Induced full potential stem cells, methods of making and methods of use

Abstract

Factors that induce in vitro totipotent stem cells that are functionally and molecularly similar to totipotent embryonic cells are provided. Provided are a cell culture medium composition for inducing cell totipotency of an isolated cell in vitro and an isolated chemically induced full potential stem cell (ciTPSC) obtained using the same. The composition includes a totipotent chemical inducer (CDT) from each of the following groups in an amount effective to induce untreated cells to be fully potential stem cells (TPS): (1) an HDAC inhibitor, (2) a Dot1L inhibitor, (3) a RAR [gamma] agonist, and (4) optionally, a GSK inhibitor. The ciTPSC may be used, for example, in cell therapy and tissue engineering.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to PCT application number PCT / CN2022 / 080149, filed on March 10, 2022, entitled “Induced fully potent stem cells, preparation methods and use methods”, which is incorporated herein by reference. Technical Field

[0002] The present invention generally relates to compositions and methods for inducing in vitro totipotent stem cells that are functionally and molecularly similar to cells from totipotent embryos. Background Art

[0003] During development, early blastomeres are totipotent cells that have the potential to produce a complete individual (including both embryonic and extraembryonic components) at the single-cell level. The full potential of early blastomeres is gradually restricted during the blastocyst stage, and they differentiate into ectoderm, trophectoderm, and primitive endoderm. The blastocyst can differentiate into different self-renewing stem cells, including pluripotent stem cells, trophoblast stem cells, and extraembryonic endoderm cells, which retain the developmental potential of ectoderm, trophectoderm, and primitive endoderm, respectively. Importantly, the developmental potential of these stem cell types is lineage restricted in that they have difficulty crossing embryonic and extraembryonic lineage boundaries, particularly in vivo (Papaioannou, VE, McBurney, MW, Gardner, RL & Evans, MJ Fate of teratocarcinoma cells injected into early mouse embryos. Nature 258, 70-73, doi:10.1038 / 258070a0 (1975); Gardner, RL & Rossant, J. Investigation of the fate of 4-5 day post-coitum mouse inner cell mass cellsby blastocyst injection. J Embryol Exp Morphol 52, 141-152 (1979); Beddington, RS & Robertson, EJ An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. Development 105, 733-737 (1989)). Compared with these blastocyst-derived stem cell types, totipotent embryonic cells have the greatest developmental potential, and there is great scientific interest in capturing such stem cells from totipotent embryos in vitro, whose unrestricted potential will have broad applications in stem cell biology and regenerative medicine.

[0004] Previous studies have shown that there is a rare 2-cell embryonic (2C)-like subpopulation in mouse embryonic stem (ES) cell culture that expresses 2-cell blastomere molecular markers and has embryonic and extraembryonic developmental potential (Macfarlan, TS et al. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487, 57-63, doi:10.1038 / nature11244 (2012); Rodriguez-Terrones, D. et al. A molecular roadmap for the emergence of early-embryonic-like cells in culture, Nature genetics 50, 106-119, doi:10.1038 / s41588-017-0016-5 (2018)). The main limitation of 2C-like cells is that they are only a small population in mouse embryonic stem cell culture and are in a transient intermediate state that cannot be stabilized in vitro. In recent years, our team and others have shown that small molecule combinations can expand the developmental potential of pluripotent stem cells to extraembryonic lineages. Importantly, extended pluripotent stem cells (EPS) can be induced into blastocyst-like structures (WO2017025061A1). Despite their expanded developmental potential, these cells remain transcriptomically distinct from 2-cell embryos. In addition, their extraembryonic differentiation potential remains limited compared to totipotent embryos (Posfai, E. et al. Evaluating totipotency using criteria of increasing stringency. Nature cell biology 23, 49-60, doi:10.1038 / s41556-020-00609-2(2021)). Therefore, capturing and maintaining truly totipotent stem cells in vitro remains a major challenge.

[0005] Therefore, one object of the present invention is to provide totipotent stem cells in vitro.

[0006] An object of the present invention is also to provide a composition for inducing pluripotency of isolated pluripotent stem cells in vitro.

[0007] An object of the present invention is also to provide a method for inducing totipotency of isolated pluripotent stem cells in vitro.

[0008] Another object of the present invention is to provide methods for using cells having totipotency. Summary of the invention

[0009] Identified can be used for inducing the mixture of multiple factors of isolated cell totipotency in vitro, it is referred to as totipotency chemical inducer (CDT) herein.CDT can be directly set up totipotent-like stem cells in vitro by embryo (such as 2-cell embryo) and by pluripotent cells (such as expanded pluripotent stem cells), called pletipotent stem cells (TPS).The induced totipotent-like stem cells can be maintained stably in vitro for a long time, with molecular characteristics similar to 2-cell to 4-cell blastomeres.In addition, they can produce embryonic pedigrees and extraembryonic pedigrees in vivo at the single cell level, and form blastocyst-like structures in vitro.

[0010] CDT includes: (1) HDAC inhibitor, (2) Dot1L inhibitor, (3) RARγ agonist, (4) optionally GSK inhibitor. In a preferred embodiment, the HDAC inhibitor is an Hdac1 inhibitor and / or an Hdac2 inhibitor, for example, selected from VPA ("V"), TSA, MS275, Scriptaid, SAHA, LBH589, FK228, PXD101, sodium butyrate, LAQ824, CUDC-101, JNJ-26481585, SB939, PCI-24781, ACY-1215, CI994, CUDC-907, RGFP109, Resminostat, curcumin, divalproex sodium, 4-PBA, GSK3117391, CAY1 0433, CM-675 and MGCD0103; Dot1L inhibitor is selected from, for example, EPZ004777 ("E"), EPZ5676 and SGC0946; RARγ agonist is selected from, for example, CD1530 ("D"), AM580, ch55, parovarotene, CD3254, CD5789, CD437, TTNPB, AGN205327 and RA; GSK inhibitor is a GSK3 inhibitor, for example, selected from CHIR99021 ("C"), AZD2858, LY2090314, BIO, CHIR 98014, SB415286, AZD1080, BRD3731, A 1070722, BIP-135 and SB216763.

[0011] Also provided is a method for inducing the pluripotency of isolated pluripotent stem cells by culturing donor cells or cells derived from donor cells using CDT disclosed herein. The cells to be induced (i.e., donor cells) are contacted with CDT for a time effective to induce pluripotency. In a preferred embodiment, the cells are cultured in a medium containing CDT for a period of time between 15-150 days, for example 15, 30, 45, 60, 75, 90, 105, 120, 135, 150 days.

[0012] Also disclosed is an isolated chemically induced pluripotent stem cell (ciTPSC). The isolated chemically induced pluripotent stem cell disclosed herein is identified as a pluripotent stem cell based on the following characteristics: i) optionally after 10 or more passages, compared to untreated corresponding cells, there is a cell expression of any one or more, preferably all pluripotent marker genes selected from Zscan4, Zfp352, Tcstv1, Tcstv3, MERVL, Dux, Dub1a, Eif1al6, Eif1al9, Gm4340 and Tdpoz4, and / or ii) compared to untreated corresponding cells, any one or more, preferably all pluripotent marker genes selected from Oct4, Nanog and Sox2 are downregulated in cell expression. Upregulation or downregulation is determined by comparing the levels of factors measured in the corresponding cells from which the ciTPSC is obtained. The ciTPSC disclosed herein can be distinguished from mouse embryonic cells, ESCs or expanded pluripotent stem cells at least by the method used to produce them, that is, by their origin. While mouse embryonic cells or ESCs are naturally occurring cells, on the other hand, when ciTPSCs are obtained by treating donor cells with the combination of factors described herein, ciTPSCs are not naturally occurring.

[0013] ciTPSCs can be cultured or induced to differentiate into desired cell types. ciTPSCs and their progeny can be used in many applications, including but not limited to cell therapy and tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 . Identification of chemical cocktails that induce totipotent stem-like cells in vitro.

[0015] a. Representative images showing the induction of TPS cells by EPS cells under 4S conditions. Scale bar, 100 μm. Similar images were obtained in at least 5 independent experiments.

[0016] b. Karyotype analysis of TPS cells after long-term culture under 4S conditions. Similar images were obtained in 3 independent experiments.

[0017] c. Q-PCR analysis of the expression of totipotency and pluripotency marker genes in TPS cells transformed from EPS cells (EPS-TPS #1) and TPS cells derived from 2-cell embryos (2C-TPS #1 and #2). N = 3 biological replicates. Similar results were obtained in at least 2 independent experiments.

[0018] d. Heat map showing the relative expression of representative totipotency and pluripotency genes in TPS and EPS cells.

[0019] e. Representative immunofluorescence analysis of the expression of trophectoderm (CDX2) and ectoderm (OCT4) markers in TPS-induced products in chimeric blastocysts. These images show different focal planes of the same embryo. Scale bar, 20 μm. Td, tdTomato. Similar images were obtained in at least 2 independent experiments.

[0020] f. UMAP plot showing the major clusters of TPS-derived teratomas. The red dashed lines indicate the extraembryonic cell lineages.

[0021] g. Heat map showing the mean expression of representative marker genes for each cluster from TPS-derived teratomas.

[0022] h. Representative images showing the mosaicism of TPS-induced products in the genital ridges of E13.5 embryos. For each image, the upper sample is from a chimeric conceptus and the lower sample is from a non-chimeric conceptus. Scale bar, 2 mm. Similar images were obtained in at least 2 independent experiments.

[0023] i. Representative morphology of 4S-treated outgrowths derived from 2-cell embryos expressing tdTomato. Scale bar, 100 μm. BF, bright field. Td, tdTomato. Similar images were obtained in at least 2 independent experiments.

[0024] j. Representative immunofluorescence analysis of ZSCAN4 expression in TPS cells derived from 2-cell embryos expressing tdTomato. Scale bar, 50 μm. Td, tdTomato. Similar images were obtained in at least 2 independent experiments.

[0025] Figure 2 . TPS cells have the same transcriptomic and epigenetic characteristics as 2-cell blastomeres.

[0026] a. UMAP diagram showing the expression of representative pluripotency marker genes in TPS, ES and EPS cells at the single cell level. Different cell types are represented by different colors.

[0027] b. Latent semantic indexing (LSI) analysis comparing the developmental progression of epiblast from fertilized eggs to postimplantation (E5.25-E7.5) with TPS cells, ES cells, EPS cells, TBLCs, and spontaneous 2C-like cells. 2C-TPS-sub, TPS 2C subpopulation. TBLC-sup, subpopulation from TBLCs. Detailed methods for constructing mouse embryonic developmental trajectories are provided in the methods section. Sequencing data for different stem cell types were obtained from GSE33923 (Macfarlan et al., 2012), GSE168728 (Shen et al., 2021), GSE74155 (Chen et al., 2016), and GSE145609 (Posfai et al., 2021).

[0028] c. Quadratic programming-based deconvolution analysis showing transcriptomic similarities between 2-cell embryos and different in vitro cell types. Embryonic cells at different developmental stages were included as controls. EPS-TPS-sub, TPS 2C subpopulation from EPS-TPS cells. 2C-TPS-sub, TPS 2C subpopulation from 2C-TPS cells. TBLC-sup, subpopulation from TBLC. 2CLC, spontaneous 2C-like cells.

[0029] d. ssGSEA analysis showing the similarities between embryonic cells and in vitro cell types at different developmental stages. EPS-TPS-sub, TPS 2C subpopulation derived from EPS-TPS cells. 2C-TPS-sub, TPS 2C subpopulation derived from 2C-TPS cells. TBLC-sup, subpopulation derived from TBLC. 2CLC, spontaneous 2C-like cells.

[0030] e. Comparison of regulon activities between TPS 2C subpopulation (TPS-sub) and EPS cells. Regulons enriched in TPS 2C subpopulation and EPS cells are highlighted.

[0031] f. Analysis of regulon activity at different developmental stages. The activities of the top 20 regulons up-regulated and down-regulated in the TPS 2C subpopulation (TPS-sub) were quantified.

[0032] g. Identification of open and closed peaks enriched in TPS cells. ES cells and EPS cells were used for comparison. Orange (high) indicates open peaks enriched in TPS, and blue (low) indicates closed peaks enriched in TPS.

[0033] h. Average signal intensity of TPS-enriched open and closed peaks in TPS, ES, EPS cells, and 2-cell embryos. Orange represents TPS-enriched open peaks, and blue represents TPS-enriched closed peaks.

[0034] i. Comparison of global DNA CpG methylation levels in TPS, ES, EPS cells, and embryonic cells at different developmental stages (2-cell, 4-cell, E6.5 and E7.5 embryos, and E3.5 inner cell mass (ICM)).

[0035] Figure 3 . At the single-cell level, TPS cells can give rise to both embryonic and extraembryonic lineages in vivo.

[0036] a. Representative images showing the mosaicism of a single TPS-derived cell in the embryonic and extraembryonic regions of an E7.5 conceptus. Scale bar, 200 μm. BF, bright field. Td, tdTomato. Similar images were obtained in at least 3 independent experiments.

[0037] b. Representative immunofluorescence analysis of the expression of embryonic (OCT4) markers and extraembryonic ectoderm (EOMES) markers in chimeric cells of origin from a single TPS cell in an E7.5 conceptus. Stained sections were from the same analyzed conceptus. Scale bar, 200 μm. Similar images were obtained in at least 3 independent experiments.

[0038] c. Figure 3 b Magnified image showing the contribution of a single TPS-induced product to the extraembryonic ectodermal region. Scale bar, 50 μm.

[0039] d. Representative images showing the contribution of a single TPS-derived cell in E10.5 embryos, yolk sac, and placenta. Scale bar, 500 μm. Td, tdTomato. BF, bright field. Similar images were obtained in at least 3 independent experiments.

[0040] e. Representative immunofluorescence analysis of E10.5 chimeric placentas generated by injection of a single TPS cell (tdTomato marker). 2-cell embryo-derived TPS cells were used. The left image shows the original image and the right image shows the enlarged image. Anti-TD, anti-tdTomato antibodies. Scale bars: left, 2 mm; right, 20 μm. Similar images were obtained in at least 3 independent experiments.

[0041] f. UMAP plot showing 19 major clusters of E17.5 chimeric placenta. Pink dashed lines indicate extraembryonic cell lineages.

[0042] g. UMAP plot showing the expression of tdTomato in E17.5 chimeric placenta. Td+, tdTomato positive cells. Td-, tdTomato negative cells.

[0043] h, Heat map showing the average expression of specific marker genes for each cluster from 3f.

[0044] Figure 4 . Inducing TPS cells to form blastocyst-like structures in vitro.

[0045] a. Representative morphology of induced blastoids from TPS cells. Scale bar, 100 μm. BF, bright field. Td, tdTomato. Similar images were obtained in at least 3 independent experiments.

[0046] b. Representative immunofluorescence analysis of trophectoderm (CDX2) and ectoderm (OCT4) marker genes in TPS-blastocysts. The lower panel shows an enlarged image of the upper panel. Arrows indicate the expression of OCT4 and CDX2. Scale bars, upper panel, 100 μm; lower panel, 50 μm. Similar images were obtained in at least 3 independent experiments.

[0047] c. UMAP plot showing the expression of representative marker genes for ectoderm, trophectoderm, primitive endoderm, and totipotency in TPS-blastocyst-like cells at the single-cell level. EPI-like, ectoderm-like cells. PE-like, primitive endoderm-like cells. TE-like, trophectoderm-like cells.

[0048] d. Heat map showing the expression of 262 representative marker genes of ectoderm, primitive endoderm, and trophectoderm in TPS blastocyst-like cells. EPI-like, ectoderm-like cells. PE-like, primitive endoderm-like cells. TE-like, trophectoderm-like cells.

[0049] e. Comparison of LSI analysis of cells from E4.5 blastocysts and TPS-blastocysts. The left panel shows the cell lineage assignment diagram, and the right panel shows the blastocyst and TPS-blastocyst diagram. EPI, epiblast. PE, primitive endoderm. TE, trophectoderm.

[0050] f. Comparison of LSI analysis of embryonic cells from different developmental stages with blastocyst-like cells from different studies. Single-cell RNA sequencing data from blastocyst-like cells from this study, Rivron et al., 2018, and Sozen et al., 2019 were analyzed. ICM, E3.5 inner cell mass. E4.5, E4.5 epiblast. E5.5, E5.5 epiblast. E6-7, E6-7 epiblast. PE, primitive endoderm from E4.5 to E7.5. TEpre, trophectoderm of E3.5 to E4.5 blastocyst. TEpost, extraembryonic ectoderm from E5.25 to E6.5. EPI-like, epiblast-like cells. PE-like, primitive endoderm-like cells. TE-like, trophectoderm-like cells.

[0051] g. ssGSEA analysis showing similarities between embryonic cells from different developmental stages and blastocyst-like cells from different studies. Single-cell RNA sequencing data of blastocyst-like cells from this study, Rivron et al., 2018, and Sozen et al., 2019 were analyzed. SB-EP represents cells from EPS / TS-blastocyst-like. SB-Lif represents cells from ES / TS-blastocyst-like.

[0052] h. Deconvolution analysis based on quadratic programming shows transcriptomic similarities between preimplantation trophectoderm and trophectoderm-like cells from different studies.

[0053] i. Representative images of decidua formation in mouse uterus 4 days after TPS-derived blastocyst transfer at 2.5 dpc are shown. Scale bars, left, 5 mm; right, 500 μm. Similar images were obtained in at least 3 independent experiments.

