Figure 2

Cells progress to nephron progenitors at Day 6. At differentiation day 6, cells were assessed by immunofluorescence and flow cytometry for nephron progenitor markers, including PAX2 and WT1 (A, C), and SIX2 (B, C). In (C), red dots represent isotype control treated cells used to identify the gated regions and blue dots represent cells stained for the indicated marker. Numbers indicate the fraction of stained cells (blue) in the gated regions. (D) Quantitative RT-PCR was used to monitor the expression of PAX2, WT1, SIX2 relative to the housekeeping gene GAPDH during IMR90-4 iPSC differentiation to podocytes using the protocol illustrated in Fig. 1A. Expression levels were normalized to undifferentiated IMR90-4 iPSCs. Data are presented as mean ± SEM of three independent experiments. Scale bars, 100 µm. Immunofluorescence labelling and flow cytometry were performed ten times from different differentiations on different days. Three technical replicates were used each time for flow cytometry. qPCR was performed three replicates each time and three times were performed from three different differentiations.