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• W4283267968 abstract "•A human 8C-specific reporter is developed to isolate 8CLCs from human prEpiSC cultures•A chemical-based culture condition increases and maintains the 8CLC population•8CLCs can self-organize to form blastocyst-like structures In human embryos, major zygotic genome activation (ZGA) initiates at the eight-cell (8C) stage. Abnormal ZGA leads to developmental defects and even contributes to the failure of human blastocyst formation or implantation. An in vitro cell model mimicking human 8C blastomeres would be invaluable to understanding the mechanisms regulating key biological events during early human development. Using the non-canonical promoter of LEUTX that putatively regulates human ZGA, we developed an 8C::mCherry reporter, which specifically marks the 8C state, to isolate rare 8C-like cells (8CLCs) from human preimplantation epiblast-like stem cells. The 8CLCs express a panel of human ZGA genes and have a unique transcriptome resembling that of the human 8C embryo. Using the 8C::mCherry reporter, we further optimize the chemical-based culture condition to increase and maintain the 8CLC population. Functionally, 8CLCs can self-organize to form blastocyst-like structures. The discovery and maintenance of 8CLCs provide an opportunity to recapitulate early human development. In human embryos, major zygotic genome activation (ZGA) initiates at the eight-cell (8C) stage. Abnormal ZGA leads to developmental defects and even contributes to the failure of human blastocyst formation or implantation. An in vitro cell model mimicking human 8C blastomeres would be invaluable to understanding the mechanisms regulating key biological events during early human development. Using the non-canonical promoter of LEUTX that putatively regulates human ZGA, we developed an 8C::mCherry reporter, which specifically marks the 8C state, to isolate rare 8C-like cells (8CLCs) from human preimplantation epiblast-like stem cells. The 8CLCs express a panel of human ZGA genes and have a unique transcriptome resembling that of the human 8C embryo. Using the 8C::mCherry reporter, we further optimize the chemical-based culture condition to increase and maintain the 8CLC population. Functionally, 8CLCs can self-organize to form blastocyst-like structures. The discovery and maintenance of 8CLCs provide an opportunity to recapitulate early human development. After fertilization, the quiescent zygotic genome initiates bursts of transcription termed zygotic genome activation (ZGA) (Jukam et al., 2017Jukam D. Shariati S.A.M. Skotheim J.M. Zygotic genome activation in vertebrates.Dev. Cell. 2017; 42: 316-332https://doi.org/10.1016/j.devcel.2017.07.026Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar; Lee et al., 2014Lee M.T. Bonneau A.R. Giraldez A.J. Zygotic genome activation during the maternal-to-zygotic transition.Annu. Rev. Cell. Dev. Biol. 2014; 30: 581-613https://doi.org/10.1146/annurev-cellbio-100913-013027Crossref PubMed Scopus (308) Google Scholar; Ortega et al., 2018Ortega N.M. Winblad N. Plaza Reyes A. Lanner F. Functional genetics of early human development.Curr. Opin. Genet. Dev. 2018; 52: 1-6https://doi.org/10.1016/j.gde.2018.04.005Crossref PubMed Scopus (11) Google Scholar; Palfy et al., 2017Pálfy M. Joseph S.R. Vastenhouw N.L. The timing of zygotic genome activation.Curr. Opin. Genet. Dev. 2017; 43: 53-60https://doi.org/10.1016/j.gde.2016.12.001Crossref PubMed Scopus (32) Google Scholar; Schulz and Harrison, 2019Schulz K.N. Harrison M.M. Mechanisms regulating zygotic genome activation.Nat. Rev. Genet. 