Fig. 5: Generating human iUB and iCD organoids from human pluripotent stem cells independent of reporters. | Nature Communications

Fig. 5: Generating human iUB and iCD organoids from human pluripotent stem cells independent of reporters.

From: Generation of patterned kidney organoids that recapitulate the adult kidney collecting duct system from expandable ureteric bud progenitors

Fig. 5

a Schematic of the stepwise differentiation from any hPSC line into iUB and iCD organoid without using reporter. (TeSR mTeSR1 medium, CR CloneR, ME (v2) mesendoderm stage medium version 2, UB-I UB Stage I medium, UB-II UB Stage II medium). b–h Characterizations of iUB or iCD organoids derived from the WNT11-GFP/PAX2-mCherry hESC line independent of its reporters. b qRT-PCR analyses of the FACS purified KIT+ precursor (orange), KIT+ precursor-derived iUB organoids cultured for 33 (D33, gray), 49 (D49, yellow), and 66 days (D66, light blue) for various UB markers as indicated. Undifferentiated H1 hESCs (dark blue) and human fetal kidney (green, 11.2-week gestational age) were used as controls. c–e Whole-mount immunostaining of the expandable iUB organoid for various UB markers as indicated. The four panels on the right represent the boxed region in the left panel. Scale bars, left panel, 100 µm; right four panels, 40 µm. f Quantification of percentages of iUB cells stained positive for different UB markers in Fig. 5c–e. Each column represents counts from three different fields of view (n = 3). g qRT-PCR analyses of the iUB organoid (blue) and iUB-derived iCD organoid (orange), for UB (WNT11, RET), PC (AQP2, AQP3, AQP4), and IC markers (FOXI1). h Whole-mount immunostaining of the iUB-derived iCD organoid for PC marker AQP3 and broad CD marker CDH1. Scale bars, 50 µm. i Flow cytometry analysis of KIT+ precursor cells differentiated from wild-type H1 hESC. j Bright field image of a typical iUB derived from wild-type H1 hESC. Scale bar, 250 µm. k qRT-PCR analyses of the iUB organoids derived from the dual-reporter hESC line (W/P reporter, orange, cultured for 33 days) or wild-type H1 hESC line (H1, gray, cultured for 30 days) for various UB markers. Undifferentiated H1 hESCs (blue) and human fetal kidney (yellow, 11.2-week gestational age) were used as controls. All data are presented as mean ± s.d. In b, g, k, the significance was determined by two-tailed unpaired Student’s t tests; n = 3. Source data are provided as a Source Data file.

Back to article page