Tissue Context Drives Regional Differences in Trophoblast Differentiation in Fetal Membranes and Chorionic Villi

Trophoblasts within the fetal membrane primarily serve as protective barriers, shielding the developing fetus from the external environment, while those in chorionic villi facilitate interactions between the maternal and fetal compartments. Although these distinct trophoblast populations perform specialized roles based on their placental location, the mechanisms governing their development and differentiation remain largely unknown. In this study, we derived trophoblast organoids from both the smooth chorion of fetal membranes and chorionic villi from matched human placentas to create parallel in vitro models of these distinct trophoblast populations. Using comparative transcriptional profiling with both bulk and single-cell RNA sequencing, we identified subtle transcriptional variations, while overall gene expression patterns and cellular composition remained highly conserved. Comparative single-cell RNA sequencing and differentiation trajectory analysis of organoids derived from chorion and villous tissues, along with their respective tissue counterparts, revealed region-specific gene expression patterns that were only partially conserved in organoids. The similarity between these in vitro models suggests that regional differences in trophoblast expression observed in vivo are driven by environmental and regional cues. These findings highlight the critical role of local environmental factors in shaping trophoblast function and offer insights into the conserved mechanisms that support placental integrity and fetal development, while emphasizing the influence of region-specific cues in vivo.