Figure 3.

Organoid forming progenitor cells are enriched in the Wnt^low epithelial population. A, Schematic of organoid assay experimental setup. B, Organoid forming efficiency (Organoid count/ Number of seeded cells × 100, %) of sorted Wnt^neg/low/high fractions in the organoid assay. Data are shown as mean ± SEM (N = 20). Statistics were performed using the mean of triplicate or quadruplicate wells. **P < .01, one-way ANOVA with Bonferroni post-test. C, Representative IF images of alveolar (white arrows), bronchiolar (red arrow), and bronchioalveolar (pink arrow) organoids derived from the Wnt^low cells labeled by IF for SFTPC (white), ACT (red), GFP (green), and DAPI (blue). Scale bars = 50 μm. D, Quantification of each type of organoids formed by Wnt^low cells. Data are shown as mean ± SEM (N = 3). E,F, Pathways enriched in the Wnt^lowcells related to, E, epithelial progenitor and, F, stem cell functions. Plotted values are the -Log₁₀FDR values. Data shown on the right end of each bar: the numbers of significantly changed genes (both Wnt^low vs Wnt^neg and Wnt^low vs Wnt^high)/total number of genes in each pathway. ACT, acetylated α-tubulin; ANOVA, analysis of variance; DAPI, 4⁰,6-diamidino-2-phenylindole; FDR, false discovery rate; GFP, green fluorescent protein; IF, immunofluorescence