[0054] j. Representative immunofluorescence analysis of PTGS2 expression in TPS-derived decidua. Scale bar on the left, 500 μm. Scale bar on the right, 100 μm. Similar images were obtained in at least 2 independent experiments.

[0055] Figure 5 . Exploration of the mechanism of induction and maintenance of totipotency in TPS cells.

[0056] a. Western blot analysis showing histone H3, H4 acetylation and H3K79me2 levels in EPS and TPS cells. Similar results were obtained in at least 2 independent experiments.

[0057] b. Q-PCR analysis of the expression levels of canonical RAR downstream target genes in EPS and TPS cells. N = 3 biological replicates.

[0058] c. Q-PCR analysis of the expression levels of representative pluripotency marker genes on day 3 after treatment with different small molecule combinations. Under 4S conditions, EPZ004777, VPA, CD1530, and CHIR 99021 were replaced by small molecules targeting DOT1L, HDAC, RA signaling, and GSK3β, respectively. N = 2 technical replicates. Similar results were obtained in at least 2 independent experiments. EPS, EPS cells. Basal, EPS cells cultured in basal medium under 4S conditions. EPZ rep, VPA rep, CD1530 rep, and CHIR rep represent small molecules targeting DOT1L, HDAC, RA, and GSK3β, respectively.

[0059] d. Q-PCR analysis of Hdac1 and Hdac2 expression in EPS cells after Hdac1 / 2 knockdown by shRNA. shHdac, Hdac1 / 2 shRNA. N = 2 biological replicates.

[0060] e. Q-PCR analysis of the expression levels of representative pluripotency marker genes at day 3 after Hdac1 / 2 knockdown in EPS cells during TPS cell induction. EPS, EPS cells, 4S-V, 4S condition without VPA. 4S-V+shHdac, Hdac1 / 2 knockdown instead of VPA. N = 2 biological replicates.

[0061] f. Q-PCR analysis of Dot1l expression in EPS cells after shRNA knockdown of Dot1l. shDot1l, Dot1l shRNA. N = 3 biological replicates.

[0062] g. Q-PCR analysis of the expression levels of representative pluripotency marker genes at day 3 after Dot1l knockdown in EPS cells during TPS cell induction. EPS, EPS cells, 4S-E, 4S condition without EPZ004777. 4S-V+shDot, Dot1l knockdown instead of EPZ004777. N = 3 biological replicates.

[0063] h. Q-PCR analysis of the effect of inhibition of RAR signaling on the induction (left) and maintenance (right) of pluripotency in TPS cells. Expression of representative pluripotency marker genes analyzed. RARγi, LY2955303. RARα / βi, LE135. RXRi, UVI3003. N = 3 biological replicates.

[0064] i. Q-PCR analysis of the effect of the 4S chemical mixture on maintenance of totipotency in early preimplantation embryos. Small molecules were treated for 2 days starting from the 2-cell embryo stage. DMSO was used as a negative control. 2C, 2-cell embryos. N = 2 biological replicates.

[0065] j. Q-PCR analysis of the effect of Dux knockdown on the maintenance of pluripotency in TPS cells. Expression of representative pluripotency marker genes was analyzed. Dux KD, dux knockdown. N = 2 biological replicates.

[0066] k. Q-PCR analysis of the effect of Dux knockdown on the induction of pluripotency from EPS cells. Expression of representative pluripotency marker genes was analyzed. Dux KD, dux knockdown. N = 2 biological replicates.

[0067] 1. Q-PCR analysis of the effect of p53 knockdown on the induction of pluripotency from EPS cells. Expression of representative pluripotency marker genes was analyzed. p53 KD, p53 knockdown. N = 2 biological replicates.

[0068] Figure 6. Identification of small molecules that can increase the percentage of cells expressing pluripotency marker genes. a. Representative images showing the effect of transient overexpression of exogenous Dux on the expression of the Zscan4 reporter gene in day 3 activated EPS cells. Scale bar, 200 μm. CTR, control. DUX OE, exogenous Dux overexpression. b. Representative flow cytometric analysis of the effect of transient overexpression of exogenous Dux on the expression of the Zscan4 reporter gene in day 3 activated EPS cells. The percentage of Zscan4-Emerald-GFP positive cells is shown. CTR, control. DUX OE, exogenous Dux overexpression. c. Q-PCR analysis of the expression of exogenous Dux (left panel) and pluripotency and pluripotency marker genes (right panel) when Dux is transiently overexpressed exogenously. exo-Dux, exogenous Dux overexpression. N = 2 biological replicates. d. Representative images showing the effects of VPA, EPZ004777, and CD1530 on the expression of Zscan4 or MERVL reporter genes in activated EPS cells. Scale bar, 200 μm. V, VPA. E, EPZ004777. CD, CD1530. CTR, N2B27 medium supplemented with CHIR 99021. eg. Representative flow cytometry analysis of the percentage of Zscan4 or MERVL positive cells in EPS cells on day 3 after small molecule treatment. For (f), N = 2 biological replicates. V, VPA. E, EPZ004777. CD, CD1530. CTR, N2B27 medium supplemented with CHIR 99021. EPS, EPS cells. 4S-3D: EPS cells treated with 4 small molecules (VPA, CHIR 99021, EPZ004777, CD1530) for 3 days. h. Representative images showing the effect of CHIR 99021 on cell proliferation during induction of TPS cells. Scale bar, 200 μm. 4S-C, 4S conditions without CHIR 99021. i. Cell number analysis showing that CHIR 99021 promotes cell proliferation during transformation of TPS cells. N = 3 biological replicates. 4S-C, 4S conditions without CHIR 99021. j. Representative immunofluorescence analysis of ZSCAN4 expression in TPS cells. Scale bar, 200 μm. K. Q-PCR analysis of totipotency and pluripotency marker gene expression during induction of TPS cells from EPS cells of different passages. EPS, EPS cells. EPS-TPS-P1 / 3 / 5 / 19: transformed cells at passages 1 / 3 / 5 / 19. N = 3 biological replicates. 1. Representative images of Zscan4-EmeraldGFP-positive cells cultured using basal medium and 4S medium are shown. Scale bar, 200 μm. Basal, basal medium under 4S conditions.m. Q-PCR analysis of expression levels of representative totipotency and pluripotency marker genes in sorted Zscan4-positive cells cultured in 4S-medium at passages 1 and 5 (4S-P1, 4S-P5). N = 2 technical replicates. Similar results were obtained in at least 2 independent experiments. n. Q-PCR analysis of expression levels of representative totipotency and pluripotency marker genes in TPS cells transformed from 2i / LIF-ES (mc2i-ES) and LCDM-EPS (mc6-1-EPS), respectively. N = 2 technical replicates. Similar results were obtained in at least 2 independent experiments.

[0069] Figure 7 . In vitro analysis of the extraembryonic developmental potential of TPS cells. a. Representative immunofluorescence analysis showing the expression of TS markers (CDX2, EOMES, TFAP2C, SOX2) and the pluripotency marker OCT4 in TS-like cells. Scale bar, 50 μm. b. Representative immunofluorescence analysis showing the expression of PE markers (GATA6, PDGFRα, SOX7, SOX17) and the pluripotency marker OCT4 in PE-like cells. Scale bar, 50 μm.

[0070] Figure 8 . In vitro immunofluorescence analysis of blastocysts with chimeric TPS-induced products. ac, representative immunofluorescence images showing the contribution of TPS-induced products in the trophectoderm (CDX2, EOMES, CK8, TFAP2C) and primitive endoderm (PDGFRα) of mouse blastocysts. For fluorescent images, the left image shows the original image and the right image shows the enlarged image. BF, bright field. Td, tdTomato. Scale bar, 20 μm.

[0071] Fig. 9 . Analysis of the process of induction of TPS cells from 2-cell embryos. Representative immunofluorescence analysis of ZSCAN4 and OCT4 expression in outgrowths cultured under 4S conditions. Outgrowths cultured under 2i / LIF conditions served as controls. Scale bar, 100 μm. Similar images were obtained in at least 2 independent experiments.

[0072] Fig.10. Transcriptomic analysis of TPS cells and EPS cells with 2-cell blastomeres. a. Hierarchical clustering analysis of TPS and EPS cell lines. b. Volcano plot showing comparison of transcriptomic differences between TPS cells and EPS cells. Representative upregulated genes are shown in red. c. Gene ontology analysis of differentially expressed genes between TPS cells and EPS cells. d. Box plot showing upregulated and downregulated gene expression in TPS cells at different developmental stages during preimplantation development (Deng et al., 2014). e. Heat map showing expression of 2-cell embryo-specific pluripotency signatures in TPS cells and EPS cells.

[0073] Fig.11 . Further analysis of the molecular characteristics of TPS cells. a. UMAP plot showing the expression of representative pluripotency marker genes in TBLC at the single-cell level. Different cell types are represented by different colors. PSC, pluripotent stem cells. Meso-like and Endo-like, fibroblast feeder. Intermediate, intermediate cells in TBLC. TBLC, TBLC subpopulation with high expression of pluripotency marker genes. b. ATAC-seq peaks near representative pluripotent and pluripotent marker genes in 2-cell embryos (2 cells), mouse ES cells, EPS cells, and TPS cells. c. WGBS analysis of CpG methylation in the loci of representative pluripotency marker genes in 2-cell embryos (2 cells), E6.5 and E7.5 embryos (E6.5 and E7.5), EPS cells, TPS cells, and ES cells.

[0074] Fig.12. Further analysis of the in vivo mosaicism of TPS cells. a. Representative images showing the mosaicism of a single blastomere of an 8-cell embryo expressing EGFP in embryonic and extraembryonic regions in an E7.5 embryo. Left: bright field; right: EGFP. Scale bar, 500 μm. Similar images were obtained in at least 3 independent experiments. b. Representative immunofluorescence analysis showing the expression of the visceral endoderm marker SOX17 in a single TPS-derived cell in an E7.5 embryo. Td, tdTomato. Scale bar, 200 μm. Similar images were obtained in at least 3 independent experiments. c. Representative images showing the contribution of a single blastomere of an 8-cell embryo expressing tdTomato in an E10.5 embryo, yolk sac, and placenta. For each image, the sample on the left is from a chimeric conceptus and the sample on the right is from a non-chimeric conceptus. Scale bar, 5 mm. Similar images were obtained in at least 3 independent experiments. d. Representative flow cytometric analysis of mosaicism of a single TPS-derived cell in E10.5 embryos, yolk sacs, and placentas. Td, tdTomato. WT, wild type. Similar images were obtained in at least 3 independent experiments. e. Representative immunofluorescence analysis of E10.5 chimeric placentas generated by injection of a single TPS cell transformed by EPS cells (tdTomato marker). Anti-TD, anti-tdTomato antibody. The right panel shows a magnified image of the left panel. Scale bars: left panel, 2 mm; right panel, 20 μm. Similar images were obtained in at least 3 independent experiments. f. Representative immunofluorescence analysis of E10.5 chimeric placentas generated by injection of a single blastomere of an 8-cell embryo expressing EGFP. The right panel shows a magnified image of the left panel. Scale bars: left panel, 2 mm; right panel, 20 μm. Similar images were obtained in at least 3 independent experiments. g. Representative immunofluorescence analysis of wild-type E10.5 placentas. Anti-TD, anti-tdTomato antibody. The right panel shows a magnified image of the left panel. Scale bars: left panel, 2 mm; right panel, 20 μm. Similar images were obtained in at least 3 independent experiments.

[0075] Fig.13. Further analysis of mosaicism in E17.5 conceptus. a. Representative images of the contribution of a single TPS-derived cell (tdTomato-labeled) to the embryo, yolk sac, and placenta in E17.5 mouse conceptus. BF: bright field. Td, tdTomato. For each image, the sample on the left is from a non-chimeric conceptus and the sample on the right is from a chimeric conceptus. Scale bar, 10 mm. Similar images were obtained in at least 3 independent experiments. b. Representative flow cytometric analysis of the mosaicism of tdTomato-positive TPS-derived cells in GFP-positive recipient E10.5 placentas. CTR, control. TD, tdTomato. Similar images were obtained in at least 2 independent experiments. c. Representative Q-PCR analysis of the expression of trophoblast marker genes by tdTomato-positive TPS-derived cells in GFP-positive recipient E18.5 placental tissues. N = 2 technical replicates. tdTomato-positive cells and GFP-negative cells were purified by flow cytometry. GFP-positive cells and tdTomato-negative cells were also sorted as controls. Similar results were obtained in at least 2 independent experiments.

[0076] Fig.14. Further mechanistic exploration of the induction and maintenance of pluripotency in TPS cells. a. Average ATAC-seq signal intensity of genomic regions containing RARγ motifs in TPS cells, ES cells, EPS cells, and 2-cell embryos (2-cell). b. Q-PCR analysis of the expression levels of representative pluripotency and pluripotency marker genes at day 3 after treatment with different small molecule combinations. Under 4S conditions, EPZ004777, VPA, CD1530, and CHIR 99021 were replaced by small molecules targeting DOT1L, HDAC, RA signaling, and GSK3β, respectively. EPS, EPS cells. Basal, EPS cells cultured in basal medium under 4S conditions. EPZ rep, VPA rep, CD1530 rep, and CHIR rep represent small molecules targeting DOT1L, HDAC, RA, and GSK3β, respectively. N = 2 technical replicates. Similar results were obtained in at least 2 independent experiments. c. Western blot analysis of protein expression of β-catenin in ES cells, EPS cells, and TPS cells is shown. ES, ES cells. EPS, EPS cells. 2C-TPS, TPS cells derived from 2-cell embryos. EPS-TPS, TPS cells transformed from EPS cells. d. Representative images showing that TPS cells can be grown in the absence of CHIR 99021. 4S-C, 4S conditions without CHIR 99021. Scale bar, 50 μm. e. Q-PCR analysis of the expression levels of totipotency and pluripotency marker genes in TPS cells cultured under CHIR 0 and CHIR 3 conditions. CHIR 0, 4S conditions without CHIR 99021; CHIR 3, 4S conditions. N = 2 technical replicates. Similar results were obtained in at least 2 independent experiments.

[0077] Fig.15. Analysis of the role of Oct4 and LIF-stat3 signaling in regulating TPS cell proliferation and expression of pluripotency markers. a. Q-PCR analysis showing the effect of Oct4 knockout on the expression levels of representative pluripotency and pluripotency marker genes in TPS cells. Oct4 ko batch 1 and batch 2 represent different batches of experiments. N = 3 biological replicates. b. Representative images showing the effect of Oct4 knockout on TPS cell proliferation. Scale bar, 500 μm. DOX-2D, 2 days after addition of doxorubicin. c. Representative images showing the effect of inhibition of LIF signaling on TPS cell proliferation. JAK inhibitor (JAKI) was used. Scale bar, 500 μm. Similar results were obtained in at least 2 independent experiments. d. Q-PCR analysis of the expression levels of representative pluripotency and pluripotency marker genes in TPS cells after inhibition of LIF signaling by Jak inhibitors AG490 and Niclosamide. Cells at passage 4 and passage 6 were analyzed. N = 2 technical replicates. Similar results were obtained in at least 2 independent experiments.

[0078] Fig.16 . Cell sorting gating strategy. a. By sorting Figure 6 Representative images of gating strategies for small molecule screening of ZSCAN4 or MERVL positive cells in e. b. Sorting Fig.12 d and Fig.13 b Representative gating strategy images of Td-labeled TPS-derived cells in the mosaic shown. DETAILED DESCRIPTION

[0079] A chemical mixture called pluripotent stem cells (TPS) that can induce new omnipotent cells with embryonic and extraembryonic developmental potential at the single cell level has been identified. As confirmed by the studies described herein, a single TPS cell can form extraembryonic and embryonic tissues at embryonic day 7.5 (E7.5) and E10.5. The studies described herein show that TPS cells can be effectively induced and stably maintained in vitro. Therefore, TPS cells provide a new cell resource for disease modeling, for example, using humanized animal models to study early development, and generating patient-specific cells for regenerative medicine.

[0080] I. Definition

[0081] The term "cell potency" as used herein refers to the ability of a cell to differentiate into other cell types. The more cell types a cell can differentiate into, the stronger its potency.

[0082] The term "chemically induced pluripotent stem cell" ("ciTPSC") as used herein refers to a pluripotent cell that has embryonic and extraembryonic developmental potential at the single cell level in an in vivo chimera assay by contacting a donor cell with a compound. For example, ciTPSC derived from human expanded pluripotent stem cells (EPS) show embryonic and extraembryonic developmental potential at the single cell level after contact with CDT. Preferably, the ciTPSC used herein has the same characteristics as a 2-cell mouse embryo in terms of pluripotency markers, transcriptome, chromatin accessibility, and DNA methylation patterns, and can be induced to form a blastocyst-like structure similar to a preimplantation mouse blastocyst.

[0083] The term "corresponding cell" is used to refer to a cell of the same type and from the same organism as the donor cell from which the ciTPSC was derived. For example, a corresponding cell of a ciTPSC derived from a mouse embryonic cell is a mouse embryonic cell that has not been contacted with a CDT.

[0084] As used herein, the term "donor cell" refers to a cell that is contacted with a CD4 T cell to induce / confer pluripotency.

[0085] The term "totipotency" used herein in relation to ciTPSCs refers to the ability of ciTPSCs to have both embryonic and extraembryonic developmental potentials at the single-cell level.

