Method for chemically inducing pluripotent stem cell and induction composition

Abstract

Provided is a chemical reprogramming method for obtaining an induced pluripotent stem cell from a somatic cell of a primate. Further provided is a composition containing a chemical inducer that can be used in the method.

Description

Method and composition for chemically inducing pluripotent stem cells Technical Field

[0001] The present invention relates to a method for inducing the generation of pluripotent stem cells from somatic cells, and an inducing agent used in the method. Background Art

[0002] Human somatic cells can restore cell plasticity and be reprogrammed to a pluripotent state by introducing and expressing pluripotency transcription factors or exposing them to a combination of chemical reagents. Compared with classic transcription factor reprogramming methods (such as OKSM), chemical reprogramming methods do not introduce exogenous gene loads into the genome. However, chemical reprogramming usually requires a significantly longer induction time, generally 28 to 32 days, and the induction efficiency varies greatly on different starting cells, and it is very common to be less than 1%.

[0003] Studies have shown that epigenetic status may be one of the main factors affecting the gradual recovery of cells from a differentiated state to a pluripotent state. Many compounds and factors with epigenetic regulatory properties have enhanced the generation of iPS cells, reflecting the importance of epigenetic remodeling in the cell reprogramming process. Histone acetylation is a major epigenetic modification known to play a role in a range of cellular physiological activities, including cell proliferation, cell differentiation / dedifferentiation, and so on. It has been reported that histone deacetylase (HDAC) 1 / 2 inhibitors can inhibit the dedifferentiation of nasopharyngeal carcinoma cells and regulate cell plasticity (Xie J et al., Targeting cancer cell plasticity by HDAC inhibition to reverse EBV-induced dedifferentiation in nasopharyngeal carcinoma. Signal Transduct Target Ther. 2021 Sep 4; 6(1): 333), and can also promote the dedifferentiation of breast cancer cells into cancer stem cells, accelerating cell proliferation and invasion (Debeb BG et al., Histone deacetylase inhibitors stimulate dedifferentiation of human breast cancer cells through WNT / β-catenin signaling. Stem Cells. 2012 Nov; 30(11): 2366-77).It has also been reported that small molecule inhibitors that inhibit the deacetylation activity of the NuRD complex (such as valproic acid and sodium butyrate) can promote reprogramming (see Huangfu D et al., Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol, 2008, 26(7): 795-797; Mali P et al., Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes. Stem Cells, 2010, 28(4): 713-720); WM-8041, a selective inhibitor of acetyltransferase KAT6A / B, can significantly increase the β-galactosidase activity of mouse fibroblasts (MEFs) and induce cell senescence. WM-8041 and its derivative WM-1119 can significantly inhibit the proliferation of lymphoma cells (Baell, JB et al., Inhibitors of histone The acetyltransferases KAT6A / B induce senescence and arrest tumor growth. Nature 560, 253–257 (2018)). Ayyub Ebrahimi's team reported the use of small molecule inhibitors SGC-CBP30 and I-CBP112 targeting the bromodomain of CBP / p300 to regulate transcription factor reprogramming of human fibroblasts. They found that the early addition of such inhibitors can effectively increase the number of iPS cells generated by canonical Nakayama factor (OSKM) reprogramming and can partially replace the induction reprogramming effect of Nakayama factors KLF4 and MYC (Ebrahimi A et al., Bromodomain inhibition of the coactivators CBP / EP300 facilitate cellular reprogramming. Nat Chem Biol 15, 519–528 (2019)).However, in experiments targeting another class of CBP / p300 inhibitors, A485 (which specifically inhibits the activity of the acetyltransferase catalytic domain), no such increase was observed, nor was SGC-CBP30 and I-CBP112 found to significantly shorten the reprogramming time. Furthermore, Ebrahimi A reported that no induced pluripotent stem cells could be obtained when SGC-CBP30 was used for chemical reprogramming.

[0004] Therefore, in order to be able to prepare pluripotent stem cells or other differentiated cells with pluripotent stem cells as precursors in a short time, large quantities and high purity in the industry, there is still a need for chemical reprogramming methods that can effectively improve reprogramming efficiency and shorten reprogramming time. Summary of the Invention

[0005] Through in-depth research, the inventors of the present application have discovered that during the process of chemical reprogramming of somatic cells, especially in the early stages of reprogramming, the addition of acetyltransferase inhibitors can significantly accelerate the speed of somatic cell reconstruction of pluripotency and shorten the time required for chemically induced reprogramming, without compromising or even further improving the reprogramming efficiency.

[0006] Therefore, an object of the present invention is to provide a method for chemically reprogramming induced pluripotent stem cells from somatic cells derived from primates, the method comprising the steps of exposing the somatic cells to one or more histone acetyltransferase inhibitors under conditions such that the somatic cells form a cell population having increased differentiation potential compared to the somatic cells, and further allowing the cell population to dedifferentiate into pluripotent stem cells.

[0007] In some embodiments, the cell population with increased differentiation potential formed by the chemical reprogramming methods disclosed herein primarily comprises epithelial-like cells positive for the pluripotency marker Lin28A, and the cells have elevated expression levels of epithelial cell markers and decreased expression levels of somatic cell markers compared to the somatic cells used as starting cells. In some embodiments, the epithelial cell marker is selected from one or more of KRT8, KRT18, or KRT19. In some embodiments, the epithelial cell marker is KRT8, KRT18, and KRT19. In some embodiments, the somatic cell marker is selected from one or more of VIM, SNAI2, COL5A1, PRKX1, COL6A2, ZEB1, MMP1, TWIST1, and COL1A1. In some embodiments, the somatic cell marker is VIM. In some embodiments, the somatic cell marker is SNAI2. In some embodiments, the somatic cell marker is COL5A1. In some embodiments, the somatic cell marker is PRKX1. In some embodiments, the somatic cell marker is COL6A2. In some embodiments, the somatic cell marker is ZEB1. In some embodiments, the somatic cell marker is MMP1. In some embodiments, the somatic cell marker is TWIST1. In some embodiments, the somatic cell marker is COL1A1.

[0008] In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cells in the cell population with increased differentiation potential formed by the chemical reprogramming method disclosed herein are epithelial-like cells that are positive for the pluripotency marker Lin28A, and have increased expression levels of epithelial cell markers and decreased expression levels of somatic cell markers compared to the somatic cells used as the starting cells.

[0009] In some embodiments, the expression level of epithelial cell markers in the cell population with increased differentiation potential formed by the chemical reprogramming methods disclosed herein is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, or at least about 300% compared to the somatic cells used as the starting cells. In some embodiments, the expression level of somatic cell markers in the cell population with increased differentiation potential formed by the chemical reprogramming methods disclosed herein is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, or at least about 300% compared to the somatic cells used as the starting cells.

[0010] In some embodiments, histone acetyltransferase inhibitors disclosed herein are specific for the catalytically active center of histone acetyltransferase. In other embodiments, histone acetyltransferase inhibitors disclosed herein are specific for the bromodomain of histone acetyltransferase. In some embodiments, histone acetyltransferase inhibitors disclosed herein can inhibit the acetyltransferase activity of any member of the GNAT family. In some embodiments, histone acetyltransferase inhibitors disclosed herein can inhibit the acetyltransferase activity of any member of the MYST family.

[0011] In some preferred embodiments, the histone acetyltransferase inhibitor used in the chemical reprogramming method disclosed herein is a CBP / P300 inhibitor, preferably one or more of A485, ICBP112, GEN049, CBP / P300 IN 12 or SGCCBP30, more preferably A485. In some preferred embodiments, the histone acetyltransferase inhibitor used in the chemical reprogramming method disclosed herein is a MOZ inhibitor, preferably one or more of CBP / P300 IN 8, WM8014 or WM1119, more preferably WM8014. In some preferred embodiments, the histone acetyltransferase inhibitor used in the chemical reprogramming method disclosed herein is a combination of a CBP / P300 inhibitor and a MOZ inhibitor, and the combination can be selected from A485 and WM8014, ICBP112 and WM8014, GEN049 and WM8014, CBP / P300 IN 12 and WM8014, SGCCBP30 and WM8014, A485 and WM1119, ICBP112 and WM1119, GEN049 and WM1119, CBP / P300 IN 12 and WM1119, SGCCBP30 and WM1119, A485 and CBP / P300 IN 8, ICBP112 and WM8014, GEN049 and WM8014 The combination of 12 and CBP / P300 IN 8, or SGCCBP30 and CBP / P300 IN 8, preferably the combination of A485 and WM8014.

[0012] In some embodiments, the concentration of the histone acetyltransferase inhibitor used in the chemical reprogramming method disclosed herein is 0.01 to 10 μM. In a preferred embodiment, the concentration of the histone acetyltransferase inhibitor used is 0.05 to 5 μM. In a more preferred embodiment, the concentration of the histone acetyltransferase inhibitor used is 0.1 to 5 μM. In a most preferred embodiment, the concentration of the histone acetyltransferase inhibitor used is 0.1 to 2 μM. In some embodiments, the chemical reprogramming method disclosed herein uses A485 at a concentration of 0.1 to 2 μM or WM8014 at a concentration of 0.1 to 5 μM or a combination of the two. In a preferred embodiment, the chemical reprogramming method disclosed herein uses A485 at a concentration of 0.5 to 1 μM or WM8014 at a concentration of 0.5 to 1 μM or a combination of the two. In a more preferred embodiment, the chemical reprogramming method disclosed herein uses A485 at a concentration of 0.5 μM or WM8014 at a concentration of 1 μM or a combination of the two.

[0013] In some embodiments, the chemical reprogramming method disclosed herein exposes the somatic cells to the histone acetyltransferase inhibitor for a period of less than 8 days. In a preferred embodiment, the chemical reprogramming method disclosed herein exposes the somatic cells to the histone acetyltransferase inhibitor for a period of 5 to 8 days. In a more preferred embodiment, the chemical reprogramming method disclosed herein exposes the somatic cells to the histone acetyltransferase inhibitor for a period of 8 days, 7 days, 6 days, or 5 days.

[0014] Chemical reprogramming method disclosed herein shortens the time required for somatic cells to become the cell mass for increasing differentiation potential. In some embodiments, compared with the situation that somatic cells are not exposed to histone acetyltransferase inhibitors, method disclosed herein can shorten the time required for somatic cells to become the cell mass for increasing differentiation potential by at least 8 days, preferably 8 to 12 days. In some embodiments, in chemical reprogramming method disclosed herein, somatic cells are made to form the conditions for the cell mass for increasing differentiation potential compared with the somatic cells including contacting the somatic cells with a composition that can increase cell differentiation potential, the composition includes glycogen synthase kinase (GSK) 3 β inhibitors, TGF β receptor inhibitors and retinoic acid receptor (RAR) agonists. In a preferred embodiment, the composition includes glycogen synthase kinase (GSK) 3 β inhibitors, TGF β receptor inhibitors, retinoic acid receptor (RAR) agonists and serine-threonine kinase (Akt) inhibitors. In a preferred embodiment, the composition comprises a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a serine-threonine kinase (Akt) inhibitor, and any one selected from a G protein coupled receptor Smoothened agonist, a Dot1L inhibitor, a Menin-MLL interaction inhibitor, a SAH hydrolase inhibitor, a Jak1 / Jak2 inhibitor, or any combination thereof. For example, in some more preferred embodiments, the composition comprises a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a serine-threonine kinase (Akt) inhibitor, a G protein coupled receptor Smoothened agonist, and a Dot1L inhibitor. In some more preferred embodiments, the composition comprises a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a serine-threonine kinase (Akt) inhibitor, a Dot1L inhibitor, and a Jak1 / Jak2 inhibitor. In some more preferred embodiments, the composition comprises a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a serine-threonine kinase (Akt) inhibitor, a Dot1L inhibitor, and a SAH hydrolase inhibitor. In some more preferred embodiments, the composition comprises a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a serine-threonine kinase (Akt) inhibitor, a Jak1 / Jak2 inhibitor, and a Menin-MLL interaction inhibitor. In a further preferred embodiment, the composition further comprises any one or any combination thereof selected from a c-Jun kinase inhibitor, a histone methyltransferase inhibitor, or an LPA1 receptor antagonist.In a most preferred embodiment, the composition comprises a glycogen synthase kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a G protein-coupled receptor Smoothened agonist, a Dot1L inhibitor, a Menin-MLL interaction inhibitor, a SAH hydrolase inhibitor, a Jak1 / Jak2 inhibitor, a c-Jun kinase inhibitor, a serine-threonine kinase (Akt) inhibitor, a histone methyltransferase inhibitor, and an LPA1 receptor antagonist.

[0015] In some embodiments, the glycogen kinase (GSK) 3β inhibitor disclosed herein is selected from CHIR99021, TD114-2, CHIR98014, GSK 3I inhibitor XV, BIO, SB-216763, or any combination thereof. In a preferred embodiment, the GSK3β inhibitor disclosed herein is CHIR99021. In some embodiments, the concentration of the GSK3β inhibitor disclosed herein is 3 to 12 μM. In a preferred embodiment, the concentration of the GSK3β inhibitor disclosed herein is 5 μM.

[0016] In some embodiments, the TGFβ receptor inhibitor disclosed herein is selected from 616452, A8301, SB505124, GW 788388, SB 525334, or any combination thereof. In a preferred embodiment, the TGFβ receptor inhibitor disclosed herein is 616452. In some embodiments, the concentration of the TGFβ receptor inhibitor disclosed herein is 2 to 50 μM. In a preferred embodiment, the concentration of the TGFβ receptor inhibitor disclosed herein is 10 μM.

[0017] In some embodiments, the retinoic acid receptor (RAR) agonist disclosed herein is selected from TTNPB, Ch 55, AM580, or any combination thereof. In a preferred embodiment, the RAR agonist disclosed herein is TTNPB. In some embodiments, the concentration of the RAR agonist disclosed herein is 0.5 to 10 μM. In a preferred embodiment, the concentration of the RAR agonist disclosed herein is 2 μM.

[0018] In some embodiments, the G protein-coupled receptor smoothened agonist disclosed herein is selected from SAG, Purmorphamine, Hg-Ag1.5, or any combination thereof. In a preferred embodiment, the G protein-coupled receptor smoothened agonist disclosed herein is SAG. In some embodiments, the concentration of the G protein-coupled receptor smoothened agonist disclosed herein is 0.2 to 2 μM. In a preferred embodiment, the concentration of the G protein-coupled receptor smoothened agonist disclosed herein is 0.5 μM.

[0019] In some embodiments, the Dot1L inhibitor disclosed herein is selected from EPZ5676, SGC 0946, EPZ004777, or any combination thereof. In a preferred embodiment, the Dot1L inhibitor disclosed herein is EPZ5676. In some embodiments, the concentration of the Dot1L inhibitor disclosed herein is 0.2 to 10 μM. In a preferred embodiment, the concentration of the Dot1L inhibitor disclosed herein is 2 μM.

