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• W3011805855 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Naïve human pluripotent stem cells (hPSCs) provide a unique experimental platform of cell fate decisions during pre-implantation development, but their lineage potential remains incompletely characterized. As naïve hPSCs share transcriptional and epigenomic signatures with trophoblast cells, it has been proposed that the naïve state may have enhanced predisposition for differentiation along this extraembryonic lineage. Here we examined the trophoblast potential of isogenic naïve and primed hPSCs. We found that naïve hPSCs can directly give rise to human trophoblast stem cells (hTSCs) and undergo further differentiation into both extravillous and syncytiotrophoblast. In contrast, primed hPSCs do not support hTSC derivation, but give rise to non-self-renewing cytotrophoblasts in response to BMP4. Global transcriptome and chromatin accessibility analyses indicate that hTSCs derived from naïve hPSCs are similar to blastocyst-derived hTSCs and acquire features of post-implantation trophectoderm. The derivation of hTSCs from naïve hPSCs will enable elucidation of early mechanisms that govern normal human trophoblast development and associated pathologies. eLife digest The placenta is one of the most important human organs, but it is perhaps the least understood. The first decision the earliest human cells have to make, shortly after the egg is fertilized by a sperm, is whether to become part of the embryo or part of the placenta. This choice happens before a pregnancy even implants into the uterus. The cells that commit to becoming the embryo transform into ‘naïve pluripotent’ cells, capable of becoming any cell in the body. Those that commit to becoming the placenta transform into ‘trophectoderm’ cells, capable of becoming the two types of cell in the placenta. Placental cells either invade into the uterus to anchor the placenta or produce hormones to support the pregnancy. Once a pregnancy implants into the uterus, the naïve pluripotent cells in the embryo become ‘primed’. This prevents them from becoming cells of the placenta, and it poses a problem for placental research. In 2018, scientists in Japan reported conditions for growing trophectoderm cells in the laboratory, where they are known as “trophoblast stem cells”. These cells were capable of transforming into specialized placental cells, but needed first to be isolated from the human embryo or placenta itself. Dong et al. now show how to reprogram other pluripotent cells grown in the laboratory to produce trophoblast stem cells. The first step was to reset primed pluripotent cells to put them back into a naïve state. Then, Dong et al. exposed the cells to the same concoction of nutrients and chemicals used in the 2018 study. This fluid triggered a transformation in the naïve pluripotent cells; they started to look like trophoblast stem cells, and they switched on genes normally active in trophectoderm cells. To test whether these cells had the same properties as trophoblast stem cells, Dong et al. gave them chemical signals to see if they could mature into placental cells. The stem cells were able to transform into both types of placental cell, either invading through a three-dimensional gel that mimics the wall of the uterus or making pregnancy hormones. There is a real need for a renewable supply of placental cells in pregnancy research. Animal placentas are not the same as human ones, so it is not possible to learn everything about human pregnancy from animal models. A renewable supply of trophoblast stem cells could aid in studying how the placenta forms and why this process sometimes goes wrong. This could help researchers to better understand miscarriage, pre-eclampsia and other conditions that affect the growth of an unborn baby. In the future, it may even be possible to make custom trophoblast stem cells to study the specific fertility issues of an individual. Introduction Mammalian pluripotency spans a continuum of discrete but interconvertible states, each with a distinct set of molecular and functional attributes. Prior to implantation, the pluripotent epiblast compartment within the inner cell mass (ICM) of the blastocyst