FIG. 3.

Equal efficiency of BMP4/A/P differentiation of hESCs into trophoblastic progenitors for male and female cells. (A) Schematic showing the differentiation strategy of hESCs into progenitors of trophoblastic cells, and time points for RNA collection. (B) IF for CDX2 and KRT7 expression during days 1–3 of BMP4/A/P differentiation of HUES9 (top) and in undifferentiated HUES9 cells (bottom). (C) Co-IF for CDX2 and KRT7 protein in day 2 differentiated BMP4/A/P cells (HUES9). (D) Flow cytometry analysis of CDX2 protein levels on day 2 of BMP4/A/P-differentiated HUES9 (red line) compared with an undifferentiated control (blue line). (E) Representative flow plots (n = 3) for markers of trophoblastic cells (KRT7, left; GATA3, right) in male (HUES7, thick blue line) and female (HUES9, thick pink line) cells differentiated for 5 days with BMP4/A/P. Random differentiation for female and male (thin pink and blue lines, respectively) is included along with a negative control FMO (fluorescence minus one) (gray line). Gating strategy is described in the Materials and Methods section. BMP4, bone morphogenic protein 4; IF, immunofluorescence. Color images available online at www.liebertpub.com/scd