Figure 2.
Hpb-CM directly induces fetal reversion of intestinal organoids. (A) SI organoids were stimulated with ENR (control) or Hpb-CM medium for 24 h after 3 d in culture. (B) Frequency of organoids with a spheroid morphology. (C) qPCR analysis of SI organoids stimulated with Hpb-CM for 24 h from the plating of fresh crypts. (D) Confocal photomicrographs of SI organoids from CluGFP crypts (Ayyaz et al., 2019) stimulated with Hpb-CM for 24 h on day 3 of culture. (E) Volcano plot of differentially expressed genes from RNA-seq analysis comparing Hpb-CM–treated organoids to control organoids, false discovery rate < 0.05. For a complete list of differentially expressed genes, see Table S1. (F and G) GSEA of fetal-associated transcripts (F) and differentiated epithelial cell markers (G). NES, normalized enrichment score. (H–J) Representative photomicrographs (H) and qPCR analysis (I and J) of SI organoids stimulated with Nippo-CM for 24 h. (K–N) qPCR analysis of SI established organoids stimulated with Hpb-CM for 24 h in the presence or absence of ENR. Scale bar, 50 μm. Data shown are representative of two or more independent experiments, n = 3 biological replicates; statistical tests: t test (B, C, I, and J), two-way ANOVA (K–N); *, P < 0.05; **, P < 0.01; ***, P < 0.005.