Impacts of reprogramming method and culture conditions in trophoblast differentiation of induced pluripotent stem cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE64272 | GEO | 2015/08/31
SECONDARY ACCESSION(S): PRJNA270612
REPOSITORIES: GEO
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Project description:Trophoblast differentiation from human ESC has been achieved by exposing the cells to BMP4 with or without supplementation of ALK4/5/7 inhibitor (A83-01) and FGF2 signaling inhibitor (PD173074) (BAP). Here the two differentiation conditions, BMP4 and BAP were applied to two sets of human PSC lines, H1 ESC and iPSC that latter was generated by DOX-inducible lentiviral (V) transductions of umbilical cord mesenchymal cells. The V-iPSC showed residual transgene expressions from the viral vectors in DOX-free culture condition. When the both ESC and iPSC lines were differentiated simultaneously, similar time dependent morphological changes were observed but BMP4 treated V-iPSC showed a minor yet consistent lag in the differentiation progression compared to BMP4 treated hESC. Although both differentiated ESC and V-iPSC showed dominant trophoblast phenotypes, the BMP4 treated V-iPSC also expressed gene markers consistent with the presence of mesoendoderm. The BAP condition provided more efficient differentiation than BMP4 alone, and the BAP-differentiated iPSC and ESC never expressed mesoendoderm markers. Five samples, one control of undifferentiated V-iPSC (FGF2) and four differentiated samples included two H1 ESC (treated with BMP4 or BMP4+A83-01) and similarly treated two V-iPSC were analyzed. Trophoblast differentiation was conducted with BMP4 (10 ng/ml) with or without supplementation of ALK4/5/7 inhibitor (A83-01; 1 μM) to H1 ESC and V-iPSC, respective reagents were added to FGF2-free MEF-CM from the second day culture of following passages for up to six additional days.
2015-08-31 | E-GEOD-64272 | biostudies-arrayexpress
Project description:Early onset preeclampsia (EOPE) affects about 0.4% of pregnancies and is characterized by impaired trophoblast (TB) invasion, resulting in a poorly perfused placenta. Here we have used an in vitro model for mimicking early trophoblast development to determine how early placental development differs between normal pregnancies and those affected by EOPE. Induced pluripotent stem cells (iPSC) were generated from umbilical cords of infants born to mothers who had EOPE and from controls. These iPSC were converted to TB, by exposing them to BMP4 and inhibitors of ACTIVIN A and FGF2 signaling (BAP treatment), under 5 % and 20 % O2, hypothesizing that 20% O2 would act as a stressor. Two embryonic stem cell lines (ESC; H1 and H9), 8 control (CTL) iPSC and 14 EOPE iPSC were tested to assess how well the differentiated TB cells invaded through a Matrigel-coated membrane.
2018-12-31 | GSE119265 | GEO
Project description:Purpose: Syncytiotrophoblast (STB) is a multi-nucleated, terminally differentiated syncytium that covers the surface of the villous placenta and forms the major interface with maternal blood. It releases placental hormones and plays a primary role in exchange of gases, nutrients and waste products. Alterations in STB development and turnover have been implicated in placental diseases, including preeclampsia (PE). In vitro cell models are badly needed to study STB development and physiology due to inaccessibility to placental tissues during gestation. To establish in vitro STB model system, we generate STB and its mononucleated precursors from human embryonic stem cells (hESC) and profile for RNA content by RNAseq. Methods: H1 Human ESC (WA01) were treated with BMP4, the ALK4/5/7 inhibitor (A83-01), and the FGF2 signaling inhibitor (PD173074) (BAP) to direct them to the trophoblast lineage and provided both STB and extravillous trophoblast. Syncytial areas emerged at day 8 BAP treatment ranged in diameter from ~40 µm to > 100 µm. The intact syncytial areas were isolated by sieving successively through 70 μm and 40 μm mesh cell strainers. The captured cells are recovered by inverting the strainer and rinsing with culture medium to separate large (>70 μm) and middle size cell sheets (40-70 μm). The fraction that passes through both sieves represents cells of smallest diameter (< 40 μm), presumably cytotrophoblast. Total 12 RNA samples from triplicate three size-fractioned BAP treated and three untreated hESC cultured in a FGF2 supplemented medium in parallel were analyzed. Results: The larger > 70 μm areas stained positively for STB markers while ultrastructural analysis clearly revealed multi-nuclear cells with an extensive cytoplasm containing many prominent secretion granules. The larger STB areas also had a larger DNA content that > 70 μm fraction contained 37 times more nuclear content and 40-70 µm fraction did 16 times more. Compared to the < 40 μm cell fraction, these larger cells over-expressed a full repertoire of genes characteristic of STB, e.g. CGA, CGB, PGF, ERVW1, GCM1. The smallest cell fraction had a DNA content consistent with mononuclear diploid cells, contained few secretory granules, and were only weakly positive for STB markers. Conclusion: The data are consistent with the > 70 μm cells being mature STB, while the intermediate fraction may represent a precursor population. Human ESC directed along the trophoblast lineage by BAP treatment offers a useful model for following STB formation in vitro and suggest that this protocol may have utility in studying the basis of certain placental diseases, especially preeclampsia, where placental tissue isolated at term or after pregnancy terminations can only offer limited information. Three size fraction mRNA profiles of syncytial areas emerged at day 8 BAP treatment of hESC were generated by deep sequencing along with untreated hESC, in triplicate, using Illumina HiSeq 2500.