[0086] The term "epigenetic" as used herein refers to covalent modifications of DNA that are not based on mutations but can still be passed down from generation to generation in certain circumstances. Genes that are activated or inhibited without any DNA sequence changes are epigenetically controlled. Epigenetic modifications are stable, but reversible changes in gene expression may occur without permanent changes in the DNA sequence. Many types of epigenetic processes have been identified, including methylation, acetylation, phosphorylation, ubiquitination, sumolyation of histones, and DNA methylation.

[0087] As used herein, the term "induced pluripotent stem cell" (iPSC) is a type of pluripotent stem cell that is artificially derived from non-pluripotent cells. CiPSCs are also iPSCs; however, they differ from some iPSCs in that they have not been genetically engineered to confer pluripotency.

[0088] As used herein, the term "humanized animal model" refers to a non-human mammal implanted with functional human cells or tissues or expressing a human transgene.

[0089] As used herein, "ability to generate extraembryonic lineages in vivo" can be determined, for example, by measuring expression of trophectoderm markers and / or contribution to both trophectoderm (TE) and ICM (inner cell mass) following microscopic injection in a chimerism assay, as described herein under Materials and Methods.

[0090] The term "isolated" or "purified" when referring to ciTPSCs means chemically induced pluripotent stem cells that are at least 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% free of contaminating cell types that are not pluripotent stem cells. Isolated TPSCs may also be substantially free of soluble naturally occurring molecules.

[0091] As used herein, "media" and "culture medium" refer to the cell culture environment. A culture medium is typically an isotonic solution and can be a liquid, gel, or semisolid, for example, providing a matrix for cell attachment or support. A culture medium, as used herein, can include components for the nutrients, chemical, and structural support necessary for culturing cells.

[0092] The term "pluripotency" (or multipotency) as used herein refers to stem cells that have the potential to differentiate into any of the following three germ layers: endoderm (e.g., stomach lining, gastrointestinal tract, lungs), mesoderm (e.g., muscle, bone, blood, urogenital system), or ectoderm (e.g., epidermal tissue and nervous system). Pluripotent stem cells have lower plasticity and a higher degree of differentiation, and can become one of several cell types in a specific organ. For example, multipotent hematopoietic stem cells can develop into erythroid progenitor cells, white blood cells, or platelet-producing cells. Adult stem cells are multipotent stem cells. Adipose-derived stem cells are multipotent.

[0093] "Pluripotent cells" and "pluripotent stem cells" are used interchangeably herein.

[0094] The term "small molecule" refers to a molecule, such as an organic compound or an organometallic compound, having a molecular weight of less than 2000 Daltons, more preferably less than 1500 Daltons, and most preferably less than 1000 Daltons.

[0095] II. Composition

[0096] A mixture of multiple factors that can be used to induce the omnipotence of isolated pluripotent stem cells in vitro has been identified. CDT is capable of inducing omnipotent-like stem cells that can be maintained stably in vitro for a long time, known as pletipotent stem cells (TPS). CDT can be used to provide a population of ciTPSCs that contain at least 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the cell types (such as non-TPS cells) that are free of contamination.

[0097] The CDT comprises: (1) an HDAC inhibitor, (2) a Dot1L inhibitor, (3) a RARγ agonist, and (4) optionally, a GSK inhibitor. The composition comprises a CDT in an amount effective to induce untreated cells to be fully potent stem cells (TPS). It is within the capabilities of a person of ordinary skill in the art to determine equivalent effective concentrations of other members of the group of HDAC inhibitors, Dot1L inhibitors, RARγ agonists, and optionally, GSK inhibitors based on the effective concentrations disclosed for a particular species within the genus using in vitro assays.

[0098] An optional compound useful in the methods disclosed herein is a GSK inhibitor, e.g., a GSK3 inhibitor or one selected from the group consisting of CHIR99021 ("C"), AZD2858, LY2090314, BIO, CHIR 98014, SB415286, AZD1080, BRD3731, A1070722, BIP-135, and SB216763.

[0099] A. Chemical inducers of omnipotence

[0100] 1. Small molecules

[0101] Compounds that induce omnipotence in vitro, i.e., chemical inducers of omnipotence (CDTs), may include small molecules with a molecular weight of less than 2,000 Daltons, more preferably less than 1,500 Daltons, and most preferably less than 1,000 Daltons, either alone or in combination with proteins. The molecular weight of the small molecule may be less than or equal to 900 Daltons, or less than or equal to 500 Daltons. Larger molecules may be used for chemically induced reprogramming, preferably targeting the same pathways as the small molecules identified here. Within the capabilities of those of ordinary skill in the art, substances having the same or similar functions in signal transduction pathways (e.g., shRNAs) may be substituted for, for example, HDAC inhibitors, Dot1L inhibitors, RARγ agonists, and GSK inhibitors, and such substances may therefore be included in some embodiments as CDTs. In some embodiments, CDTs consist of the listed compounds.

[0102] (i) HDAC inhibitors

[0103] The HDAC inhibitor is preferably an Hdac1 and / or Hdac2 inhibitor, for example, selected from the group consisting of VPA ("V"), TSA, MS275, Scriptaid, SAHA, LBH589, FK228, PXD101, sodium butyrate, LAQ824, CUDC-101, JNJ-26481585, SB939, PCI-24781, ACY-1215, CI994, CUDC-907, RGFP109, Resminostat, Curcumin, Divalproex Sodium, 4-PBA, GSK3117391, CAY10433, CM-675 and MGCD0103. VPA can be used in a concentration range of 10-1000 μM. For example, VPA is present in the composition at a concentration of 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μM.

[0104] (ii) Dot1L inhibitors

[0105] Suitable Dot1L inhibitors are, for example, EPZ004777 ("E"), EPZ5676 and SGC0946. EPZ004777 ("E") can be used in a concentration range of 0.1-10 μM. For example, the concentration of E in the composition is 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 μM.

[0106] (iii) RARγ agonists

[0107] Suitable Dot1L inhibitors are, for example, CD1530 ("D"), AM580, ch55, Palovarotene, CD3254, CD5789, CD437, TTNPB, AGN205327, and RA. CD1530 ("D") can be used in a concentration range of 0.1-5 μM. For example, the concentration of E in the composition is 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 μM.

[0108] (iv) GSK inhibitors

[0109] The GSK inhibitor preferably inhibits GSK3, and is preferably selective for GSK3. Suitable GSK inhibitors are CHIR99021 ("C"), AZD2858, LY2090314, BIO, CHIR 98014, SB415286, AZD1080, BRD3731, A1070722, BIP-135, and SB216763. The CDT composition includes CHIR99021 at a concentration range of 0.5-10 μM, preferably between 1 and 5 μM, and even more preferably between 1.5 and 3 μM. For example, CDT may include CHIR99021 at a concentration of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 μM. Concentrations falling between these numbers are contemplated, as those of ordinary skill in the art can easily fine-tune the desired effective amount.

[0110] B. Cells to be induced (donor cells)

[0111] Totipotent stem cells are obtained by culturing isolated embryonic cells or pluripotent cells. In some embodiments, pluripotent cells can be obtained from mammals such as any mammal (e.g., cattle, sheep, pigs, dogs, cats, horses, primates), preferably people. In some embodiments, donor cells are obtained from non-human mammals. Sources include bone marrow, fibroblasts, fetal tissue (such as fetal liver tissue), peripheral blood, umbilical cord blood, pancreas, skin or any organ or tissue. In some embodiments, donor cells can be separated from non-human embryos, such as mouse embryos.

[0112] In a preferred embodiment, ciTPSCs are obtained from EPS cells. However, other pluripotent cells, such as embryonic stem cells or induced pluripotent stem cells (iPSCs) can be used, for example, first induced into extended pluripotent stem cells (e.g., by the method disclosed in WO2017025061A1), and then the extended pluripotent stem cells are cultured with CDT. iPSCs include cells obtained by genetic engineering and / or pure chemical reprogramming. In other embodiments, ciTPSCs are obtained from blastocysts.

[0113] Preferably, iPSCs are obtained from chemically induced fibroblasts, adipose-derived stem cells, neural stem cells, or intestinal epithelial cells. In some embodiments, CiPSCs are obtained from chemically induced neonatal (e.g., foreskin) or adult fibroblasts. However, iPSCs can be obtained from other cell types, including but not limited to: pluripotent stem cells, cells of hematological origin, cells of embryonic origin, cells of skin origin, fibroblasts, adipocytes, epithelial cells, endothelial cells, mesenchymal cells, parenchymal cells, neural cells, and connective tissue cells.

[0114] Pluripotent cells that can be used in the methods disclosed herein are known in the art and have been described, including methods for maintaining cells in culture. Mouse embryonic stem (ES) cells are isolated from the inner cell mass of the blastocyst and maintained in a naive inner cell mass-like structure in vitro by providing exogenous stimulation of leukemia inhibitory factor (LIF) and small molecule inhibition of ERK1 / ERK2 and GSK3β signaling (referred to as 2i / LIF conditions). Hallmarks of naive pluripotency include driving transcription of Oct4 (also known as Pou5f1) through its distal enhancer, retaining the state before X chromosome inactivation, and an overall reduction in DNA methylation and deposition of H3K27me3 repressive chromatin marks on promoters of developmental regulatory genes. After exiting 2i / LIF, naive mouse embryonic stem cells can drift toward a primed pluripotent state similar to the post-implantation epiblast. Although human embryonic stem cells share some of the same molecular features as naive mouse embryonic stem cells, they also share many of the same epigenetic properties as primed mouse epiblast stem cells (EpiSCs). These include the predominant use of proximal enhancer elements to maintain OCT4 expression, a clear trend toward X chromosome inactivation in most female human embryonic stem cells, increased DNA methylation, and significant deposition of H3K27me3 and bivalent domains on lineage regulatory genes. Gafni et al., Nature, 504(7479): 282-286 (2013) disclose the induction of genetically unmodified human naive pluripotent stem cells from established primed human embryonic stem cells, from somatic cells by induced pluripotent stem cell (iPS) cell reprogramming, or directly from blastocysts.

[0115] Donor cells can be separated by decomposing suitable organs or tissues as cell sources using technology well known to those skilled in the art.For example, tissue or organ can be mechanically decomposed and / or treated with digestive enzymes and / or chelating agents that weaken the connection between adjacent cells, so that the tissue is dispersed to form a suspension of independent cells without obvious cell damage.Enzymolysis can be completed by chopping the tissue and processing the chopped tissue with one or more enzymes (such as trypsin, chymotrypsin, collagenase, elastase and / or hyaluronidase, DNA enzyme, pronase, dispase etc.).Mechanical destruction can also be completed by many methods, including but not limited to, using a grinder, a blender, a sieve, a homogenizer, a pressure element or an ultrasonic generator.

[0116] In some embodiments, donor cells can include established human embryonic stem cells, for example, commercially available human embryonic stem cells. The ciTPSC obtained by culturing human embryonic stem cells in vitro can be used for example for regenerative medicine. In some embodiments, donor cells do not include human embryonic stem cells or human embryonic cells. In some embodiments, donor cells are non-human embryonic stem cells or non-human embryonic cells.

[0117] C. Chemically induced pluripotent stem cells (ciTPSC)

[0118] ciTPSCs are identified as stem cells with full potential based on properties including: i) the presence of cellular expression of any one or more totipotency marker genes selected from Zscan4, Zfp352, Tcstv1, Tcstv3, MERVL, Dux, Dub1a, Eif1al6, Eif1al9, Gm4340 and Tdpoz4 as compared to corresponding untreated cells, optionally after 10 or more passages, and / or ii) downregulation of cellular expression of any one or more pluripotency marker genes selected from Oct4, Nanog and Sox2 as compared to corresponding untreated cells; and / or (iii) functionally based on the ability of the cells to have embryonic and extraembryonic developmental potential at the single-cell level, and / or to be induced into a blastocyst-like structure similar to a preimplantation mouse blastocyst, and / or to have the same characteristics as a 2-cell mouse embryo in terms of totipotency markers, transcriptome, chromatin accessibility and DNA methylation pattern, for example as shown below.

[0119] III. Preparation method

[0120] A. Induction of pluripotency in donor cells

[0121] ciTPSCs are produced by contacting cells to be induced (herein, donor cells) with a medium containing CDT for a sufficient time to cause the cells to be reprogrammed into chemically induced pluripotent stem cells (ciTPSCs). The donor cells are contacted with CDT disclosed herein in an amount effective to induce and / or reprogram cells into pluripotent stem cells. One skilled in the art can easily determine the concentration of CDT compounds disclosed herein required to provide complete reprogramming by using the methods outlined in the examples below or other methods known in the art.

[0122] B. Isolation of ciTPSCs

[0123] For example, by extracting from a culture source (e.g., by density gradient centrifugation and / or flow cytometry), a substantially purified ciTPSC population can be obtained. Purity can be measured by any suitable method. Pluripotent cells can be purified by, for example, flow cytometry (such as FACS analysis) 99%-100%. Human induced pluripotent stem cells can be separated by the following methods, for example, using a molecule (e.g., an antibody, an antibody derivative, a ligand or an Fc peptide fusion molecule) that binds to a marker or a combination of markers on the induced donor cells, thereby positively selecting cells that bind to the molecule (i.e., positive selection). Other examples of positive selection methods include methods that preferentially promote the growth of desired cell types in a mixed population of desired and undesired cell types. Alternatively, by using a molecule that binds to a marker that is not present on the desired cell type but is present on the undesired cell type, undesired cells containing such markers can be removed from the desired cells (i.e., negative selection). Other negative selection methods include preferentially killing undesired cell types or inhibiting the growth of undesired cell types in a mixed population of desired and undesired cell types. Thus, by using negative selection, positive selection, or a combination thereof, enriched populations of cells can be prepared.

[0124] C. Cultivation and maintenance of ciTPSCs (and their progeny)

[0125] ciTPSC can be expanded and stored in culture for subsequent recovery and use. Once a culture of cells or a mixed culture of stem cells is established, the cell population is expanded in vitro in a mitotic manner by passage to fresh culture medium according to cell density under conditions conducive to cell proliferation, with or without tissue formation. Such a culture method may include, for example, passage of cells in a culture medium lacking specific growth factors that induce differentiation (e.g., IGF, EGF, FGF, VEGF and / or other growth factors). When a sufficient cell density is reached, the cultured cells can be transferred to fresh culture medium.

[0126] In preferred embodiments, the cell culture medium used to maintain ciTPSCs is, for example, N2B27 basal medium or 1640 basal medium supplemented with CDT disclosed herein. According to some embodiments of the present invention, the culture medium can maintain ciTPSCs for 2 to more than 50 generations in culture. For example, the culture medium can maintain ciTPSCs for 2, 3, 4, 5, 6, 7, 8, 9, or 10 generations in culture, preferably more than 10 generations, such as about 20 generations in culture, for example, at least about 25, about 30, about 35, about 40, about 45, about 50 generations in culture. In preferred embodiments, ciTPSCs maintain a normal karyotype for 2, 3, 4, 5, 6, 7, 8, 9, 10, more than 10, such as about 20 generations in culture.

[0127] IV. How to use

[0128] Identifying readily available sources of totipotent stem cells that can generate desired cell types or morphologies is very important for therapeutic treatments, tissue engineering, and research. The availability of totipotent stem cells is extremely useful in transplantation, tissue engineering, regulation of angiogenesis, angiogenesis, organ regeneration, humanized animal models, cell replacement or cell therapy, and disease prevention. Such stem cells can also be used to introduce genes into a subject as part of a gene therapy protocol.

[0129] A. Provide differentiated somatic cells (redifferentiated cells)

[0130] Once established, cultures of totipotent stem cells can be used to generate progeny cells, such as fibroblasts that can generate new tissues. ciTPSCs can be induced to differentiate into cells of any of the three germ layers, such as skin cells and hair cells (including epithelial cells, keratinocytes, melanocytes, adipocytes), cells that form bone, muscle, and connective tissue (such as myocytes, chondrocytes, osteocytes, alveolar cells), parenchymal cells (such as liver cells, kidney cells, adrenal cells, and pancreatic islet cells), blood cells, retinal cells (and other cells involved in sensory perception, such as those that form hair cells in the ear or taste buds on the tongue), and neural tissue including nerves. Redifferentiated cells can be expanded in culture and stored for later recovery and use.

[0131] B. Cell therapy

[0132] The therapeutic use of induced totipotent stem cells includes transplanting totipotent stem cells, cell populations, or their progeny into individuals to treat various pathological conditions, including diseases and conditions caused by cancer, wounds, tumors, injuries, viral infections, diabetes, etc. Treatment may require the use of cells to generate new tissues and use the tissues generated thereby, according to any method currently known in the art. These cells can be implanted, injected, or otherwise directly administered to the site of tissue damage so that they generate new tissues in vivo. In one embodiment, administration includes administration of genetically modified ciTPSCs or their progeny.

[0133] In a preferred embodiment, ciTPSCs are obtained from autologous cells, i.e., the donor cells are autologous. However, these cells can be obtained from heterologous cells. In one embodiment, the donor cells are obtained from a donor that is genetically related to the recipient. In another embodiment, the donor cells are obtained from a donor that is not genetically related to the recipient. If the human ciTPSCs are derived from a heterologous (non-autologous / allogeneic) source compared to the recipient subject, concomitant immunosuppressive therapy is usually administered, such as administration of the immunosuppressant cyclosporine or FK506. However, due to the immature state of human-induced donor cells, such immunosuppressive therapy may not be required. Therefore, in one embodiment, human-induced donor cells can be administered to the recipient in the absence of immunomodulatory (e.g., immunosuppressive) treatment. Alternatively, the cells can be encapsulated in a membrane that allows fluid exchange but prevents cell / cell contact. Transplantation of microencapsulated cells is known in the art, for example, Balladur et al., Surgery, 117: 189-94, 1995; and Dixit et al., Cell Transplantation 1: 275-79 (1992).