2019; 20: 221-234https://doi.org/10.1038/s41576-018-0087-xCrossref PubMed Scopus (117) Google Scholar). The major ZGA occurs at the late two-cell (L2C) stage in mice and eight-cell (8C) stage in humans, which is essential for subsequent lineage segregation (Jukam et al., 2017Jukam D. Shariati S.A.M. Skotheim J.M. Zygotic genome activation in vertebrates.Dev. Cell. 2017; 42: 316-332https://doi.org/10.1016/j.devcel.2017.07.026Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar; Lee et al., 2014Lee M.T. Bonneau A.R. Giraldez A.J. Zygotic genome activation during the maternal-to-zygotic transition.Annu. Rev. Cell. Dev. Biol. 2014; 30: 581-613https://doi.org/10.1146/annurev-cellbio-100913-013027Crossref PubMed Scopus (308) Google Scholar; Ortega et al., 2018Ortega N.M. Winblad N. Plaza Reyes A. Lanner F. Functional genetics of early human development.Curr. Opin. Genet. Dev. 2018; 52: 1-6https://doi.org/10.1016/j.gde.2018.04.005Crossref PubMed Scopus (11) Google Scholar; Palfy et al., 2017Pálfy M. Joseph S.R. Vastenhouw N.L. The timing of zygotic genome activation.Curr. Opin. Genet. Dev. 2017; 43: 53-60https://doi.org/10.1016/j.gde.2016.12.001Crossref PubMed Scopus (32) Google Scholar; Schulz and Harrison, 2019Schulz K.N. Harrison M.M. Mechanisms regulating zygotic genome activation.Nat. Rev. Genet. 2019; 20: 221-234https://doi.org/10.1038/s41576-018-0087-xCrossref PubMed Scopus (117) Google Scholar). The onset of ZGA depends on intricately coordinated mechanisms in which transcription factors and epigenetic regulation (e.g., DNA methylation, histone post-translational modification) play crucial roles (Ortega et al., 2018Ortega N.M. Winblad N. Plaza Reyes A. Lanner F. Functional genetics of early human development.Curr. Opin. Genet. Dev. 2018; 52: 1-6https://doi.org/10.1016/j.gde.2018.04.005Crossref PubMed Scopus (11) Google Scholar; Palfy et al., 2017Pálfy M. Joseph S.R. Vastenhouw N.L. The timing of zygotic genome activation.Curr. Opin. Genet. Dev. 2017; 43: 53-60https://doi.org/10.1016/j.gde.2016.12.001Crossref PubMed Scopus (32) Google Scholar; Schulz and Harrison, 2019Schulz K.N. Harrison M.M. Mechanisms regulating zygotic genome activation.Nat. Rev. Genet. 2019; 20: 221-234https://doi.org/10.1038/s41576-018-0087-xCrossref PubMed Scopus (117) Google Scholar). Advances in low-input multi-omics techniques have recently led to the discoveries of several mammalian genome activators. Intriguingly, DUX4/DUX family genes, which are specific to placental mammals, are critical drivers of ZGA in both mice and humans (De Iaco et al., 2017De Iaco A. Planet E. Coluccio A. Verp S. Duc J. Trono D. DUX-family transcription factors regulate zygotic genome activation in placental mammals.Nat. Genet. 2017; 49: 941-945https://doi.org/10.1038/ng.3858Crossref PubMed Scopus (244) Google Scholar; Hendrickson et al., 2017Hendrickson P.G. Doráis J.A. Grow E.J. Whiddon J.L. Lim J.W. Wike C.L. Weaver B.D. Pflueger C. Emery B.R. Wilcox A.L. et al.Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons.Nat. Genet. 2017; 72: 483-484https://doi.org/10.1097/ogx.0000000000000471Crossref Google Scholar; Whiddon et al., 2017Whiddon J.L. Langford A.T. Wong C.J. Zhong J.W. Tapscott S.J. Conservation and innovation in the DUX4-family gene network.Nat. Genet. 2017; 49: 935-940https://doi.org/10.1038/ng.3846Crossref PubMed Scopus (149) Google Scholar). Recent studies also have shown that the orthologous genes have distinct functions in regulating ZGA in mammals. For instance, OCT4 knockdown results in downregulation of hundreds of ZGA genes in human embryos, implicating its essential roles in human ZGA (Fogarty et al., 2017Fogarty N.M.E. McCarthy A. Snijders K.E. Powell B.E. Kubikova N. Blakeley P. Lea R. Elder K. Wamaitha S.E. Kim D. et al.Genome editing reveals a role for OCT4 in human embryogenesis.Nature. 