Establishment of Human Trophoblast Progenitor Cell Lines from the Chorion

Abstract

Placental trophoblasts are key determinants of in utero development. Mouse trophoblast stem cells (mTSCs), 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 trophoblast 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 FGF and an inhibitor of activin/nodal signaling, which is required for human embryonic SC self-renewal. Colonies of polarized cells with a limited lifespan emerged. Trypsin dissociation yielded continuously self-replicating monolayers. Colonies and monolayers formed the two major human trophoblast lineages—multinucleate syncytiotrophoblasts and invasive cytotrophoblasts (CTBs). Transcriptional profiling experiments revealed the factors associated with the self-renewal or differentiation of human chorionic trophoblast 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.

Activin/Nodal Signaling Switches the Terminal Fate of Human Embryonic Stem Cell-derived Trophoblasts

“…Role of Activin/Nodal Signaling in Trophoblast Differentiation-It was previously shown that SB431542 treatment leads to trophoblast differentiation (17,33). However, the study was questioned due to the use of ambiguous markers for characterizing hESC-derived trophoblasts (43).…”

“…2 , embryonic carcinoma cells (30,31), induced pluripotent stem cells (28,32), other multipotent cells (33), and mouse embryonic stem cells (mESCs) (34 -37). Treatment with bone morphogenetic protein (BMP) and/or inhibitors of the activin/nodal pathway have been used for deriving trophoblasts (1, 6 -8, 10, 12-15, 17, 19, 22-29, 33, 38).…”

Isolation and characterisation of a novel trophoblast side-population from first trimester placentae

“…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.…”

“…It has previously been reported that a TBPC population was isolated from the chorion of first trimester placentae (Genbacev et al 2011). To determine how similar our villus-derived side-population trophoblasts were to this previously published chorionic TBPC population, we reanalysed the data to compare genes that were significantly (P!0.05) up-or down-regulated more than twofold in side-population trophoblasts relative to cytotrophoblasts with genes up-or down-regulated more than twofold in TBPC relative to cytotrophoblasts.…”

Notch1 controls development of the extravillous trophoblast lineage in the human placenta

“…Bipotential trophoblast progenitor cells have been derived from the chorionic mesenchyme differentiating into EVTs and STBs (14,15), whereas others identified a specific precursor of the EVT lineage in villous explant cultures (16). Placental structures, trophoblast cell types, and expression patterns of key regulatory transcription factors diverge between mouse and man, thereby hindering comparison of putative regulatory mechanisms (17).…”