HUMAN FETAL KIDNEY ORGANOIDS MODEL EARLY NEPHROGENESIS AND NOTCH-DRIVEN CELL FATE [scRNA-Seq 2]

Pluripotent stem cell–derived kidney organoids (PSC-KOs) are widely used to model human kidney development and disease. However, organoids from human fetal kidney tissue (hFKOs), which could benchmark PSC-KOs, remain underdeveloped. Here, using a chemically defined serum-free medium, we established a prolonged culture protocol for hFKOs that self-organize into polarized renal epithelium, reinitiate from NCAM1+ progenitors, and recapitulate nephrogenic and ureteric bud lineages. Bulk transcriptomics, single-cell RNA sequencing, pseudotime analysis, and immunostaining revealed diverse cell populations, with a preserved epithelial progenitor pool and tubular differentiation axis—both more robust than in PSC-KOs. hFKOs were enriched for NOTCH signaling genes, enabling single-cell analysis of pharmacological NOTCH inhibition. This revealed a maturation block with increased nephron progenitors and a bias toward distal over early proximal tubule fates. We also identified a novel prominin-1–expressing cell state that evades NOTCH inhibition to generate both proximal and distal tubules. Overall, hFKOs provide a developmentally faithful model and offer critical insights into kidney morphogenesis, advancing the fields of stem cell biology and regenerative medicine. Overall design: Two samples of human fetal kidney organoids (hFKOs) at P0 after 2-3 weeks of culture, one hFKO sample is treated with the ?-secretase inhibitor DAPT the other hFKOs sample is treated with DMSO, serves as a control. hFKOs were derived from freshly dissociated week 20 human fetal kidney. NOTCH inhibition in hFKOs: After 3 days of initial culture, DAPT, reconstituted in DMSO, was added to the culture medium at 5 uM final concentration; this medium was refreshed every 2 days. hFKOs were grown for 2–3 weeks under NOTCH inhibition next to control wells in which the culture medium was supplemented with DMSO only.