Nat Protoc. 2025 Aug 29. doi: 10.1038/s41596-025-01236-7. Online ahead of print.
ABSTRACT
Nephron progenitor cells (NPCs) have a central role in kidney organogenesis: they self-renew and differentiate into nephrons, the functional units of the kidney. Human pluripotent stem cells (hPSCs) can transiently produce induced nephron progenitor-like cells (iNPCs), which then differentiate into nephron organoids. Here, we describe a protocol to purify and expand the hPSC-derived iNPCs in a regular monolayer culture format with an optimized iNPC culture medium. Under this culture condition, iNPCs are programmed to a state with their transcriptome much closer to primary human NPCs than the transient hPSC-derived iNPCs. By following this protocol, iNPC lines can be derived from any hPSC lines, exhibiting a stable cell proliferation rate and retaining NPC marker gene expression over long-term culture. We also describe a protocol to generate nephron organoids from the iNPC lines. These iNPC-derived nephron organoids show minimal off-target cell types compared to hPSC-derived kidney organoids, with enhanced podocyte maturity. This protocol consists of a modified 10-d protocol to generate iNPCs from hPSCs, an iNPC expansion phase with a unique chemically defined iNPC expansion medium called 'hNPSR-v2' and a stepwise 21-d differentiation protocol to generate nephron organoids from iNPCs on an air-liquid interface. Experience in culturing and differentiating hPSCs is required to conduct this protocol, which can be executed within 1.5-2 months.
PMID:40883437 | DOI:10.1038/s41596-025-01236-7