Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP pathway activation in human colon organoids
Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide a unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using organoids from human healthy tissue. Culture of normal patient-derived colon organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the g-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 and DBZ showing only weak signals of cell differentiation. We also investigated the effects of 1a,25-dihydroxyvitamin D3 (calcitriol) on differentiation. Calcitriol attenuated the effects of the BMP4 and DBZ on normal colon organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features. Overall design: We have performed global transcriptomic analyses (RNA-seq) in colon normal organoids derived from 6 patients that were incubated in proliferation medium (PROL) or differentiation medium (B+D) containing Bone Morphogenetic Protein 4 (BMP4, 20 ng/mL) and the Notch inhibitor dibenzazepine (DBZ, 2.5 µM) for 48 h. Additionally, organoids cultured in B+D medium were treated with calcitriol (1,25(OH)2D3, 100 nM) or vehicle (ethanol) to investigate the effect of vitamin D on the differentiation of colon normal organoids.