Chemical reprogramming method for blood cells

Abstract

Provided is a composition for pretreating a cell, which can improve the responsiveness of the cell to a reprogramming reagent, thereby increasing the success rate of reprogramming and improving the reprogramming efficiency. Also provided are a method for pretreating a cell using the composition, a method for reprogramming a cell using the composition, and a pluripotent stem cell obtained using the method.

Description

Chemical reprogramming methods for blood cells Technical Field

[0001] This invention relates to a cell pretreatment composition that can improve the success rate and efficiency of reprogramming. The invention also relates to a method for pretreating somatic cells using the said pretreatment composition, particularly for blood-derived cells, a kit comprising the said composition, a culture medium, pluripotent stem cells obtained using the said composition and method, and the use of said pluripotent stem cells. Background Technology

[0002] Human somatic cells can be reprogrammed into a pluripotent state by restoring cellular plasticity through the introduction and expression of pluripotent transcription factors or by exposure to a combination of chemical reagents. Compared with classical transcription factor reprogramming methods (such as OKSM), chemical reprogramming methods can regulate cell fate more flexibly, precisely, and simply, and do not introduce exogenous gene load into the genome, making them more suitable for personalized treatment. It has been reported that human pluripotent stem cells (hCiPS) obtained using chemical reprogramming methods can successfully induce various human cells. Among them, type 1 diabetic patients who received islets derived from hCiPS were able to get rid of insulin dependence after transplantation, showing great therapeutic potential (see Wang S et al., Transplantation of chemically induced pluripotent stem-cell-derived islets under abdominal anterior rectus sheath in a type 1 diabetes patient. Cell. 2024 Oct31; 187(22):6152-6164.e18).

[0003] Blood is the most ideal source of somatic cells compared to other tissues because of its abundance, convenience, and accessibility. Blood collection procedures are less invasive than other tissue sampling methods, and frozen blood stored in blood banks can also serve as a source of samples for research and therapeutic purposes.

[0004] To date, reprogramming methods for blood cells have significant limitations. For example, non-integrating methods using Sendai virus or episomes are significantly less efficient than methods that integrate exogenous transcription factors into the genome. Furthermore, non-integrating methods require multiple passages after transfection to obtain cell lines free of exogenous reprogramming agents. Reprogramming methods using mRNA have extremely low transfection efficiency for blood cells, and successful reprogramming of blood cells has not yet been reported (Schlaeger, T., Daheron, L., Brickler, T. et al. A comparison of non-integrating reprogramming methods. Nat Biotechnol 33, 58–63 (2015)).

[0005] Unlike transgenic methods that require the exogenous introduction of pluripotency transcription factors, chemical reprogramming methods can induce somatic cells to become pluripotent cells stepwise using only different combinations of small molecules. Chemical reprogramming typically involves combining small molecules targeting key developmental pathways with epigenetic regulators that can modulate gene transcription activity. This downregulates the somatic cell's genetic program and upregulates one or more key pluripotency-related transcription factors, ultimately reconstructing the cell's pluripotency network without the introduction of exogenous genes, thus transforming the cell's identity from a somatic cell to a pluripotent cell. During this process, changes in the genetic program will cause regular changes in cell morphology, with the formation of a dense monolayer of LIN28A-positive epithelial-like cells as a key milestone, marking the successful erasure of the initial somatic cell identity and the activation of pluripotency-related transcription factor expression. LIN28A-positive epithelioid cells can be further dedifferentiated into more plastic intermediate states, such as extraembryonic endoderm-like cells (XEN-like cells). These can be stably passaged in vitro (see, for example, Chinese application CN202411422045.8), further dedifferentiated to establish stem cell lines (see, for example, PCT application PCT / CN2021 / 085936), or directly redifferentiated into various target cell types (see, for example, Chinese application CN201780090055.8). Therefore, obtaining LIN28A-positive epithelioid cells from somatic cells is the core technology of chemical reprogramming methods. No specific successful cases of obtaining LIN28A-positive epithelioid cells from blood cells through chemical reprogramming have been reported.

[0006] Therefore, there is a need for a method that can robustly and efficiently generate LIN28A-positive epithelioid cells from blood cells, and subsequently generate CiPS cells. Summary of the Invention

[0007] Existing chemical reprogramming methods for inducing pluripotent stem cells from blood cells have extremely low overall efficiency. Although these methods can successfully downregulate the expression of characteristic genes in blood cells, the activation of pluripotency-related transcription factors is far lower than expected, resulting in cells failing to quickly restore pluripotency, low proliferation rates, and an extremely limited number of LIN28A-positive epithelial-like cells, thus restricting the possibility of large-scale production of CiPS.

[0008] Through in-depth research, the inventors of this application discovered that pretreating blood cells with a small molecule combination containing inhibitors targeting the SWI / SNF complex before chemical reprogramming allows the blood cells to better respond to subsequent reprogramming reagents and conditions, entering a state more conducive to reprogramming. In this state, relevant characteristic genes of blood cells are significantly suppressed or downregulated, but the expression of pluripotent transcription factors is not yet significantly activated. Reprogramming the pretreated blood cells resulted in a significant increase in the number of LIN28A-positive epithelial-like cells and a significant improvement in the generation efficiency of CiPS clones.

[0009] This pretreatment makes the chemical reprogramming of blood cells a viable industrial practice. Compared to other known methods, chemical reprogramming of blood cells offers a more convenient and less invasive way to obtain CiPS cells, significantly increasing the accessibility of CiPS-based regenerative medicine. This method also demonstrates robustness between blood cells derived from different individuals, laying the foundation for personalized cell therapy or organ transplantation. Furthermore, the compositions and methods of this application are not limited to blood cells and are effective for a variety of somatic cells, including fibroblasts.

[0010] Therefore, a first aspect of this application is to provide a method for pretreating somatic cells, comprising: incubating somatic cells with a pretreatment composition to obtain a cell population with enhanced responsiveness to a reprogramming agent, wherein the pretreatment composition contains an inhibitor targeting the SWI / SNF complex.

[0011] In some embodiments, the pretreatment composition further comprises small molecules and / or epigenetic regulators targeting major developmental pathways. In some embodiments, the small molecules targeting major developmental pathways are selected from glycogen synthase kinase (GSK) 3β inhibitors, TGFβ receptor inhibitors, cAMP activators, or any combination thereof. In a preferred embodiment, the small molecules targeting major developmental pathways are selected from glycogen synthase kinase (GSK) 3β inhibitors, TGFβ receptor inhibitors, cAMP activators, retinoic acid receptor (RAR) agonists, G protein-coupled receptor smoothed agonists, or any combination thereof. In a more preferred embodiment, the small molecules targeting major developmental pathways comprise a combination of glycogen synthase kinase (GSK) 3β inhibitors, TGFβ receptor inhibitors, cAMP activators, retinoic acid receptor (RAR) agonists, and G protein-coupled receptor smoothed agonists.

[0012] In some embodiments, the epigenetic regulator is selected from Dot1L inhibitors, Menin-MLL interaction inhibitors, histone acetyltransferase inhibitors, or any combination thereof. In a preferred embodiment, the epigenetic regulator is selected from Dot1L inhibitors, Menin-MLL interaction inhibitors, SETD2 inhibitors, histone acetyltransferase inhibitors, or any combination thereof. In a more preferred embodiment, the epigenetic regulator comprises a combination of Dot1L inhibitors, Menin-MLL interaction inhibitors, SETD2 inhibitors, and histone acetyltransferase inhibitors.

[0013] In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex and a glycogen synthase kinase (GSK) 3β inhibitor. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex and a TGFβ receptor inhibitor. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex and a cAMP activator. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex and a retinoic acid receptor (RAR) agonist. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex and a G protein-coupled receptor smoothed agonist. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex and a Dot1L inhibitor. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex and a Menin-MLL interaction inhibitor. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex and a SETD2 inhibitor. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex and a histone acetyltransferase inhibitor.

[0014] In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex, a glycogen synthase kinase (GSK) 3β inhibitor, and a histone acetyltransferase inhibitor. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex, a TGFβ receptor inhibitor, and a histone acetyltransferase inhibitor. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex, a cAMP activator, and a histone acetyltransferase inhibitor. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex, a retinoic acid receptor (RAR) agonist, and a histone acetyltransferase inhibitor. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex, a G protein-coupled receptor smoothed agonist, and a histone acetyltransferase inhibitor. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex, a Dot1L inhibitor, and a histone acetyltransferase inhibitor. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex, a Menin-MLL interaction inhibitor, and a histone acetyltransferase inhibitor. In some embodiments, the pretreatment composition comprises an inhibitor targeting the SWI / SNF complex, a SETD2 inhibitor, and a histone acetyltransferase inhibitor.

[0015] In a preferred embodiment, the pretreatment composition further comprises a compound capable of further enhancing the responsiveness of somatic blood cells to the reprogramming agent, said compound being selected from PRC2 complex inhibitors, LATS kinase inhibitors, or a combination of both.

[0016] In a preferred embodiment, the pretreatment composition further comprises a compound capable of reducing the gene mutation rate, the compound being selected from a combination of p53 protein agonists, or DNA-dependent protein kinase (DNA-PK) inhibitors and antioxidants.

[0017] In some embodiments, the pretreated somatic cells are selected from blood cells, fibroblasts, epithelial cells, and endothelial cells. In a preferred embodiment, the pretreated somatic cells are blood cells. In some embodiments, the blood cells are obtained from peripheral blood, umbilical cord blood, refrigerated blood samples, fingertip blood, venous blood, arterial blood, capillary blood, whole blood, blood components, or bone marrow. In some embodiments, the somatic cells are nucleated blood cells. In some embodiments, the blood cells are selected from hematopoietic stem cells, colony-forming unit cells (CFU-C), bone marrow stem cells, erythroid progenitor cells, lymphoid stem cells, lymphocytes, plasma cells, monocytes, granulocytes, neutrophils, eosinophils, basophils, T cells, B cells, and any combination thereof. In a preferred embodiment, the somatic cells are erythroid progenitor cells.

[0018] In some embodiments, the cells are incubated with the pretreatment composition for no more than 20 days. In some embodiments, the cells are incubated with the pretreatment composition for 1 to 20 days. In some embodiments, the cells are incubated with the pretreatment composition for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0019] In some embodiments, the inhibitor targeting the SWI / SNF complex specifically inhibits the activity of SMARCA2 and / or SMARCA4. In some embodiments, the inhibitor is selected from small molecule compounds, antisense nucleic acids, gene editors, or directed degraders. In some embodiments, the small molecule compound specifically inhibiting the SWI / SNF complex is selected from SGC-SMARCA-BRDVIII, PFI-3, FHT-2344, or any combination thereof. In some embodiments, the antisense nucleic acid specifically inhibiting the SWI / SNF complex is selected from siRNA, shRNA, or nucleic acid aptamers. In some embodiments, the gene editor specifically inhibiting the SWI / SNF complex is selected from CRISPR or TALEN. In some embodiments, the directed degrader specifically inhibiting the SWI / SNF complex is selected from PROTAC, lysosome-targeted chimera (LYTAC), or autophagy-targeted chimera (AUTAC). In some embodiments, the PROTAC specifically inhibiting the SWI / SNF complex is selected from AU-15330, AU-24118, or ACBI1. In some embodiments, the concentration of the inhibitor targeting the SWI / SNF complex is 0.01–100 μM. In a preferred embodiment, the concentration of the inhibitor targeting the SWI / SNF complex is 0.1–10 μM. In a more preferred embodiment, the concentration of the inhibitor targeting the SWI / SNF complex is 0.5–5 μM. In the most preferred embodiment, the concentration of the inhibitor targeting the SWI / SNF complex is 0.5–2 μM.

[0020] In some embodiments, the GSK3β inhibitor is selected from CHIR99021, TD114-2, CHIR98014, GSK 3β inhibitor XV, BIO, SB-216763, or any combination thereof. In a preferred embodiment, the GSK3β inhibitor is CHIR99021. In some embodiments, the concentration of the GSK3β inhibitor is 0.05–100 μM.

[0021] In some embodiments, the TGFβ receptor inhibitor is selected from 616452, A8301, SB505124, GW 788388, SB 525334, or any combination thereof. In a preferred embodiment, the TGFβ receptor inhibitor is 616452. In some embodiments, the concentration of the TGFβ receptor inhibitor is 0.02–500 μM.

[0022] In some embodiments, the cAMP activator is selected from Forskolin, IBMX, Rolipra, 8BrcAMP, Prostaglandin E2 (PGE2), NKH 477, bis(butyryl)-cAMP (DBcAMP), Sp-8-Br-cAMPs, or any combination thereof. In a preferred embodiment, the cAMP activator is Forskolin. In some embodiments, the concentration of the cAMP activator is 0.05–500 μM.

[0023] In some embodiments, the retinoic acid receptor (RAR) agonist is selected from TTNPB, Ch 55, AM580, or any combination thereof. In a preferred embodiment, the RAR agonist is TTNPB. In some embodiments, the concentration of the RAR agonist is 0.02–200 μM.

[0024] In some embodiments, the G protein-coupled receptor smoothed agonist is selected from SAG, Purmorphamine, Hg-Ag1.5, or any combination thereof. In a preferred embodiment, the G protein-coupled receptor smoothed agonist is SAG. In some embodiments, the concentration of the smoothed agonist is 0.01–50 μM.

[0025] In some embodiments, the Dot1L inhibitor is selected from EPZ5676, SGC 0946, EPZ004777, or any combination thereof. In a preferred embodiment, the Dot1L inhibitor is EPZ5676. In some embodiments, the concentration of the Dot1L inhibitor is 0.05–100 μM.

[0026] In some embodiments, the Menin-MLL interaction inhibitor is selected from VTP50469, MI3454, WDR5-IN-4, or any combination thereof. In a preferred embodiment, the Menin-MLL interaction inhibitor is VTP50469. In some embodiments, the concentration of the Menin-MLL interaction inhibitor is 0.01–100 μM.

[0027] In some embodiments, the SETD2 inhibitor is selected from SETD2-IN-1, EPZ-719, MMSET-IN-1, or any combination thereof. In a preferred embodiment, the SETD2 inhibitor is SETD2-IN-1. In some embodiments, the concentration of the SETD2 inhibitor is 0.01–100 μM.

[0028] In some embodiments, the histone acetyltransferase inhibitor is selected from A485, CBP / P300 IN 8, WM8014, GEN049, WM1119, CBP / P300 IN 12, SGC / CBP30, ICBP112, PROTAC CBP / P300 Degrader-1, or any combination thereof. In a preferred embodiment, the histone acetyltransferase inhibitor is PROTAC CBP / P300 Degrader-1. In some embodiments, the concentration of the histone acetyltransferase inhibitor is 0.01–100 μM.

