Capturing human trophoblast development with naive pluripotent stem cells in vitro

•Trophectoderm can be generated from naive human pluripotent stem cells•Human trophoblast stem cells represent post-implantation cytotrophoblast•Trajectory of trophoblast from pre- to post-implantation is drawn by our model•ACE is essential for specification and maintenance of cytotrophoblast stem cells Trophoblasts are extraembryonic cells that are essential for maintaining pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblasts (CTs), syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs), composing the placenta. Here we show that naïve, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naive PSC-derived TE and CTs (nCTs) recreated human and monkey TE-to-CT transition. nCTs self-renewed as CT stem cells and had the characteristics of proliferating villous CTs and CTs in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (BMP4, A83-01, and PD173074 [BAP]-treated primed PSCs [pBAPs]), pBAPs were distinct from nCTs and human placenta-derived CT stem cells, exhibiting properties consistent with the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naive human PSCs. Our system provides a platform to study the molecular mechanisms underlying trophoblast development and related diseases. Trophoblasts are extraembryonic cells that are essential for maintaining pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblasts (CTs), syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs), composing the placenta. Here we show that naïve, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naive PSC-derived TE and CTs (nCTs) recreated human and monkey TE-to-CT transition. nCTs self-renewed as CT stem cells and had the characteristics of proliferating villous CTs and CTs in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (BMP4, A83-01, and PD173074 [BAP]-treated primed PSCs [pBAPs]), pBAPs were distinct from nCTs and human placenta-derived CT stem cells, exhibiting properties consistent with the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naive human PSCs. Our system provides a platform to study the molecular mechanisms underlying trophoblast development and related diseases. Trophoblasts compose the placenta and are essential extraembryonic cells from blastocyst through birth. Abnormal differentiation of trophoblasts during early pregnancy is thought to cause placenta-related complications (Burton and Jauniaux, 2017Burton G.J. Jauniaux E. The cytotrophoblastic shell and complications of pregnancy.Placenta. 2017; 60: 134-139Crossref PubMed Scopus (59) Google Scholar; Malhotra et al., 2016Malhotra S.S. Banerjee P. Gupta S.K. Regulation of trophoblast differentiation during embryo implantation and placentation: Implications in pregnancy complications.J. Reprod. Health Med. 2016; 2: S26-S36Crossref Google Scholar). However, it is difficult to analyze trophoblasts in the human uterus, especially during the first few weeks after implantation, because of technical difficulties and ethics restrictions. Trophoblast development and physiology have been described based on mice, and early lethality (embryonic days 9.5 (E9.5)−14.5) in knockout lines is frequently associated with severe placental malformation (Perez-Garcia et al., 2018Perez-Garcia V. Fineberg E. Wilson R. Murray A. Mazzeo C.I. Tudor C. Sienerth A. White J.K. Tuck E. Ryder E.J. et al.Placentation defects are highly prevalent in embryonic lethal mouse mutants.Nature. 2018; 555: 463-468Crossref PubMed Scopus (160) Google Scholar). However, human and mouse placentation differ in many ways. Fibroblast growth factor receptor 2 (FGFR2) is expressed in mouse trophectoderm (TE) of blastocysts, whereas FGFR1, FGFR2, and FGFR3 are not expressed in human TE (Kunath et al., 2014Kunath T. Yamanaka Y. Detmar J. MacPhee D. Caniggia I. Rossant J. Jurisicova A. Developmental differences in the expression of FGF receptors between human and mouse embryos.Placenta. 