Establishment of Human Trophoblast Progenitor Cell Lines from the Chorion

Center for Reproductive Sciences, University of California San Francisco, San Francisco, California, USA.
Stem Cells (Impact Factor: 6.52). 09/2011; 29(9):1427-36. DOI: 10.1002/stem.686
Source: PubMed

ABSTRACT Placental trophoblasts are key determinants of in utero development. Mouse trophoblast (TB) stem cells, which were first derived over a decade ago, are a powerful cell culture model for studying their self-renewal or differentiation. Our attempts to isolate an equivalent population from the trophectoderm of human blastocysts generated colonies that quickly differentiated in vitro. This finding suggested that the human placenta has another progenitor niche. Here, we show that the chorion is one such site. Initially, we immunolocalized pluripotency factors and TB fate determinants in the early gestation placenta, amnion, and chorion. Immunoreactive cells were numerous in the chorion. We isolated these cells and plated them in medium containing fibroblast growth factor which is required for human embryonic stem cell self-renewal, and an inhibitor of activin/nodal signaling. Colonies of polarized cells with a limited lifespan emerged. Trypsin dissociation yielded continuously self-replicating monolayers. Colonies and monolayers formed the two major human TB lineages-multinucleate syncytiotrophoblasts and invasive cytotrophoblasts (CTBs). Transcriptional profiling experiments revealed the factors associated with the self-renewal or differentiation of human chorionic TB progenitor cells (TBPCs). They included imprinted genes, NR2F1/2, HMGA2, and adhesion molecules that were required for TBPC differentiation. Together, the results of these experiments suggested that the chorion is one source of epithelial CTB progenitors. These findings explain why CTBs of fully formed chorionic villi have a modest mitotic index and identify the chorionic mesoderm as a niche for TBPCs that support placental growth.

