Figure 1.

Consistent differentiation of ileum- and colon-derived ASC organoids

(A) Schematic for time courses. Cultures were maintained for 10 days in total, switching to differentiation medium on day 3. Samples were collected on days 2, 4, 7, and 10. Day-2 cultures, maintained only in proliferation medium, were used as a baseline for gene expression in subsequent analysis.

(B) Stem/proliferative, secretory, and absorptive cell markers used to assess differentiation.

(C) Assessing differentiation for each culture and donor. A ratio of gene expression (qPCR analysis) on day 4, 7, or 10 to gene expression on day 2 was calculated for each marker. Data were visualized as volcano plots for each culture, representing p value versus fold change in gene expression. Both ileum and colon cultures were present for all donors except for donor 4 (ileum only) and donor 5 (colon only). Each individual culture was analyzed using 2–3 independent time courses (details outlined in experimental procedures); each time course included three cell-culture (technical) replicates. Gray vertical lines represent fold change of 1. Circles represent ileum cultures and diamonds represent colon cultures. Each symbol represents a marker for one sample (distinct replicate and time point). Colors represent each class of cell types (stem/proliferative, secretory, or absorptive) assessed.