[0134] Using methods known in the art, ciTPSC and its descendants can be used to make tissue engineering structures. Tissue engineering constructs can be used for various purposes, including as prosthetic devices to repair or replace damaged organs or tissues. They can also be used as in vivo delivery systems for proteins or other molecules secreted by the cells of the construct or are generally used as drug delivery systems. Tissue engineering constructs are also used as in vitro models of tissue function, or as models for testing the effects of various treatments or drugs. Tissue engineering techniques often involve selecting suitable culture matrices to maintain and promote tissue growth. In general, these matrices should be three-dimensional and should be processable to form a scaffold of the desired shape of the target tissue.

[0135] ciTPSCs can be induced to differentiate into cells from any of the three germ layers, such as skin cells and hair cells (including epithelial cells, keratinocytes, melanocytes, adipocytes), cells that form bone, muscle and connective tissue (such as myocytes, chondrocytes, osteocytes, alveolar cells), parenchymal cells (such as liver cells, kidney cells, adrenal cells and pancreatic islet cells (e.g., α cells, δ cells, PP cells and β cells)), blood cells (e.g., leukocytes, erythrocytes, macrophages and lymphocytes), retinal cells (and other cells involved in sensory perception, such as those that form hair cells in the ear or taste buds on the tongue), and neural tissue including nerves.

[0136] ciTPSCs can be formulated for administration, delivery, or contact with a subject, tissue, or cell to promote dedifferentiation in vivo or in vitro / ex vivo. Other factors can be incorporated, such as growth factors, other factors that induce differentiation or dedifferentiation, secreted products, immunomodulators, anti-inflammatory agents, regression factors, biologically active compounds that promote innervation, vascularization, or enhance lymphatic networks, and drugs.

[0137] The induced pluripotent cells can be administered to a patient by means of a composition comprising a ciTPSC population or ciTPSC progeny population alone or on or in a carrier or support structure. In many embodiments, a carrier will not be required. The cells can be administered by injection into or at a site where the cells are needed. In these cases, the cells will typically be washed to remove the cell culture medium and will be suspended in a physiological buffer.

[0138] C. Animal Models and Organ Regeneration

[0139] Isolated ciTPSCs can be used to generate animal models that incorporate ciTPSCs from a desired species (donor) into a second animal (recipient) of the same or different species. The donor animal can be a mammal, such as a human, mouse, rat, pig, cattle, sheep, goat, horse, dog, chimpanzee, gorilla, orangutan, monkey, marmoset, etc. In some preferred embodiments, the donor mammal is human and the recipient mammal is non-human, for providing a humanized animal model. In other embodiments, the donor animal and the recipient animal are size matched. The recipient can be any animal other than human, such as pigs, rats, mice, cattle, sheep, goats, horses, dogs, chimpanzees, gorillas, orangutans, monkeys, marmosets, and bonobos. ciTPSCs can be used for organ regeneration in non-human mammals; ciTPSCs can be used to generate desired organs in mammals, where the mammal has an abnormality associated with insufficient development of the organ at a developmental stage.

[0140] The method comprises transplanting ciTPSC into a blastocyst stage fertilized egg of a recipient non-human mammal; developing the fertilized egg in the uterus of a non-human surrogate parent mammal to obtain litters, and obtaining organs from the litters using methods known in the art. Examples of organs that can be produced include, but are not limited to, solid organs with fixed shapes, such as kidneys, hearts, pancreas, cerebellum, lungs, thyroid glands, hair, and thymus. The recipient embryo can be from any animal other than humans, such as pigs, rats, mice, cattle, sheep, goats, horses, dogs, chimpanzees, gorillas, orangutans, monkeys, marmosets, etc.

[0141] Methods for generating humanized mouse models are known in the art. Examples of recipient embryos with abnormalities associated with the development of a target organ that can be used to regenerate the organ include animals with Sall1 knockouts associated with abnormalities associated with insufficient kidney development during development (Nishinakamura, et al., Development, 128: 3105-3115 (2001)); animals with Pdx1 knockouts associated with abnormalities associated with insufficient pancreatic development during development (Offield, et al., Development, 122: 983-995 (1996)); animals with Wnt-1 (int-1) knockouts associated with abnormalities associated with insufficient cerebellum development during development (McMahon et al., Cell, 62: 1073-1085, (1990)); animals with T / ebp knockouts associated with abnormalities associated with insufficient lung and thyroid development during development (Kimura, et al., Genes and Development, 10: 60-69, 1996); or a dominant negative transgenic mutant animal model can be used, which overexpresses a defect in the intracellular domain of the fibroblast growth factor (FGF) receptor (FGFR) and results in defects in multiple organs such as the kidney and lung (Celli, et al., EMBO J., 17: 1642-655, (1998)). Alternatively, nude mice can be used to produce hair or thymus.

[0142] IV. Kit

[0143] A kit is provided comprising a chemical inducer of pluripotency (CDT) disclosed herein. The CDT is as described above. These can be in a form with defined concentrations to facilitate addition to a cell culture medium to produce the desired concentration. The kit can include instructions for providing the desired concentration range and administration time according to the donor cell type. The kit can also include a cell culture medium premixed with the CDT for culturing donor cells to induce pluripotency.

[0144] The present invention will be further understood by reference to the following non-limiting examples.

[0145] Example

[0146] method

[0147] Mouse

[0148] The mouse strain B6-Tg (C57BL / 6-td Tomato) was purchased from Beijing Weitongda Biotechnology Co., Ltd. Other mouse strains ICR and EGFP were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center. All animal experiments were performed in accordance with NIH guidelines. All mouse experiments were approved by the Institutional Animal Care and Use Committee of Peking University. Mice were placed in a temperature-controlled room (22 ± 1 °C) with a humidity of 40-60% and a 12-h light-dark cycle from 06:00 to 18:00.

[0149] Culture of mouse TPS cells

[0150] All cell lines were cultured at 37°C in 20% O2 and 5% CO2. Mouse TPS cells were cultured in 4S medium containing the following basal medium: N2B27 basal medium to promote cell proliferation [50% DMEM / F12 (Gibco, 11330-032), 50% Neurobasal (Gibco, 21103-049), 0.5% N2 supplement (Gibco, 17502-048), 1% B27 supplement (Gibco, 12587-010), 1% GlutaMAX (Gibco, 35050-061), 1% non-essential amino acids (Gibco, 11140-050)], or 1640 basal medium to enhance pluripotency [RPMI Medium 1640 basic (Gibco, 22400-089), 0.5% N2 supplement (Gibco, 17502-048), 1% B27 supplement (Gibco, The basal medium was supplemented with 5% knockout serum replacement (Gibco, 10828-028), 0.2% sodium pyruvate (Gibco, 11360-070), 0.14% sodium DL-lactate solution (Sigma, L7900) and 0.1% Chemically Defined Lipid Concentrate (Gibco, 11905-031), VPA (50 μM; Selleck, S3944), CHIR-99021 (3 μM; Selleck, S1263), EPZ004777 (1 μM, Selleck, S7353) and CD1530 (0.2 μM - 0.5 μM; Tocris, 2554). Mouse TPS cells were cultured on mitomycin C-inactivated mouse embryonic fibroblasts (feeder layer, 3×104 Pieces / cm 2 ) on a 4S medium. The medium was changed every day. TPS cells were passaged every 3 days using 0.05% trypsin-EDTA (Gibco, 25300-062) and seeded at a split ratio of 1:3 to 1:10. To generate TPS cells, EPS cells or ES cells were separated and cultured on a feeder layer in 4S medium for the first three generations. EPS cells or ES cells needed to be seeded at a lower split ratio of 1:3 to increase cell survival. After 5 passages, TPS cells were generated for further experiments.

[0151] Induction of mouse TPS cells from 2-cell embryos

[0152] TPS cells were derived from 2-cell embryos of B6-Tg mice. For 2-cell embryos, the zona pellucida was removed using Acidic Tyrode's Solution (Sigma, T1788). When the zona pellucida disappeared, the 2-cell embryos were washed three times with M2 medium (Sigma, M7167) and then seeded on a feeder layer in 4S medium. After 2-3 days, fresh 4S medium was replaced. After 6 days, the outgrowth was picked and cut into small pieces. After 4 days, the outgrowth was picked again and digested with 0.05% trypsin-EDTA. Single cells were seeded on a feeder layer in 4S medium supplemented with Y27632 (10 μM; Tocris, 1254). TPS clones gradually emerged. During the first 5 passages, it is recommended to seed cells in 4S medium supplemented with Y27632. The next day, Y27632 was removed. Newly established cell lines were passaged every three days using 0.05% trypsin-EDTA for further analysis.

[0153] Induction of TPS cells from EPS cells

[0154] All cell lines were cultured at 37°C, 20% O2, and 5% CO2. Mouse EPS cells and ES cells were cultured on feeder layers (3 × 10 4 cells / cm 2). N2B27 medium was prepared by including: 50% DMEM / F12 (Gibco, 11330-032), 50% Neurobasal (Gibco, 21103-049), 0.5% N2 supplement (Gibco, 17502-048), 1% B27 supplement (Gibco, 12587-010), 1% GlutaMAX (Gibco, 35050-061), 1% non-essential amino acids (Gibco, 11140-050). N2B27-LCDM medium was prepared by including: recombinant human LIF (10 ng / ml, peprotech, 300-05), CHIR-99021 (3 μM; Selleck, S1263), (S)-(+)-Dimethindene maleate (2 μM, Tocris, 1425), minocycline hydrochloride (2 μM, Tocris, 3268). N2B27-2i / LIF medium was prepared by including: recombinant human LIF (10 ng / ml), CHIR99021 (3 μM), PD0325901 (1 μM, Selleck, S1036).

[0155] Mouse EPS cells and ES cells cultured in N2B27-LCDM medium or N2B27-2i / LIF medium for 2 days were used for TPS generation. Mouse EPS cells and ES cells were washed with DMEM / F12, then incubated with 0.05% trypsin-EDTA (Gibco, 25300-062) for 3 minutes, and then added with DMEM (Gibco, 11965092) supplemented with 10% FBS (Hyclone, SH30070.03). The cells were pipetted up and down several times to form single cells. The cells were centrifuged and resuspended in 4S medium containing N2B27 to promote cell proliferation or RPMI Medium 1640 basic (Gibco, 22400-089) to enhance the pluripotency characteristics, which was supplemented with 5% knockout serum replacement (Gibco, 10828-028), 0.2% sodium pyruvate (Gibco, 11360-070), 0.14% sodium DL-lactate solution (Sigma, L7900) and 0.1% chemically defined lipid concentrate (Gibco, 11905-031), VPA (50 μM; Selleck, S3944), CHIR-99021, EPZ004777 (1 μM, Selleck, S7353) and CD1530 (0.2 μM - 0.5 μM; Tocris, 2554). EPS cells and ES cells were seeded on feeder layers in 4S medium at a split ratio of 1:10. 4S medium was changed every day. TPS cells were passaged every 3 days with 0.05% trypsin-EDTA and seeded at a split ratio of 1:3 to 1:10. For the first three passages, TPS cells needed to be passaged at a high density (1:3) to improve cell survival. After 5 passages, TPS cells gradually proliferated well in 4S medium and were generated for further experiments.

[0156] Transient overexpression of exogenous Dux in Zscan4-Emerald GFP and MERVL-tdTomato reporter cells

[0157] Plasmids containing exogenous Dux driven by the CMV promoter were transfected into Zscan4-Emerald GFP and MERVL-tdTomato reporter cells by nuclear transfection. Transfected cells were cultured with LCDM medium. Three days after transfection, cells were harvested for further analysis.

[0158] Flow cytometry

[0159] The cells were gently digested into single cells using 0.05% trypsin-EDTA. The suspension was filtered through a 40 μm cell strainer. The samples were then analyzed on a CytoFLEX (Beckman Coulter). Data were analyzed using CytExpert software.

[0160] Cell sorting and culture of sorted cells

[0161] Cell sorting was performed on a BD FACSAria SORP. MERVL-tdTomato-positive cells or Zscan4-Emerald GFP-positive cells were sorted and cultured with TPS medium or basal medium at 2 × 10 4 cells / cm 2 The cells were seeded at a density of 1:10 on the feeder layer. The culture medium was changed every day. The sorted cells were passaged every 3 days using 0.05% trypsin-EDTA (Gibco, 25300-062) and seeded at a split ratio of 1:3 to 1:10.

[0162] Karyotype analysis

[0163] On the day of sampling, cells were prepared to reach 50 ± 70% confluence. After incubation with fresh medium for 2 hours, colcemid solution was added to the medium at a final concentration of 0.02 mg / ml and incubated for 1 hour. The cells were then washed in PBS, trypsinized, and spun down. To obtain a single cell suspension, the pellet was resuspended in a hypotonic solution (0.56% KCl) and allowed to stand at room temperature for 6 minutes. After spinning and removing the hypotonic solution, 5 mL of ice-cold fixative (3:1 methanol: acetic acid) was added dropwise to the suspension, allowed to stand at room temperature for 5 minutes, and then spun down. The fixation procedure was further repeated three additional times. Finally, the pellet was resuspended in a final volume of 1 mL of fixative. The cells were then dropped onto slides washed with 5% acetic acid ± ethanol (ice-cold) and stained with Giemsa staining. For each analysis, at least 30 ± 40 metaphases were examined. The number of chromosomes and the presence of chromosomal structural abnormalities were examined.

[0164] Quantitative PCR analysis

[0165] Total RNA was isolated using the Direct-zol RNA Kit (ZYMO Research, R2052). RNA was converted to cDNA using the TransScript FirstStrand cDNA Synthesis SuperMix (TransGen Biotech, AT311). Quantitative PCR analysis was performed using the KAPA SYBR FAST qPCR Kit (KAPA Biosystems, KK4601) and the Bio RADCFX Connect Real-Time System. The primers used for Q-PCR analysis are listed in Table 1. Data were analyzed using the Δ-ΔCT method.

[0166] Table 1-1: qPCR primers used in this study

[0167]

[0168]

[0169]

[0170]

[0171] Table 1-2: Oligonucleotides used in this study

[0172]

[0173] Table 1-3: Gene sequences used in this study

[0174]

[0175]

[0176] Induction of trophoblast stem-like cells from TPS cells in vitro

[0177] TPS cells were digested into single cells and inoculated onto feeder cells cultured in TS conditioned medium: RPMI-1640 (Gibco, 11879-020) supplemented with 309 20% ES qualified FBS, 1% L-glutamine (Gibco, 25030-081), 1% sodium pyruvate (Gibco, 11360-070), heparin (1 ug / ml; Macklin, H811552-500KU), mouse FGF4 (25 ng / ml; Bioteche, 5846-F4), human bFGF (20 ng / ml; Novoprotein, C046). After 3-4 days, cells were passaged with TS medium. Flat TS-like colonies gradually appeared after passage. The culture medium was then changed to serum-free FAXY-TS medium: 50% Neurobasal, 50% DMEM / F12, 0.5% N2 supplement, 1% B27 supplement, 1% L-glutamine, 1-thioglycerol (1.5×10-4 M; Sigma, M6145), human bFGF (25 ng / ml; Novoprotein, C046), recombinant human activin A (20 ng / ml; Novoprotein, C687), XAV939 (10 μM; Selleck, S1180), and Y27632 (5 μM; Tocris, 1254).

[0178] Induction of primitive endoderm-like cells from TPS cells in vitro

[0179] In N2B27 medium, 2.5 × 10 4 cells / cm 2 TPS cells were differentiated into primitive endoderm-like cells by plating on matrigel-coated plates for 3 days in N2B27 medium supplemented with mouse FGF4 (50 ng / ml; Bioteche, 5846-F4), retinoic acid (10 nM; Sigma, R2625), 8-bromo-cAMP (1 mM; Selleck, S7857), CHIR-99021 (3 μM, Selleck, S1263). After the emergence of XEN-like cells, XEN cells were maintained in eXEN medium: RPMI-1640 supplemented with 20% ES-qualified FBS, 1% sodium pyruvate, 1% L-glutamine, mouse FGF4 (25 ng / ml; Bioteche, 5846-F4) and heparin (1 μg / ml; Macklin H811552-500 KU).

[0180] Immunofluorescence

[0181] Cells were fixed in 4% paraformaldehyde (DingGuo, AR-0211) for 20 minutes at room temperature and blocked with PBS (Corning, 21-040-CVR) containing 0.2% Triton X-100 (Sigma-Aldrich, T8787) and 3% normal donkey serum (Jackson ImmunoResearch, 017-000-121) for 1 hour at room temperature. Cells were incubated with primary antibodies overnight at 4°C. After washing the primary antibody three times with PBS, the secondary antibody (Jackson ImmunoResearch) was incubated for 1 hour at room temperature. Cell nuclei were stained with DAPI (Roche Life Science, 10236276001) for 3 minutes at room temperature and washed 3 times with PBS. The following primary antibodies were used: anti-ZSCAN4 (1:5000; MilliporeSigma, AB4340), anti-MuERVL-Gag (1:500; Epigentek, A-2801-100), anti-OCT4 (1:500; Abcam, ab181557), anti-OCT4 (1:200; Abcam, ab27985), anti-SOX2 (1:200; MilliporeSigma, AB5603), anti-EOMES (1:500; Abcam, ab183991), anti-TFAP2C (1:500; Abcam, ab218107), anti-PDGFRA (1:200; R&D, AF1062), anti-SOX17 (1:200; R&D, AF1924), and anti-SOX7 (1:200; R&D, AF2766), anti-GATA6 (1:200; R&D,AF1700), anti-CDX2 (1:200; BioGenex, MU392A).

