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• W2775775321 abstract "•Human TS cells have the capacity to give rise to the three major trophoblast lineages•Human TS and primary trophoblast cells have similar transcriptomes and methylomes•Human TS cells injected into mice mimic trophoblast invasion during implantation•Signaling pathways maintaining human and mouse TS cells are substantially different Trophoblast cells play an essential role in the interactions between the fetus and mother. Mouse trophoblast stem (TS) cells have been derived and used as the best in vitro model for molecular and functional analysis of mouse trophoblast lineages, but attempts to derive human TS cells have so far been unsuccessful. Here we show that activation of Wingless/Integrated (Wnt) and EGF and inhibition of TGF-β, histone deacetylase (HDAC), and Rho-associated protein kinase (ROCK) enable long-term culture of human villous cytotrophoblast (CT) cells. The resulting cell lines have the capacity to give rise to the three major trophoblast lineages, which show transcriptomes similar to those of the corresponding primary trophoblast cells. Importantly, equivalent cell lines can be derived from human blastocysts. Our data strongly suggest that the CT- and blastocyst-derived cell lines are human TS cells, which will provide a powerful tool to study human trophoblast development and function. Trophoblast cells play an essential role in the interactions between the fetus and mother. Mouse trophoblast stem (TS) cells have been derived and used as the best in vitro model for molecular and functional analysis of mouse trophoblast lineages, but attempts to derive human TS cells have so far been unsuccessful. Here we show that activation of Wingless/Integrated (Wnt) and EGF and inhibition of TGF-β, histone deacetylase (HDAC), and Rho-associated protein kinase (ROCK) enable long-term culture of human villous cytotrophoblast (CT) cells. The resulting cell lines have the capacity to give rise to the three major trophoblast lineages, which show transcriptomes similar to those of the corresponding primary trophoblast cells. Importantly, equivalent cell lines can be derived from human blastocysts. Our data strongly suggest that the CT- and blastocyst-derived cell lines are human TS cells, which will provide a powerful tool to study human trophoblast development and function. The placenta is a multifunctional organ essential for fetal development and survival. Trophoblast cells are specialized cells in the placenta that mediate the interactions between the fetus and mother at the feto-maternal interface. In the human placenta, there are three major trophoblast subpopulations: the cytotrophoblast (CT), extravillous cytotrophoblast (EVT), and syncytiotrophoblast (ST) (Bischof and Irminger-Finger, 2005Bischof P. Irminger-Finger I. The human cytotrophoblastic cell, a mononuclear chameleon.Int. J. Biochem. Cell Biol. 2005; 37: 1-16Crossref PubMed Scopus (133) Google Scholar, James et al., 2012James J.L. Carter A.M. Chamley L.W. Human placentation from nidation to 5 weeks of gestation. Part I: What do we know about formative placental development following implantation?.Placenta. 2012; 33: 327-334Crossref PubMed Scopus (126) Google Scholar). CT cells are an undifferentiated and proliferative population that can give rise to EVT and ST cells. CT cells aggregate into cell columns at the tips of villi, where they differentiate into EVT cells. EVT cells can be subdivided based on their anatomical locations (Cierna et al., 2016Cierna Z. Varga I. Danihel Jr., L. Kuracinova K. Janegova A. Danihel L. Intermediate trophoblast–A distinctive, unique and often unrecognized population of trophoblastic cells.Ann. Anat. 2016; 204: 45-50Crossref PubMed Scopus (15) Google Scholar). Those that invade the decidualized endometrium are called interstitial EVT cells. Those that invade and remodel the spiral arteries are known as endovascular EVTs. Other subtypes likely exist because EVT cells have also been found in uterine glands, veins, and lymphatics (Moser et al., 2010Moser G. Gauster M. Orendi K. Glasner A. Theuerkauf R. Huppertz B. Endoglandular trophoblast, an alternative route of trophoblast invasion? Analysis with novel confrontation co-culture models.Hum. Reprod. 2010; 25: 1127-1136Crossref PubMed Scopus (95) Google Scholar, Windsperger et al., 2017Windsperger K. Dekan S. Pils S. Golletz C. Kunihs V. Fiala C. Kristiansen G. Knöfler M. Pollheimer J. Extravillous trophoblast invasion of venous as well as lymphatic vessels is altered in idiopathic, recurrent, spontaneous abortions.Hum. Reprod. 2017; 32: 1208-1217Crossref PubMed Scopus (67) Google Scholar). Multinucleated ST cells are formed by fusion of CT cells and produce large quantities of placental hormones and other factors to maintain pregnancy. ST cells are directly in contact with maternal blood and mediate the exchange of gases and nutrients. All of the trophoblast lineages arise from the trophectoderm (TE) cells of the blastocyst, and their coordinated proliferation and differentiation is essential for a successful pregnancy. Impaired trophoblast development and function are thought to lead to various pregnancy complications, including miscarriage, preeclampsia, and intrauterine growth restriction (Moffett and Loke, 2006Moffett A. Loke C. Immunology of placentation in eutherian mammals.Nat. Rev. Immunol. 2006; 6: 584-594Crossref PubMed Scopus (642) Google Scholar, Norwitz, 2006Norwitz E.R. Defective implantation and placentation: laying the blueprint for pregnancy complications.Reprod. Biomed. Online. 2006; 13: 591-599Abstract Full Text PDF PubMed Scopus (190) Google Scholar). Mouse trophoblast stem (TS) cells, which were first derived from blastocysts and the extra-embryonic ectoderm (ExE) of post-implantation embryos (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 (1078) Google Scholar), are the best in vitro model for molecular and functional analysis of mouse trophoblast cells. In the presence of fibroblast growth factor 4 (FGF4) and transforming growth factor β1 (TGF-β1)/Activin, mouse TS cells self-renew indefinitely without losing their ability to differentiate into all trophoblast lineages. A number of transcription factors, including Cdx2, Eomes, Elf5, Esrrb, and Gata3, have been identified as essential for maintaining the undifferentiated state of mouse TS cells (Latos and Hemberger, 2016Latos P.A. Hemberger M. From the stem of the placental tree: trophoblast stem cells and their progeny.Development. 2016; 143: 3650-3660Crossref PubMed Scopus (67) Google Scholar). Although it has been assumed that TE cells of human blastocysts and CT cells of early human placentas contain a stem cell population, attempts to derive human TS cells from these cells have so far been unsuccessful (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 (65) Google Scholar, Soncin et al., 2015Soncin F. Natale D. Parast M.M. Signaling pathways in mouse and human trophoblast differentiation: a comparative review.Cell. Mol. Life Sci. 2015; 72: 1291-1302Crossref PubMed Scopus (86) Google Scholar). In this study, we analyzed the transcriptomes of primary trophoblast cells to infer how CT cells are maintained in their undifferentiated state in vivo. Using this knowledge, we optimized the culture conditions and derived human TS cells from CT cells and blastocysts. Our culture system will provide a powerful tool to study human trophoblast development and function. We isolated CT, EVT, and ST cells from first-trimester placentas (Figures S1A–S1C) and performed RNA sequencing (RNA-seq) (Table S1). We identified 377, 228, and 289 genes that were predominantly expressed in CT, EVT, and ST cells, respectively (fragments per kilobase per million [FPKM] > 10 in the cell type with the highest expression, fold change > 4, adjusted p < 0.01) (Figure 1A). We confirmed that widely used lineage markers such as ITGA6 and TP63 (CT), ITGA5 and HLA-G (EVT), and CGB and CSH1 (ST) (Bischof and Irminger-Finger, 2005Bischof P. Irminger-Finger I. The human cytotrophoblastic cell, a mononuclear chameleon.Int. J. Biochem. Cell Biol. 2005; 37: 1-16Crossref PubMed Scopus (133) Google Scholar, Reis-Filho et al., 2003Reis-Filho J.S. Simpson P.T. Martins A. Preto A. Gartner F. Schmitt F.C. Distribution of p63, cytokeratins 5/6 and cytokeratin 14 in 51 normal and 400 neoplastic human tissue samples using TARP-4 multi-tumor tissue microarray.Virchows Arch. 2003; 443: 122-132Crossref PubMed Scopus (204) Google Scholar) were included in the gene lists. We then conducted functional annotation of the gene lists using ConsensusPathDB (Herwig et al., 2016Herwig R. Hardt C. Lienhard M. Kamburov A. Analyzing and interpreting genome data at the network level with ConsensusPathDB.Nat. Protoc. 