[0029] In some embodiments, the PRC2 complex inhibitor is selected from UNC6852, EPZ6438, and PROTAC EZH2Degrader-1, or any combination thereof. In a preferred embodiment, the PRC2 complex inhibitor is UNC6852. In some embodiments, the concentration of the PRC2 complex inhibitor is 0.01–100 μM.

[0030] In some embodiments, the LATS kinase inhibitor is NIBR-LTSi. In some embodiments, the concentration of the LATS kinase inhibitor is 0.01–100 μM.

[0031] In some embodiments, the YAP activator is PY-60. In some embodiments, the concentration of the YAP activator is 0.01–100 μM.

[0032] In some embodiments, the p53 protein agonist is selected from C16-ceramide, Kevetrin or its salts (such as Kevetrin hydrochloride), and SCH29074. In some embodiments, the concentration of the p53 protein agonist is 0.01–100 μM.

[0033] In some embodiments, the DNA-dependent protein kinase (DNA-PK) inhibitor is NU7441. In some embodiments, the concentration of the DNA-dependent protein kinase (DNA-PK) inhibitor is 0.01–100 μM.

[0034] In some embodiments, the antioxidant is glutathione (GSH). In some embodiments, the concentration of the antioxidant is 0.01–1000 μM.

[0035] In some embodiments, the pretreatment composition comprises 0.01–100 μM of a SWI / SNF complex inhibitor. In some embodiments, the pretreatment composition comprises 0.01–100 μM of AU15330, AU24118, or ACBII. In some embodiments, the pretreatment composition comprises a combination of AU15330 and AU24118, a combination of AU15330 and ACBII, or a combination of AU24118 and ACBII in a total amount of 0.01–100 μM. In some embodiments, the pretreatment composition comprises a combination of AU15330, AU24118, and ACBII in a total amount of 0.01–100 μM.

[0036] In some embodiments, the pretreatment composition contains a SWI / SNF complex inhibitor at concentrations of 0.01–100 μM, 0.02–80 μM, 0.03–50 μM, 0.04–40 μM, 0.05–30 μM, 0.06–20 μM, or 0.07–10 μM. In some embodiments, the pretreatment composition contains a SWI / SNF complex inhibitor at concentrations of 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM. The amounts of SWI / SNF complex inhibitors are 0.1 to 10 μM, ranging from μM to 1.9 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, or 100 μM. In a preferred embodiment, the pretreatment composition contains 0.1 to 10 μM of SWI / SNF complex inhibitor. In a more preferred embodiment, the pretreatment composition contains 0.5 to 5 μM of SWI / SNF complex inhibitor. In an even more preferred embodiment, the pretreatment composition contains 0.5 to 2 μM of SWI / SNF complex inhibitor.

[0037] In some embodiments, the pretreatment composition comprises 0.01–100 μM of a SWI / SNF complex inhibitor and 0.01–100 μM of a histone acetyltransferase inhibitor. In some embodiments, the pretreatment composition comprises 0.01–100 μM AU15330 and 0.01–100 μM PROTAC CBP / P300 Degrader-1. In some embodiments, the pretreatment composition comprises 0.01–100 μM AU24118 and 0.01–100 μM PROTAC CBP / P300 Degrader-1. In some embodiments, the pretreatment composition comprises 0.01–100 μM ACBII and 0.01–100 μM PROTAC CBP / P300 Degrader-1. In some embodiments, the pretreatment composition comprises a combination of AU15330 and AU24118 in a total amount of 0.01–100 μM and PROTAC CBP / P300 Degrader-1. In some embodiments, the pretreatment composition comprises a combination of AU15330 and ACBII in a total amount of 0.01–100 μM and PROTAC CBP / P300 Degrader-1. In some embodiments, the pretreatment composition comprises a combination of AU24118 and ACBII in a total amount of 0.01–100 μM and PROTAC CBP / P300 Degrader-1. In some embodiments, the pretreatment composition comprises a combination of AU15330, AU24118 and ACBII in total amounts of 0.01 to 100 μM, and PROTAC CBP / P300 Degrader-1 in total amounts of 0.01 to 100 μM.

[0038] In some embodiments, the pretreatment composition comprises 0.01–100 μM of a SWI / SNF complex inhibitor, 0.02–500 μM of a TGFβ receptor inhibitor, and 0.01–100 μM of a histone acetyltransferase inhibitor. In some embodiments, the pretreatment composition comprises 0.01–100 μM AU15330, 0.02–500 μM 616452, and 0.01–100 μM PROTAC CBP / P300 Degrader-1. In some embodiments, the pretreatment composition comprises 0.01–100 μM AU24118, 0.02–500 μM 616452, and 0.01–100 μM PROTAC CBP / P300 Degrader-1. In some embodiments, the pretreatment composition comprises 0.01–100 μM ACBII, 0.02–500 μM 616452, and 0.01–100 μM PROTAC CBP / P300 Degrader-1. In some embodiments, the pretreatment composition comprises a combination of AU15330 and AU24118 in total amounts of 0.01–100 μM, 0.02–500 μM 616452, and 0.01–100 μM PROTAC CBP / P300 Degrader-1. In some embodiments, the pretreatment composition comprises a combination of AU15330 and ACBII in total amounts of 0.01–100 μM, 0.02–500 μM 616452, and 0.01–100 μM PROTAC CBP / P300 Degrader-1. In some embodiments, the pretreatment composition comprises a combination of AU24118 and ACBII in total amounts of 0.01–100 μM, 616452 in total amounts of 0.02–500 μM, and PROTAC CBP / P300 Degrader-1 in total amounts of 0.01–100 μM. In some embodiments, the pretreatment composition comprises a combination of AU15330, AU24118, and ACBII in total amounts of 0.01–100 μM, 616452 in total amounts of 0.02–500 μM, and PROTAC CBP / P300 Degrader-1 in total amounts of 0.01–100 μM.

[0039] In some embodiments, the pretreatment composition comprises 0.01–100 μM of a SWI / SNF complex inhibitor, 0.02–500 μM of a TGFβ receptor inhibitor, 0.01–100 μM of a histone acetyltransferase inhibitor, and 0.05–100 μM of a GSK3β inhibitor. In some embodiments, the pretreatment composition comprises 0.01–100 μM of AU15330, 0.02–500 μM of 616452, 0.01–100 μM of PROTAC CBP / P300 Degrader-1, and 0.05–100 μM of CHIR99021. In some embodiments, the pretreatment composition comprises 0.01–100 μM AU24118, 0.02–500 μM 616452, 0.01–100 μM PROTAC CBP / P300 Degrader-1, and 0.05–100 μM CHIR99021. In some embodiments, the pretreatment composition comprises a combination of AU15330 and AU24118 in total amounts of 0.01–100 μM, 616452 in total amounts of 0.02–500 μM, PROTAC CBP / P300 Degrader-1 in total amounts of 0.01–100 μM, and CHIR99021 in total amounts of 0.05–100 μM. In some embodiments, the pretreatment composition comprises a total amount of 0.01–100 μM of a combination of AU24118 and ACBII, 0.02–500 μM of 616452, 0.01–100 μM of PROTAC CBP / P300 Degrader-1, and 0.05–100 μM of CHIR99021. In some embodiments, the pretreatment composition comprises a total amount of 0.01–100 μM of AU15330, a combination of AU24118 and ACBII, 0.02–500 μM of 616452, 0.01–100 μM of PROTAC CBP / P300 Degrader-1, and 0.05–100 μM of CHIR99021.

[0040] In some embodiments, the pretreatment composition comprises 0.01–100 μM of a SWI / SNF complex inhibitor, 0.02–500 μM of a TGFβ receptor inhibitor, 0.01–100 μM of a histone acetyltransferase inhibitor, 0.05–100 μM of a GSK3β inhibitor, and 0.01–100 μM of a Menin-MLL interaction inhibitor. In some embodiments, the pretreatment composition comprises 0.01–100 μM of AU15330, 0.02–500 μM of 616452, 0.01–100 μM of PROTAC CBP / P300 Degrader-1, 0.05–100 μM of CHIR99021, and 0.01–100 μM of VTP50469. In some embodiments, the pretreatment composition comprises 0.01–100 μM AU24118, 0.02–500 μM 616452, 0.01–100 μM PROTAC CBP / P300 Degrader-1, 0.05–100 μM CHIR99021, and 0.01–100 μM VTP50469. In some embodiments, the pretreatment composition comprises 0.01–100 μM ACBII, 0.02–500 μM 616452, 0.01–100 μM PROTAC CBP / P300 Degrader-1, 0.05–100 μM CHIR99021, and 0.01–100 μM VTP50469. In some embodiments, the pretreatment composition comprises a combination of AU15330 and AU24118 in total amounts of 0.01–100 μM, 616452 in total amounts of 0.02–500 μM, PROTAC CBP / P300 Degrader-1 in total amounts of 0.01–100 μM, CHIR99021 in total amounts of 0.05–100 μM, and VTP50469 in total amounts of 0.01–100 μM. In some embodiments, the pretreatment composition comprises a combination of AU24118 and ACBII in total amounts of 0.01–100 μM, 616452 in total amounts of 0.02–500 μM, PROTAC CBP / P300 Degrader-1 in total amounts of 0.01–100 μM, CHIR99021 in total amounts of 0.01–100 μM, and VTP50469 in total amounts of 0.01–100 μM.In some embodiments, the pretreatment composition comprises a combination of AU15330, AU24118 and ACBII in total amounts of 0.01 to 100 μM, 616452 in total amounts of 0.02 to 500 μM, PROTAC CBP / P300 Degrader-1 in total amounts of 0.01 to 100 μM, CHIR99021 in total amounts of 0.01 to 100 μM, and VTP50469 in total amounts of 0.01 to 100 μM.

[0041] In some embodiments, the pretreatment composition comprises 0.01–100 μM of a SWI / SNF complex inhibitor, 0.02–500 μM of a TGFβ receptor inhibitor, 0.01–100 μM of a histone acetyltransferase inhibitor, 0.05–100 μM of a GSK3β inhibitor, 0.01–100 μM of a Menin-MLL interaction inhibitor, and 0.05–500 μM of a cAMP activator. In some embodiments, the pretreatment composition comprises 0.01–100 μM of AU15330, 0.02–500 μM of 616452, 0.01–100 μM of PROTAC CBP / P300 Degrader-1, 0.05–100 μM of CHIR99021, 0.01–100 μM of VTP50469, and 0.05–500 μM of trichomoniasisin. In some embodiments, the pretreatment composition comprises 0.01–100 μM AU24118, 0.02–500 μM 616452, 0.01–100 μM PROTAC CBP / P300 Degrader-1, 0.05–100 μM CHIR99021, 0.01–100 μM VTP50469, and 0.05–500 μM trichotillomansi. In some embodiments, the pretreatment composition comprises 0.01–100 μM ACBII, 0.02–500 μM 616452, 0.01–100 μM PROTAC CBP / P300 Degrader-1, 0.05–100 μM CHIR99021, 0.01–100 μM VTP50469, and 0.05–500 μM trichotillomansi. In some embodiments, the pretreatment composition comprises a combination of AU15330 and AU24118 in total amounts of 0.01–100 μM, 616452 in total amounts of 0.02–500 μM, PROTAC CBP / P300 Degrader-1 in total amounts of 0.01–100 μM, CHIR99021 in total amounts of 0.01–100 μM, VTP50469 in total amounts of 0.05–500 μM, and trichomoniasis. In some embodiments, the pretreatment composition comprises a total amount of 0.01–100 μM of a combination of AU15330 and ACBII, 0.02–500 μM of 616452, 0.01–100 μM of PROTAC CBP / P300 Degrader-1, 0.05–100 μM of CHIR99021, 0.01–100 μM of VTP50469, and 0.05–500 μM of trichomoniasis.In some embodiments, the pretreatment composition comprises a total amount of 0.01–100 μM of a combination of AU24118 and ACBII, 0.02–500 μM of 616452, 0.01–100 μM of PROTAC CBP / P300 Degrader-1, 0.05–100 μM of CHIR99021, 0.01–100 μM of VTP50469, and 0.05–500 μM of laryngin. In some embodiments, the pretreatment composition comprises a combination of AU15330, AU24118 and ACBII in total amounts of 0.01–100 μM, 616452 in total amounts of 0.02–500 μM, PROTAC CBP / P300 Degrader-1 in total amounts of 0.01–100 μM, CHIR99021 in total amounts of 0.01–100 μM, VTP50469 in total amounts of 0.05–500 μM, and trichomoniasis.

[0042] In some embodiments, the pretreatment composition comprises 0.01–100 μM of a SWI / SNF complex inhibitor, 0.02–500 μM of a TGFβ receptor inhibitor, 0.01–100 μM of a histone acetyltransferase inhibitor, 0.05–100 μM of a GSK3β inhibitor, 0.01–100 μM of a Menin-MLL interaction inhibitor, 0.05–500 μM of a cAMP activator, and 0.05–100 μM of a Dot1L inhibitor. In some embodiments, the pretreatment composition comprises 0.01–100 μM AU15330, 0.02–500 μM 616452, 0.01–100 μM PROTAC CBP / P300 Degrader-1, 0.05–100 μM CHIR99021, 0.01–100 μM VTP50469, 0.05–500 μM saliva extract, and 0.05–100 μM EPZ5676. In some embodiments, the pretreatment composition comprises 0.01–100 μM AU24118, 0.02–500 μM 616452, 0.01–100 μM PROTAC CBP / P300 Degrader-1, 0.05–100 μM CHIR99021, 0.01–100 μM VTP50469, 0.05–500 μM saliva extract, and 0.05–100 μM EPZ5676. In some embodiments, the pretreatment composition comprises 0.01–100 μM ACBII, 0.02–500 μM 616452, 0.01–100 μM PROTAC CBP / P300 Degrader-1, 0.05–100 μM CHIR99021, 0.01–100 μM VTP50469, 0.05–500 μM saliva extract, and 0.05–100 μM EPZ5676. In some embodiments, the pretreatment composition comprises a combination of AU15330 and AU24118 in total amounts of 0.01–100 μM, 616452 in total amounts of 0.02–500 μM, PROTAC CBP / P300 Degrader-1 in total amounts of 0.01–100 μM, CHIR99021 in total amounts of 0.01–100 μM, VTP50469 in total amounts of 0.05–500 μM, and EPZ5676 in total amounts of 0.05–100 μM.In some embodiments, the pretreatment composition comprises a total amount of 0.01–100 μM of a combination of AU15330 and ACBII, 0.02–500 μM of 616452, 0.01–100 μM of PROTAC CBP / P300 Degrader-1, 0.05–100 μM of CHIR99021, 0.01–100 μM of VTP50469, 0.05–500 μM of saliva extract, and 0.05–100 μM of EPZ5676. In some embodiments, the pretreatment composition comprises a combination of AU24118 and ACBII in total amounts of 0.01–100 μM, 616452 in total amounts of 0.02–500 μM, PROTAC CBP / P300 Degrader-1 in total amounts of 0.01–100 μM, CHIR99021 in total amounts of 0.01–100 μM, VTP50469 in total amounts of 0.05–500 μM, and EPZ5676 in total amounts of 0.05–100 μM. In some embodiments, the pretreatment composition comprises a combination of AU15330, AU24118 and ACBII in total amounts of 0.01–100 μM, 616452 in total amounts of 0.02–500 μM, PROTAC CBP / P300 Degrader-1 in total amounts of 0.01–100 μM, CHIR99021 in total amounts of 0.01–100 μM, VTP50469 in total amounts of 0.05–500 μM, and EPZ5676 in total amounts of 0.05–100 μM.