2017; 550: 67-73https://doi.org/10.1038/nature24033Crossref PubMed Scopus (212) Google Scholar; Gao et al., 2018Gao L. Wu K. Liu Z. Yao X. Yuan S. Tao W. Yi L. Yu G. Hou Z. Fan D. et al.Chromatin accessibility landscape in human early embryos and its association with evolution.Cell. 2018; 173: 248-259.e15https://doi.org/10.1016/j.cell.2018.02.028Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), whereas OCT4 is not involved in ZGA in mice (Gao et al., 2018Gao L. Wu K. Liu Z. Yao X. Yuan S. Tao W. Yi L. Yu G. Hou Z. Fan D. et al.Chromatin accessibility landscape in human early embryos and its association with evolution.Cell. 2018; 173: 248-259.e15https://doi.org/10.1016/j.cell.2018.02.028Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). On the other hand, misexpression of these ZGA regulators has serious consequences for embryonic development. OCT4 depletion at the pre-ZGA stage compromises human blastocyst formation but weakly affects mouse preimplantation development (Fogarty et al., 2017Fogarty N.M.E. McCarthy A. Snijders K.E. Powell B.E. Kubikova N. Blakeley P. Lea R. Elder K. Wamaitha S.E. Kim D. et al.Genome editing reveals a role for OCT4 in human embryogenesis.Nature. 2017; 550: 67-73https://doi.org/10.1038/nature24033Crossref PubMed Scopus (212) Google Scholar; Gao et al., 2018Gao L. Wu K. Liu Z. Yao X. Yuan S. Tao W. Yi L. Yu G. Hou Z. Fan D. et al.Chromatin accessibility landscape in human early embryos and its association with evolution.Cell. 2018; 173: 248-259.e15https://doi.org/10.1016/j.cell.2018.02.028Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). DUX4 misexpression in human skeletal muscle cells, where it activates aberrant expression of ZGA genes, causes an untreatable form of muscular dystrophy called facioscapulohumeral dystrophy (Geng et al., 2012Geng L.N. Yao Z. Snider L. Fong A.P. Cech J.N. Young J.M. van der Maarel S.M. Ruzzo W.L. Gentleman R.C. Tawil R. Tapscott S. DUX4 activates germline genes, retroelements, and immune mediators: implications for facioscapulohumeral dystrophy.Dev. Cell. 2012; 22: 38-51https://doi.org/10.1016/j.devcel.2011.11.013Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar; Karpukhina et al., 2021Karpukhina A. Tiukacheva E. Dib C. Vassetzky Y.S. Control of DUX4 expression in facioscapulohumeral muscular dystrophy and cancer.Trends Mol. Med. 2021; 27: 588-601https://doi.org/10.1016/j.molmed.2021.03.008Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar; Lemmers et al., 2010Lemmers R.J.L.F. van der Vliet P.J. Klooster R. Sacconi S. Camaño P. Dauwerse J.G. Snider L. Straasheijm K.R. Jan van Ommen G. Padberg G.W. et al.A unifying genetic model for facioscapulohumeral muscular dystrophy.Science. 2010; 329: 1650-1653https://doi.org/10.1126/science.1189044Crossref PubMed Scopus (497) Google Scholar). Besides transcription factors, proper epigenetic regulation is also critical for ZGA. Previous studies suggest that failed H3K9me3 reprogramming impedes ZGA in mammalian embryos generated by somatic cell nuclear transfer, compromising blastocyst formation (Chen et al., 2020Chen M. Zhu Q. Li C. Kou X. Zhao Y. Li Y. Xu R. Yang L. Yang L. Gu L. et al.Chromatin architecture reorganization in murine somatic cell nuclear transfer embryos.Nat. Commun. 2020; 11: 1813https://doi.org/10.1038/s41467-020-15607-zCrossref PubMed Scopus (26) Google Scholar; Chung et al., 2015Chung Y.G. Matoba S. Liu Y. Eum J.H. Lu F. Jiang W. Lee J.E. Sepilian V. Cha K.Y. Lee D.R. Zhang Y. Histone demethylase expression enhances human somatic cell nuclear transfer efficiency and promotes derivation of pluripotent stem cells.Cell Stem Cell. 2015; 17: 758-766https://doi.org/10.1016/j.stem.2015.10.001Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar; Deng et al., 2020Deng M. Liu Z. Chen B. Wan Y. Yang H. Zhang Y. Cai Y. Zhou J. Wang F. Aberrant DNA and histone methylation during zygotic genome activation in goat cloned embryos.Theriogenology. 