Isolation and characterization of quiescent intestinal stem cells

Critical to the identification of putative stem cell populations is the ability to evaluate their functional potential. Within this work I describe the development of a robust culture system which enables regeneration of the intestinal epithelium from isolated crypts and single intestinal stem cells. To discriminate putative quiescent intestinal stem cells (ISCs) from rapidly cycling Lgr5+ ISCs a tetracycline inducible H2BGFP labeling strategy was employed. Distinct populations of label and non-label retaining cells were separated via fluorescent activated cell sorting (FACS). Analysis of stem cell potential, using the intestinal in vitro culture system developed herein, revealed that CD166high label retaining cells (LRCs) and non-LRCs contain functional intestinal stem cells (ISCs). Gene expression analysis of these populations confirmed the expression of stem cell genes including Lgr5. Coupled with the identification of long term BrdU retaining Lgr5+ cells, this work demonstrates the existence of both active and quiescent Lgr5+ ISCs.