constitutes a naïve or ground state of pluripotency (Nakamura et al., 2016; Nichols and Smith, 2009; Stirparo et al., 2018). This naïve state can be captured in vitro in the form of mouse embryonic stem cells (mESCs). After implantation, transcription factors associated with naïve pluripotency are downregulated and pluripotent cells become primed for differentiation in response to signals from the surrounding extraembryonic tissues. Human embryonic stem cells (hESCs) derived under conventional conditions are thought to represent a primed pluripotent state, and were shown to correspond transcriptionally to the late post-implantation epiblast in a non-human primate model (Nakamura et al., 2016; Nichols and Smith, 2009). Much effort has been made in recent years to develop strategies for capturing hESCs in a naïve pluripotent state (Chan et al., 2013; Gafni et al., 2013; Hanna et al., 2010; Qin et al., 2016; Takashima et al., 2014; Theunissen et al., 2014; Ware et al., 2014; Zimmerlin et al., 2016). In particular, two transgene-free culture systems, 5i/L/A and t2i/L/Gö, were shown to induce defining transcriptional and epigenetic features of the human pre-implantation epiblast (Huang et al., 2014; Liu et al., 2017; Stirparo et al., 2018; Takashima et al., 2014; Theunissen et al., 2016; Theunissen et al., 2014). The isolation of naïve hESCs provides a cellular experimental platform to interrogate aspects of human pre-implantation development that are difficult to study in primed hESCs. For example, naïve hESCs have offered insights into the mechanisms governing X-linked dosage compensation (Sahakyan et al., 2017), the role of human-specific transposable elements that are expressed in the pre-implantation embryo (Pontis et al., 2019; Theunissen et al., 2016), and the mechanisms leading to activation of naïve-specific enhancers (Pastor et al., 2018). Naïve hESCs may also afford a platform for dissecting cell fate decisions in the early human embryo (Dong et al., 2019). While naïve hESCs are unresponsive to direct application of embryonic inductive cues, they acquire the capacity to undergo efficient multi-lineage differentiation upon treatment with a Wnt inhibitor, a process called ‘capacitation’ (Rostovskaya et al., 2019). This process is thought to reflect the requirement for dismantling of the naïve transcriptional program upon implantation in vivo, and the acquisition of a differentiation-competent formative phase (Smith, 2017). Molecular profiling of naïve hESCs has suggested that these cells may harbor a predisposition towards human extraembryonic cell fates. Gene expression studies revealed a pronounced upregulation in naïve relative to primed hESCs of trophoblast-associated transcription factors, including ELF3, GCM1, and TFAP2C (Theunissen et al., 2016). In addition, chromatin accessibility studies indicated that naïve hESCs share a broad panel of open chromatin sites with first-trimester placental tissues (Pontis et al., 2019). Intriguingly, embryonic and extraembryonic lineage markers are briefly co-expressed in the late morula and early blastocyst according to single cell RNA-seq (scRNA-seq) studies of human pre-implantation embryos (Petropoulos et al., 2016). This is precisely the stage of human development that displays the closest correspondence to naïve hESCs based on the expression patterns of transposable elements (Theunissen et al., 2016). Thus, we surmised that current methodologies for inducing naïve human pluripotency may yield a pre-implantation identity that is competent for both embryonic and extraembryonic differentiation. Here, using three independent methodologies, we find that naïve hPSCs have enhanced capacity for differentiation along the trophoblast lineage relative to primed hPSCs. In particular, we show that when cultured in human trophoblast stem cell (hTSC) media (Okae et al., 2018), naïve hPSCs can directly give rise to hTSCs, as confirmed by morphological, molecular, and transcriptomic criteria. We have also profiled the chromatin accessibility landscape of hTSCs for the first time, thus providing a valuable resource to identify potential regulatory elements and transcriptional determinants of human trophoblast development. Results Naïve hESCs exhibit increased trophoblast