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


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
    • "More recently, Genbacev et al. (2011) isolated a population of continuously self-replicating TBPCs from the chorionic membrane of first trimester placentae. Our side-population trophoblasts show a very different gene expression profile compared to TBPC (Fig. 4), which up-regulate genes involved in neuronal development, stem cell pluripotency, integrin signalling and thrombin signalling (Genbacev et al. 2011). It is possible this may be a result of differences in the gestational age of the placentae from which the reference cytotrophoblast population was isolated in the two studies. "

    Placenta; 09/2015
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    • "To obtain transcriptomic profiles of previously isolated trophoblast progenitor cells (TBPC) and human embryonic stem cells (hESC) (Genbacev et al. 2011), Affymetrix CEL files were obtained from the National Center for Biotechnology Information's (NCBI) GEO (reference GSE30127). These data were reanalysed using the process described above to obtain a directly comparable set of profiles between the two experiments ; in addition, lists of differentially expressed transcripts from this analysis were checked against the analysis in the original publication and found to be in agreement. "
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    ABSTRACT: The placenta is responsible for all nutrient and gas exchange between mother and baby during pregnancy. The differentiation of specialised placental epithelial cells called trophoblasts is essential for placental function, but we understand little about how these populations arise. Mouse trophoblast stem cells have allowed us to understand many of the factors that regulate murine trophoblast lineage development, but the human placenta is anatomically very different from the mouse, and it is imperative to isolate a human trophoblast stem cell to understand human placental development. Here we have developed a novel methodology to isolate a Hoechst side-population of trophoblasts from early gestation placentae and compared their transcriptome to differentiated trophoblast populations (cytotrophoblasts and extravillous trophoblasts) using microarray technology. Side-population trophoblasts clustered as a transcriptomically distinct population but were more closely related to cytotrophoblasts than extravillous trophoblasts. Side-population trophoblasts up-regulated of a number of genes characteristic of trophectoderm and murine trophoblast stem cells in comparison to cytotrophoblasts or extravillous trophoblasts, and could be distinguished from both of these more mature populations by a unique set of 22 up-regulated genes, which were enriched for morphogenesis, organ development and regulation of growth functions. Cells expressing two of these genes (LAMA2 and COL6A3) were distributed throughout the cytotrophoblast layer at the trophoblast/mesenchymal interface. Comparisons to previously published trophoblast progenitor populations suggest that the side-population trophoblasts isolated in this work are a novel human trophoblast population. Future work will determine whether these cells exhibit functional progenitor/stem cell attributes.
    Reproduction 08/2015; DOI:10.1530/REP-14-0646 · 3.17 Impact Factor
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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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