Developmental landmarks and cellular transitions during extravillous trophoblast cell differentiation

Human trophoblast stem (TS) can be captured, maintained in vitro under specific conditions, and differentiated into extravillous trophoblast (EVT) cells. The regulatory mechanisms that govern the self-renewal and differentiation of human TS cells into EVT cells are largely unknown. In this study, bulk RNA-sequencing (RNA-seq) and single cell RNA-seq (scRNA-seq) were performed on human TS cells maintained in the stem state and on cells progressing from the stem state into EVT cells (differentiation days 3, 6, and 8). Distinct bulk and single cell transcript profiles were identified for each day of analysis. Day 3 of EVT cell differentiation represented a striking transition point and was readily distinguished from stem state and days 6 and 8 of EVT cell differentiation. Analysis of scRNA-seq led to the identification of several unique cell populations, trophoblast cell developmental state-specific regulons, and trajectories. We elucidated functional roles of key regulators of EVT cell development: cyclin B1, CCAAT/enhancer-binding protein beta, and A Disintegrin And Metalloproteinase (ADAM) metallopeptidase with thrombospondin type 1 motif 20. Collectively, we have defined developmental landmarks and transitional cell populations during EVT cell differentiation. These findings provide a valuable resource and foundation for future investigations into regulatory mechanisms controlling TS cell differentiation into the EVT cell lineage.