2014; 35: 1079-1088Crossref PubMed Scopus (61) Google Scholar). Trophoblast stem (TS) cells cultured in FGF4 have been established in mice (Tanaka et al., 1998Tanaka S. Kunath T. Hadjantonakis A.K. Nagy A. Rossant J. Promotion of trophoblast stem cell proliferation by FGF4.Science. 1998; 282: 2072-2075Crossref PubMed Scopus (1042) Google Scholar) and used to investigate trophoblast gene functions (Kuckenberg et al., 2010Kuckenberg P. Buhl S. Woynecki T. van Fürden B. Tolkunova E. Seiffe F. Moser M. Tomilin A. Winterhager E. Schorle H. The transcription factor TCFAP2C/AP-2gamma cooperates with CDX2 to maintain trophectoderm formation.Mol. Cell. Biol. 2010; 30: 3310-3320Crossref PubMed Scopus (109) Google Scholar; Nishioka et al., 2009Nishioka N. Inoue K. Adachi K. Kiyonari H. Ota M. Ralston A. Yabuta N. Hirahara S. Stephenson R.O. Ogonuki N. et al.The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass.Dev. Cell. 2009; 16: 398-410Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar; Niwa et al., 2005Niwa H. Toyooka Y. Shimosato D. Strumpf D. Takahashi K. Yagi R. Rossant J. Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation.Cell. 2005; 123: 917-929Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar; Perez-Garcia et al., 2018Perez-Garcia V. Fineberg E. Wilson R. Murray A. Mazzeo C.I. Tudor C. Sienerth A. White J.K. Tuck E. Ryder E.J. et al.Placentation defects are highly prevalent in embryonic lethal mouse mutants.Nature. 2018; 555: 463-468Crossref PubMed Scopus (160) Google Scholar; Senner and Hemberger, 2010Senner C.E. Hemberger M. Regulation of early trophoblast differentiation - lessons from the mouse.Placenta. 2010; 31: 944-950Crossref PubMed Scopus (49) Google Scholar), but in humans, TS cells cannot be established in FGF4 (Kunath et al., 2014Kunath T. Yamanaka Y. Detmar J. MacPhee D. Caniggia I. Rossant J. Jurisicova A. Developmental differences in the expression of FGF receptors between human and mouse embryos.Placenta. 2014; 35: 1079-1088Crossref PubMed Scopus (61) Google Scholar). In 2018, several groups reported successful culturing of trophoblasts from 5- to 9-week human placentas or blastocysts for long periods in vitro as two-dimensional TS cells or three-dimensional trophoblast organoids (Haider et al., 2018Haider S. Meinhardt G. Saleh L. Kunihs V. Gamperl M. Kaindl U. Ellinger A. Burkard T.R. Fiala C. Pollheimer J. et al.Self-Renewing Trophoblast Organoids Recapitulate the Developmental Program of the Early Human Placenta.Stem Cell Reports. 2018; 11: 537-551Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar; Okae et al., 2018Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of Human Trophoblast Stem Cells.Cell Stem Cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar; Turco et al., 2018Turco M.Y. Gardner L. Kay R.G. Hamilton R.S. Prater M. Hollinshead M.S. McWhinnie A. Esposito L. Fernando R. Skelton H. et al.Trophoblast organoids as a model for maternal-fetal interactions during human placentation.Nature. 2018; 564: 263-267Crossref PubMed Scopus (219) Google Scholar). However, whether these cell lines resemble early-stage trophoblasts during peri-implantation development has not been confirmed. Another approach to study early trophoblast development is to employ pluripotent stem cells (PSCs). Primed human PSCs have been induced to trophoblast lineages by bone morphogenetic protein 4 (BMP4) (Xu et al., 2002Xu R.H. Chen X. Li D.S. Li R. Addicks G.C. Glennon C. Zwaka T.P. Thomson J.A. BMP4 initiates human embryonic stem cell differentiation to trophoblast.Nat. Biotechnol. 2002; 20: 1261-1264Crossref PubMed Scopus (860) Google Scholar). Subsequently, trophoblast-like cells have been generated with cocktails of BMP4, the ALK4/5/7 inhibitor A83-01, and the FGFR inhibitor PD173074 (BAP medium) (Amita et al., 2013Amita M. Adachi K. Alexenko A.P. Sinha S. Schust D.J. Schulz L.C. Roberts R.M. Ezashi T. Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4.Proc. Natl. Acad. Sci. USA. 2013; 110: E1212-E1221Crossref PubMed Scopus (149) Google Scholar; Yang et al., 2015Yang Y. Adachi K. Sheridan M.A. Alexenko A.P. Schust D.J. Schulz L.C. Ezashi T. Roberts R.M. Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure.Proc. Natl. Acad. Sci. USA. 2015; 112: E2337-E2346Crossref PubMed Scopus (49) Google Scholar). However, primed PSC-derived cells differed in human leukocyte antigen (HLA) class I and epigenetic properties from in vivo trophoblasts (Bernardo et al., 2011Bernardo A.S. Faial T. Gardner L. Niakan K.K. Ortmann D. Senner C.E. Callery E.M. Trotter M.W. Hemberger M. Smith J.C. et al.BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages.Cell Stem Cell. 2011; 9: 144-155Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Human trophoblast-like cells have also been reportedly generated from expanded potential stem cells (EPSCs) (Gao et al., 2019Gao X. Nowak-Imialek M. Chen X. Chen D. Herrmann D. Ruan D. Chen A.C.H. Eckersley-Maslin M.A. Ahmad S. Lee Y.L. et al.Establishment of porcine and human expanded potential stem cells.Nat. Cell Biol. 2019; 21: 687-699Crossref PubMed Scopus (138) Google Scholar). Recently, naive human PSCs were established. These cells have gene expression patterns consistent with human pre-implantation epiblast (EPI) and naive mouse PSCs, representing an earlier stage than conventional primed PSCs (Guo et al., 2016Guo G. von Meyenn F. Santos F. Chen Y. Reik W. Bertone P. Smith A. Nichols J. Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass.Stem Cell Reports. 2016; 6: 437-446Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar; Stirparo et al., 2018Stirparo G.G. Boroviak T. Guo G. Nichols J. Smith A. Bertone P. Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast.Development. 2018; 145: dev158501Crossref PubMed Scopus (102) Google Scholar; Takashima et al., 2014Takashima Y. Guo G. Loos R. Nichols J. Ficz G. Krueger F. Oxley D. Santos F. Clarke J. Mansfield W. et al.Resetting transcription factor control circuitry toward ground-state pluripotency in human.Cell. 2014; 158: 1254-1269Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar; Theunissen et al., 2014Theunissen T.W. Powell B.E. Wang H. Mitalipova M. Faddah D.A. Reddy J. Fan Z.P. Maetzel D. Ganz K. Shi L. et al.Systematic identification of culture conditions for induction and maintenance of naive human pluripotency.Cell Stem Cell. 2014; 15: 471-487Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar). Although human trophoblasts arise from the morula as TE, single-cell RNA sequencing (scRNA-seq) data suggest that the fate determination of human cells might be late (Meistermann et al., 2019Meistermann D. Loubersac S. Reignier A. Firmin J. Campion V.F. Kilens S. Lelièvre Y. Lammers J. Feyeux M. Hulin P. et al.Spatio-temporal analysis of human preimplantation development reveals dynamics of epiblast and trophectoderm.bioRxiv. 2019; https://doi.org/10.1101/604751Crossref Scopus (0) Google Scholar; 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: 1012-1026Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). There are reports showing that naive human PSCs can differentiate into TS cells (Castel et al., 2020Castel G. Meistermann D. Bretin B. Firmin J. Blin J. Loubersac S. Bruneau A. Chevolleau S. Kilens S. Chariau C. et al.Induction of Human Trophoblast Stem Cells from Somatic Cells and Pluripotent Stem Cells.Cell Rep. 2020; 33: 108419Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar; Cinkornpumin et al., 2020Cinkornpumin J.K. Kwon S.Y. Guo Y. Hossain I. Sirois J. Russett C.S. Tseng H.W. Okae H. Arima T. Duchaine T.F. et al.Naive Human Embryonic Stem Cells Can Give Rise to Cells with a Trophoblast-like Transcriptome and Methylome.Stem Cell Reports. 2020; 15: 198-213Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar; Dong et al., 2020Dong C. Beltcheva M. Gontarz P. Zhang B. Popli P. Fischer L.A. Khan S.A. Park K.M. Yoon E.J. Xing X. et al.Derivation of trophoblast stem cells from naïve human pluripotent stem cells.eLife. 