[0182] Teratoma assay

[0183] Mouse TPS cells were collected by trypsinization before injection. 6 The cells were mixed with Matrigel and injected subcutaneously into immunodeficient NPG mice. Teratomas generally form within 2 to 6 weeks, and animals are killed before the tumor diameter exceeds 1.5 cm. The teratomas are then digested into single cells using collagenase IV. Red blood cells are then removed using red blood cell (RBC) lysis buffer (Thermo Fisher Scientific, 00-4333-57). The digested teratomas were processed for 10x Genomics scRNA-seq.

[0184] Chimerism assay by single-cell microinjection

[0185] Digest the cells with 0.05% trypsin-EDTA and filter the digested cells through a 40 μm cell strainer and centrifuge at 1,200-1,500 rpm for 3 minutes at room temperature. Remove the supernatant and resuspend the cells in medium supplemented with Y-27632 (10 μM; Tocris, 1254) and place on ice before injection. After placing on ice, the digested cells should be injected within 1 hour, otherwise digest another batch of cells for the remaining injections.

[0186] Single cells were microinjected into 8-cell ICR diploid mouse embryos. The injected embryos were cultured in a medium with Y-27632 (10 μM; Tocris, 1254) for the first 4 hours. To produce chimeric blastocysts, the embryos were transferred to KSOM medium (Merck, MR-106-D). To produce single-cell derived in vivo chimeric conceptuses, chimeric embryos were cultured overnight at 37°C in a humidified incubator at 5% CO2 in KSOM medium supplemented with Y-27632 (10 μM; Tocris, 1254). The injected embryos were transferred to the uterine horns of pseudopregnant females after 0.5 mating. Fetal tissue, yolk sac, and placenta were stripped from the conceptuses at the E7.5, E10.5, E13.5, or E17.5 developmental stages.

[0187] Chimerism assay by multi-cell microinjection

[0188] To generate chimeric blastocysts, TPS cells were cultured in FAXY basal medium [50% Neurobasal, 50% DMEM / F12, 0.5% N2 supplement, 1% B27 supplement, 1% L-glutamine, 1-thioglycerol (1.5×10−4 M; Sigma, M6145), supplemented with human BMP4 (100 ng / ml; Stemimmune, HST-B4-0100) and bFGF (100 ng / ml; Novoprotein, C046)] for 2 days. The cells were first digested with 0.05% trypsin-EDTA and then filtered through a 40 μm filter. Afterwards, centrifugation was performed at 1200-15000 rpm for 3 minutes. The cells were suspended with medium and placed on ice. 10-15 digested cells were microinjected into each 2 / 8 cell ICR or GFP diploid mouse embryo. The injected embryos were transferred to KSOM medium and cultured in a humidified incubator at 37°C with 5% CO 2 for 48 to 72 hours until they developed to E4.5, fixed and immunostained, and more than 20 embryos were analyzed.

[0189] Mosaicism determination of single blastomeres of 8-cell embryos

[0190] The zona pellucida of the immunofluorescent 8-cell embryos was removed with Acidic Tyrode's Solution (Sigma, T1788). When the zona pellucida disappeared, the 2-cell embryos were washed three times with M2 medium (Sigma, M7167). TM Select (Gibco, A1217702) digested three times, and then digested twice with DNase I (Gibco, 90083). TM After final digestion with Select and DNase I at 37°C for 5 minutes, blastomeres were separated. Single immunofluorescent blastomeres were aggregated with wild-type 8-cell embryos with zona pellucida removed in AggreWell (Stemcell, 34415). Aggregated embryos were cultured overnight at 37°C in KSOM in a humidified incubator with 5% CO2. Aggregated embryos were transferred to the uterine horns of pseudopregnant females after 0.5 mating. Fetal tissue, yolk sac, and placenta were stripped from the conceptus at the E7.5 or E10.5 developmental stage.

[0191] Analysis of the contribution of TPS-induced products to the gonads of E13.5 fetuses

[0192] TPS cells were microinjected into 8-cell ICR diploid mouse embryos. The injected embryos were transferred to the uterine horns of pseudopregnant females at 0.5 post-mating. E13.5 embryos were collected for analysis of tdTomato and OCT4-GFP expression.

[0193] Immunofluorescence of E4.5 embryos

[0194] E4.5 embryos were washed three times in drops of PBS and then fixed in drops of 4% paraformaldehyde (DingGuo, AR-0211) for 20 minutes at room temperature. Embryos were blocked for 1 hour at room temperature with PBS (Corning, 21-040-CVR) containing 0.2% Triton X-100 (Sigma-Aldrich, T8787) and 3% normal donkey serum (Jackson ImmunoResearch, 017-000-121). Primary antibodies were diluted in blocking solution and embryos were incubated overnight at 4°C. Embryos were washed three times in drops of PBS and then incubated with secondary antibodies (Jackson ImmunoResearch) for 1 hour at room temperature. After the final wash, blastocysts were transferred to confocal dishes in drops of PBS covered with paraffin for imaging. Confocal microscopy was performed using a Leica TCS-SP8. The following primary antibodies were used: anti-tdTomato (1:2000; SICGEN, AB8181-200), anti-OCT4 (1:200; Santa CruzBiotechnology, sc-8626), anti-PDGFRA (1:200; R&D, AF1062), anti-CDX2 (1:200; BIOGENEX, MU392A), anti-EOMES (1:500; Abcam, ab183991), anti-TFAP2C (1:500; Abcam, ab218107), and anti-CK8 (1:200; Abcam, ab53280).

[0195] Immunofluorescence of E7.5 embryos

[0196] E7.5 embryos were dissected in PBS and then embedded in OCT (SAKURA, 4583). Embryos were frozen by liquid nitrogen vapor. Tissue sections of 10-15 mm thickness were cut in a cryostat and transferred to slides. The tissue was circled with a PAP pen and then fixed in 4% paraformaldehyde (DingGuo, AR-0211) for 15 minutes at room temperature and blocked with PBS (Corning, 21-040-CVR) containing 0.2% Triton X-100 (441 Sigma-Aldrich, T8787) and 3% normal donkey serum (Jackson ImmunoResearch, 017-000-121) for 1 hour at room temperature. After blocking, the placental tissue was incubated with the primary antibody overnight at 4°C. After washing the primary antibody three times with PBS, the secondary antibody (Jackson ImmunoResearch) was incubated for 1 hour at room temperature. Nuclei were stained with DAPI (Roche Life Science, 10236276001) at room temperature for 3 min and washed three times with PBS. Confocal microscopy was performed using Leica TCS-SP8 and Leica TCS-SP8 DIVE. The following primary antibodies were used: anti-tdTomato (1:2000; SICGEN, AB8181-200), anti-OCT4 (1:500; Abcam, ab181557), anti-EOMES (1:500; Abcam, ab183991), and anti-SOX17 (1:200; R&D, AF1924).

[0197] Immunofluorescence of E10.5 placenta

[0198] E10.5 chimeric embryos were dissected in PBS. Fetuses, yolk sacs, and placentas were separated using pointed forceps. Placentas were embedded in OCT (SAKURA, 4583) and frozen with liquid nitrogen vapor. Tissues were circled with a PAP pen and then fixed with 4% paraformaldehyde (DingGuo, AR-0211) for 15 min at room temperature and blocked with PBS (Corning, 21-040-CVR) containing 0.2% Triton X-100 (Sigma-Aldrich, T8787) and 3% normal donkey serum (Jackson ImmunoResearch, 017-000-121) for 1 h at room temperature. After blocking, placental tissues were incubated with primary antibodies overnight at 4°C.

[0199] Secondary antibodies (Jackson ImmunoResearch) were incubated for 1 hour at room temperature. After washing three times with PBS, the primary antibodies were used. Cell nuclei were stained with DAPI (Roche Life Science, 10236276001) for 3 minutes at room temperature and washed three times with PBS. Confocal microscopy imaging was performed using Leica TCS-SP8 and Leica TCS-SP8 DIVE. The following primary antibodies were used: anti-tdTomato (1:2000; SICGEN, AB8181-200), anti-TFAP2C (1:500; Abcam, ab218107), anti-CK8 (1:500; Abcam, ab53280), and anti-MCT4 (1:200; Millipore, AB3314P).

[0200] Flow cytometric analysis of chimeric placental tissue

[0201] Chimeric GFP-positive placental tissue was gently dissociated and digested into single cells using 0.1% collagenase IV (Gibco, 17104019; dissolved in Ca2+ / Mg2+-free PBS with 10% FBS and 100 μg / ml DNase I). The suspension was filtered through a cell strainer (100 mm). Samples were then analyzed on an Arial Sorp (BD Biosciences) or CytoFLEX (BeckmanCoulter) to detect the presence of tdTomato and GFP expression. Data analysis was performed using FlowJo software (Ashland).

[0202] Production of blastoid

[0203] TPS cells were dissociated into single cells using 0.05% trypsin-EDTA. Cells were suspended in induction medium on AggreWell (Stemcell, 34415), which contained a 1:1:1 mixture of the following three media: TS conditioned medium; N2B27 basal medium; and KSOM plus human BMP4 (100 ng / ml; Stemimmune, HST-B4-0100), human bFGF (100 ng / ml; Novoprotein, C046), mouse FGF4 (25 ng / ml; Biotech, 5846-F4), LPA (10μM; Sigma, 857228P) and Y27632 (10μM; Tocris, 1254). 3×10 cells were seeded per well. 4 After 4 to 6 days, blastocysts are formed.

[0204] Immunofluorescence of blastocysts

[0205] The blastocysts were washed three times in a drop of PBS and then fixed in a drop of 4% paraformaldehyde (DingGuo, AR-0211) at room temperature for 20 minutes. The embryos were blocked for 1 hour at room temperature using PBS (Corning, 21-485 040-CVR) containing 0.2% Triton X-100 (Sigma-Aldrich, T8787) and 3% normal donkey serum (Jackson ImmunoResearch, 017-000-121). The primary antibody was diluted with blocking solution and the embryos were incubated overnight at 4°C. The embryos were washed three times in a drop of PBS and then incubated with a secondary antibody (Jackson ImmunoResearch) at room temperature for 1 hour. After the final wash, the blastocysts were transferred to a confocal dish in a drop of PBS covered with paraffin for imaging. Confocal microscopy imaging was performed using a Leica TCS-SP8. The following primary antibodies were used: anti-OCT4 (1:500; Abcam, ab181557), anti-CDX2 (1:200; BioGenex, MU392A).

[0206] Immunoblotting

[0207] To detect histone proteins, a histone protein extraction kit (beibokit, BB-3117) was used to extract the proteins from 5x10 6 To detect β-catenin protein, 5 × 10 cells were isolated using RIPA lysis buffer (Beyotime Technology Technology, P0013B) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, 78443) and a phosphatase inhibitor cocktail (Thermo Fisher Scientific, 78428). 6Whole cell protein extracts were isolated from 10 cells. The amount of protein was determined using a bicinchoninic acid (BCA) assay kit (Applygen, P1511). The blots were incubated in 5% skim milk powder / TBST at room temperature for 1 hour and then incubated with the following antibodies in 5% BSA or 5% skim milk powder / TBST at 4°C overnight: anti-histone H3 (1:1000, Abcam, ab1791), anti-acetyl histone H3 (1:1000, Millipore, 06-599), anti-acetyl histone H4 (1:1000, Millipore, 06-866), anti-dimethyl histone H3 (Lys79) (1:1000, Millipore, 04-835), anti-β-catenin (1:2000, CELL SIGNALING, 8480), anti-GAPDH (1:4000, Applygen, C1312). The primary antibody was washed with TBST, and the samples were further incubated with secondary antibodies for 1 hour at room temperature with shaking. The following secondary antibodies were used: goat anti-rabbit IgG, HRP-linked antibody (1: 3000; ZSGB-BIO, ZB-2301) and goat anti-mouse IgG, HRP-linked antibody (1: 3000; ZSGB-BIO, ZB- 2305). The blots were developed using immunoblotting luminol reagent (Santa Cruz, sc-2048).

[0208] Generation of p53 and Dux knockout mEPS and mTPS cell lines

[0209] mEPS or mTPS cells carrying the Zscan4-Emerald GFP reporter gene were co-transfected with the px330 plasmid encoding Cas9 and sgRNA (Addgene, 42230) by nucleofection (4D - Nucleofector System, Lonza). Cells from the entire well were collected and total RNA was extracted for further analysis by Q-PCR.

[0210] shRNA knockdown

[0211] Hdac1 / 2 or Dot1l shRNA was transfected into EPS cells by nucleofection (4D- Nucleofector System, Lonza). EPS cells were then cultured in LCDM for 2 days. On day 3, EPS cells were transfected with the same shRNA by nucleofection, and cells were plated under 4S conditions or 4S conditions without VPA / EPZ004777. The medium was changed every day. Puromycin (0.8ug / ml) was added to the medium to enrich for cells expressing shRNA. After 3 days, cells were collected and total RNA was isolated for Q-PCR analysis.

[0212] Induced Oct4 knockout in TPS cells

[0213] The Oct4 conditional knockout mouse embryonic stem cell line ZHBtc4 (Niwa, H., Miyazaki, J. &Smith, AG Quantitative expression of Oct-3 / 4 defines differentiation,dedifferentiation or self-renewal of ES cells. Nature genetics 24, 372-376,doi:10.1038 / 74199 (2000)) was transformed into TPS cells using 4S conditions for more than 5 generations. Tetracycline (1 μg / ml) was added to the 4S conditions to induce Oct4 knockout in ZHBtc4-TPS cells. After 3 days of treatment, the proliferation of these cells was detected. The treated and untreated cells were then collected and total RNA was isolated for Q-PCR analysis.

[0214] Analysis of the Effects of a 4S Chemical Mixture on the Maintenance of Totipotency in Mouse Preimplantation Embryos

[0215] Mouse middle 2-cell embryos were collected and treated with 4S chemical mixture (VPA 500 μM, EPZ004777 1 μM, CD1530 0.5 μM, CHIR 99021 3 μM) added to KSOM medium. KSOM medium supplemented with DMSO was used as a control. After 48 hours, the embryos were collected and lysed with Trizol reagent. Total RNA was extracted from the lysate, which was further used to synthesize cDNA. cDNA was prepared and amplified using the Smart-seq2 method. The amplified cDNA product was diluted 10 times as required for qPCR template. Quantitative PCR analysis was performed on the Bio RAD CFX Connect real-time system using the KAPA SYBR FAST qPCR kit (KAPA Biosystems, KK4601). The primers used for Q-PCR are listed in Table 1.

[0216] Analysis of the effects of HDAC inhibition, DOT1L inhibition, RAR activation / inhibition, and LIF signaling on the induction of pluripotency in TPS cells using small molecules

[0217] EPS cells were seeded onto feeder cells. To replace VPA, EPZ004777, CD1530, and CHIR 99021, these small molecules were removed from the 4S condition and small molecules targeting the same target were added. After 3 days of induction, cells were collected for further analysis. Small molecules that replaced VPA included TSA (1 nM; Selleck, S1045), MS275 (1 μM; Selleck, S1053), Scriptaid (1 μM; Selleck, S8043), and MGCD0103 (20 nM; Selleck, S1122). Small molecules that replaced EPZ004777 included EPZ5676 (3 μM; Selleck, S5676) and SGC0946 (0.05μM; Selleck S7079). Small molecules that displace CD1530 include AM580 (0.05 μM; Selleck, S2933), ch55 (0.05 μM; MCE, HY-107397), parovastatin (0.01 μM; MCE, HY-14799), CD3254 (50 nM; MCE, CD3254), and RA (0.5 μM; MCE, HY-14649). Small molecules that replaced CHIR 99021 included AZD2858 (2 μM; MCE, HY-15761), LY2090314 (1 μm; MCE, HY-16294), BIO (2 μm; MCE, HY-10580), CHIR98014 (2 μm; MCE, HY-13076), and SB216763 (10 μM; MCE, HY-216763). To inhibit RAR signaling during the culture of TPS cells, RARγ inhibitor: LY2955303 (1 μm, MCE, HY-107765); RARα / β inhibitor: LE135 (2 μm, MCE, HY-107436); RXR inhibitor: UVI3003 (1 μm, MCE, HY-107500) were added to 4S medium, respectively. To inhibit LIF-Stat3 signaling during TPS cell culture, AG490 (10 μM; MCE, HY-12000), niclosamide (1 μM; MCE, HY-B0497), and JAK inhibitor (10 μM; Millipore, 420097) were added to 4S medium, respectively. After 4-6 passages, cells were collected for further analysis.

