Derivation of human trophoblast stem cells from placentas at birth

Trophoblast stem cells (TSCs) were initially obtained from cytotrophoblast (CTB) of the first trimester placenta or blastocyst stage embryo. The cells were cultured in a medium called trophoblast stem cell medium (TSCM), which contains several components, including epidermal growth factor (EGF), the inhibitor CHIR99021 for glycogen synthase kinase-beta (GSK3-beta), the inhibitors A83-01 and SB431542 for transforming growth factor-beta (TGF-beta), valproic acid (VPA), and the inhibitor Y-27632 for Rho-associated protein kinase (ROCK). In this study, we demonstrate that human trophoblast stem cells (hTSCs) can be obtained from cytotrophoblasts (CTBs) isolated from the term placenta. This is achieved by culturing the CTBs in a medium using TSCM, which is supplemented with a low concentration of the mitochondrial pyruvate uptake inhibitor UK5099 and lipid-rich albumin (referred to as TUA medium). It is important to note that hTSCs cannot be obtained from term CTBs utilizing TSCM alone, or without the presence of either UK5099 or lipid-rich albumin. Overall design: RNA sequencing was conducted on the following cells/conditions: two primary hTSC lines from the term placenta (T1 and T2 cell lines), cultured in TUA medium, TSCM from TUA medium; and cultured in TSCMa medium (TSCM supplemented with lipid-rich albumin). 3 biological replicates were used for each cell type/condition, only 2 replicates were used for T2 TSCMa medium (3 x 5 + 2 = 17 samples total)