2020; 9: e52504Crossref PubMed Scopus (84) Google Scholar). However, because TE, which is the starting point for trophoblast development from pre-implantation, was not identified, the process of trophoblast development from the pre-implantation stage and the in vivo identity of human TS cells remain unclear. In the present study, we induced TE from naive human PSCs to establish an in vitro model of the trophoblast lineage. Naive PSC-derived TE differentiated successfully into cytotrophoblasts (CTs), which had a similar character as trophoblasts of cynomolgus monkey (Macaca fascicularis) embryos in vivo, trophoblasts of cultured human embryos, and placenta-derived TS cells. Furthermore, our analysis unveiled the counterpart of in vitro human TS cells as some parts of villous CTs (VCTs) and CTs in cell columns (CCCs) of in vivo first-trimester placenta. Finally, using this model, we investigated trophoblast development from TE to CTs during peri-implantation stages. Human trophoblasts arise from the morula as TE. After implantation, TE differentiates into CTs, syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs) (Figure 1A). We investigated whether naive human PSCs could differentiate into TE (Figures S1A and S1B). Using previously reported scRNA-seq data (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: 1012-1026Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar; Stirparo et al., 2018Stirparo G.G. Boroviak T. Guo G. Nichols J. Smith A. Bertone P. Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast.Development. 2018; 145: dev158501Crossref PubMed Scopus (102) Google Scholar), we identified TACSTD2 and ENPEP as cell surface marker genes of TE in pre-implantation human blastocysts (Figure 1B). TACSTD2 is expressed in TE of early (E5) and late blastocysts (E7), whereas ENPEP expression is increased in TE of late blastocysts. Next we analyzed signaling receptors and inferred the necessary signaling pathways to develop an appropriate TE induction medium. We examined Nodal, FGF, Janus kinases (JAK)/ signal transducer and activator of transcription proteins (STAT), and BMP signaling receptors because these are known to be related to early development in mice and humans. Expression was evaluated by fragments per kilobase of transcript per million mapped reads (FPKM) and F values (Figure S1C). The genes with the highest F value in each signaling pathway were ACVR1B, FGFR1, IL6R, and TGFBR3. TGFBR3 was highly expressed in TE of human embryos (Stirparo et al., 2018Stirparo G.G. Boroviak T. Guo G. Nichols J. Smith A. Bertone P. Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast.Development. 2018; 145: dev158501Crossref PubMed Scopus (102) Google Scholar; Table S1), but ACVR1B, FGFR1, and IL6R were not expressed (Figure S1D). FGFR2, the receptor for FGF4, which is important for mouse TS cell induction (Kunath et al., 2014Kunath T. Yamanaka Y. Detmar J. MacPhee D. Caniggia I. Rossant J. Jurisicova A. Developmental differences in the expression of FGF receptors between human and mouse embryos.Placenta. 2014; 35: 1079-1088Crossref PubMed Scopus (61) Google Scholar), was not expressed. These receptors exhibited similar patterns in cynomolgus monkey TE but not in mouse TE (Figures S1E and S1F; Nakamura et al., 2015Nakamura T. Yabuta Y. Okamoto I. Aramaki S. Yokobayashi S. Kurimoto K. Sekiguchi K. Nakagawa M. Yamamoto T. Saitou M. SC3-seq: a method for highly parallel and quantitative measurement of single-cell gene expression.Nucleic Acids Res. 2015; 43: e60Crossref PubMed Scopus (73) Google Scholar, Nakamura et al., 2016Nakamura T. Okamoto I. Sasaki K. Yabuta Y. Iwatani C. Tsuchiya H. Seita Y. Nakamura S. Yamamoto T. Saitou M. A developmental coordinate of pluripotency among mice, monkeys and humans.Nature. 