Supplementary Figure 3: Protocol adjustment in hiPSCs

(a) Comparison of CHIR dose in T expression on day 4. A slightly higher dose of CHIR was required for sustained T expression in hiPSCs on day 4. (b) Immunocytochemistry for T, TBX6 or FOXF1 on day 4 of differentiation. hESCs were differentiated with CHIR 8 μM, and hiPSCs were differentiated with CHIR 10 μM. Notably, FOXF1 was negative in hESCs, but was positive in hiPSCs. (c) The tested protocol and representative immunocytochemistry in hiPSCs. Noggin at >5 ng/ml suppressed FOXF1 expression on day 4. To induce WT1 expression on day 7, Noggin 5 ng/ml was optimal. (d) The differentiation protocol and staining for FOXF1 on day 4, and for WT1 on day 7 in hESCs. Either additional Noggin or BMP4 significantly suppressed WT1 expression on day 7, and BMP4 induced FOXF1 expression on day 4, suggesting endogenous BMP4 signal is optimal in hESCs with CHIR treatment alone. (e) The protocol and staining with WT1 and HOXD11 on day 7 in hiPSCs. CHIR 10 μM + Noggin 5 ng/ml followed by activin 10 ng/ml showed the most efficient differentiation into WT1+HOXD11+ cells in hiPSCs. Scale bars: 100 μm.