2020; 148: 27-36https://doi.org/10.1016/j.theriogenology.2020.02.036Crossref PubMed Scopus (19) Google Scholar; Matoba et al., 2014Matoba S. Liu Y. Lu F. Iwabuchi K.A. Shen L. Inoue A. Zhang Y. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation.Cell. 2014; 159: 884-895https://doi.org/10.1016/j.cell.2014.09.055Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Together, abnormal ZGA contributes to substantial lethality of human embryos, and even may bring about long-term detrimental consequences on health. Therefore, understanding the regulatory mechanism of human ZGA is important for human reproduction and health. The studies on human embryos, however, are experimentally challenged and ethically restricted. The derivation of pluripotent stem cells facilitates research about mammalian early development. A rare cell subpopulation is identified in mouse embryonic stem cell (mESC) culture, which is similar to the mouse late two-cell embryo, thus called 2C-like cells (2CLCs) (Macfarlan et al., 2012Macfarlan T.S. Gifford W.D. Driscoll S. Lettieri K. Rowe H.M. Bonanomi D. Firth A. Singer O. Trono D. Pfaff S.L. Embryonic stem cell potency fluctuates with endogenous retrovirus activity.Nature. 2012; 487: 57-63https://doi.org/10.1038/nature11244Crossref PubMed Scopus (624) Google Scholar). The ZGA-like transcriptional program is reactivated in 2CLCs (Iturbide and Torres-Padilla, 2020Iturbide A. Torres-Padilla M.E. A cell in hand is worth two in the embryo: recent advances in 2-cell like cell reprogramming.Curr. Opin. Genet. Dev. 2020; 64: 26-30https://doi.org/10.1016/j.gde.2020.05.038Crossref PubMed Scopus (10) Google Scholar; Macfarlan et al., 2012Macfarlan T.S. Gifford W.D. Driscoll S. Lettieri K. Rowe H.M. Bonanomi D. Firth A. Singer O. Trono D. Pfaff S.L. Embryonic stem cell potency fluctuates with endogenous retrovirus activity.Nature. 2012; 487: 57-63https://doi.org/10.1038/nature11244Crossref PubMed Scopus (624) Google Scholar; Rodriguez-Terrones et al., 2018Rodriguez-Terrones D. Gaume X. Ishiuchi T. Weiss A. Kopp A. Kruse K. Penning A. Vaquerizas J.M. Brino L. Torres-Padilla M.E. A molecular roadmap for the emergence of early-embryonic-like cells in culture.Nat. Genet. 2018; 50: 106-119https://doi.org/10.1038/s41588-017-0016-5Crossref PubMed Scopus (76) Google Scholar). Moreover, based on the 2CLC model, we and two other groups establish the mouse totipotent-like stem cell lines that faithfully recapitulate mouse two-cell embryos and have more robust developmental potency than mESCs (Shen et al., 2021Shen H. Yang M. Li S. Zhang J. Peng B. Wang C. Chang Z. Ong J. Du P. Mouse totipotent stem cells captured and maintained through spliceosomal repression.Cell. 2021; 184: 2843-2859.e20https://doi.org/10.1016/j.cell.2021.04.020Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar; Xu et al., 2022Xu Y. Zhao J. Ren Y. Wang X. Lyu Y. Xie B. Sun Y. Yuan X. Liu H. Yang W. et al.Derivation of totipotent-like stem cells with blastocyst-like structure forming potential.Cell Res. 2022; 32: 513-529https://doi.org/10.1038/s41422-022-00668-0Crossref PubMed Scopus (3) Google Scholar; Yang et al., 2022Yang M. Yu H. Yu X. Liang S. Hu Y. Luo Y. Izsvák Z. Sun C. Wang J. Chemical-induced chromatin remodeling reprograms mouse ESCs to totipotent-like stem cells.Cell Stem Cell. 2022; 29: 400-418.e13https://doi.org/10.1016/j.stem.2022.01.010Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). This