2016; 537: 57-62Crossref PubMed Scopus (260) Google Scholar). Based on the above findings and previous reports (Amita et al., 2013Amita M. Adachi K. Alexenko A.P. Sinha S. Schust D.J. Schulz L.C. Roberts R.M. Ezashi T. Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4.Proc. Natl. Acad. Sci. USA. 2013; 110: E1212-E1221Crossref PubMed Scopus (149) Google Scholar; Cinkornpumin et al., 2020Cinkornpumin J.K. Kwon S.Y. Guo Y. Hossain I. Sirois J. Russett C.S. Tseng H.W. Okae H. Arima T. Duchaine T.F. et al.Naive Human Embryonic Stem Cells Can Give Rise to Cells with a Trophoblast-like Transcriptome and Methylome.Stem Cell Reports. 2020; 15: 198-213Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar; Dong et al., 2020Dong C. Beltcheva M. Gontarz P. Zhang B. Popli P. Fischer L.A. Khan S.A. Park K.M. Yoon E.J. Xing X. et al.Derivation of trophoblast stem cells from naïve human pluripotent stem cells.eLife. 2020; 9: e52504Crossref PubMed Scopus (84) Google Scholar; Okae et al., 2018Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of Human Trophoblast Stem Cells.Cell Stem Cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), we cultured naive human PSCs in a cocktail of A83-01, PD0325901 (PD03; a MEK inhibitor), JAK inhibitor I, and BMP4 for TE induction. Combinations of these factors partially upregulated trophoblast genes (Figure S1G). To optimize the four factors, we performed screening (Figure S1H–S1J; Table S2). The condition shown in Figure 1C induced TACSTD2+ENPEP+ cells most efficiently (Figure S1J). Under this condition, TACSTD2+ENPEP− cells were observed on days 1 and 2, and around 60% of TACSTD2+ENPEP+ cells were induced on day 3 (Figure 1D). Cell morphology on day 3 was polygonal and flat, whereas tightly packed three-dimensional colonies of naive PSCs were not seen (Figures 1E and S1A). TE marker proteins were expressed on day 3 (Figure 1F; Blakeley et al., 2015Blakeley P. Fogarty N.M. del Valle I. Wamaitha S.E. Hu T.X. Elder K. Snell P. Christie L. Robson P. Niakan K.K. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq.Development. 2015; 142: 3151-3165Crossref PubMed Scopus (283) Google Scholar; Chen et al., 2009Chen A.E. Egli D. Niakan K. Deng J. Akutsu H. Yamaki M. Cowan C. Fitz-Gerald C. Zhang K. Melton D.A. Eggan K. Optimal timing of inner cell mass isolation increases the efficiency of human embryonic stem cell derivation and allows generation of sibling cell lines.Cell Stem Cell. 2009; 4: 103-106Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). We sorted TACSTD2+ENPEP+ cells on day 3 by flow cytometry and confirmed that TACSTD2+ENPEP+ cells expressed trophoblast markers, whereas expression of pluripotent genes was decreased (Figure 1G). The top 25 EPI-specific genes (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: 1012-1026Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar) were highly expressed in naive PSCs, but expression declined with differentiation (Figure 1H; Table S3). Expression of almost all TE-specific genes was increased on days 2 and 3 (Figure 1H), suggesting that naive PSCs could differentiate into TE-like cells (nTEs). We confirmed downregulation of phosphorylated (p)SMAD2 under A83-01, of pERK1/2 under PD03, and of pSTAT3 under JAKi and transient upregulation of SMAD1/5 under BMP4 (Figure S1K). Furthermore, upregulation or downregulation of downstream genes in each signaling pathway suggested that these factors worked functionally under our condition (Figure S1L). TACSTD2+ENPEP+ cells were induced in three naive human embryonic stem cell (ESC)/induced pluripotent stem cell (iPSC) lines (H9 ESCs, 409B2 iPSCs, and adipose-derived iPS cells (AdiPS cells)) established by two different naive induction procedures (chemical reset and 5i/L/A) (Figure S1M; Guo et al., 2017Guo G. von Meyenn F. Rostovskaya M. Clarke J. Dietmann S. Baker D. Sahakyan A. Myers S. Bertone P. Reik W. et al.Epigenetic resetting of human pluripotency.Development. 