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Three-dimensional spheroid and organoid cultures using cells from placental tissues or trophoblast stem cells have emerged as models that recapitulate cell\u2013cell interactions and aspects of the physiological environment. Trophoblast organoids typically contain self-renewing cytotrophoblasts located toward the periphery in contact with basement membrane, whereas cells within the core fuse into hormone-producing syncytiotrophoblasts. Removing cells from matrix and\/or culturing them in suspension can reverse this organization. In both cases, spontaneous syncytialization occurs, but the underlying mechanisms remain poorly understood. To identify signaling pathways associated with spontaneous trophoblast differentiation in three-dimensional conditions, human trophoblast stem cells were either maintained in the stem state as two-dimensional (2D) monolayers or aggregated in suspension without an exogenous matrix to form three-dimensional cultures. Expression and localization of trophoblast subpopulation markers were compared between two- and three-dimensional cultures. Three-dimensional cultures displayed increased spontaneous differentiation into the syncytiotrophoblast lineage along the periphery, accompanied by reduced proliferation of cytotrophoblasts. Consistent with this observation, BeWo choriocarcinoma cells exhibited increased chorionic gonadotropin levels in three-dimensional culture, whereas this response was not observed in HTR8\/SVneo immortalized trophoblasts. Gene expression and pathway enrichment analyses identified Hippo signaling as altered in three-dimensional trophoblast stem cell cultures, with reduced expression of several genes associated with YAP1\/TAZ-TEAD transcriptional activity. Inhibition of LATS1\/2, which activates YAP1\/TAZ-TEAD signaling, reduced spontaneous syncytiotrophoblast formation. These findings suggest that reduced YAP1\/TAZ-TEAD activity may facilitate self-organized syncytialization in three-dimensional trophoblast cultures.<\/jats:p>","DOI":"10.1093\/molehr\/gaag054","type":"journal-article","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T11:35:05Z","timestamp":1788867305000},"source":"Crossref","is-referenced-by-count":0,"title":["Hippo signaling regulates spontaneous human trophoblast syncytialization in three-dimensional cultures"],"prefix":"10.1093","volume":"32","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0968-689X","authenticated-orcid":false,"given":"Michael","family":"Zheng","sequence":"first","affiliation":[{"name":"Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario , London, ON,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lin","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Cell Biology, 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