UNC-CH Department of Cell and Molecular Physiology -

Figure 1. Identification of SP cells from neonatal mouse jejunum. Hoechst 33342 staining of isolated jejunal mucosa was analyzed by flow cytometry. The SP fraction was analyzed further by sorting with FITC-labeled CD45 antibodies to distinguish cells of hematopoietic origin. From Dekaney et al. (2005)

In recent years our group has pursued two areas of research. The goal of the first was to understand the hormonal factors that control maturation of the epithelium of the small intestine. This is a clinically relevant problem because immaturity of the intestine accounts for the high susceptibility of infants to diarrhea and for feeding intolerance in preterm infants. Using rodent models, we showed that there are two distinct regulatory pathways for intestinal maturation: a) a timing mechanism intrinsic to the intestinal tissue; and b) a pathway elicited by glucocorticoid hormones. Further studies have begun to elucidate the regulatory genes that mediate each pathway. Our data suggest that glucocorticoid stimulation of mature markers such as sucrase-isomaltase is secondary to rapid induction or activation of transcription factors. A microarray approach identified several other classes of immediate early genes of glucocorticoid action, and the functional significance of these is under investigation.

The primary goal of our current work is to identify and characterize intestinal stem cells. Although the presence of these cells deep in the crypts of Lieberkuhn has been inferred for many years, to date they have never been isolated. We have deployed a novel sorting approach in order to isolate a “side population” (SP) of putative intestinal stem cells (Fig 1). The CD45-negative SP cells are cytokeratin positive, confirming their epithelial origin. Initial gene expression studies by quantitative RT-PCR showed the CD45-negative SP fraction to be enriched for the stem cell marker Musashi-1. More recent microarray analyses showed a broad pattern of gene expression consistent with a progenitor phenotype. Moreover, in situ hybridization of 36 transcripts enriched in the CD45-negative SP and 12 transcripts de-enriched, confirmed that SP sorting selects cells from the lower crypt (Fig 2). We are currently exploring a graft model to assess the capacity of CD45-negative intestinal SP cells for proliferation and differentiation. In addition, refinement of the isolation procedure is being pursued based on membrane markers identified by microarray. We believe that ultimately the isolation and transplantation techniques developed in this project should have two applications: a) new therapies for various conditions in which the bowel is damaged; and b) use of the intestine as a site for gene therapy.