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Available from: Gabriel Alan Goldfien, Mar 05, 2014
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    • "Using an embryoid body selection procedure self-renewing cytotrophoblast stem (CTBS) cell lines were successfully established from hES cells [26]. More recently, human trophoblast progenitor cells (TBPCs) have been established from 7 to 8 week chorionic tissue [27], while isolation and culture of cytotrophoblasts from term placenta always results in a decline in proliferation and rapid syncytialisation [28] [29]. Here we describe our attempts to derive human TS cells from in vitro cultured blastocysts under a variety of conditions. "
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    ABSTRACT: Fibroblast growth factor (FGF) signaling is essential for early trophoblast expansion and maintenance in the mouse, but is not required for trophectoderm specification during blastocyst formation. This signaling pathway is stably activated to expand the trophoblast stem cell compartment in vivo, while in vitro, FGFs are used for the derivation of trophoblast stem (TS) cells from blastocysts and early post-implantation mouse embryos. However, the function of FGFs during human trophoblast development is not known.Methods We sought to derive TS cells from human blastocysts in a number of culture conditions, including in the presence of FGFs and stem cell factor (SCF). We also investigated the expression of FGF receptors (FGFRs) in blastocysts, and the expression of FGFR2 and activated ERK1/2 in first trimester human placentae.ResultsWe found that SCF, but not FGF2/4, improved the quality of blastocyst outgrowths, but we were unable to establish stable human TS cell lines. We observed CDX2 expression in the trophectoderm of fully blastocysts, but rarely observed transcription of FGFRs. FGFR2 protein was not detected in human blastocysts, but was strongly expressed in mouse blastocysts. However, we found robust FGFR2 expression and activated ERK1/2 in the cytotrophoblast layer of early human placenta.DiscussionOur data suggests that initiation of FGF-dependent trophoblast expansion may occur later in human development, and is unlikely to drive maintenance of a TS cell compartment during the peri-implantation period. These findings suggest that cytotrophoblast preparations from early placentae may be a potential source of FGF-dependent human TS cells.
    Placenta 09/2014; 35(12). DOI:10.1016/j.placenta.2014.09.008 · 2.71 Impact Factor
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    • "In our analysis, while JEG3 cells did not express any of the so-called " stemness- " associated transcription markers, HTR8/SVneo cells expressed most of them (OCT4a neg , NANOG weak , and SOX2 weak ). Interestingly, a recent characterization of trophoblast progenitor cells derived from first trimester placentae reveals that undifferentiated trophoblast progenitor cells that form embryoid bodies and that are capable of multipassage also display OCT4 neg , NANOG weak , and SOX2 weak phenotypes [39]. Due to the association of this transcription marker expression profile with the presence of progenitor-like functions in HTR8/SVneo cells and in trophoblast progenitor cells, it is enticing to conclude that NANOG and SOX2 are responsible for the observed functions. "
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    ABSTRACT: Introduction: JEG3 is a choriocarcinoma--and HTR8/SVneo a transformed extravillous trophoblast--cell line often used to model the physiologically invasive extravillous trophoblast. Past studies suggest that these cell lines possess some stem or progenitor cell characteristics. Aim was to study whether these cells fulfill minimum criteria used to identify stem-like (progenitor) cells. In summary, we found that the expression profile of HTR8/SVneo (CDX2+, NOTCH1+, SOX2+, NANOG+, and OCT-) is distinct from JEG3 (CDX2+ and NOTCH1+) as seen only in human-serum blocked immunocytochemistry. This correlates with HTR8/SVneo's self-renewal capacities, as made visible via spheroid formation and multi-passagability in hanging drops protocols paralleling those used to maintain embryoid bodies. JEG3 displayed only low propensity to form and reform spheroids. HTR8/SVneo spheroids migrated to cover and seemingly repopulate human chorionic villi during confrontation cultures with placental explants in hanging drops. We conclude that HTR8/SVneo spheroid cells possess progenitor cell traits that are probably attained through corruption of "stemness-" associated transcription factor networks. Furthermore, trophoblastic cells are highly prone to unspecific binding, which is resistant to conventional blocking methods, but which can be alleviated through blockage with human serum.
    11/2013; 2013(9):243649. DOI:10.1155/2013/243649
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    • "Our understanding of the significance of hESC and iPSC differentiation [33] will be enhanced by better definition of the potential heterogeneity of trophoblast populations which arise from hESC differentiation paradigms. Recently, methods to derive a trophoblast stem/progenitor population from the chorionic plate have been reported [36]. While the role of these cells in this chorionic plate niche remains to be further elucidated, a direct comparison of these chorion-derived cells with hESC-derived trophoblasts will also be very informative. "
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    ABSTRACT: The human embryo is not a feasible experimental system for the detailed study of implantation and early placentation, so surrogate systems have been sought for investigating the determination of the trophectoderm lineage, its differentiation into trophoblasts of the early implantation site, and subsequently the morphogenesis of the definitive placenta. An alternative to the use of embryos for studying early placental development was revealed by work with human embryonic stem cells (hESC), demonstrating BMP2/4-stimulated trophoblast differentiation, and spontaneous formation from embryoid bodies (EBs). These cells display a trophoblastic transcriptome, as well as a placental protein and steroid hormone secretory profile, and invasive and chemotactic behavior resembling human placental trophoblasts. With EB-derived trophoblasts, two-dimensional and three-dimensional paradigms and other modifications of the culture environment, including extracellular matrix and aggregation with placental fibroblasts, impact on trophoblast differentiation. Recent studies have questioned the identity of the trophoblasts directed by BMP treatment of hESC, and careful attention to culture conditions is needed to interpret different results among research groups. Although the precise placental counterpart of the hESC-derived trophoblast remains unclear, hESC-derived trophoblasts remain an intriguing platform for modeling early implantation.
    Placenta 12/2012; 34. DOI:10.1016/j.placenta.2012.11.019 · 2.71 Impact Factor
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