Methods for facilitating somatic cell reprogramming

Abstract

This invention relates to a method for promoting somatic cell reprogramming to generate iPSC clones. Specifically, it provides a small molecule composition for rapidly promoting CD34+ blood cell reprogramming to generate iPSC clones.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a small molecule composition and method for rapidly promoting somatic cell reprogramming. Background Technology

[0002] Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell that exhibits characteristics similar to human embryonic stem cells. This is achieved by reprogramming human adult cells (such as skin fibroblasts or blood cells) with stem cell-specific transcription factors, leading to dedifferentiation. iPSC-derived cell products possess the potential for large-scale, in vitro production, minimizing the cost of cell-based drugs and ensuring accessibility for widespread clinical application in humans, thus demonstrating significant clinical value.

[0003] Current induced pluripotent stem cell (iPSC) reprogramming techniques, in addition to using classic stem transcription factors such as Oct3 / 4, Sox2, Myc, Klf4, LIN28, and NANOG, have also revealed other stem transcription factors, such as Nr5a2, Glis1, Sall4, Dppa2, and Utf1, which can also induce the reprogramming of mature human somatic cells into pluripotent stem cells. However, iPSCs obtained using classic reprogramming factors (Oct3 / 4, Sox2, Myc, Klf4, and LIN28) have been studied in greater depth regarding their phenotypic characteristics and differentiation properties, and have been applied more widely.

[0004] Currently, iPSC technology mainly uses integrative viruses (lentiviruses and retroviruses), non-integrative viruses (Sendai virus), RNA, and non-integrative plasmids to introduce stemness factors into cells for reprogramming. Integrative viruses can cause random integration into the cell genome, significantly impacting cellular genetic stability; Sendai virus, compared to non-viral systems, still presents certain safety risks, and its preparation is relatively complex; RNA reprogramming technology is relatively complex to operate, inefficient, and only applicable to certain cell types; non-integrative plasmids (episomal plasmid vectors) carrying stemness factors for iPSC reprogramming have a wider range of applications. Their plasmid preparation is simple, and the reprogramming efficiency is high and stable, making it a relatively important reprogramming method (PMID:23193063, DOI:10.1002 / stem.1293). However, this plasmid system (e.g., the commercially available Epi5) has limitations. TMThe Episomal iPSC Reprogramming Kit (ThermoFisher, catalog number A15960) contains the reprogramming factors mp53DD and EBNA1, and its clinical application carries potential safety risks (https: / / doi.org / 10.1016 / j.stem.2020.09.014). Furthermore, these plasmid systems require a lengthy reprogramming time of at least 16-20 days to generate iPSC clones from CD34+ blood cells.

[0005] In recent years, the technology of reprogramming iPSCs entirely using small chemical molecules has gradually emerged (https: / / doi.org / 10.1038 / s41586-022-04593-5). This technology mainly uses inhibitors and / or activators related to developmental signaling pathways, small molecules related to cell epigenetic regulation, etc., to induce differentiated and mature somatic cells to be reprogrammed into iPSCs in stages. This technology involves the use of a large number of small molecule inhibitors and / or activators, and the reprogramming cycle is relatively long (30-50 days), making the process of obtaining iPSCs relatively complex. Moreover, long-term small molecule action may have potential adverse effects on the metabolism and genetic stability of iPSCs.

[0006] To address the shortcomings of existing iPSC-induced reprogramming methods, which have long reprogramming cycles, this invention is provided. Summary of the Invention

[0007] In one aspect, the present invention provides a composition for promoting somatic cell reprogramming to produce iPSCs, the composition comprising an EZH2 methyltransferase inhibitor (e.g., DZNeP), a histone deacetylase (HDAC) inhibitor (e.g., TSA), and a MEK inhibitor (PD0325901).

[0008] In one or more embodiments, the composition further comprises an inhibitor of DOT1L (protein methyltransferase). Preferably, the inhibitor of protein methyltransferase DOT1L is a competitive inhibitor of S-adenosylmethionine (SAM) of DOT1L.

[0009] In one or more embodiments, the composition further comprises one or more selected from the following: methyltransferase H3K36me3 activator (e.g., vitamin B12), PKC inhibitor (e.g., Go 6983), ROCK inhibitor (e.g., Y23672).

[0010] In one or more embodiments, the EZH2 methyltransferase inhibitor includes DZNeP.

[0011] In one or more embodiments, the histone deacetylase inhibitor includes TSA.

[0012] In one or more embodiments, the MEK inhibitor includes PD0325901.

[0013] In one or more embodiments, the DOT1L inhibitor includes EPZ5676.

[0014] In one or more embodiments, the methyltransferase H3K36me3 activator includes vitamin B12.

[0015] In one or more embodiments, the PKC inhibitor includes Go 6983.

[0016] In one or more embodiments, the ROCK inhibitor includes Y23672.

[0017] In one or more embodiments, the composition comprises DZNeP, TSA, PD0325901, and EPZ5676.

[0018] In one or more embodiments, the composition further comprises one or more of B12, Go 6983 and Y23672; in some such embodiments, the composition further comprises (1) B12, (2) B12 and Go 6983, or (3) B12, Go 6983 and Y23672.

[0019] In one or more embodiments, the composition does not contain one or more of B12, Go 6983, and Y23672. In some such embodiments, the composition does not contain (1) B12, (2) B12 and Go 6983, or (3) B12, Go 6983, and Y23672.

[0020] In one or more embodiments, the concentration of EPZ5676 in the composition ranges from 0.05 μM to 1 μM.

[0021] In one or more embodiments, the DZNeP concentration in the composition ranges from 0.001 μM to 0.1 μM, preferably from 0.01 μM to 0.1 μM.

[0022] In one or more embodiments, the TSA concentration in the composition ranges from 0.001 μM to 0.025 μM, preferably from 0.005 μM to 0.025 μM.

[0023] In one or more embodiments, the concentration of PD0325901 in the composition ranges from 0.25 μM to 1 μM.

[0024] In one or more embodiments, the composition is introduced into somatic cells (e.g., added to culture medium) on days 2, 3, 4, 5, 6 or 7 after electroporation, up to day 8, 10, 11 or 12; preferably, the composition is introduced into somatic cells (e.g., added to culture medium) on day 3 or 4 after electroporation; more preferably, the small molecule composition is introduced into somatic cells (e.g., added to culture medium) on day 3 after electroporation.

[0025] In one or more embodiments, the somatic cells include peripheral mononuclear blood cells, fibroblasts, blood cells, and mesenchymal cells; preferably, the somatic cells are CD34+ blood cells.

[0026] In another aspect, the present invention provides a rapid induction medium for iPSCs, the medium comprising the small molecule composition described herein.

[0027] In another aspect, the present invention provides a kit comprising the composition or culture medium described herein.

[0028] The present invention provides a method for preparing iPSCs or inducing somatic cell reprogramming to generate iPSC clones, the method comprising: 1) introducing a reprogramming factor into somatic cells; 2) culturing the cells obtained in step 1) in a culture medium containing the composition described in any embodiment of the present invention; and 3) selecting cells with embryonic stem cell-like morphology generated in step 2).

[0029] In one or more embodiments, the method further includes: step 4) further culturing the cells selected in step 3) in a culture medium.

[0030] In one or more embodiments, the reprogramming factor comprises: a gene specifically expressed in embryonic cells, or the product of such genes.

[0031] In one or more embodiments, the reprogramming factor contains at least the Oct3 / 4 gene.

[0032] In one or more embodiments, the reprogramming factor comprises the Oct3 / 4 gene and one, two, or three family genes selected from the Sox family, Myc family, and K1f family.

[0033] In one or more embodiments, the Sox family genes are selected from: Sox1 gene, Sox2 gene, Sox3 gene, Sox15 gene, Sox17 gene and Sox18 gene.