[0043] In some embodiments, the pretreatment composition further comprises PY-60 at a concentration of 0.01–100 μM. In some embodiments, the pretreatment composition comprises a YAP agonist at concentrations of 0.02–80 μM, 0.03–50 μM, 0.04–40 μM, 0.05–30 μM, 0.06–20 μM, or 0.07–10 μM. In some embodiments, the pretreatment composition comprises a YAP agonist at concentrations of 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, ... 1.9μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 11μM, 12μM, 13μM, 14μM, 15μM , 16μM, 17μM, 18μM, 19μM, 20μM, 30μM, 40μM, 50μM, 60μM, 70μM, 80μM, 90μM or 100μM.

[0044] In some embodiments, the pretreatment composition contains a p53 protein agonist at concentrations from 0.01 to 100 μM. In some embodiments, the pretreatment composition contains a p53 protein agonist at concentrations of 0.02–80 μM, 0.05–50 μM, or 0.1–10 μM. In some embodiments, the pretreatment composition contains a p53 protein agonist at concentrations of 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, or 1.8 μM. , 1.9μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 11μM, 12μM, 13μM, 14μM, 15μ M, 16μM, 17μM, 18μM, 19μM, 20μM, 30μM, 40μM, 50μM, 60μM, 70μM, 80μM, 90μM or 100μM.

[0045] In some embodiments, the pretreatment composition contains a DNA-dependent protein kinase (DNA-PK) inhibitor in the range of 0.01 to 100 μM. In some embodiments, the pretreatment composition contains a DNA-dependent protein kinase (DNA-PK) inhibitor in the range of 0.01–50 μM, 0.05–25 μM, or 0.1–10 μM. In some embodiments, the pretreatment composition contains a DNA-dependent protein kinase (DNA-PK) inhibitor in the amounts of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μM.

[0046] In some embodiments, the pretreatment composition contains an antioxidant in the range of 0.01 to 1000 μM. In some embodiments, the pretreatment composition contains an antioxidant in the range of 1–800 μM, 10–500 μM, or 100–300 μM. In some embodiments, the pretreatment composition contains a DNA-dependent protein kinase (DNA-PK) inhibitor in the range of 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μM.

[0047] In some embodiments, somatic cells are not exposed to animal-derived components during pretreatment. In some embodiments, somatic cells are not exposed to fetal bovine serum during pretreatment. In some embodiments, somatic cells are not exposed to serum-derived components, such as serum albumin, during pretreatment. In some embodiments, the pretreatment composition does not contain animal-derived components (such as fetal bovine serum or serum-derived components). In some embodiments, the pretreatment composition contains a serum substitute.

[0048] In some embodiments, somatic cells are not exposed to animal-derived components during the induced reprogramming process. In some embodiments, somatic cells are not exposed to fetal bovine serum during the induced reprogramming process. In some embodiments, somatic cells are not exposed to serum-derived components, such as serum albumin, during the induced reprogramming process. In some embodiments, the culture medium used for induced reprogramming does not contain animal-derived components (such as fetal bovine serum or serum-derived components). In some embodiments, the culture medium used for induced reprogramming contains a serum substitute.

[0049] In some implementations, the serum substitute may be selected from KnockOut. TM SR (KSR for short), N2, B27, PhysiologixTMXF SR, StemSure TM Serum Substitute Supplement or any combination thereof. In some embodiments, the pretreatment composition comprises 1% to 10% B27 and 1% to 10% KSR.

[0050] In some implementations, somatic cells may be exposed to animal-derived components, including but not limited to animal serum or serum-derived components such as fetal bovine serum or serum albumin, at any stage or throughout the pretreatment and / or induction of reprogramming.

[0051] A second aspect of this application is to provide a pretreatment composition disclosed herein for pretreating somatic cells to be reprogrammed, said composition comprising an inhibitor targeting the SWI / SNF complex.

[0052] The third aspect of this application is to provide the use of an inhibitor targeting the SWI / SNF complex for (1) increasing the responsiveness of somatic cells (e.g., blood cells) to a reprogramming agent; (2) increasing the number of LIN28A-positive epithelial-like cells obtained per unit number of somatic cells (e.g., blood cells); (3) shortening the time for somatic cells (e.g., blood cells) to transform into LIN28A-positive epithelial-like cells; (4) increasing the number of extraembryonic endoderm-like cells obtained per unit number of somatic cells (e.g., blood cells); (5) shortening the time for somatic cells (e.g., blood cells) to transform into extraembryonic endoderm-like cells; (6) increasing the number of chemically reprogrammed pluripotent stem cells (CiPS) obtained per unit number of somatic cells (e.g., blood cells); and / or (7) shortening the time for somatic cells (e.g., blood cells) to transform into chemically reprogrammed pluripotent stem cells (CiPS).

[0053] A fourth aspect of this application provides a method for reprogramming somatic cells, comprising: (A) a step of pretreating somatic cells, (B) a step of reprogramming the pretreated cells, and (C) a step of obtaining XEN-like cells or pluripotent stem cells; wherein the pretreating step is either using the pretreating method disclosed herein with the somatic cells or contacting the somatic cells with the pretreating composition disclosed herein. In a preferred embodiment, the reprogramming method disclosed herein does not include the step of introducing pluripotent transcription factors into somatic cells.

[0054] The fifth aspect of this application is to provide pluripotent stem cells obtained by the reprogramming method described herein.

[0055] The sixth aspect of this application is to provide the use of the pluripotent stem cells described herein for (1) the preparation of animal models, (2) the screening of drug candidates, (3) the preparation of drugs, (4) the acquisition of differentiated target cells, or (5) the treatment of diseases.

[0056] The seventh aspect of this application is to provide a method for reducing the gene mutation rate of chemically reprogrammed pluripotent stem cells (CiPS), the method comprising contacting somatic cells with a composition comprising a combination of a p53 protein agonist or a DNA-dependent protein kinase (DNA-PK) inhibitor and an antioxidant.

[0057] In some embodiments, the p53 protein agonist is selected from C16-ceramide, Kevetrin or its salts (such as Kevetrin hydrochloride), and SCH29074. In some embodiments, the concentration of the p53 protein agonist is 0.01–100 μM.

[0058] In some embodiments, the DNA-dependent protein kinase (DNA-PK) inhibitor is NU7441. In some embodiments, the concentration of the DNA-dependent protein kinase (DNA-PK) inhibitor is 0.01–100 μM.

[0059] In some embodiments, the antioxidant is glutathione (GSH). In some embodiments, the concentration of the antioxidant is 0.01–1000 μM. Attached Figure Description

[0060] Figures 1A and 1B show the changes in cell state and number after inducing blood cells under stage I conditions using a three-stage chemical reprogramming method.

[0061] Figure 2 shows the changes in umbilical cord blood mononuclear cells after treatment with exemplary pretreatment conditions and chemical reprogramming stage 1 conditions. Figure 2A shows the expression levels of EPC marker genes GATA1, KLF1, HBA, HBB, and HBG; Figure 2B shows the morphology of adherent cells (left image shows the cell state after pretreatment, right image shows the cell state after pretreatment + stage 1); Figure 2C shows the results of RT-PCR detection of Lin28A gene expression (left image) and LIN28A protein expression detected by immunofluorescence staining (right image); Figure 2D shows the changes in the expression level of the epithelial marker gene KRT8.

[0062] Figure 3 shows the results of a single subtraction experiment using the pretreatment reagent combination;

[0063] Figures 4A and 4B show the effects of adding and not adding the SWI / SNF complex inhibitor on cell proliferation and activation of LIN28A expression, respectively.

[0064] Figures 5A and 5C show the number of CiPS induced from CBMCs (Figure 5A) or PBMCs (Figure 5C) from different donor sources using exemplary preprocessing and reprogramming methods, respectively.

[0065] Figures 5B and 5D show the effects of changing the processing time on the number of CiPS. For example, 6+8+4+6 represents 6 days of pretreatment, 8 days of stage 1 induction, 4 days of stage 2 induction, and 6 days of stage 3 induction.

[0066] Figure 5E shows the results of induction of three PBMCs from different sources under fresh isolation and cryopreservation followed by thawing conditions.

[0067] Figure 5F shows the results of CiPS induction using fingertip blood from three different donors;

[0068] Figure 6A compares the efficiency of three PBMCs in generating CiPS using the exemplary pretreatment disclosed herein combined with chemical reprogramming and the efficiency of generating iPS cells using the classic OSKM four-factor method combined with P53 interference.

[0069] Figure 6B compares the gene mutation rates of CiPS generated by combining the exemplary pretreatment disclosed herein with chemical reprogramming with iPS generated using the classic OSKM four-factor method.

[0070] Figure 7 shows the identification results of CiPS cells obtained by the exemplary methods disclosed herein, including: bright-field morphology of CiPS cells (left) and immunofluorescence staining (right) (Figure 7A); protein expression levels of key stem cell genes (Figure 7B); RT-PCR detection of CiPS cell stem cell gene expression and RNA-seq analysis of whole-genome expression, showing low similarity between CBMC and H1, and high similarity between CiPS cells and H1; RNA-seq analysis of gene expression in some blood cells and pluripotent stem cells (Figure 7C); teratoma formation (Figure 7D); and karyotype analysis (Figure 7E).

[0071] Figure 8A shows a phase-contrast microscope bright-field photograph illustrating the adherence morphology and proliferation of cells in each group;

[0072] Figure 8B shows the statistical analysis results of the number of adherent cells in each group after 6 days of pretreatment;

[0073] Figure 8C shows the statistical analysis results of the number of pluripotent stem cell clones finally induced in each group;

[0074] Figure 8D shows fluorescence micrographs of the cell states of B27+KSR group 2 (+PY-60) at various stages. S1D3 and S1D6 are cells on day 3 and day 6 of pretreatment, respectively, S2D4 is cells on day 4 of culture in Stage I medium, and so on.

[0075] Figure 9A shows the results of flow cytometry analysis of sorted T cells using an allotype antibody control and an anti-CD3 antibody (CD3), respectively.

[0076] Figure 9B shows the results of immunofluorescence staining of the CiPS clones obtained by the method of Example 10;

[0077] Figure 10A shows the results of B cell sorting by flow cytometry using CD19 monoclonal antibody;

[0078] Figure 10B shows the results of immunofluorescence staining of the CiPS clones obtained by the method of Example 11;

[0079] Figures 11A and 11B show the measurement results of the gene mutation rate of iPS clones obtained by different methods, with each point in the figure representing a single clone. Detailed Implementation

[0080] definition

[0081] As used in the specification of this invention, the following words and phrases are generally considered to have the meanings set forth below, unless otherwise specified in the context in which they are used.

[0082] As used herein, the terms “comprising” or “including” mean that a composition contains the described components, but does not exclude other components. “Mainly composed of”, when used to define a composition, should exclude any other components that are obviously important to the composition. Therefore, compositions defined herein as mainly composed of these components do not exclude trace contamination from separation and purification methods and pharmaceutically acceptable carriers such as phosphate-buffered saline, preservatives, etc. “Composed of” should exclude other components.

[0083] Unless otherwise stated herein, the listing of numerical ranges herein is intended only as a shorthand method for individually referring to each individual value falling within that range, and each individual value is incorporated into the specification as if it were listed separately herein. For example, in this specification, if the concentration range is stated as 0.01 to 100 μM, it is intended to explicitly list values ​​such as 0.02 to 99 μM, 0.4 to 50 μM, or 1 to 20 μM. These are merely examples of specific intentions, and all possible combinations of values ​​between and including the listed minimum and maximum values ​​will be considered as explicitly stated in this disclosure. The use of the term “about” to describe a particular listed quantity or range of quantities means indicating that a value very close to the listed quantity is included in that quantity, such as a value that can or naturally be taken into account due to manufacturing tolerances, instrumental errors in the formation of the measurement, and human errors. For example, a numerical value described herein as “about” means covering ±10% of the indicated value. In some cases, “about” may mean ±20%, or ±5%, or ±1%. Values ​​or parameters described in this document in the manner of “about” include the value or parameter itself.

[0084] As used herein, the term "reprogramming" refers to a method of enhancing cell potential or dedifferentiating cells into a lower or higher differentiation state. For example, reprogrammed cells possess greater developmental plasticity and differentiation potential (i.e., the ability to differentiate into more cell types) compared to the same cells in their unreprogrammed state. In other words, reprogrammed cells are cells with a lower differentiation state or higher differentiation potential compared to the same cells in their unreprogrammed state. In some embodiments, reprogramming can be achieved by contacting differentiated cells with a reprogramming agent. "Chemical reprogramming" refers to situations where the contacted reprogramming agent includes or is limited to small molecule reagents. In preferred embodiments, chemical reprogramming does not require the exogenous transfer of polynucleotides encoding pluripotent transcription factors and / or recombinant proteins.

[0085] As used herein, reprogramming agents are agents that can enhance the developmental potential of cells, alone or in combination with other agents. Reprogramming agents include, but are not limited to, polynucleotides, peptides, and small molecules that can enhance cell developmental potential. Exemplary reprogramming reagents include, for example, the Zhongshan factor (OKSM), small molecule reagents (see, for example, Chinese patent applications CN201310292339.9, CN201580084912.4, CN201780090055.8, CN202280018680.2, CN202380038304.4, CN202411422045.8, PCT international applications PCT / CN2022 / 077048, PCT / CN2023 / 113082, PCT / CN2023 / 113080, etc., the full text of which is incorporated herein by reference), or combinations thereof (see, for example, Chinese patent application CN2012298969878, the full text of which is incorporated herein by reference).