[0020] In some embodiments, the Menin-MLL interaction inhibitor disclosed herein is selected from VTP50469, MI3454, WDR5-IN-4, or any combination thereof. In a preferred embodiment, the Menin-MLL interaction inhibitor disclosed herein is VTP50469. In some embodiments, the concentration of the Menin-MLL interaction inhibitor disclosed herein is 0.1 to 5 μM. In a preferred embodiment, the concentration of the Menin-MLL interaction inhibitor disclosed herein is 0.5 μM.

[0021] In some embodiments, the SAH hydrolase inhibitor disclosed herein is selected from DZNEP, (-)Neplanocin A, adenozine periodate oxidized, 3-deazaadenosine, or any combination thereof. In a preferred embodiment, the SAH hydrolase inhibitor disclosed herein is DZNEP. In some embodiments, the concentration of the SAH hydrolase inhibitor disclosed herein is 0.01 to 0.2 μM. In a preferred embodiment, the concentration of the SAH hydrolase inhibitor disclosed herein is 0.05 μM.

[0022] In some embodiments, the Jak1 / Jak2 inhibitor disclosed herein is selected from ruxolitinib, tofacitinib, AZD1480, baricitinib, fuzotinib, or any combination thereof. In a preferred embodiment, the Jak1 / Jak2 inhibitor disclosed herein is ruxolitinib. In some embodiments, the concentration of the Jak1 / Jak2 inhibitor disclosed herein is 0.1 to 10 μM. In a preferred embodiment, the concentration of the Jak1 / Jak2 inhibitor disclosed herein is 1 μM.

[0023] In some embodiments, the c-Jun kinase inhibitor disclosed herein is selected from JNKIN8, JNKIN7, JNKIN5, JNKIN12, or any combination thereof. In a preferred embodiment, the c-Jun kinase inhibitor disclosed herein is JNKIN8. In some embodiments, the concentration of the c-Jun kinase inhibitor disclosed herein is 0.05 to 2 μM. In a preferred embodiment, the concentration of the c-Jun kinase inhibitor disclosed herein is preferably 0.2 μM.

[0024] In some embodiments, the serine-threonine kinase (Akt) inhibitor disclosed herein is selected from HY-10249A (CAS No. 842148-40-7), PF-AKT400, Capivasertib, Afuresertib, or any combination thereof. In a preferred embodiment, the Akt inhibitor disclosed herein is HY-10249A (CAS No. 842148-40-7). In some embodiments, the concentration of the Akt inhibitor disclosed herein is 0.1 to 10 μM. In a preferred embodiment, the concentration of the Akt inhibitor disclosed herein is 1 μM.

[0025] In some embodiments, the histone methyltransferase inhibitor disclosed herein is selected from SETD2-IN-1, EPZ-719, MMSET-IN-1, or any combination thereof. In a preferred embodiment, the histone methyltransferase inhibitor disclosed herein is SETD2-IN-1. In some embodiments, the concentration of the histone methyltransferase inhibitor disclosed herein is 0.01 to 2 μM. In a preferred embodiment, the concentration of the histone methyltransferase inhibitor disclosed herein is 0.2 μM.

[0026] In preferred embodiments, the LPA1 receptor antagonist disclosed herein is selected from AM095, AM966, Ki16425, or any combination thereof. In a more preferred embodiment, the LPA1 receptor antagonist disclosed herein is AM095. In some embodiments, the concentration of the LPA1 receptor antagonist disclosed herein is 0.1 to 5 μM. In a preferred embodiment, the concentration of the LPA1 receptor antagonist disclosed herein is 0.5 to 1 μM. In a more preferred embodiment, the LPA1 receptor antagonist used in the chemical reprogramming method disclosed herein is 0.5 to 1 μM AM095.

[0027] In some embodiments of the chemical reprogramming methods disclosed herein, the conditions for causing somatic cells to form a cell population having increased differentiation potential compared to the somatic cells are contacting the somatic cells with a composition comprising 3-12 μM CHIR99021, 2-50 μM 616452, and 0.5-10 μM TTNPB. In a preferred embodiment, the conditions are contacting the somatic cells with a composition comprising 3-12 μM CHIR99021, 2-50 μM 616452, 0.5-10 μM TTNPB, and 0.1-10 μM HY-10249A (CAS No. 842148-40-7). In a more preferred embodiment, the conditions are such that the somatic cells are contacted with a composition comprising 3-12 μM CHIR99021, 2-50 μM 616452, 0.5-10 μM TTNPB, 0.1-10 μM HY-10249A (CAS No. 842148-40-7), and any one selected from 0.2-2 μM SAG, 0.2-10 μM EPZ5676, 0.1-5 μM VTP50469, 0.01-0.2 μM DZNEP, 0.1-10 μM ruxolitinib, or any combination thereof. For example, in some more preferred embodiments, the composition comprises 3-12 μM CHIR99021, 2-50 μM 616452, 0.5-10 μM TTNPB, 0.1-10 μM HY-10249A (CAS No. 842148-40-7), 0.2-2 μM SAG, and 0.2-10 μM EPZ5676. In some more preferred embodiments, the composition comprises 3-12 μM CHIR99021, 2-50 μM 616452, 0.5-10 μM TTNPB, 0.1-10 μM HY-10249A (CAS No. 842148-40-7), 0.2-10 μM EPZ5676, and 0.1-10 μM ruxolitinib. In some more preferred embodiments, the composition comprises 3-12 μM CHIR99021, 2-50 μM 616452, 0.5-10 μM TTNPB, 0.1-10 μM HY-10249A (CAS No. 842148-40-7), 0.2-10 μM EPZ5676, and 0.01-0.2 μM DZNEP. In some more preferred embodiments, the composition comprises 3-12 μM CHIR99021, 2-50 μM 616452, 0.5-10 μM TTNPB, 0.1-10 μM HY-10249A (CAS No. 842148-40-7), 0.1-10 μM ruxolitinib, and 0.1-5 μM VTP50469.In a further preferred embodiment, the composition comprises 3-12 μM CHIR99021, 2-50 μM 616452, 0.5-10 μM TTNPB, 0.1-10 μM HY-10249A (CAS No. 842148-40-7), 0.2-2 μM SAG, 0.2-10 μM EPZ5676, 0.1-5 μM VTP50469, 0.01-0.2 μM DZNEP, 0.1-10 μM ruxolitinib, and any one selected from 0.05-2 μM JNKIN8, 0.01-2 μM SETD2-IN-1, or 0.1 to 5 μM AM095, or any combination thereof. In a further preferred embodiment, the composition comprises 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1 and 0.2 μM JNKIN8. In a most preferred embodiment, the composition comprises 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8 and 0.5 μM095.

[0028] In a preferred embodiment, the chemical reprogramming method disclosed herein is to add a histone acetyltransferase inhibitor to a composition capable of increasing cell differentiation potential, and then contact the somatic cells with the added composition.

[0029] In some embodiments, the chemical reprogramming method disclosed herein is a method for obtaining induced pluripotent stem cells from human somatic cells.

[0030] In some embodiments, the chemical reprogramming method disclosed herein is a method for obtaining induced pluripotent stem cells from somatic cells selected from fibroblasts, blood cells, epithelial cells, or endothelial cells. In a preferred embodiment, the chemical reprogramming method disclosed herein is a method for obtaining induced pluripotent stem cells from fibroblasts. In some preferred embodiments, the fibroblasts are selected from adipose fibroblasts, skin fibroblasts, or umbilical cord mesenchymal stem cells.

[0031] In a preferred embodiment, the chemical reprogramming method for obtaining induced pluripotent stem cells from somatic cells from primates disclosed herein comprises contacting the somatic cells with the following composition for 5 to 8 days (e.g., contacting for 5 days, 6 days, 7 days or 8 days) to obtain a cell population having increased differentiation potential compared to the somatic cells, and further dedifferentiating the cell population into pluripotent stem cells, wherein the composition comprises 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8 and 0.5 μM A-485; or comprises 5 μM CHIR999021, 10μM 616452, 2μM TTNPB, 0.5μM SAG, 2μM EPZ5676, 0.02μM DZNep, 1μM Ruxolitinib, 0.5μM VTP50469, 1μM HY-10249A (CAS No.842148-40-7), 0.2μM SETD2IN1, 0.2μM JNKIN8, and 1μM WM8014; or containing 5μM CHIR999021, 10μM 616452, 2μM TTNPB, 0.5μM SAG, 2μM EPZ5676, 0.02μM DZNep, 1μM Ruxolitinib, 0.5μM VTP50469, 1μM HY-10249A(CAS No.842148-40-7), 0.2μM SETD2IN1, 0.2μM JNKIN8, 0.5μM A-485 and 1μM WM8014; or 5μM CHIR999021, 10μM 616452, 2μM TTNPB, 0.5μM SAG, 2μM EPZ5676, 0.02μM DZNep, 1μM Ruxolitinib, 0.5μM VTP50469, 1μM HY-10249A (CAS No.842148-40-7), 0.2μM SETD2IN1, 0.2μM JNKIN8, 0.5μM A-485 and 0.5μM AM095; or 5μM CHIR999021, 10μM 616452, 2μM TTNPB, 0.5μM SAG, 2μM EPZ5676, 0.02μM DZNep, 1μM Ruxolitinib, 0.5μM VTP50469, 1μM HY-10249A (CAS No. 842148-40-7), 0.2μM SETD2IN1, 0.2μM JNKIN8, 1μM WM8014 and 0.5μM AM095; or 5μM CHIR999021, 10μM 616452, 2μM TTNPB, 0.5μM SAG, 2μM EPZ5676, 0.02μM DZNep, 1μM Ruxolitinib, 0.5μM VTP50469, 1μM HY-10249A (CAS No.842148-40-7), 0.2μM SETD2IN1, 0.2μM JNKIN8, 0.5μM A-485, 1μM WM8014 and 0.5μM AM095, the cell population with increased differentiation potential is epithelial-like cells that are positive for the pluripotency marker Lin28A, and have increased expression levels of epithelial cell markers and decreased expression levels of somatic cell markers compared to the somatic cells.

[0032] Another object of the present invention is to provide the first composition, which includes a combination of reagents and one or more histone acetyltransferase inhibitors that increase somatic cell differentiation potential. In some embodiments, the composition includes glycogen synthesis kinase (GSK) 3β inhibitors, TGFβ receptor inhibitors, retinoic acid receptor (RAR) agonists and histone acetyltransferase inhibitors. In a preferred embodiment, the composition includes glycogen synthesis kinase (GSK) 3β inhibitors, TGFβ receptor inhibitors, retinoic acid receptor (RAR) agonists, serine-threonine kinase (Akt) inhibitors and histone acetyltransferase inhibitors. In a preferred embodiment, the composition includes glycogen synthesis kinase (GSK) 3β inhibitors, TGFβ receptor inhibitors, retinoic acid receptor (RAR) agonists, serine-threonine kinase (Akt) inhibitors and histone acetyltransferase inhibitors. In a preferred embodiment, the composition includes glycogen synthesis kinase (GSK) 3β inhibitors, TGFβ receptor inhibitors, retinoic acid receptor (RAR) agonists, serine-threonine kinase (Akt) inhibitors, histone acetyltransferase inhibitors and any one or their arbitrary combination selected from G protein coupled receptor Smoothened agonists, Dot1L inhibitors, Menin-MLL interaction inhibitors, SAH hydrolase inhibitors, Jak1 / Jak2 inhibitors. For example, in some more preferred embodiments, the composition comprises a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a serine-threonine kinase (Akt inhibitor, a histone acetyltransferase inhibitor, a G protein-coupled receptor Smoothened agonist and a Dot1L inhibitor. In some more preferred embodiments, the composition comprises a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a serine-threonine kinase (Akt inhibitor, a histone acetyltransferase inhibitor, a Dot1L inhibitor and a Jak1 / Jak2 inhibitor. In some more preferred embodiments, the composition comprises a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a serine-threonine kinase (Akt inhibitor, a histone acetyltransferase inhibitor, a Dot1L inhibitor and a SAH hydrolase inhibitor. In some more preferred embodiments, the composition comprises a glycogen synthase kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a serine-threonine kinase (Akt) inhibitor, a histone acetyltransferase inhibitor, a Jak1 / Jak2 inhibitor, and a Menin-MLL interaction inhibitor. In further preferred embodiments, the composition further comprises any one or any combination selected from a c-Jun kinase inhibitor, a histone methyltransferase inhibitor, or an LPA1 receptor antagonist.In a most preferred embodiment, the composition comprises a glycogen synthase kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a serine-threonine kinase (Akt) inhibitor, a histone acetyltransferase inhibitor, a G protein-coupled receptor Smoothened agonist, a Dot1L inhibitor, a Menin-MLL interaction inhibitor, a SAH hydrolase inhibitor, a Jak1 / Jak2 inhibitor, a c-Jun kinase inhibitor, a histone methyltransferase inhibitor, and an LPA1 receptor antagonist.

[0033] In some embodiments, the glycogen kinase (GSK) 3β inhibitor disclosed herein is selected from CHIR99021, TD114-2, CHIR98014, GSK 3I inhibitor XV, BIO, SB-216763, or any combination thereof. In a preferred embodiment, the GSK3β inhibitor disclosed herein is CHIR99021. In some embodiments, the concentration of the GSK3β inhibitor disclosed herein is 3 to 12 μM. In a preferred embodiment, the concentration of the GSK3β inhibitor disclosed herein is 5 μM.

[0034] In some embodiments, the TGFβ receptor inhibitor disclosed herein is selected from 616452, A8301, SB505124, GW 788388, SB 525334, or any combination thereof. In a preferred embodiment, the TGFβ receptor inhibitor disclosed herein is 616452. In some embodiments, the concentration of the TGFβ receptor inhibitor disclosed herein is 2 to 50 μM. In a preferred embodiment, the concentration of the TGFβ receptor inhibitor disclosed herein is 10 μM.

[0035] In some embodiments, the retinoic acid receptor (RAR) agonist disclosed herein is selected from TTNPB, Ch 55, AM580, or any combination thereof. In a preferred embodiment, the RAR agonist disclosed herein is TTNPB. In some embodiments, the concentration of the RAR agonist disclosed herein is 0.5 to 10 μM. In a preferred embodiment, the concentration of the RAR agonist disclosed herein is 2 μM.

[0036] In some embodiments, the G protein-coupled receptor smoothened agonist disclosed herein is selected from SAG, Purmorphamine, Hg-Ag1.5, or any combination thereof. In a preferred embodiment, the G protein-coupled receptor smoothened agonist disclosed herein is SAG. In some embodiments, the concentration of the G protein-coupled receptor smoothened agonist disclosed herein is 0.2 to 2 μM. In a preferred embodiment, the concentration of the G protein-coupled receptor smoothened agonist disclosed herein is 0.5 μM.

[0037] In some embodiments, the Dot1L inhibitor disclosed herein is selected from EPZ5676, SGC 0946, EPZ004777, or any combination thereof. In preferred embodiments, the Dot1L inhibitor disclosed herein is EPZ5676. In some embodiments, the concentration of the Dot1L inhibitor disclosed herein is 0.2 to 10 μM. In preferred embodiments, the concentration of the Dot1L inhibitor disclosed herein is 2 μM.