2017; 144: 2748-2763Crossref PubMed Scopus (130) Google Scholar; Theunissen et al., 2014Theunissen T.W. Powell B.E. Wang H. Mitalipova M. Faddah D.A. Reddy J. Fan Z.P. Maetzel D. Ganz K. Shi L. et al.Systematic identification of culture conditions for induction and maintenance of naive human pluripotency.Cell Stem Cell. 2014; 15: 471-487Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar). PD03 could be replaced with an FGFR inhibitor, PD173074 (Figure S1N). We analyzed whether nTEs differentiate into post-implantation CTs (Figure 2A). TACSTD2 and ENPEP were expressed in CTs at 5 gestational weeks (Figure S2A). SIGLEC6, which has been reported to be expressed in trophoblasts of early pregnancy (Rumer et al., 2012Rumer K.K. Post M.D. Larivee R.S. Zink M. Uyenishi J. Kramer A. Teoh D. Bogart K. Winn V.D. Siglec-6 is expressed in gestational trophoblastic disease and affects proliferation, apoptosis and invasion.Endocr. Relat. Cancer. 2012; 19: 827-840Crossref PubMed Scopus (16) Google Scholar), was detected in CTs (CCCs and VCTs) and STs at 5 gestational weeks (Figure 2B) but not in TE (Figure S2B). Flow cytometry confirmed that first-trimester placentas (9 and 11 gestational weeks) and the placenta-derived TS cell line CT30 (Okae et al., 2018Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of Human Trophoblast Stem Cells.Cell Stem Cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar) expressed TACSTD2, ENPEP, and SIGLEC6 (Figure 2C, S2C, and S2D). Expression of mouse Tacstd2, Enpep, and Siglec5 (the mouse ortholog for human SIGLEC6) was conserved in mouse pre-implantation TE (Figure S2E). We thus chose to use these three genes as markers for CTs. As expected, nTEs on day 3 were SIGLEC6− (Figure 2D). To determine the induction condition from TE to CTs, we observed protein expression and signaling transduction in human chorionic villi (7 gestational weeks) (Figure S2F). Consistent with previous studies (Haider et al., 2018Haider S. Meinhardt G. Saleh L. Kunihs V. Gamperl M. Kaindl U. Ellinger A. Burkard T.R. Fiala C. Pollheimer J. et al.Self-Renewing Trophoblast Organoids Recapitulate the Developmental Program of the Early Human Placenta.Stem Cell Reports. 2018; 11: 537-551Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar; Okae et al., 2018Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of Human Trophoblast Stem Cells.Cell Stem Cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar; Turco et al., 2018Turco M.Y. Gardner L. Kay R.G. Hamilton R.S. Prater M. Hollinshead M.S. McWhinnie A. Esposito L. Fernando R. Skelton H. et al.Trophoblast organoids as a model for maternal-fetal interactions during human placentation.Nature. 2018; 564: 263-267Crossref PubMed Scopus (219) Google Scholar), non-phosphorylated β-catenin was localized in the cell membrane of CCCs and VCTs, suggesting that Wnt is active. The pEGF receptor was strongly expressed in the cell membranes of CCCs, VCTs, and STs, whereas pSMAD2 was not accumulated in the nuclei of CCCs, VCTs, or STs (Figure S2G). We therefore cultured nTEs on day 3 in A83-01, CHIR99021, and epidermal growth factor (EGF) (ACE) (Figure 2A). In ACE, nTEs differentiated into TACSTD2+, ENPEP+, and SIGLEC6+ CT-like cells (naive CTs [nCTs]) on day 10 (Figure 2D). TACSTD2+ENPEP+SIGLEC6+ cells were sorted on day 10 and continued to be cultured in ACE. nCTs continuously expressed TACSTD2, ENPEP, and SIGLEC6 (Figure 2D), and the morphology of nCTs was maintained for more than 10 passages and resembled that of placenta-derived TS cells (Figure 2E). Their chromosomes were normal (Figure S2H). nCTs expressed trophoblast markers over 10 passages (Figures 2F and 2G) and could be maintained for more than 35 passages and over 75 days like CT stem cells (Figure S2I; Video S1). In previous reports, Ki67 was expressed in CTs (Chang and Parast, 2017Chang C.W. Parast M.M. Human trophoblast stem cells: Real or not real?.Placenta. 