Identification of intestinal epithelial stem cell populations by differential immunofluorescence

Abstract

Distinct levels of the transcription factor, SOX9, differentially mark the progenitor and stem cell lineages of the intestinal epithelium. Although immunostaining can be used to detect SOX9 in tissue sections, there is not an established method for quantifying differential fluorescence intensities in individual cells. Our hypothesis was that SOX9 immunofluorescence intensity could be correlated to Sox9 transcriptional activity at the cellular level. Using a Sox9EGFP mouse model as a proxy for Sox9 transcriptional activity, confocal image analysis and NIH ImageJ software were used to measure the average fluorescence intensity values of EGFP and SOX9 for individual colonic crypt cells. Our results show that SOX9 immunofluorescence was proportional to fluorescence of the EGFP reporter, and that higher levels of Sox9EGFP and SOX9 were expressed in the crypt base, consistent with the proposed location of the colonic epithelial stem cells. This validated method was extended to human colorectal biopsies where heterogeneous expression of Sox9 was observed, suggesting phenotypic differences between tumor cell populations. This semi-quantitative imaging method could be used to analyze differential gene expression in individual cells, and potentially characterize tumor cell stemness based on expression levels of candidate biomarkers. Research was supported by the NIH, UNC GI Cancer SPORE, and the APS UGSRF.