Reporter‐based fate mapping in human kidney organoids confirms nephron lineage relationships and reveals synchronous nephron formation

Abstract

Nephron formation continues throughout kidney morphogenesis in both mice and humans. Lineage tracing studies in mice identified a self‐renewing Six2‐expressing nephron progenitor population able to give rise to the full complement of nephrons throughout kidney morphogenesis. To investigate the origin of nephrons within human pluripotent stem cell‐derived kidney organoids, we performed a similar fate‐mapping analysis of the SIX2‐expressing lineage in induced pluripotent stem cell (iPSC)‐derived kidney organoids to explore the feasibility of investigating lineage relationships in differentiating iPSCs in vitro. Using CRISPR/Cas9 gene‐edited lineage reporter lines, we show that SIX2‐expressing cells give rise to nephron epithelial cell types but not to presumptive ureteric epithelium. The use of an inducible (CreERT2) line revealed a declining capacity for SIX2+ cells to contribute to nephron formation over time, but retention of nephron‐forming capacity if provided an exogenous WNT signal. Hence, while human iPSC‐derived kidney tissue appears to maintain lineage relationships previously identified in developing mouse kidney, unlike the developing kidney in vivo, kidney organoids lack a nephron progenitor niche capable of both self‐renewal and ongoing nephrogenesis.

Kidney organoids: accurate models or fortunate accidents

“…Indeed, we have now definitively shown that the nephron epithelium arising in our kidney organoids comes from a SIX2-expressing nephron progenitor, as anticipated based upon our understanding of nephron formation in the mouse (Fig. 3OP; Howden et al 2019).…”

“…However, induction at day 18 resulted in the identification of a stromal SIX2-derived population but no evidence of ongoing nephrogenesis ( Fig. 3P; Howden et al 2019). Indeed, it is possible that there are distinct SIX2-expressing populations arising at this time that may represent off-target cell types, such a muscle progenitors.…”

“…As a result, loxP-Cre mediated excision of the EGFP cassette present within the GAPDH safe harbor locus will cause a switch in cell color from green to red (mCherry). Howden et al (2019). (P) High-resolution immunofluorescence of nephrons within kidney organoids subjected to early (day 7 + 10) or date (day 7 + 18) 4-OHT.…”

“…Glomeruli (NPHS1; white), proximal tubules (LTL; blue), nephron epithelium (EpCAM; green). Adapted from Howden et al (2019). Scale bars, 50 µM.…”

“…Cell types within kidney organoids were initially defined based on morphology and expression of established markers of mouse renal cell types by immunofluorescence. The application of single-cell profiling has enabled a deeper analysis of kidney organoid cell types, dramatically expanding the complement of markers used to identify organoid renal cell types and identifying varying proportions of off-target neural, glial, muscle progenitor, and melanocyte populations (Wu et al 2018;Combes et al 2019b;Harder et al 2019;Howden et al 2019;Subramanian et al 2019). Pseudotime analysis of organoid nephron cell types suggests that organoid nephron formation replicates the expected trajectory from nephron progenitor to podocyte and tubular endpoints (Combes et al 2019b).…”