[0086] It has been reported that small molecule combinations of VC6T (VPA, CHIR99021, 616452, Tranylcypromine) can allow reprogramming using a single pluripotent transcription factor, Oct4. Adding cAMP agonists (FSK, prostaglandin E2, Rolipram, etc.) and epigenetic regulators (DZNep, 5-azacytidine, sodium butyrate, RG108, etc.) to VC6T can eliminate the need for exogenous transgenes, achieving complete chemical reprogramming, but the reprogramming efficiency is low, generally not exceeding 1‰–1.5‰. In the optimization of reprogramming methods, it was found that adding different reprogramming reagent combinations in stages can significantly improve reprogramming efficiency, leading to the proposal of reprogramming methods such as two-stage, three-stage, and four-stage methods.

[0087] The following are some reagent combinations suitable for reprogramming; however, the reprogramming reagent combinations that can be used in conjunction with the pretreatment methods of this application are not limited to these. Methods for screening suitable combinations of reprogramming reagents can be found in, for example, Hou P et al., Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science. 2013 Aug 9; 341(6146):651-4; Guan, J., Wang, G., Wang, J. et al., Chemical reprogramming of human somatic cells to pluripotent stem cells. Nature 605, 325–331(2022); Liuyang S et al., Highly efficient and rapid generation of human pluripotent stem cells by chemical reprogramming. Cell Stem Cell. 2023 Apr 6; 30(4):450-459.e9; Wang, Y. et al., Arapid chemical reprogramming system to generate human pluripotent stem cells. Nat Chem Biol (2025), etc.

[0088] As used herein, the term "differentiation" is the process by which undifferentiated (i.e., non-specialized) or weakly specialized cells acquire the characteristics of specialized cells (e.g., blood cells). Differentiated cells are cells that have already reached a more specialized (i.e., specialized) position within a cell lineage. The term "specialization," when applied to the differentiation process, refers to a point in the differentiation pathway where a cell has progressed to a point where, under normal circumstances, it would continue to differentiate into a specific cell type or a subpopulation of that cell type, and where, under normal circumstances, it can no longer differentiate into different cell types or revert to a less differentiated cell type.

[0089] The term "differentiation potential" refers to a cell's ability to differentiate into other cell types. Cells with higher differentiation potential can differentiate into more cell types. Generally, cell differentiation potential is considered to decrease in the order of totipotency, pluripotency, specialization, unipotency, and somatic cells. Cell differentiation potential can be assessed using methods commonly known in the art, such as cell morphology, expression levels of pluripotency markers or differentiation markers, and the cell's ability to form three germ layers and chimeras.

[0090] As used herein, the term "pluripotency" refers to the ability of a cell to form all lineages of the body or embryo itself. For example, embryonic stem cells are a typical example of pluripotent stem cells, capable of forming every type of cell derived from the ectoderm, mesoderm, and endoderm. Pluripotency is a continuous developmental potential that encompasses everything from incompletely pluripotent or partially pluripotent cells (such as ectoderm stem cells or EpiSCs) that cannot produce a complete organism, to more primitive, multipotent cells (such as embryonic stem cells) that can produce a complete organism. Pluripotency can be determined in part by assessing the following pluripotency characteristics: (i) morphology of pluripotent stem cells; (ii) potential for unlimited self-renewal; (iii) expression of pluripotent stem cell markers, including but not limited to SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and / or CD50; (iv) ability to differentiate into the three somatic cell lineages of ectoderm, mesoderm and endoderm; (v) ability to form teratomas composed of the three somatic cell lineages; and (vi) ability to form embryoid bodies composed of cells from the three somatic cell lineages.

[0091] As used in this article, the term "pluripotent stem cell morphology" refers to the classic morphological characteristics of healthy and normal embryonic stem cells, including but not limited to small round shape, high nucleus-to-cytoplasm ratio, prominent nucleolus, and typical intercellular spacing.

[0092] As used herein, the term "blood cell" broadly refers to all nucleated cells present in the blood. As non-limiting examples of blood cells, hematopoietic stem cells, their various downstream blood progenitor cells or cells, and all types of nucleated blood cells that further differentiate into them can be cited. Therefore, "blood cells" in this article include pluripotent hematopoietic stem cells (blood embryonic cells), bone marrow progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells, lymphocyte progenitor cells, myeloid cells (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineage cells (T cells, B cells, NK cells). Furthermore, it also includes cells that can be isolated from the blood, such as vascular endothelial cells.

[0093] As used in this article, the term "hematopoietic stem cell" refers to cells that are specialized in the hematopoietic lineage but are capable of further differentiation into various blood progenitor cells or blood cells.

[0094] As used herein, the term "erythroid progenitor cells (EPC)" refers to CD71+ hematopoietic cells that produce mature red blood cells. Methods for isolating and purifying EPCs from blood are known in the art, see, for example, the methods described in Tan HK et al., Human finger-prick induced pluripotent stem cells facilitate the development of stem cell banking. Stem Cells Transl Med. 2014 May; 3(5): 586-98.

[0095] As used herein, the term "finger-prick blood" generally refers to a droplet of capillary blood formed on the skin surface using a device such as a lancet. Finger-prick blood can be collected using capillaries. Depending on the specific circumstances of the subject (e.g., a newborn), capillary blood can also be formed and collected from other body parts such as the earlobe, upper arm, abdomen, and sole of the foot using essentially the same method. The volume of finger-prick blood is typically no more than 100 microliters. In some embodiments, the blood may be a relatively large amount of peripheral blood collected, such as 10 to 100 milliliters. In some embodiments, the collected peripheral blood may also be a small or trace amount, such as 1 to 10 milliliters.

[0096] As used herein, the term "SWI / SNF complex" refers to a complex of multiple proteins with a molecular weight of approximately 2 M Daltons. In this specification, specific examples of SWI / SNF complexes primarily refer to complexes containing SMARCA2 (also known as Brm, GenBank accession number X72889) or SMARCA4 (also known as BRG1, GenBank accession number U29175) from humans and other mammals as DNA-dependent adenosine triphosphatase (ATPase) subunits. They may also include other homologues from humans and mammals such as yeast SWI / SNF or fruit fly Brahma.

[0097] SWI / SNF complexes are broadly classified into two types: those containing SMARCA2 and those containing SMARCA4. Other components of the SWI / SNF complex in mammals include, but are not limited to, INI1, BAF250, BAF60a, BAF60b, BAF60c, BAF170, BAF57, BAF155, BAF53, and β-actin (Vignali, M. et al., (2000) Mol. Cell. Biol. 20, 1899-1910). An example of the constituents of the SWI / SNF complex is shown in Table 1 below.

[0098] Table 1: Composition of the SWI / SNF complex

[0099] (Quoted from "Jingfu Medical University Chronicle 124(12), 825-838, 2015", with some modifications)

[0100] The SWI / SNF complex possesses chromatin remodeling function through DNA-dependent adenosine triphosphatase. Chromatin remodeling refers to the process of altering the chromatin structure of DNA. Chromatin structure refers to the structure with nucleosomes as the basic unit, formed by the folding and compression of these nucleosomes. Nucleosomes are mainly composed of DNA and histones, formed by wrapping 165 bp (approximately two cycles) of DNA around a histone octamer composed of four histone molecules (H2A, H2B, H3, and H4). This highly folded DNA hinders transcription, replication, repair, and recombination. The SWI / SNF complex has the function of using the energy from ATP hydrolysis to move and / or remove nucleosomes, thereby altering chromatin structure. In this process, it plays an important role in gene expression and / or regulation, together with histone modifying enzymes (histone acetyltransferases, deacetylases, phosphorylases, methyltransferases, etc.). For example, during the transcription of mRNA from DNA, the SWI / SNF complex alters the chromatin structure and exposes the target DNA sequence through the aforementioned chromatin reconstruction, thereby allowing transcriptional regulatory factors to access the DNA.

[0101] Methods for detecting the inhibitory activity targeting the SWI / SNF complex include: methods for detecting the binding activity of the SWI / SNF complex to the inhibitor; methods for detecting the transcriptional activity (upregulation or downregulation) of downstream transcriptional control factors of the SWI / SNF complex in the presence or absence of the inhibitor, but are not limited to these. Specific detection methods include, but are not limited to, immunoprecipitation, density gradient centrifugation, electrophoretic mobility shift analysis, ChIP (chromatin immunoprecipitation) assay, Farwestern blotting, yeast two-hybrid assay, proximity ligation assay (PLA), fluorescence resonance energy transfer (FRET), bioluminescent resonance energy transfer (BRET), assays based on reporter molecules (luciferase, green fluorescent protein, alkaline phosphatase, etc.), quantitative RT-PCR (qRT-PCR), microarrays, and Northern Hybridization, etc. Examples of inhibitors of the SWI / SNF complex can be found in, for example, PCT international application numbers PCT / JP2018 / 005527 and PCT / IB2019 / 056847, the full text of which is incorporated herein by reference. See also, for example, Xiao, L. et al., Targeting SWI / SNF ATPases in enhancer-addicted prostate cancer. Nature 601, 434–439 (2022); Kofink, C. et al., A selective and orally bioavailable VHL-recruiting PROTAC achieves SMARCA2 degradation in vivo. Nat Commun 13, 5969 (2022); He T, et al., Development of an orally bioavailable mSWI / SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer. Proc Natl Acad Sci US A. 2024 Apr 9;121(15):e2322563121.

[0102] In some embodiments, the concentration of the inhibitor targeting the SWI / SNF complex is 0.01–100 μM. In some embodiments, the concentration of the inhibitor targeting the SWI / SNF complex is 0.1–20 μM. In some embodiments, the concentration of the inhibitor targeting the SWI / SNF complex is 10–100 μM. In some embodiments, the concentration of the inhibitor targeting the SWI / SNF complex is 0.1–10 μM. In some embodiments, the concentration of the inhibitor targeting the SWI / SNF complex is 0.5–8 μM. In some embodiments, the concentration of the inhibitor targeting the SWI / SNF complex is 0.5–5 μM. In some embodiments, the concentration of the inhibitor targeting the SWI / SNF complex is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 μM.

[0103] As used herein, the term "epigenetic regulator" refers to an agent that alters the epigenetic state (e.g., methylation state, acetylation state) of cellular DNA upon or after contact with or application. In some embodiments, epigenetic regulators include DNA methyltransferase inhibitors, histone acetyltransferase inhibitors, histone deacetyltransferase inhibitors, histone methyltransferase inhibitors, histone demethyltransferase inhibitors, etc.

[0104] The term "small molecule" generally refers to molecules with a molecular weight of less than 2,000 Daltons, preferably less than 1,500 Daltons, and more preferably less than 1,000 Daltons. This includes, but is not limited to, organic compounds or organometallic compounds.

[0105] As used herein, the term “GSK3β inhibitor” refers to a small molecule that reduces the activity of glycogen synthase kinase 3β. Non-limiting examples of GSK3β inhibitors include CHIR99021, TD114-2, CHIR98014, GSK 3I inhibitor XV, BIO, or SB-216763, as well as those described in WO2011 / 149762, WO13 / 067362, Chambers et al., Nat Biotechnol. 2012 Jul 1, 30(7):715-20, Kriks et al., Nature. 2011 Nov 6, 480(7378):547-51, and Calder et al., J Neurosci. 2015 Aug 19, 35(33):11462-81, all of which are incorporated herein by reference in their entirety.

[0106] In a preferred embodiment, the GSK3β inhibitor may be selected from CHIR99021 or its active derivatives, or pharmaceutically acceptable salts thereof. “CHIR99021” refers to a small molecule inhibitor of the IUPAC name 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile, also known as “aminopyrimidine” or “3-[3-(2-carboxyethyl)-4-methylpyrrole-2-methylene]-2-indolone”.

[0107] In some embodiments, the concentration of the GSK3β inhibitor is less than 100 μM, less than 80 μM, less than 60 μM, less than 40 μM, or less than 20 μM. In some embodiments, the concentration of the GSK3β inhibitor is 0.05–100 μM. In some embodiments, the concentration of the GSK3β inhibitor is 0.1–50 μM. In some embodiments, the concentration of the GSK3β inhibitor is 1–20 μM. In some embodiments, the concentration of the GSK3β inhibitor is 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM.

[0108] As used herein, the term “TGF-β1 receptor inhibitor” may also be referred to as “TGFβ / Activin-Nodal inhibitor”. TGFβ receptors are single-pass serine / threonine kinase receptors, including type I, II, and III receptors, namely TGF-β receptor 1, TGF-β receptor 2, and TGF-β receptor 3. Non-limiting examples of TGF-β1 inhibitors include those disclosed in WO / 2010 / 096496, WO / 2011 / 149762, WO / 2013 / 067362, WO / 2014 / 176606, WO / 2015 / 077648, Chambers et al., Nat Biotechnol. 2009 Mar, 27(3):275-80, Kriks et al., Nature. 2011 Nov 6, 480(7378):547-51, and Chambers et al., Nat Biotechnol. 2012 Jul 1, 30(7):715-20, all of which are incorporated herein by reference in their entirety. In some embodiments, the TGF-β1 inhibitor may be selected from 616452, A8301, SB505124, GW 788388, and SB 525334. "616452" is a small molecule compound with the CAS number 446859-33-2, which is a selective inhibitor of TGF-β receptor I / activin-like kinase 5 (ALK5).

[0109] In some embodiments, the concentration of the TGFβ receptor inhibitor is less than 500 μM, less than 400 μM, less than 300 μM, less than 200 μM, less than 100 μM, or less than 50 μM. In some embodiments, the concentration of the TGFβ receptor inhibitor is 0.02–500 μM. In some embodiments, the concentration of the TGFβ receptor inhibitor is 0.1–200 μM. In some embodiments, the concentration of the TGFβ receptor inhibitor is 1–100 μM. In some embodiments, the concentration of the TGFβ receptor inhibitor is 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, or 100 μM.

[0110] As used herein, the term "cAMP activator" refers to a compound that exhibits adenylate cyclase activation and promotes intracellular cAMP synthesis. cAMP (cyclic adenosine monophosphate) participates in various intracellular signal transductions as a second messenger. In cells, cAMP is generated by cyclizing adenosine triphosphate (ATP) using adenylate cyclase. Non-limiting examples of cAMP activators include trichodin (CAS No.: 66575-29-9) and its derivatives (see, for example, Japanese Patent Application Publication No. 2002-348243). In some embodiments, the cAMP activator may be selected from Forskolin, IBMX, Rolipra, 8BrcAMP, Prostaglandin E2 (PGE2), NKH 477, bis(butyryl)-cAMP (DBcAMP), Sp-8-Br-cAMPs, or any combination thereof.

