Leveraging chorionic villus biopsies for the derivation of patient-specific trophoblast stem cells

Human trophoblast stem (TS) cells are an informative in vitro model for the generation and testing of biologically meaningful hypotheses. The goal of this project was to derive patient-specific TS cell lines from clinically available chorionic villus sampling (CVS) biopsies. Cell outgrowths were captured from human CVS tissue specimens cultured in modified human TS cell medium. Cell colonies emerged early during the culture and cell lines were established and passaged for several generations. Karyotypes of the newly established CVS-derived trophoblast stem (TSCV) cell lines were determined and compared to initial genetic diagnoses from freshly isolated chorionic villi. Phenotypes of TSCV cells in the stem state and following differentiation were compared to cytotrophoblast-derived TS (TSCT) cells. TSCV and TSCT cells uniformly exhibited similarities in the stem state and following differentiation into syncytiotrophoblast. These shared features included morphology and gene expression. TSCV cell differentiation into extravillous trophoblast cells exhibited cell line dependent phenotypes. CVS tissue specimens provide a valuable source for TS cell derivation. They expand the genetic diversity of available TS cells and are associated with defined clinical outcomes. TSCV cell lines provide a new set of experimental tools for investigating trophoblast cell lineage development. Overall design: Chorionic villus tissue specimens were cultured in Complete TS Cell Medium (Okae et al., 2018) on type IV collagen-coated dishes and cell outgrowths formed and expanded from attached tissue specimens. Human TS cell colonies emerged after first passage and lines were expanded with subsequent passaging. TS cells were propogated in tissue culture treated dishes pre-coated with type IV collagen in Complete TS Cell Medium. EVT cell differentiation (2018 protocol) was induced by plating human TS cells onto 6-well plates pre-coated with 1 µg/mL collagen IV at a density of 80,000 cells per well. Cells were cultured in EVT Differentiation Medium and analyzed on day 8 of EVT cell differentiation (Okae et al., 2018). Alternatively, EVT cell differentiation (2022 protocol) was induced by plating human TS cells into 6 cm petri dishes at a density of 1x105 TS cells per dish and cultured for two days in Spheroid Medium. After two days spheroids collected and replated in EVT Differentiation Medium and Matrigel. Cells were analyzed on day 7 of EVT cell differentiation. Syncytiotrophoblast cell differentiation was induced by plating TS cells into 6 cm petri dishes at a density of 300,000 cells per dish in Three-dimensional (3D) Syncytiotrophoblast Differentiation Medium (Okae et al., 2018). Cells were analyzed on day 6 of syncytiotrophoblast cell differentiation.