibitors in Examples 1-10 Embodiment 11 Example 11 provides a method for inducing somatic cells into pluripotent stem cells.

[0038] The difference between the above embodiment and embodiment 1 is that human adipose-derived mesenchymal stem cells (hADMSC) are replaced by human skin fibroblasts (hASF).

[0039] Comparative Example 1 Comparative Example 1 provides a method for inducing somatic cells into pluripotent stem cells.

[0040] The above embodiment is different from embodiment 1 in that the retinoic acid receptor agonist is TTNPB, the receptor tyrosine kinase inhibitor is ABT869, the SMO agonist is SAG, and the Janus kinase 1 and Janus kinase 2 inhibitor is ruxolitinib.

[0041] Comparative Example 2 Comparative Example 2 provides a method for inducing somatic cells into pluripotent stem cells.

[0042] The above embodiment is different from embodiment 1 in that the retinoic acid receptor agonist is SR1078, the glycogen kinase inhibitor is BIP-135, and the Menin-MLL interaction inhibitor is M-1121.

[0043] Cell morphology The cells obtained before and after induction in Example 1 were observed under a microscope. Figure 1-5 shown.

[0044] By comparison Figure 1 and Figure 5 It can be seen that after four stages of induction, the cell morphology of human adipose-derived mesenchymal stem cells (hADMSC) gradually changed from spindle-shaped to rounded, and the cells gradually changed from uniformly adherent and dispersed to tightly packed into a quasi-circular shape with gradually clear boundaries, indicating that hADMSC gradually differentiated into mature pluripotent stem cells after four stages of induction.

[0045] Immunohistochemistry Immunohistochemical detection was performed on the pluripotent stem cells obtained in Example 1-11 and Comparative Example 1-2 to detect the expression of each specific antibody OCT4, SOX2 and NANOG antibody. The results are shown in Table 2.

[0046] Immunohistochemical detection method is: (1) Characteristic cell clusters were selected from primary pluripotent stem cells and placed in IPS-specific culture medium. The cells were cultured at 37°C and 5% CO2 for 24 h, and their adhesion was observed. The medium was changed every three days. When the cell confluence reached about 90%, the cells were digested with IPS-specific digestion solution. After termination, the cells were collected.

[0047] (2) Fix the cells with 10% neutral formalin, and centrifuge to obtain the cell pellet to make wax blocks. Dewax the wax blocks and slice them into water, perform antigen retrieval, wash once with distilled water, block with 3% hydrogen peroxide for 10 min, wash five times with BPS buffer, add primary antibody and incubate at 37°C for 90 min, wash five times with BPS buffer, add secondary antibody and incubate for 30 min, wash five times with BPS buffer, develop with DAB colorimetric solution for 5 min, wash with running water for 1 min, stain with hematoxylin solution for 5 min, wash with running water for 1 min, differentiate for 2 s, wash with running water for 1 min, blue with bluing solution for 30 s, wash with running water for 1 min, dehydrate for 1 min each with gradient, make transparent with xylene for 3 min, and seal the slides.

[0048] (3) Detect the expression of specific antibodies OCT4, SOX2 and NANOG by optical microscopy. Note: The higher the expression of specific antibodies, the higher the conversion efficiency of this method.

[0049] Table 2 Detection results of antibody expression levels of pluripotent stem cells obtained in Example 1-11 and Comparative Example 1-2 According to the test results in Table 2, the pluripotent stem cells obtained in Examples 1-11 and Comparative Example 1 can express specific antibodies OCT4, SOX2 and NANOG, but the expression of the specific antibody OCT4 of the pluripotent stem cells obtained in Examples 1-11 is 85.3-92.7% (≥85%), the expression of the specific antibody SOX2 is 30.7-34.1% (≥30%), and the expression of the specific antibody NANOG is 90.4-95.7% (≥90%); while the expression of the specific antibody OCT4 of the pluripotent stem cells obtained in Comparative Example 1 is only 80.5-81.2%, the expression of the specific antibody SOX2 is only 26.3-27.3%, and the expression of the specific antibody NANOG is only 85.7-86.1%. Therefore, it is shown that the culture medium obtained by the small molecules screened in this application can efficiently and stably induce somatic cells to reprogram pluripotent stem cells, and has a good prospect for use.

[0050] The detection results of Examples 1-10 show that the expression level of the specific antibody OCT4 of the pluripotent stem cells obtained in Example 1-3 is 85.3-87.1%, the expression level of the specific antibody SOX2 is 30.7-31.2%, and the expression level of the specific antibody NANOG is 90.4-92.1%; the expression level of the specific antibody OCT4 of the pluripotent stem cells obtained in Examples 4-8 is 90.5-92.7% (≥90%), the expression level of the specific antibody SOX2 is 32.4-34.1% (≥32%), and the expression level of the specific antibody NANOG is 94.2-95.7% (≥94%); the expression level of the specific antibody OCT4 of the pluripotent stem cells obtained in Examples 9-10 is 87.8-88.3%, the expression level of the specific antibody SOX2 is 31.2-31.6%, and the expression level of the specific antibody NANOG is 91.8-92.2%. Therefore, it is shown that the present application further uses a mixture of Mirdametinib and LY2228820 as a mitogen-activated protein kinase inhibitor, which can increase the efficiency of converting human somatic cells into pluripotent stem cells.

