Differentiation of human iPSCs into functional podocytes.

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.

Towards optimisation of induced pluripotent cell culture: Extracellular acidification results in growth arrest of iPSC prior to nutrient exhaustion.

Human induced pluripotent stem cells (iPSC) have the potential to radically reduce the number of animals used in both toxicological science and disease elucidation. One initial obstacle culturing iPSC is that they require daily medium exchange. This study attempts to clarify why and propose some practical solutions. Two iPSC lineages were fed at different intervals in a full growth area (FGA) or a restricted growth area (RGA). The FGA consisted of a well coated with Matrigel™ and the RGA consisted of a coated coverslip placed in a well. Glucose, lactate, extracellular pH and cell cycle phases were quantified. Without daily feeding, FGA cultured iPSC had significantly reduced growth rates by day 2 and began to die by day 3. In contrast, RGA cultured cells grew to confluence over 3days. Surprisingly, glucose was not exhausted under any condition. However, extracellular pH reached 6.8 after 72h in FGA cultures. Artificially reducing medium pH to 6.8 also inhibited glycolysis and initiated an increase in G0/G1 phase of the cell cycle, while adding an additional 10mM bicarbonate to the medium increased glycolysis rates. This study demonstrates that iPSC are highly sensitive to extracellular acidification, a likely limiting factor in maintenance of proliferative and pluripotent status. Culturing iPSC in RGA prevents rapid extracellular acidification, while still maintaining pluripotency and allowing longer feeding cycles.

Activated platelets release sphingosine 1-phosphate and induce hypersensitivity to noxious heat stimuli in vivo.

At the site of injury activated platelets release various mediators, one of which is sphingosine 1-phosphate (S1P). It was the aim of this study to explore whether activated human platelets had a pronociceptive effect in an in vivo mouse model and whether this effect was based on the release of S1P and subsequent activation of neuronal S1P receptors 1 or 3. Human platelets were prepared in different concentrations (10(5)/µl, 10(6)/µl, 10(7)/µl) and assessed in mice with different genetic backgrounds (WT, S1P1 (fl/fl), SNS-S1P1 (-/-), S1P3 (-/-)). Intracutaneous injections of activated human platelets induced a significant, dose-dependent hypersensitivity to noxious thermal stimulation. The degree of heat hypersensitivity correlated with the platelet concentration as well as the platelet S1P content and the amount of S1P released upon platelet activation as measured with LC MS/MS. Despite the significant correlations between S1P and platelet count, no difference in paw withdrawal latency (PWL) was observed in mice with a global null mutation of the S1P3 receptor or a conditional deletion of the S1P1 receptor in nociceptive primary afferents. Furthermore, neutralization of S1P with a selective anti-S1P antibody did not abolish platelet induced heat hypersensitivity. Our results suggest that activated platelets release S1P and induce heat hypersensitivity in vivo. However, the platelet induced heat hypersensitivity was caused by mediators other than S1P.

Alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media.

The search for alternatives to the use of fetal bovine serum (FBS) in cell and tissue culture media has become a major goal in terms of the 3R principles in order to reduce or to avoid harvesting of FBS from bovine fetuses, and, in terms of Good Manufacturing Practice (GMP), to ensure safe and animal product-free conditions for biomedical tissue engineering and (adult) stem cell therapy, respectively. In the present study, we investigated the feasibility of using platelet lysates (PL) as a substitute for FBS, based on the fact that most of the potent mitogenic factors present in serum are derived from activated thrombocytes. Platelet lysates were obtained from outdated human donor platelet concentrates. Methods were established to activate human donor platelets in order to achieve a maximum yield of platelet a-granule growth factors. Platelet lysates were successfully introduced to grow and maintain anchorage-dependent and -independent human and animal cell lines. For cell culture experiments, cells were either grown in culture media supplemented with 10% FBS, 5% PL, or under serum-free conditions. Growth experiments, viability assays, and platelet lysate-induced activation of ERK1/2 mitogen-activated protein kinase revealed platelet lysates as a valuable alternative to FBS in cell culture media.