2017; 60: S57-S60Crossref PubMed Scopus (17) Google Scholar; Mühlhauser et al., 1993Mühlhauser J. Crescimanno C. Kaufmann P. Höfler H. Zaccheo D. Castellucci M. Differentiation and proliferation patterns in human trophoblast revealed by c-erbB-2 oncogene product and EGF-R.J. Histochem. Cytochem. 1993; 41: 165-173Crossref PubMed Scopus (134) Google Scholar). Almost all nCTs expressed Ki67 (Figure 2H). Interestingly, CCCs and some VCTs of human chorionic villi expressed Ki67 at 5 gestational weeks, but the number of Ki67+ cells decreased by 9 gestational weeks and had almost disappeared at 11 gestational weeks, indicating that our nCTs correspond to the early stage of CCCs and VCTs (Figures 2I and S2J). We also tested each counteracting signaling molecule of ACE compounds. Activin A, XAV939, and gefitinib (an EGF receptor tyrosine kinase inhibitor) each reduced cell growth and viability (Figure S2K), indicating that all three ACE compounds are necessary to maintain nCTs. https://www.cell.com/cms/asset/0c819e27-eee6-4bc2-86ca-46c348185362/mmc7.mp4Loading ... Download .mp4 (6.08 MB) Help with .mp4 files Video S1. Time-lapse video of culturing nTE-derived cytotrophoblast stem cells, related to Figures 2 and S236 h. Furthermore, TACSTD2+ENPEP+SIGLEC6+ cells of placental chorionic villi at 7 and 9 gestational weeks were sorted and cultured in ACE (Figure S3A). Chorionic villus-derived (7 and 9 gestational weeks) CT cells (cCTs) cultured in ACE for more than 10 passages had a similar morphology as nCTs and TS cells (Figures S3A and S3B). cCTs expressed CT markers similarly as nCTs after 10 passages (Figures S3C and S3D) and were karyotypically stable (Figure S3E). cCTs differentiated into ST-like cells in forskolin (Figures S3F–S3H) and into EVT-like cells in A83-01 and Neuregulin-1 with Geltrex (Figures S3I and S3J). Next we analyzed the global transcriptome of nTEs; nCTs; TACSTD2+ENPEP+SIGLEC6+ placental chorionic villi at 7, 9, and 11 gestational weeks; and CT30 cells, which we cultured under a condition reported previously (human TS condition, SAVECY) (Okae et al., 2018Okae H. Toh H. Sato T. Hiura H. Takahashi S. Shirane K. Kabayama Y. Suyama M. Sasaki H. Arima T. Derivation of Human Trophoblast Stem Cells.Cell Stem Cell. 2018; 22: 50-63.e6Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), and also the published RNA-seq data of first-trimester human yolk sacs (Cindrova-Davies et al., 2017Cindrova-Davies T. Jauniaux E. Elliot M.G. Gong S. Burton G.J. Charnock-Jones D.S. RNA-seq reveals conservation of function among the yolk sacs of human, mouse, and chicken.Proc. Natl. Acad. Sci. USA. 2017; 114: E4753-E4761Crossref PubMed Scopus (40) Google Scholar). Unbiased hierarchical clustering (UHC) suggested that nCTs, CT30 cells, and placental chorionic villi have more similar gene expression profiles than nTEs and naive PSCs, which correspond to the pre-implantation developmental stage (Figure 2J). Principal-component analysis (PCA) revealed that various nCT lines have gene expression patterns similar to CT30 cells and chorionic villi (Figure 2K). nTEs were distant from nCTs and placental chorionic villi. During differentiation, TE marker genes were downregulated even in TE induction medium (Figure S3K). Monitoring CDX2 expression using CDX2-GFP H9 ESCs (Figure S3L), CDX2-GFP+ cells lost GFP expression after 7 or 10 days in TE induction medium, CT induction medium, or other conditions except for A83-01, CHIR99021, and lysophosphatidic acid (ACL), which was applied for mouse polar-like TS cells (Frias-Aldeguer et al., 2020Frias-Aldeguer J. Kip M. Vivié J. Li L. Alemany A. Korving J. Darmis F. van Oudenaarden A. Geijsen N. Rivron N.C. Embryonic signals perpetuate polar-like trophoblast stem cells and pattern the blastocyst axis.bioRxiv. 2020; https://doi.org/10.1101/510362Crossref Scopus (0) Google Scholar; Figure S3M). However, half of the sorted cells in ACL lost GFP expression on day 7. Under counteracting signaling molecules in ACE, expression of C