[0038] In some embodiments, the Menin-MLL interaction inhibitor disclosed herein is selected from VTP50469, MI3454, WDR5-IN-4, or any combination thereof. In a preferred embodiment, the Menin-MLL interaction inhibitor disclosed herein is VTP50469. In some embodiments, the concentration of the Menin-MLL interaction inhibitor disclosed herein is 0.1 to 5 μM. In a preferred embodiment, the concentration of the Menin-MLL interaction inhibitor disclosed herein is 0.5 μM.

[0039] In some embodiments, the SAH hydrolase inhibitor disclosed herein is selected from DZNEP, (-)Neplanocin A, adenozine periodate oxidized, 3-deazaadenosine, or any combination thereof. In a preferred embodiment, the SAH hydrolase inhibitor disclosed herein is DZNEP. In some embodiments, the concentration of the SAH hydrolase inhibitor disclosed herein is 0.01 to 0.2 μM. In a preferred embodiment, the concentration of the SAH hydrolase inhibitor disclosed herein is 0.05 μM.

[0040] In some embodiments, the Jak1 / Jak2 inhibitor disclosed herein is selected from ruxolitinib, tofacitinib, AZD1480, baricitinib, fuzotinib, or any combination thereof. In a preferred embodiment, the Jak1 / Jak2 inhibitor disclosed herein is ruxolitinib. In some embodiments, the concentration of the Jak1 / Jak2 inhibitor disclosed herein is 0.1 to 10 μM. In a preferred embodiment, the concentration of the Jak1 / Jak2 inhibitor disclosed herein is 1 μM.

[0041] In some embodiments, the c-Jun kinase inhibitor disclosed herein is selected from JNKIN8, JNKIN7, JNKIN5, JNKIN12, or any combination thereof. In a preferred embodiment, the c-Jun kinase inhibitor disclosed herein is JNKIN8. In some embodiments, the concentration of the c-Jun kinase inhibitor disclosed herein is 0.05 to 2 μM. In a preferred embodiment, the concentration of the c-Jun kinase inhibitor disclosed herein is preferably 0.2 μM.

[0042] In some embodiments, the serine-threonine kinase (Akt) inhibitor disclosed herein is selected from HY-10249A (CAS No. 842148-40-7), PF-AKT400, Capivasertib, Afuresertib, or any combination thereof. In a preferred embodiment, the Akt inhibitor disclosed herein is HY-10249A (CAS No. 842148-40-7). In some embodiments, the concentration of the Akt inhibitor disclosed herein is 0.1 to 10 μM. In a preferred embodiment, the concentration of the Akt inhibitor disclosed herein is 1 μM.

[0043] In some embodiments, the histone methyltransferase inhibitor disclosed herein is selected from SETD2-IN-1, EPZ-719, MMSET-IN-1, or any combination thereof. In a more preferred embodiment, the histone methyltransferase inhibitor disclosed herein is SETD2-IN-1. In some embodiments, the concentration of the histone methyltransferase inhibitor disclosed herein is 0.01 to 2 μM. In a preferred embodiment, the concentration of the histone methyltransferase inhibitor disclosed herein is 0.2 μM.

[0044] In some embodiments, the histone acetyltransferase inhibitor disclosed herein is a CBP / P300 inhibitor or a MOZ inhibitor. In a preferred embodiment, the histone acetyltransferase inhibitor disclosed herein is selected from A485, ICBP112, GEN049, CBP / P300 IN 12, SGC-CBP30, CBP / P300 IN 8, WM8014, WM1119 or any combination thereof. In a more preferred embodiment, the histone acetyltransferase inhibitor disclosed herein is selected from A485 or WM8014 or a combination of the two. In some embodiments, the composition disclosed herein comprises a histone acetyltransferase inhibitor at a concentration of 0.01 to 10 μM. In a preferred embodiment, the composition disclosed herein comprises a histone acetyltransferase inhibitor at a concentration of 0.05 to 5 μM. In a more preferred embodiment, the composition disclosed herein comprises a histone acetyltransferase inhibitor at a concentration of 0.1 to 5 μM. In a most preferred embodiment, the composition disclosed herein comprises a histone acetyltransferase inhibitor at a concentration of 0.1 to 2 μM. In some embodiments, the compositions disclosed herein comprise A485 at a concentration of 0.1 to 2 μM or WM8014 at a concentration of 0.1 to 5 μM, or a combination thereof. In preferred embodiments, the compositions disclosed herein comprise A485 at a concentration of 0.5 to 1 μM or WM8014 at a concentration of 0.5 to 1 μM, or a combination thereof. In more preferred embodiments, the compositions disclosed herein comprise A485 at a concentration of 0.5 μM or WM8014 at a concentration of 1 μM, or a combination thereof.

[0045] In a preferred embodiment, the composition comprises an LPA1 receptor antagonist selected from AM095, AM966, Ki16425, or any combination thereof. In a more preferred embodiment, the composition comprises an LPA1 receptor antagonist of AM095. In some embodiments, the composition comprises an LPA1 receptor antagonist at a concentration of 0.1 to 5 μM. In a preferred embodiment, the composition comprises an LPA1 receptor antagonist at a concentration of 0.5 to 1 μM. In a more preferred embodiment, the composition comprises an LPA1 receptor antagonist of 0.5 to 1 μM AM095.

[0046] In a most preferred embodiment, the composition disclosed herein comprises 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8 and 0.5 μM A-485. In some preferred embodiments, the compositions disclosed herein comprise 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, and 1 μM WM8014. In some preferred embodiments, the compositions disclosed herein comprise 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, 1 μM WM8014, and 0.5 μM A-485. In some preferred embodiments, the compositions disclosed herein comprise 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, 0.5 μM A-485, and 0.5 μM AM095.In some preferred embodiments, the compositions disclosed herein comprise 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, 1 μM WM8014, and 0.5 μM AM095. In some preferred embodiments, the compositions disclosed herein comprise 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, 0.5 μM A-485, 1 μM WM8014, and 0.5 μM AM095.

[0047] Another object of the present invention is to provide a second composition for further enhancing the differentiation potential of the cell population with increased differentiation potential disclosed herein, making it closer to pluripotent stem cells, the composition comprising a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a G protein-coupled receptor Smoothened agonist, a c-Jun kinase inhibitor, a SAH hydrolase inhibitor, and a DNA methyltransferase (DNMT) inhibitor. In a preferred embodiment, the composition comprises a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a G protein-coupled receptor Smoothened agonist, a c-Jun kinase inhibitor, a SAH hydrolase inhibitor, a DNA methyltransferase (DNMT) inhibitor, and any one selected from ROCK1 inhibitors, Dot1L inhibitors, Menin-MLL interaction inhibitors, MAPK inhibitors, Jak1 / Jak2 inhibitors, histone acetyltransferase inhibitors, serine-threonine kinase (Akt inhibitors, and casein kinase inhibitors, or any combination thereof. In a more preferred embodiment, the composition comprises a glycogen synthase kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a G protein-coupled receptor Smoothened agonist, a c-Jun kinase inhibitor, a SAH hydrolase inhibitor, a DNA methyltransferase (DNMT) inhibitor, a ROCK1 inhibitor, a Dot1L inhibitor, a Menin-MLL interaction inhibitor, a MAPK inhibitor, a Jak1 / Jak2 inhibitor, a histone acetyltransferase inhibitor, a serine-threonine kinase (Akt) inhibitor, and a casein kinase inhibitor.

[0048] In some embodiments, the glycogen kinase (GSK) 3β inhibitor disclosed herein is selected from CHIR99021, TD114-2, CHIR98014, GSK 3I inhibitor XV, BIO, SB-216763, or any combination thereof. In a preferred embodiment, the GSK3β inhibitor disclosed herein is CHIR99021. In some embodiments, the concentration of the GSK3β inhibitor disclosed herein is 3 to 12 μM. In a preferred embodiment, the concentration of the GSK3β inhibitor disclosed herein is 5 μM.

[0049] In some embodiments, the TGFβ receptor inhibitor disclosed herein is selected from 616452, A8301, SB505124, GW 788388, SB 525334, or any combination thereof. In a preferred embodiment, the TGFβ receptor inhibitor disclosed herein is 616452. In some embodiments, the concentration of the TGFβ receptor inhibitor disclosed herein is 2 to 50 μM. In a preferred embodiment, the concentration of the TGFβ receptor inhibitor disclosed herein is 10 μM.

[0050] In some embodiments, the retinoic acid receptor (RAR) agonist disclosed herein is selected from TTNPB, retinoic acid (RA), Ch55, AM580, or any combination thereof. In a preferred embodiment, the RAR agonist disclosed herein is TTNPB and / or RA. In some embodiments, the concentration of the RAR agonist disclosed herein is 0.5 to 10 μM. In a preferred embodiment, the concentration of the RAR agonist disclosed herein is 2 to 5 μM.

[0051] In some embodiments, the G protein-coupled receptor smoothened agonist disclosed herein is selected from SAG, Purmorphamine, Hg-Ag1.5, or any combination thereof. In a preferred embodiment, the G protein-coupled receptor smoothened agonist disclosed herein is SAG. In some embodiments, the concentration of the G protein-coupled receptor smoothened agonist disclosed herein is 0.2 to 2 μM. In a preferred embodiment, the concentration of the G protein-coupled receptor smoothened agonist disclosed herein is 0.5 μM.

[0052] In some embodiments, the c-Jun kinase inhibitor disclosed herein is selected from JNKIN8, JNKIN7, JNKIN5, JNKIN12, or any combination thereof. In a preferred embodiment, the c-Jun kinase inhibitor disclosed herein is JNKIN8. In some embodiments, the concentration of the c-Jun kinase inhibitor disclosed herein is 0.1 to 2.5 μM. In a preferred embodiment, the concentration of the c-Jun kinase inhibitor disclosed herein is preferably 0.5 μM.

[0053] In some embodiments, the SAH hydrolase inhibitor disclosed herein is selected from DZNEP, (-)Neplanocin A, adenozine periodate oxidized, 3-deazaadenosine, or any combination thereof. In a preferred embodiment, the SAH hydrolase inhibitor disclosed herein is DZNEP. In some embodiments, the concentration of the SAH hydrolase inhibitor disclosed herein is 0.04 to 1 μM. In a preferred embodiment, the concentration of the SAH hydrolase inhibitor disclosed herein is 0.2 μM.

[0054] In some embodiments, the ROCK1 inhibitor is selected from Y27632, Thiazovivin, Fasudil, Chroman 1, or any combination thereof. In a preferred embodiment, the ROCK1 inhibitor is Y27632. In some embodiments, the concentration of the ROCK1 inhibitor disclosed herein is 2 to 50 μM. In a preferred embodiment, the concentration of the ROCK1 inhibitor disclosed herein is 10 μM.

[0055] In some embodiments, the Dot1L inhibitor disclosed herein is selected from EPZ5676, SGC 0946, EPZ004777, or any combination thereof. In a preferred embodiment, the Dot1L inhibitor disclosed herein is EPZ5676. In some embodiments, the concentration of the Dot1L inhibitor disclosed herein is 0.2 to 10 μM. In a preferred embodiment, the concentration of the Dot1L inhibitor disclosed herein is 2 μM.

[0056] In some embodiments, the Menin-MLL interaction inhibitor disclosed herein is selected from VTP50469, MI3454, WDR5-IN-4, or any combination thereof. In a preferred embodiment, the Menin-MLL interaction inhibitor disclosed herein is VTP50469. In some embodiments, the concentration of the Menin-MLL interaction inhibitor disclosed herein is 0.1 to 5 μM. In a preferred embodiment, the concentration of the Menin-MLL interaction inhibitor disclosed herein is 0.5 μM.

[0057] In some embodiments, the MAPK inhibitor disclosed herein is selected from BIRB796, AZD8330, or TAK-733, or any combination thereof. In preferred embodiments, the MAPK inhibitor disclosed herein is BIRB796. In some embodiments, the concentration of the MAPK inhibitor disclosed herein is 0.2 to 5 μM. In preferred embodiments, the concentration of the MAPK inhibitor disclosed herein is 1 μM.

[0058] In some embodiments, the Jak1 / Jak2 inhibitor disclosed herein is selected from ruxolitinib, tofacitinib, AZD1480, baricitinib, fuzotinib, or any combination thereof. In a preferred embodiment, the Jak1 / Jak2 inhibitor disclosed herein is ruxolitinib. In some embodiments, the concentration of the Jak1 / Jak2 inhibitor disclosed herein is 0.1 to 10 μM. In a preferred embodiment, the concentration of the Jak1 / Jak2 inhibitor disclosed herein is 1 μM.

[0059] In some embodiments, the histone acetyltransferase inhibitor disclosed herein is selected from SGC / CBP300, A485, CBP / P300IN 8, WM8014, GEN049, WM1119, CBP / P300 IN 12, ICBP112, or any combination thereof. In a preferred embodiment, SGC / CBP300. In some embodiments, the concentration of the histone acetyltransferase inhibitor disclosed herein is 0.4 to 10 μM. In a preferred embodiment, the concentration of the histone acetyltransferase inhibitor disclosed herein is 2 μM.

[0060] In some embodiments, the serine-threonine kinase (Akt) inhibitor disclosed herein is selected from HY-10249A (CAS No. 842148-40-7), PF-AKT400, Capivasertib, Afuresertib, or any combination thereof. In a preferred embodiment, the Akt inhibitor disclosed herein is HY-10249A (CAS No. 842148-40-7). In some embodiments, the concentration of the Akt inhibitor disclosed herein is 0.04 to 1 μM. In a preferred embodiment, the concentration of the Akt inhibitor disclosed herein is 0.2 μM.

[0061] In some embodiments, the casein kinase inhibitor disclosed herein is selected from CX-4945, gallic acid (Ellagic Acid), TTP 22, or any combination thereof. In a preferred embodiment, the casein kinase inhibitor disclosed herein is CX-4945. In some embodiments, the concentration of the casein kinase inhibitor disclosed herein is 0.2 to 5 μM. In a preferred embodiment, the concentration of the Akt inhibitor disclosed herein is 1 μM.

[0062] In some embodiments, the DNA methyltransferase (DNMT) inhibitor disclosed herein is selected from GSK3689032, 5-aza-C, Decitabine, or any combination thereof. In a preferred embodiment, the DNA methyltransferase (DNMT) inhibitor disclosed herein is GSK3689032. In some embodiments, the concentration of the DNMT inhibitor disclosed herein is 0.01 to 0.1 μM. In a preferred embodiment, the concentration of the DNMT inhibitor disclosed herein is 0.02 μM.

[0063] In a most preferred embodiment, the composition comprises 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 10 μM Y-27632, 0.5 μM JNKIN8, 1 μM Ruxolitinib, 2 μM BIRB796, 2 μM SGC-CBP30, 2 μM EPZ5676, 0.2 μM DZNep, 0.5 μM VTP50469, 0.5 μM 5-iodotubercidin, 5 μM RA, 0.02 μM GSK3685032, 0.2 μM HY-10249A (CAS No. 842148-40-7) and 1 μM CX-4945.