[0034] In one or more embodiments, the Myc family gene is selected from: c-Myc gene, N-Myc gene and L-Myc gene.

[0035] In one or more embodiments, the Klf family genes are selected from: Klf1 gene, Klf2 gene, Klf4 gene and Klf5 gene.

[0036] In one or more implementations, the reprogramming factor includes:

[0037] (a) A combination of Oct3 / 4 genes, Sox family genes, Klf4 genes and L-Myc genes, or a combination of the products of these genes;

[0038] (b) A combination of the Oct3 / 4 gene, the Sox2 gene, the L-Myc family genes and the Klf4 gene, or a combination of the products of these genes;

[0039] (c) A combination of Oct3 / 4 genes, Sox2 genes, L-Myc genes and Klf family genes, or a combination of the products of these genes.

[0040] In one or more embodiments, the reprogramming factor further comprises at least one gene selected from Lin28 and Sall1.

[0041] In one or more implementations, the reprogramming factor includes:

[0042] Oct3 / 4, Sox2 and Klf4;

[0043] Oct3 / 4, Sox2, Klf4 and Lin28;

[0044] Oct3 / 4, Sox2, Klf4 and Sall1;

[0045] Oct3 / 4, Sox2, Klf4, and L-Myc;

[0046] Oct3 / 4, Sox2, Klf4, L-Myc and Lin28; or

[0047] Oct3 / 4, Sox2, Klf4, L-Myc and Sall1.

[0048] In one or more embodiments, the sequence of the OCT3 / 4 protein is shown in SEQ ID NO:1, the sequence of the SOX2 protein is shown in SEQ ID NO:2, the sequence of the KLF4 protein is shown in SEQ ID NO:3, the sequence of the L-MYC protein is shown in SEQ ID NO:4, and the sequence of the LIN28 protein is shown in SEQ ID NO:5.

[0049] In one or more embodiments, the reprogramming factor does not include substances that downregulate p53 expression and substances that upregulate EBNA1 expression.

[0050] In one or more embodiments, pluripotent stem cells express the reprogramming factor.

[0051] In one or more embodiments, the gene can be introduced into somatic cells using a recombinant vector. The vector can be a viral vector or a non-viral vector.

[0052] In one or more embodiments, the somatic cell is a mammalian somatic cell. In one or more embodiments, the somatic cell is a primate somatic cell. In one or more embodiments, the somatic cell is a human somatic cell. In one or more embodiments, the somatic cell is a peripheral mononuclear blood cell, fibroblast, blood cell, or mesenchymal cell. In one or more embodiments, the somatic cell is an umbilical cord blood cell. In one or more embodiments, the somatic cell is a CD34+ umbilical cord blood cell.

[0053] In one or more embodiments, the somatic cells are somatic cells collected from a patient.

[0054] In one or more embodiments, the introduction method in 1) includes electroporation, microinjection, gene gun method, DEAE-dextran method, calcium phosphate coprecipitation transfection method, and artificial liposome method. Preferably, the introduction method is electroporation.

[0055] In another aspect, the present invention provides induced pluripotent stem cells obtained by the method described in any embodiment herein.

[0056] The present invention provides a stem cell therapy comprising: using somatic cells collected from healthy individuals or patients to induce differentiation into induced pluripotent stem cells obtained by the method described in any embodiment herein, and transplanting the resulting somatic cells or tissues, organs or body fluids prepared by the final differentiation of the induced pluripotent stem cells into an autologous or allogeneic patient.

[0057] The present invention also provides a method for determining the physiological activity or toxicity of compounds, drugs or toxins using cells obtained by differentiating and inducing induced pluripotent stem cells obtained by any of the embodiments described herein.

[0058] Advantages of the present invention

[0059] 1. This invention can avoid the use of reprogramming factors mp53DD and EBNA1, thereby avoiding potential risks such as tumorigenesis and teratogenicity, and improving the safety of the final cell product for clinical application.

[0060] 2. This invention combines the effects of reprogramming factors and small molecules to accelerate the generation of iPSC clones by day 8 of reprogramming, shortening the time for iPSC clone generation by at least 50%.

[0061] 3. Reduce reprogramming costs and facilitate commercialization. Attached Figure Description

[0062] Figure 1 Small molecule combinatorial optimization experimental procedures;

[0063] Figure 2 Optimization of small molecule compositions during iPSC-induced reprogramming;

[0064] Figure 3 Results of concentration studies on small molecule combinations;

[0065] Figure 4 The experimental procedure of Example 3;

[0066] Figure 5 Bright-field photography of the iPSC reprogramming process under experimental condition 1 (the area circled in dashed lines represents the iPSC clone);

[0067] Figure 6 Bright-field photography of the iPSC reprogramming process under experimental condition 2;

[0068] Figure 7 Bright-field photography of the iPSC reprogramming process under experimental condition 3 (the area circled in dashed lines represents the iPSC clone);

[0069] Figure 8 Bright-field photography of the iPSC reprogramming process under experimental condition 4 (the area circled in dashed lines represents the iPSC clone);

[0070] Figure 9 Immunofluorescence staining results on days 8 and 12 of the iPSC reprogramming process under experimental condition 1;

[0071] Figure 10 Immunofluorescence staining results on days 8 and 12 of the iPSC reprogramming process under experimental condition 2;

[0072] Figure 11 Immunofluorescence staining results on days 8 and 12 of the iPSC reprogramming process under experimental condition 3;

[0073] Figure 12 Statistics on the number of iPSC clones on day 12 of electrical reprogramming under conditions 1-3;

[0074] Figure 13 The flow cytometry results of the iPSC clones generated under conditions 1-3 in Example 2;

[0075] Figure 14 The results of the three germ layer differentiation detection of iPSC clones generated under conditions 1-3 in Example 2;

[0076] Figure 15Typical karyotype analysis results of iPSC clones generated under conditions 1-3 in Example 2. Detailed Implementation

[0077] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0078] This article provides methods and small molecule compositions for cell transformation using chemical reprogramming factors. These methods and compositions can rapidly generate cells with differentiation potential, transforming cell populations containing at least one cell type into cell populations containing another cell type with higher differentiation potential. The various cells generated by these methods and / or compositions can include pluripotent stem cells or other intermediate cells with a higher potential to become pluripotent stem cells relative to untransformed cells. Compared to existing methods, these methods and compositions can generate stem cells or cells in various differentiation states in a shorter time. Therefore, these methods and compositions can meet the needs for cell resources in basic research, therapeutics, agriculture, and the food industry.

[0079] In this document, somatic cells are mammalian somatic cells. In one or more embodiments, the somatic cells are primate somatic cells. In one or more embodiments, the somatic cells are human somatic cells. In one or more embodiments, the somatic cells are peripheral mononuclear blood cells, fibroblasts, blood cells, or mesenchymal cells. In one or more embodiments, the somatic cells are umbilical cord blood cells. In one or more embodiments, the somatic cells are CD34+ umbilical cord blood cells. In one or more embodiments, the somatic cells are somatic cells collected from a patient.

[0080] One of the objectives of this invention is to provide a small molecule composition that promotes the reprogramming of CD34+ (umbilical cord) blood cells to generate iPSC clones. The small molecule composition contains an EZH2 methyltransferase inhibitor, a histone deacetylase (HDAC) inhibitor, a MEK inhibitor, and a competitive inhibitor of S-adenosylmethionine (SAM) of protein methyltransferase DOT1L.

[0081] In one or more embodiments, the EZH2 methyltransferase inhibitor comprises DZNeP. In one or more embodiments, the histone deacetylase inhibitor comprises TSA. In one or more embodiments, the MEK inhibitor comprises PD0325901. In one or more embodiments, the DOT1L inhibitor comprises EPZ5676. In one or more embodiments, the methyltransferase H3K36me3 activator comprises vitamin B12. In one or more embodiments, the PKC inhibitor comprises Go6983. In one or more embodiments, the ROCK inhibitor comprises Y23672.

