Next generation trophoblast organoids with apical-out trophoblast polarity and central villous core-like cavity

Human trophoblast organoid serves as a revolutionary tool for studying trophoblast cell development and maternal-fetal interactions. However, currently reported trophoblast organoids exhibited opposite trophoblast polarity to in vivo or a solid cell spheroid lacking of central villous core-like cavity, both of which fail to model the physiological structure of in vivo placenta villi. Here, using human naïve embryonic stem cells, we constructed a trophoblast organoid with both peripheral apical-out bilaminar trophoblast cells and central villous core-like cavity, named ppTO. Besides, ppTO simulated the typical molecular and secretory features of in vivo trophoblast. Single-cell RNA sequencing (scRNA-seq) analysis demonstrated that the development of ppTO simulated in vivo trophoblast syncytialization process and pan-trophoblast transcriptome features. Using ppTO, we revealed that the syncytialization process relied on a suppressed HIPPO signaling for initiating syncytialization and followed by a reactivated HIPPO signaling for STB maturation. By comparative study with reported organoids, we revealed the important role of the expression and orientation of polarity molecule aPKC in the formation and spatial patterning of STB within trophoblast organoids. In summary, we develop a trophoblast organoid simulating the physiological structure and molecular features of placental villous trophoblast for studying the mechanisms underlying trophoblast development and function. Overall design: The single cells from a new trophoblast organoid (ppTO) modeling the real structure of placenta villi with peripheral apical-out trophoblast cells and a central villous core-like cavity for scRNA-seq