Intestinal Stem Cells - Biological Properties and Potential for Therapeutic Application
|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)|
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 both in vitro culture models and in vivo transplantation models 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.
|Figure 2. In situ hybridization of transcripts differentially expressed in the CD45–/SP cells. Hybridization patterns of representative transcripts that are enriched (A and B) and deenriched (C and D) in the CD45–/SP cell population relative to intact jejunum are shown. Transcripts enriched in the CD45–/SP cells, such as Notch1 ( 4-fold; A) and Fgfr3 ( 4-fold; B) localize to the crypt base/stem cell zone, while deenriched transcripts such as Mtf1 ( 4-fold; C) and Pcp4 ( 4-fold; D) localize outside of this zone. From Gulati et al. (2008)|