[0082] In one or more embodiments, the composition comprises DZNeP, TSA, PD0325901, and EPZ5676. In one or more embodiments, the composition further comprises one or more of B12, Go 6983, and Y23672; in some such embodiments, the composition further comprises (1) B12, (2) B12 and Go 6983, or (3) B12, Go 6983, and Y23672. In one or more embodiments, the composition does not comprise one or more of B12, Go 6983, and Y23672. In some such embodiments, the composition does not comprise (1) B12, (2) B12 and Go 6983, or (3) B12, Go 6983, and Y23672.

[0083] In one or more embodiments, the concentration of EPZ5676 in the small molecule composition ranges from 0.05 μM to 1 μM, preferably 0.1 to 0.5 μM, more preferably 0.1 to 0.25 μM, and even more preferably 0.1 μM. In one or more embodiments, the concentration of DZNeP in the small molecule composition ranges from 0.01 μM to 0.1 μM, preferably 0.01 to 0.05 μM, more preferably 0.01 to 0.025 μM, and even more preferably 0.01 μM. In one or more embodiments, the concentration of TSA in the small molecule composition ranges from 0.005 μM to 0.025 μM, preferably 0.005 to 0.02 μM, more preferably 0.005 to 0.01 μM, and even more preferably 0.005 μM. In one or more embodiments, the concentration of PD0325901 in the small molecule composition ranges from 0.25 μM to 1 μM, preferably 0.5 to 1 μM, more preferably 0.8 to 1 μM, and even more preferably 1 μM. In one or more embodiments, the concentration of EPZ5676 in the small molecule composition ranges from 0.05 μM to 1 μM, the concentration of DZNeP ranges from 0.01 μM to 0.1 μM, the concentration of TSA ranges from 0.005 μM to 0.025 μM, and the concentration of PD0325901 ranges from 0.25 μM to 1 μM; preferably, the concentration of EPZ5676 is 0.1 to 0.5 μM, the concentration of DZNeP is 0.01 to 0.05 μM, and the concentration of TSA is 0.005 μM. -0.02 μM, PD0325901 concentration is 0.5-1 μM; preferably, EPZ5676 concentration is 0.1-0.25 μM, DZNeP concentration is 0.01-0.025 μM, TSA concentration is 0.005-0.01 μM, PD0325901 concentration is 0.8-1 μM; preferably, EPZ5676 concentration is 0.1 μM, DZNeP concentration is 10 nM, TSA concentration is 5 nM, PD0325901 concentration is 1 μM.

[0084] In one or more embodiments, the small molecule composition contains B12 at a concentration of about 0.1 μM. In one or more embodiments, the small molecule composition contains Go 6983 at a concentration of about 0.5 μM. In one or more embodiments, the small molecule composition contains Y23672 at a concentration of about 10 μM.

[0085] A second objective of this invention is to provide a reprogramming factor for inducing somatic cell reprogramming to generate iPSC clones. The reprogramming factor includes OCT3 / 4, SOX2, KLF4, L-MYC, and LIN28. The OCT3 / 4 sequence is shown in SEQ ID NO:1; the SOX2 sequence is shown in SEQ ID NO:2; the KLF4 sequence is shown in SEQ ID NO:3; the L-MYC sequence is shown in SEQ ID NO:4; and the LIN28 sequence is shown in SEQ ID NO:5. Preferably, the reprogramming factor does not include mp53DD and EBNA1.

[0086] The term "CD34+ blood cells" refers to a population of cells with the specific surface marker CD34, primarily found in bone marrow, peripheral blood, and umbilical cord blood. These cells typically include hematopoietic stem cells and hematopoietic progenitor cells, and possess the ability to self-renew and differentiate into various blood cell types.

[0087] The terms “induced pluripotent stem cells,” “iPSCs,” and “iPSC” are used interchangeably to refer to a type of pluripotent stem cell that is artificially obtained by reprogramming non-pluripotent cells (e.g., somatic cells) to have self-renewal capacity and the potential to differentiate into cells of the three germ layers. Reprogramming refers to the process of obtaining induced pluripotent stem cells through exogenous gene expression, compound induction, epigenetic modification, and other means.

[0088] The reprogramming factor fragments of this invention can typically be obtained using PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, particularly the open reading frame sequences, and the relevant sequences can be amplified using commercially available DNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art. When the sequences are long, two or more PCR amplifications are often required, followed by splicing the amplified fragments together in the correct order. Once the relevant sequences are obtained, they can be obtained in large quantities using recombination. Typically, they are cloned into a vector, transformed into cells, and then isolated from the proliferated host cells using conventional methods.

[0089] Furthermore, the relevant sequences can also be synthesized artificially, especially when the fragment length is short. Typically, long sequences are obtained by first synthesizing multiple small fragments and then ligating them. Currently, it is possible to obtain the DNA sequence encoding the reprogramming factor of this invention entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0090] This invention also provides a recombinant vector comprising the gene of this invention. As a preferred embodiment, the recombinant vector contains a multiple cloning site or at least one restriction enzyme site downstream of the promoter. When it is necessary to express the target gene of this invention, the target gene is ligated into a suitable multiple cloning site or restriction enzyme site, thereby operatively linking the target gene to the promoter. As another preferred embodiment, the recombinant vector comprises (from 5' to 3' direction): a promoter, a target gene, and a terminator. If desired, the recombinant vector may further comprise elements selected from the group consisting of: a 3' polynucleotide signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; an resistance selection marker (dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); an enhancer; or an operator.

[0091] The methods used to prepare recombinant vectors are well known to those skilled in the art. Expression vectors can be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host.

[0092] Those skilled in the art can use well-known methods to construct expression vectors containing the genes described in this invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. When constructing recombinant expression vectors using the genes of this invention, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide.

[0093] Vectors containing the genes of this invention can be used to transform suitable host cells to reprogram them. Host cells can be peripheral mononuclear blood cells, fibroblasts, umbilical cord blood cells, and preferably, CD34+ (umbilical cord) blood cells. Those skilled in the art will understand how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (such as *E. coli*), it can be treated with CaCl2 or electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.).

[0094] In another aspect, the present invention provides a rapid induction medium for iPSCs, the medium comprising the small molecule composition described herein. The concentration of each small molecule described herein in the composition is the same as the concentration of the small molecule in the medium.

[0095] In this document, the basal medium can be any medium suitable for stem cell growth, particularly PSC growth, including but not limited to: Dulbecco modified Eagle medium (DMEM), essential minimum medium (MEM), basal Eagle medium (BME), RPMI 1640, F10, F12, α-essential minimum medium (αMEM), Glasgow essential minimum medium (GMEM), Iscove modified Dulbecco medium, Neurobasal medium, and DMEM / F12 and combinations thereof. Preferably, the basal medium is a DMEM / F12 medium containing N-2 and B-27. Exemplarily, the basal medium also contains NEAA, GlutaMAX, β-Mercaptoethanol, and Basic FGF.

[0096] In one or more embodiments, the iPSC rapid induction medium described herein comprises an EZH2 methyltransferase inhibitor, a histone deacetylase (HDAC) inhibitor, a MEK inhibitor, and a competitive inhibitor of the protein methyltransferase DOT1L on S-adenosylmethionine (SAM). In one or more embodiments, the EZH2 methyltransferase inhibitor comprises DZNeP. In one or more embodiments, the histone deacetylase inhibitor comprises TSA. In one or more embodiments, the MEK inhibitor comprises PD0325901. In one or more embodiments, the DOT1L inhibitor comprises EPZ5676.

