Fig 1: Activin A promotes ExMC specification from trophoblasts(A) Heatmap of differentially expressed genes (DEGs, padj < 0.05, |log2 fold change (FC)| > 1.5) between ExMC and trophoblast subtypes (PrSyn and Peri-TB) identified in scRNA-seq.(B) Upstream regulator analysis by IPA with DEGs identified in (A) as input.(C) Expression of marker genes quantified by RT-qPCR in optimized ExMC induction protocols. Error bars from n = 3 for each condition calculated as standard error of the mean (SEM). A non-paired two-sided t test was used to determine significance. ns, not significant; **p < 0.01, ***p < 0.001.(D) PCA of RNA-seq data at day 6 of differentiation of KOLF2.C1 iPSC under the three conditions indicated in the schematic, together with ExMC and trophoblast cell clusters identified from in vitro cultured blastocyst (Xiang) and in vivo implanted human embryos (Tyser).(E) Heatmap of DEGs between BS- and BSA-induced iPSC differentiation (as in D), with three biological replicates for each condition.(F) Immunofluorescence of three lineage-specific markers (PRR9 and HLA-G for trophoblasts and HAND1 for ExMC) on day 6 of differentiation. White arrowheads indicate HAND1+ ExMC in BS condition. Scale bar, 100 mm.(G) Expression of ExMC markers on ExMC derived from hTSCs quantified by time-course RT-qPCR. Error bars from n = 3 for each condition calculated as 95% confidence interval.