Characterization and isolation of stem cell enriched human hair follicle bulge cells

The human hair follicle bulge is an important niche for keratinocyte stem cells (KSC). Elucidation of human bulge cell biology could be facilitated by analysis of global gene expression profiles and identification of unique cell surface markers. The lack of distinctive bulge morphology in human hair follicles has hampered studies of bulge cells and KSC. In this study, we determined the distribution of label-retaining cells to carefully define the human anagen bulge. Using navigated-laser capture microdissection, bulge cells and outer root sheath cells from other follicle regions were obtained and analyzed with cDNA microarrays. Gene transcripts encoding inhibitors of WNT and Activin/BMP signaling were over-represented in the bulge while genes responsible for cell proliferation were under-represented, consistent with quiescent non-cycling KSC in anagen follicles. Positive markers for bulge cells included CD200, PHLDA1, follistatin, and frizzled homolog 1 while CD24, 34, 71 and 146 were preferentially expressed by non-bulge keratinocytes. Importantly, CD200+ cells (CD200hi24lo34lo71lo146lo) obtained from hair follicle suspensions demonstrated high colony forming efficiency in clonogenic assays, indicating successful enrichment of living human bulge stem cells. Keywords: Affymetrix micrarray analysis of human hair follicles In this study, we developed methods to precisely identify the anatomical location of the human bulge region, and then collected bulge ORS cells by navigated-LCM (N-LCM). The global gene expression profile of bulge ORS cells was determined by microarray analysis and compared to that of other defined ORS cell subpopulations to identify genes that were differentially expressed in the bulge area. By combining positive and negative selection markers identified on bulge cells by microarray analysis and confirmed by immunohistochemistry, we enriched living human bulge cells and assessed their biological behavior in an in vitro colony forming keratinocyte stem cell assay. This report describes the systematic transcriptional profiling of human bulge cells and the first demonstration of directed enrichment of putative human hair follicle stem cells.