potential during embryoid body formation As a first step toward examining the trophoblast potential of naïve and primed hESCs, we measured the expression levels of trophoblast-associated markers during embryoid body (EB) formation (Figure 1A), which provides a rapid assessment of spontaneous differentiation capacity into early lineages (Allison et al., 2018). Previous studies reported limited induction of embryonic lineage markers in EBs formed from naïve hESCs, but did not examine the expression of trophoblast-associated genes (Liu et al., 2017; Rostovskaya et al., 2019). We generated naïve hESCs in 5i/L/A (Theunissen et al., 2014) from two genetic backgrounds, H9 and WIBR3, confirmed their upregulation of naïve-specific markers and downregulation of primed-specific markers (Figure 1—figure supplement 1A), and aggregated them to form EBs in growth factor- and inhibitor-free media for 12 days (Figure 1A; Figure 1—figure supplement 1B). The mRNA expression levels of six trophoblast markers, ELF5, KRT7, TFAP2C, GATA3, TEAD4, and CDX2 (Hemberger et al., 2010; Lee et al., 2016; Ng et al., 2008; Strumpf et al., 2005), were measured by quantitative real time PCR (qRT-PCR) analysis (Figure 1B; Figure 1—figure supplement 1C). Significantly enriched expression of trophoblast markers was observed in EBs derived from naïve compared to primed hESCs (Figure 1B; Figure 1—figure supplement 1C). Furthermore, many of the examined trophoblast markers were already elevated in naïve versus primed hESCs prior to EB formation (Figure 1B; Figure 1—figure supplement 1C), consistent with our prior transcriptome analysis (Theunissen et al., 2016). These findings support the notion that naïve hESCs harbor increased spontaneous trophoblast differentiation potential compared to primed hESCs. Figure 1 with 2 supplements see all Download asset Open asset Trophoblast potential of different hPSC states under spontaneous and BMP4-mediated differentiation conditions. (A) The experimental scheme for assessing spontaneous trophoblast differentiation potential of primed and naïve hPSCs by using EB formation assay. (B) Quantitative gene expression analysis for trophoblast marker genes ELF5, KRT7, TFAP2C, and GATA3 in H9 primed and naïve hPSCs and EBs generated from the respective hPSCs. Error bars indicate ±1 SD of technical replicates. ‘*' indicates a p-value<0.05, ‘**' indicates a p-value<0.01, and ‘***' indicates a p-value<0.001. (C) The experimental scheme for assessing BMP4-mediated trophoblast differentiation potential of naive hESCs (top). Phase contrast image of H9 naïve hESCs and H9 naïve hESCs following the 6 day step I protocol for BMP4-mediated CTB differentiation (bottom). The scale bars indicate 75 μm. (D) The experimental scheme of re-priming naïve hESCs and assessing their BMP4-mediated trophoblast differentiation potential (top). Phase contrast images of H9 re-primed hESCs and H9 re-primed hESCs following the 6 day step I protocol for BMP4-mediated CTB differentiation (bottom). The scale bars indicate 75 μm. (E) Immunofluorescence staining for CDX2, ZO1, and KRT7 in CTBs generated from H9 primed and re-primed hESCs. The scale bars indicate 75 μm. (F) The experimental scheme of capacitating naïve hESCs and assessing their BMP4-mediated trophoblast differentiation potential (top). Phase contrast images of H9 capacitated hESCs and H9 capacitated hESCs following the 6 day step I protocol for BMP4-mediated CTB differentiation (bottom). The scale bars indicate 75 μm. (G) Quantitative gene expression analysis for trophoblast marker genes GATA3, CDX2, KRT7, MMP2, hCGB, and SDC1 in H9 primed and capacitated hESCs and trophoblasts differentiated from the respective hPSCs. Error bars indicate ± 1 SD of technical replicates. ‘***' indicates a p-value<0.001. Naïve hESCs require capacitation or re-priming to respond to BMP4-directed trophoblast differentiation We next assessed the trophoblast potential of naïve relative to primed hESCs using a protocol for directed trophoblast differentiation that utilizes a low dose of bone morphogenetic protein 4 (BMP4) (Horii et al., 2016). It has long been known that primed hPSCs acquire certain trophoblast characteristics upon stimulation with BMP4 (Amita et al., 2013; Horii et al., 2016; Xu et al., 