unique cell type with 2C-like transcriptome holds promise to identify ZGA regulators and explore the regulatory mechanisms of early mouse development. However, to date the early blastomere-like cells have not been captured from pluripotent stem cell cultures in other species, including humans. Clearly, one suitable cell model like 2CLCs is crucial for dissecting the mechanisms that regulate key biological events (e.g., ZGA, lineage segregation) during early human development. Thus, it is worth exploring whether such early human blastomere-like cells can be cultured in vitro. In this study, based on single-cell RNA-sequencing (scRNA-seq) analysis, we developed an 8C-specific mCherry reporter (8C::mCherry) and successfully captured the rare human 8C-like cells (8CLCs) from human preimplantation epiblast-like stem cells, in parallel with the other two latest reports (Mazid et al., 2022Mazid M.A. Ward C. Luo Z. Liu C. Li Y. Lai Y. Wu L. Li J. Jia W. Jiang Y. et al.Rolling back human pluripotent stem cells to an eight-cell embryo-like stage.Nature. 2022; 605: 315-324https://doi.org/10.1038/s41586-022-04625-0Crossref PubMed Scopus (8) Google Scholar; Taubenschmid-Stowers et al., 2022Taubenschmid-Stowers J. Rostovskaya M. Santos F. Ljung S. Argelaguet R. Krueger F. Nichols J. Reik W. 8C-like cells capture the human zygotic genome activation program in vitro.Cell Stem Cell. 2022; 29: 449-459.e6https://doi.org/10.1016/j.stem.2022.01.014Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). These 8CLCs express multiple signature genes and retrotransposons of human 8C embryos and exhibit a human ZGA-like transcriptional program. By chemical screening, we further optimized the culture condition to improve and maintain the 8CLC population in cultures. Finally, we evaluated the developmental potential of 8CLCs by the blastoid generation assay. In mice, 2CLCs were discovered specifically from naive pluripotent stem cells (PSCs) representing the preimplantation epiblast (pre-EPI). We asked whether the PSCs representing the human pre-EPI might contain 8CLCs. To address this question, we first generated human pre-EPI-like stem cells (prEpiSCs) by chemical screening. Using the LTR7/HERVH-based, pluripotency reporter (pT2-LTR7-GFP) (Wang et al., 2014Wang J. Xie G. Singh M. Ghanbarian A.T. Rasko T. Szvetnik A. Cai H. Besser D. Prigione A. Fuchs N.V. et al.Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells.Nature. 2014; 516: 405-409https://doi.org/10.1038/nature13804Crossref PubMed Scopus (250) Google Scholar) (Figure 1A ), we conducted chemical screening on human embryonic stem cells (hESCs) cultured in the N2B27 medium with a selected small molecule pool targeting multiple signaling pathways enriched in hESCs but underrepresented in the human pre-EPI (Figure S1A and STAR Methods). As interleukin (IL)6R rather than LIFR is expressed in human pre-EPI (Figure S1A), the cytokine IL6 (but not LIF) was added into the N2B27 medium. First, we screened the chemicals one by one, and selected those that could enhance the reporter GFP signal and increase the GFP+ cell number in human PSC (hPSC) culture (Figure 1B). In the second round of screening, we identified the essential compounds to maintain the GFP+ cells in hPSC culture. This strategy revealed that combined inhibition of MEK (GSK1120212), WNT (XAV939), and PKC (Go6983) signaling pathways as well as transiently inhibiting Src (A419259) and deposition of H3K27me3 marks (DZNep) were suitable for stable maintenance of GFP+ cells in hPSC culture (Figures 1C and S1B). Of note, the bone morphogenetic protein (BMP) signaling pathway seems to be required for prEpiSC induction from