2016-04-06 | E-GEOD-72712 | biostudies-arrayexpress
Project description:Purpose: Syncytiotrophoblast (STB) is a multi-nucleated, terminally differentiated syncytium that covers the surface of the villous placenta and forms the major interface with maternal blood. It releases placental hormones and plays a primary role in exchange of gases, nutrients and waste products. Alterations in STB development and turnover have been implicated in placental diseases, including preeclampsia (PE). In vitro cell models are badly needed to study STB development and physiology due to inaccessibility to placental tissues during gestation. To establish in vitro STB model system, we generate STB and its mononucleated precursors from human embryonic stem cells (hESC) and profile for RNA content by RNAseq. Methods: H1 Human ESC (WA01) were treated with BMP4, the ALK4/5/7 inhibitor (A83-01), and the FGF2 signaling inhibitor (PD173074) (BAP) to direct them to the trophoblast lineage and provided both STB and extravillous trophoblast. Syncytial areas emerged at day 8 BAP treatment ranged in diameter from ~40 µm to > 100 µm. The intact syncytial areas were isolated by sieving successively through 70 μm and 40 μm mesh cell strainers. The captured cells are recovered by inverting the strainer and rinsing with culture medium to separate large (>70 μm) and middle size cell sheets (40-70 μm). The fraction that passes through both sieves represents cells of smallest diameter (< 40 μm), presumably cytotrophoblast. Total 12 RNA samples from triplicate three size-fractioned BAP treated and three untreated hESC cultured in a FGF2 supplemented medium in parallel were analyzed. Results: The larger > 70 μm areas stained positively for STB markers while ultrastructural analysis clearly revealed multi-nuclear cells with an extensive cytoplasm containing many prominent secretion granules. The larger STB areas also had a larger DNA content that > 70 μm fraction contained 37 times more nuclear content and 40-70 µm fraction did 16 times more. Compared to the < 40 μm cell fraction, these larger cells over-expressed a full repertoire of genes characteristic of STB, e.g. CGA, CGB, PGF, ERVW1, GCM1. The smallest cell fraction had a DNA content consistent with mononuclear diploid cells, contained few secretory granules, and were only weakly positive for STB markers. Conclusion: The data are consistent with the > 70 μm cells being mature STB, while the intermediate fraction may represent a precursor population. Human ESC directed along the trophoblast lineage by BAP treatment offers a useful model for following STB formation in vitro and suggest that this protocol may have utility in studying the basis of certain placental diseases, especially preeclampsia, where placental tissue isolated at term or after pregnancy terminations can only offer limited information.
2016-04-06 | GSE72712 | GEO
Project description:The mechanisms controlling trophoblast proliferation and differentiation during embryo implantation are fundamentally important in defining a successful pregnancy, yet poorly understood. Human trophoblast stem cells (TSC) and BMP4/A83-01/PD17034-treated pluripotent stem cell derived trophoblast cells (BAP) are two widely employed, contemporary models to study trophoblast development and function but how faithfully they mimic the identity of early stage trophoblast cells has not been fully examined. Here, we evaluate the transcriptomic profiles of trophoblast cells from these two widely used cell models and directly compare them with trophoblast cells from peri-implantation stage human embryos during extended embryo culture (EEC) between embryonic d 8 to d 12. Cells from BAP and TSC models all grouped closely with trophoblast cells from EEC, with the trophoblast sublineages, including cytotrophoblast (CTB), syncytiotrophoblast (STB), and extravillous-like migratory trophoblast (MTB), clustered together following dimensional analysis and unsupervised hierarchical clustering. However, subtle differences amongst models exist for each trophoblast sublineages. TSC-CTB is more similar to EEC-CTB from d 8 embryos with transcripts associated with increased Wnt signaling activities important for trophoblast stemness. BAP-CTB are more similar to EEC-CTB from d 10 and 12 embryos and upregulate transcripts important for trophoblast differentiation and invasion. For STB, EEC-STB showed enriched GO terms related to oxygen-dependent metabolism, while BAP and TSC-derived STB both prioritize lipid and membrane synthesis as well as actin reorganization and cell spreading. EEC-MTB transcripts indicate an increased level of invasiveness and motility compared to TSC-MTB with increased proliferative capacity. We also provide a pool of novel lineage markers in early trophoblast that were upregulated (>4-fold) across all three models and validate the presence of 11 proteins prominent in peri-implantation human embryos. Together, our comprehensive analysis revealed that both commonly used BAP and TSC models resemble features of peri-implantation trophoblasts, while still maintaining subtle transcriptomic differences in sublineages.
