A CELL CULTURE MODEL FOR SMALL INTESTINAL STEM CELL FUNCTION AND DIFFERENTIATION

Abstract

Aim: The stem cells located in small intestinal crypts are responsible for the continuous renewal of the epithelium and thus have a crucial role in maintaining tissue homeostasis. Since at present there is no cell culture model for small bowel epithelial stem cell function and differentiation, our aim is to establish culture conditions that allow intestinal stem cell propagation and proliferation as well as differentiation into all four epithelial cell types present in the small bowel. Methods: Single crypt epithelial cells and small organoids were isolated from mouse small intestine and cultured under specific conditions on collagen with fibroblasts seeded within the gel as feeder layer. Cultures were observed daily and studied immunohistologically. Results: The isolated small intestinal epithelial cells form organized clusters that remained viable for at least 10 weeks. The clusters were characterized by a large central lumen and well-deposited basement membrane. A portion of the cells were proliferative as evinced by Ki-67 positivity. In addition the clusters also harboured polarized columnar cells that had obviously differentiated as indicated by alkaline phosphatase activity and mucin profile typical for small intestine. When these parental clusters were broken into single cells that were cultured under identical conditions they formed daughter clusters with similar characteristics as the parental ones. Summary: At this point we have cultured the clonogenic cells derived from mouse small bowel for 10 weeks - the longest period ever published. During this time the cells form clusters containing both differentiated cells and undifferentiated proliferating cells that can also be subcultured. Conclusion: We have developed a long-term cell culture system that models the in vivo small intestinal crypt villus axis in terms of stem cell proliferation and differentiation. If applicable to human, the development of this culture system will eventually allow the use of small intestinal stem cells in regenerative medicine as a source for transplantation in diseased patients to correct dysregulated or prolonged mucosal regeneration.