Fig. 5: Sertoli-like cell differentiation from hiPSCs.

a Differentiation kinetics of human hiPSCs derived from a healthy 46,XY male, starting with hiPSC, and sequential alteration of the culture medium (M1, M2, and M3) to induce differentiation into Sertoli-like cells. For greater clarity, the x-axis starts after 30 h in M1. The labels M1-36h, M2-06h, M2-12h, M2-24h, M2-48h, and M3-48h indicate the time cells spent in specific media, while the labels 36, 42, 48, 60, 72, 84, 108, and 132 h indicate time elapsed since the initiation of differentiation, triggered by the replacement of mTeSR with M1 medium. The expression levels of key genes, SRY (blue), SOX9 (red), and WNT4 (green) were quantified using qRT-PCR with the ∆∆CT method. Normalization was performed using the 18S rRNA RPL19 gene, and the end of M1 served as the calibration point. b Comparison of SRY, SOX9, and WNT4 expression between wild-type and mutant E250-∆33 hiPSC cell lines. The expression levels were quantified using qRT-PCR with the ∆∆CT method using the wild-type condition as the calibrator for the visual representation. Gene expression is visually represented with wild-type clones shown in dark and mutant clones light blue for SRY, in red and light brown for SOX9, and in dark and light green for WNT4. The data were pooled with two to five biological replicates performed, with each experiment having three to six technical replicates. The statistical analysis was performed using a linear mixed model. At the initial timepoints (M1-36h, M2-06h), data were shown within a box, to highlight a 40 to 70% reduction in relative expression of SRY in mutant cells compared to wild-type control cells (detailed in the Supplementary Information file).