[0111] In some embodiments, the concentration of the cAMP activator is less than 500 μM, less than 400 μM, less than 300 μM, less than 200 μM, less than 100 μM, or less than 50 μM. In some embodiments, the concentration of the cAMP activator is 0.05–500 μM. In some embodiments, the concentration of the cAMP activator is 0.1–200 μM. In some embodiments, the concentration of the cAMP activator is 1–100 μM. In some embodiments, the concentration of the cAMP activator is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 4 9, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 97, 99 or 100μM.

[0112] The retinoic acid receptor (RAR) belongs to the nuclear receptor superfamily and its transcriptional activity is activated by retinoic acid as a ligand. As used herein, the term "retinoic acid receptor (RAR) agonist" refers to a compound that can promote RAR activity, such as TTNPB (arachidonic acid, CAS No.: 71441-28-6), retinoic acid, Ch55, AM580, or any combination thereof.

[0113] In some embodiments, the concentration of the RAR agonist is below 200 μM, below 100 μM, or below 50 μM. In some embodiments, the concentration of the RAR agonist is 0.02–200 μM. In some embodiments, the concentration of the RAR activator is 0.1–100 μM. In some embodiments, the concentration of the RAR activator is 0.5–40 μM. In some embodiments, the concentration of the RAR activator is 1–20 μM. In some embodiments, the concentration of the RAR activator is 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 μM.

[0114] As used herein, the term "G protein-coupled receptor smoothed agonist" is used interchangeably with "SMO agonist" and refers to a compound that can promote the activity of smoothed receptors. Smoothed receptors are members of the G protein-coupled receptor family and play a key role in the Hedgehog signaling pathway. Non-limiting examples of smoothed agonists include, for example, SAG, Purmorphamine, Hg-Ag1.5, or any combination thereof. The term "SAG" refers to the compound molecule with CAS numbers 912545-86-9 and 364590-63-6 (hydrochloride) and the name 3-chloro-N-[(1r,4r)-4-(methylamino)cyclohexyl]-N-[3-(pyridin-4-yl)benzylmethyl]benzo[b]thiophene-2-carboxamide.

[0115] In some embodiments, the concentration of the smoothed agonist is less than 50 μM, less than 40 μM, less than 30 μM, less than 20 μM, or less than 10 μM. In some embodiments, the concentration of the smoothed agonist is 0.01–50 μM. In some embodiments, the concentration of the smoothed activator is 0.05–40 μM. In some embodiments, the concentration of the smoothed activator is 0.1–30 μM. In some embodiments, the concentration of the smoothed activator is 0.2–20 μM. In some embodiments, the concentration of the smoothed activator is 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 μM.

[0116] As used herein, the term "Dot1L inhibitor" refers to a compound that can reduce or inhibit the expression or enzymatic activity of DOT1L. DOT1L, also known as DOT1-like (telomere silencing interferon 1-like) histone H3K79 methyltransferase (Saccharomyces cerevisiae), is a protein found in humans and other eukaryotes. DOT1L methylates histone H3 lysine 79 (H3K79), a conserved epigenetic marker in many eukaryotic epigenomes that gradually increases with aging. Non-limiting examples of Dot1L inhibitors include EPZ5676, SGC 0946, EPZ004777, or any combination thereof.

[0117] In some embodiments, the concentration of the Dot1L inhibitor is below 100 μM, below 80 μM, below 60 μM, below 40 μM, below 20 μM, or below 10 μM. In some embodiments, the concentration of the Dot1L inhibitor is 0.05–100 μM. In some embodiments, the concentration of the Dot1L inhibitor is 0.1–50 μM. In some embodiments, the concentration of the Dot1L inhibitor is 0.5–25 μM. In some implementations, the concentration of the Dot1L inhibitor is 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 μM.

[0118] As used herein, the term "Menin-MLL interaction inhibitor" refers to a compound that reduces or inhibits the interaction between menin and the transcriptional activator MLL. Menin is the product of the MEN1 tumor suppressor gene. Non-limiting examples of Menin-MLL interaction inhibitors include VTP50469, MI3454, WDR5-IN-4, or any combination thereof. See also, for example, U.S. Patent Nos. 9,212,180 and 9,216,993; U.S. Patent Application Publications Nos. 2011 / 0065690, 2014 / 0275070, 2016 / 0045504, and 2016 / 0046647; and International Publications Nos. WO 2017 / 192543 and WO 2018 / 183857.

[0119] In some embodiments, the concentration of the Menin-MLL interaction inhibitor is less than 100 μM, less than 80 μM, less than 60 μM, less than 40 μM, less than 20 μM, or less than 10 μM. In some embodiments, the concentration of the Menin-MLL interaction inhibitor is 0.01–100 μM. In some embodiments, the concentration of the Menin-MLL interaction inhibitor is 0.1–50 μM. In some embodiments, the concentration of the Menin-MLL interaction inhibitor is 0.5–25 μM. In some embodiments, the concentration of the Menin-MLL interaction inhibitor is 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM.

[0120] As used herein, non-limiting examples of SETD2 inhibitors may include SETD2-IN-1, EPZ-719, MMSET-IN-1, or any combination thereof. In some embodiments, the concentration of the SETD2 inhibitor is less than 100 μM, less than 80 μM, less than 60 μM, less than 40 μM, less than 20 μM, or less than 10 μM. In some embodiments, the concentration of the SETD2 inhibitor is from 0.01 to 100 μM. In some embodiments, the concentration of the SETD2 inhibitor is from 0.1 to 50 μM. In some embodiments, the concentration of the SETD2 inhibitor is from 0.5 to 25 μM. In some embodiments, the concentration of the SETD2 inhibitor is 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μM.

[0121] The terms “histone acetyltransferase inhibitor” or “HAT inhibitor” or “HATi” are used interchangeably herein and refer to substances capable of detectably reducing the expression or activity of histone acetyltransferases. Non-limiting examples of histone acetyltransferase inhibitors include A485, CBP / P300 IN 8, WM8014, GEN049, WM1119, CBP / P300 IN 12, SGC / CBP30, ICBP112, PROTAC CBP / P300 Degrader-1, or any combination thereof. See also, for example, the 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; the A485 derivative 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 the WM8014 derivative described in Baell, JB et al., Inhibitors of histone acetyltransferases KAT6A / B induce senescence and arrest tumour growth. Nature 560, 253–257 (2018).

[0122] In some embodiments, the concentration of the histone acetyltransferase inhibitor is less than 100 μM, less than 80 μM, less than 60 μM, less than 40 μM, less than 20 μM, or less than 10 μM. In some embodiments, the concentration of the histone acetyltransferase inhibitor is 0.01–100 μM. In some embodiments, the concentration of the histone acetyltransferase inhibitor is 0.1–50 μM. In some embodiments, the concentration of the histone acetyltransferase inhibitor is 0.5–25 μM. In some embodiments, the concentration of the histone acetyltransferase inhibitor is 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM.

[0123] As used herein, the term "PRC2 inhibitor" refers to a substance capable of detectably reducing the expression or enzymatic activity of the PRC2 complex, also known as polycomb inhibitory complex 2, a multiprotein complex that facilitates epigenetic silencing of target genes. The PRC2 complex consists of three core subunits: zeste gene enhancer homolog 2 (EZH2), embryonic ectoderm developmental protein (EED), and zeste 12 inhibitor (SUZ12). Two additional non-essential subunits, AEBP2 and RbAp48, contribute to the enzymatic activity of the PRC2 complex. The PRC2 complex catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3) to silence target genes. Non-limiting examples of PRC2 inhibitors include UNC6852, EPZ6438, and PROTAC EZH2 Degrader-1, or any combination thereof. See also, for example, compounds described in PCT International Application No. PCT / US2019 / 015677.

[0124] In some embodiments, the concentration of the PRC2 complex inhibitor is less than 100 μM, less than 80 μM, less than 60 μM, less than 40 μM, less than 20 μM, or less than 10 μM. In some embodiments, the concentration of the PRC2 complex inhibitor is 0.01–100 μM. In some embodiments, the concentration of the PRC2 complex inhibitor is 0.1–50 μM. In some embodiments, the concentration of the PRC2 complex inhibitor is 0.5–25 μM. In some embodiments, the concentration of the PRC2 complex inhibitor is 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM.

[0125] As used herein, the term "LATS kinase inhibitor" refers to a substance that can detectably reduce the expression or enzymatic activity of LATS kinases. A non-limiting example of a LATS kinase inhibitor is NIBR-LTSi. LATS kinases are serine / threonine protein kinases that directly phosphorylate the transcriptional regulator YAP, leading to YAP cytoplasmic retention and inactivation. A non-limiting example of a LATS kinase inhibitor is NIBR-LTSi.

[0126] In some embodiments, the concentration of the LATS kinase inhibitor is less than 100 μM, less than 80 μM, less than 60 μM, less than 40 μM, less than 20 μM, or less than 10 μM. In some embodiments, the concentration of the LATS kinase inhibitor is 0.01–100 μM. In some embodiments, the concentration of the LATS kinase inhibitor is 0.1–50 μM. In some embodiments, the concentration of the LATS kinase inhibitor is 0.5–25 μM. In some embodiments, the concentration of the LATS kinase inhibitor is 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM.

[0127] As used herein, the term "pretreatment" refers to the process of exposing blood cells to other reagents (such as the pretreatment compositions described herein) before administering reprogramming agents, thereby making the cells more suitable for reprogramming agent treatment. In some embodiments, the expression of blood-related genes is downregulated in pretreated cells compared to before pretreatment, including but not limited to KLF1, GATA1, HBA, HBB, HBG, etc. In some embodiments, the expression levels of blood-related genes after pretreatment are reduced 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 before treatment. In some embodiments, the expression levels of pluripotent transcription factors in pretreated cells are substantially the same as those in untreated blood cells. In some embodiments, the expression levels of pluripotent transcription factors are slightly increased after pretreatment compared to before treatment. In some embodiments, the expression levels of pretreated pluripotent transcription factors (e.g., Lin28A) increase by less than approximately 10%, less than approximately 9%, less than approximately 8%, less than approximately 7%, less than approximately 6%, less than approximately 5%, less than approximately 4%, less than approximately 3%, less than approximately 2%, or less than approximately 1% compared to before treatment. In some embodiments, the cell state after pretreatment shows typical changes under a microscope, such as a change from a suspension state to an adherent state, and the formation of proliferating cell aggregates.

[0128] The pretreatment methods described herein can increase the responsiveness of blood cells to reprogramming reagents compared to before treatment. The term "responsiveness to reprogramming reagents" refers to the ability of cells to transform into a lower differentiation state (dedifferentiation) after exposure to reprogramming reagents. Response ability can be determined by, for example, the number of dedifferentiated intermediate cells (such as LIN28A-positive epithelial-like cells and / or extraembryonic endoderm-like cells) obtained from a unit number of differentiated cells, or the number of ultimately dedifferentiated cells (such as pluripotent stem cells) obtained; or the time taken for differentiated cells to become intermediate cells or pluripotent stem cells; or the concentration or amount of reprogramming reagent required for transformation into intermediate cells or pluripotent stem cells.

[0129] In some embodiments, blood cells are separated from other blood components (such as plasma) before pretreatment. In some embodiments, blood cells undergo cell amplification before pretreatment. In some embodiments, blood cells can be pretreated without cell amplification.

[0130] As used herein, the term "LIN28A-positive epithelioid cells" refers to a class of cells in a critical intermediate state during reprogramming. Compared to somatic cells (such as blood cells) used as initial materials, LIN28A-positive epithelioid cells exhibit elevated expression levels of epithelial cell markers and decreased or even eliminated expression levels of somatic cell markers (such as blood cell-related markers). In some embodiments, the epithelial cell markers are selected from one or more of KRT8, KRT18, or KRT19. In some embodiments, the epithelial cell markers are KRT8, KRT18, and KRT19. In some embodiments, the blood cell markers are selected from one or more of GATA1, KLF1, HBA, HBB, and HBG. In some embodiments, the blood cell marker is GATA1. In some embodiments, the blood cell marker is KLF1. In some embodiments, the blood cell marker is HBA. In some embodiments, the blood cell marker is HBB. In some embodiments, the blood cell marker is HBG. In some implementations, LIN28A-positive epithelioid cells are essentially a pure cell population in terms of LIN28A marker expression.

[0131] 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%, and at least about 95% of the cells formed by the chemical reprogramming method disclosed herein are epithelial-like cells positive for the pluripotency marker Lin28A, and have elevated levels of expression of epithelial cell markers and decreased levels of expression of somatic cell markers compared to somatic cells used as starting cells.

[0132] As used herein, the term "substantially identical" means that two or more comparative objects have substantially the same quantity or level of the characteristics being compared, for example, taking into account the error range of the measurement methods used. The term "substantially pure" with respect to a particular cell population means a population of cells that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure relative to the cells constituting the total cell population.

[0133] The preferred embodiments for carrying out the present invention will now be described. It should be noted that the embodiments described below are examples illustrating representative embodiments of the present invention, but the present invention is not limited to these embodiments.

[0134] Two or more of the methods described below can be combined, and such combinations are also included in this invention.

[0135] Example

[0136] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0137] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are the average values ​​of three replicate experiments.

[0138] Experimental materials, reagents, instruments and experimental methods

[0139] Cell lines:

[0140] Cell line hCBMC-XU0625034 was obtained from SAILYBIO;

[0141] Cell lines hCBMC-1210A, hCBMC-1210B, hCBMC-1210D, hPBMC-LP200728, hPBMC-LP200729B, hPBMC-Z0684, hPBMC-YB3136, hPBMC-YB3286, hPBMC-YB3288, hPBMC-YB3290, hPBMC-YB3297, hPBMC-Z0004, hPBMC-Z0005, hPBMC-Y1904, hPBMC-YB3265, hPBMC-YB3271, hPBMC-YB3272, and hPBMC-Z0566 were all obtained from ORIBIOTECH.

[0142] The cell lines hPBMC-1220C and hPBMC-1220N were obtained from Peking University.

[0143] Culture medium:

[0144] EPC medium: Serum-free amplification medium II (SFEM II, STEMCELL) supplemented with 1% penicillin-streptomycin (Gibco), 100 ng / ml SCF (Stemmune LLC), 2 U / ml EPO (Stemmunome LLC), a combination of 100 ng / ml SCF and 2 U / ml EPO, 40 ng / ml IGF-1 (Stemmune LLC), 10 ng / ml IL-3 (Stemmune LLC), 250X cholesterol lipid concentrate (Gibco), and 1 μM dexamethasone (MCE).

[0145] Blood cell culture medium: 44% RPMI 1640 medium and 44% complete mTeSR™ Plus medium, supplemented with 10% FBS, 1% KSR, 1% penicillin-streptomycin, 50 μg / mL Vc2p and 1 ng / mL IL-1β;

[0146] HEK293T cell culture medium: Dulbecco's Modified Eagle medium (DMEM, Gibco) with 10% fetal bovine serum (FBS, Vistech) and 1% glutamine (Gibco) added.

