DERIVATION OF HUMAN FETAL KIDNEY ORGANOIDS MIMICKING NOTCH-DEPENDENT EPITHELIAL MATURATION AND TUBULE DEVELOPMENT [bulk RNA-Seq]

Pluripotent stem cell–derived kidney organoids (PSC-KOs) have been put forward as a preferred culture system in which to study human kidney development and disease. In contrast, the development of human fetal kidney tissue–derived organoids (hFKOs) that could serve as a benchmark for PSC-KOs has not advanced very far. Herein, utilizing a chemically defined serum-free medium, we established a long-term culture protocol for hFKOs that self-organize into polarized renal epithelium and faithfully recapitulate nephrogenic and ureteric bud cell lineages. Bulk transcriptomics, single-cell RNA sequencing, and pseudotime analysis along with immunostaining revealed diverse populations among hFKOs, with a preserved differentiation axis mostly at higher levels and to a greater extent than for PSC-KOs. Accordingly, compared to PSC-KOs, hFKOs were significantly enriched for the NOTCH signaling pathway genes, allowing single-cell analysis of changes following NOTCH inhibition. We observed a preferential increase of mature distal tubules at the expense of early proximal tubules and defined a new adaptive prominin 1–expressing (PROM1+, or CD133+) cell state that escapes NOTCH inhibition to generate proximal and distal tubules. Overall, the concomitant understanding of developmental processes using hFKOs is a key development for the fields of kidney stem cell biology and regenerative medicine. Overall design: Characterization of human fetal kidney organoids (hFKOs) at P0 and P5-P6 and iPSC-derived kidney organoids (iPSC-KOs) for comparison via bulk RNA sequencing. P0 (3 weeks cluture) of week 18 human fetal kidney, with 3 replicates. P5 and P6 (5 months of culture) of week 18 human fetal kidney, with 4 replicates. iPSC-derived kidney organoids day 10 and 18 of Freedman et al. differentiation protocol, WTC-11 cell line, 3 replicates.