2016; 11: 1889-1907Crossref PubMed Scopus (233) Google Scholar; Figure 1A). Intriguingly, genes related to the Wingless/Integrated (Wnt) and epidermal growth factor (EGF) signal transduction pathways (“regulation of FZD by ubiquitination” and “EGFR1”) were overrepresented in the CT highest (CThighest) gene list. Wnt and EGF signaling are required for proliferation of various epithelial stem cells, including skin stem cells and intestinal stem cells (Fatehullah et al., 2016Fatehullah A. Tan S.H. Barker N. Organoids as an in vitro model of human development and disease.Nat. Cell Biol. 2016; 18: 246-254Crossref PubMed Scopus (837) Google Scholar, Hsu et al., 2014Hsu Y.C. Li L. Fuchs E. Emerging interactions between skin stem cells and their niches.Nat. Med. 2014; 20: 847-856Crossref PubMed Scopus (364) Google Scholar). Consistently, the top-ranked pathway for the CThighest genes was “hair follicle development,” which included some genes important for the maintenance of hair follicle stem cells (TP63, FGFR2, and CTNNB1 [encoding β-catenin]). These data imply that CT cells might be maintained under conditions similar to those of the other epithelial stem cells. Based on the results described above, we tried to culture CT cells in a medium containing CHIR99021 (a Wnt activator) and EGF, but the cells did not adhere to the culture plate and died within several days. We then tested several inhibitors and growth factors (Figure 1B) that are known to enhance in vitro proliferation of various epithelial stem cells (Fatehullah et al., 2016Fatehullah A. Tan S.H. Barker N. Organoids as an in vitro model of human development and disease.Nat. Cell Biol. 2016; 18: 246-254Crossref PubMed Scopus (837) Google Scholar). In the presence of all of these inhibitors and growth factors, highly proliferative cell lines were derived from CT cells (condition 1 in Figure 1B and Figure S1D). Among the inhibitors and growth factors, Y27632 (a Rho-associated protein kinase [ROCK] inhibitor) was found to be essential for cell attachment and was added to all culture media in subsequent experiments. CHIR99021 was indispensable for cell proliferation, and its absence led to differentiation of CT cells into HLA-G-positive EVT-like cells (Figure S1E). EGF, A83-01, and SB431542 (TGF-β inhibitors) and valproic acid (VPA) (a histone deacetylase [HDAC] inhibitor) significantly enhanced proliferation of CT cells (Figure 1B). Eventually, we found that CHIR99021, EGF, TGF-β inhibitors, VPA, and Y27632 together were sufficient for long-term culture of CT cells (Figure 1C). We were able to derive proliferative CT cells from as few as 1,000 CT cells (five cell lines from five independent experiments) but failed to derive TS cells from single CT cells (n = 200). CHIR99021, EGF, TGF-β inhibitors, and VPA were all important for the long-term maintenance of proliferative CT cells (Figure S1F). VPA could be replaced by trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) (Figure S1G). Although either A83-01 or SB431542 could support the derivation of proliferative CT cells (Figure S1H), we retained both inhibitors in consideration of their different specificities (Vogt et al., 2011Vogt J. Traynor R. Sapkota G.P. The specificities of small molecule inhibitors of the TGFß and BMP pathways.Cell. Signal. 2011; 23: 1831-1842Crossref PubMed Scopus (200) Google Scholar). The culture conditions tested in this study are summarized in Table S2. We successfully derived proliferative CT cell lines from all first-trimester placental samples tested (n = 8) (Table S3). In contrast, we were unable to derive such cells from term placentas (placentas obtained after elective caesarean section, n = 5) under the same conditions. The proliferative CT cells had a normal karyotype (Figure S1I) and continued to proliferate for at least 5 months (∼150 population doublings) (Figure 1D). These cells expressed a pan-trophoblast marker, KRT7 (Figure 1E), but HLA-ABC expression was very low (Figure 1F), which is a hallmark of CT cells (King et al., 2000King A. Thomas L. Bischof P. Cell culture models of trophoblast II: trophoblast cell lines–a workshop report.Placenta. 