Assessment of Intestinal Stem Cell Survival Using the Microcolony Formation Assay

The microcolony assay originally described by Withers and Elkind in 1970 (1 ) has been a useful method for investigating the effects of radiation and various other genotoxic and cytotoxic damaging agents on the intestinal epithelial stem cell population and to assess the ability of a variety of compounds to protect the epithelial stem cell population from the lethal effects of chemical and physical agents (e.g., 2-7 ). Epithelial stem cells are located near the base of each intestinal crypt and play an important role in normal epithelial renewal and differentiation, epithelial injury-repair, and in neoplastic transformation (8 -11 ). In the adult mouse small intestine these functionally anchored clonogenic stem cells divide rarely to produce a daughter stem cell (self renewal) as well as a more rapidly replicating transit cell. Transit cells, in turn, undergo a number of rapid cell divisions in the proliferative zone located in the lower half of each crypt. Their progeny subsequently differentiate into the mature epithelial cell types found in the small intestine as they migrate away from the proliferative zone in each intestinal crypt (8 -11 ). Following intestinal injury and disruption of the epithelium, epithelial cells adjacent to the wound first migrate over the injured area to reestablish continuity of the epithelium. Stem cells subsequently proliferate to increase their numbers and to give rise to the more rapidly proliferating transit cell population. The transit cell population then expands rapidly to form a regenerative crypt. If the injury has completely destroyed some crypts, the surviving regenerative crypts can subsequently branch and divide to restore near normal numbers of viable crypts (3 ).