[0097] In one or more embodiments, the culture medium further comprises one or more of the following: a methyltransferase H3K36me3 activator, a PKC inhibitor, and a ROCK inhibitor. In one or more embodiments, the methyltransferase H3K36me3 activator comprises vitamin B12. In one or more embodiments, the PKC inhibitor comprises Go 6983. In one or more embodiments, the ROCK inhibitor comprises Y23672.

[0098] In one or more embodiments, the culture medium comprises DZNeP, TSA, PD0325901, and EPZ5676. In one or more embodiments, the culture medium further comprises one or more of B12, Go 6983, and Y23672; in some such embodiments, the composition further comprises (1) B12, (2) B12 and Go 6983, or (3) B12, Go 6983, and Y23672. In one or more embodiments, the culture medium does not comprise one or more of B12, Go 6983, and Y23672. In some such embodiments, the culture medium does not comprise (1) B12, (2) B12 and Go 6983, or (3) B12, Go 6983, and Y23672.

[0099] In one or more embodiments, the concentration of EPZ5676 in the culture medium ranges from 0.05 μM to 1 μM, preferably 0.1 to 0.5 μM, more preferably 0.1 to 0.25 μM, and even more preferably 0.1 μM. In one or more embodiments, the concentration of DZNeP in the culture medium ranges from 0.01 μM to 0.1 μM, preferably 0.01 to 0.05 μM, more preferably 0.01 to 0.025 μM, and even more preferably 0.01 μM. In one or more embodiments, the concentration of TSA in the culture medium ranges from 0.005 μM to 0.025 μM, preferably 0.005 to 0.02 μM, more preferably 0.005 to 0.01 μM, and even more preferably 0.005 μM. In one or more embodiments, the concentration of PD0325901 in the culture medium ranges from 0.25 μM to 1 μM, preferably 0.5 to 1 μM, more preferably 0.8 to 1 μM, and even more preferably 1 μM. In one or more embodiments, the concentrations of EPZ5676, DZNeP, TSA, and PD0325901 in the culture medium range from 0.05 μM to 1 μM; preferably, the concentrations are 0.1-0.5 μM for EPZ5676, 0.01-0.05 μM for DZNeP, and 0.005-0.025 μM for TSA. The concentrations are as follows: 0.02 μM, PD0325901 concentration is 0.5-1 μM; preferably, EPZ5676 concentration is 0.1-0.25 μM, DZNeP concentration is 0.01-0.025 μM, TSA concentration is 0.005-0.01 μM, PD0325901 concentration is 0.8-1 μM; preferably, EPZ5676 concentration is 0.1 μM, DZNeP concentration is 10 nM, TSA concentration is 5 nM, PD0325901 concentration is 1 μM.

[0100] In one or more embodiments, the culture medium contains B12 at a concentration of about 0.1 μM. In one or more embodiments, the culture medium contains Go 6983 at a concentration of about 0.5 μM. In one or more embodiments, the culture medium contains Y23672 at a concentration of about 10 μM.

[0101] This invention provides a method for preparing iPSCs or inducing somatic cell reprogramming to generate iPSC clones, the method comprising: 1) introducing a reprogramming factor into somatic cells; 2) culturing the cells obtained in step 1) in a culture medium containing the composition described in any embodiment herein; and 3) selecting cells generated in step 2) that have embryonic stem cell-like morphology. In one or more embodiments, the method further comprises: step 4) further culturing the cells selected in step 3) in a culture medium.

[0102] In one or more embodiments, the composition is introduced into somatic cells (e.g., added to culture medium) on days 2, 3, 4, 5, 6 or 7 after electroporation, up to day 8, 10, 11 or 12; preferably, the composition is introduced into somatic cells (e.g., added to culture medium) on day 3 or 4 after electroporation; more preferably, the small molecule composition is introduced into somatic cells (e.g., added to culture medium) on day 3 after electroporation.

[0103] In embodiments involving gene modification (introducing reprogramming factors into somatic cells), the method converts somatic cells into pluripotent stem cells in less than about 30 days, calculated from the start of the introduction of reprogramming factors into somatic cells. That is, the pluripotent stem cell conversion period is less than about 30 days, for example, less than 29 days, 28 days, 27 days, 26 days, 25 days, 24 days, 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days or less.

[0104] In embodiments that do not involve gene modification (introducing reprogramming factors into somatic cells), the method converts somatic cells into pluripotent stem cells in less than about 30 days, calculated from the time the composition described herein comes into contact with somatic cells. That is, the pluripotent stem cell conversion period is less than about 30 days, for example, less than 29 days, 28 days, 27 days, 26 days, 25 days, 24 days, 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days or less.

[0105] In one or more embodiments, the somatic cells may or may not contain genetic modifications. In one or more embodiments, the pluripotent stem cells may or may not contain genetic modifications. In some cases, the genetic modification contains a foreign nucleic acid sequence. In some cases, the foreign nucleic acid sequence encodes a polypeptide. In some cases, the foreign nucleic acid sequence contains a sequence of a non-coding nucleic acid molecule. In some cases, the genetic modification contains an alteration to the genome sequence.

[0106] In this document, the reprogramming factor for gene modification comprises: genes specifically expressed in embryonic cells, or products of such genes. In one or more embodiments, the reprogramming factor comprises at least the Oct3 / 4 gene. In one or more embodiments, the reprogramming factor comprises the Oct3 / 4 gene and one, two, or three family genes selected from the Sox family, Myc family, and K1f family. In one or more embodiments, the Sox family genes are selected from: Sox1, Sox2, Sox3, Sox15, Sox17, and Sox18 genes. In one or more embodiments, the Myc family genes are selected from: c-Myc, N-Myc, and L-Myc genes. In one or more embodiments, the Klf family genes are selected from: Klf1, Klf2, Klf4, and Klf5 genes. In one or more embodiments, the reprogramming factor further comprises at least one gene selected from Lin28 and Sall1. The reprogramming factor may also include substances that downregulate p53 expression and substances that upregulate EBNA1 expression.

[0107] In one or more embodiments, the reprogramming factor comprises: (a) a combination of the Oct3 / 4 gene, the Sox family gene, the Klf4 gene, and the L-Myc gene, or a combination of the products of these genes; (b) a combination of the Oct3 / 4 gene, the Sox2 gene, the L-Myc family gene, and the Klf4 gene, or a combination of the products of these genes; (c) a combination of the Oct3 / 4 gene, the Sox2 gene, the L-Myc gene, and the Klf family gene, or a combination of the products of these genes. In one or more embodiments, the reprogramming factor comprises: Oct3 / 4, Sox2, and Klf4; Oct3 / 4, Sox2, Klf4, and Lin28; Oct3 / 4, Sox2, Klf4, and Sall1; Oct3 / 4, Sox2, Klf4, and L-Myc; Oct3 / 4, Sox2, Klf4, L-Myc, and Lin28; or Oct3 / 4, Sox2, Klf4, L-Myc, and Sall1.

[0108] In this document, the sequences of the OCT3 / 4 proteins are shown in SEQ ID NO:1, the sequences of the SOX2 proteins are shown in SEQ ID NO:2, the sequences of the KLF4 proteins are shown in SEQ ID NO:3, the sequences of the L-MYC proteins are shown in SEQ ID NO:4, and the sequences of the LIN28 proteins are shown in SEQ ID NO:5.

[0109] The present invention also provides stem cell therapy, comprising: a method / step of inducing the differentiation of somatic cells collected from healthy persons or patients into induced pluripotent stem cells as described in claim 98, and transplanting the resulting somatic cells or tissues, organs or body fluids prepared by the final differentiation of the induced pluripotent stem cells into the allogeneic or autologous patient.

[0110] The present invention also provides cells obtained by differentiating and inducing induced pluripotent stem cells obtained by any of the embodiments described herein, and methods for using these cells to determine the physiological activity or toxicity of compounds, drugs or toxins.