[0064] Another object of the present invention is to provide a third composition, which is used to promote the cell population with increased differentiation potential disclosed herein to ultimately form pluripotent stem cells, and the composition includes glycogen synthesis kinase (GSK) 3β inhibitors, ROCK1 inhibitors, MAPK inhibitors and BRAF inhibitors. In some embodiments, the composition includes glycogen synthesis kinase (GSK) 3β inhibitors, ROCK1 inhibitors, MAPK inhibitors, BRAF inhibitors and histone deacetylase inhibitors. In some embodiments, the composition includes glycogen synthesis kinase (GSK) 3β inhibitors, ROCK1 inhibitors, MAPK inhibitors, BRAF inhibitors, histone deacetylase inhibitors and any one or any combination thereof selected from YAP activators, monoamine oxidase inhibitors, SAH hydrolase inhibitors, Dot1L inhibitors. In a preferred embodiment, the composition includes glycogen synthesis kinase (GSK) 3β inhibitors, ROCK1 inhibitors, MAPK inhibitors, BRAF inhibitors, histone deacetylase inhibitors, YAP activators, monoamine oxidase inhibitors, SAH hydrolase inhibitors and Dot1L inhibitors.

[0065] In some embodiments, the glycogen kinase (GSK) 3β inhibitor disclosed herein is selected from CHIR99021, TD114-2, CHIR98014, GSK 3I inhibitor XV, BIO, SB-216763, or any combination thereof. In a preferred embodiment, the GSK3β inhibitor disclosed herein is CHIR99021. In some embodiments, the concentration of the GSK3β inhibitor disclosed herein is 0.2 to 5 μM. In a preferred embodiment, the concentration of the GSK3β inhibitor disclosed herein is 1 μM.

[0066] In some embodiments, the ROCK1 inhibitor disclosed herein is selected from Y27632, Thiazovivin, Fasudil, Chroman1, or any combination thereof. In a preferred embodiment, the ROCK1 inhibitor disclosed herein is Y27632. In some embodiments, the concentration of the ROCK1 inhibitor disclosed herein is 2 to 50 μM. In a preferred embodiment, the concentration of the ROCK1 inhibitor disclosed herein is 10 μM.

[0067] In some embodiments, the MAPK inhibitor disclosed herein is selected from BIRB796, AZD8330, or TAK-733, or any combination thereof. In preferred embodiments, the MAPK inhibitor disclosed herein is BIRB796. In some embodiments, the concentration of the MAPK inhibitor disclosed herein is 0.2 to 5 μM. In preferred embodiments, the concentration of the MAPK inhibitor disclosed herein is 1 μM.

[0068] In some embodiments, the BRAF inhibitor disclosed herein is selected from SB590885, AZ 628, Agerafenib, Temuterkib, or any combination thereof. In a preferred embodiment, the BRAF inhibitor disclosed herein is SB590885. In some embodiments, the concentration of the BRAF inhibitor disclosed herein is 0.1 to 2.5 μM. In a preferred embodiment, the concentration of the BRAF inhibitor disclosed herein is 0.5 μM.

[0069] In some embodiments, the histone deacetylase inhibitor disclosed herein is selected from VPA, MS275, CI 994, HDACi IV, or any combination thereof. In a preferred embodiment, the histone deacetylase inhibitor disclosed herein is VPA. In some embodiments, the concentration of the histone deacetylase inhibitor disclosed herein is 40 to 1000 μM. In a preferred embodiment, the concentration of the histone deacetylase inhibitor disclosed herein is 200 μM.

[0070] In some embodiments, the YAP activator disclosed herein is selected from PY60 and / or LAST-In-1. In a preferred embodiment, the YAP activator disclosed herein is PY60. In some embodiments, the concentration of the YAP activator disclosed herein is 0.4 to 10 μM. In a preferred embodiment, the concentration of the YAP activator disclosed herein is 2 to 10 μM.

[0071] In some embodiments, the monoamine oxidase inhibitor disclosed herein is selected from tranylcypromine, S2101, GSK2879552, or any combination thereof. In a preferred embodiment, the monoamine oxidase inhibitor disclosed herein is tranylcypromine. In some embodiments, the concentration of the monoamine oxidase inhibitor disclosed herein is 2 to 50 μM. In a preferred embodiment, the concentration of the monoamine oxidase inhibitor disclosed herein is 10 μM.

[0072] In some embodiments, the SAH hydrolase inhibitor disclosed herein is selected from DZNEP, (-)Neplanocin A, adenozine periodate oxidized, 3-deazaadenosine, or any combination thereof. In a preferred embodiment, the SAH hydrolase inhibitor disclosed herein is DZNEP. In some embodiments, the concentration of the SAH hydrolase inhibitor disclosed herein is 0.04 to 1 μM. In a preferred embodiment, the concentration of the SAH hydrolase inhibitor disclosed herein is 0.2 μM.

[0073] In some embodiments, the Dot1L inhibitor disclosed herein is selected from EPZ5676, SGC 0946, EPZ004777, or any combination thereof. In a preferred embodiment, the Dot1L inhibitor disclosed herein is EPZ5676. In some embodiments, the concentration of the Dot1L inhibitor disclosed herein is 0.4 to 10 μM. In a preferred embodiment, the concentration of the Dot1L inhibitor disclosed herein is 2 μM.

[0074] In a most preferred embodiment, the composition comprises 1 μM CHIR999021, 10 μM Y-27632, 1 μM PD0325901 and 0.5 μM SB590885; or

[0075] 1 μM CHIR999021, 10 μM Y-27632, 1 μM PD0325901, 0.5 μM SB590885, 200 μM VPA, 10 μM tranylcypromine, 2 μM EPZ5676, 0.2 μM DZNep, and 10 μM PY60; or

[0076] Contains 1 μM CHIR999021, 10 μM Y-27632, 1 μM PD0325901, 0.5 μM SB590885, 200 μM VPA, and 2 μM PY60.

[0077] Therefore, the present invention provides a chemical reprogramming method for obtaining induced pluripotent stem cells from somatic cells from primates, comprising: exposing the somatic cells to a first composition disclosed herein, optionally for 5 to 8 days, to obtain a cell population having increased differentiation potential compared to the somatic cells; and first treating the cell population with a second composition disclosed herein, optionally for 1 to 8 days, and then treating the cell population with a third composition disclosed herein, optionally for 4 to 8 days, to obtain the induced pluripotent stem cells.

[0078] In a preferred embodiment, the methods disclosed herein comprise: exposing somatic cells to a composition comprising 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, and 0.5 μM A-485; or exposing somatic cells to a composition comprising 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, and 0.5 μM A-485. VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, and 1 μM WM8014; or a composition comprising 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, 0.5 μM A-485, and 1 μM WM8014; or a composition comprising 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, 0.5 μM A-485, and 0.5 μM AM095; or a composition comprising 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. No.842148-40-7), 0.2μM SETD2IN1, 0.2μM JNKIN8, 0.5μM A-485, 1μM WM8014 and 0.or a composition comprising 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, 1 μM WM8014 and 0.5 μM AM095, and after 5 to 8 days, a cell population with increased differentiation potential compared to the somatic cells is obtained, the cell population is first treated with 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 10 μM Y-27632, 0.5 μM JNKIN8, 1 μM Ruxolitinib, 2 μM BIRB796, 2 μM SGC-CBP30, 2 μM EPZ5676, 0.2 μM DZNep, 0.5 μM VTP50469, 0.5 μM 5-iodotubercidin, 5 μM RA, 0.02 μM GSK3685032, 0.2 μM HY-10249A (CAS No. 842148-40-7) and 1 μM CX-4945 were treated for 1 to 8 days, and then treated with a composition containing 1 μM CHIR999021, 10 μM Y-27632, 1 μM PD0325901, 0.5 μM SB590885, 200 μM Induced pluripotent stem cells were obtained by treating cells with a composition containing VPA, 10 μM tranylcypromine, 2 μM EPZ5676, 0.2 μM DZNep, and 10 μM PY60 for 2 to 4 days, followed by treatment with a composition containing 1 μM CHIR999021, 10 μM Y-27632, 1 μM PD0325901, 0.5 μM SB590885, and 200 μM VPA for 2 to 4 days.

[0079] In some embodiments, by the chemical reprogramming methods disclosed herein, somatic cells can be converted into pluripotent stem cells within about 10 days to about 30 days. In some embodiments, by the chemical reprogramming methods disclosed herein, somatic cells can be converted into pluripotent stem cells within about 10 days to about 24 days. In some embodiments, by the chemical reprogramming methods disclosed herein, somatic cells can be converted into pluripotent stem cells within about 10 days to about 18 days. In some embodiments, by the chemical reprogramming methods disclosed herein, somatic cells can be converted into pluripotent stem cells at about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days.

[0080] In some embodiments, the chemical reprogramming methods disclosed herein may further comprise the step of purifying the pluripotent stem cells. In some embodiments, the pluripotent stem cells are purified based on a pluripotency marker selected from OCT4, SOX2, NANOG, FGF4, ZFP57, DPPA5, REX1, DPPA4, TDGF1, TRA-1-60, TRA-1-81, SSEA4, KLF4, KLF17, DPPA3, DNMT3L, UTF, or any combination thereof. In some embodiments, the purity of the obtained pluripotent stem cell population is about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 100% based on the pluripotency marker used for purification.

[0081] Another object of the present invention is to provide a histone acetyltransferase inhibitor for use in chemically reprogramming somatic cells into induced pluripotent stem cells, wherein the inhibitor is used before the somatic cell becomes a cell population with the following characteristics, the cell population mainly comprising epithelial cells positive for pluripotency marker LIN28A, and compared with the somatic cells as starting cells, there are epithelial cell surface markers with elevated expression levels and somatic cell markers with reduced expression levels. In some embodiments, the epithelial cell surface markers are selected from one or more of KRT8, KRT18 or KRT19. In some embodiments, the somatic cell markers are selected from one or more of VIM, SNAI2, COL5A1, PRKX1, COL6A2, ZEB1, MMP1, TWIST1, COL1A1. In a preferred embodiment, the histone acetyltransferase inhibitor is a CBP / P300 inhibitor. In a preferred embodiment, the histone acetyltransferase inhibitor is a monocytic leukemia zinc finger protein (MOZ) inhibitor. In a preferred embodiment, the histone acetyltransferase inhibitor is a combination of a CBP / P300 inhibitor and a MOZ inhibitor. In a preferred embodiment, the histone acetyltransferase inhibitor is selected from A485, CBP / P300 IN 8, WM8014, GEN049, WM1119, CBP / P300 IN 12, SGCCBP30, ICBP112 or their combination in any. In a preferred embodiment, the histone acetyltransferase inhibitor is selected from WM8014 or A485 or the two combinations.

[0082] By in the process of chemical reprogramming, especially the early stage of reprogramming uses the inhibitor of histone acetyltransferase, somatic cell can be efficiently and quickly converted into pluripotent stem cell.The reprogramming efficiency of reprogramming method can be calculated by the quantity of the pluripotent stem cell produced by the somatic cell of predetermined amount, that is, the quantity of the pluripotent stem cell produced is measured divided by the ratio (such as percentage) of the total somatic cell for reprogramming induction, referred to herein as the pluripotent stem cell conversion efficiency of chemical reprogramming method.For example, if method disclosed herein can produce 1 pluripotent stem cell by 1000 somatic cells, the pluripotent stem cell conversion efficiency of the method is calculated as 0.1%.

[0083] The reprogramming efficiency of the reprogramming method can also be calculated by the time taken for the somatic cell as the starting cell to be contacted with a composition disclosed herein (e.g., a composition comprising a histone acetyltransferase inhibitor) until the number of pluripotent stem cells produced does not significantly increase, referred to herein as the conversion time of pluripotent stem cells. In some embodiments, the conversion time required for the method disclosed herein is at most about 24 days. In a preferred embodiment, the conversion time required for the method disclosed herein is at most about 21 days. In a more preferred embodiment, the conversion time required for the method disclosed herein is at most about 18 days. In a further preferred embodiment, the conversion time required for the method disclosed herein is at most about 15 days. In a further preferred embodiment, the conversion time required for the method disclosed herein is at most about 13 days. In a further preferred embodiment, the conversion time required for the method disclosed herein is at most about 12 days. In a further preferred embodiment, the conversion time required for the method disclosed herein is at most about 11 days. In a most preferred embodiment, the conversion time required for the method disclosed herein is about 10 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 shows the Gene Ontology (GO) analysis results of the transcriptomes of cell lines with different chemical reprogramming conversion efficiencies. Figure 1A shows the GO analysis results of seven cell lines with different conversion efficiencies. Figure 1B shows the gene types enriched in cell lines with low conversion efficiencies. Figure 1C is a schematic diagram of the screening process of the candidate small molecule library.

[0085] Figure 2 shows the changes in the number of CiPS clones obtained by adding different histone acetyltransferase inhibitors during the early stages of chemically induced reprogramming. The left and right panels respectively show the induction results for the "8+4+8" and "6+4+8" methods described in Example 2. "NC" indicates a control without the addition of any HATi. HATi was added to a final concentration of 0.5 μM in all test groups.

[0086] Figure 3 shows the reprogramming results of Example 3. "SI+WM8014" indicates the group to which 1 μM WM8014 was additionally added to the early induction medium; "SI+A485" indicates the group to which 0.5 μM A485 was additionally added to the early induction medium; and "SI+A485+WM8014" indicates the group to which a combination of 1 μM WM8014 and 0.5 μM A485 was additionally added to the early induction medium.

[0087] Figure 4 shows the reprogramming results of Example 4. "SII+RA" indicates the group to which 5 μM RA was additionally added to the Stage II induction medium; "SII+GSK3689032" indicates the group to which 0.02 μM GSK3689032 was additionally added to the Stage II induction medium; and "S1+GSK3689032+RA" indicates the group to which 0.02 μM GSK3689032 and 5 μM RA were additionally added to the Stage II induction medium.

[0088] Figure 5 shows the reprogramming results of Example 5. "SIII+PY60" indicates the group to which 10 μM PY60 alone was additionally added to the Stage III induction medium; "SIII+PY60, VPA200-500" indicates the group to which 10 μM PY60 was additionally added to the Stage III induction medium, and the VPA concentration was reduced to 200 μM during the first three days of incubation with the Stage III induction medium; and "SIII+PY60, VPA200-200" indicates the group to which 2 μM PY60 was additionally added to the Stage III induction medium, and the VPA concentration was reduced to 200 μM during the first six days of incubation with the Stage III induction medium.

[0089] FIG6 shows the number of CiPS clones obtained by the exemplary reprogramming method of the present invention under different induction time combinations.

[0090] FIG. 7 shows the number of CiPS clones obtained by an exemplary reprogramming method of the present invention when incubated with Stage II induction medium for different lengths of time.

[0091] FIG8 shows the number of CiPS clones obtained by an exemplary reprogramming method of the present invention when incubated with Stage III induction medium for different lengths of time.