hESCs, as BMP4 promoted, whereas the BMP inhibitor (K02288), blocked this induction process (Figure S1C). We observed that once GFP+ hPSCs were stabilized (indicated by bright domed-shape colonies without differentiation), DZNep and A419259 could be removed from the medium for the long-term culturing, while GFP+ hPSC colonies were differentiated or died once GSK1120212, XAV939, and Go6983 were withdrawn from the medium (Figure S1D). With this strategy, we derived an hPSC line mimicking human pre-EPI, which was featured by HERVH hypertranscription and high expression of pre-EPI-specific genes (Figures 1E and 1F, S1E, and S1F), thus termed pre-EPI-like hPSCs (prEpiSC). However, unlike other naive hPSCs (e.g., 5iLA, t2iLGo hPSCs) (Pastor et al., 2016Pastor W.A. Chen D. Liu W. Kim R. Sahakyan A. Lukianchikov A. Plath K. Jacobsen S.E. Clark A.T. Naive human pluripotent cells feature a methylation landscape devoid of blastocyst or germline memory.Cell Stem Cell. 2016; 18: 323-329https://doi.org/10.1016/j.stem.2016.01.019Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), most of prEpiSCs expressed SSEA4 instead of SSEA1 (Figure 1E), consistent with that SSEA4 is expressed in human inner cell mass (ICM) (Henderson et al., 2002Henderson J.K. Draper J.S. Baillie H.S. Fishel S. Thomson J.A. Moore H. Andrews P.W. Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens.Stem Cells. 2002; 20: 329-337https://doi.org/10.1634/stemcells.20-4-329Crossref PubMed Scopus (400) Google Scholar). Unlike the primed hPSCs, prEpiSCs could be passaged as single cells and formed dome-shaped colonies under either feeder-dependent or feeder-free conditions (Figure 1D). Importantly, while other naive hPSCs usually exhibit quite abnormal karyotypes after 10 to 12 passages (Pastor et al., 2016Pastor W.A. Chen D. Liu W. Kim R. Sahakyan A. Lukianchikov A. Plath K. Jacobsen S.E. Clark A.T. Naive human pluripotent cells feature a methylation landscape devoid of blastocyst or germline memory.Cell Stem Cell. 2016; 18: 323-329https://doi.org/10.1016/j.stem.2016.01.019Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), no karyotype aberration was observed in most of prEpiSCs even after 80 passages (normal/abnormal: 359/6) (Figure S1G). In addition, prEpiSCs could be readily and directly differentiated into three germ layers (Figure S1H). To further characterize prEpiSCs, we performed comparative epigenome and transcriptome analyses between prEpiSCs and epiblast cells from human embryos as well as other hPSC lines. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis revealed that prEpiSCs displayed a similar chromatin accessibility landscape to human ICM and 5iLA naive hESCs (Figure 1G). However, the analysis for the genome-wide histone mark landscape revealed that the global H3K4me3 occupancy in prEpiSCs was comparable to that in ICM rather than naive and primed hPSCs (Figure 1H). In line with the chromatin state, the principal component analysis (PCA) showed that prEpiSCs transcriptomically resembled preimplantation epiblast cells (day 6), but were distinctive from postimplantation epiblast cells (day 8–12) (Figures 1I and 1J) (Zhou et al., 2019Zhou F. Wang R. Yuan P. Ren Y. Mao Y. Li R. Lian Y. Li J. Wen L. Yan L. et al.Reconstituting the transcriptome and DNA methylome landscapes of human implantation.Nature. 