[0111] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. The invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the raw materials or processing techniques used in the following embodiments are conventional commercially available raw materials or conventional processing techniques in the art.

[0112] Example

[0113] Experimental materials:

[0114] Umbilical cord blood CD34+ blood cell culture medium (Table 1)

[0115] reagents brand Item number Working concentration <![CDATA[StemSpan TM -AOF]]> STEMCELL Technologies 100-0130 NA Recombinant human IL-6 PeproTech 200-06 50ng / ml Recombinant human IL-3 PeproTech 200-03 50ng / ml Recombinant human SCF PeproTech 300-07 50ng / ml Recombinant human Flt3-L PeproTech 300-19 50ng / ml Recombinant human GM-CSF PeproTech 300-03 50ng / ml Recombinant human TPO PeproTech 300-18 50ng / ml StemRegenin 1 (SR1) Selleck S2858 0.75uM

[0116] B27 / N2 culture medium (Table 2)

[0117]

[0118]

[0119] 50×: Diluted 50 times

[0120] 100×: Diluted 100 times

[0121] iPSC rapid induction medium (Table 3)

[0122]

[0123] Method 1: Plasmid preparation method

[0124] Material

[0125] Reagents (Table 4)

[0126] reagents brand Item number restriction endonuclease EcoRI Thermo FD0274 DNA Marker Xitu Biology GQ001 PrimeSTAR Max DNA Polymerase TaKaRa R045 A 5xIn-FusionHD Enzyme TaKaRa ST0344 Agarose Gel DNA Recovery Kit AxyGEN AP-GX-250 E. coli stbl3 competent cell Xitu Biology MG216 SYBR Safe DNA gel stain invitrogen S33102 NucleoBond Xtra Midi Plus EF MN 740420.50

[0127] Instruments (Table 5)

[0128] instrument brand model DNA synthesizer Lingkun LK-192 PCR instrument BioRad T100 Gel imaging system Tanon 2500R Nanodrop Thermo Nano2000C Small benchtop high-speed centrifuge Thermo PICO17 Large capacity low speed centrifuge Thermo ST40R Gel electrophoresis apparatus BioRad Powerpac HC Electronic balance METTLER XS104 Pure water system HealForce SMART-RO Biosafety cabinet NUAIRE NU-440-600E isothermal incubator shaker Minquan MQDB3R constant temperature water bath Jinghong D-80

[0129] Experimental methods

[0130] 1. Divide the target sequence into 3-4kb segments, with adjacent segments overlapping by 20bp at the ends. Each segment is decomposed into overlapping primer sequences of 50-60nt using gene synthesis software, and the corresponding fragments are synthesized by PCR splicing.

[0131] 2. Recombination reaction: The segmented synthetic fragments are linked and cyclized using a recombinase. Reaction system:

[0132] Element volume Synthetic fragment (50 ng / ul) 1ul each 5*HD InFusion enzyme 2ul ddH2O Add to 10ul

[0133] Gently pipette to mix, briefly centrifuge, and incubate at 50°C for 15 minutes.

[0134] 3. Transformation: Add 10 μL of the recombinant reaction product to 100 μL of stbl3 competent cells, gently tap the tube wall a few times to mix, place on ice for 5 min, spread the transformed bacterial solution evenly on LB agar plates containing 100 ug / ml ampicillin, and incubate upside down in a constant temperature incubator for 12-16 h.

[0135] 4. Sequencing alignment: Select 3-5 clones from each plate for Sanger sequencing identification, and compare the sequencing results with the designed plasmid map.

[0136] 5. Plasmid extraction: The correctly sequenced bacterial culture was transferred to 100 ml of LB liquid medium containing 100 ug / ml ampicillin and cultured overnight at 37°C. Plasmid extraction was performed using the MN endotoxin-free plasmid extraction kit. The extracted plasmid was quantified using Nanodrop and then diluted to 3000 ng / ul with endotoxin-free ultrapure water.

[0137] Plasmid information:

[0138]

[0139] Method 2: Method for reprogramming CD34+ blood cells into iPSCs

[0140] Umbilical cord blood CD34+ blood cell culture and electroporation:

[0141] Day 3, CD34+ blood cell resuscitation: cells were resuspended at a density of 0.5E6 / ml in umbilical cord blood CD34+ blood cell culture medium (Table 1) and placed in a 37°C cell culture incubator for static culture.

[0142] Day-2 / -1 Half-change of medium: With the pipette close to the liquid surface, carefully discard half of the supernatant and add fresh CD34+ blood cell culture medium to continue culturing.

[0143] Day 0 Electroporation: Preheat the culture medium, collect and centrifuge CD34+ blood cells, resuspend and count them, and take 2.4E5 cells per cell and electroporate with Lonza electroporation buffer (solution 16.4ul + supplement 3.6ul) (P3 Primary Cell 4D-Nucleofector™ X Kit). The mixture of S, Lonza, Cat#V4XP-3032) and reprogramming plasmids (reprogramming plasmids 1-5; where plasmids 1-3: 0.4 μL / reaction, plasmid 4: 0.3 μL / reaction, plasmid 5: 0.24 μL / reaction) was added to the electroporation tank, and the electroporation program was EO-115 (4D-NucleofectorTM, Lonza, Cat#AAF-1002B / AAF-1002X). After electroporation, the electroporated cells were aspirated with preheated culture medium and transferred to a six-well plate coated with Lamnin521 (BioLamina, Cat#LN521). The plate was then placed in a 5% CO2, 37°C cell culture incubator and cultured for 24 hours.

[0144] iPSC reprogramming-induced culture:

[0145] On Day 1, all cell supernatant was transferred to a 15ml centrifuge tube, centrifuged at 300g for 5min, the supernatant was discarded, and the corresponding volume of B27 / N2 medium (Table 2) was added and the cells were resuspended. The cell suspension was then transferred to the corresponding cell culture plate and placed in a 5% CO2, 37℃ cell culture incubator for further culture.

[0146] Day 3 Half-medium change: With the pipette close to the liquid surface, discard half of the culture medium in each well and add the corresponding volume of B27 / N2 culture medium (Table 2) to continue culturing.

[0147] Day 5 Half-medium change: With the pipette close to the liquid surface, discard half of the culture medium in each well and add the corresponding volume of B27 / N2 culture medium (Table 2) to continue culturing.

[0148] Reprogramming.

[0149] Day 7 Half-medium change: With the pipette close to the liquid surface, discard half of the culture medium in each well and add the corresponding volume of iPSC medium (mTesR1, STEMCELL Technologies, Cat#85850) to continue culturing.

[0150] Day 8: Complete medium change: Transfer all cell supernatant to a 15ml centrifuge tube, centrifuge at 300g for 5 minutes, discard the supernatant, add the appropriate volume of mTesR1 to resuspend the cells, and transfer the cell suspension from the centrifuge tube to the corresponding cell culture plate. Replace with fresh mTesR1 medium every 1-2 days until iPSC clones appear.

[0151] iPSC cell culture:

[0152] Laminin 521 coating of culture plates: Dilute laminin 521 to 7.5 μg / ml with DPBS containing calcium and magnesium ions for coating culture plates: 1 ml / well (6-well plate, Nunc, catalog number: 140675), incubate at 37°C for 2 hours. iPSC cell culture medium is mTeSR containing 1% P / S. TM 1. iPSC cell digestion and passage: Discard the old iPSC culture medium, wash once with DPBS (calcium and magnesium ions-free), then add 1 ml of ReLeSR™ (STEMCELL Technologies, Cat#100-0484), incubate at 37°C for 5 min, then discard the ReLeSR™, add 2 ml of iPSC cell culture medium to each well, gently disperse the cells at the bottom of the culture plate, and transfer 25-50 μL of cell suspension into a cell culture plate coated with laminin 521. Place the plate in a 37°C cell culture incubator, shake well, and incubate statically. Change the iPSC culture medium daily with fresh medium. When the cell density increases to approximately 70-80%, proceed with the next round of digestion and passage.