2000; 21: S113-S119Crossref PubMed Scopus (124) Google Scholar). They also expressed TP63 and TEAD4 (CT markers) and GATA3 (a mononuclear trophoblast marker) (Figure 1G). The proliferative CT cells were designated CT-derived TS cells (TSCT cells) because they had the ability to differentiate into EVT- and ST-like cells as detailed below. To analyze the differentiation potential of TSCT cells, we first cultured TS cells in a basal medium containing only Y27632. Most of the cells differentiated into multinucleated ST-like cells, but some cells remained mononucleated (Figure S2A). The culture conditions did not support the survival of the differentiated cells, and most of them died within 5 days. Therefore, additional factor(s) may be required for the efficient and directed differentiation of TSCT cells. Matrigel is widely used to induce outgrowth of EVT cells from placental explants (Miller et al., 2005Miller R.K. Genbacev O. Turner M.A. Aplin J.D. Caniggia I. Huppertz B. Human placental explants in culture: approaches and assessments.Placenta. 2005; 26: 439-448Crossref PubMed Scopus (177) Google Scholar). A recent study also revealed that decidua-derived NRG1 promotes EVT formation in placental explant cultures (Fock et al., 2015Fock V. Plessl K. Draxler P. Otti G.R. Fiala C. Knöfler M. Pollheimer J. Neuregulin-1-mediated ErbB2-ErbB3 signalling protects human trophoblasts against apoptosis to preserve differentiation.J. Cell Sci. 2015; 128: 4306-4316Crossref PubMed Scopus (24) Google Scholar). In addition, CT cells preferentially differentiated into EVT-like cells under condition 2 shown in Figure 1B (see also Figure S1E). Among the inhibitors and growth factors contained in condition 2, the TGF-β inhibitors were found to promote differentiation of CT cells into EVT-like cells, and A83-01 was more potent than SB431542 (Figure S2B). In a culture system containing NRG1, A83-01, and Matrigel (Figure 2A), TSCT cells underwent epithelial-mesenchymal transition (Figure 2B; Figure S2C) and gave rise to EVT-like cells that strongly expressed HLA-G (Figure 2C). The resulting cells were named EVT-TSCT cells. We confirmed that NRG1, A83-01, and Matrigel were all important for the induction of EVT-TSCT cells (Figure S2D). ITGA6 and CDH1 (CT markers), SDC1 (an ST marker), and VIM (a stromal marker) expression was low or undetectable in EVT-TSCT cells (Figure 2G). CGB (an ST marker) is expressed at low levels in EVT cells (Pröll et al., 2000Pröll J. Bensussan A. Goffin F. Foidart J.M. Berrebi A. Le Bouteiller P. Tubal versus uterine placentation: similar HLA-G expressing extravillous cytotrophoblast invasion but different maternal leukocyte recruitment.Tissue Antigens. 2000; 56: 479-491Crossref PubMed Scopus (52) Google Scholar) and, consistently, was detectable in EVT-TSCT cells (Figure 2G). Previous studies on choriocarcinoma cell lines revealed that cyclic AMP (cAMP) enhances ST formation (Strauss et al., 1992Strauss 3rd, J.F. Kido S. Sayegh R. Sakuragi N. Gåfvels M.E. The cAMP signalling system and human trophoblast function.Placenta. 1992; 13: 389-403Crossref PubMed Scopus (103) Google Scholar). Thus, we treated TSCT cells with forskolin, a cAMP agonist (Figure 2A). In the presence of forskolin, the cells started to make aggregates and efficiently fused to form large syncytia (Figures 2D and 2E; Figure S2E). The ST markers CGB and SDC1 were highly expressed in these syncytia, whereas ITGA6, CDH1, HLA-G, and VIM were poorly expressed (Figure 2G). The ST-like syncytia were designated ST(2D)-TSCT cells. It has also been reported that 3D culture enhances differentiation of choriocarcinoma cells into ST-like cells (McConkey et al., 2016McConkey C.A. Delorme-Axford E. Nickerson C.A. Kim K.S. Sadovsky Y. Boyle J.P. Coyne C.B. A three-dimensional culture system recapitulates placental syncytiotrophoblast development and microbial resistance.Sci. Adv. 2016; 2: e1501462Crossref PubMed Scopus (73) Google Scholar). Therefore, we cultured the proliferative CT cells in low adhesion plates (Figure 2A). These cells formed cyst-like structures (Figure 2F), expressed CGB and SDC1 (Figure 