2019; 572: 660-664https://doi.org/10.1038/s41586-019-1500-0Crossref PubMed Scopus (104) Google Scholar). Interestingly, prEpiSCs expressed a large number of 8C-specific genes (e.g., LEUTX, TPRX1, ZSCAN4, DUXA, KLF17, MBD3L2, ZSCAN5B) that were defined by the scRNA-seq data of early human embryos (Figures 1K, 1L, and Table S1) (Petropoulos et al., 2016Petropoulos S. Edsgärd D. Reinius B. Deng Q. Panula S.P. Codeluppi S. Plaza Reyes A. Linnarsson S. Sandberg R. Lanner F. Single-cell RNA-seq reveals lineage and X chromosome dynamics in human preimplantation embryos.Cell. 2016; 165: 1012-1026https://doi.org/10.1016/j.cell.2016.03.023Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar; Yan et al., 2013Yan L. Yang M. Guo H. Yang L. Wu J. Li R. Liu P. Lian Y. Zheng X. Yan J. et al.Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells.Nat. Struct. Mol. Biol. 2013; 20: 1131-1139https://doi.org/10.1038/nsmb.2660Crossref PubMed Scopus (943) Google Scholar). In particular, multiple DUX4-regulated genes were reactivated in prEpiSCs (Figure 1M) (Hendrickson et al., 2017Hendrickson P.G. Doráis J.A. Grow E.J. Whiddon J.L. Lim J.W. Wike C.L. Weaver B.D. Pflueger C. Emery B.R. Wilcox A.L. et al.Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons.Nat. Genet. 2017; 72: 483-484https://doi.org/10.1097/ogx.0000000000000471Crossref Google Scholar). Notably, the DUX4-regulated ZGA genes (e.g., ZSCAN4, LEUTX, DUXA, TPRX1) were expressed in prEpiSCs at much higher levels than those in human naive and formative pluripotent cells (Figure 1N). According to the above results, we hypothesized that human 8CLCs might exist in the prEpiSC culture. To test this hypothesis, we performed scRNA-seq analysis on prEpiSCs. We identified one main cell cluster and two rare clusters from prEpiSCs (Figure 2A ). The main cell cluster (cluster0) expressed multiple pluripotent genes at comparable levels to the epiblast from human blastocysts, suggesting the similarity between prEpiSCs and human preimplantation epiblast. Intriguingly, one rare cell cluster (cluster2) expressed multiple 8C-specific genes (e.g., ZSCAN4, LEUTX, KLF17, TPRX1, H3Y1), while the other rare cell cluster (cluster1) expressed pluripotent genes at quite low levels (Figure 2B). To address the identities of these two rare cell clusters, we performed t-distributed stochastic neighbor embedding (tSNE) analysis by integrating these rare cell clusters into scRNA-seq datasets of early human embryos. Some cells from cluster2 were grouped with 8C embryos, thus named 8CLCs (Figure 2C). On the other hand, some cells from cluster1 were grouped with medium trophectoderm (TE), thus named TE-like cells (TELCs) (Figure 2C). The expression of multiple markers for the 8C embryo and TE was detected in 8CLCs and TELCs, respectively (Figures 2D and 2E). In line with the scRNA-seq analysis, immunofluorescence analysis revealed that LEUTX was heterogeneously expressed in prEpiSC colonies, while ZSCAN4 seemed to be homogeneously expressed (Figure 2F). Collectively, these results demonstrate that 8CLCs are present in prEpiSC culture. To explore whether the existence of 8CLCs is dependent on culture conditions and pluripotent states, we analyzed the scRNA-seq data of naive and primed hPSCs cultured in different conditions (Kinoshita et al., 2021Kinoshita M. Barber M. Mansfield W. Cui Y. Spindlow D. Stirparo G.G. Dietmann S. Nichols J. Smith A. Capture of mouse and human stem cells with features of formative pluripotency.Cell Stem Cell. 2021; 28: 2180https://doi.org/10.1016/j.stem.2021.11.002Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar; Liu et al., 2020Liu X. Ouyang J.F. Rossello F.J. Tan J.P. Davidson K.C. Valdes D.S. Schröder J. Sun Y.B.Y. Chen J. Knaupp A.S. et al.Reprogramming roadmap reveals route to human induced trophoblast stem cells.Nature. 