2002). However, when naïve hESCs were subjected to a protocol for BMP4-directed differentiation into cytotrophoblast (CTB) progenitors (Horii et al., 2016), the cells did not survive (Figure 1C). This recalcitrance to BMP4 is reminiscent of the delayed response of naïve hPSCs to embryonic inductive cues (Liu et al., 2017; Rostovskaya et al., 2019). We examined whether naïve hESCs would gain the capacity for BMP4-directed differentiation upon returning to the primed state, a process referred to as ‘re-priming’ (Theunissen et al., 2016). Indeed, naïve hESCs treated with StemPro for five passages re-gained competence for BMP4-directed trophoblast differentiation, as shown by the expression of several CTB markers (Figure 1D,E; Figure 1—figure supplement 2A,B). When these CTBs were further differentiated using feeder-conditioned medium supplemented with BMP4, we observed the expected induction of extravillous trophoblast (EVT) and syncytiotrophoblast (STB) marker genes, as observed for differentiation from primed hESCs (Horii et al., 2016; Figure 1G; Figure 1—figure supplements 1D and 2A,B). We also examined whether capacitation of naïve hESCs using the Wnt inhibitor, XAV939 (Rostovskaya et al., 2019), would confer responsiveness to BMP4-directed trophoblast differentiation. Capacitated cells could not only readily undergo Step I CTB differentiation, but did so more efficiently than primed hESCs based on analysis of trophoblast-specific transcripts and proteins (Figure 1F,G; Figure 1—figure supplements 1D and 2C). These results indicate that an efficient response to BMP4-directed trophoblast differentiation requires exit from naïve human pluripotency, and appears to be most efficient when initiated from the formative phase. Human trophoblast stem cells can be derived from naïve, but not primed, hPSCs The above experiments indicate that naïve hESCs efficiently upregulate trophoblast markers during spontaneous EB differentiation, but require transition into a formative pluripotent state to become competent for BMP4-directed trophoblast differentiation. This led us to consider whether naïve hESCs might be directly responsive to alternative conditions for trophoblast differentiation that are independent of BMP4. A recent study reported the derivation of hTSCs from blastocysts and first-trimester placental tissues in the presence of recombinant epidermal growth factor (EGF) and Wnt activator (CHIR), and inhibitors of transforming growth factor beta (TGFβ), histone deacetylase (HDAC), and Rho-associated kinase (ROCK) (Okae et al., 2018). We seeded isogenic lines of naïve and primed hPSCs on Collagen IV and examined their response to hTSC media (Figure 2A). Naïve hPSCs acquired a typical hTSC-like morphology within several passages and could be expanded for at least 20 passages while maintaining a high proliferation rate (Figure 2B; Figure 2—figure supplement 1A). Similar results were obtained using three independent naïve hPSC lines derived from H9 hESCs, WIBR3 hESCs, and AN1 induced pluripotent stem cells (iPSCs). In contrast, the parental primed hPSCs did not acquire an hTSC-like morphology, even after prolonged culture in hTSC media (Figure 2B). These observations indicate that naïve hPSCs are capable of adapting to the specific culture environment of hTSCs, whereas primed hPSCs are not. Figure 2 with 2 supplements see all Download asset Open asset Examining the response of naïve and primed hPSCs to conditions for hTSC derivation. (A) The experimental scheme for deriving hTSCs from primed and naïve hPSCs. (B) Phase contrast images of H9 and AN_1.1 hTSC-like cells derived from naïve hPSCs, as well as H9 and AN_1.1 primed hPSCs following culture in hTSC medium. All H9 cells were at passage 8, and all AN_1.1 cells were at passage 10. The scale bars indicate 75 μm. (C) Flow cytometry analysis for TSC markers ITGA6 and EGFR in H9 naïve hPSCs, H9 hTSC-like cells derived from naïve hPSCs, H9 primed hPSCs, and H9 primed hPSCs cultured in hTSC medium. (D) Quantitative gene expression analysis for trophoblast marker genes ELF5, KRT7, GATA3, TFAP2C, TEAD4, and CDX2 in H9 primed and naïve hPSCs, H9 hTSC-like cells derived from naïve hPSCs, and H9 primed hPSCs cultured in hTSC medium. Error bars indicate ±1 SD of technical