Abstract
The renal proximal tubule is a main target for drug-induced toxicity. The prediction of proximal tubular toxicity during drug development remains difficult. Any in vitro methods based on induced pluripotent stem cell-derived renal cells had not been developed, so far. Here, we developed a rapid 1-step protocol for the differentiation of human induced pluripotent stem cells (hiPSC) into proximal tubular-like cells. These proximal tubular-like cells had a purity of >90% after 8 days of differentiation and could be directly applied for compound screening. The nephrotoxicity prediction performance of the cells was determined by evaluating their responses to 30 compounds. The results were automatically determined using a machine learning algorithm called random forest. In this way, proximal tubular toxicity in humans could be predicted with 99.8% training accuracy and 87.0% test accuracy. Further, we studied the underlying mechanisms of injury and drug-induced cellular pathways in these hiPSC-derived renal cells, and the results were in agreement with human and animal data. Our methods will enable the development of personalized or disease-specific hiPSC-based renal in vitro models for compound screening and nephrotoxicity prediction.
Highlights
Primary cells stem cell-based approaches would be preferred
Differentiation of human induced pluripotent stem cells (hiPSC) into HPTC-like cells. iPS(Foreskin)-4 cells were differentiated by cultivating the cells in matrigel-coated multi-well plates with renal epithelial cell growth medium (REGM) supplemented with bone morphogenetic protein (BMP)[2] and BMP7 (Supplementary Fig. S1, for details see Methods)
OCT3/4, NANOG, SOX2 and DNMT3B were down-regulated after day (d) 1
Summary
Differentiation of hiPSC into HPTC-like cells. iPS(Foreskin)-4 cells were differentiated by cultivating the cells in matrigel-coated multi-well plates with renal epithelial cell growth medium (REGM) supplemented with bone morphogenetic protein (BMP)[2] and BMP7 (Supplementary Fig. S1, for details see Methods). In the kidney AQP1 and GGT expression is characteristic for proximal tubular epithelial cells[21,22] These results showed a profound change between d7 and d9, and on d9 the cells expressed high levels of all of the tested nephron progenitor and PTC markers. We addressed whether PTC-specific nephrotoxicity of drugs could be predicted with hiPSC-derived HPTC-like cells This question was addressed with the IL6/IL8-based assay[5,7] (for overall procedure see Supplementary Fig. S1). All results remained negative with respect to acarbose and ethylene glycol up to the highest concentration tested (1000 μ g/ml, Fig. 5 and Table 2) This was consistent with the fact that these compounds are not toxic for PTC in humans.
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