Differentiation of Urine-derived Progenitor Cells into Podocytes

Urine-derived renal progenitor cells (UdRPCs) of a 51-year-old male (UM51) were isolated as described in Rahman et al. [24 (link)]. Further, the UM51 cell line was immortalized with an hTERT-expressing plasmid (UM51 hTERT) [28 (link)]. The cell lines were cultured on 0.2% type 1 collagen (Thermo Fisher Scientific, Waltham, MA, USA) coated 6- or 12-well plates at 37 °C under hypoxic conditions. The cells were cultured in Proliferation Medium (PM) composed of 50% DMEM high-glucose (Gibco) and 50% keratinocyte growth basal medium (Lonza, Basel, Switzeland) supplemented with 5% fetal bovine serum (Gibco), 0.5% Non-Essential Amino Acid (Gibco), 0.25% Glutamax (Gibco), and 0.5% penicillin and streptomycin (Gibco). For further differentiation into podocytes, the cells were seeded at a low density (50,000 cells per 6-well) and cultured for 24 h in PM. On the next day, the medium was exchanged to Advanced RPMI 1640 (Gibco) supplemented with 0.5% fetal bovine serum, 1% penicillin and streptomycin, and 30 μM retinoic acid (Sigma-Aldrich Chemistry, Steinheim, Germany). After 7 days, the typical podocyte morphology was observed. Losartan (Sigma-Aldrich Chemistry) and ANG II (Sigma-Aldrich Chemistry) were diluted using Advanced RPMI 1640 to a final concentration of 100 μM ANG II or 1 μM losartan. First, the cells were incubated for 24 h with 1 μM losartan and then for 24 h with ANG II.