Keratinocyte stem cells are best identified by their ability to retain a BrdU label, and we have successfully FACS-sorted pure populations of these label-retaining cells (LRC). Unfortunately, the detection of BrdU requires that cells be fixed and permeablized, making them unsuitable for biological studies requiring living cells. In order to obtain living cells for biological studies, we have tried to identify unique cell surface markers on keratinocyte LRC, using both microarray analysis of global gene expression and proteomic mass spectrometry analysis of membrane proteins. For microarray analysis, we have developed """"""""navigated"""""""" laser capture microdissection to isolate enriched populations of LRC in the human hair follicle and have identified panels of genes, including genes that encode membrane proteins, that are differentially expressed in the LRC population of the human hair follicle bulge area (CD200, Frizzled1 receptor, and follistatin) as compared to non-LRC keratinocytes (CD24, CD34, CD71, CD146). In collaboration with the Biomedical Proteomics Program at FCRF, we have developed high-throughput mass spectrometry methods able to identify membrane proteins on LRC, and we are now using semi-quantitative mass spectrometry to compare the relative levels of the plasma membrane proteins prepared from LRC keratinocytes to control keratinocyte populations (transit amplifying basal keratinocytes). We have also assessed stem cell behavior in other putative KSC populations including side population or SP cells. These cells can be identified by unique fluorescent emission characteristics due to their ability to exclude HO33342 nuclear dye. We have recently described a SP population of keratinocytes and have completed characterization of SP keratinocytes long-term repopulating ability, using our recently described in vivo competitive repopulation stem cell assay.
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