Fig. 4. NZC-derived kidney organoid generation.

A) Schematic and timeline of NZC-derived organoid protocol. A heterogeneous population of nephrogenic zone cells (NZCs), including stromal cells, nephron progenitor cells (NPCs), endothelial cells (ECs), and ureteric bud (UB) tip cells, are isolated from embryonic mouse kidneys. NZCs may be expanded on monolayer for up to 2 passages to increase the organoid yield or proceed directly to the aggregation step. Organoids are aggregated on a filter floating on APEL2 media then treated with a 1 h CHIR pulse before culture with 200 ng/ml FGF9 for 5 days before being switched to growth-factor free media. Mouse illustration from http://www.emouseatlas.org B) Immunofluorescence of an E17.5 mouse kidney with E-Cad⁺ UB, Six2⁺ NPCs, and CD31/EMCN ⁺ ECs. The nephrogenic zone is demarcated with a yellow dotted line. B′) After pancreatin and collagenase A digestion, the NPCs, ECs, and stromal cells of the nephrogenic zone are no longer present. C-D) Immunofluorescence showing the NZCs after monolayer culture. C) Foxd1Cre; YFP + stromal cells, Six2⁺ NPCs and D) Hoxb7Cre; YFP ⁺ UB cells. E) Brightfield images of NZC organoids 2–7 days after aggregation. F-J) Immunofluorescence of various kidney cell types present in NZC organoids. F) Whole mount immunofluorescence of an NZC organoid with E-Cad⁺ epithelium. 80 μm scale. G-J) Immunofluorescence of sectioned NZC organoids, oriented with the air-liquid interface on top displaying Aqp1⁺ proximal tubule and descending loop of Henle and NPHS1⁺ podocytes (G), LTL⁺ proximal tubule and Umod⁺ loop of Henle (H), Meis 1/2/3⁺ stroma (I), and Aqp3⁺ and pan-cytokeratin⁺ UB (J). GH 100 μm scale. DAPI marks nuclei. Representative immunofluorescence images shown in A-C were derived from n ≥3 organoids from at least 2 different litters.