2H), and secreted a large amount of human chorionic gonadotropin (hCG) (Figure 2I). Forskolin and EGF synergistically enhanced the formation of the cyst-like structures (Figure S2F). These cyst-like structures were designated ST(3D)-TSCT cells. Expression of ST markers was higher in ST(3D)-TSCT cells than in ST(2D)-TSCT cells (Figure S2G). TSCT cells maintained their ability to differentiate into EVT- and ST-like cells after 50 passages (Figures S2H and S2I). We also cultured single TSCT cells (n = 50) and isolated 10 clonal lines (Figure 2J). We randomly selected three clonal lines and confirmed that they could differentiate into both EVT- and ST-like cells (Figures 2K and 2L), suggesting that individual TSCT cells were bipotent. We next investigated whether cells similar to TSCT cells could be derived directly from human blastocysts. Sixteen blastocysts were cultured under the same conditions (Figure 3A), and eight cell lines were established. These cell lines, designated blastocyst-derived TS cells (TSblast cells), were morphologically similar to TSCT cells (Figure 3B). A normal karyotype was confirmed in all six TSblast cell lines examined (Figure S3A; Table S3). TSblast cells continued to proliferate for at least 5 months (Figure 3B). As in the case of TSCT cells, TSblast cells expressed KRT7, TP63, GATA3, and TEAD4 (Figures 3C and 3D), and HLA-ABC expression was very low (Figure S3B). Furthermore, TSblast cells had the ability to differentiate into EVT-TSblast (Figures 3E and 3F), ST(2D)-TSblast (Figures 3G–3I), and ST(3D)-TSblast cells (Figures 3J and 3K) just as TSCT cells did (Figure 2A), although two TSblast lines (4 and 7) differentiated into EVT-like cells less efficiently than the other TSblast and TSCT lines (Figures S3C and S3D). The differentiation ability of TSblast cells was maintained after 55 passages (Figures S3E and S3F). We also cultured single TSblast cells (n = 50) and isolated 8 clonal lines. We randomly selected three clonal lines and confirmed that they could differentiate into both EVT- and ST-like cells (Figures 3L and 3M). To determine whether TSCT and TSblast cells had gene expression patterns similar to primary trophoblast cells, we performed RNA-seq of TSCT and TSblast cells and their derivatives (Figure 4A). A preliminary investigation suggested that ST(2D) and ST(3D) cells had very similar transcriptome profiles, but ST(3D) cells were a little more similar to primary ST cells (Figure S4A). Therefore, we chose ST(3D) cells as the model of ST cells. We compared the RNA-seq data with those of primary trophoblast cells and placenta-derived stromal cells. Hierarchical clustering revealed that TSCT and TSblast cells had very similar gene expression patterns to each other, both before and after their differentiation (R > 0.98) (Figure 4A). Importantly, the gene expression profiles of CT cells were closest to those of TSCT and TSblast cells (Figure 4A). The profiles of TSCT- and TSblast-derived EVT- and ST-like cells were closely related to those of primary EVT and ST cells, respectively (Figure 4A). Furthermore, most of the genes predominantly expressed in CT, EVT, or ST cells (the genes shown in Figure 1A) showed similar expression patterns in TSCT and TSblast cells and their derivatives (Figure 4B). We then focused on some representative lineage markers. All CT markers we examined exhibited the expected expression patterns, although some genes, such as LRP5, TP63, and ELF5, showed lower expression in TSCT and TSblast cells (Figure 4C). Most EVT and ST markers also showed comparable expression patterns and levels in the primary and cultured cells, with a few exceptions (e.g., CD9 and CSH1) (Figures 4D and 4E). Although TSCT and TSblast cells had gene expression profiles similar to those of primary cells, they were not exactly the same, presumably reflecting the artificial in vitro conditions. Gene set enrichment analysis (GSEA) revealed that genes associated with various gene ontology (GO) terms were differentially expressed between the primary and cultured cells. Notably, genes related to ribosome biogenesis were especially enriched in TS cells (Figure S4B), which might contribute to TS cell proliferation because ribosomes drive cell proliferation and growth. We also