2020; 586: 101-107https://doi.org/10.1038/s41586-020-2734-6Crossref PubMed Scopus (48) Google Scholar; Messmer et al., 2019Messmer T. von Meyenn F. Savino A. Santos F. Mohammed H. Lun A.T.L. Marioni J.C. Reik W. Transcriptional heterogeneity in naive and primed human pluripotent stem cells at single-cell resolution.Cell Rep. 2019; 26: 815-824.e4https://doi.org/10.1016/j.celrep.2018.12.099Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). We first analyzed the scRNA-seq data (Sahakyan et al., 2017Sahakyan A. Kim R. Chronis C. Sabri S. Bonora G. Theunissen T.W. Kuoy E. Langerman J. Clark A.T. Jaenisch R. Plath K. Human naive pluripotent stem cells model X chromosome dampening and X inactivation.Cell Stem Cell. 2017; 20: 87-101https://doi.org/10.1016/j.stem.2016.10.006Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) of hESCs to interrogate any cell subset that expresses 8C-specific genes. The cell subpopulation expressing multiple 8C-specific genes was discovered in neither feeder-dependent nor feeder-free hESCs (Figures S2A–S2C). We also analyzed the human extended PSCs (hEPSCs) that were reported to be capable of differentiation into both embryonic and extraembryonic lineages (Liu et al., 2021aLiu B. Chen S. Xu Y. Lyu Y. Wang J. Du Y. Sun Y. Liu H. Zhou H. Lai W. et al.Chemically defined and xeno-free culture condition for human extended pluripotent stem cells.Nat. Commun. 2021; 12: 3017https://doi.org/10.1038/s41467-021-23320-8Crossref PubMed Scopus (4) Google Scholar), but could not identify one subpopulation expressing 8C-specific genes (Figure S2C). Though the transcription of a few 8C-sepcific genes could be detected (Figures S3A and S3B), the unique 8C-like subpopulation could not be discovered from 5iLA and t2iLGo naive hPSCs. However, similar to prEpiSCs, 8CLCs were also identified from PXGL naive hPSCs (Figures S3C–S3G), in accordance with the latest studies (Mazid et al., 2022Mazid M.A. Ward C. Luo Z. Liu C. Li Y. Lai Y. Wu L. Li J. Jia W. Jiang Y. et al.Rolling back human pluripotent stem cells to an eight-cell embryo-like stage.Nature. 2022; 605: 315-324https://doi.org/10.1038/s41586-022-04625-0Crossref PubMed Scopus (8) Google Scholar; Taubenschmid-Stowers et al., 2022Taubenschmid-Stowers J. Rostovskaya M. Santos F. Ljung S. Argelaguet R. Krueger F. Nichols J. Reik W. 8C-like cells capture the human zygotic genome activation program in vitro.Cell Stem Cell. 2022; 29: 449-459.e6https://doi.org/10.1016/j.stem.2022.01.014Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). These data indicate that the existence of 8CLCs is dependent on the culture condition and pluripotent states of hPSCs. It is a challenge to characterize 8CLCs in detail and dissect the transition between 8CLCs and hPSCs, because of the quite low proportion of 8CLCs in prEpiSCs. Thus, we tried to develop an 8C-specific reporter to monitor and enrich the 8CLC population from the prEpiSC culture. By analyzing the scRNA-seq data of early human embryos (Petropoulos et al., 2016Petropoulos S. Edsgärd D. Reinius B. Deng Q. Panula S.P. Codeluppi S. Plaza Reyes A. Linnarsson S. Sandberg R. Lanner F. Single-cell RNA-seq reveals lineage and X chromosome dynamics in human preimplantation embryos.Cell. 2016; 165: 1012-1026https://doi.org/10.1016/j.cell.2016.03.023Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar; Yan et al., 2013Yan L. Yang M. Guo H. Yang L. Wu J. Li R. Liu P. Lian Y. Zheng X. Yan J. et al.Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells.Nat. Struct. Mol. Biol. 2013; 20: 1131-1139https://doi.org/10.1038/nsmb.2660Crossref PubMed Scopus (943) Google Scholar), we confirmed LEUTX as one of the most specific markers for human 8C embryos (Figures 3A, 3B, and S4A) (Jou" @default.
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• W4283267968 title "Recapitulating early human development with 8C-like cells" @default.
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