[0092] FIG. 9 shows the results of immunofluorescence staining of OCT4 and SOX2 on a CiPS cell population obtained by an exemplary reprogramming method of the present invention.

[0093] FIG. 10 shows the results of counting CiPS clones obtained by the exemplary reprogramming method of the present invention.

[0094] Figure 11 shows the conversion curve when hADSC cell reprogramming is induced using an exemplary method of the present invention, wherein the X-axis represents the number of days from the start of induction, and the Y-axis represents the number of CiPS clones generated.

[0095] Figure 12 shows a conversion curve when HD cell reprogramming is induced using an exemplary method of the present invention, wherein the X-axis represents the number of days from the start of induction and the Y-axis represents the number of CiPS clones generated.

[0096] FIG. 13 shows the number of CiPS clones obtained from two different hADSC cell lines using the exemplary method of the present invention at different induction time combinations.

[0097] FIG. 14 shows changes in the reprogramming efficiency of somatic cells in which the endogenous histone acetyltransferase gene was knocked out. DETAILED DESCRIPTION

[0098] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0099] Unless otherwise noted, the experimental methods in the following examples are conventional methods and were performed according to the techniques and conditions described in literature in the field or according to product specifications. The materials, reagents, and instruments used in the following examples are all commercially available unless otherwise noted. Quantitative tests in the following examples are the average of three replicates unless otherwise noted.

[0100] definition

[0101] As used in the description of the present invention, the following words and phrases are generally deemed to have the meanings set forth below, unless the context in which the word or phrase is used indicates otherwise.

[0102] As used herein, the terms "comprising" or "including" mean that the compositions and methods include the recited components, but do not exclude other components. "Consisting essentially of," when used to define compositions and methods, shall mean excluding any other components that are clearly essential to the combination. Thus, compositions defined herein as consisting essentially of these components will not exclude trace contamination from isolation and purification methods, as well as pharmaceutically acceptable carriers, such as phosphate-buffered saline, preservatives, and the like. "Consisting of shall mean excluding trace components of other ingredients used to administer the compositions and substantive methods of the invention. Embodiments defined by these provisional terms are within the scope of the invention.

[0103] Unless otherwise specified herein, the enumeration of numerical ranges herein is intended only to be used as a shorthand method for individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually enumerated herein. For example, in this specification, if a concentration range is stated as 1 to 10 μM, then values ​​such as 2 to 9 μM, 5 to 6 μM, or 1 to 5 μM are intended to be explicitly enumerated. These are only examples of specific intents, and all possible numerical combinations between and including the lowest and highest values ​​enumerated are considered to be explicitly stated in this disclosure. Using the word "about" to describe a specific enumerated amount or range of amounts means indicating that a value very close to the enumerated amount is included in the amount, such as values ​​that can or naturally be considered due to manufacturing tolerances, instruments and human errors when forming measurements, etc. For example, the numerical value described herein with "about" means covering ±10% of the indicated value. In some cases, "about" can mean ±20%, or ±5%, or ±1%. The values ​​or parameters described herein in an "about" manner include the value or parameter itself.

[0104] As used herein, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the technical features being referred to. Unless expressly stated otherwise, a feature designated "first" or "second" may explicitly or implicitly include at least one of such features.

[0105] As used herein, unless expressly stated otherwise, "plurality" means at least two, such as two, three, etc.

[0106] The terms "histone acetyltransferase" and "HAT" are used interchangeably herein and refer to enzymes that can catalyze the acetylation of lysine residues within histone tails. Eukaryotic transcription is regulated by chromatin structure, and its changes are mediated by conserved post-translational histone tail modifications. Histone tail modifications include, but are not limited to, acetylation, methylation, phosphorylation, and ubiquitination. HATs reduce the interaction of histones with DNA by acetylation of lysine residues within the histone tails, thereby converting condensed chromatin into a more relaxed structure to promote higher levels of gene transcription. In some embodiments, histones include H3K27ac, H3K27ac, H3K9ac, H3K14ac, H3K18ac, H3K23ac, or H4K5ac.

[0107] The terms "histone acetyltransferase inhibitor" or "HAT inhibitor" or "HATi" are used interchangeably herein and refer to substances that can detectably reduce the expression or activity of a histone acetyltransferase. An inhibitor can reduce the expression or activity of a histone acetyltransferase by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control in the absence of an inhibitor. In some cases, the expression or activity of a histone acetyltransferase is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or lower than the expression or activity in the absence of an inhibitor. As non-limiting examples of histone acetyltransferase inhibitors, for example, compounds with HATi activity described in Mengyuan Huang et al., Histone acetyltransferase inhibitors: An overview in synthesis, structure-activity relationship and molecular mechanism. European Journal of Medicinal Chemistry, Vol. 178, No. 15, 2019, pp. 259-286; A485 derivatives described in Lasko, L et al., Discovery of a selective catalytic p300 / CBP inhibitor that targets lineage-specific tumours. Nature 550, 128–132 (2017); and WM8014 derivatives described in Baell, JB et al., Inhibitors of histone acetyltransferases KAT6A / B induce senescence and arrest tumour growth. Nature 560, 253–257 (2018).

[0108] The term "exposing" when describing the treatment of cells means bringing the cells into direct contact with the specified substance.

[0109] The term "differentiation potential" refers to the ability of a cell to differentiate into other cell types. Cells with higher differentiation potentials are capable of forming more cell types. It is generally believed that the differentiation potential of a cell decreases in the order of totipotency, pluripotency, multipotency, unipotency, and somatic cells. The differentiation potential of a cell can be assessed by methods generally known in the art. For example, it can be assessed based on cell morphology, expression levels of pluripotency markers or differentiation markers, the ability of the cell to form three germ layers and chimeras, etc.

[0110] The term "cell population" generally refers to a group of cells. A cell population can be composed of cells (e.g., pluripotent stem cells) with a common phenotype, or can include at least a portion of cells with a common phenotype. When cells are substantially similar or identical in one or more provable features, it is believed that they have a common phenotype, and the provable features include but are not limited to morphological appearance, the presence, absence or level of expression of specific cellular components or products (e.g., RNA, proteins or other markers), the activity of certain biochemical pathways, proliferation capacity and / or kinetics, differentiation potential and / or the response to differentiation signals or the behavior during in vitro culture (e.g., adhesion, non-adhesion, monolayer growth, proliferation kinetics, etc.). Therefore, this provable feature can define a cell population or its fraction.

[0111] As used herein, the term "cell surface marker" or similar expressions refers to a protein, carbohydrate, lipid, or combination thereof on the surface of a cell that can be used to distinguish between cell populations.

[0112] The following describes a preferred embodiment of the present invention. It should be noted that the embodiments described below are examples showing representative embodiments of the present invention, but the present invention is not limited to these examples.

[0113] Two or more of the embodiments described below may be combined, and such combinations are also included in the present invention.

[0114] Example

[0115] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0116] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in literature in the field or according to product specifications. The materials, reagents, and instruments used in the following examples are all commercially available unless otherwise specified. Relevant nucleic acid chains, genes, and enzymes can also be obtained using conventional molecular biology experimental techniques based on information from public databases.

[0117] Unless otherwise specified, nucleotide sequences referred to herein are written from left to right in the order from 5' to 3' terminus, and amino acid sequences are written from left to right in the order from amino terminus to carboxyl terminus. In the event of any discrepancy between the sequence described in the specification and the sequence listing, the sequence described in the specification shall prevail.

[0118] Experimental materials, reagents, instruments and experimental methods

[0119] Plasmid construction:

[0120] The plasmids used herein can be constructed from commercially available original plasmids using conventional gene cloning and recombination methods in the art (see, for example, Molecular Cloning Laboratory Manual (3rd Edition) (Science Press) and Microbiology Experiments (4th Edition) (Higher Education Press)). The coding gene sequences involved can be obtained from public databases using conventional molecular biology experimental techniques. The plasmids used herein were synthesized by GenScript.

[0121] Plasmid and primer information: gRNA cloning vector: pLentiCRISPR v2 (purchased from GenScript Biotech Co., Ltd.)

[0122] Lentiviral infection:

[0123] 15 μg of the lentiviral vector containing the target sequence was co-transfected with pMDLg / pRRE, RSV / Rev, and VSV-G (purchased from Addgene, 5 μg each) using the PEI method into 293T cells and incubated overnight. After 12 hours, the medium was replaced and the cells were cultured for another 36 hours. The supernatant containing the lentiviral particles was collected. The collected supernatant was filtered through a 0.45 μm filter (Millipore). 1 x 10 4 hADSCs were usually infected with 20-50 μl of viral supernatant and 10 ng / μl polybrene (Sigma-Aldrich) added, and the virus was withdrawn 12-24 hours after infection.

[0124] Table 1: Cell lines:

[0125] The hCiPSCs and human ES cell lines (H1 and H9) were maintained in mTeSR at 37°C and 5% CO2. TM In the Plus medium, the cell plate was coated with Matrigel in advance. When the cell confluence reached about 85%, it was passaged with ReLeSR at a passage ratio of about 1:10 to 1:20. After passage, the separated cell clusters were added to mTeSR with Y-27632 (10μM) TM After culturing in Plus medium for 24 h, the medium was replaced with fresh mTeSR without Y-27632. TM Plus culture medium.

[0126] The human adult adipose-derived mesenchymal cells (hADSCs) and human adult dermal fibroblasts (hASFs) cell lines were maintained in Mesenchymal Stem Cell Growth Media 2 (PromoCell) at 37° C. and 5% CO 2 .

[0127] Table 2: Antibodies:

[0128] Table 3: Chemical reagents:

[0129] CiPSC clone counting:

[0130] After immunofluorescence staining (primarily with OCT4 antibodies) of the cell culture plate to be counted, count the number of OCT4-positive colonies in the entire well under a fluorescence microscope. Typically, a dense colony of ≥5 cells is counted as one CiPSC.

[0131] Immunofluorescence staining:

[0132] After discarding the original culture medium from the cell culture plate to be stained, wash it 2-3 times with PBS. Add 4% paraformaldehyde to cover the cells and fix them at room temperature for 30 minutes. Then, aspirate the liquid and gently rinse the cells 3 times with PBS. TM The cells were permeabilized in PBS containing 0.1% Triton X-100 and 2% normal donkey serum at 37°C for 1 hour, and the liquid was then aspirated. TM Add the appropriate ratio of primary antibody to a solution of X-100 and 2% normal donkey serum in PBS and incubate the cells overnight at 4°C. Then, aspirate the liquid and gently rinse the cells three times with PBS. Add the appropriate ratio of secondary antibody to a solution of 2% normal donkey serum in PBS and incubate overnight at 4°C in the dark. Stain DNA with DAPI solution. After staining, aspirate the liquid and gently rinse the cells three times with PBS. The cells can be stored at 4°C for 1–2 weeks or directly observed under a fluorescence microscope.

[0133] Triblastoma differentiation (teratoma)

[0134] About 2x 10 6 hCiPSCs were resuspended in Matrigel and injected subcutaneously into 2- to 3-month-old immunodeficient male NPG mice (Beijing Vitalstar Biotechnology Co., Ltd.). After teratomas formed (usually 6-7 weeks), the teratomas were embedded in paraffin and stained with hematoxylin and eosin. All animal experiments were approved by the Institutional Animal Care and Use Committee of Peking University and performed in accordance with the Peking University Animal Care Guidelines.

[0135] Gene Ontology (GO) analysis

[0136] GO analysis was performed using the enrichGO function in the R package clusterProfiler according to the method described in Yu, GC et al., clusterProfiler: an R Package for Comparing Biological Themes Among Gene Clusters. Omics 16, 284-287 (2012).

[0137] Example 1

[0138] This example describes epigenetic analysis of chemically reprogrammed cells.

[0139] (1) Chemically induced reprogramming of human fibroblasts

[0140] According to the method described by Liuyang S et al. (2023), chemically reprogrammed induced pluripotent stem cells (CiPS) were formed from human adipose-derived mesenchymal stem cells (hADSCs) and human skin fibroblasts (hASFs) (see Liuyang S et al., Cell Stem Cell. 2023 Apr 6; 30(4): 450-459.e9).

[0141] Briefly, hADSCs or hASFs were cultured at 1 × 10 4 Cells / well were seeded into 12-well plates, or 0.5-0.6×10 4Cells were seeded into 24-well plates and cultured overnight. The next day, the cells were replaced with Stage I induction medium (Knockout DMEM supplemented with 2% B27 supplement, 1% KSR, 1% GlutaMAX, 1% NEAA, 1% penicillin-streptomycin, 50 μg / mL Vc2p, 5 mM LiCl, 1 mM NAM, 20 ng / mL BMP4, 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.05 μM DZNep, 1 μM ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM JNKIN8, and 0.2 μM SETD2-IN-1) and cultured under hypoxic (5% O2) conditions until a monolayer of epithelial-like cells appeared and reached approximately 100% confluence. hADSC cells typically require 8-10 days, while hASF cells typically require 9-16 days. Immunofluorescence staining of the resulting epithelial-like monolayer confirmed LIN28A staining. RT-PCR analysis revealed elevated expression of the epithelial cell marker KRTs and decreased expression of fibroblast markers.

[0142] Then, the medium was replaced with Stage II induction medium (Knockout DMEM supplemented with 2% B27 supplement, 1% GlutaMAX, 1% NEAA, 1% penicillin-streptomycin, 50 μg / mL Vc2p, 200 ng / mL bFGF, 5 μM CHIR99021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 0.5 μM JNKIN8, 2 μM EPZ5676, 0.2 μM DZNep, 1 μM ruxolitinib, 2 μM BIRB796, 2 μM SGC-CBP30, 0.5 μM Dorsormorphin, 0.5 μM VTP50469, 0.5 μM 5ITU, 2 μM 5-Azacytidine, and 0.2 μM HY-10249A (CAS)). No.842148-40-7)) were cultured under normoxia (21% O2) for 8 to 12 days until the monolayer of cells became multilayered and raised colonies appeared and gradually grew larger. Then, the culture medium was changed to Stage III induction medium (Knockout DMEM supplemented with 2% B27 supplement, 1% GlutaMAX, 1% NEAA, 1% penicillin-streptomycin, 5% KSR, 50 μg / mL Vc2p, 20 ng / mL recombinant human heregulin β-1 (HRG), 1 μM CHIR99021, 10 μM Y-27632, 1 μM PD0325901, and 0.5 μM SB590885) and cultured under normoxia for 8 to 12 days to form CiPSC colonies. During the first 4 days of this stage, VPA (1000 μM), tranylcypromine (10 μM), DZNep (0.2 μM), and EPZ5676 (2 μM) were also added to the culture medium. From days 5 to 8, tranylcypromine, DZNep, and EPZ5676 were no longer added to the culture medium, and the VPA concentration was halved to 500 μM.

[0143] Obtained CiPS clones were immunostained for OCT4, with OCT4 positivity used as an indicator of hCiPSC clones. The conversion efficiency of the reprogramming method was calculated by dividing the number of OCT4-positive clones by the number of hADSCs or hASFs initially seeded.

