Trophoblast organoids with physiological polarity model placental structure and function.

Human trophoblast organoids (TOs) are a three-dimensional ex vivo culture model that can be used to study various aspects of placental development, physiology, and pathology. Previously, we showed that TOs could be derived and cultured from full-term human placental tissue and used as models of trophoblast innate immune signaling and teratogenic virus infections (Yang et al., 2022). However, a remaining challenge of TOs cultured in 'domes' of Matrigel or other extracellular matrix is their inverted polarity, with proliferative cytotrophoblasts (CTBs) on the outer surface of organoids and the multi-nucleated syncytiotrophoblast (STB) primarily localized within the inner surface, which is in direct contrast to the orientation that occurs in vivo . Here, we developed a method to culture TOs under conditions that recapitulate the cellular orientation of chorionic villi in vivo . We show that standard TOs containing the STB layer inside the organoid (STB in ) develop into organoids containing the STB on the outer surface (STB out ) when cultured in suspension with gentle agitation. STB out organoids secrete higher levels of hormones and cytokines from the STB, including human chorionic gonadotropin (hCG) and interferon (IFN)-λ2. Using membrane capacitance measurements, we also show that the outermost surface of STB out organoids contain large syncytia comprised of >60 nuclei compared to STB in organoids that contain small syncytia (<6 nuclei) and mononuclear cells. The growth of TOs under conditions that mimic the cellular orientation of chorionic villi in vivo thus allows for the study of a variety of aspects of placental biology under physiological conditions.