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Next, we carefully compared four protocols for the differentiation of iPSC-derived podocytes derived from the DYR0100 iPSC line (Fig. 4). The differentiation efficiency of the accelerated protocol [14] was compared to Ciampi [17], Rauch [41], and Musah [12] protocols. Each protocol involves differentiation in different defined mediums, with major variation in the length of time required for each step and the composition of the various media (Fig. 4A). Our protocol begins and ends with the same media as described in the Musah protocol, but was shortened by adding defined steps to reach the nephron progenitor cell type through modulation of the Wnt pathway. Whereas in the Musah protocol the concentration of the Wnt activator and glycogen-synthase kinase-3 (GSK-3) inhibitor CHIR99021 was held constant [12], in our protocol CHIR99021 was increased from 3 to 8 μM for two days. Next, we withdrew CHIR99021 to stop Wnt activation, similar to the 13-day Ciampi protocol [17]. To induce the nephron progenitor cell type, cells were cultured for two days in media including FGF9 stabilized by heparin without Wnt activation. This period required 4 days, whereas the Musah protocol required 13 days (Fig. 4A). The other lengthy protocol, Rauch, does not rely on Wnt activation [41].

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