[0218] Bulk RNA-seq analysis

[0219] Total RNA was isolated from mouse TPS and EPS cells using the RNeasy mini kit (Qiagen, 74106). RNA sequencing libraries were constructed by using magnetic beads with oligonucleotide (DT)-enriched mRNA. Afterwards, fragmentation buffer was added to break the mRNA into short fragments. Using mRNA as a template, cDNA strands were synthesized with 6-base random primers. Buffer, dNTPs, DNA polymerase I, and RNase H were then added to synthesize double-stranded cDNA, which was purified with AMPURE XP beads. The purified double-stranded cDNA was repaired, a-tailed, and sequenced, and then the fragment size was selected by AMPURE XP beads. Finally, PCR amplification was performed, and the PCR products were purified with AMPURE XP beads to obtain the final library. The fragmented and randomly primed 2×100 bp paired-end library was sequenced using Illumina HiSeq 2500. The generated sequencing reads were plotted against the mouse genome build GRCm38.p6 for mice using STAR v2.7.3a. The read counts for each gene were calculated and the expression values ​​of each gene were normalized using TPM. Hierarchical clustering analysis was performed using the PCA function of the FactoMineR package and the ward.D algorithm in R software. Differentially expressed genes were analyzed using DESeq2 and filtered by adjusted P < 0.05. Gene ontology analysis was performed using the topGO package and org.Mm.eg.db. Single-cell RNA sequencing data from preimplantation mouse embryos (GSE45719 (Deng, QL, Ramskold, D., Reinius, B. & Sandberg, R.Single-Cell RNA-Seq Reveals Dynamic, Random Monoallelic Gene Expression inMammalian Cells. Science 343, 193-196, doi:10.1126 / science.1245316 (2014))) were reanalyzed and the raw counts were normalized using TPM. The TPM matrix was used to calculate the average expression of differentially expressed genes between TPS cells and EPS cells in preimplantation embryos at different developmental stages. Box plots are used to illustrate the expression of these genes during preimplantation development. In order to identify the omnipotence characteristics of 2-cell embryos, the default parameters of the expression matrix from the single-cell RNA sequencing data (GSE45719) of mouse preimplantation embryos were used to construct Seurat objects. The "vst" algorithm was used to identify the top 2000 most variable genes, which were further standardized and normalized to perform PCA analysis. Using the genes from the first 11 PCA components, KNN maps and UMAP maps were constructed.The epiblast population was identified from blastocyst cells by co-expression of Oct4, Nanog, and Sox2. Differentially expressed genes between 2-cell embryos and epiblast cells were identified using Findmarker (logfc.threshold = 1, min.pct = 0.6, test.use = 'DESeq2'). A total of 2399 genes were identified as 2-cell embryo enriched genes (padj < 0.0001).

[0220] 10x Genomics Single Cell RNA Sequencing

[0221] Cells were washed and resuspended in 1X PBS (without calcium and magnesium) containing 10% fetal bovine serum. Cell viability was determined by Count Star and the viable cell density was adjusted to 300-600 viable cells per microliter. The cell suspension was loaded onto the Chromium Single Cell Controller (10xGenomics). Single cell gel beads were generated in emulsion using the Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (10x Genomics, 1000075) and Chromium Single Cell BChip Kit (10x Genomics, 1000074) according to the manufacturer's protocol. Briefly, single cells were suspended in PBS containing 0.04% BSA. Approximately 6000 cells were added to each channel and target cells (approximately 3000 cells) were recovered. The captured cells were lysed and the released RNA was barcoded by reverse transcription in a separate GEM. Reverse transcription was performed at 53°C for 45 minutes on a S1000TM Touch Thermal Cycler (Bio Rad), then at 85°C for 5 minutes and kept at 4°C. cDNA was generated, then amplified, and quality assessed using an Agilent 4200 (performed by CapitalBioTechnology, Beijing). Single-cell RNA-seq libraries were constructed using the Single Cell 3' Libraryand Gel Bead Kit V3.1 according to the manufacturer's instructions. Finally, the library was sequenced using an Illumina Novasek6000 sequencer with a sequencing depth of at least 100,000 reads per cell, using a paired-end 150 bp (PE150) read strategy (performed by CapitalBio Technology, Beijing).

[0222] 10x Genomics ATAC sequencing

[0223] Cells were washed and resuspended in 1X PBS (calcium and magnesium free) containing 10% FBS. Cell viability was determined by Count Star. Nuclei were isolated and washed by the method provided by 10X Genomics: "Nuclei Isolationfor Single Cell ATAC Sequencing" (CG000169). The nuclei were then resuspended with ice-cold diluted nuclear buffer (10x Genomics; 2000153). The volume of nuclear dilution buffer used to resuspend the nuclei was based on the number of starting cells and the concentration of the final target nuclei. Countstar (Rigel S2) was used to count the nuclei. Then, the nuclei were immediately subjected to single cell ATAC-seq library construction. The nuclei were segmented into nanoliters of GEM using the ChromiumChip E Single Cell Kit (Product Code 1000156) and the Chromium Single Cell ATAC Library & Gel Bead Kit (Product Code 1000110). ~750,000 10x barcodes were sampled to individually and uniquely index the transposed DNA of each individual nucleus. Libraries were then generated (performed by CapitalBio Technology, Beijing). These libraries were sequenced using an Illumina Novaseq sequencer with a sequencing depth of at least 25k reads per nucleus using a paired-end 50 bp (PE50) read strategy.

[0224] Whole-genome bisulfite sequencing (WGBS)

[0225] Genomic DNA was extracted by DNeasy blood and tissue kit (QIAGEN, 69504). 100 ng genomic DNA was fragmented into about 200 bp by Covaris. Then, DNA was subjected to bisulfite conversion using EZ DNA Methylation-GoldTM kit (Zymo, D5005). Bisulfite-converted DNA was captured using Accel-NGS Methyl-Seq DNA Library kit (Swift Biosciences, 30024). Library samples were subjected to IlluminaNova-seq 6000 sequencing system.

[0226] Single-cell RNA-seq preprocessing

[0227] For all our samples, single-cell RNA-seq data were collected and mapped to the mouse reference genome mm10 using Cell Ranger 3.1.0. We used Seurat 3.2.3 (Stuart, T. et al. Comprehensive Integration of Single-Cell Data. Cell 177, 1888-1902 e1821, doi:10.1016 / j.cell.2019.05.031 (2019)) for preprocessing in the R environment version 3.6.3. Specifically, quality control was first performed to remove cells with the following characteristics: (i) total UMI counts less than 2000, (ii) the number of detected genes less than 1500, or (iii) mitochondrial UMI counts greater than 20%. Normalization was performed using the function NormalizeData with default parameters. We performed the standard Seurat clustering pipeline using the following functions: FindVariableFeatures with 2000 genes, ScaleData, RunPCA, FindNeighbors with the first 10 PCs, and FindClusters with a resolution of 1.2. We then checked the scores for bimodality for each sample using the doubletCells function of the R package scran. We found that the scores were evenly distributed across clusters, indicating that there were no detected doublets between clusters, so we kept all cells for subsequent analysis. We performed batch effect correction using the R package Harmony (Korsunsky, I. et al. Fast, sensitive and accurate integration of single-cell data with Harmony. Nat Methods 16, 1289-1296, doi:10.1038 / s41592-019-0619-0 (2019)). Uniform manifold approximation and projection (UMAP) dimensionality reduction was performed on the first 10 corrected PCs using the function RunUMAP. Differentially expressed genes (DEGs) were analyzed based on normalized gene expression using the FindMarkers function.

[0228] Transcriptional integration analysis of embryonic developmental states and different stem cells

[0229] Public dataset collection

[0230] We collected 12 public datasets to construct mouse embryonic development trajectories as references (GSE45719 from Deng et al., 2014 (Deng, QL, Ramskold, D., Reinius, B. & Sandberg, R. Single-Cell RNA-Seq Reveals Dynamic, Random Monoallelic Gene Expression in Mammalian Cells. Science 343, 193-196, doi:10.1126 / science.1245316 (2014)); GSE109071 from Cheng et al., 2019 (Cheng, SL et al. Single-Cell RNA-Seq Reveals Cellular Heterogeneity of Pluripotency Transition and X Chromosome Dynamics during Early Mouse Development. Cell Reports 26, 2593-+, doi:10.1016 / j.celrep.2019.02.031 (2019)); GSE44183 from Xue et al., 2013 (Xue, ZG et al. Genetic programs in human and mouse early embryos revealed by single-cell RNAsequencing. Nature 500, 593-+, doi:10.1038 / nature12364 (2013)); GSE71434 from Zhang et al., 2016 (Zhang, BJ et al. Allelic reprogramming of the histonemodification H3K4me3 in early mammalian development. Nature 537, 553-+, doi:10.1038 / nature19361 (2016)); GSE66582 from Wu et al., 2016 (Wu, JY et al. The landscape of accessible chromatin in mammalian preimplantation embryos. Nature 534, 652-+, doi:10.1038 / nature18606 (2016)); GSE70605 from Liu et al., 2016 (Liu, XY et al. Distinct features of H3K4me3 and H3K27me3 chromatin domainsin pre-implantation embryos. Nature 537, 558-+, doi:10.1038 / nature19362(2016)); GSE100597 from Mohammed et al., 2017 (Mohammed, H. et al. Single-Cell Landscape of Transcriptional Heterogeneity and Cell Fate Decisions during Mouse Early Gastrulation. Cell Reports 20, 1215-1228, doi:10.1016 / j.celrep.2017.07.009 (2017)); GSE84892 from Posfai et al., 2017 (Posfai, E. et al. Position-and Hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo. eLife 6, 24, doi:10.7554 / eLife.22906 (2017)); DRP005519 from Israel et al., 2019 (Israel, S. et al. An integrated genome-wide multi-omics analysis of gene expression dynamics in the preimplantation mouse embryo. Sci Rep 9, 13356, doi:10.1038 / s41598-019-49817-3 (2019)); GSE121708 from Argelaguet et al., 2019 (Argelaguet, R. et al. Multi-omics profiling of mouse gastrulation at single-cell resolution. Nature 576, 487-+, doi:10.1038 / s41586-019-1825-8 (2019)); GSE136714 is from Wang et al., 2021 (Wang, Y. et al. Single-cell multiomics sequencing reveals the functional regulatory landscape of early embryos. Nat. Commun. 12, 14, doi:10.1038 / s41467-021-21409-8 (2021)); GSE145609 is from Posfai et al., 2021 (Posfai, E. et al. Evaluating totipotency using criteria of increasing stringency. Nat. Cell Biol. 23, 32, doi:10.1038 / s41556-020-00609-2 (2021))). From these datasets, we used cells from the zygote to E7.5 EPI, as well as extraembryonic lineages. Cells of the epiblast lineages after embryonic day 5 were defined as postimplantation epiblast.

[0231] We also collected 4 stem cell datasets for comparison with our data (GSE33923 from Macfarlan et al., 2012 (Macfarlan, TS et al. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487, 57-+, doi:10.1038 / nature11244 (2012)); GSE168728 from Shen et al., 2021 (Shen, H. et al. Mouse totipotent stem cells captured and maintained through spliceosomalrepression. Cell 184, 2843-+, doi:10.1016 / j.cell.2021.04.020 (2021)); GSE74155 from Chen et al., 2016 (Chen, G. et al. Single-cell analyses of X Chromosomeinactivation dynamics and pluripotency during differentiation. Genome Res.26, 1342-1354, doi:10.1101 / gr.201954.115 (2016)); GSE145609 from Posfai et al., 2021 (Posfai, E. et al. Evaluating totipotency using criteria of increasing stringency. Nat. Cell Biol. 23, 32, doi:10.1038 / s41556-020-00609-2 (2021))). From these data sets, ESCs, EPSCs, EPiSCs to be derived, 2CLCs, and TBLCs were used for subsequent analysis.

[0232] The raw sequencing data of all public datasets were downloaded and preprocessed, and then the normalized expression data were obtained by following the same steps using the same measurement as our data (logTP10K, log-normalized transcripts per 10,000).

[0233] Embryonic development trajectory construction

[0234] To integrate multiple datasets together to form a continuous developmental trajectory, we performed latent semantic indexing (LSI) projection as described previously (Granja, JM et al. Single-cell multiomic analysis identifies regulatory programs in mixed-phenotype acute leukemia. NatBiotechnol 37, 1458-1465, doi:10.1038 / s41587-019-0332-7 (2019)). Specifically, we used GSE45719 and GSE109071 as “stem” datasets and merged them directly. We chose these two datasets because: (i) they were both sequenced using Smart-Seq2, (ii) they both had sufficient cell numbers for each developmental state to identify biological differences along the trajectory, and (iii) they were widely used for comparison in previous studies. We performed LSI dimensionality reduction on the cells of these two datasets using the top 2000 variable genes. The other 10 datasets were then projected into the LSI space. The coordinates of all cells in the LSI space were used only for visualization.

[0235] Stem cell data integration

[0236] To compare stem cells and embryonic developmental stages from multiple datasets, we projected the stem cell data into the above LSI space. Due to the low sequencing depth per cell on the 10x platform, we combined cells from our data with GSE168728 into a pseudo-bulk sample. The first two LSI dimensions of stem cell samples and cells of the ectoderm lineage were used for visualization. Cells at the same developmental stage are represented as a point at the median of their coordinates.

[0237] Calculation of embryonic development stage characteristics

[0238] We used the entropy weight method (EWM) to calculate the gene signature for each embryonic development stage by combining multiple DEG fold change values ​​with the comprehensive score of each gene. For multiple datasets, DEGs were calculated using DESeq2 (Love, MI, Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550, doi:10.1186 / s13059-014-0550-8(2014)).

[0239] Single sample gene set enrichment analysis

[0240] We used the R package GSVA to perform single-sample gene set enrichment analysis (ssGSEA) to quantify the similarities between stem cell samples and embryonic development stages. As a control, cells along the embryonic development trajectory were pooled and subjected to the same analysis. The enrichment score for each feature was further normalized by dividing by the value that could be achieved with the positive control for better visualization.

[0241] Embryo stage identity analysis

[0242] To assess the degree of similarity between stem cell samples and a given embryonic stage, we performed quadratic programming-based deconvolution analysis using the R package quadprog as previously described (Treutlein, B. et al. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq. Nature 534, 391-395, doi:10.1038 / nature18323 (2016)).

[0243] Transcriptional integration analysis of blastocysts and blastocystoids

[0244] We constructed the blastocyst developmental trajectory using cells from a subset of E3.5 in the whole embryonic developmental trajectory following the same steps as described above. Our blastocyst-like data were collected and preprocessed identically to those described above. Two public datasets were collected for comparison (GSE99786 from Rivron et al., 2018 (Rivron, NC et al. Blastocyst-like structures generated solely from stem cells. Nature 557, 106-+, doi:10.1038 / s41586-018-0051-0 (2018)); GSE134240 from Sozen et al., 2019 (Sozen, B. et al. Self-Organization of Mouse Stem Cells into an Extended Potential Blastoid. Dev. Cell 51, 698-+, doi:10.1016 / j.devcel.2019.11.014(2019))). LSI projection, ssGSEA, and identity score analyses were performed as described above.

[0245] Single-cell gene regulatory network analysis

[0246] According to the standard pySCENIC pipeline ( https: / / pyscenic.readthedocs.io / en / latest / index.html ), we performed Single-CellrEgulatory Network Inference and Clustering (SCENIC) analysis on our data (Aibar, S. et al. SCENIC:single-cell regulatory network inference and clustering. Nat Methods 14,1083-1086, doi:10.1038 / nmeth.4463 (2017)). The R package AUCell was used to calculate the regulatory sub-activity of cells in other datasets.

[0247] Single-cell ATAC-seq analysis

[0248] For all our samples, single-cell ATAC-seq data were collected using Cell Ranger ATAC 1.2.0 and mapped to the mouse reference genome mm10. Downstream analysis was performed according to the standard ArchR (Granja, JM et al. ArchR is a scalable software package for integrative single-cell chromatinaccessibility analysis. Nat Genet 53, 403-411, doi:10.1038 / s41588-021-00790-6(2021)) pipeline ( https: / / www.archrproject.com ) is executed.

[0249] We collected public ATAC-seq data of mouse preimplantation embryos (GSE66581 from Wu et al., 2016) for comparative analysis.

[0250] WGBS analysis

[0251] We followed the standard Bismark(Krueger, F. & Andrews, SR Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27, 1571-1572, doi:10.1093 / bioinformatics / btr167 (2011)) workflow to perform mapping, deduplication, and methylation extraction for all our samples ( https: / / github.com / Felix Krueger / Bismark ). Only CpG sites were used for further analysis and visualization.

[0252] We collected public methylome data of mouse embryos (GSE56697, from Wang et al., 2014 (Wang, L. et al. Programming and Inheritance of Parental DNA Methylomes in Mammals. Cell 157, 979-991, doi:10.1016 / j.cell.2014.04.017 (2014))) for comparative analysis.

[0253] Data availability

[0254] The RNA-seq, WGBS, scRNA-seq, and scATAC-seq data generated during this study are available at GEO:GSE183522.

