Fig. 3: Differentiation capacity of TSCs is uncompromised by CIN.

A Immunostaining of STs derived from nTSC, nbTSC, ccTSC, and pdTSC. Despite the presence of aneuploidy (passage number >10) and CIN, all four TSC types differentiated into STs expressing the ST marker SDC1 and the trophoblast marker TFAP2A. Scale bar, 100 μm. B Immunostaining of EVTs derived from nTSC, nbTSC, ccTSC, and pdTSC. All four TSC types differentiated into EVTs expressing the EVT marker HLA-G, with spontaneous ST differentiation indicated by SDC1 expression. nTSC and nbTSC showed more efficient EVT induction, with a higher nucleus/cytoplasm ratio. Passage number of all TSC types >10. Scale bar, 100 μm. C qRT-PCR analysis of ST, EVT, and general trophoblast markers in STs and EVTs derived from all four TSC types. Data are presented as mean ± SD (n = 3 independent biological replicates). Significance was analyzed by two-sided Student’s t-test. *P < 0.05 compared to their parental TSCs. Source data are provided as a Source Data file. D EVTs derived from nTSCs and nbTSCs formed finger-like projections upon replating, while EVTs from ccTSCs and pdTSCs did not display this characteristic. Arrows indicate finger-like projections. Scale bar, 100 μm. The experiment was independently repeated more than three times with similar results. E EVT differentiation was sufficiently induced only from nTSC-TO and nbTSC-TO, but not from ccTSC-TO and pdTSC-TO. Scale bar, 100 μm. The experiment was independently repeated more than three times with similar results. The schematic graph was created using BioRender https://biorender.com/pfs10ip. F Immunofluorescent staining showed that EVT-TOs derived from nbTSCs fully expressed the EVT marker HLA-G, whereas EVT-TOs derived from ccTSCs did not. Both types expressed the trophoblast marker TFAP2A. Scale bar, 100 μm. The experiment was independently repeated more than three times with similar results.