Cells with features of totipotency derived from human ESC and iPSC by transient BMP4 exposure
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE62065 | GEO | 2015/06/11
SECONDARY ACCESSION(S): PRJNA263146
REPOSITORIES: GEO
Similar Datasets
Project description:Human pluripotent stem cells (hPSC) exposed to BMP4 (B) and inhibitors of ACTIVIN signaling (A83-01; A) and FGF2 (PD173074; P) in absence of FGF2 (BAP conditions) differentiate into colonies primarily comprised of trophoblast. In an attempt to isolate trophoblast stem cells, colonies of hESC were exposed to BAP for 24 h at which time they had begun to transition into a CDX2-positive state. Cultures were then dissociated into single cells by trypsin and grown on a gelatin substratum. Under these conditions, organized CDX2+/KRT7- colonies began to emerge within a few days. The self-renewing cell lines were not TBSC, but met standard criteria for pluripotency. They were named H1BP cells. They differed from the progenitor hPSC in morphology, ability to be clonally propagated from single cells onto gelatin, requirements for FGF2, and transcriptome profile. RNA was isolated from control human embryonic stem cells H1 cells, H1 cells exposed to BAP conditions for 24 and 48 h, H1BP colonies picked individually at two different passage numbers (p7 and p18), and H1BP cells that had been allowed to differentiate spontaneously in standard non-conditioned hESC medium in absence of FGF2. In order to collect RNA from cells, medium was removed and RNA STAT60 (I ml; Tel-Test, Friendswood, TX) was immediately added to culture dish and RNA extracted by following the manufacturer’s instructions. The samples of RNA were submitted to University Texas Southwestern Medical Center Microarray Core Facility (https://microarray.swmed.edu/) and microarray analysis performed with Illumina HumanHT-12 v4 expression BeadChips. Raw intensity data were background subtracted by using BeadStudio software and analyzed further by GeneSpring 12.6 software (Agilent Technologies Inc., Santa Clara CA), according to the advanced workflow protocol: percentile shift and filter by flags (detected).
2015-06-11 | E-GEOD-62065 | biostudies-arrayexpress
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: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.
2015-08-31 | GSE64272 | GEO
Project description:Abnormalities at any stage of trophoblast development may result in pregnancy-related com-plications. Many of these adverse outcomes are discovered later in pregnancy, but the underly-ing pathomechanisms constitute during the first trimester. Acquiring developmentally relevant material to elucidate the disease mechanisms is difficult. Human pluripotent stem cell (hPSC) technology can provide a renewable source of relevant cells. BMP4, A83-01, and PD173074 (BAP) treatment drives trophoblast commitment of hPSCs towards syncytiotrophoblast (STB) but lacks extravillous trophoblast (EVT) cells. EVTs mediate key functions during placentation, remodel-ing of uterine spiral arteries, and maintenance of immunological tolerance. We optimized the protocol for more efficient generation of HLA-Gpos EVT-like trophoblasts from primed hiPSCs. Increasing the concentrations of A83-01 and PD173074, while decreasing bulk cell density re-sulted in the increase of HLA-G of up to 71%. Gene expression profiling support the advance-ments of our treatment regarding the generation of trophoblast cells. The reported differentia-tion protocol will allow the on-demand access to human trophoblast cells enriched for HLA-Gpos EVT-like cells, allowing the elucidation of placenta-related disorders and to investigate the im-munological tolerance towards the fetus, overcoming the difficulties in obtaining primary EVTs without the need for a complex differentiation pathway via naïve pluripotent or trophoblast stem cells.
2023-09-14 | GSE234949 | 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 first lineage decisions during mouse development lead to establishment of embryonic and extraembryonic tissues. The transcription factor Cdx2 plays a central role by repressing pluripotency genes, such as Oct4 and promoting trophoblast fate at the blastocyst stage. Here we show that the transcription factor Gata3 is coexpressed with Cdx2 in the blastocyst and that overexpression of Gata3 in embryonic stem cells is sufficient to induce expression of trophoblast genes. Gata3 expression in the blastocyst does not depend on Cdx2, nor do Gata3 overexpressing cell lines require Cdx2 for expression of a subset of trophoblast genes. In the embryo, expression of Gata3, like Cdx2, depends on Tead4, and expression of both factors becomes restricted to nascent trophoblast by an Oct4-independent mechanism. These observations place Tead4 at the top of a trophoblast hierarchy, with Gata3 and Cdx2 acting downstream to induce expression of common and independent targets in this lineage. This SuperSeries is composed of the following subset Series: GSE12985: Differentiation time course of trophoblast stem cells GSE12986: Expression of Cdx2 or Gata3 in R1 mouse embryonic stem cells
2010-02-21 | E-GEOD-12999 | biostudies-arrayexpress
Project description:Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. We have identified a CDX2+/p63+ cytotrophoblast (CTB) subpopulation in the early post-implantation human placenta, which is significantly reduced later in gestation. CTB differentiate into different trophoblast subtypes, which are responsible for gas/nutrient exchange (syncytiotrophoblast/STB) and invasion and maternal vascular remodeling (extravillous trophoblast/EVT). Study of early human placental development is severely hampered by lack of a representative trophoblast stem cell (TSC) model, with the capacity for self-renewal and the ability to differentiate into both STB and EVT. We describe a reproducible protocol, using defined media containing BMP4, by which human embryonic stem cells (hESC) can be differentiated into CDX2+/p63+ CTB-like cells. These cells can be replated to further differentiate into STB- and EVT-like cells, based on marker expression, hormone secretion and invasive ability. Differentiation of hPSC-derived CTB in hypoxia leads to reduced hCG secretion and STB-associated gene expression, instead inducing EVT differentiation in a hypoxia-inducible factor-dependent manner.
2016-06-16 | GSE76090 | GEO
Project description:To identify whether Cdx2 or Gata3 can activate trophoblast specific gene expression when expressed in R1 ES cells. To assess the dependency of Gata3 activity on Cdx2, Gata3 was also expressed in Cdx2-null ES cells. Keywords: gene expression a fusion construct of either Cdx2 or Gata3 and the tamoxifen responsive estrogen receptor ligand binding domain was expressed in R1 ES cells. Cell were induced with tamoxifen for 6 days to activate the transcription factor and cultured under trophoblast stem cell conditions
2010-02-21 | E-GEOD-12986 | biostudies-arrayexpress
Project description:The first lineage decisions during mouse development lead to establishment of embryonic and extraembryonic tissues. The transcription factor Cdx2 plays a central role by repressing pluripotency genes, such as Oct4 and promoting trophoblast fate at the blastocyst stage. Here we show that the transcription factor Gata3 is coexpressed with Cdx2 in the blastocyst and that overexpression of Gata3 in embryonic stem cells is sufficient to induce expression of trophoblast genes. Gata3 expression in the blastocyst does not depend on Cdx2, nor do Gata3 overexpressing cell lines require Cdx2 for expression of a subset of trophoblast genes. In the embryo, expression of Gata3, like Cdx2, depends on Tead4, and expression of both factors becomes restricted to nascent trophoblast by an Oct4-independent mechanism. These observations place Tead4 at the top of a trophoblast hierarchy, with Gata3 and Cdx2 acting downstream to induce expression of common and independent targets in this lineage. This SuperSeries is composed of the SubSeries listed below.
2010-02-01 | GSE12999 | GEO