[0153] Method 3: Immunofluorescence identification method during iPSC reprogramming

[0154] Reagents (Table 6)

[0155] Product Name factory Item number 4% PFA (paraformaldehyde) Source Leaf Bio R20497-10x10ml Donkey Serum Solarbio SL050 D-PBS(-) NACALAI TESQUE 14249-24 MS@Oct4 Santacruz Sc-5279 Rb@Nanog(D73G4)XP CST 4903 Ms@TRA-1-60(S) CST 4746 Ms@SSEA4(MC813) CST 4755 Donkey anti-mouse 488 ThermoFisher A-21202 Donkey anti rabbit Cy3 Jackson ImmunoResearch 711-165-152 DAPI Solution DOJINDO D523 Immunofluorescence mounting tablets DAKO S3023 Sodium hypochlorite (Pulax-S) AS ONE 2-2171-02

[0156] Antibody dilution ratios (Table 7)

[0157]

[0158]

[0159] method:

[0160] Umbilical cord blood CD34+ cells were electroporated and reprogrammed to day 8 and day 12 for immunofluorescence identification.

[0161] 1. Sample preparation: Fix with 4% PFA for 30 min, wash three times.

[0162] 2. Prepare blocking solution: PBST (0.5% Triton-X + 5% Donkey serum + PBS)

[0163] 3. Prepare primary antibody: primary antibody (antibody dilution ratio table) + blocking buffer

[0164] 4. Prepare the secondary antibody: secondary antibody (antibody dilution ratio table) + blocking buffer

[0165] 5. Prepare DAPI: 1000× + sealing solution

[0166] 6. Incubate the blocking solution at room temperature for 1 hour.

[0167] 7.1 Resistance to overnight storage at 4℃

[0168] 8. Wash three times with 500 μL PBST, 5 min each time.

[0169] 9.2 Anti-RT 45min

[0170] 10. DAPI 5min

[0171] 11. Wash three times with 500 μL PBST, 5 min each time.

[0172] 12. Seal the film and take photos.

[0173] Method 4: iPSC cell flow cytometry detection method

[0174] Reagents (Table 8)

[0175] Product Name factory Item number ACCUTASE STEMCELL Technologies 07920 AF 488 anti-Oct4 Antibody biolegend 653706 PE Mouse anti-Human Nanog BD 560483 PE Mouse Anti-SSEA-4 BD 560128 PE Mouse anti-Human TRA-1-60 BD 560193 PE Mouse IgG1,κIsotype BD 550617 AF488Mouse IgG2b,κIsotype biolegend 400329 PE Mouse IgG3,κIsotype BD 556659 PE Mouse IgM,κIsotype BD 555584 Stain Buffer (BSA) BD 554657 FOXP3 / TRN FACTOR STAIN BUFFER eBioscience 00-5523-00

[0176] method:

[0177] 1. Digest iPSCs into single cells using Accutase (37°C, 5 minutes), count the cells, and transfer the required number of iPSCs into 1.5 mL centrifuge tubes. The required total cell suspension volume (mL) = 2E5 (cells) / viable cell density (cells / mL), 2E5 per sample.

[0178] 2. Add 1 mL of Staining Buffer to each tube to resuspend the cells, and centrifuge at 180 G for 5 minutes at room temperature.

[0179] 3. Add 0.5 mL of 4% PFA (paraformaldehyde) to each tube, pipette and aspirate 3-5 times to mix, incubate on wet ice for 10 minutes, then centrifuge to remove the supernatant.

[0180] 4. Add 0.5 mL of Foxp3 fixation / membrane breaking working solution to each tube, pipette and aspirate 3-5 times to mix, incubate on wet ice for 20 minutes, then centrifuge at 180 G for 5 minutes at room temperature.

[0181] 5. Add 1 mL of 1× membrane rupture buffer to each tube, pipette and aspirate 3-5 times to mix, then centrifuge.

[0182] 6. Add 50uL of blocking buffer to each tube, mix well, and incubate at room temperature for 40 minutes.

[0183] 7. Add 1 mL of 1× membrane rupture buffer to each tube, pipette and aspirate 3-5 times to mix, then centrifuge.

[0184] 8. Add Anti-Oct4, anti-Human Nanog, Anti-SSEA-4 and anti-Human TRA-1-60 antibodies and their corresponding isotype antibodies to the corresponding centrifuge tubes and incubate at room temperature for 60 minutes.

[0185] 9. Wash 2-3 times with 1× membrane rupture buffer and then discard the supernatant.

[0186] 10. Resuspend the cells in 200 μL of Staining Buffer, aspirate 3-5 times with a pipette to mix, and store on ice in the dark.

[0187] 11. Flow cytometry analysis was performed using a Cytoflex S flow cytometer (Beckman, DL230324-0146). Method 5: Flow cytometry detection method for in vitro differentiation of iPSC cells into three germ layers.

[0188] Reagents (Table 9)

[0189]

[0190]

[0191] The differentiation function of iPSCs was verified using an in vitro trigerm layer differentiation kit. When the iPSC confluence reached 70-80%, the iPSCs were digested into single cells by incubation with ACCUTASE digestive enzyme at 37°C for 5-10 minutes. Following the kit instructions, the appropriate number of iPSCs and differentiation reagents were seeded into 12-well plates (endoderm: 8E5 / 1.5ml / well; mesoderm: 2E5 / 1.5ml / well; ectoderm: 8E5 / 1.5ml / well). When the iPSCs reached the trigerm layer differentiation endpoint, the cells were digested into single cells by digestive enzyme, and 2E5 cells were counted and collected into centrifuge tubes. The cells were centrifuged at 300×g for 3 min. The supernatant was discarded, and the cells were resuspended in 200 μL of PBS containing 2% FBS. The cells were centrifuged at 300×g for 3 min, and the supernatant was discarded again. 200 μL of membrane permeabilization and fixation working solution (eBioscience) was added. TM Resuspend cells in Foxp3 / Transcription Factor Staining Buffer Set. Incubate at room temperature, protected from light, for 30-60 min. Centrifuge at 300×g for 3 min and discard the supernatant. Add 200 μL of permeabilization buffer, centrifuge at 300×g for 3 min, and repeat the washing twice. Resuspend cells in 100 μL of permeabilization buffer, add flow cytometry fluorescent antibody reagents PE-Pax-6, Sox17, and Brachyury, and incubate at room temperature, protected from light, for 30 min. Add 200 μL of permeabilization buffer, centrifuge at 300×g for 3 min, and repeat the washing twice. Resuspend cells in 200 μL of PBS containing 2% FBS. Unstained cells serve as a negative control. After adjusting the FSC and SSC voltages of the flow cytometer, detect the expression of Pax6, Sox17, and Brachyury in the cells using the corresponding fluorescence channels.

[0192] As shown in the figure, the results showed that the expression rates of Pax6, Sox17 and Brachyury were 99.9%, 59.8% and 99.8%, respectively, indicating that iPSCs have the ability to differentiate into endoderm (Sox17), mesoderm (Brachyury) and ectoderm (Pax6).

[0193] Method 6: iPSC karyotype analysis method

[0194] Reagents (Table 10)

[0195]

[0196] Step 1: Cell preparation:

[0197] (1) The cells were cultured in an incubator until harvest. The culture conditions were 37°C and 5% CO2.

[0198] (2) Add 20 μL of colchicine solution (20 μg / mL) to the culture flask, invert the culture flask, mix well, and continue to culture for 20 minutes until harvest.

