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Podocytes play a critical role in glomerular barrier function, both in health and disease. However, in vivo terminally differentiated podocytes are difficult to be maintained in in vitro culture. Induced pluripotent stem cells (iPSCs) offer the unique possibility for directed differentiation into mature podocytes. The current differentiation protocol to generate iPSC-derived podocyte-like cells provides a robust and reproducible method to obtain podocyte-like cells after 10 days that can be employed in in vitro research and biomedical engineering. Previous published protocols were improved by testing varying differentiation media, growth factors, seeding densities, and time course conditions. Modifications were made to optimize and simplify the one-step differentiation procedure. In contrast to earlier protocols, adherent cells for differentiation were used, the use of fetal bovine serum (FBS) was reduced to a minimum, and thus ß-mercaptoethanol could be omitted. The plating densities of iPSC stocks as well as the seeding densities for differentiation cultures turned out to be a crucial parameter for differentiation results. Conditionally immortalized human podocytes served as reference controls. iPSC-derived podocyte-like cells showed a typical podocyte-specific morphology and distinct expression of podocyte markers synaptopodin, podocin, nephrin and WT-1 after 10 days of differentiation as assessed by immunofluorescence staining or Western blot analysis. qPCR results showed a downregulation of pluripotency markers Oct4 and Sox-2 and a 9-fold upregulation of the podocyte marker synaptopodin during the time course of differentiation. Cultured podocytes exhibited endocytotic uptake of albumin. In toxicological assays, matured podocytes clearly responded to doxorubicin (Adriamycin™) with morphological alterations and a reduction in cell viability after 48 h of incubation.

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The search for alternatives to fetal bovine serum (FBS) has become a major goal in the field of cell and tissue culture research. Although the supplementation of culture media with FBS is routine practice, FBS bears a number of disadvantages: unknown composition, high lot-to-lot variablity, ethical concerns about the harvest from bovine fetuses, and possible shortage in global supply. Several strategies have been developed to reduce or replace FBS in cell culture media (Bjare 1992;Even et al. 2006;Gstraunthaler 2003;van der Valk et al. 2004van der Valk et al. , 2010). Here we report on the use of human platelet lysates (PL) as a serum replacement (Alden et al. 2007;Bernardo et al. 2007;Bieback et al. 2009;Doucet et al. 2005;Johansson et al. 2003;Kocaoemer et al. 2007;Müller et al. 2009; Schallmoser et al. 2009). PL in DMEM support growth, proliferation and differentiation, as assessed by dome formation, of proximal tubule-like LLC-PK 1 (porcine kidney) and HK-2 (human kidney) cells, as well as PL-supplemented DMEM/Ham F-12 for distal tubule-like MDCK (dog kidney) cells. In addition to adherent epithelial cell lines, anchorage-independent Raji human lymphoma cells were investigated. PL fully supported growth and proliferation of Raji cells in RPMI-1640 medium in suspension. In order to biochemically determine the proliferative potential of PL, the stimulation of extracellular signal-regulated MAP kinase (ERK1/2) was determined. Addition of PL to quiescent LLC-PK 1 cultures resulted in specific phosphorylation, and thus activation, of ERK1/2 within minutes. The time course is identical with ERK1/2 activation upon addition of FBS. The data show the high potential of PL as a valuable substitute for FBS in mammalian cell and tissue culture.