[0144] (2) Biological property analysis (Go analysis)

[0145] Using the above method, successful CiPS clones were observed in all tested somatic cells, but the conversion efficiency varied significantly between different cell lines, ranging from approximately 1% to 20%. Therefore, we selected hADSC cell lines with varying hCiPSC conversion efficiencies and analyzed their transcriptomes using Gene Ontology (GO) to identify genes positively or negatively correlated with the conversion efficiency of somatic cell reprogramming. The results are shown in Figure 1.

[0146] As shown in Figure 1B, a series of regulatory enzyme genes related to histone modification are highly expressed in cell lines with low transformation efficiency (Figure 1b), suggesting that these epigenetic regulatory factors may be a major obstacle to low reprogramming transformation efficiency. Based on this, a candidate chemical library was constructed using small molecules with histone modification regulatory activity.

[0147] Example 2

[0148] This example describes that histone acetyltransferase inhibitors can improve the conversion efficiency of somatic cell chemical reprogramming and shorten the total conversion time of somatic cell chemical reprogramming.

[0149] hADSC cells were chemically reprogrammed using a method substantially the same as in Example 1, except that in Method 1, the time for culturing cells with Stage I induction medium was shortened to 8 days, the time for culturing cells with Stage II induction medium was shortened to 4 days, and the time for culturing cells with Stage III induction medium was shortened to 8 days, with a total culture period of 20 days as a control group ("8+4+8"); whereas in Method 2, the time for culturing cells with Stage I induction medium was further shortened to 6 days, the time for culturing cells with Stage II induction medium was 4 days, and the time for culturing cells with Stage III induction medium was 8 days, with a total culture period of 18 days as a control group ("6+4+8").

[0150] In the test groups, chemical small molecules from the candidate library were added to the stage I induction medium, and the number of OCT4-positive CiPS clones in the control and test groups was measured on the last day of culture to screen for small molecule compounds that can significantly improve the efficiency of somatic cell reprogramming. The results showed that in both the control and test groups, a single layer of LIN28A-positive epithelial-like cells was observed during culture using the stage I induction medium (results not shown). If culture is continued until approximately 100% confluence is reached, it usually takes 5 to 8 days for the hADSC cell test group and 6 to 10 days for the hASF cell test group, which greatly shortens the induction time compared to the control group.

[0151] In addition, in the test groups to which multiple histone acetyltransferase inhibitors (HATi) were added, a significant increase in reprogramming efficiency was observed, and exemplary results are shown in Figures 2A and 2B. In the figure, A485, CBP / P300IN8, and ICBP112 belong to CBP / P300 complex (KAT3A / B) inhibitors, and WM8014 and WM1119 belong to MOZ (KAT6A) inhibitors. As shown in Figure 2A, using the "8+4+8" culture method, about 150 CiPS clones were produced per 5,000 somatic cells in the control group, with a conversion efficiency of about 3%. In the test groups to which histone acetyltransferase inhibitors (HATi) were added, except for the slightly lower conversion efficiency (about 2.4%) of the MOZ inhibitor WM1119, the conversion efficiency of the remaining four HATi was above 5%, achieving a statistically significant increase. As shown in Figure 2B, when the induction time, especially the initial induction period for initiating somatic cell reprogramming, is further shortened ("6+4+8"), the conversion efficiency of the control group decreases dramatically, with only approximately one CiPS clone produced per 5,000 somatic cells, a conversion efficiency of approximately 0.02%. At the same time, most of the test groups with added HATi were still able to generate CiPS clones with a conversion efficiency at least an order of magnitude higher than the control. Without being bound by theory, this may be because the addition of histone acetyltransferase inhibitors relaxes the somatic cell chromatin, which is originally in a relatively closed state, thereby more conducive to initiating gene expression of early reprogramming regulatory factors.

[0152] Example 3

[0153] This example describes how a combination of different types of histone acetyltransferase inhibitors synergistically improves the reprogramming efficiency of somatic cells.

[0154] hADSC cells were chemically reprogrammed using a method substantially the same as in Example 1, except that the cell culture time with Stage I induction medium was shortened to 6 days, the cell culture time with Stage II induction medium was shortened to 8 days, and the cell culture time with Stage III induction medium was shortened to 8 days. A total of 22 days of culture was used as a control ("6+8+8").

[0155] The following reagents or reagent combinations were added to the Stage I induction medium as test groups:

[0156] Test group 1: KAT6A inhibitor WM8014 was added alone to a concentration of 1 μM;

[0157] Test group 2: KAT3A / B inhibitor A485 was added alone to a concentration of 0.5 μM;

[0158] Test Group 3: A combination of WM8014 and A485 was added to a concentration of 1 μM for WM8014 and 0.5 μM for A485.

[0159] On the last day of culture, the number of OCT4-positive CiPS clones in the control and each test group was measured to calculate the reprogramming efficiency of somatic cells. The results are shown in Figure 3. As can be seen, when the induction time of Stage I was shortened to 6 days, the combination of inhibitors A485 and WM8014 was still able to successfully induce LIN28A-positive monolayer epithelial-like cells, and ultimately achieved a conversion efficiency of approximately 24%, which was approximately 15 times higher than the control group, indicating that the combination of inhibitors A485 and WM8014 in the early stage of reprogramming can achieve a synergistic improvement.

[0160] Example 4

[0161] This example describes how to improve the efficiency of generating induced pluripotent stem cells from LIN28A-positive epithelial-like cell monolayers by optimizing the combination of small molecules.

[0162] hADSC cells were chemically reprogrammed using a method substantially the same as in Example 1, except that a combination of two histone acetyltransferase inhibitors (1 μM WM8014 and 0.5 μM A485) was added to the Stage I induction medium, the cell culture time with Stage I induction medium was shortened to 6 days, the cell culture time with Stage II induction medium was shortened to 4 days, and the cell culture time with Stage III induction medium was shortened to 8 days, with a total culture of 18 days as a control ("6+4+8").

[0163] The following reagents or reagent combinations were added to the stage II induction medium as test groups:

[0164] Test group 1: RA was added alone to a concentration of 5 μM;

[0165] Test group 2: GSK3685032 was added alone to a concentration of 0.02 μM;

[0166] Test Group 3: A combination of RA and GSK3685032 was added to a concentration of RA of 5 μM and a concentration of GSK3685032 of 0.02 μM.

[0167] On the last day of culture, the number of OCT4-positive CiPS clones in the control and test groups was measured to calculate the somatic cell reprogramming efficiency. The results are shown in Figure 4. As can be seen, when the induction time is shortened, the addition of either the RA receptor activator or the DNMT inhibitor GSK3685032 alone can statistically significantly improve the conversion of LIN28A-positive monolayer epithelial-like cells to pluripotent stem cells compared to the control group, and the combination of the two also has a significant synergistic effect on the conversion efficiency of CiPS cells.

[0168] Example 5

[0169] This example describes how to further improve the efficiency of generating induced pluripotent stem cells from LIN28A-positive epithelial-like cell monolayers by optimizing the combination of small molecules.

[0170] hADSC cells were chemically reprogrammed by a method substantially the same as in Example 1, except that a combination of two histone acetyltransferase inhibitors (1 μM WM8014 and 0.5 μM A485) was added to the Stage I induction medium, and the culture time was shortened to 8 days, a combination of an RA receptor agonist (5 μM RA) and a DNMT inhibitor (0.02 μM GSK3685032) was added to the Stage II induction medium, and the culture time was shortened to 5 days, and the cells were cultured in the Stage III induction medium for 8 days, with a total culture time of 21 days as a control ("8+5+8").

[0171] The following reagents or reagent combinations were added to the stage III induction medium as test groups:

[0172] Test group 1: YAP activator PY60 was added alone to a concentration of 10 μM;

[0173] Test group 2: YAP activator PY60 was added to a concentration of 10 μM, and the concentration of histone deacetylase inhibitor VPA was reduced from 1000 μM to 200 μM in the first three days;

[0174] Test group 3: PY60 was added to a concentration of 2 μM, and the concentration of VPA was reduced to 200 μM for the first six days.

[0175] On the last day of culture, the number of OCT4-positive CiPS colonies in the control and test groups was measured to calculate the somatic cell reprogramming efficiency. The results are shown in Figure 5. As can be seen, the addition of a YAP activator to the stage III induction medium, as well as further reduction of the concentration of a histone deacetylase inhibitor, resulted in a statistically significant increase in the conversion of LIN28A-positive epithelial-like cells into pluripotent stem cells compared to the control group, increasing the conversion efficiency by up to approximately three times that of the control.

[0176] Example 6

[0177] This example describes several exemplary induction reprogramming methods of the present invention, and compares the conversion efficiency at different induction times to determine the shortest time for effectively obtaining CiPS clones using the reprogramming methods disclosed herein.

[0178] hADSCs or hASFs were cultured at a rate of 0.5 to 0.7 × 10 4 Cells were seeded into 24-well plates and cultured overnight. The next day, the cells were replaced with Stage I induction medium (Knockout DMEM supplemented with 2% B27 supplement, 1% KSR, 1% GlutaMAX, 1% NEAA, 1% penicillin-streptomycin, 50 μg / mL Vc2p, 5 mM LiCl, 1 mM NAM, 20 ng / mL BMP4, 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.05 μM DZNep, 1 μM ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM JNKIN8, 0.2 μM SETD2-IN-1, 0.5 μM AM095, 0.5 μM A485, and 1 μM WM8014), and cultured under hypoxic (5% O2) conditions until a monolayer of epithelial-like cells was observed (culture could also be continued until the confluence of the monolayer of epithelial-like cells reached approximately 100%).

[0179] Then, the culture medium was replaced with Stage II induction medium (Knockout DMEM supplemented with 2% B27 supplement, 1% GlutaMAX, 1% NEAA, 1% penicillin-streptomycin, 50 μg / mL Vc2p, 200 ng / mL bFGF, 5 μM CHIR99021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 0.5 μM JNKIN8, 2 μM EPZ5676, 0.2 μM DZNep, 1 μM ruxolitinib, 2 μM BIRB796, 2 μM SGC-CBP30, 0.5 μM Dorsormorphin, 0.5 μM VTP50469, 0.5 μM 5ITU, 2 μM 5-Azacytidine, 0.2 μM HY-10249A (CAS No.842148-40-7), 0.02 μM GSK3685032, 5 μM RA, and cultured under normoxia (21% O2) until the monolayer cells became multilayers and raised colonies appeared and gradually grew larger.

[0180] Then, the culture medium was replaced with Stage III induction medium (Knockout DMEM supplemented with 2% B27 supplement, 1% GlutaMAX, 1% NEAA, 1% penicillin-streptomycin, 5% KSR, 50 μg / mL Vc2p, 20 ng / mL recombinant human heregulin β-1 (HRG), 1 μM CHIR99021, 10 μM Y-27632, 1 μM PD0325901, and 0.5 μM SB590885). A combination of 200 μM VPA, 10 μM tranylcypromine, 0.2 μM DZNep, 2 μM EPZ5676, and 10 μM PY60 (VTZEP) was first added, and then replaced with 200 μM VPA and 2 μM PY60. The cells were cultured under normoxic conditions until CiPSC colonies were formed. It should be noted that the medium can be changed to Stage III medium and cultured to expand the hCiPSC clones, but the number of clones will hardly increase at this time.

[0181] Table 4: Specific training plans for each test group

[0182] *The above groups are the average values ​​of more than 3 repetitions.

[0183] With OCT4 and SOX2 double positive as the indication of hCiPSC clone, the conversion efficiency of the reprogramming method is calculated by dividing the number of double positive clones in each well by the number of hADSC or hASF cells initially inoculated, and the results are shown in Table 4, Figures 6 to 8 and 13. As can be seen, by adding histone acetyltransferase inhibitors in the early stages of reprogramming, the various stages of somatic cell chemical reprogramming to form pluripotent stem cells (including the stage of LIN28A positive epithelial cells with increased differentiation potential formed by somatic cells and the stage of induced pluripotent stem cells formed by the epithelial cells) are significantly shortened, thereby significantly shortening the overall duration of somatic cell reprogramming, and CiPS clones can be obtained in at least 10 days. This can greatly save culture costs, improve production efficiency, and reduce contamination risks in the process of industrialized production of CiPS cells, and is extremely beneficial. In addition, when the time of induction culture is appropriately extended, the conversion efficiency of the method of the present invention can also be stabilized at more than 1%, and even achieve an efficient conversion of about 30%.

[0184] Example 7

[0185] This example describes the establishment of hCiPS cell lines using pluripotent stem cells obtained using the exemplary reprogramming methods of the present invention.

[0186] The system establishment method is as follows:

[0187] Preparation of hCiPSCs establishment culture medium

[0188] Knockout DMEM supplemented with 2% B27 supplement, 1% GlutaMAX, 1% NEAA, 1% penicillin-streptomycin, and 2 mg / mL AlbuMAX TM -II or 4% KSR, 50μg / mL Vc2p, 20ng / mL HRG, 100ng / mL bFGF, 1μM CHIR99021, 0.5μM PD0325901 and 10μM Y-27632. 4% KSR can also be used instead of AlbuMAX TM -II.

[0189] hCiPSCs lineage establishment

[0190] Somatic cells (e.g., hADSCs or hASFs) were cultured using the same method as in Example 6. The cells were then digested with Accutase, centrifuged at 400 g for 3 minutes, resuspended in the above-mentioned establishment medium, diluted at a ratio of 1:3 to 1:12, and plated onto culture plates containing Laminin-521 matrix.

[0191] The cells were cultured in a 21% O2, 5% CO2, 37°C environment with daily medium changes. After 10-12 days of culture, distinct hCiPSC colonies were observed. After 5 minutes of digestion with ReLeSRTM, the hCiPSC colonies were cut into small pieces using a glass needle and transferred to a 48-well culture plate covered with Matrigel matrix. The cells were then incubated with mTeSRTM supplemented with 10 μM Y-27632. TM Plus medium. After 24 hours, fresh mTeSR TM Replace the medium with Plus medium, which does not contain Y-27632. Thereafter, the medium should be changed every 2 days.

[0192] The cell population at the end of culture was subjected to immunofluorescence staining for OCT4 and SOX2, and the results are shown in Figure 9. Figure 9 indicates that the pluripotent stem cells obtained by the chemical reprogramming method disclosed herein can be used to establish hCiPS cell lines.

[0193] Example 8

[0194] This example compares the differences in the effects of generating CiPS cells from a human adipose-derived mesenchymal cell line (hADSC) and a human skin-derived fibroblast cell line (hASF) using the medium described in Example 1 (original protocol) and the medium described in Example 6 (rapid protocol), respectively.

[0195] (1) The exemplary scheme of the present invention can achieve higher CiPS cell conversion efficiency in various cell lines

[0196] On the final day of 16 or 20 days of induction culture using the original and rapid protocols, respectively, the number of CiPS clones obtained was determined by OCT4 immunofluorescence staining, as shown in Table 5 and Figure 10. Comparison of these results demonstrates that the exemplary methods herein can consistently yield a large number of iPS clones across batches; however, similar control methods were unable to produce clones or had difficulty generating clones within the same timeframe.

