Isolation and characterization of a putative intestinal stem cell fraction from mouse jejunum.

BACKGROUND & AIMS: Although there have been many recent advances regarding the biology of intestinal stem cells, the field has been hampered significantly by the lack of a method to isolate these cells. Therefore, the aim of this study was to explore the hypothesis that viable intestinal stem cells can be isolated as a side population (SP) by fluorescence-activated cell sorting after staining with the DNA-binding dye Hoechst 33342. METHODS: Preparations of individual cells from either whole mucosa or epithelium of mouse jejunum were stained with Hoechst 33342 and propidium iodide and then sorted using fluorescence-activated cell sorting. Cells were characterized using fluorochrome-labeled antibodies to surface markers, intracellular markers, and annexin V to detect early apoptosis. Total RNA was isolated from sorted fractions and used for quantitative real-time reverse-transcription polymerase chain reaction to evaluate the expression of cell lineage markers and the intestinal stem-cell marker, Musashi-1. RESULTS: Adult and neonatal jejunum contain a viable population of cells that shows the SP phenotype and is sensitive to verapamil. This population of cells (from both mucosal and epithelial preparations) includes a CD45-negative fraction corresponding to nonhematopoietic cells, which shows minimal expression of surface markers typically found on stem cells from other tissues and of intracellular markers found in mesenchymal cells. Additionally, these cells were enriched for Musashi-1 and beta1-integrin, were cytokeratin positive, and survived in culture for up to 14 days. CONCLUSIONS: The CD45-negative SP fraction, although not pure, represents the successful isolation of a viable population significantly enriched in small intestinal epithelial stem cells.

Development of the fetal intestine in mice lacking the glucocorticoid receptor (GR).

During rodent development there are two surges of circulating corticosterone: one just prior to birth and then one in the third postnatal week. Prior studies have shown that the latter controls the rate of intestinal development in the postnatal period. To date, a role for the earlier surge in the prenatal phase of intestinal development has not been investigated. We hypothesized that the late fetal surge of circulating corticosterone is involved in both morphologic and functional maturation of the intestinal epithelium, and thus that such maturation would be delayed if glucocorticoid action was abrogated. The hypothesis was tested by studying intestinal development in mice lacking a functional glucocorticoid receptor (GR). After GR+/- mice were bred onto a C57Bl/6 background, heterozygote matings yielded the expected ratios of -/-, +/-, and +/+ offspring. Analysis of GR mRNA in intestines of +/+ and -/- fetuses confirmed expression in wild-type mice but not in the GR-null mice. Intestinal histology of GR+/+ and -/- littermates at E13.5, E15.5, and E18.5 showed no effect of GR genotype on morphologic development. Further studies at E18.5 showed that GR-/- mice have normal functional maturation of the intestinal epithelium as assessed by: lactase activity in the enterocyte lineage, normal numbers of goblet and enteroendocrine cells, and normal numbers of proliferating cells in the intestinal crypts. Neither the minerolocorticoid receptor (MR) nor the pregnane X receptor (PXR) showed compensatory up-regulation in GR-/- mice. We conclude that, in contrast to our original hypothesis, the rodent intestine passes through a phase of glucocorticoid independence (late fetal) prior to becoming responsive to glucocorticoids in the postnatal period. These findings have implications for the clinical use of corticosteroids to enhance intestinal maturation in preterm infants.