We previously established a trophoblast differentiation protocol from primed human pluripotent stem cells (PSC). To induce this lineage, we use a combination of Bone Morphogenetic Protein-4 (BMP4) and the WNT inhibitor IWP2. This protocol has enabled us to obtain a pure population of trophectoderm (TE)-like cells that could subsequently be terminally differentiated into syncytiotrophoblasts (STB) and extravillous trophoblasts (EVT). However, the resulting TE-like cells could only be terminally differentiated to a variable mixture of STB and EVT, with a bias toward the STB lineage. Recently, methods have been developed for derivation and culture of self-renewing human trophoblast stem cells (TSC) from human embryos and early gestation placental tissues. These primary TSCs were further able to differentiate into either STB or EVT with high efficiency using the lineage specific differentiation protocols. Based partly on these protocols, we have developed methods for establishing self-renewing TSC-like cells from PSC, and for efficient lineage-specific terminal differentiation. Here, we describe in detail the protocols to derive and maintain PSC-TSC, from both embryonic stem cells (ESC) and patient-derived induced pluripotent stem cells (iPSC), and their subsequent terminal differentiation to STB and EVT. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Trophoblast Differentiation into TE-like Cells Basic Protocol 2: Conversion of PSC-Derived TE-like Cells to TSC Basic Protocol 3: Passaging PSC-Derived TSC in iCTB Complete Medium Basic Protocol 4: STB Differentiation from PSC-derived TSC Basic Protocol 5: EVT Differentiation from PSC-derived TSC Support Protocol 1: Geltrex-coated tissue culture plate preparation Support Protocol 2: Collagen IV-coated tissue culture plate preparation Support Protocol 3: Fibronectin-coated tissue culture plate preparation.
Keywords: extravillous trophoblast; human pluripotent stem cell; human trophoblast stem cell; syncytiotrophoblast; trophoblast differentiation.
© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.