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Derivation of functional trophoblast stem cells from human primed pluripotent stem cells [scRNA-seq]

ABSTRACT: Trophoblast stem cells (TSC) have recently been derived from human embryos, and early first trimester placenta; however, aside from ethical challenges, the unknown disease potential of these cells limits their scientific utility. We have previously established a BMP4-based two-step protocol for differentiation of primed human pluripotent stem cells (hPSC) into functional trophoblast; however, those trophoblast could not be maintained in a self-renewing TSC-like state. Here, we use the first step from this protocol, followed by a switch to a newly-developed TSC media, to derive bona-fide TSC. We show that these cells resemble placenta- and naïve hPSC-derived TSC, based on their transcriptome, as well as in vitro and in vivo differentiation potential. We conclude that primed hPSC can be used to generate functional TSC, through a simple protocol which can be applied to a widely-available set of existing hPSC, including induced pluripotent stem cells derived from patients with known birth outcomes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE182790 | GEO | 2022/05/16

REPOSITORIES: GEO

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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. Human embryonic stem cells (hESC) (WA09/H9) were maintained on Geltrex-coated plates (BD Biosciences ) in StemPro (Thermo Fisher) + bFGF (12 ng/ml). Undifferentiated hESC (D-2) were switched to minimal media (EMIM (Erb et al., 2011) containing KO DMEM/F12 (Gibco), 1% Insulin-Transferrin-Selenium Mix (Sigma-Aldrich), 1% NEAA (Gibco), 2mM L-Glutamine (Corning), 0.1mM 2-mercaptoethanol (Gibo), and 2% BSA (Gemini Bio Products)) for 2 days then treated with BMP4 (StemRD, 10ng/ml) for 3 days in minimal media+BMP4 (D3). At D3, cells were replated onto Geltrex in Feeder Conditioned Media +BMP4 and cultured in normoxia (20% O2) or hypoxia (2% O2) for 2 days (D3+2) to assess the effect of hypoxia. Cells were infected with lentiviral shRNA for shScramble (control) or shARNT, the beta subunit of the Hypoxia Inducible Factor (HIF) complex to assess the effect of ARNT knockdown. Each sample includes biological triplicates.

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Derivation of functional trophoblast stem cells from human primed pluripotent stem cells [scRNA-seq]

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