Fig 1.
Effect of initial cell plating density of undifferentiated iPSC for podocyte differentiation efficiency.
Initial cell density of approximately 100 000 cells/cm2 (right column) is optimal for podocyte differentiation. For differentiation 9000 cells/cm2 were directly seeded into differentiation medium. Representative images of the SBAD3 iPSC line are shown.
Fig 2.
Time course of specific gene expression during differentiation of iPSC into podocyte-like cells, assessed by qPCR.
The pluripotency markers Oct4 and Sox-2 gradually decreased to zero at day 13, while podocyte marker synaptopodin (SYNPO) mRNA specifically increased 9-fold during the course of differentiation. Data are given as means ± SEM of 7 independent series of experiments, pooled from the 3 iPSC lines (SBAD3, SBNEO, SFC018) (*P < 0.05; ** P < 0.01; *** P < 0.001).
Fig 3.
Morphological changes of cells during differentiation of human iPSC into podocyte-like cells.
Phase contrast images of iPSC during differentiation. Podocyte-like foot processes are indicated with an asterisks and interdigitations between foot process with an arrow.
Fig 4.
Differentiated conditionally immortalized human podocytes served as reference controls.
The podocyte cell line (Saleem et al., 2002) was cultured under non-permissive conditions. Phase contrast images of cell morphology (left) and synaptopodin immunofluorescence staining (middle). Right: Staining of cell nucleus with Hoechst 33342.
Fig 5.
Immunostaining of podocyte-like cells derived from the iPSC line SBAD3 with podocyte markers and F-actin staining.
iPSC were differentiated on glass cover slips fixed and stained for synaptopodin, WT-1, podocin, and F-actin as described in methods. Red and green colours were applied post capture. Staining in two other iPSC lines are provided in S1 and S2 Figs.
Fig 6.
Time course of induction of podocyte-specific markers during differentiation.
Differentiating, SBAD3 iPSC were lysed at days 0 (undifferentiated iPSC, d0) and at days 6 and 13 and processed for Western blot analysis. Representative blots are shown. Expression levels for two other iPS cell lines are shown S3 Fig.
Fig 7.
Albumin endocytosis by SBAD3 iPSC-derived podocyte-like cells on day 10 of differentiation.
Cells were grown and differentiated in 12-well plates and incubated with FITC-albumin for 1 h at 4 °C and 37 °C and for 24 h at 37 °C. Representative images of two independent experiments are shown.
Fig 8.
Effect of doxorubicin exposure on the viability and morphology of iPSC derived podocytes compared to a podocyte cell line.
SBAD3, SBNEO and SFC018 iPSC-derived podocyte-like cells and human conditionally immortalized podocytes were incubated for 48 h in 6-well plates. Cell viability was assessed by resazurin assay (A). Results are expressed as means ± SEM, *P<0.05; **P<0.01. Phase contrast micrographs (B). Original magnification: iPSC-derived podocytes 50 X and the cell line 10 X.