[0255] Results and Discussion

[0256] Identification of a chemical cocktail for establishing totipotent-like stem cells in vitro

[0257] To identify small molecules that can induce pluripotency characteristics in mouse EPS cells, we used cell lines carrying MERVL-TdTomato or Zscan4-Emerald GFP reporters. As a positive control, Dux overexpression was performed in these reporter cells ( Figure 6ac). Screening was performed using N2B27 medium, and the Wnt signaling agonist CHIR99021 was added to promote cell proliferation. In the initial screening, we focused on identifying small molecules that could increase the percentage of Zscan4-Emerald GFP+ cells and identified more than 50 primary hits. Using MERVL-tdTomato reporter cells, these candidates were further evaluated and small molecules that were also able to induce MERVL-tdTomato were selected to further determine their optimal doses for activating Zscan4-Emerald and MERVL-tdTomato expression without significant toxicity. After titration, we found that CD1530 was most effective in inducing Zscan4-GFP and MERVL-tdTomato expression ( Figure 6 df). Therefore, we tested the effect of combining CD1530 with other selected candidates to induce pluripotent marker genes and found that VPA and EPZ004777 had synergistic effects with CD1530 in inducing pluripotent markers ( Figure 6 g). We also tested whether CHIR99021 could be omitted from the mixture and found that during transformation, cell proliferation was greatly reduced under this condition ( Figure 6 hi). Therefore, CHIR 99021 was included in the chemical mixture.

[0258] We further analyzed whether cells cultured under the conditions of 4 small molecules (4S) can stably maintain and retain their pluripotent characteristics in vitro. Cells cultured under 4S can be maintained and passaged for more than 15 generations with normal karyotype ( Figure 1 ab). After long-term culture (more than 10 generations), 4S-treated cells still maintained the expression of pluripotent markers such as Zscan4, Zfp352, Tcstv1, Tcstv3, MERVL element, Dux, Dub1a, Eif1al6, Eif1al9, Gm4340, and Tdpoz4 ( Figure 1 c), and the protein expression of ZSCAN4 could be detected in these cells ( Figure 6 j). Meanwhile, pluripotency markers including Oct4, Nanog, and Sox2 were significantly downregulated in these cells ( Figure 1 c). We also compared the expression of pluripotency marker genes in 4S-treated cells of different passages and found that cells after passage 5 showed relatively stable upregulation of pluripotency marker genes, which means that at least 5 passages are required to obtain stable pluripotency characteristics in 4S-treated cells ( Figure 6 k). Consistent with the Q-PCR analysis, bulk RNA sequencing analysis also confirmed the upregulation of multiple pluripotency marker genes and the downregulation of multiple pluripotency marker genes ( Figure 1 d).

[0259] To analyze the stability of cells expressing pluripotent marker genes, we also enriched these cells using the Zscan4-Emerald GFP reporter gene and cultured them in vitro for 5 generations. The cells treated with 4S proliferated well after sorting, while the purified cells without 4 small molecules grew poorly ( Figure 6 l). Further Q-PCR analysis showed that the expression of pluripotency marker genes in 4S-treated cells could be maintained after purification and culture ( Figure 6 m). We also tested whether 4S conditions could induce the expression of pluripotency marker genes in mouse ES cells and found that after five passages, ES cells treated with 4S upregulated multiple pluripotency marker genes ( Figure 6 n). Together, these results suggest that 4S-treated cells acquire totipotent molecular features that are distinct from conventional pluripotent cell types.

[0260] Since the 4S-treated cells showed omnipotent molecular features, we further explored whether they had in vitro extraembryonic developmental potential. To this end, the 4S-treated cells were cultured and passaged in mouse trophoblast stem cell (TS) medium. After 2 to 4 passages, multiple flat mouse TS-like colonies appeared, which could be further passaged in mouse TS medium. Immunofluorescence analysis showed that these TS-like cells expressed TS markers including EOMES, TFAP2C CDX2, and SOX2 ( Figure 7 a), indicating that they have acquired characteristics of the trophoblast lineage. Consistent with these results, 4S-treated cells can also be induced to express markers of primitive endoderm (PE) after being cultured in a medium that induces PE differentiation ( Figure 7 b). Next, we evaluated the developmental potential of 4S-treated cells by performing in vitro chimera experiments. 4S-treated cells carrying fluorescent reporter genes were injected into 8-cell mouse embryos cultured in vitro for 48 h. Immunofluorescence analysis of chimeric mouse embryos showed that induced products from 4S-treated cells expressed markers of trophectoderm (TFAP2C, CK8, CDX2, EOMES), ectoderm (OCT4), and primitive endoderm (PDGFRα) ( Figure 1 e and Figure 8 ).

[0261] To further explore the developmental potential of 4S-treated cells in vivo, we injected these cells into immunodeficient mice and obtained teratomas, which were analyzed using single-cell RNA sequencing analysis. Notably, in addition to the presence of multiple embryonic lineages in teratomas, we found that teratomas derived from 4S-treated cells also contained extraembryonic lineages ( Figure 1fg). Next, we examined the in vivo chimerism of 4S-treated cells and found that 4S-treated cells after 5 passages showed relatively high chimerism (Table 2). We also tested the germline competence of 4S-treated cells by injecting 4S-treated cells into 8-cell embryos and transplanting the injected embryos in vivo to generate chimeric embryos. Analysis of E13.5 chimeric embryos showed that the induced products of these cells could efficiently form chimeras in fetal gonads, which expressed OCT4-GFP ( Figure 1 ). Together, these results indicate that cells cultured under this novel condition can be maintained long-term while retaining the ability to express pluripotency marker genes and bidevelopmental potential, and we therefore named these cells pluripotent stem cells (TPS cells).

[0262] Table 2: Overview of Chimerism

[0263]

[0264] We further explored whether TPS cells could be induced directly from 2-cell embryos. To this end, 2-cell mouse embryos without zona pellucida were plated on feeder cells and cultured under 4S conditions. We found that some embryos transformed into dense cell clumps under 4S conditions and then developed outgrowths ( Figure 1 i). Notably, immunofluorescence analysis revealed that the pluripotent marker ZSCAN4 was detectable in outgrowths, whereas the expression of the pluripotent marker OCT4 was greatly reduced compared with that in 2i / LIF-treated outgrowths ( Fig. 9 The outgrowths can be further passaged, resulting in the subsequent appearance of dome-shaped TPS colonies showing expression of ZSCAN4 ( Figure 1 j). Similar to TPS cells induced from EPS cells, TPS cells established from 2-cell embryos also expressed pluripotent marker genes ( Figure 1 c) These results indicate that 4S conditions can induce TPS cells directly from 2-cell mouse embryos.

[0265] TPS cells share transcriptomic signatures with 2-cell blastomeres

[0266] To explore the molecular characteristics of TPS cells, we used bulk RNA sequencing data to analyze the transcriptomic differences between TPS cells and EPS cells. Hierarchical clustering analysis showed that the overall transcriptome of TPS cells was different from that of EPS cells ( Fig.10 a). Identification of differentially expressed genes between TPS cells and EPS cells showed the presence of representative pluripotency marker genes such as Gm5662, Zscan4c, and Zfp352 ( Fig.10b). We further performed gene ontology (GO) analysis and found that the differentially expressed genes were mainly involved in the regulation of development ( Fig.10 c).

[0267] Next, we analyzed the expression levels of these differentially expressed genes in early preimplantation development. Notably, genes upregulated in TPS cells were also highly expressed in the 2C embryonic stage, and their expression gradually decreased in the blastocyst stage ( Fig.10 d). In contrast, genes downregulated in TPS cells are more abundantly expressed in blastocyst stage rather than 2C embryo stage ( Fig.10 d). We also used published single-cell RNA sequencing data of preimplantation mouse embryos (Deng, Q., Ramsköld, D., Reinius, B. & Sandberg, R. Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells. Science 343, 193-196, doi:10.1126 / science.1245316 (2014)) to identify 2399 genes, including pluripotency features, that were upregulated in 2-cell pluripotent embryos compared with the epiblast cells from the blastocyst. Among these genes, 1585 genes were significantly upregulated in TPS cells compared with EPS cells ( Fig.10 e) These data indicate that TPS cells have a transcriptomic signature that is unique to 2-cell embryos.

[0268] To further explore the transcriptomic characteristics of TPS cells, we performed single-cell RNA sequencing using TPS cells. As controls, single-cell RNA sequencing data of mouse EPS cells, ES cells, 2C-like cells, and the recently reported totipotent blastomere-like cells (TBLCs) were also analyzed. Consistent with bulk RNA sequencing analysis, we found that most TPS cells expressed multiple totipotent marker genes ( Figure 2 a), whose expression levels vary among the populations. Notably, differential expression of omnipotence marker genes was also observed in TBLC ( Fig.11a), which is consistent with a recent report (Lin, PY et al. ComparativeAnalyses of Single-Cell Transcriptomic Profiles between In Vitro TotipotentBlastomere-like Cells and In Vivo Early Mouse Embryonic Cells. Cells 10, doi:10.3390 / cells10113111 (2021)). We further compared the transcriptomes of these in vitro cell types with those of early embryos from 2 cells to E7.5 ( Figure 2 b), and applied a quadratic programming-based analysis technique to quantify the transcriptomic similarity of these cell types to 2-cell embryos (Treutlein, B. et al. Dissecting directreprogramming from fibroblast to neuron using single-cell RNA-seq. Nature534, 391-395, doi:10.1038 / nature18323 (2016)). TPS cells had a higher identity score than other cell types ( Figure 2 c), suggesting that TPS cells transcriptomically resemble 2-cell embryos more than other cell types.

[0269] Due to the differences in the expression of pluripotent marker genes in the TPS cell population, we further explored the subpopulations present in the TPS cell population and identified a subset of cells (9.14% on average) in the TPS cell population that were transcriptomically similar to those of mid- to late-stage 2-cell embryos ( Figure 2 b). Importantly, the identity score of this TPS subset (TPS 2C subset) was the highest among all in vitro cell types and subsets analyzed ( Figure 2 c), which was also supported by single sample gene set enrichment analysis (ssGSEA) ( Figure 2 d). We also analyzed the transcriptomic differences between the TPS 2C subpopulation and 2-cell embryos. GO analysis showed that the genes differentially expressed between this TPS2C subpopulation and 2-cell embryos were mainly involved in the regulation of mitotic cell cycle, proteasome protein catabolism, nucleoside triphosphate metabolism, and oxidative phosphorylation (data not shown). We further analyzed the regulatory network of the TPS 2C subpopulation using SCENIC and compared it with the regulatory network of EPS cells. Notably, in this group, the enrichment level of totipotency regulators was upregulated, while the enrichment level of pluripotency regulators was downregulated ( Figure 2e-f). Together, these data indicate that TPS cells share the same totipotent transcriptional signature as 2-cell embryos and that the TPS cell subpopulation is transcriptomically similar to mid- to late-stage 2C embryos.

[0270] TPS cells have the same epigenetic signature as 2-cell blastomeres

[0271] To characterize the epigenetic signature of TPS cells, we first performed an assay for transposase-accessible nuclear chromatin using sequencing (ATAC-seq) of these cells at the single-cell level. EPS cells and ES cells were also analyzed as controls. We identified 1,857 open peaks and 8,903 closed peaks that were uniquely enriched at annotated or putative enhancers and promoters in TPS cells compared to EPS cells and ES cells ( Figure 2 g). The unique open loci in TPS cells include multiple genes specifically expressed in 2-cell embryos, such as Zfp352, Zscan4c, and Tcstv1 ( Fig.11 b). We further analyzed the distribution of TPS-specific open and closed peaks in 2-cell embryos and mouse ES cells. Obviously, TPS-specific open loci were also highly open in 2-cell embryos, while TPS-specific closed loci were in a more closed state in 2-cell embryos ( Figure 2 h). We further analyzed the global DNA methylation levels of TPS cells, EPS cells, and ES cells using whole-genome bisulfite sequencing (WGBS). Compared with EPS cells and ES cells, the global DNA methylation level of TPS cells was greatly reduced ( Figure 2 i), which is similar to 2-cell embryos. In addition, we also observed that DNA methylation levels were reduced in the loci of representative pluripotent genes such as Zfp352, Zscan4d, and Tcstv1 ( Fig.11 c) Taken together, these results suggest that TPS cells share epigenetic features with 2-cell blastomere cells in terms of accessible nuclear chromatin distribution and DNA methylation profile.

[0272] TPS cells are able to give rise to both embryonic and extraembryonic lineages in vivo

[0273] To rigorously analyze the developmental potential of TPS cells, we first tested their ability to generate embryonic and extraembryonic lineages in vivo in E7.5 mouse embryos. To this end, single tdTomato reporter-labeled TPS cells were injected into 8-cell embryos that were transferred in vivo. As a control, a single 8-cell blastomere ( Fig.12 a). Remarkably, we observed contributions from a single TPS-derived cell to both embryonic and extraembryonic regions in E7.5 mouse chimeric embryos ( Figure 3a and Table 2). Immunofluorescence analysis further confirmed that extraembryonic chimeric cells expressed EOMES in the extraembryonic ectoderm (ExE) region. Figure 3 bc), indicating that they adopted an extraembryonic trophoblast fate. We also noted that cells that integrated into the visceral endoderm (VE) region expressed the VE marker SOX17 ( Fig.12 b), implying their VE identity.

[0274] Next, we analyzed the developmental potential of single TPS cells in E10.5 mouse conceptuses (Table 2). Injected mouse embryos were transferred in vivo and recovered at E10.5. Significantly, compared with blastomeres of 8-cell embryos ( Fig.12 c) Similarly, extensive chimeric contributions of a single TPS-derived cell to the embryo, placenta, and yolk sac were observed ( Figure 3 d), which was also confirmed by FACS analysis ( Fig.12 d). To further confirm the trophoblastic identity of the chimeric cells in the placenta, we performed immunofluorescence analysis. Similar to a single 8-cell blastomere ( Fig.12 f) Positive control, the induced products of TPS cells integrated into the trophoblast of the chimeric placenta and expressed trophoblast markers CK8, MCT4 and TFAP2C ( Figure 3 e and Fig.12 e), indicating that these chimeric cells can further differentiate into the trophoblast lineage. As a negative control, we did not detect tdTomato expression in non-injected placentas ( Fig.12 g).

[0275] To further analyze the trophoblastic properties of chimeric cells in the late developing placenta, we generated E17.5-E18.5 chimeric conceptuses using TPS cells ( Fig.13 a). We performed single-cell RNA sequencing analysis using cells from chimeric E17.5 placentas ( Figure 3 f). Of the 9765 analyzed cells from chimeric placentas, 1924 cells expressed tdTomato ( Figure 3 g), indicating that they are induced products of TPS cells. Notably, these TPS-induced products contain extraembryonic lineages expressing trophoblast subtype markers, such as Ctsq (trophoblast giant cells), Prl3a1 (sponge trophoblast cells), Pla2g4d (glycogen trophoblast cells), and Lgr5 (labyrinth trophoblast progenitor cells) ( Figure 3 h). In addition, these TPS-derived trophoblasts were transcriptomically similar to in vivo trophoblasts ( Figure 3 f). These results were further supported by Q-PCR analysis using FACS-purified chimeric TPS-induced products from E18.5 placentas ( Fig.13 bc). To exclude potential artifacts of cell fusion in the placenta, we injected TPS cells labeled with the tdTomato reporter gene into 8-cell mouse embryos expressing GFP. Importantly, most chimeric tdTomato-positive cells in the placenta did not show GFP signal ( Fig.13 bc). Together, the chimeric analyses provide strong evidence that TPS cells have extraembryonic and embryonic developmental potential in vivo at the single-cell level.

[0276] Induction of TPS cells into blastocyst-like structures in vitro

[0277] Since TPS cells have embryonic and extraembryonic developmental potential, we explored whether they could be induced into blastocyst-like structures (blastocysts) in vitro. FGF, BMP and Yap signaling are very important for preimplantation development, especially extraembryonic lineage development, and we tried to treat TPS cells with bFGF, FGF4, BMP4 and LPA. TPS cells were seeded onto AggreWell microplates and treated with these factors to generate small aggregates. Significantly, these aggregates can further form cavities and are morphologically similar to preimplantation blastocysts ( Figure 4 a). Immunofluorescence analysis showed that blastocysts induced from TPS cells expressed trophectoderm (CDX2) and ectoderm (OCT4) markers ( Figure 4 b).

[0278] To analyze the transcriptomic characteristics of TPS-blastocysts, we performed single-cell RNA sequencing analysis. Cluster analysis divided the 914 cells analyzed into three clusters, which expressed markers of ectoderm, primitive endoderm, and trophectoderm, including Oct4 (ectoderm), Sox17 (primitive endoderm), and Gata2 (trophectoderm) ( Figure 4 c). It is also noteworthy that expression of pluripotency marker genes is almost absent in the three clusters ( Figure 4 c), indicating that cells in TPS-blastocysts have lost totipotent characteristics. Next, 262 representative lineage markers of ectoderm, primitive endoderm, and trophectoderm were analyzed in TPS-blastocysts. Consistent with the cluster analysis, the three clusters were well clustered in the ectoderm, primitive endoderm, and trophectoderm-like lineages, respectively ( Figure 4 d). We further compared the transcriptomic features of these TPS-blastocyst-like cells with those of E4.5 blastocysts and found that most of the TPS-blastocyst-like cells clustered with blastocyst cells belonging to the same lineage ( Figure 4 e).