[0197] Table 5:

[0198] *: The starting cell lines HDF-6027, 6665, and 38040 are hASF cell lines, and the rest are hADSC cell lines; each starting cell line was reprogrammed 8 times.

[0199] (2) Using the exemplary culture medium of the present invention can obtain a faster conversion curve

[0200] For hADSC cell lines, the rapid protocol was used for 10, 11, 12, 14, 16, 18, or 20 days of culture, or the original protocol was used for 10, 11, 12, 14, 16, 18, 20, 24, or 28 days of culture; for hASF cell lines, the rapid protocol was used for 18, 20, 22, or 24 days of culture, or the original protocol was used for 18, 20, 22, 26, or 30 days of culture. On the last day of each culture protocol, the number of CiPS clones induced in the rapid protocol and the control original protocol was determined by OCT4 immunofluorescence staining. The specific results are shown in Tables 6 and 7 below and in Figures 11 and 12. As can be seen from the figures, the use of the exemplary methods disclosed herein can not only convert somatic cells into pluripotent stem cells more quickly, but also achieve a higher conversion rate in a shorter time.

[0201] Table 6: Reprogramming effect of hASDC cells at different culture days

[0202] *: “-” indicates not measured; the control and test groups were repeated eight times each.

[0203] Table 7: Reprogramming effect of hASF cells at different culture days

[0204] *: “-” indicates not measured; the control and test groups were repeated eight times each.

[0205] Without being bound by theory, we believe that this is mainly due to the addition of histone acetyltransferase inhibitors in the critical early stage of reprogramming, which enables the induced somatic cells to be more quickly and comprehensively in a state susceptible to reprogramming induction, thereby greatly shortening the time required for chemical reprogramming and significantly improving the conversion efficiency of iPS clones.

[0206] Example 9

[0207] This example investigates the effect of histone acetyltransferase inhibitors in promoting the generation of CiPS cells from urine-derived cells.

[0208] According to the method described in Zhou, T. et al., Generation of human induced pluripotent stem cells from urine samples. Nat Protoc 7, 2080–2089 (2012), urine-derived cells were prepared, the main cell type of which was endothelial cells. The prior art has not reported the ability to generate pluripotent stem cells from such cells through chemically induced reprogramming.

[0209] The urine-derived endothelial cells were induced and cultured using the culture medium protocol described in Example 1 (original protocol) and the culture medium protocol described in Example 6 (rapid protocol), respectively. The only difference was that the corresponding Stage I, II, and III culture media were used for 25 days, 8 days, and 9 days, respectively. On the last day of culture, the number of CiPS clones induced in the rapid protocol and the original protocol as a control was determined by OCT4 immunofluorescence staining. Among them, 40 OCT4-positive pluripotent stem cell monoclones were successfully induced from urine-derived cells using the method described in Example 6, with an induction efficiency of approximately 0.8%; while the control group failed to induce any monoclones.

[0210] Example 10

[0211] This example investigated the effect of histone acetyltransferase inhibitors on promoting the generation of CiPS cells from dental pulp-derived mesenchymal cells.

[0212] To obtain human dental pulp-derived mesenchymal stem cells (DPMSCs), intact human teeth were collected from patients undergoing tooth extraction at Peking University Hospital with informed consent. Under sterile conditions, the root surface was cleaned with povidone-iodine, and the pulp was extracted within 2 hours of extraction. The tissue was stored in a 15-ml tube containing Mesenchymal Stem Cell Growth Medium 2 (Promo Cell) supplemented with 2% penicillin-streptomycin and transported to the laboratory for cell isolation. The pulp tissue was minced into small fragments and then digested with 3 mg / ml type I collagenase at 37°C for 40 minutes. After neutralization with 10% FBS, the cells were centrifuged, resuspended in Promo Cell medium, and plated into one well of a 6-well plate in an incubation chamber at 37°C under 21% O₂ and 5% CO₂. Non-adherent cells were removed 48 hours after initial plating. The medium was changed every 3 days until the cells reached 80–90% confluence. In a 6-well plate, 3 × 10 5 Cells were plated at a density of 100 μg / mL and passaged every 2-3 days after confluence. The cells were cultured and expanded in Promo Cell medium. For chemical reprogramming, hDPMSCs were digested and passaged with 0.25% Trypsin-EDTA and plated in 15% FBS-DMEM medium. 1-1.5×10 cells were cultured per well in a 12-well plate. 4 The cells were plated at a density of 0.5-0.6×10 per well in a 24-well plate. 4 Cells were plated at a density of 1:1.

[0213] The dental pulp-derived cells were induced and cultured using the culture medium protocol described in Example 1 (original protocol) and the culture medium protocol described in Example 6 (rapid protocol), respectively. The specific components and culture days of Stage I, II, and III culture media were slightly adjusted. When obvious human pluripotent stem cell monoclones were observed, the number of CiPS clones induced in the two protocols was determined by OCT4 immunofluorescence staining. It was found that the rapid protocol with the addition of histone acetyltransferase inhibitors was able to effectively induce OCT4-positive pluripotent stem cell monoclones from dental pulp mesenchymal stem cells, while the control group failed to induce any monoclones.

[0214] Example 11

[0215] This example describes the changes in the reprogramming induction efficiency of somatic cells after knocking out the endogenous histone acetyltransferase gene from the genome.

[0216] According to the gene editing method described in Sanjana NE et al., Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014 Aug; 11(8): 783-4, the endogenous CREBBP gene, KAT6A gene, and / or EP300 gene in the hADSC cell genome were knocked out using sgRNAs represented by SEQ ID Nos: 1 to 6, respectively. The cells in which successful knockout was verified were reprogrammed using the same method as in Example 1, except that the induction time in the third stage was adjusted to 6 days.

[0217] Control group: wild-type hADSC cells with normal expression of endogenous histone acetyltransferase genes;

[0218] Test group 1: hADSC cells with double knockout of CREBBP and KAT6A genes;

[0219] Test group 2: hADSC cells with double knockout of EP300 and KAT6A genes;

[0220] Test group 3: hADSC cells with triple knockout of CREBBP gene, EP300 gene and KAT6A gene.

[0221] On the last day of culture, the number of OCT4-positive CiPS colonies in the control and test groups was measured to calculate the somatic cell reprogramming efficiency. The results are shown in Figure 14. As can be seen, inhibiting the expression of endogenous histone acetyltransferase genes, particularly CBP / P300 and MOZ, significantly improved the conversion efficiency of CiPS.

[0222] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A method for chemically reprogramming induced pluripotent stem cells from somatic cells derived from primates, the method comprising: exposing the somatic cells to one or more histone acetyltransferase inhibitors under conditions that cause the somatic cells to form a cell population having increased differentiation potential compared to the somatic cells; as well as The cell population is further differentiated into pluripotent stem cells.

2. The method according to claim 1, wherein The histone acetyltransferase inhibitor is a CBP / P300 inhibitor or a monocytic leukemia zinc finger protein (MOZ) inhibitor or a combination thereof; preferably, the histone acetyltransferase inhibitor is selected from A485, CBP / P300 IN 8, WM8014, GEN049, WM1119, CBP / P300 IN 12, SGC / CBP30, ICBP112 or any combination thereof; Optionally, the concentration of the histone acetyltransferase inhibitor is 0.01 to 10 μM, preferably 0.05 to 5 μM, more preferably 0.1 to 5 μM, most preferably 0.1 to 2 μM; Preferably, the histone acetyltransferase inhibitor is A485 at a concentration of 0.1 to 2 μM or WM8014 at a concentration of 0.1 to 5 μM.

3. The method according to claim 1 or 2, wherein The conditions that allow the somatic cells to form a cell population having increased differentiation potential compared to the somatic cells include contacting the somatic cells with a composition comprising a glycogen synthase kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, and a retinoic acid receptor (RAR) agonist; Preferably, the composition comprises a glycogen synthase kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, and a serine-threonine kinase (Akt) inhibitor; More preferably, the composition comprises a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a serine-threonine kinase (Akt) inhibitor, and any one selected from a G protein-coupled receptor Smoothened agonist, a Dot1L inhibitor, a Menin-MLL interaction inhibitor, a SAH hydrolase inhibitor, a Jak1 / Jak2 inhibitor, or any combination thereof; Further preferably, the composition further comprises any one selected from c-Jun kinase inhibitors, histone methyltransferase inhibitors, LPA1 receptor antagonists, or any combination thereof; Still further preferably, the composition comprises a glycogen synthase kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a serine-threonine kinase (Akt) inhibitor, a G protein-coupled receptor Smoothened agonist, a Dot1L inhibitor, a Menin-MLL interaction inhibitor, a SAH hydrolase inhibitor, a Jak1 / Jak2 inhibitor, a c-Jun kinase inhibitor, a histone methyltransferase inhibitor and an LPA1 receptor antagonist; Optionally, the concentration of the glycogen synthase kinase (GSK) 3β inhibitor contained in the composition is 3 to 12 μM, preferably 5 μM; the concentration of the TGFβ receptor inhibitor is 2 to 50 μM, preferably 10 μM; the concentration of the retinoic acid receptor (RAR) agonist is 0.5 to 10 μM, preferably 2 μM; the concentration of the G protein coupled receptor Smoothened agonist is 0.2 to 2 μM, preferably 0.5 μM; the concentration of the Dot1L inhibitor is 0.2 to 10 μM, preferably 2 μM. M; the concentration of Menin-MLL interaction inhibitor is 0.1-5 μM, preferably 0.5 μM; the concentration of SAH hydrolase inhibitor is 0.01-0.2 μM, preferably 0.05 μM; the concentration of Jak1 / Jak2 inhibitor is 0.1-10 μM, preferably 1 μM; the concentration of serine-threonine kinase (Akt) inhibitor is 0.1-10 μM, preferably 1 μM; the concentration of histone methyltransferase inhibitor is 0.01-2 μM, preferably 0.2 μM; The concentration of c-Jun kinase inhibitor is 0.05 to 2 μM, preferably 0.2 μM; the concentration of LPA1 receptor antagonist is 0.1 to 5 μM, preferably 0.5 to 1 μM; Optionally, the glycogen kinase (GSK) 3β inhibitor is selected from CHIR99021, TD114-2, CHIR98014, GSK 3I inhibitor XV, BIO, SB-216763 or any combination thereof, preferably CHIR99021; Optionally, the TGFβ receptor inhibitor is selected from 616452, A8301, SB505124, GW 788388, SB 525334 or any combination thereof, preferably 616452; Optionally, the retinoic acid receptor (RAR) agonist is selected from TTNPB, Ch 55, AM580 or any combination thereof, preferably TTNPB; Optionally, the G protein coupled receptor Smoothened agonist is selected from SAG, Purmorphamine, Hg-Ag1.5 or any combination thereof, preferably SAG; Optionally, the Dot1L inhibitor is selected from EPZ5676, SGC 0946, EPZ004777 or any combination thereof, preferably EPZ5676; Optionally, the Menin-MLL interaction inhibitor is selected from VTP50469, MI3454, WDR5-IN-4 or any combination thereof, preferably VTP50469; Optionally, the SAH hydrolase inhibitor is selected from DZNEP, (-)Neplanocin A, Adenozine periodate oxidized, 3-deazaadenosine, or any combination thereof, preferably DZNEP; Optionally, the Jak1 / Jak2 inhibitor is selected from Ruxolitinib, Tofacitinib, AZD1480, Baricitinib, Fedratinib or any combination thereof, preferably Ruxolitinib; Optionally, the c-Jun kinase inhibitor is selected from JNKIN8, JNKIN7, JNKIN5, JNKIN12 or any combination thereof, preferably JNKIN8; Optionally, the serine-threonine kinase (Akt) inhibitor is selected from HY-10249A (CAS No. 842148-40-7), PF-AKT400, Capivasertib, Afuresertib or any combination thereof, preferably HY-10249A (CAS No. 842148-40-7); Optionally, the histone methyltransferase inhibitor is selected from SETD2-IN-1, EPZ-719, MMSET-IN-1 or any combination thereof, preferably SETD2-IN-1; Optionally, the LPA1 receptor antagonist is selected from AM095, AM966, Ki16425 or any combination thereof, preferably AM095.

4. The method according to any one of claims 1 to 3, wherein The primate is selected from humans.

5. The method according to any one of claims 1 to 4, wherein The somatic cells are selected from fibroblasts, blood cells, epithelial cells, and endothelial cells, preferably fibroblasts; Optionally, the fibroblasts are selected from adipose fibroblasts, skin fibroblasts, umbilical cord mesenchymal stem cells or dental pulp mesenchymal stem cells, and the endothelial cells are selected from urine-derived endothelial cells.

6. The method according to any one of claims 1 to 5, wherein The cell population with increased differentiation potential comprises cells having the following characteristics: Epithelial-like cells positive for the pluripotency marker Lin28A; and Compared to the somatic cells, the expression levels of epithelial cell surface markers are increased, and the expression levels of somatic cell markers are decreased; Optionally, the epithelial cell surface marker is selected from one or more of KRT8, KRT18 or KRT19; Optionally, the somatic cell marker is selected from one or more of VIM, SNAI2, COL5A1, PRKX1, COL6A2, ZEB1, MMP1, TWIST1, and COL1A1.

7. The method according to any one of claims 1 to 6, wherein The fibroblasts are exposed to the histone acetyltransferase inhibitor for a period of less than 8 days, preferably 5 to 8 days.

