Generation of 3D Trophoblast Organoids from Human Naïve Pluripotent Stem Cells.

The human placenta is a transient organ that functions to support the needs of the fetus throughout gestation. Trophoblasts are the major epithelial cells found within the placenta and comprise a variety of distinct cell types with specialized roles in fetal-maternal communication. Our understanding of human trophoblast development remains limited due to ethical and legal restrictions on accessing first-trimester placental tissues, as well as the inability of common animal models to replicate primate placental development. It is therefore important to advance in vitro models of human trophoblast development as a basis for studying pregnancy-associated complications and diseases. In this chapter, we describe a protocol for generating 3D trophoblast organoids from naïve human pluripotent stem cells (hPSCs). The resulting stem-cell-derived trophoblast organoids (SC-TOs) contain distinct cytotrophoblast (CTB), syncytiotrophoblast (STB), and extravillous trophoblast (EVT) cell types, which closely correspond to trophoblast identities in the human post-implantation embryo. We discuss methods for characterizing SC-TOs by immunofluorescence, flow cytometry, mRNA and microRNA expression profiling, and placental hormone secretion. Furthermore, SC-TOs can undergo differentiation into specialized 3D EVT organoids, which display robust invasion when co-cultured with human endometrial cells. Thus, the protocol described herein offers an accessible 3D model system of human placental development and trophoblast invasion.