found that DNA replication-related genes were significantly depleted in TS-derived EVT-like cells (Figure S4C), consistent with our observation that TS cells differentiating into EVT-like cells gradually lost their proliferative capacity. Several genes such as CYP19A1, EDNRB, IL2RB, and PTN are reported to have placenta-specific promoters (Cohen et al., 2011Cohen C.J. Rebollo R. Babovic S. Dai E.L. Robinson W.P. Mager D.L. Placenta-specific expression of the interleukin-2 (IL-2) receptor β subunit from an endogenous retroviral promoter.J. Biol. Chem. 2011; 286: 35543-35552Crossref PubMed Scopus (31) Google Scholar, Rawn and Cross, 2008Rawn S.M. Cross J.C. The evolution, regulation, and function of placenta-specific genes.Annu. Rev. Cell Dev. Biol. 2008; 24: 159-181Crossref PubMed Scopus (182) Google Scholar). We found that these placenta-specific promoters were active in ST(3D)-TSCT and ST(3D)-TSblast cells (Figure S4D). As shown in Figure 4C, FGFR2 was predominantly expressed in CT cells and undifferentiated TSCT and TSblast cells. We found that, of the two major isoforms of FGFR2 (FGFR2b and FGFR2c), FGFR2b was expressed almost exclusively (Figure S4E). This is intriguing because the essential role of Fgfr2c was reported in mouse trophoblast cells (Arman et al., 1998Arman E. Haffner-Krausz R. Chen Y. Heath J.K. Lonai P. Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development.Proc. Natl. Acad. Sci. USA. 1998; 95: 5082-5087Crossref PubMed Scopus (519) Google Scholar). Furthermore, CDX2, EOMES, ESRRB, and SOX2, which encode transcription factors required for mouse TS cell self-renewal (Latos and Hemberger, 2016Latos P.A. Hemberger M. From the stem of the placental tree: trophoblast stem cells and their progeny.Development. 2016; 143: 3650-3660Crossref PubMed Scopus (67) Google Scholar), were poorly expressed (< 1 FPKM) in CT, TSCT, and TSblast cells (Table S1). Trophoblast cells have unique DNA methylation patterns characterized by large partially methylated domains (PMDs) (Schroeder et al., 2013Schroeder D.I. Blair J.D. Lott P. Yu H.O. Hong D. Crary F. Ashwood P. Walker C. Korf I. Robinson W.P. LaSalle J.M. The human placenta methylome.Proc. Natl. Acad. Sci. USA. 2013; 110: 6037-6042Crossref PubMed Scopus (205) Google Scholar), placenta-specific promoter hypomethylation (Robinson and Price, 2015Robinson W.P. Price E.M. The human placental methylome.Cold Spring Harb. Perspect. Med. 2015; 5: a023044Crossref PubMed Scopus (52) Google Scholar), and placenta-specific germline differentially methylated regions (gDMRs) (Court et al., 2014Court F. Tayama C. Romanelli V. Martin-Trujillo A. Iglesias-Platas I. Okamura K. Sugahara N. Simón C. Moore H. Harness J.V. et al.Genome-wide parent-of-origin DNA methylation analysis reveals the intricacies of human imprinting and suggests a germline methylation-independent mechanism of establishment.Genome Res. 2014; 24: 554-569Crossref PubMed Scopus (240) Google Scholar). To examine whether these unique methylation patterns were maintained in TSCT and TSblast cells, we performed whole-genome bisulfite sequencing (WGBS) of TSCT and TSblast cells and compared the data with those of CT cells (Hamada et al., 2016Hamada H. Okae H. Toh H. Chiba H. Hiura H. Shirane K. Sato T. Suyama M. Yaegashi N. Sasaki H. Arima T. Allele-Specific Methylome and Transcriptome Analysis Reveals Widespread Imprinting in the Human Placenta.Am. J. Hum. Genet. 2016; 99: 1045-1058Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), human embryonic stem cells (ESCs) (Lister et al., 2011Lister R. Pelizzola M. Kida Y.S. Hawkins R.D. Nery J.R. Hon G. Antosiewicz-Bourget J. O’Malley R. Castanon R. Klugman S. et al.Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells.Nature. 2011; 471: 68-73Crossref PubMed Scopus (1216) Google Scholar), and cord blood cells (Okae et al., 2014Okae H. Chiba H. Hiura H. Hamada H. Sato A. Utsunomiya T. Kikuchi H. Yoshida H. Tanaka A. Suyama M. Arima T. Genome-wide analysis of DNA methylation dynamics during early human development.PLoS Genet. 