Colonic epithelial stem cells are believed to be located at the crypt base where they have previously been shown to express musashi-1. The colonic stem cell niche, which includes extracellular matrix and myofibroblasts (together with other cell types), is likely to be important in maintaining the function of the progenitor cells. The aims of our studies were to characterize stem cells in isolated and disaggregated human colonic crypt epithelial cells and investigate their interactions with monolayers of primary human colonic myofibroblasts. In unfractionated preparations of disaggregated colonic crypts, musashi-1 positive cells preferentially adhered to colonic myofibroblasts, despite the presence of excess blocking anti-1 integrin antibody. These adherent epithelial cells remained viable for a number of days and developed slender processes. Cells with side population characteristics (as demonstrated by ability to expel the dye Hoechst 33342) were consistently seen in the isolated colonic crypt epithelial cells. These side population cells expressed musashi-1, 1 integrin, BerEP4 and CD133. Sorted side population crypt epithelial cells also rapidly adhered to primary colonic myofibroblasts. In conclusion, in preparation of isolated and disaggregated human colonic crypts, cells with stem cell characteristics preferentially adhere to primary human colonic myofibroblasts in a 1 integrin-independent fashion.