[0199] Step 2: Chromosome preparation:

[0200] (1) Obtain cells co-cultured with colchicine, centrifuge at 1,100 rpm for 10 minutes, and discard the supernatant. Add 0.075M potassium chloride solution to the centrifuge tube and place it in a 37℃ water bath for 15-17 minutes for hypotonic treatment;

[0201] (2) Use 1 mL of fresh fixative (acetic acid: methanol = 1:3) for pre-fixation, repeat the fixation twice before dropping the slide; after centrifugation, add fresh fixative to the cell slurry, and reserve an appropriate amount of fixative according to the cell count (600 μL / 80,000 cells). Gently tap the tube wall with your finger to mix the cells and prepare a cell suspension.

[0202] (3) Drop 75-100 μL of cell suspension onto a clean, damp, and cold glass slide, air dry, and bake the prepared glass slide in an oven at 90°C for 2 hours.

[0203] Step 3: G-band staining:

[0204] (1) Digest the baked slide in trypsin separation solution for 33 seconds, then place it in FBS solution for 1 minute to stop the trypsin action, and finally immerse it in Giemsa staining solution for 2.5 minutes. Rinse the slide with tap water and blow dry.

[0205] (2) Place 2 drops of neutral resin mounting medium on each slide, cover with a glass slide, and wait for the mounting medium to dry completely before scanning.

[0206] Step 4: Result Analysis and Interpretation:

[0207] (1) The slides were scanned using a Leica GSL120 fully automated fluorescence microscope system. Each slide was examined at 100x magnification, and 500 fields of view containing metaphase cells were collected.

[0208] (2) Use Cytovision software to examine chromosome morphology.

[0209] (3) Select 50 metaphase cells with banding levels of 400-550 or higher and perform chromosome banding analysis.

[0210] Example 1: Optimization and screening of small molecule compositions during iPSC-induced reprogramming

[0211] To expedite the iPSC induction and reprogramming process, following Method 2 (method for reprogramming CD34+ blood cells into iPSCs), one of the experimental conditions A, B, C, D, or E from Table 10 was added on days 2, 3, 4, 5, 6, and 7 after electroporation reprogramming. The number of CD34+ blood cells for each condition was 0.24E5. Cells were cultured until day 10 after electroporation reprogramming, and the number of iPSC clones generated under each experimental condition was counted on day 12. Figure 1 ).like Figure 2 The experimental results showed that under experimental condition B, the addition of the small molecule combination "DZNeP (10 nM) + TSA (5 nM) + PD0325901 (1 uM) + EPZ5676 (0.1 uM)" (i.e. iPSC rapid induction medium (Table 3)) on days 3 and 4 of electroporation reprogramming resulted in a better number of iPSC clones (iPSC clone statistics criteria: typical iPSC morphological characteristics, homogeneous cells, tight intercellular connections, and more than 200 cells per iPSC clone), namely 8 and 11 clones respectively; while the condition without the addition of the small molecule did not produce any iPSC clones.

[0212] Small molecule combinations (Table 10)

[0213]

[0214]

[0215] Example 2: Study on the concentration of small molecule combinations in iPSC rapid induction medium

[0216] Following Example 1, experimental condition B (iPSC rapid induction medium (Table 3)) was added on day 3 of electroporation reprogramming, and the effect of changes in the concentration of the small molecule combination "DZNeP+TSA+PD0325901+EPZ5676" on iPSC cloning was investigated simultaneously. Experimental conditions are shown in Table 11. iPSC cloning was statistically analyzed on day 12 of electroporation reprogramming, and the results are as follows... Figure 3 The results showed that, compared with the control conditions, iPSC clones could be obtained on day 12 of electroporation reprogramming with EPZ5676 at 0.05 uM-1 uM, DZNeP at 0.01 uM-0.1 uM, TSA at 0.005 uM-0.025 uM, and PD0325901 at 0.25 uM-1 uM. Among them, the concentration of the small molecule combination "DZNeP (10 nM) + TSA (5 nM) + PD0325901 (1 uM) + EPZ5676 (0.1 uM)" obtained the optimal number of iPSC clones.

[0217] Small molecule concentration conditions (Table 11)

[0218]

[0219] Example 3: Further experimental verification of experimental condition B in Example 1

[0220] Considering the experimental results of Example 1, to further accelerate iPSC reprogramming, the small molecule combinations from Experimental Condition B (iPSC rapid induction medium (Table 3)) were added on days 2 and 3 of electroporation reprogramming in Example 1 for further experimental verification (e.g., Figure 4 The experimental conditions for Example 2 are shown in Table 12, with a total of 4 conditions. Condition 1 is the control condition, derived from the classic electroporation reprogramming method of Yamanaka (Keisuke Okita, et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells. 2013 Mar; 31(3):458-66. doi:10.1002 / stem.1293.). Condition 2 is set by removing the two factors shP53 RNA and EBNA1, thereby increasing the safety of iPSCs obtained by electroporation reprogramming in clinical applications. The other two conditions are: Condition 3 is the addition of iPSC rapid induction medium (Table 3) on the 3rd day after electroporation based on Condition 2, and Condition 4 is the addition of iPSC rapid induction medium (Table 3) on the 2nd day after electroporation. On day 8 of electroporation reprogramming, the iPSC induction reprogramming medium was replaced with mTesR1 (STEMCELL Technologies, Cat#85850), and samples were taken for immunofluorescence identification of iPSC clones. On day 12 of electroporation reprogramming, immunofluorescence identification of iPSC clones and iPSC clone count were performed (iPSC clone count criteria: typical iPSC morphological characteristics, homogeneous cells, tight intercellular connections, and more than 200 cells per iPSC clone). Bright-field photography was performed throughout the iPSC electroporation reprogramming process, and the results are as follows. Figure 5-8 The results showed that condition 1 (control condition) produced obvious iPSC clones on day 16 of electroporation reprogramming, while no obvious iPSC clones were observed during the 16-day electroporation reprogramming process in condition 2; obvious iPSC clone formation was observed starting on day 8 of electroporation reprogramming in both conditions 3 and 4. Since condition 4 did not produce iPSC clones earlier than condition 3, the identification experimental data of condition 3 will be the focus of subsequent analysis. Figure 9-11Immunofluorescence staining results of NANOG and SSEA4, key biomarkers of stem cells, on days 8 and 12 of electroporation reprogramming under conditions 1-3. The results showed that in conditions 1 and 2, the NANOG and SSEA4 immunofluorescence staining was relatively dispersed, and no typical iPSC clone was formed between cells, indicating that no iPSC clones were generated at this stage. However, in condition 3, on day 8, the NANOG and SSEA4 immunofluorescence staining overlapped, and the cell connections were relatively tight, forming a typical iPSC clone morphology. The fluorescence results on day 12 showed that the iPSC clones further proliferated and enlarged; similarly, the NANOG and SSEA4 immunofluorescence staining completely overlapped, presenting a typical iPSC clone morphology. The number of iPSC clones in conditions 1-3 was counted on day 12 of electroporation reprogramming (e.g., ...). Figure 12 The results showed that no clones were generated under conditions 1 and 2, while approximately 21 iPSC clones (n=2) were generated under condition 3. In summary, adding iPSC rapid induction medium (Table 3) on the 2nd or 3rd day after electroporation reprogramming in this technical protocol can significantly accelerate the iPSC induction and reprogramming time, ultimately advancing the generation of iPSC clones to the 8th day after electroporation, which is half the iPSC reprogramming time (16 days) under the control conditions.

[0221] Experimental conditions for Example 2 (Table 12)

[0222]

[0223]

[0224] Example 4: Flow cytometry analysis of iPSC clones generated under conditions 1-3 in Example 2

[0225] The iPSC clones generated under conditions 1-3 in Example 2 were subjected to flow cytometry detection according to method 4, and the detection results are as follows: Figure 13 Flow cytometry results showed that condition 3 had expression rates of 100%, 99.9%, 96.7%, and 95.5% for the key stem cell biomarkers SSEA4, TRA-1-60, OCT4, and NANOG, respectively, which were superior to conditions 1 and 2.