2024-08-21 | GSE232526 | GEO
Project description:Human embryonic stem cells (hESC) can be differentiated into progenitors resembling trophoblast upon exposure to BMP4. Among the earliest transcription factors that are activated after the BMP4 stimulation are GATA2, GATA3, TFAP2A and TFAP2C. Using trophoblast progenitors at day 3 of BMP4-induced differentiation, here we profile the chromatin binding landscape of these 4 early transcription factors to analyse their putative targets and cross-connectivity in regualtion of trophoblast commitment.
2017-10-18 | GSE105081 | GEO
Project description:Human embryonic stem cells (hESC) can be differentiated into progenitors resembling trophoblast upon exposure to BMP4. Putative trophpblast progenitors express APA cell surface marker Using trophoblast progenitors at day 2.5 of BMP4-induced differentiation, here we profile H3K4me3 and H3K27me3 chromatin marks that mark active and inactive genes, respectively. Double occupancy indicates bivalency
2017-10-20 | GSE105258 | GEO
Project description:The excitement and controversy surrounding the potential role of human embryonic stem (ES) cells in transplantation therapy have often overshadowed their potentially more important use as a basic research tool for understanding the development and function of human tissues. Human ES cells can proliferate without a known limit and can form advanced derivatives of all three embryonic germ layers. What is less widely appreciated is that human ES cells can also form the extra-embryonic tissues that differentiate from the embryo before gastrulation. The use of human ES cells to derive early human trophoblast is particularly valuable, because it is difficult to obtain from other sources and is significantly different from mouse trophoblast. Here we show that bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor-beta (TGF-beta) superfamily, induces the differentiation of human ES cells to trophoblast. DNA microarray, RT-PCR, and immunoassay analyses demonstrate that the differentiated cells express a range of trophoblast markers and secrete placental hormones. When plated at low density, the BMP4-treated cells form syncytia that express chorionic gonadotrophin (CG). These results underscore fundamental differences between human and mouse ES cells, which differentiate poorly, if at all, to trophoblast. Human ES cells thus provide a tool for studying the differentiation and function of early human trophoblast and could provide a new understanding of some of the earliest differentiation events of human postimplantation development. Groups of assays that are related as part of a time series. Using regression correlation
2005-11-03 | E-GEOD-3553 | biostudies-arrayexpress
Project description:Complete Hydatidiform Mole (HM) is a gestational trophoblastic disease resulting in hyper proliferation of trophoblast cells and absence of embryo development. Mutations in NLRP7 gene is a major cause of familial recurrent complete HM, where no embryonic tissue is present during the pregnancy. NLRP7 is a novel protein and its function is poorly understood. Lack of NLRP7 in the mouse genome and challenges in studying human embryogenesis as well as human trophoblast differentiation has prevented elucidation of the pathophysiology of this disease and the function of NLRP7 in HM. Here, we established an in vitro model of HM using NLRP7 deficient patient-derived human induced pluripotent stem cells (iPSCs). Whole transcriptome profiling during trophoblast differentiation revealed that NLRP7 deficiency results in precocious downregulation of pluripotency factors, activation of trophoblast genes and promotes maturation of differentiated extraembryonic cell types such as syncytiotrophoblasts. We further showed that these phenotypes are dependent on BMP4 signaling and BMP pathway inhibition prevented excessive trophoblast differentiation of HM-iPSCs. Taken together, this novel human iPSC model of a genetic placental disease recapitulates aspect of trophoblast biology and sheds light on early human embryogenesis by identifying NLRP7 as an important regulator of this process.
2021-09-09 | GSE125592 | GEO
Project description:To test the regulatory effects of Dmrt5 on gene expression, we designed tetracycline inducible lines of Dmrt5 transgenic mouse ESCs. Overexpression of Dmrt5 was induced upon addition of Doxycycline (Dox). To evaluate the effects of Dmrt5 on gene expression in different stages of in vitro differentiated NPC derived from mouse embryonic stem cells (ESC), we analyzed gene expression profiles at differentiation day 7 and day 9 with or without Dox. The data revealed that overexpression of Dmrt5 in in vitro differentiated neural progenitor cells (NPC) regulates gene expression. Addition of Dox to the medium of the control cell line rtTA did not significantly alter gene expression profile, demonstrating that the observed effects were through induction of Dmrt5, but not simply through Dox. A tetracycline-inducible rtTA-Dmrt5 ES cell line and the parental rtTA cell line were used for the experiment. These ES cells were differentiated using a monolayer differentiation (MD) protocol in N2B27 medium. At MD day 5, cells were treated with Dox or without. RNA was extracted from cells harvested at MD day 7 and day 9. Two replicates for each group were used for the experiment.
2011-05-18 | E-GEOD-25179 | biostudies-arrayexpress