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Single nucleus RNA sequence (snRNAseq) analysis of the spectrum of trophoblast lineages generated from human pluripotent stem cells in vitro

ABSTRACT: We report the single nuclear RNA sequencing analysis for the profiling of trophoblast lineages generated from human embryoinc stem cells (hESCs) in vitro under 20 % and 5 % O2 concentration conditions. The hESCs were exposed to bone morphogenetic protein 4 (BMP4) in presence of inhibitors of ACTIVIN/TGFB (A83–01) and FGF2 (PD1730) signaling formerly called BAP treatment for 8 days after passaging, which generated a mixture of cytotrophoblast, syncytiotrophoblast, and HLA-G positive cells with similarities to extravillous trophoblast under both high and low O2 conditions (20 % and 5 %, respectively). A single nuclei RNA sequence approach from both O2 conditions determined two major groupings of cell clusters, one comprised of five and the second of four subcluster cell populations respectively. Of these, two subclusters resembled extravillous trophoblast, two carried the hallmark transcripts of syncytiotrophoblast, while the remaining five were likely different kinds of mononucleated cytotrophoblast.

ORGANISM(S): Homo sapiens

PROVIDER: GSE171768 | GEO | 2021/07/01

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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Single nucleus RNA sequence (snRNAseq) analysis of the spectrum of trophoblast lineages generated from human pluripotent stem cells in vitro

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