Abstract

The ability to differentiate human pluripotent stem cells (hPSC) into kidney organoids provides a next generation cell-based assay and platforms for basic research studies, patient-specific disease modeling and nephrotoxic drug screening. We developed the STEMdiff™ Kidney Organoid Kit, containing specialized serum-free media to enable efficient and reproducible differentiation across multiple human embryonic and induced pluripotent stem cell lines (H1, H9, WLS-1C and STiPS-M001), previously maintained in mTeSR™1. Cells were seeded into Corning® Matrigel® coated 96-well plates. After 24 hours, adherent cells were overlaid with an additional layer of Corning® Matrigel®, which resulted in the formation of cavitated hPSC spheroids within the next 48 hours. On the following day, differentiation of cavitated hPSC spheroids was initiated by switching media from mTeSR™1 to the STEMdiff™ Kidney Organoid Kit. During the next 18 days of differentiation, cells were directed through stages of late primitive streak, posterior intermediate mesoderm, and metanephric mesoderm to give rise to kidney organoids that are composed of podocytes, proximal and distal tubules. All tested hPSC lines were highly efficient in generating self-organizing kidney organoids that form convoluted tubular structures with typical nephron-like segmentation. Kidney organoids reliably establish with efficiencies of 98.7 ± 4.1 (mean ± SD; n = 1 - 3 per cell line) organoids per cm2. Organoids, analyzed on day 18, express markers of podocytes (PODXL, NPHS1), proximal (LTL, CUBN) and distal tubules (CDH1, GATA3) (n = 8). In summary, STEMdiff™ Kidney Organoid Kit supports highly efficient and robust differentiation of hPSCs into kidney organoids in high-throughput-compatible microwell plates, which can be used for nephrotoxic compound screening.