replicates. ‘***' indicates a p-value<0.001. (E) Immunofluorescence staining for TSC markers KRT7, TEAD4, and TP63 in H9 hTSC-like cells derived from naïve hPSCs. The scale bars indicate 75 μm. (F) Flow cytometry analysis for naïve hPSC markers SUSD2 and CD75 in H9 and AN_1.1 naïve hPSCs and hTSC-like cells derived from naïve hPSCs. (G) Quantitative gene expression analysis for naïve hPSC marker KLF17 in H9 primed and naïve hPSCs, hTSC-like cells derived from naïve hPSCs, and primed hPSCs cultured in hTSC medium. Error bars indicate ±1 SD of technical replicates. ‘***' indicates a p-value<0.001. (H) Quantitative gene expression analysis for the embryonic germ layer markers PAX6, VIM, and SOX17 in H9 primed and naïve hPSCs, hTSC-like cells derived from naïve hPSCs, and primed hPSCs cultured in hTSC medium. Error bars indicate ± 1 SD of technical replicates. ‘*' indicates a p-value<0.05, ‘***' indicates a p-value<0.001, and ‘n.s.’ indicates a p-value>0.05. We proceeded to further characterize the naïve hPSC-derived hTSC-like cells (from here referred to as naïve hTSCs). We found that naïve hTSCs uniformly express ITGA6 and EGFR, two commonly used cell surface markers that mark both CTBs and hTSCs (Bischof and Irminger-Finger, 2005; Horii et al., 2016; Okae et al., 2018), in contrast to primed hPSCs that had been expanded in hTSC media (Figure 2C; Figure 2—figure supplement 1B). The naïve hTSCs also expressed significantly higher levels of ELF5, KRT7, GATA3, TFAP2C, and TEAD4 transcripts (Figure 2D; Figure 2—figure supplement 1C). Notably, naïve hTSCs exhibited almost no CDX2 expression, which is consistent with hTSCs derived from blastocysts or first-trimester placental tissues (Okae et al., 2018; Figure 2D; Figure 2—figure supplement 1C). Naïve hTSCs also expressed the hTSC markers KRT7, TEAD4, and TP63 at the protein level (Lee et al., 2016; Li et al., 2013; Figure 2E; Figure 2—figure supplement 1D). Flow cytometry analysis for CD75 and SUSD2, two naïve-specific cell surface markers (Bredenkamp et al., 2019a; Collier et al., 2017), confirmed the loss of naïve identity in naïve hTSCs (Figure 2F), which was further corroborated by downregulation of KLF17 transcript (Figure 2G; Figure 2—figure supplement 1E). These findings indicate that naïve hPSCs directly give rise to a uniform population of cells that closely resemble hTSCs based on gross morphology, surface markers, and trophoblast-specific gene expression. Conversely, primed hPSCs did not upregulate trophoblast markers when cultured in hTSC media, but instead exhibited increased expression of VIM and PAX6, suggesting the acquisition of a neuroectodermal fate (Figure 2H; Figure 2—figure supplement 1F). To confirm that the ability of naïve hPSCs to give rise to hTSC-like cells is not specific to the 5i/L/A culture condition, we attempted to derive naïve hTSCs from naïve hPSCs cultured in an alternative naïve medium, PXGL (Bredenkamp et al., 2019b; Figure 2—figure supplement 1G). Both morphological and molecular analyses indicated that naïve hTSCs derived from PXGL-cultured naïve hPSCs closely resemble those derived from 5i/L/A-cultured naïve hPSCs (Figure 2—figure supplement 1G–J). This suggests that naïve hTSC-derivation is likely an intrinsic property of the naïve state of human pluripotency, regardless of the specific culture condition used. Additionally, we investigated whether naïve hPSCs can give rise to naïve hTSCs on a clonal level, which would preclude that hTSCs arise from a small population of pre-existing trophoblast-like cells in the culture. We therefore picked and expanded three single-cell H9 naïve hPSC clones (Figure 2—figure supplement 2A), and confirmed their naïve molecular characteristics and lack of hTSC marker expression (Figure 2—figure supplement 2B,C). Subsequently, naïve hTSCs were derived from these clonally expanded naïve hPSCs. All of these clonally derived naïve hTSCs exhibit a typical hTSC morphology (Figure 2—figure supplement 2A), and are negative for naïve hPSC markers but positive for hTSC markers (Figure 2—figure supplement 2B,C). These results lend further support to the notion that the ability to give rise to hTSCs is an intrinsic property of naïve hPSCs. hTSCs derived from naïve hPSCs have bipotent differentiation potential Since naïve hTSCs exhibit numerous characteristics of hTSCs derived from blastocysts or placental tissues, we sought to examine their differentiation potential into specialized trophoblast cells. First, we performed directed differentiation of naïve hTSCs into EVTs, which invade the endometrium to increase blood flow between the mother and fetus (James et al., 2012; Watson and Cross, 2005). Following application of a culture system for EVT differentiation from hTSCs that contains Neuregulin 1 (NRG1), the TGF-β inhibitor A83-01, and Matrigel (Okae et al., 2018), naïve hTSCs acquired a characteristic EVT-like morphology (Figure 3A,B). We tested whether the naïve hTSC-derived EVT-like cells expressed two EVT-specific protein markers, HLA-G and MMP2 (Horii et al., 2016; Lee et al., 2016). Flow cytometry analysis demonstrated that about 90% of EVT-like cells were positive for HLA-G (Figure 3C). Similarly, expression and secretion of MMP2 was detected by ELISA and immunofluorescence staining (Figure 3D; Figure 3—figure supplement 1A). The induction of HLA-G and MMP2 in naïve hTSC-derived EVT-like cells was also confirmed at the mRNA level by qRT-PCR (Figure 3E; Figure 3—figure supplement 1B). Finally, since invasiveness is a prominent property of EVTs (Burton et al., 2009; James et al., 2012; McEwan et al., 2009), we performed a transwell-based Matrigel invasion assay. The results indicate that our EVT-like cells have invasive potential, in contrast to the naïve hTSCs from which they were derived (Figure 3F; Figure 3—figure supplement 1C). Figure 3 with 1 supplement see all Download asset Open asset Directed differentiation of EVTs and STBs from naïve hPSC-derived hTSCs. (A) The experimental scheme for EVT differentiation from naïve hPSC-derived hTSCs. (B) Phase contrast image of H9 and AN_1.1 hTSC-like and EVT-like cells. The scale bars indicate 75 μm. (C) Flow cytometry analysis for EVT marker HLA-G in H9 and AN_1.1 hTSC-like cells and EVT-like cells. (D) Levels of MMP2 secreted by H9 and AN_1.1 hTSC-like cells and EVT-like cells as measured by ELISA. Error bars indicate ±1 SD of technical replicates. (E) Quantitative gene expression analysis for EVT marker gene MMP2 in H9 and AN_1.1 hTSC-like cells and EVT-like cells. Error bars indicate ± 1 SD of technical replicates. ‘***' indicates a p-value<0.001. (F) Matrigel invasion assay of H9 and AN_1.1 hTSC-like cells and EVT-like cells. (G) The experimental scheme for STB differentiation from naïve hPSC-derived hTSCs. (H) Phase contrast image of H9 and AN_1.1 hTSC-like and 3D STB-like cells. The scale bars indicate 75 μm. (I) Quantitative gene expression analysis for STB marker genes CGB and SDC1 in H9 and AN_1.1 hTSC-like cells and 3D STB-like cells. Error bars indicate ±1 SD of technical replicates. ‘*' indicates a p-value<0.05, and ‘**' indicates a p-value<0.01. (J) Immunofluorescence staining for STB markers hCG and SDC1 in H9 3D STB-like cells. The scale bars indicate 75 μm. (K) Immunofluorescence staining for STB markers hCG and SDC1 in AN_1.1 3D STB-like cells. The scale bars indicate 75 μm. (L) Quantitative gene expression analysis for TSC marker gene TEAD4 in H9 and AN_1.1 hTSC-like cells and 3D STB-like cells. Error bars indicate ± 1 SD of technical replicates. ‘***' indicates a p-value<0.001. Second, we performed directed differentiation of naïve hTSCs into STBs, which are multinucleated cells that produce placental hormones and mediate maternal-fetal communication. We applied two protocols for differentiation of hTSCs into STBs involving either 2D or 3D culture in the presence of Forskolin (Okae et al., 2018; Figure 3G). The naïve hTSC-derived 2D STB-like cells showed characteristic morphological features, including multinucleation (Figure 3—figure supplement 1D). They also expressed the STB marker human chorionic gonadotropin (hCG) (Figure 3—figure supplement 1E). The naïve hTSC-derived 3D STB-like cells exhibited a cyst-like morphology typical for 3D STBs (Okae et al., 2018; Figure 3H), and expressed the STB markers hCG and SDC1 based on immunofluorescence and qRT-PCR analysis (Jokimaa et al., 1998; Strumpf et al., 2005; Figure 3I–K). Finally, qRT-PCR analysis confirmed downregulation of the hTSC-specific marker TEAD4 (Figure 3L), indicating the loss