[0226] Example 5: Functional experiment on the trigerm layer differentiation of iPSC clones generated under conditions 1-3 in Example 2.

[0227] To verify whether the iPSC clones generated under conditions 1-3 in Example 2 have the ability to differentiate into three germ layers, three germ layer differentiation and flow cytometry detection were performed according to method 5. The experimental results are as follows: Figure 14The results showed that the iPSC clones generated under conditions 1-3 in Example 2 all had the ability to differentiate into three germ layers, and the flow cytometry results of the three germ layer differentiation under condition 3 were not significantly different from those under the control condition.

[0228] Example 6: Karyotype analysis of iPSC clones generated under conditions 1-3 in Example 2

[0229] To verify the genetic stability of the iPSC clones generated under conditions 1-3 in Example 2, karyotype analysis of the iPSCs was performed according to method 6. The experimental results are as follows: Figure 15 See Table 13. The results showed that experimental conditions 1-3 all yielded normal iPSC karyotypes; however, further analysis according to the karyotype analysis methods in the Chinese Pharmacopoeia revealed that condition 1 (the reprogrammed plasmid contained shP53 RNA and EBNA1 factor) produced a greater number of iPSCs with structural abnormalities, while the number of karyotype abnormalities in experimental conditions 2 and 3 remained within the standard range of 100 karyotype abnormalities in cells at the mitotic phase. This suggests that the iPSCs obtained using the reprogramming method in this study have higher safety.

[0230] Table 13. Statistical results of the number of abnormal chromosome karyotypes in 100 cells in the mitotic phase.

[0231]

[0232] Partial sequence

[0233] SEQ ID NO:1OCT3 / 4:MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSEVWGIPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAGVGVESNSDGASPEPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVL FGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETLVQARKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGEAFPPVSVTTLGSPMHSN

[0234] SEQ ID NO:2SOX2

[0235] MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLHMSQHYQSGPVPGTAINGTLPLSHM

[0236] SEQ ID NO:3KLF4

[0237] MNNSPFTMAVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMKRLPPVLPGRPYDLAAATVATDLESGGAGAACGGSNLAPLPRRETEEFNDLLDLDFILSNSLTHPPESVAATVSSSASASSSSSPSSSGPASAPSTCSFTYPIRAGNDPGVAPGGTGGGLLYGRESAPPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKASLSAPGSEYGSPSVISVSKGSPDGSHPVVVAPYNGGPPRTCPKIKQEAVSSCTHLGAGPPLSNGHRPAAHDFPLGRQLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYPSFLPDQMQPQVPPLHYQELMPPGSCMPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCDRAFSRSDHLALHMKRHF

[0238] SEQ ID NO:4L-MYC

[0239] MDYDSYQHYFYDYDCGEDFYRSTAPSEDIWKKFELVPSPPTSPPWGLGPGAGDPAPGIGPPEPWPGGCTGDEAESRGHSKGWGRNYASIIRRDCMWSGFSARERLERAVSDRLAPGAPRGNPPKASAAPDCTPSLEAGNPAPAAPCPLGEPKTQACSGSESPSDSENEEIDVVTVEKRQSLGIRKPVTITVRADPLDPCMKHFHISIHQQQHNYAARFPPESCSQEEASERGPQEEVLERDAAGEKEDEEDEEIVSPPPVESEAAQSCHPKPVSSDTEDVTKRKNHNFLERKRRNDLRSRFLALRDQVPTLASCSKAPKVVILSKALEYLQALVGAEKRMATEKRQLRCRQQQLQKRIAYLTGY

[0240] SEQ ID NO:5LIN28

[0241] MNNSPFTMGSVSNQQFAGGCAKAAEEAPEEAPEDAARAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDPPVDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKGKSMQKRRSKGDRCYNCGGLDHHAKECKLPPQPKKCHFCQSISHMVASCPLKAQQGPSAQGKPTYFREEEEEIHSPTLLPEAQN

Claims

1. A composition for promoting somatic cell reprogramming to produce iPSCs, said composition comprising an EZH2 methyltransferase inhibitor, a histone deacetylase inhibitor, and a MEK inhibitor. Preferably, the composition further comprises one or more of the following: methyltransferase H3K36me3 activator, DOT1L inhibitor, PKC inhibitor, ROCK inhibitor, More preferably, the EZH2 methyltransferase inhibitor is DZNeP, the histone deacetylase inhibitor is TSA, the MEK inhibitor is PD0325901, the methyltransferase H3K36me3 activator is vitamin B12, the DOT1L inhibitor is EPZ5676, the PKC inhibitor is Go6983, and the ROCK inhibitor is Y23672.

2. The composition according to claim 1, characterized in that, The concentration range of DZNeP is 0.01uM-0.1uM, the concentration range of EPZ5676 is 0.05uM-1uM, the concentration range of TSA is 0.005uM-0.025uM, and the concentration range of PD0325901 is 0.25uM-1uM.

3. A rapid induction medium for iPSCs, characterized in that, The culture medium comprises the composition of claim 1.

4. A reagent kit, characterized in that, The kit includes the culture medium as described in claim 3.

5. A method for preparing iPSCs or inducing somatic cell reprogramming to generate iPSC clones, comprising: 1) Introduce reprogramming factors into somatic cells; 2) Culture the cells obtained in step 1) in a culture medium containing the composition of claim 1; 3) Select cells with embryonic stem cell-like morphology generated in step 2). Preferably, the method further includes: step 4) further culturing the cells selected in step 3) in a culture medium.

6. The method of claim 5, wherein the reprogramming factor comprises: a gene specifically expressed in embryonic cells, or a product of such genes. Preferably, The reprogramming factor contains at least the Oct3 / 4 gene. The reprogramming factor includes the Oct3 / 4 gene and one, two, or three gene families selected from the Sox family, Myc family, and K1f family. The Sox family genes are selected from: Sox1, Sox2, Sox3, Sox15, Sox17, and Sox18 genes. The Myc family genes are selected from: c-Myc gene, N-Myc gene, and L-Myc gene. The Klf family genes are selected from: Klf1, Klf2, Klf4, and Klf5 genes. More preferably, the reprogramming factor comprises: (a) a combination of the Oct3 / 4 gene, the Sox family gene, the Klf4 gene, and the cL-Myc gene, or a combination of the products of these genes; (b) a combination of the Oct3 / 4 gene, the Sox2 gene, the L-Myc family gene, and the Klf4 gene, or a combination of the products of these genes; and (c) a combination of the Oct3 / 4 gene, the Sox2 gene, the cL-Myc gene, and the Klf family gene, or a combination of the products of these genes. More preferably, the reprogramming factor further comprises at least one gene selected from Lin28 and Sall1.

7. The method as described in claim 6, characterized in that, The reprogramming factor includes: Oct3 / 4, Sox2 and Klf4, Oct3 / 4, Sox2, Klf4 and Lin28, Oct3 / 4, Sox2, Klf4 and Sall1, Oct3 / 4, Sox2, Klf4 and L-Myc, Oct3 / 4, Sox2, Klf4, L-Myc and Lin28, or Oct3 / 4, Sox2, Klf4, L-Myc and Sall1.

8. Induced pluripotent stem cells obtained by the method of claim 6 or 7.

9. A stem cell therapy, comprising: Methods / steps for using somatic cells collected from healthy individuals or patients to differentiate and induce induced pluripotent stem cells as described in claim 8, and transplanting the resulting somatic cells, tissues, organs, or body fluids prepared by the final differentiation of the induced pluripotent stem cells to allogeneic or autologous patients.

10. A method for determining the physiological activity or toxicity of a compound, drug, or toxin using cells obtained from induced pluripotent stem cells as described in claim 8.

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