Fig. 1

NR5A1 induction has a minimal effect on the upregulation of bipotential gonadal and female markers. A A schematic representation of the gonadal differentiation protocol from hESCs to steroidogenic-like cells with the matching developmental stages and administered growth factors, inhibitors, and small molecules. B RT-qPCR analysis of markers associated with gonadal development showed that NR5A1 induction was successful. Bipotential gonadal markers (NR5A1, GATA4, WT1) were upregulated by day 8, female gonadal markers (RSPO1, WNT4) were upregulated by day 10 as a result of gonadal differentiation even without NR5A1 induction and INHA was significantly upregulated after NR5A1 induction. Data are reported as mean ± SEM, n=3 biological replicates. Two-way ANOVA; 0.12 (ns), 0.033 (*), 0.002 (**), < 0.001 (***). C Confocal images of immunofluorescence staining showed expression of bipotential markers NR5A1 and GATA4, and the female gonadal marker WNT4 in non-induced (-DOX -TMP) and induced (+DOX +TMP) conditions at day 10 of gonadal differentiation. Images were taken with a ×40 objective. Scale bars 50 µm. ActA, activin A; BMP, bone morphogenetic protein; CHIR, CHIR-99021; DM, dorsomorphin; hESCs, human embryonic stem cells; IM, intermediate mesoderm; PS, primitive streak; d, day of differentiation; DOX, doxycycline hyclate; TMP, trimethoprim