A CRISP(e)R view on kidney organoids allows generation of an induced pluripotent stem cell–derived kidney model for drug discovery

Development of physiologically relevant cellular modelswith strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular modelusing induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytescan be monitored in live cells due to CRISPR/ Cas9-mediated fluorescent taggingof kidney lineage markers(SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoidsgrown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature ( CD31) within renal cortical structures with microvilli, tight junctionsand podocytefoot processes visualized by electron microscopy. Additionally, the organoidsresemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation.