Human fetal kidney organoids enriched for Notch dependent early epithelial differentiation

The gold standard source for kidney development research is the fetal kidney. Transgenic mice have been utilized as a robust model for congenital abnormalities of the kidney and urinary tract, however, they do not precisely mimic human fetal kidney (hFK) development. In recent years, pluripotent stem cell derived-kidney organoids have shown great potential which is hindered by unspecific differentiation and incomplete maturation. In contrast, three-dimensional culture methods of hFK have been hardly reported. Utilizing a chemically defined serum free media we generated hFK organoids (hFKOs) expressing key markers of early nephron epithelium, differentiating renal epithelium, mature tubular markers and self-organize into polarized renal epithelium similarly to the native tissue. In addition, bulk RNA sequencing of hFKOs disclosed cellular lineages of early nephron epithelium at the renal vesical, comma and s-shape stage, at levels that far exceed those in iPSC-derived kidney organoids and adult kidney tubuloids. Furthermore, single-cell RNA sequencing (scRNA-seq) and pseudotime analysis present diverse populations in hFKOs with a preserved differentiation axis, spanning nephron progenitors, glomeruli, developing and mature nephron segments. Importantly, hFKOs were highly enriched for markers of the NOTCH signaling pathway. Inhibition of NOTCH dampened hFKO growth and downregulated expression of LHX1 & JAG1, hindering proper differentiation of proximal tubules. scRNA-seq reveals diminished proximal tubule population post NOTCH inhibition and an increase in the nephron progenitor population. Our results suggest that NOTCH is crucial for proximal-distal segmentation allowing proper proximal tubule differentiation. Thus, hFKOs can be utilized for modelling NOTCH pathologies such as Alagille syndrome.