[0147] Stage I induction medium: Knockout DMEM supplemented with 2% B27, 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 AKT kinase inhibitor, 0.2 μM JNKIN8 and 0.2 μM SETD2-IN-1;

[0148] Stage II induction medium: Knockout DMEM supplemented with 2% B27, 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).

[0149] Stage III induction medium: Knockout DMEM supplemented with 2% B27, 1% GlutaMAX, 1% NEAA, 1% penicillin-streptomycin, 5% KSR, 50 μg / mL Vc2p, 20 ng / mL recombinant human modulator β-1 (HRG), 1 μM CHIR99021, 10 μM Y-27632, 1 μM PD0325901 and 0.5 μM SB590885.

[0150] Laboratory animals:

[0151] NOD.Cg-Prkdc scid Il2rg tm1Vst / Vst mice were obtained from Beijing Vitalstar Biotechnology Company.

[0152] Antibody:

[0153] Chemical reagents:

[0154] Preparation of peripheral blood mononuclear cells (PBMCs)

[0155] Peripheral blood and fingertip blood were obtained with the informed written consent of the donors and the approval of the Peking University Ethics Committee (IRB 00001052-1970), from which hPBMCs were isolated. All studies followed the principles of the Declaration of Helsinki.

[0156] Dilute 20 ml of venous blood with twice the volume of PBS (Korning). Load 35 ml of the diluted blood onto 15 ml of human lymphocyte separation medium (Dakewe) in a 50 ml conical tube and centrifuge at 800 g for 20 minutes at room temperature. Harvest the white intermediate layer of cells as hPBMCs. Wash twice and lyse the red blood cells with ACK lysis buffer (Gibco). After counting, resuspend the hPBMCs in EPC medium or freeze for future use.

[0157] In a sterile environment, approximately 50–100 μl of fingertip capillary blood was collected from each donor. After treatment with ACK lysis buffer (to remove red blood cells), the fingertip blood cells were resuspended in 500 μl of EPC medium. The resuspended fingertip blood cells were then seeded into one well of a 24-well low-attachment cell culture plate (Corning).

[0158] Expanding cultured erythroid progenitor cells (EPC)

[0159] PBMCs, umbilical cord blood mononuclear cells (hCBMCs), or fingertip blood cells were collected at 5 x 10⁻⁶ ppm. 6 ~1x10 7 Cells were seeded at a density of 10 cells / well into 6-well low-attachment cell culture plates (Corning) and cultured in EPC medium for 7 days. Medium was added every two days during this period. On day 7, cells were collected by centrifugation, resuspended in fresh EPC medium, and the density was adjusted to 5 x 10⁶ cells / well. 6 ~1x10 7 Cells / ml. Continue culturing to day 12-14. After detecting the CD71 positivity rate by flow cytometry (CytoFLEX, BECKMAN COULTER), it was used for chemical reprogramming.

[0160] Example 1

[0161] This example describes a pretreatment process that promotes the reprogramming of blood cells into pluripotent stem cells.

[0162] Following the three-stage method described by Liuyang S et al. (Cell Stem Cell. 2023 Apr 6; 30(4):450-459.e9), chemical reprogramming was performed on expanded cultured hPBMCs or hCBMCs. The results showed that during Stage I culture, only a few cells transitioned from suspension to adherence. Until the end of Stage I, cells failed to proliferate effectively and instead began to senescent or undergo apoptosis (Figures 1A and 1B). Therefore, Stage II and III induction cultures were not continued.

[0163] In contrast, before implementing the above three-stage method, pretreating the expanded blood cells with a blood cell culture medium containing the following pretreatment reagent combination can induce the blood cells into chemically reprogrammed pluripotent stem cells (CiPS).

[0164] Pretreatment reagent combination: 5μM CHIR999021, 20μM 616452, 2μM TTNPB, 0.5μM SAG, 50μM laryngin, 4μM EPZ5676, 1μM VTP50469, 0.4μM SETD2-IN-1, 1μM PROTAC CBP / P300 Degrader-1, 1μM AU15330, 0.5μM AU24118, 2μM UNC6852, 2μM Compound 150d, 1μM BI-7237 and 2μM NIBR-LTSi.

[0165] Specifically, the expanded hPBMCs or hCBMCs were divided into groups of 8 x 10. 4 One cell / well was inoculated into a 48-well plate with the above-mentioned pretreated medium and cultured for 6 days under hypoxic (5% O2) conditions.

[0166] Then, following the three-stage reprogramming method described by Liuyang S et al. (2023), the pretreated cells were cultured sequentially in Stage I, II, and III media for 4–8 days, 2–8 days, and 4–10 days, respectively, to obtain pluripotent stem cell clones. Various markers and cell states of the initial somatic cells, pretreated cells, and cells that completed Stage 1 induction were detected, and the results are shown in Figures 2A to 2D.

[0167] As can be seen, compared with untreated cells, pretreated blood cells had their blood cell identity erased more effectively. Significant downregulation of EPC-related marker genes such as GATA1, KLF1, HBA, HBB, and HBG was observed (Figure 2A), and a higher proportion of cells transitioned from a suspension to an adherent state after pretreatment and reprogramming initiation (Figure 2B). RT-PCR and immunofluorescence staining results showed only a slight increase in LIN28A expression in pretreated cells (Figure 2C), while one or more of the epithelial cell markers KRT8, KRT18, or KRT19 were not observed to be expressed (Figure 2D). This indicates that while pretreatment effectively erased blood cell identity, it did not induce cells to the crucial intermediate state for reprogramming (i.e., LIN28A). + The cells undergo an effective transformation (single-layered epithelial-like cells) by adding a reprogramming reagent.

[0168] Example 2

[0169] This embodiment uses single-subtraction experiments to determine the influence of each reagent in the pretreatment reagent combination.

[0170] Chemical reprogramming was induced from expanded hPBMC cells according to the pretreatment + three-stage induction method in Example 1.

[0171] The pretreatment reagent combination used in the positive control group (NC) was simplified to: 5 μM CHIR999021, 20 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 50 μM laryngin, 4 μM EPZ5676, 1 μM VTP50469, 0.4 μM SETD2-IN-1, 1 μM PROTAC CBP / P300 Degrader-1, 1 μM AU15330, and 2 μM UNC6852. Experimental groups 1–11 had CHIR999021, 616452, TTNPB, SAG, laryngin, EPZ5676, VTP50469, SETD2-IN-1, PROTAC CBP / P300 Degrader-1, AU15330, or UNC6852 removed from the simplified pretreatment combination. The number of Oct4-positive pluripotent stem cell clones formed in each group was measured, and the results are shown in Figure 3.

[0172] Figure 3 shows that group 10 (i.e., the group without AU15330) ultimately failed to obtain CiPS clones. This indicates that for cells that are difficult to reprogram (such as blood-derived cells), the signaling of the SWI / SNF complex-related pathway likely plays a central role. Specifically inhibiting the signaling of this pathway can greatly improve the cell's responsiveness to chemical reprogramming agents, making it easier to reprogram cells that are otherwise difficult to reprogram.

[0173] Furthermore, group 9 (i.e., the group with the histone acetyltransferase inhibitor removed) also failed to obtain CiPS clones, highlighting the importance of histone acetyltransferase inhibitors. While other single-subtraction experimental groups were able to form CiPS clones, the number was less than that of the positive control group.

[0174] Example 3

[0175] This embodiment describes the study of the influence mechanism of SWI / SNF complex-related pathways on reprogramming.

[0176] Before inducing expanded cord blood mononuclear cells (CBMCs) using the same three-stage reprogramming method as in Example 1, pretreatment with a reagent combination (CHIR999021, 20 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 50 μM trichomoniasis, 4 μM EPZ5676, 1 μM VTP50469, 0.4 μM SETD2-IN-1, 1 μM PROTAC CBP / P300 Degrader-1, 1 μM AU15330) served as a positive control. The reagent combination (CHIR999021, 20 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 50 μM trichomoniasis, 4 μM EPZ5676, 1 μM VTP50469, 0.4 μM SETD2-IN-1, 1 μM PROTAC CBP / P300) served as a positive control. Cells were treated with Degrader-1 as the experimental group. The cell numbers (Fig. 4A) and the expression level of the pluripotency marker LIN28A (Fig. 4B) of the two groups were detected in the initial stage, pretreatment stage and reprogramming-induced Stage I stage.

[0177] As shown in Figure 4, although no significant differences were observed between the AU15330-added group and the unadded group during the pretreatment stage (e.g., in terms of cell proliferation and LIN28A expression activation), cells pretreated with AU15330 were significantly more responsive to the induction conditions of Stage 1, exhibiting a clear transformation into pluripotent cells, including rapid cell proliferation, LIN28A expression activation, and ultimately, LIN28A-positive monolayer epithelial-like cells. This suggests that SWI / SNF complex inhibitors are key pretreatment components for achieving hematologic reprogramming.

[0178] While not wanting to be limited by the following theories, the inventors speculate that the mechanism by which SWI / SNF inhibitors promote CiPS formation may be that the SWI / SNF complex plays an important role in maintaining the open state of chromosomes. Inhibiting the activity of key elements of SWI / SNF (such as catalytic subunits) will cause the originally open chromosome state in somatic cells to close, thereby downregulating or shutting down the expression of genes that maintain the differentiation state (such as blood cell-related genes), making them more susceptible to subsequent reprogramming.

[0179] In this regard, we verified that different SWI / SNF inhibitors all had the effect of promoting reprogramming. The expanded CBMC cells were pretreated for 6 days as follows, and then cultured in the same three-stage induction medium as in Example 1 for 12 days, 4 days and 6 days respectively for reprogramming.

[0180] Control group: 5μM CHIR999021, 20μM 616452, 2μM TTNPB, 0.5μM SAG, 50μM laryngin, 4μM EPZ5676, 1μM VTP50469, 0.4μM SETD2-IN-1, 1μM PROTAC CBP / P300Degrader-1

[0181] Group 1: Control group + 1 μM AU15330;

[0182] Group 2: Control group + 0.5 μM ACBI1;

[0183] Each condition is repeated six times.

[0184] Table 1:

[0185] Furthermore, we verified that the combination of SWI / SNF inhibitors accelerated reprogramming. The expanded CBMC cells were pretreated for 6 days, then cultured in the same three-stage induction medium as in Example 1 for 4, 4, and 6 days, respectively, for reprogramming.

[0186] Single SWI / SNF inhibitor group: 5μM CHIR999021, 20μM 616452, 2μM TTNPB, 0.5μM SAG, 50μM laryngin, 4μM EPZ5676, 1μM VTP50469, 0.4μM SETD2-IN-1, 1μM PROTAC CBP / P300 Degrader-1, 1μM AU15330;

[0187] The combination of SWI / SNF inhibitors included: 5 μM CHIR999021, 20 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 50 μM saliva extract, 4 μM EPZ5676, 1 μM VTP50469, 0.4 μM SETD2-IN-1, 1 μM PROTAC CBP / P300 Degrader-1, 1 μM AU15330, and 0.5 μM AU24118.

[0188] Repeat each set 3 times.

[0189] Table 2:

[0190] Furthermore, we verified that different concentrations of SWI / SNF inhibitors all had the effect of accelerating reprogramming. The expanded CBMC cells were pretreated for 8 days, then cultured in the same three-stage induction medium as in Example 1 for 3, 3, and 6 days, respectively, for reprogramming.

[0191] Control group: 5μM CHIR999021, 20μM 616452, 2μM TTNPB, 0.5μM SAG, 50μM laryngin, 4μM EPZ5676, 1μM VTP50469, 0.4μM SETD2-IN-1, 1μM PROTAC CBP / P300 Degrader-1

[0192] AU15330 0.5 group: control group + 0.5 μM AU15330;

[0193] AU15330 1.0 group: control group + 1.0 μM AU15330;

[0194] AU15330 2.0 group: control group + 2.0 μM AU15330.

[0195] Repeat each set 3 times.

[0196] Table 3:

[0197] Example 4

[0198] This embodiment provides an exemplary pretreatment recipe that can improve reprogramming success rate and reprogramming efficiency.

[0199] Expanded PBMCs or CBMCs were used. Cells in each group were pretreated for 6–10 days using the same method as in Example 1, followed by three-stage reprogramming. The pretreatment reagent combinations used in each group are shown below. The control group was identical to the pretreatment experimental group, except that the pretreatment step was omitted, and reprogramming induction was performed directly on PBMCs or CBMCs. The final number of CiPS clones was determined by OCT4 immunofluorescence staining.

[0200] Group 1: 1 μM AU15330;

[0201] Group 2: 1μM PROTAC CBP / P300 Degrader-1;

[0202] Group 3: 1 μM AU15330 and 1 μM PROTAC CBP / P300 Degrader-1;

[0203] Group 4: 1 μM PROTAC CBP / P300 Degrader-1 and 20 μM 616452;

[0204] Group 5: 1 μM AU15330 and 20 μM 616452; Group 6: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1 and 20 μM 616452;

[0205] Group 7: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1 and 1 μM VTP50469;

[0206] Group 8: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1 and 5 μM CHIR999021;

[0207] Group 9: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1 and 50 μM saliva-containing tincture;

[0208] Group 10: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1 and 4 μM EPZ5676;

[0209] Group 11: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 5 μM CHIR999021 and 50 μM hairy throat extract;

[0210] Group 12: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 4 μM EPZ5676 and 1 μM VTP50469;

[0211] Group 13: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 20 μM 616452 and 1 μM VTP50469;

[0212] Group 14: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 20 μM 616452 and 5 μM CHIR999021;

[0213] Group 15: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 20 μM 616452 and 50 μM salivain;

[0214] Group 16: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 20 μM 616452 and 4 μM EPZ5676;

[0215] Group 17: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 5 μM CHIR999021, 20 μM 616452 and 1 μM VTP50469;

[0216] Group 18: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 5 μM CHIR999021, 20 μM 616452 and 50 μM salivain;

[0217] Group 19: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 5 μM CHIR999021, 20 μM 616452 and 4 μM EPZ5676;

[0218] Group 20: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 50 μM laryngin, 20 μM 616452 and 1 μM VTP50469;

[0219] Group 21: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 4 μM EPZ5676, 20 μM 616452 and 1 μM VTP50469;

[0220] Group 22: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 5 μM CHIR999021, 20 μM 616452, 50 μM saliva extract and 4 μM EPZ5676;

[0221] Group 23: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 5 μM CHIR999021, 50 μM laryngin, 20 μM 616452 and 1 μM VTP50469;

[0222] Group 24: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 5 μM CHIR999021, 4 μM EPZ5676, 20 μM 616452 and 1 μM VTP50469;

[0223] Group 25: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 4 μM EPZ5676, 50 μM saliva extract, 20 μM 616452 and 1 μM VTP50469;

[0224] Group 26: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 5 μM CHIR999021, 4 μM EPZ5676, 50 μM saliva extract, 20 μM 616452 and 1 μM VTP50469;

[0225] Group 26: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 20 μM 616452 and 20 μM EPZ6438;

[0226] Group 27: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 20 μM 616452, 5 μM CHIR999021 and 20 μM EPZ6438;

[0227] Group 28: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 20 μM 616452, 5 μM CHIR999021, 50 μM saliva extract and 20 μM EPZ6438;

[0228] Group 29: 1 μM AU15330, 1 μM PROTAC CBP / P300 Degrader-1, 20 μM 616452, 5 μM CHIR999021, 50 μM saliva extract, 4 μM EPZ5676, 1 μM VTP50469 and 20 μM EPZ6438.

