Fig. 4: Derivation of TS cells from term placentas.
a Images of TSTerm#2 cells and their derivative EVTs and STBs. TSTerm#2 cells derived from ITGA6-positive term CTBs were treated with FSK (ST medium) for five days or A83-01 and NRG1 (EVT medium) for 10 days for differentiation into STBs (ST-TSTerm#2) or EVTs (EVT-TSTerm#2), respectively. Magnification of the boxed syncytium in ST-TSTerm#2 cells is presented at right. b Expression of trophoblast stemness markers in TSTerm#2 cells. c Expression of STB markers and formation of syncytium in ST-TSTerm#2 cells. d Expression of EVT markers in EVT-TSTerm#2 cells. The HLA-G-positive EVT-TSTerm#2 cells were subjected to transwell invasion assays. Scale bar, 100 μm. e Regulation of GCM1 and ΔNp63α expression in TSTerm#2 cells and ST-TSTerm#2 by hypoxia. Arrow and arrowhead denote the ΔNp63α band and a non-specific band, respectively. f Suppression of GCM1 autoregulation by hypoxia. TSTerm#1 cells were transfected with pGL4-SV40 or E1bLUCGCM1-2K and incubated under normoxia or hypoxia for 48 h. Cells were then harvested for luciferase assays. g Interaction of GCM1 and ΔNp63α in TSTerm cells. TSTerm#2 cells were cultured under normoxia for 96 h and then subjected to immunofluorescence microscopy (IFM) or PLA using GCM1 Ab, ΔNp63 mAb, and normal rabbit IgG (R-IgG). Arrows point to cells coexpressing ΔNp63α and GCM1 in IFM or exhibiting PLA puncta. Average PLA signal per nucleus was calculated as total PLA puncta divided by the number of all nuclei in the field. Scale bar, 20 μm. h Suppression of STB differentiation by ΔNp63α. GFP-positive mock or ΔNp63α-expressing TSTerm#2 cells were treated with or without FSK for 72 h, followed by cell fusion assays and analysis of GCM1, hCGβ and SYN1 transcripts or proteins. Data are presented as mean ± SD of independent experiments (n = 3 in e–h). Differences were assessed by two-way ANOVA with Tukey’s post hoc test (e, h (left)) or unpaired two-tailed Student’s t-test (f, g, h (right)). Source data are provided in the Source Data file.
