Visualization of Differentiated Cells in 3D and 2D Intestinal Organoid Cultures

The intestinal epithelium maintains self-renewal and differentiation capacities via coordination of key signaling pathways, including the Wnt, bone morphogenetic protein (BMP), epidermal growth factor (EGF), and Notch signaling pathways. Based on this understanding, a combination of stem cell niche factors, EGF, Noggin, and the Wnt agonist R-spondin was shown to enable the growth of mouse intestinal stem cells and the formation of organoids with indefinite self-renewal and full differentiation capacity. Two small-molecule inhibitors, including a p38 inhibitor and a TGF-beta inhibitor, were added to propagate cultured human intestinal epithelium but at the cost of differentiation capacity. There have been improvements in culture conditions to overcome these issues. Substitution of the EGF and a p38 inhibitor with insulin-like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2) enabled multilineage differentiation. Monolayer culture with mechanical flow to the apical epithelium promoted the formation of villus-like structures with mature enterocyte gene expression. Here, we summarize our recent technological improvements in human intestinal organoid culture that will deepen the understanding of intestinal homeostasis and diseases.