Maintenance of functional stem cells in isolated and cultured adult intestinal epithelium

We have previously described a method far the primary culture of adult larg e intestinal epithelium, suggesting that stem cells had survived both the i solation and the culture procedures. However, as no markers for such cells exist, cofirmation of stem cell survival is difficult- only the functional properties can be used to define them. Unfortunately, many of these (e,g., differentiation, crypt regeneration) do not occur in culture, probably due to suboptimal conditions. To address this problem both freshly isolated and cultured small and large intestinal crypts were grown subcutaneously in an immunocompromized mouse. All initially formed cysts lined by a simple epit helium which gradually became multicellular and formed invaginations contai ning many mitoses and apoptoses. Epithelial differentiation, as assayed by Goblet cell mucin production, was also apparent. Mucin maturation was also typical of the normal intestine. The lumen was frequently filled with mucin and apoptotic bodies. Interestingly, in grafts displaying pronounced crypt -like morphology the regions of proliferation were situated toward the base of the structure and the Goblet cells toward the lumen, i.e., a typical cr ypt-like morphology. Hence, functional adult stem cells appear to survive i solation and tissue culture, permitting organotypic regeneration, possibly involving homeobox gene expression. This may now allow direct stem cell cha racterization and experimental manipulation, such as transfection, and may ultimately permit transplantation and therapeutic gene therapy. (C) 1999 Ac ademic Press.