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. 2022 Oct 8;13:5943. doi: 10.1038/s41467-022-33623-z

Fig. 1. Extended monolayer culture in CDBLY supports nephron progenitors and preserves nephrogenic capacity.

Fig. 1

a Schematic depicting the extended differentiation protocol in CDBLY2. b Brightfield and confocal immunofluorescence images of extended monolayer differentiations in E6, CDBLY and NPSR, and resulting organoids. Immunofluorescence depicts nephrons (EPCAM; green), podocytes of glomeruli (NPHS1; grey), and proximal tubules (LTL; blue). Scale bars represent 100 µm (monolayers) and 200 µm (organoids). c Brightfield images of extended monolayer differentiations using CDBLY variations and resulting organoids, with inset confocal immunofluorescence images highlighting organoid nephron alignment and patterning. Immunofluorescence depicts nephrons (EPCAM; green), podocytes of glomeruli (NPHS1; grey), proximal tubules (HNF4A; blue), and Loop of Henle (SLC12A1; red). Scale bars represent 100 µm (monolayer brightfields and organoid immunofluorescence) and 200 µm (organoid brightfields). d qRT-PCR analysis of standard and extended monolayer differentiations. Error bars represent SEM from n = 3 biological replicates. Statistical significance was determined using an unpaired t test. Asterisks represent two-tailed P values adjusted for multiple comparisons using the Holm–Sidak method, alpha = 0.05 (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 [SIX1: P = 0.002059; SIX2: P = 0.000075; WNT4: P = 0.017459; GATA3: P = 0.001246]). Source data are provided as a Source data file.