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. 2025 Dec 27;16:8. doi: 10.1186/s13578-025-01508-5

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© The Author(s) 2025

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

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Fig. 2 — PGR knockdown impairs self-renewal and differentiation in trophoblast stem cells and organoids. A Schematic workflow for deriving primary TSCs and induced TOs from clinical human placental villi samples. B Representative images of primary TSCs and induced TOs, with immunofluorescence staining showing syncytiotrophoblast marker CGB (red, inner layer), epithelial marker EPCAM (green), and nuclear marker DAPI (blue) in TOs derived from primary TSCs. C Experimental workflow for establishing PGR-knockdown (shPGR) and control (shLUC) TSCs and TOs, followed by extravillous trophoblast (EVT) differentiation. D Morphological comparison of primary TSCs, TOs, and EVT-differentiated TOs between shPGR and shLUC groups. E–F Real-time qPCR analysis of self-renewal markers (MKI67 and TEAD4) in TSCs and TOs with PGR knockdown (TSC-shPGR, TO-shPGR) versus controls (TSC-shLUC, TO-shLUC). G Real-time qPCR analysis of EVT differentiation marker (HLA-G and MMP2) expression in EVT-differentiated TOs (TO#EVT-shPGR vs TO-EVT#shLUC). H Western blot analysis of TEAD4 protein expression in TO-shPGR (n = 3) and TO-shLUC (n = 3) samples (left), with corresponding quantitative densitometry (right). I Western blot analysis of MMP2 protein expression in TO#EVT-shPGR (n = 3) and TO#EVT-shLUC (n = 3) samples (left), with corresponding quantitative densitometry (right). J-K Immunofluorescence detection of EVT marker HLA-G (green), epithelial marker EPCAM (green), and nuclear staining (DAPI, blue) in EVT-differentiated TOs, with quantitative analysis of HLA-G mean fluorescence intensity(K). Data represent means ± SEM. *p < 0.05, **p < 0.01. ***p < 0.001. ****p < 0.0001. Scale bar, 200 µm. Abbreviations: TSC, trophoblast stem cell; TO, trophoblast organoid; EVT, extravillous trophoblast; CGB, chorionic gonadotropin beta; EPCAM, epithelial cell adhesion molecule; DAPI, 4′,6-diamidino-2-phenylindole