2014; 10: e1004868Crossref PubMed Scopus (170) Google Scholar) (Figure 5A). TSCT and TSblast cells showed almost identical global DNA methylation patterns (R = 0.97). Although the average methylation levels of TSCT (33.7%) and TSblast cells (33.6%) were substantially lower than that of CT cells (52.3%), their methylation patterns were similar to each other (R ≥ 0.80). Most of the PMDs defined in a previous study (Schroeder et al., 2013Schroeder D.I. Blair J.D. Lott P. Yu H.O. Hong D. Crary F. Ashwood P. Walker C. Korf I. Robinson W.P. LaSalle J.M. The human placenta methylome.Proc. Natl. Acad. Sci. USA. 2013; 110: 6037-6042Crossref PubMed Scopus (205) Google Scholar) maintained the intermediate methylation levels in CT cells but were hypomethylated in TSCT and TSblast cells (Figures 5B and 5C). Actively transcribed regions showed higher methylation levels compared with other regions in CT, TSCT, and TSblast cells (Figure S5A), consistent with previous findings in the human placenta (Schroeder et al., 2013Schroeder D.I. Blair J.D. Lott P. Yu H.O. Hong D. Crary F. Ashwood P. Walker C. Korf I. Robinson W.P. LaSalle J.M. The human placenta methylome.Proc. Natl. Acad. Sci. USA. 2013; 110: 6037-6042Crossref PubMed Scopus (205) Google Scholar). Therefore, the placenta-specific DNA methylome was largely maintained in TSCT and TSblast cells, although the cause and significance of the PMD hypomethylation remain unclear. We next analyzed the ELF5 promoter, which is hypomethylated in trophoblast cells but hypermethylated in many other cell types (Hemberger et al., 2010Hemberger M. Udayashankar R. Tesar P. Moore H. Burton G.J. ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.Hum. Mol. Genet. 2010; 19: 2456-2467Crossref PubMed Scopus (140) Google Scholar). We found that the ELF5 promoter was hypomethylated in both TSCT and TSblast cells (Figure 5D). In addition to the ELF5 promoter, we identified 55 promoters with methylation patterns similar to that of the ELF5 promoter (methylation level < 20% in CT cells and > 80% in ESCs and blood cells), which included some promoters that are known to be specifically hypomethylated in the placenta (e.g., the promoters of INSL4 and DSCR4) (Du et al., 2011Du Y. Zhang J. Wang H. Yan X. Yang Y. Yang L. Luo X. Chen Y. Duan T. Ma D. Hypomethylated DSCR4 is a placenta-derived epigenetic marker for trisomy 21.Prenat. Diagn. 2011; 31: 207-214Crossref PubMed Scopus (15) Google Scholar, Macaulay et al., 2011Macaulay E.C. Weeks R.J. Andrews S. Morison I.M. Hypomethylation of functional retrotransposon-derived genes in the human placenta.Mamm. Genome. 2011; 22: 722-735Crossref PubMed Scopus (36) Google Scholar). We found that most of these promoters (48 of 55) maintained less than 20% methylation levels in TSCT and TSblast cells (Figure 5E; Table S4). We confirmed the hypomethylation of three selected promoters (DSCR4, ELF5, and ZNF750) in all TSCT and TSblast lines established in this study (Figure S5B). We also identified 5 promoters with the opposite pattern, and all of them had more than 80% methylation levels in TSCT and TSblast cells (Figure 5E; Table S4). A number of placenta-specific gDMRs, which maintain allele-specific DNA methylation in a placenta-specific manner, have been identified. We focused on placenta-specific gDMRs associated with imprinted genes (n = 33) (Table S5). Most of the gDMRs maintained the expected intermediate methylation levels (30%–70%) in CT (33 of 33), TSCT (26 of 33), and TSblast cells (24 of 33) but not in ESCs (0 of 33) or blood cells (0 of 33). We analyzed allele-specific DNA methylation in two TSCT cell lines using targeted bisulfite sequencing (see STAR Methods for details). The allelic methylation patterns were successfully obtained for ten placenta-specific gDMRs, and nine of them maintained maternal allele-specific DNA methylation in TSCT cells (Figure 5F). We did not analyze the allelic DNA methylation patterns in TSblast cells because the maternal genotype was not available. However, nine of the ten gDMRs maintained i" @default.
• W2775775321 created "2017-12-22" @default.
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• W2775775321 date "2018-01-01" @default.
• W2775775321 modified "2023-10-17" @default.
• W2775775321 title "Derivation of Human Trophoblast Stem Cells" @default.
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• W2775775321 doi "https://doi.org/10.1016/j.stem.2017.11.004" @default.
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