8. A composition comprising a combination of agents that increase the differentiation potential of somatic cells and one or more histone acetyltransferase inhibitors, Preferably, the agent combination comprises a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor and a retinoic acid receptor (RAR) agonist, more preferably comprises a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, and any one or any combination thereof selected from G protein-coupled receptor Smoothened agonists, Dot1L inhibitors, Menin-MLL interaction inhibitors, SAH hydrolase inhibitors, Jak1 / Jak2 inhibitors, c-Jun kinase inhibitors, serine-threonine kinase (Akt) inhibitors, and histone methyltransferase inhibitors, and further preferably further comprises one or more LPA1 receptor antagonists; Preferably, the composition comprises a glycogen synthase kinase (GSK) 3β inhibitor at a concentration of 3 to 12 μM, preferably 5 μM; a TGFβ receptor inhibitor at a concentration of 2 to 50 μM, preferably 10 μM; a retinoic acid receptor (RAR) agonist at a concentration of 0.5 to 10 μM, preferably 2 μM; a G protein-coupled receptor Smoothened agonist at a concentration of 0.2 to 2 μM, preferably 0.5 μM; a Dot1L inhibitor at a concentration of 0.2 to 10 μM, preferably 2 μM; a Menin-MLL interaction inhibitor at a concentration of 0.1 to 5 μM, preferably 0.5 μM; and a SAH hydrolase inhibitor at a concentration of 0.01 to 0.2 μM. , preferably 0.05 μM; the concentration of Jak1 / Jak2 inhibitors is 0.1-10 μM, preferably 1 μM; the concentration of c-Jun kinase inhibitors is 0.05-2 μM, preferably 0.2 μM; the concentration of serine-threonine kinase (Akt) inhibitors is 0.1-10 μM, preferably 1 μM; the concentration of histone methyltransferase inhibitors is 0.01-2 μM, preferably 0.2 μM; the concentration of LPA1 receptor antagonists is 0.1 to 5 μM, preferably 0.5 to 1 μM; the concentration of histone acetyltransferase inhibitors is 0.01 to 10 μM, preferably 0.05 to 5 μM, more preferably 0.1 to 5 μM, and most preferably 0.1 to 2 μM; Optionally, the glycogen kinase (GSK) 3β inhibitor is selected from CHIR99021, TD114-2, CHIR98014, GSK 3I inhibitor XV, BIO, SB-216763 or any combination thereof, preferably CHIR99021; Optionally, the TGFβ receptor inhibitor is selected from 616452, A8301, SB505124, GW 788388, SB 525334 or any combination thereof, preferably 616452; Optionally, the retinoic acid receptor (RAR) agonist is selected from TTNPB, Ch 55, AM580 or any combination thereof, preferably TTNPB; Optionally, the G protein coupled receptor Smoothened agonist is selected from SAG, Purmorphamine, Hg-Ag1.5 or any combination thereof, preferably SAG; Optionally, the Dot1L inhibitor is selected from EPZ5676, SGC 0946, EPZ004777 or any combination thereof, preferably EPZ5676; Optionally, the Menin-MLL interaction inhibitor is selected from VTP50469, MI3454, WDR5-IN-4 or any combination thereof, preferably VTP50469; Optionally, the SAH hydrolase inhibitor is selected from DZNEP, (-)Neplanocin A, Adenozine periodate oxidized, 3-deazaadenosine, or any combination thereof, preferably DZNEP; Optionally, the Jak1 / Jak2 inhibitor is selected from ruxolitinib, tofacitinib, AZD1480, baricitinib, fuzotinib or any combination thereof, preferably ruxolitinib; Optionally, the c-Jun kinase inhibitor is selected from JNKIN8, JNKIN7, JNKIN5, JNKIN12 or any combination thereof, preferably JNKIN8; Optionally, the serine-threonine kinase (Akt) inhibitor is selected from HY-10249A (CAS No. 842148-40-7), PF-AKT400, Capivasertib, Afuresertib or any combination thereof, wherein HY-10249A (CAS No. 842148-40-7) is preferred; Optionally, the histone methyltransferase inhibitor is selected from SETD2-IN-1, EPZ-719, MMSET-IN-1 or any combination thereof, preferably SETD2-IN-1; Optionally, the histone acetyltransferase inhibitor is a CBP / P300 inhibitor or a monocytic leukemia zinc finger protein (MOZ) inhibitor or a combination thereof, preferably selected from A485, CBP / P300 IN 8, WM8014, GEN049, WM1119, CBP / P300IN 12, SGCCBP30, ICBP112 or any combination thereof, more preferably selected from WM8014 or A485 or a combination thereof; Optionally, the LPA1 receptor antagonist is selected from AM095, AM966, Ki16425 or any combination thereof, preferably AM095; Most preferably, the composition is any one selected from the following: a composition comprising 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, and 0.5 μM A-485; or a composition comprising 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, and 1 μM WM8014; or a composition comprising 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, 0.5 μM A-485, and 1 μM WM8014; or a composition comprising 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, 0.5 μM A-485, and 0.5 μM AM095; or a composition comprising 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, 1 μM WM8014, and 0.5 μM AM095; or A composition comprising 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 2 μM EPZ5676, 0.02 μM DZNep, 1 μM Ruxolitinib, 0.5 μM VTP50469, 1 μM HY-10249A (CAS No. 842148-40-7), 0.2 μM SETD2IN1, 0.2 μM JNKIN8, 0.5 μM A-485, 1 μM WM8014, and 0.5 μM AM095.

9. A composition comprising a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a G protein-coupled receptor Smoothened agonist, a c-Jun kinase inhibitor, a SAH hydrolase inhibitor, and a DNA methyltransferase (DNMT) inhibitor, preferably comprising a glycogen synthesis kinase (GSK) 3β inhibitor, a TGFβ receptor inhibitor, a retinoic acid receptor (RAR) agonist, a G protein-coupled receptor Smoothened agonist, a c-Jun kinase inhibitor, a SAH hydrolase inhibitor, a DNA methyltransferase (DNMT) inhibitor, and any one selected from a ROCK1 inhibitor, a Dot1L inhibitor, a Menin-MLL interaction inhibitor, a MAPK inhibitor, a Jak1 / Jak2 inhibitor, a histone acetyltransferase inhibitor, a serine-threonine kinase (Akt) inhibitor, and a casein kinase inhibitor, or any combination thereof; Preferably, the concentration of the glycogen synthase kinase (GSK) 3β inhibitor contained in the composition is 3-12 μM, preferably 5 μM; the concentration of the TGFβ receptor inhibitor is 2-50 μM, preferably 10 μM; the concentration of the retinoic acid receptor (RAR) agonist is 0.5-10 μM, preferably 2-5 μM; the concentration of the G protein-coupled receptor Smoothened agonist is 0.2-2 μM, preferably 0.5 μM; the concentration of the c-Jun kinase inhibitor is 0.1-2.5 μM, preferably 0.5 μM; the concentration of the SAH hydrolase inhibitor is 0.04-1 μM, preferably 0.2 μM; the concentration of the DNA methyltransferase (DNMT) inhibitor is 0.01-0.1 μM, Preferably 0.02 μM; ROCK1 inhibitor concentration is 2-50 μM, preferably 10 μM; Dot1L inhibitor concentration is 0.2-10 μM, preferably 2 μM; Menin-MLL interaction inhibitor concentration is 0.1-5 μM, preferably 0.5 μM; MAPK inhibitor concentration is 0.4-10 μM, preferably 2 μM; Jak1 / Jak2 inhibitor concentration is 0.1-10 μM, preferably 1 μM; histone acetyltransferase inhibitor concentration is 0.4-10 μM, preferably 2 μM; serine-threonine kinase (Akt) inhibitor concentration is 0.04-1 μM, preferably 0.2 μM; casein kinase inhibitor concentration is 0.2-5 μM, preferably 1 μM; Optionally, the glycogen kinase (GSK) 3β inhibitor is selected from CHIR99021, TD114-2, CHIR98014, GSK 3I inhibitor XV, BIO, SB-216763 or any combination thereof, preferably CHIR99021; Optionally, the TGFβ receptor inhibitor is selected from 616452, A8301, SB505124, GW 788388, SB 525334 or any combination thereof, preferably 616452; Optionally, the retinoic acid receptor (RAR) agonist is selected from TTNPB, retinoic acid (RA), Ch 55, AM580 or any combination thereof, preferably TTNPB and / or RA; Optionally, the G protein coupled receptor Smoothened agonist is selected from SAG, Purmorphamine, Hg-Ag1.5 or any combination thereof, preferably SAG; Optionally, the c-Jun kinase inhibitor is selected from JNKIN8, JNKIN7, JNKIN5, JNKIN12 or any combination thereof, preferably JNKIN8; Optionally, the SAH hydrolase inhibitor is selected from DZNEP, (-)Neplanocin A, Adenozine periodate oxidized, 3-deazaadenosine, or any combination thereof, preferably DZNEP; Optionally, the ROCK1 inhibitor is selected from Y27632, Thiazovivin, Fasudil, Chroman 1 or any combination thereof, preferably Y27632; Optionally, the Dot1L inhibitor is selected from EPZ5676, SGC 0946, EPZ004777 or any combination thereof, preferably EPZ5676; Optionally, the Menin-MLL interaction inhibitor is selected from VTP50469, MI3454, WDR5-IN-4 or any combination thereof, preferably VTP50469; Optionally, the MAPK inhibitor is selected from BIRB796, SB203580, SB202190 or any combination thereof, preferably BIRB796; Optionally, the Jak1 / Jak2 inhibitor is selected from ruxolitinib, tofacitinib, AZD1480, baricitinib, fuzotinib or any combination thereof, preferably ruxolitinib; Optionally, the histone acetyltransferase inhibitor is selected from SGC / CBP300, A485, CBP / P300 IN 8, WM8014, GEN049, WM1119, CBP / P300 IN 12, ICBP112 or any combination thereof, preferably SGC / CBP300; Optionally, the serine-threonine kinase (Akt) inhibitor is selected from HY-10249A (CAS No. 842148-40-7), PF-AKT400, Capivasertib, Afuresertib or any combination thereof, preferably HY-10249A (CAS No. 842148-40-7); Optionally, the casein kinase inhibitor is selected from CX-4945, gallic acid (Ellagic Acid), TTP 22 or any combination thereof, preferably CX-4945; Optionally, the DNA methyltransferase (DNMT) inhibitor is selected from GSK3689032, 5-aza-C, Decitabine or any combination thereof, preferably GSK3689032; Most preferably, the composition comprises 5 μM CHIR999021, 10 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 10 μM Y-27632, 0.5 μM JNKIN8, 1 μM Ruxolitinib, 2 μM BIRB796, 2 μM SGC-CBP30, 2 μM EPZ5676, 0.2 μM DZNep, 0.5 μM VTP50469, 0.5 μM 5-iodotubercidin, 5 μM RA, 0.02 μM GSK3685032, 0.2 μM HY-10249A (CAS No. 842148-40-7) and 1 μM CX-4945.

10. A composition comprising a glycogen synthesis kinase (GSK) 3β inhibitor, a ROCK1 inhibitor, a MAPK inhibitor, and a BRAF inhibitor, preferably comprising a glycogen synthesis kinase (GSK) 3β inhibitor, a ROCK1 inhibitor, a MAPK inhibitor, a BRAF inhibitor, and a histone deacetylase inhibitor, more preferably comprising a glycogen synthesis kinase (GSK) 3β inhibitor, a ROCK1 inhibitor, a MAPK inhibitor, a BRAF inhibitor, a histone deacetylase inhibitor, and any one selected from a YAP activator, a monoamine oxidase inhibitor, a SAH hydrolase inhibitor, and a Dot1L inhibitor, or any combination thereof; Preferably, the composition comprises a glycogen synthase kinase (GSK) 3β inhibitor at a concentration of 0.2 to 5 μM, preferably 1 μM; a ROCK1 inhibitor at a concentration of 2 to 50 μM, preferably 10 μM; a MAPK inhibitor at a concentration of 0.2 to 5 μM, preferably 1 μM; a BRAF inhibitor at a concentration of 0.1 to 2.5 μM, preferably 0.5 μM; a histone deacetylase inhibitor at a concentration of 40 to 1000 μM, preferably 200 μM; a YAP activator at a concentration of 0.4 to 10 μM, preferably 2 μM; a monoamine oxidase inhibitor at a concentration of 2 to 50 μM, preferably 10 μM; a SAH hydrolase inhibitor at a concentration of 0.04 to 1 μM, preferably 0.2 μM; and a Dot1L inhibitor at a concentration of 0.4 to 10 μM, preferably 2 μM. Optionally, the glycogen kinase (GSK) 3β inhibitor is selected from CHIR99021, TD114-2, CHIR98014, GSK 3I inhibitor XV, BIO, SB-216763 or any combination thereof, preferably CHIR99021; Optionally, the ROCK1 inhibitor is selected from Y27632, Thiazovivin, Fasudil, Chroman 1 or any combination thereof, preferably Y27632; Optionally, the MAPK inhibitor is selected from PD0325901, AZD8330 or TAK-733 or any combination thereof, preferably PD0325901; Optionally, the BRAF inhibitor is selected from SB590885, AZ 628, Agerafenib, Temuterkib or any combination thereof, preferably SB590885; Optionally, the histone deacetylase inhibitor is selected from VPA, MS275, CI 994, HDACi IV or any combination thereof, preferably VPA; Optionally, the YAP activator is selected from PY60 and / or LAST-In-1, preferably PY60; Optionally, the monoamine oxidase inhibitor is selected from tranylcypromine, S2101, GSK2879552 or any combination thereof, preferably tranylcypromine; Optionally, the SAH hydrolase inhibitor is selected from DZNEP, (-)Neplanocin A, Adenozine periodate oxidized, 3-deazaadenosine, or any combination thereof, preferably DZNEP; Optionally, the Dot1L inhibitor is selected from EPZ5676, SGC 0946, EPZ004777 or any combination thereof, preferably EPZ5676; Most preferably, the composition comprises 1 μM CHIR999021, 10 μM Y-27632, 1 μM PD0325901 and 0.5 μM SB590885; or containing 1 μM CHIR999021, 10 μM Y-27632, 1 μM PD0325901, 0.5 μM SB590885, 200 μM VPA, 10 μM tranylcypromine, 2 μM EPZ5676, 0.2 μM DZNep, and 10 μM PY60; or Contains 1 μM CHIR999021, 10 μM Y-27632, 1 μM PD0325901, 0.5 μM SB590885, 200 μM VPA, and 2 μM PY60.

11. The method according to any one of claims 1 to 7, wherein The further dedifferentiation comprises treating the cell population with the composition of claim 9 or 10.

12. The method of claim 11, comprising: exposing somatic cells to the composition of claim 8 for 5 to 8 days to obtain a cell population having increased differentiation potential compared to the somatic cells; The cell population is first treated with the composition of claim 9, optionally for 1 to 8 days, and then treated with the composition of claim 10, optionally for 4 to 8 days, to obtain induced pluripotent stem cells.

13. The method of any one of claims 1 to 7 or 11 to 12, further comprising purifying the pluripotent stem cells to obtain a population of pluripotent stem cells having a purity of 50% or greater, 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% based on a pluripotency marker selected from OCT4, SOX2, NANOG, FGF4, ZFP57, DPPA5, REX1, DPPA4, TDGF1, TRA-1-60, TRA-1-81, SSEA4, KLF4, KLF17, DPPA3, DNMT3L, UTF, or any combination thereof.

14. Use of a histone acetyltransferase inhibitor for chemically reprogramming somatic cells into induced pluripotent stem cells, wherein: The inhibitor is used before somatic cells become a cell population containing cells having the following characteristics: the cells are epithelial-like cells that are positive for the pluripotency marker LIN28A, and the cells have an increased expression level of an epithelial cell surface marker and a decreased expression level of a somatic cell marker compared to the somatic cells that are the starting cells; Optionally, the epithelial cell surface marker is selected from one or more of KRT8, KRT18 or KRT19; Optionally, the somatic cell marker is selected from one or more of VIM, SNAI2, COL5A1, PRKX1, COL6A2, ZEB1, MMP1, TWIST1, and COL1A1; Optionally, the histone acetyltransferase inhibitor is a CBP / P300 inhibitor or a monocytic leukemia zinc finger protein (MOZ) inhibitor or a combination of the two, preferably selected from A485, CBP / P300 IN 8, WM8014, GEN049, WM1119, CBP / P300 IN 12, SGCCBP30, ICBP112 or any combination thereof, more preferably selected from WM8014 or A485 or a combination of the two.

Images