[0229] Example 5

[0230] This embodiment uses blood cells from different blood types to verify the effectiveness of the pretreatment method described in this application.

[0231] umbilical cord blood

[0232] hCBMCs, designated XU0625034, hCBMC-1210A, hCBMC-1210B, and hCBMC-1210D, were prepared using umbilical cord blood from four different donors. The four hCBMC samples were pretreated for 6 days using the same method as in Example 1, followed by three-stage induction reprogramming for 4, 4, and 6 days respectively, to obtain hCiPS clones. The clones were stained with OCT4 and counted; the results are shown in Figure 5A.

[0233] Using hCBMC-1210D as an example, the effects of different pretreatment and induction times on CiPS clone formation efficiency were tested. The results are shown in Figure 5B.

[0234] As can be seen, the method of this application is robust across umbilical cord blood cells from different donor sources. It can induce CiPS clones within as little as 12 days.

[0235] Peripheral blood:

[0236] Peripheral blood hPBMCs from 11 different donors were used. hPBMC samples were pretreated for 10 days using the same method as in Example 1, followed by three-stage induction reprogramming for 8, 4, and 6 days respectively to obtain hCiPS clones. The clones were stained with OCT4 and counted; the results are shown in Figure 5C.

[0237] Peripheral blood cells (hPBMCs) from two different donors (hPBMC-Z0684 and hPBMC-YB3271) were used as examples to test the effects of different pretreatment and induction times on CiPS colony formation efficiency. The results are shown in Figure 5D.

[0238] As can be seen, the method of this application exhibits extremely high reproducibility among peripheral blood cells from different donor sources.

[0239] It is worth noting that the two samples, hPBMC-LP200728 and hPBMC-LP200729B, had been cryopreserved for more than four years. Based on this, we tested the reprogramming efficiency of the method proposed in this application using fresh blood and cryopreserved blood from the same donor, and the results are shown in Figure 5E. The results indicate that although cryopreservation (especially ultra-long-term cryopreservation) is generally considered to have an adverse effect on cell viability, the method proposed in this application remains effective for blood samples that have been cryopreserved for a long time, which means convenience for industrial applications.

[0240] Finger tip blood:

[0241] Finger-prick blood samples were collected from three human individuals. Using the same method as in Example 1, the finger-prick blood samples were pretreated for 6 days, followed by three-stage induction reprogramming for 8, 6, and 6 days respectively, to obtain hCiPS clones. The clones were stained with OCT4 and counted; the results are shown in Figure 5F.

[0242] The results show that CiPS cell colonies can be successfully obtained from approximately 10–100 μL of fingertip blood using the method described in this application.

[0243] Example 6

[0244] This embodiment compares the effects of the chemical reprogramming method of this disclosure with those of transcription factor reprogramming.

[0245] 1. Chemical reprogramming has a significantly higher reprogramming efficiency than transcription factor reprogramming.

[0246] Equal amounts of peripheral blood mononuclear cells from the same individual were subjected to transcription factor reprogramming induction (OSKMP group) and the pretreatment + chemical reprogramming induction method of this application (Chemical group), respectively. After the same total induction treatment time, the number of OCT4 positive clones was measured. The results are shown in Figure 6A.

[0247] OSKMP Group: Based on the method described in Zhao Y et al., Two supporting factors greatly improve the efficiency of human iPSC generation. Cell Stem Cell. 2008 Nov 6; 3(5):475-9 (see the section on Generation of hiPS cells by OSKMP reprogramming);

[0248] Chemical group: Same as in Example 1.

[0249] Figure 6A shows that, when using peripheral blood cells as initial cells, the success rate and induction efficiency of transcription factor reprogramming to induce pluripotent stem cells are significantly lower than those of the method described in this application. Transcription factor reprogramming induced pluripotent stem cell colonies in only two samples. Furthermore, using the same amount of initial cells, the number of iPS colonies induced by the transcription factor reprogramming method was only about 1% to about 5% of that induced by the method described in this application.

[0250] 2. The gene mutation rate of iPS cells obtained by chemical reprogramming was significantly lower than that of iPS cells obtained by transcription factor reprogramming.

[0251] CiPS clones (Chemical group) were obtained from peripheral blood mononuclear cells or human adipose-derived mesenchymal cells (hADSC) using the methods disclosed herein. Whole-genome sequencing analysis was performed on the CiPS clones. The sequencing results were compared with the whole-genome sequencing results of transcription factor reprogrammed iPS clones (OSKM-iPS) obtained from public databases.

[0252] OSKM group: iPS cell whole genome sequencing data came from the HipSci project (https: / / www.hipsci.org / lines / # / assays / wgs);

[0253] Chemical group: peripheral blood mononuclear cells were reprogrammed using the same method as described in Example 1; human adipose-derived mesenchymal cells were reprogrammed using the same method as described in Example 10.

[0254] Whole-genome sequencing analysis (Rouhani et al., Substantial somatic genomic variation and selection for BCOR mutations in human induced pluripotent stem cells. Nature Genetics. 2022 Sep; 54(9):1406-16, see the sections HipSci hiPSC sequence alignment, QC and variant calling and Variant consequence annotation) was used to count the number of coding region mutations in iPS clones obtained from the OSKM group and the Chemical group, respectively. The results are shown in Figure 6B. In the figure, each point represents a CiPS cell. It can be seen that in the CiPS induced by the method disclosed herein, most clones have only 10 or fewer coding gene mutations, while most clones induced by the OSKM transcription factor reprogramming method have 20 or more mutations.

[0255] Example 7

[0256] This embodiment identifies and characterizes the hCiPS cell line established from blood cells.

[0257] Commercially available embryonic stem cell lines H1 and H9 were used as positive controls. Following the methods described in Guan, J. et al., Chemical reprogramming of human somatic cells to pluripotent stem cells. Nature 605, 325–331 (2022), the six CiPS cell lines prepared using the methods described in this application were subjected to microscopic morphological observation, immunofluorescence detection of pluripotency markers, RNA-seq-based transcriptome analysis, teratoma assay to detect trigerm layer formation capacity, and karyotype analysis. The results are shown in Figures 7A to 7E.

[0258] The results showed that the obtained cells exhibited typical human pluripotent stem cell morphology. Immunofluorescence analysis revealed high expression of core pluripotency markers OCT4, SOX2, and NANOG, as well as pluripotency-related surface markers (Fig. 7A). RT-qPCR also confirmed that the expression levels of pluripotency genes in these cells were similar to those in human embryonic cells (Fig. 7B). Transcriptome analysis further revealed that hCiPS cells derived from blood cells were highly similar to human embryonic cells (Fig. 7C). Teratoma formation assays showed that hCiPS cells differentiated into cell types of three germ layers, demonstrating their trigerm layer differentiation potential (Fig. 7D). Simultaneously, the cells maintained a normal karyotype (Fig. 7E).

[0259] Example 8

[0260] This embodiment tests the effect of implementing an exemplary pretreatment in a serum-free system.

[0261] Pretreatment was performed using the same method as described in Example 1, followed by the same three-stage reprogramming, except that 10% FBS in the blood cell culture medium was replaced as follows.

[0262] -FBS group: Contains no 10% FBS;

[0263] Group 1 (B27+KSR): Replace 10% FBS with 2% B27 and 5% KSR;

[0264] Group 2 (B27+KSR): Replace 10% FBS with 2% B27 and 5% KSR, and add PY-60 to a concentration of 5 μM.

[0265] The results are shown in Figures 8A to 8D.

[0266] As shown in Figure 8, the serum-free system containing the pretreatment composition can also successfully induce CiPS clones.

[0267] Example 9

[0268] This embodiment verifies the effectiveness of the pretreatment method disclosed herein for T cells.

[0269] PBMC cells were sorted by flow cytometry using an anti-CD3 antibody (FITC anti-human CD3 antibody, purchased from BioLegend, catalog number 300406) to obtain CD3-positive T cells for subsequent pretreatment and reprogramming experiments.

[0270] After pretreating T cells for 10 days using the same method as described in Group B27+KSR 2 of Example 8, the same three-stage reprogramming method was performed. CiPS clones were obtained, and the results of their immunofluorescence staining are shown in Figure 9.

[0271] As shown in Figure 9, the pluripotency core markers OCT4, SOX2, and NANOG were highly expressed in the obtained clones (Figure 9B).

[0272] Example 10

[0273] This embodiment investigates the success rate and reprogramming efficiency of pretreatment in promoting the generation of CiPS cells from other tissue cells.

[0274] Following the method described in Guan, J. et al., Chemical reprogramming of human somatic cells to pluripotent stem cells. Nature 605, 325–331 (2022), human adipose-derived mesenchymal cell line (hADSC) and human skin-derived fibroblast line (hASF) were obtained. Urine-derived cells, primarily endothelial cells, were prepared 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). No existing techniques have reported the ability to prepare pluripotent stem cells from urine endothelial cells through chemically induced reprogramming.

[0275] As a control group, the four-stage induction method described in Guan, J. et al., Chemical reprogramming of human somatic cells to pluripotent stem cells. Nature 605, 325–331 (2022) and the two-stage induction method described in Liuyang, SJ et al., (2023). Highly efficient and rapid generation of human pluripotent stem cells by chemical reprogramming. Cell Stem Cell 30, 450 were used to chemically reprogram urinary endothelial cells, hADSC cells, and hASF cells, respectively.

[0276] The reprogramming method for the pretreatment experimental group was the same as that for the control group, but before reprogramming induction, the experimental group was pretreated for 4 to 6 days with 5 μM CHIR999021, 20 μM 616452, 2 μM TTNPB, 0.5 μM SAG, 50 μM laryngin, 4 μM EPZ5676, 1 μM VTP50469, 0.4 μM SETD2-IN-1, 1 μM PROTAC CBP / P300 Degrader-1, 1 μM MAU15330, 0.5 μM AU24118, 2 μM UNC6852, 2 μM Compound 150d, 1 μM BI-7237 and 2 μM NIBR-LTSi.

[0277] Each group was repeated 3–4 times. The final number of CiPS clones was determined by OCT4 immunofluorescence staining. The results are shown in Table 4.

[0278] Table 4:

[0279] Example 11

[0280] This embodiment verifies the effectiveness of the pretreatment method disclosed herein for B cells.

[0281] Peripheral blood was sorted by flow cytometry using an anti-CD19 antibody (FITC anti-human CD19 Antibody, BioLegend, 302206) to obtain CD19-positive B cells for subsequent pretreatment and reprogramming experiments.

[0282] After pretreating T cells for 10 days using the same method as described in Group 2 of Example 8 (B27+KSR), the same three-stage reprogramming method was performed. CiPS clones were obtained, and the results of their immunofluorescence staining are shown in Figure 10.

[0283] Figure 10B shows that iPS monoclonal antibodies expressing the pluripotency core marker OCT4 were successfully induced from B cells using the method disclosed herein.

[0284] Example 12

[0285] This embodiment provides a method for reducing the gene mutation rate of chemically reprogrammed pluripotent stem cells (CiPS).

[0286] CiPS clones were obtained from peripheral blood mononuclear cells or human adipose-derived mesenchymal cells (hADSCs) using the methods disclosed herein. The reprogramming method for peripheral blood mononuclear cells is the method of Example 1, and the reprogramming method for human adipose-derived mesenchymal cells is the method of Example 10. The only difference is the additional addition of a p53 protein agonist, or a combination of a DNA-dependent protein kinase (DNA-PK) inhibitor and an antioxidant, during the pretreatment and Stage I phases.

[0287] As an example, add the p53 protein agonist C16-ceramide to a concentration of 5 μM, or Kevetrin to a concentration of 5 μM, or SCH29074 to a concentration of 0.1 μM.

[0288] As an example, the DNA-dependent protein kinase (DNA-PK) inhibitor NU7441 was added to a concentration of 0.25 μM, and the antioxidant glutathione was added to a concentration of 200 μM.

[0289] Whole-genome sequencing analysis was performed on the obtained CiPS clones using the method described in Example 6. The number of coding region mutations in cells belonging to the same CiPS clone was counted, and the results are shown in Figure 11. In the figure, each dot represents a CiPS cell line.

[0290] The results show that the pretreatment method of this application can be combined with various reprogramming methods to universally improve the efficiency of chemical reprogramming of various tissues and cells.

Claims

1. A method of pre-conditioning somatic cells, the method comprising incubating somatic cells with a pre-conditioning composition, obtaining a cell population with improved responsiveness to reprogramming reagents, wherein the pre-conditioning composition comprising an inhibitor targeting SWI / SNF complex.

2. The method of claim 1, wherein, the composition further comprising a small molecule targeting a major developmental related pathway and / or an epigenetic modulator; optionally, the small molecule targeting a major developmental related pathway is selected from a glycogen synthase kinase (GSK) 3 beta inhibitor, a TGF beta receptor inhibitor, a cAMP activator, or any combination thereof, preferably from a glycogen synthase kinase (GSK) 3 beta inhibitor, a TGF-beta receptor inhibitor, a cAMP activator, a retinoic acid receptor (RAR) agonist, a G-protein coupled receptor Smoothened agonist, or any combination thereof, more preferably comprising a glycogen synthase kinase (GSK) 3 beta inhibitor, a TGF-beta the epigenetic modulator is selected from a Dot1L inhibitor, a Menin-MLL interaction inhibitor, a histone acetyltransferase inhibitor, or any combination thereof, preferably from a Dot1L inhibitor, a Menin-MLL interaction inhibitor, a SETD2 inhibitor, a histone acetyltransferase inhibitor, or any combination thereof, more preferably comprising a Dot1L inhibitor, a Menin-MLL interaction inhibitor, a SETD2 3. The method of claim 1 or 2, wherein, the composition further comprising a compound capable of further enhancing the responsiveness of somatic cells to reprogramming reagents, the compound being selected from a PRC2 complex inhibitor, a LATS kinase inhibitor, a YAP activator, or any combination thereof; optionally, the composition further comprises a compound reducing the rate of genetic mutation, the compound reducing the rate of genetic mutation being selected from a p53 protein agonist, or a combination of a DNA-dependent protein kinase (DNA-PK) inhibitor and an antioxidant.