[0279] We further analyzed the similarities between the transcriptomes of TPS-blastocyst-like cells and blastocyst cells. Ectoderm cells from E4.5 to E7.5 and extraembryonic blastoderm cells from postimplantation stages (E5.25 to E6.5) were also included in the analysis. As a control, we also analyzed blastocyst-like cells generated by combining mouse TS cells with mouse ES cells or EPS cells (Rivron, NC et al. Blastocyst-like structures generated solely from stemcells. Nature 557, 106-111, doi:10.1038 / s41586-018-0051-0 (2018); Sozen, B. et al. Self-Organization of Mouse Stem Cells into an Extended PotentialBlastoid. Dev Cell 51, 698-712.e698, doi:10.1016 / j.devcel.2019.11.014(2019)). Importantly, cells resembling ectoderm, primitive endoderm, and trophectoderm in TPS-blastocysts are transcriptionally similar to the three lineages from the E4.5 blastocyst stage ( Figure 4 f). Notably, trophectoderm-like cells from previously reported blastocysts showed significant transcriptomic differences compared to trophectoderm-like cells from TPS-blastocysts and in vivo trophectoderm ( Figure 4 f). These results were further supported by ssGSEA and quadratic programming-based identity score calculations ( Figure 4 gh). Together, these data indicate that TPS-blastocysts contain three blastocyst lineages that are transcriptomically similar to E4.5 blastocysts.

[0280] We further evaluated the in vivo developmental potential of TPS-blastocysts. To this end, tdTomato reporter-labeled TPS-blastocysts were transferred into pseudopregnant mice at 2.5 days post coitum (dpc). At 6.5 dpc, TPS-blastocysts were induced to decidualize ( Figure 4 ij), which was further supported by the induction of PTGS2 expression in the decidua. These results suggest that TPS-blastocysts can implant and initiate decidualization in vivo.

[0281] Study on the mechanism of induction and maintenance of pluripotency of TPS cells

[0282] We sought to explore the mechanisms regulating pluripotency using 4S conditions. Since VPA, CD1530, and EPZ004777 showed synergistic effects in inducing the expression of pluripotency marker genes, we first focused on analyzing the molecular targets of these three compounds. Among these 3 small molecules, VPA is reported to be an HDAC inhibitor, CD1530 is a RARγ agonist, and EPZ004777 is a Dot1L inhibitor. To analyze whether these small molecules indeed target these molecular targets in TPS cells, we first analyzed the levels of histone acetylation and H3K79 methylation in TPS cells by immunoblotting. Compared with EPS cells, we found that histone H3 acetylation levels were increased, while H3K79 methylation levels were decreased ( Figure 5 a). In addition, upregulation of classical RAR downstream target genes was observed in TPS cells ( Figure 5 b). Interestingly, we found that chromatin accessibility of RARγ binding sites was more closed in TPS cells compared with EPS cells, and similar results were observed in 2-cell embryos ( Fig.14 a).

[0283] To confirm that these small molecules indeed induced pluripotency by targeting these reported targets, we first replaced VPA, EPZ004777, and CD1530 with other small molecules targeting the same molecular targets, including HDAC, DOT1L, and RAR, respectively. We found that these replacements could also support the induction of pluripotency marker genes ( Figure 5 c and Fig.14 b). Consistent with these results, knockout of Hdac1 / 2 and Dot1l could also replace VPA and EPZ004777-induced pluripotency markers, respectively ( Figure 5 dg), and small-molecule inhibition of RARγ signaling greatly reduced the expression of pluripotent markers during TPS induction and maintenance ( Figure 5 h). Interestingly, Q-PCR analysis showed that treatment of preimplantation embryos with these four small molecules could promote the expression of pluripotent marker genes in preimplantation mouse embryos after the 2-cell embryonic stage ( Figure 5 i). Taken together, these data suggest that inhibition of HDAC1 / 2 and DOT1L activity and activation of RARγ signaling are important for inducing pluripotency in TPS cells.

[0284] Next, we sought to analyze the role of CHIR 99021 in the induction and maintenance of TPS cells. Consistent with the reported role in activating Wnt signaling through inhibition of GSK3β, we found that TPS cells expressed β-catenin at the protein level ( Fig.14c). To examine whether CHIR 99021 is required for the maintenance of TPS cells, we removed CHIR 99021 from the chemical mixture during the culture of TPS cells. The omission of CHIR 99021 resulted in reduced proliferation during the first few passages (approximately 3-5 passages). After further passages, we found that CHIR 99021 was not required for the proliferation of TPS cells ( Fig.14 d). Q-PCR analysis further showed that the omission of CHIR 99021 did not affect the expression of pluripotency marker genes in TPS cells after long-term culture ( Fig.14 e). However, we also found that omitting CHIR 99021 greatly reduced the in vivo chimerism of TPS cells (Table 2). Therefore, CHIR 99021 is beneficial for promoting proliferation during TPS transformation and is important for maintaining the in vivo developmental potential of TPS cells.

[0285] Finally, we sought to explore the role of known key regulators of totipotency and pluripotency in the induction and maintenance of TPS cells. Dux is an important regulator for the induction of 2C-like cells, and p53 has been reported to be a key upstream activator for the induction of 2C-like cells. During the maintenance of TPS cells, we found that knockdown of Dux expression led to the downregulation of several totipotency markers (MERVL, Zfp352, Tcstv3), while the expression of other totipotency markers (Tcstv1, Zscan4) was not significantly affected ( Figure 5 jk). On the other hand, p53 knockout did not result in decreased expression of pluripotent markers during TPS cell induction (Figure 51). Next, we investigated the role of Oct4 and LIF signaling in TPS cell maintenance, which are key pluripotent regulators. Knockout of Oct4 in TPS cells had no significant effect on the expression of pluripotent marker genes ( Fig.15 a), but the proliferation of TPS cells decreased ( Fig.15 b). Similar to Oct4, small molecule inhibition of LIF signaling also resulted in impaired TPS cell proliferation ( Fig.15 c), while the expression of pluripotency markers was not affected ( Fig.15 d). These data collectively suggest that the regulatory mechanism of pluripotency in TPS cells is unique and different from that in 2C-like cells and warrants further exploration.

[0286] discuss

[0287] Inducing totipotent stem cells that are functionally and molecularly similar to cells from totipotent embryos in vitro is challenging. Here, we report that a chemical cocktail is able to induce totipotent-like stem cells, termed totipotent stem (TPS) cells, from 2-cell mouse embryos and expanded pluripotent stem cells, which can be stably maintained in vitro for a long time. TPS cells share features with 2-cell mouse embryos in terms of totipotency markers, transcriptome, chromatin accessibility, and DNA methylation patterns. In vivo chimerism assays demonstrated that these cells possess embryonic and extraembryonic developmental potentials at the single-cell level. Furthermore, we show that TPS cells can be induced into blastocyst-like structures that resemble preimplantation mouse blastocysts. Mechanistically, inhibition of HDAC1 / 2 and DOT1L activities and activation of RARγ signaling are important for inducing and maintaining the totipotent features of TPS cells. Our studies demonstrate the feasibility of capturing and maintaining totipotency in vitro.

[0288] In this study, TPS cells exhibited transcriptomic and epigenetic features similar to 2-cell mouse embryos. Importantly, TPS cells have the ability to generate embryonic and extraembryonic lineages in vivo at the single-cell level. When self-organized in vitro, TPS cells can form blastocyst-like structures that are transcriptomically similar to E4.5 blastocysts, can be transplanted in vivo and trigger decidualization. Mechanistic studies showed that HDAC1 / 2 and DOT1L inhibition and activation of RARγ signaling are important for inducing and maintaining the totipotent characteristics of TPS cells. These results demonstrate the feasibility of capturing and maintaining totipotent-like stem cells in vitro.

[0289] Induction of TPS cells from 2-cell mouse embryos is an important step in capturing true totipotency in vitro. A long-standing scientific question in the stem cell field is whether totipotent stem cells can be captured from totipotent embryos, which has not yet been achieved. To date, efforts to generate totipotent-like cells, including 2C-like cells and TBLCs, have relied solely on conversion from pluripotent stem cells and have not been validated on totipotent embryos. In contrast, our results demonstrate that treatment of preimplantation embryos with four small molecules can promote the expression of totipotency marker genes in preimplantation mouse embryos to maintain beyond the 2-cell embryonic stage ( Figure 5 i). In addition, 4S conditions allow the establishment of 2-cell embryo-derived TPS cells that highly express pluripotency marker genes ( Figure 1 c), with the same transcriptomic and epigenetic features as 2-cell embryos ( Figure 2 ) and has a bidirectional developmental potential that allows it to form blastocyst-like cells ( Figure 3 e and Figure 4). These findings provide proof of principle that totipotent-like stem cells can be induced directly from totipotent embryos. This represents an important step towards capturing true 2-cell totipotent embryos in vitro. At this point, TPS cells directly derived from 2-cell embryos could represent the closest in vitro model to totipotent embryos in vivo.

[0290] Another key unique feature of TPS cells is their ability to self-organize to generate blastocyst-like structures, which mimics the natural developmental process. Under stimulation from natural signaling molecules that drive early preimplantation development, TPS cells are able to self-organize into blastocyst-like structures that are transcriptomically similar to E4.5 blastocysts in vivo. Figure 4 ag). In addition, these blastocysts can implant in vivo and induce decidualization ( Figure 4 ij). Previous efforts to generate synthetic blastocysts using pluripotent cells have relied on the co-use of TS cells or reprogramming of epiblast stem cells. The main limitation of using TS cells is that they are transcriptionally similar to the post-implantation extraembryonic ectoderm (ExE) rather than the pre-implantation trophectoderm, and thus the induction process is artificial and unnatural, which results in significant transcriptomic differences between in vivo trophectoderm and TS-derived trophectoderm-like cells from blastocysts ( Figure 4 fh). Similarly, the induction of blastocyst-like cysts (iBLCs) from epiblast stem cells by reprogramming also fails to mimic natural development. In contrast, the generation of TPS-blastocysts relies on the differentiation of totipotent stem cells into the blastocyst lineage ( Figure 4 c), which is more similar to the process of blastocyst formation in vivo. On the other hand, unlike EPS-blastocysts that contain a large number of intermediates and mesoderm cells, the majority of TPS-blastocysts contain three blastocyst lineages, and the intermediates are almost absent ( Figure 4 cd). Therefore, TPS-blastocysts avoid the limitations of previously reported synthetic blastocysts and can serve as a powerful model to recapitulate the in vivo process of in vitro blastocyst formation. It will also be important to further optimize the induction method of TPS-based blastocysts and explore whether they can develop into conceptuses in vivo.

[0291] In conclusion, TPS cells provide a valuable in vitro platform for exploring the regulation of pluripotency, and elucidating the molecular targets of small molecules used under 4S conditions could provide new mechanistic insights into the regulation of pluripotency. Importantly, we found that inhibition of HDAC1 / 2 and DOT1L activity and activation of RARγ signaling are important for inducing pluripotency in TPS cells ( Figure 5ah). Interestingly, a role for RARγ signaling in the regulation of totipotency is supported by a recent study that showed that RA signaling is critical during the totipotency window of early development. It will be important to further explore the synergistic effects of these targets in maintaining embryonic totipotency. Studies in recent years have shown that 2C-like cells do not fully recapitulate the 2-cell embryo in terms of regulating 2-cell embryo-specific genes, and caution is required when using 2C-like cells as a model system to study totipotency, highlighting the need to establish new in vitro model systems to study totipotency. Although a recent study showed that spliceosome inhibition can induce TBLCs, this approach can only enable their conversion from pluripotent stem cells and cannot be captured from totipotent embryos, while our 4S conditions allow the induction of TPS cells from 2C embryos. In addition, unlike spliceosome inhibition, which broadly affects gene expression in a non-specific manner, the induction and maintenance of totipotency under 4S conditions relies on the combined effects of regulating specific signaling pathways and epigenetic regulators ( Figure 5 ah), this combined effect is more specific for the regulation of pluripotency. In fact, compared with TBLC, the transcriptomic profile of the TPS 2C subpopulation is significantly closer to that of mid-late 2C embryos ( Figure 2 bd). In addition, our 4S conditions also circumvent potential safety issues associated with spliceosome inhibition, such as tumor induction or proliferation difficulties. Therefore, TPS cells have broad potential applications as a new platform that facilitates the study of totipotency in vitro.

[0292] In conclusion, our study demonstrates the feasibility of capturing totipotent stem cells from early embryos. TPS cells will provide a useful tool for studying the regulatory mechanisms of totipotency and early preimplantation embryogenesis. Our study also opens the way for capturing totipotent stem cells from other mammalian species, including humans.

Claims

1. A cell culture medium composition for inducing cell totipotency of isolated cells in vitro, the composition comprising a chemical totipotency inducer (CDT) from each of the following groups in an amount effective to induce untreated cells to become totipotent stem (TPS) cells: (1) HDAC inhibitors; (2) Dot1L inhibitors; (3) RARγ agonists; and (4) Optionally, a GSK inhibitor.

2. The composition of claim 1, wherein the HDAC inhibitor is an Hdac1 and / or Hdac2 inhibitor.

3. The composition of claim 1, wherein the HDAC inhibitor is selected from the group consisting of VPA ("V"), TSA, MS275, Scriptaid, SAHA, LBH589, FK228, PXD101, sodium butyrate, LAQ824, CUDC-101, JNJ-26481585, SB939, PCI-24781, ACY-1215, CI994, CUDC-907, RGFP109, Resminostat, Curcumin, Divalproex Sodium, 4-PBA, GSK3117391, CAY10433, CM-675, and MGCD0103.

4. The composition of claim 1, wherein the Dot1L inhibitor is selected from the group consisting of EPZ004777 ("E"), EPZ5676, and SGC0946.

5. The composition of claim 1, wherein the RARγ agonist is selected from the group consisting of CD1530 ("D"), AM580, ch55, parovatin, CD3254, CD5789, CD437, TTNPB, AGN 205327, and RA.

6. The composition of claim 1, wherein the GSK inhibitor is a GSK3 inhibitor.

7. The composition of claim 1, wherein the GSK inhibitor is selected from the group consisting of CHIR99021 ("C"), AZD2858, LY2090314, BIO, CHIR 98014, SB415286, AZD1080, BRD3731, A1070722, BIP-135 and SB216763.

8. The composition of claim 1, comprising VPA ("V"), EPZ004777 ("E"), and CD1530 ("D"), wherein the concentration of V ranges from 10-1000 μM; the concentration of E ranges from 0.1-10 μM; and the concentration of D ranges from 0.1-5 μM.

9. The composition of claim 8, further comprising CHIR99021 ("C"), wherein the concentration of C ranges from 0.5 to 10 μM.

10. The composition of any one of claims 1-9, wherein the composition is in a kit and the CDT is present in a cell culture medium in a relative amount that induces pluripotency in pluripotent cells.

11. A method for inducing pluripotency in a donor cell, the method comprising: The donor cell or a cell derived from the donor cell is cultured with the composition of any one of claims 1-10 for a time effective to induce pluripotency.

12. The method of claim 11, wherein the donor cell or a cell derived from a donor cell is selected from the group consisting of: cells isolated from non-human embryos, embryonic stem cells, induced pluripotent stem cells, expanded pluripotent stem cells, multipotent stem cells, blood-derived cells, embryo-derived cells, skin-derived cells, fibroblasts, adipocytes, epithelial cells, endothelial cells, mesenchymal cells, parenchymal cells, neural cells, and connective tissue cells.

13. The method of claim 11, wherein the donor cell or a cell derived from a donor cell is selected from the group consisting of mouse embryonic stem cells and induced pluripotent stem cells.

14. The method of claim 11, wherein the donor cell or a cell derived from a donor cell is selected from the group consisting of a cell isolated from a 2-cell mouse embryo and an expanded pluripotent stem cell.

15. The method of claim 11, wherein the donor cell or the cell derived from the donor cell is first induced to be an expanded pluripotent stem cell, and then the expanded pluripotent stem cell is cultured with the composition.

16. The method of claim 11, wherein the donor cells are seeded as single cells or as small colonies.

17. An isolated, chemically induced pluripotent stem cell (ciTPSC) having pluripotency compared to a corresponding cell, or an isolated cell population comprising at least 10% of said isolated cell.

18. ciTPSCs obtained by the method of any one of claims 11 to 16, or an isolated cell population comprising at least 10% of the isolated cells.

19. The ciTPSC or cell population according to claim 17 or 18, wherein i) optionally after 5 or more passages, there is cellular expression of any one or more pluripotency marker genes selected from Zscan4, Zfp352, Tcstv1, Tcstv3, MERVL, Dux, Dub1a, Eif1a16, Eif1a19, Gm4340 and Tdpoz4 compared to corresponding untreated cells, and / or ii) cellular expression of any one or more pluripotency marker genes selected from Oct4, Nanog and Sox2 is downregulated compared to corresponding untreated cells.

20. The ciTPSC or cell population of claim 17 or 18, wherein the cells are capable of giving rise to both embryonic and extraembryonic lineages at the single cell level.

21. The ciTPSC or cell population of claim 17 or 18, wherein the cells are capable of forming blastocyst-like structures in vitro.

22. A therapeutic composition comprising the ciTPSC or cell population of any one of claims 17-21 formulated for administration to an individual by injection, implantation of a prosthetic device, or a tissue engineered matrix.

23. A cell culture comprising: The isolated ciTPSCs and culture medium of claim 17 or 18, wherein the culture medium comprises (1) an HDAC inhibitor, (2) a Dot1L inhibitor, (3) a RARγ agonist, and (4) optionally, a GSK inhibitor.

24. The cell culture of claim 23, wherein the culture medium effectively maintains the ciTPSCs in a pluripotent state for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 passages.

25. The cell culture of claim 24, wherein the ciTPSCs maintain expression of a pluripotency marker selected from the group consisting of Zscan4, Zfp352, Tcstv1, Tcstv3, MERVL, Dux, Dub1a, Eif1a16, Eif1a19, Gm4340, and Tdpoz4.

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