Fig. 3: Generating engineered kidney from expandable NPCs and UBs and gene editing of the UB organoid.

a Schematic of the engineered kidney reconstruction and organotypic culture procedure. b Time course images (bright field and Hoxb7-Venus) showing the branching morphogenesis of the engineered kidney reconstructed at air–liquid interface at Days 2, 4, 5, and 7. Scale bars, 200 µm. c Immunostaining of the engineered kidney (Day 7) constructed from Wnt11-RFP UB organoid and wild-type NPCs for UB/CD marker KRT8, nephron marker PODXL and WT1 (podocytes) and LTL (proximal tubule). Note that both KRT8 and PODXL were stained green. The round structures that co-stain with WT1 are podocytes of the nephron. UB-derived structures do not co-stain with WT1. Scale bar, 100 µm. d Immunostaining of the engineered kidney constructed from Hoxb7-Venus UB organoid and wild-type NPC (Day 10) for GATA3 and CDH1. Scale bars, 50 µm. e Summary of engineered kidney generation experiments. f Schematic of gene overexpression and gene knockout procedures in the UB organoid. OE overexpression, KO knockout. g Fluorescence image of GFP overexpression (GFP OE) in wild-type UB organoid. Scale bar, 200 µm. h Knockout of GFP in Rosa26-Cas9/GFP UB organoid using multiplexed sgRNAs (“GFP KO,” right panels) targeting the coding sequence of GFP. Multiplexed non-targeting sgRNAs were introduced to the organoid as control (“Ctrl KO,” left panel). Note the gene-edited single cells self-organized into typical branching organoid morphology. Scale bars, 400 µm.