4. The method of any one of claims 1 to 3, wherein, the somatic cells are selected from blood cells, fibroblasts, epithelial cells, endothelial cells, preferably blood cells; optionally, the blood cells are obtained from peripheral blood, umbilical cord blood, cryopreserved blood samples, fingertip blood, venous blood, arterial blood, capillary blood, whole blood, fractionated blood, or bone marrow; optionally, the blood cells are nucleated blood cells; optionally, the blood cells are selected from hematopoietic stem cells, colony forming unit cells (CFU-C), bone marrow stem cells, erythroid progenitor cells, lymphoid stem cells, lymphocytes, plasma cells, monocytes, granulocytes, neutrophils, eosinophils, basophils, T cells, B cells, and any combination thereof, preferably erythroid progenitor cells.

5. The method of claim 1, wherein, the incubation is performed for no more than 20 days.

6. The method of claim 1, wherein, the inhibitor targeting SWI / SNF complex specifically inhibits the activity of SMARCA2 and / or SMARCA4; optionally, the inhibitor is selected from a small molecule compound, an antisense nucleic acid, a gene editor, or a directed degrader. Optionally, the small molecule compound that specifically inhibits SWI / SNF complex is selected from SGC-SMARCA-BRDVIII, PFI-3, FHT-2344, or any combination thereof; Optionally, the antisense nucleic acid that specifically inhibits SWI / SNF complex is selected from siRNA, shRNA, or aptamer; Optionally, the gene editor that specifically inhibits SWI / SNF complex is selected from CRISPR or TALEN; Optionally, the directed degrader that specifically inhibits SWI / SNF complex is selected from PROTAC, lysosome-targeting chimera (LYTAC), or autophagy-targeting chimera (AUTAC); Optionally, the PROTAC that specifically inhibits SWI / SNF complex is selected from AU-15330, AU-24118, or ACBI1.

7. The method of any one of claims 2 to 4, wherein, The glycogen synthase kinase (GSK) 3beta inhibitor is selected from CHIR99021, TD114-2, CHIR98014, GSK 3I inhibitor XV, BIO, SB-216763, or any combination thereof, preferably CHIR99021; Optionally, the TGF beta receptor inhibitor is selected from 616452, A8301, SB505124, GW 788388, SB 525334, or any combination thereof, preferably 616452; Optionally, the cAMP activator is selected from Forskolin, IBMX, Rolipra, 8BrcAMP, Prostaglandin E2 (PGE2), NKH 477, dibutyryl-cAMP (DBcAMP), Sp-8-Br-cAMPs, or any combination thereof, preferably Forskolin; 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 SETD2 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 selected from A485, CBP / P300 IN 8, WM8014, GEN049, WM1119, CBP / P300 IN 12, SGC / CBP30, ICBP112, PROTAC CBP / P300 Degrader-1, or any combination thereof, preferably PROTAC CBP / P300 Degrader-1; Optionally, the PRC2 complex inhibitor is selected from UNC6852, EPZ6438, and PROTAC EZH2 Degrader-1, or any combination thereof, preferably UNC6852; Optionally, the LATS kinase inhibitor is selected from NIBR-LTSi; Optionally, the YAP activator is selected from PY-60; Optionally, the p53 protein agonist is selected from C16-ceramide, Kevetrin or its salts (such as Kevetrin hydrochloride), SCH29074; Optionally, the DNA-dependent protein kinase (DNA-PK) inhibitor is NU7441; Optionally, the antioxidant is selected from glutathione (GSH).

8. The method of any one of claims 1 to 7, wherein, The concentration of the inhibitor targeting SWI / SNF complex is 0.01-100 μΜ; Optionally, the concentration of the glycogen synthase kinase (GSK) 3β inhibitor is 0.05-100 μΜ; Optionally, the concentration of the TGFβ receptor inhibitor is 0.02-500 μΜ; Optionally, the concentration of the cAMP activator is 0.05-500 μΜ; Optionally, the concentration of the retinoic acid receptor (RAR) agonist is 0.02-200 μΜ; Optionally, the concentration of the G-protein coupled receptor Smoothened agonist is 0.01-50 μΜ; Optionally, the concentration of the Dot1L inhibitor is 0.05-100 μΜ; Optionally, the concentration of the Menin-MLL interaction inhibitor is 0.01-100 μΜ; Optionally, the concentration of the SETD2 inhibitor is 0.01-100 μΜ; Optionally, the concentration of the histone acetyltransferase inhibitor is 0.01-100 μΜ; Optionally, the concentration of the PRC2 complex inhibitor is 0.01-100 μΜ; Optionally, the concentration of the LATS kinase inhibitor is 0.01-100 μΜ; Optionally, the concentration of the p53 protein agonist is 0.01-100 μΜ; Optionally, the concentration of the DNA-dependent protein kinase (DNA-PK) inhibitor is 0.01-100 μΜ; Optionally, the concentration of the antioxidant is 0.01-1000 μΜ.

9. The method of claim 1, wherein, The somatic cells are not contacted with an animal-derived component during incubation; Optionally, the animal-derived component is selected from fetal bovine serum.

10. The method of claim 1, wherein, The somatic cells are contacted with an animal-derived component during incubation.

11. A pre-treatment composition for somatic cells comprising an inhibitor targeting SWI / SNF complex. Optionally, the composition further comprises a small molecule targeting a major developmentally related pathway and / or an epigenetic modulator; Optionally, the small molecule targeting a major developmentally related pathway is selected from a glycogen synthase kinase (GSK) 3 beta inhibitor, a TGF beta receptor inhibitor, a cAMP activator, or any combination thereof; Optionally, the small molecule targeting a major developmentally related pathway is selected from a glycosen synthase kinase (GSK) 3 beta inhibitor, a TGF beta receptor inhibitor, cAMP activator, a retinoic acid receptor (RAR) agonist, a G protein-coupled receptor Smoothened agonist, or any combination thereof; Optionally, the small molecule targeting a major developmentally related pathway comprises a glycogen synthase kinase (GSK) 3 beta inhibitor, a TGF beta inhibitor, a cAMP activator, a retinoic acid receptor (RAR) agonist, and a G protein-coupled receptor Smoothened agonist; Optionally, the epigenetic modulator is selected from a Dot1L inhibitor, a Menin-MLL interaction inhibitor, a histone acetyltransferase inhibitor, or any combination thereof; Optionally, the epigenetic modulator is selected from a Dot1L inhibitor, Menin-MLL interaction inhibitor, a SETD2 inhibitor, a histone acetyltransferase inhibitor, or any combination thereof; Optionally, epigenetic modulator comprises a Dot1L inhibitor, a Menin-MLL interaction inhibitor, a SETD2 inhibitor, and a histone acetyltransferase inhibitor; Optionally, the composition further comprises a compound capable of further enhancing the responsiveness of blood cells to reprogramming reagents; Optionally, the compound capable of further enhancing the responsiveness of blood cells to reprogramming reagents is selected from a PRC2 complex inhibitor, a LATS kinase inhibitor, or any combination thereof; Optionally, the composition further comprises a compound that reduces the rate of genetic mutation; Optionally, the compound that reduces the rate of genetic mutation is selected from a p53 protein agonist, or a combination of a DNA-dependent protein kinase (DNA-PK) inhibitor and an antioxidant; Optionally, the somatic cell is selected from a blood cell, a fibroblast, an epithelial cell, an endothelial cell, preferably a blood cell; Optionally, the blood cell is obtained from peripheral blood, umbilical cord blood, cryopreserved blood sample, fingertip blood, venous blood, arterial blood, capillary blood, whole blood, fractionated blood, or bone marrow; Optionally, the blood cell is a nucleated blood cell; Optionally, the blood cell is selected from a hematopoietic stem cell, a colony forming unit cell (CFU-C), a bone marrow stem cell, an erythroid progenitor cell, a lymphoid stem cell, a lymphocyte, a plasma cell, a monocyte, a granulocyte, a neutrophil, an eosinophil, a basophil, a T cell, a B cell, and any combination thereof, preferably an erythroid progenitor cell; Optionally, the inhibitor targeting the SWI / SNF complex specifically inhibits the activity of SMARCA2 and / or SMARCA4; Optionally, the inhibitor is selected from a small molecule compound, an antisense nucleic acid, a gene editor, or a directed degrader. Optionally, the small molecule compound that specifically inhibits SWI / SNF complex is selected from SGC-SMARCA-BRDVIII, PFI-3, FHT-2344, or any combination thereof; Optionally, the antisense nucleic acid that specifically inhibits SWI / SNF complex is selected from siRNA, shRNA, or aptamer; Optionally, the gene editor that specifically inhibits SWI / SNF complex is selected from CRISPR or TALEN; Optionally, the directed degrader that specifically inhibits SWI / SNF complex is selected from PROTAC, lysosome-targeting chimera (LYTAC), or autophagy-targeting chimera (AUTAC); Optionally, the PROTAC is selected from AU-15330, AU-24118, or ACBI1; Optionally, the glycogen synthase kinase (GSK) 3beta inhibitor is selected from CHIR99021, TD114-2, CHIR98014, GSK 3I inhibitor XV, BIO, SB-216763, or any combination thereof, preferably CHIR99021; Optionally, the TGF beta receptor inhibitor is selected from 616452, A8301, SB505124, GW 788388, SB 525334, or any combination thereof, preferably 616452; Optionally, the cAMP activator is selected from Forskolin, IBMX, Rolipra, 8BrcAMP, Prostaglandin E2 (PGE2), NKH 477, dibutyryl-cAMP (DBcAMP), Sp-8-Br-cAMPs, or any combination thereof, preferably Forskolin; 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 SETD2 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 selected from A485, CBP / P300 IN 8, WM8014, GEN049, WM1119, CBP / P300 IN 12, SGC / CBP30, ICBP112, PROTAC CBP / P300 Degrader-1, or any combination thereof, preferably PROTAC CBP / P300 Degrader-1; Optionally, the PRC2 complex inhibitor is selected from UNC6852, EPZ6438, and PROTAC EZH2 Degrader-1, or any combination thereof, preferably UNC6852; Optionally, the LATS kinase inhibitor is selected from NIBR-LTSi; Optionally, the YAP activator is selected from PY-60; Optionally, the p53 protein agonist is selected from C16-ceramide, Kevetrin or its salts (such as Kevetrin hydrochloride), SCH29074; Optionally, the DNA-dependent protein kinase (DNA-PK) inhibitor is NU7441; Optionally, the antioxidant is glutathione (GSH); Optionally, the concentration of the inhibitor targeting SWI / SNF complex is 0.01-100 μΜ; Optionally, the concentration of the glycogen synthase kinase (GSK) 3β inhibitor is 0.05-100 μΜ; Optionally, the concentration of the TGFβ receptor inhibitor is 0.02-500 μΜ; Optionally, the concentration of the cAMP activator is 0.05-500 μΜ; Optionally, the concentration of the retinoic acid receptor (RAR) agonist is 0.02-200 μΜ; Optionally, the concentration of the G-protein coupled receptor Smoothened agonist is 0.01-50 μΜ; Optionally, the concentration of the Dot1L inhibitor is 0.05-100 μΜ; Optionally, the concentration of the Menin-MLL interaction inhibitor is 0.01-100 μΜ; Optionally, the concentration of the SETD2 inhibitor is 0.01-100 μΜ; Optionally, the concentration of the histone acetyltransferase inhibitor is 0.01-100 μΜ; Optionally, the concentration of the PRC2 complex inhibitor is 0.01-100 μΜ; Optionally, the concentration of the LATS kinase inhibitor is 0.01-100 μΜ; Optionally, the concentration of the YAP activator is 0.01-100 μΜ; Optionally, the concentration of the p53 protein agonist is 0.01-100 μΜ; Optionally, the concentration of the DNA-dependent protein kinase (DNA-PK) inhibitor is 0.01-100 μΜ; Optionally, the concentration of the antioxidant is 0.01-1000 μΜ.

12. Use of an inhibitor targeting SWI / SNF complex for preparing pluripotent stem cells from somatic cells; Preferably, for preparing pluripotent stem cells from blood cells by chemical reprogramming. Preferably, for preparing pluripotent stem cells from blood cells by chemical reprogramming.

13. Use according to claim 12, wherein, The inhibitor is used for pre-treating somatic cells to be chemically reprogrammed.

14. The use according to claim 12, which is selected from any one of (1) increasing the responsiveness of somatic cells to reprogramming reagents; (2) increasing the number of LIN28A-positive epithelial-like cells obtained from a unit number of somatic cells; (3) shortening the time for conversion from somatic cells to LIN28A-positive epithelial-like cells; (4) increasing the number of extraembryonic endoderm-like cells obtained from a unit number of somatic cells; (5) shortening the time for conversion from somatic cells to extraembryonic endoderm-like cells; (6) increasing the number of chemically reprogrammed pluripotent stem cells (CiPS) obtained from a unit number of somatic cells; or (7) shortening the time for conversion from somatic cells to chemically reprogrammed pluripotent stem cells (CiPS), or any combination of (1) to (7).

15. A method for reprogramming somatic cells, the method comprising: A. pre-treating somatic cells; B. reprogramming the pre-treated cells obtained in step A, and C. obtaining XEN-like cells or pluripotent stem cells; wherein the pre-treatment is subjecting somatic cells to the method according to any one of claims 1 to 10, or contacting with the composition according to claim 11; Preferably, the method does not comprise a step of introducing pluripotent transcription factors into the cells.

16. A method for reducing the genetic mutation rate of chemically reprogrammed pluripotent stem cells (CiPS), the method comprising contacting somatic cells with a composition comprising a combination selected from a p53 protein agonist, or a combination of a DNA-dependent protein kinase (DNA-PK) inhibitor and an antioxidant; Optionally, the p53 protein agonist is selected from C16-ceramide, Kevetrin or a salt thereof (such as Kevetrin hydrochloride), SCH29074; Optionally, the DNA-dependent protein kinase (DNA-PK) inhibitor is NU7441; Optionally, the antioxidant is glutathione (GSH); Optionally, the concentration of the p53 protein agonist is 0.01-100 μΜ; Optionally, the concentration of the DNA-dependent protein kinase (DNA-PK) inhibitor is 0.01-100 μΜ; Optionally, the concentration of the antioxidant is 0.01-1000 μΜ.

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