Fig. 4: Generating human UB and CD organoids from primary human UPCs and dual-reporter human pluripotent stem cells.

a Schematic of the purification of primary human UPCs from the nephrogenic zone (illustrated as boxed region) of the human fetal kidney (9–13 weeks of gestational age) and the derivation of human UB organoid. b Immunostaining of the human fetal kidney nephrogenic zone for UB tip marker RET (red), broad UB lineage marker KRT8 (green), and NPC marker SIX2 (cyan). Scale bars, 50 µm. c Time course bright field images showing the growth of human UB organoid derived from primary human UPCs in a typical passage cycle at Days 1 (D1), 5 (D5), and 9 (D9). Scale bar, 200 µm. All images in (c) have been scaled to share the same scale bar with the D9 image. d qRT-PCR analyses of human UB organoid (cultured for 54 days) derived from primary human UPCs for various UB markers as indicated. Human fetal kidney from 11.2-week (11.2 weeks) gestational age was used as control. e Schematic of the stepwise differentiation from WNT11-GFP/PAX2-mCherry dual-reporter hESC line into iUB and iCD organoid. (TeSR mTeSR1 medium, Y Y27632, ME mesendoderm stage medium, UB-I UB Stage I medium, UB-II UB Stage II medium). f qRT-PCR analyses of the FACS purified mCherry⁺ cells (orange) and the iUB organoid (blue, cultured for 50 days) for various UB markers as indicated. Undifferentiated H1 hESCs (gray) and human fetal kidney (green, 11.2-week gestational age) were used as controls. g Whole-mount immunostaining of the expandable iUB organoid for UB markers SOX9 and CDH1. Scale bars, 50 µm. h Bright field (BF) and PAX2-mCherry (PAX2-mCh) images of expandable iUB organoid (left panels) and mature iCD organoid (right panels). Scale bars, 200 µm. i qRT-PCR analyses of the iUB organoid (orange, cultured for 49 days) and iUB-derived iCD organoid (blue), for UB (WNT11, ETV5), PC (AQP2, AQP4), and IC markers (FOXI1). j Summary of human UB organoid derivation from different sources and their expansion in vitro. ***, this is the culture time we achieved before our lab shutdown due to coronavirus outbreak. The maximum organoid culture time and expansion could be much greater. All data are presented as mean ± s.d. In d, f, i, the significance was determined by two-tailed unpaired Student’s t tests; NS not significant; n = 3. Source data are provided as a Source Data file.