[0051] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for inducing somatic cells into pluripotent stem cells, characterized in that: The following steps are involved: Inducing and culturing the somatic cells in sequence using medium I, medium II, medium III, and medium IV to obtain pluripotent stem cells; The culture media I to IV all use DMEM high-glucose culture media as the basic culture media; The medium I and medium II both include the following components: retinoic acid receptor agonist, glycogen kinase inhibitor, TGFβ inhibitor, selective inhibitor of cytoskeleton rearrangement-related coiled-coil protein kinase, receptor tyrosine kinase inhibitor, SMO agonist, Janus kinase 1 and Janus kinase 2 inhibitor, Menin-MLL interaction inhibitor; The retinoic acid receptor agonist is BMS961, The glycogenokinase inhibitor is CHIR 98014, The TGFβ inhibitor is SB-431542, The selective inhibitor of the cytoskeletal rearrangement-related coiled-coil protein kinase is Y27632, The receptor tyrosine kinase inhibitor is RG3635, The SMO agonist is Cyclopamine, The Janus kinase 1 and Janus kinase 2 inhibitor is Baricitinib, The Menin-MLL interaction inhibitor is MI503.

2. The method for inducing somatic cells into pluripotent stem cells according to claim 1, characterized in that: The medium No. 1 also includes the following components: FBS, GlutaMAX TM , NEAA, penicillin-streptomycin, 2-mercaptoethanol, VC, niacinamide, histone methyltransferase inhibitors, S-adenosylhomocysteine ​​hydrolase inhibitors; The histone methyltransferase inhibitor is EPZ004777, and the S-adenosyl homocysteine ​​hydrolase inhibitor is 3-deazaadenine A.

3. The method for inducing somatic cells into pluripotent stem cells according to claim 1, characterized in that: The medium II also includes the following components: FBS, GlutaMAX TM , NEAA, penicillin-streptomycin, 2-mercaptoethanol, VC, NAM, bFGF, c-Jun kinase inhibitors, DNA methyltransferase inhibitors, monoamine oxidase inhibitors, EHMT2 inhibitors, BMP receptor / AMPK inhibitors, CREB binding protein and CREB binding protein bromodomain inhibitors; The c-Jun kinase inhibitor is AS601245, The DNA methyltransferase inhibitor is Vidaza, The monoamine oxidase inhibitor is tranylcypromine, The EHMT2 inhibitor is UNC0224, The BMP receptor / AMPK inhibitor is Dorsomorphin, The CREB binding protein and CREB binding protein bromodomain inhibitor is SGC-CBP30.

4. The method for inducing somatic cells into pluripotent stem cells according to claim 1, characterized in that: The III medium also includes the following components: N2 supplement, B27 supplement, GlutaMAXTM, NEAA, penicillin-streptomycin, 2-mercaptoethanol, AlbuMAX TM -II, HRG, glycogen kinase inhibitors, TGFβ inhibitors, selective inhibitors of cytoskeletal rearrangement-associated coiled-coil protein kinases, histone deacetylase inhibitors, mitogen-activated protein kinase inhibitors, histone methyltransferase inhibitors, S-adenosylhomocysteine ​​hydrolase inhibitors, monoamine oxidase inhibitors.

5. The method for inducing somatic cells into pluripotent stem cells according to claim 1, characterized in that: The IV culture medium includes DMEM high-glucose culture medium and the following components: N2 supplement, B27 supplement, GlutaMAXTM, NEAA, penicillin-streptomycin, 2-mercaptoethanol, HRG, glycogen kinase inhibitor, selective inhibitor of cytoskeletal rearrangement-associated coiled-coil protein kinase, mitogen-activated protein kinase inhibitor, Wnt inhibitor, B-Raf inhibitor, and histone deacetylase inhibitor.

6. The method for inducing somatic cells into pluripotent stem cells according to claim 4 or 5, characterized in that: The histone deacetylase inhibitor is valproic acid; the Wnt inhibitor is Wnt-C59; and the B-Raf inhibitor is PLX4032.

7. The method for inducing somatic cells into pluripotent stem cells according to claim 4 or 5, characterized in that: The mitogen-activated protein kinase inhibitor is selected from Mirdametinib, MEK162 and LY2228820.

8. The method for inducing somatic cells into pluripotent stem cells according to claim 7, characterized in that: The mitogen-activated protein kinase inhibitor is a mixture of Mirdametinib and LY2228820 in a molar ratio of 1:(0.25-0.45).

9. The method for inducing somatic cells into pluripotent stem cells according to any one of claims 1 to 5, characterized in that: The culture conditions of the medium I are: 37°C, 5% O2, 5% CO2, induced culture for 8-10 days, and the culture medium is changed every 3-4 days; The culture conditions of the medium II, medium III and medium IV are: 37° C., 5% CO2, and the culture medium is changed every 3-4 days; the medium II is induced for 10-12 days, the medium III is induced for 12-16 days, and the medium IV is induced for 12-14 days.

10. A pluripotent stem cell obtained by the method for inducing somatic cells into pluripotent stem cells according to any one of claims 1 to 9.

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