Fig. 4

Cell differentiation analysis. a–d Gene expression analysis of lineage specific differentiation marker genes in unstained (DMSO) and CMFDA-stained cells following 3 days of differentiation. Expression levels in cells cultured in standard culture medium are similarly depicted. Data are presented as Ct-values of target genes normalized to the housekeeping gene (TBP). a CAII (enterocytes), b MUC2 (goblet cells), c LGR5 (intestinal stem cells), and d CHGA (enteroendocrine cells). A significant reduction in the expression of LGR5 (*) in CMFDA-stained cells was detected following differentiation. No statistically significant (ns) difference was observed in the expression levels CA II or MUC2. The expression of CHGA could be detected in a few samples only (both stained and unstained) but indicated increased expression levels following differentiation. e Staining with hematoxylin-eosin and cytokeratin 20 (CK20) of unstained and CMFDA-labeled organoids before and after differentiation, confirming presence of absorptive colonocytes along with secretory goblet cells in both conditions. Cell nuclei are spherical and basally located, consistent with more differentiation phenotype. Strong reaction for CK20 after differentiation. Hence, no signs that the differentiation capacity is affected by CMFDA. Black scale bar, 100 μm