of hTSC identity. These morphological and molecular data demonstrate that hTSCs derived from naïve hPSCs are capable, at least at the population level, of undergoing further differentiation into two specialized trophoblast cell types, EVT and STB. Global transcriptome and chromatin accessibility profiles of hTSCs derived from naïve hPSCs To examine whether naïve hTSCs and their differentiated derivatives possess transcriptomic signatures of the trophoblast lineage, we sequenced total RNA isolated from three types of hPSCs (naïve, capacitated, and primed), blastocyst-derived BT5 hTSCs (Okae et al., 2018), naïve hTSCs and their differentiated progeny (EVT and STB), and primed hPSCs cultured in hTSC media. Principal component analysis (PCA) revealed that the samples clustered together based on cell type, rather than genetic background (Figure 4A). Pluripotent and trophoblast identities were predominantly divided along principal component 1 (PC1), which accounted for 41% of the variation in gene expression. In addition, naïve, capacitated, and primed hPSCs formed clearly defined clusters that were separated along PC2. It is worth noting that naïve hTSCs and BT5 hTSCs clustered together very closely on the PCA plot (Figure 4A). Further examination indicated that naïve hTSCs express key trophoblast marker genes at comparable or even higher levels than the bona fide, BT5 hTSCs (Figure 4—figure supplement 1A). Whereas naïve hTSCs were well-separated from the pluripotent samples, primed hPSCs acquired a very distinct transcriptional profile in hTSC media compared to naïve hTSCs. Naïve hTSCs displayed strong upregulation of many transcripts associated with trophoblast development, whereas primed cells instead acquired neuroectodermal characteristics in hTSC media (Figure 4B and Supplementary file 1). Hence, exposure to hTSC media induces a drastically different transcriptomic profile when applied to either naïve or primed hPSCs. Figure 4 with 2 supplements see all Download asset Open asset Transcriptomic and chromatin accessibility profiling of naïve hPSC-derived hTSCs. (A) Principal component analysis (PCA) of primed, capacitated, and naïve hPSCs, BT5 hTSCs, naïve hTSCs, EVTs, STBs, and primed hPSCs cultured in hTSC medium based on RNA-seq data. Circles were drawn around samples cultured in the same media. (B) Volcano plots showing fold change (X axis) between naïve hTSCs and primed hPSCs cultured in hTSC medium. The light blue dots represent genes that are the most significantly upregulated in primed hTSCs (defined as those that have a log fold change < −1 and wherein p<0.05). The red dots represent genes that are the most significantly upregulated in naïve hPSCs cultured in hTSC medium (defined as those that have a log fold change >1 and wherein p<0.05). (C) Heatmap of RNA-seq data from naïve hPSCs, naïve hTSCs, EVTs, and STBs (DEGs with more than 2X fold change and p-adj < 0.05 are analyzed). Cluster one represents genes highly expressed in naïve hPSCs only. Cluster two represents genes highly expressed in both naïve hPSCs and naïve hTSCs. Cluster three represents genes highly expressed in naïve hTSCs only. Cluster four represents genes highly expressed in both naïve hTSCs and EVTs. Cluster five represents genes highly expressed in EVTs only. Cluster six represents genes highly expressed in STBs only. (D) Expression levels of key marker genes associated with clusters 1–6. (E) Heatmap indicating the correlation among scRNA-seq data from published day 6, day 8, day 10, and day 12 TE, EPI, and PE (Zhou et al., 2019), as well as RNA-seq data from naïve and BT5 hTSCs. Genes specifically expressed in the TE, EPI, or PE lineages were analyzed (Zhou et al., 2019). (F) Heatmap of ATAC-seq data from naïve hESCs and naïve hTSCs (Pearson correlations were calculated based on genome-wise ATAC-seq signal). Naïve hESC ATAC-seq data were retrieved from a published dataset (Pastor et al., 2018). (G) Transcription factor binding motifs enriched in the promoter and non-promoter regions of the" @default.
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• W3011805855 title "Author response: Derivation of trophoblast stem cells from naïve human pluripotent stem cells" @default.
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