Characterization of the epithelial stem cell niche of the human hair follicle

Since the discovery of epithelial hair follicle (HF) stem cells in the bulge of human HFs, the definition of useful stem cell markers in situ has become a major challenge in skin research and regenerative medicine. In the current study, we contribute to this ongoing quest by critically evaluating published IR patterns of putative epithelial HF stem cells, and by attempting to identify markers for the in situ-identification of normal human epithelial HF stem cells within their physiological niche, using highly sensitive immunohistochemical relevant methods. In view of our findings, the most useful positive in situ-markers for human bulge cells turned out to be CK15, CK19 and CD200, even though these markers were not exclusively expressed in the bulge. Contrary to previous reports, we could not confirm that s1 integrin IR is upregulated by normal human bulge keratinocytes in situ. The putative epithelial HF stem cell marker, Lhx2, also was not found to be upregulated here. However, the present study indicates that absent IR for CD34, connexin 43 and nestin may be exploited as negative markers for epithelial HF stem cells. Among the examined BM / mesenchymal markers, α6 integrin, fibronectin, nidogen, fibrillin-1 and LTBP-1 were expressed throughout the connective tissue sheath of human HFs, and no significant upregulation of these markers could be detected in or around the bulge. Instead, tenascin-C IR was significantly upregulated in the bulge, suggesting that tenascin-C constitutes a component of the bulge stem cell niche of human HFs. Therefore, this thesis reveals that the combined use of a limited panel of defined positive and negative markers helps to identify human bulge stem cells in situ. In addition, this thesis shows that a peculiar epithelial protrusion of the outer root sheath with distinct morphology (´follicular trochanter`) can serve as convenient lead structure for epithelial HF stem cell identification, at least in some patients. Finally, these analyses were concluded by exploring the functional significance of s1 integrins in human HF biology. β1 integrins and their ligands (e.g. tenascin-C, fibronectin, fibrillin-1) are of particular interest, since e.g. s1 integrin-deficient mice show severe, epithelial HF stem cell-related abnormalities in HF morphogenesis and cycling. However, the missing upregulation of s1 integrin expression in the human HF bulge did not suggest a similarly prominent role for s1 integrin in human compared to murine HF biology. Therefore, we also studied in microdissected, organ-cultured human anagen hair bulbs, whether blocking or stimulating s1 integrin-mediated signalling affects at least defined functions of the progeny of these epithelial HF stem cells, i.e. hair bulb keratinocytes and the HS produced by them. We could demonstrate that human scalp HF IR for b1 integrin was most prominent in the outermost layer of the outer root sheath and the surrounding connective tissue sheath and that sites of b1 integrin IR co-express fibronectin and tenascin-C. Functionally, two activating b1 integrin antibodies (12G10, TS2/16) and ligand-mimicking RGD peptides promoted the growth of microdissected, organ-cultured human scalp HFs in vitro (i.e. stimulated hair matrix keratinocyte proliferation and HS elongation) and modulated human HF cycling [i.e. inhibited spontaneous hair follicle regression (catagen)]. These data provide the first available evidence that b1 integrin-mediated signalling is also important in human hair growth control. In summary, the current thesis has defined both, a useful panel of positive and negative markers, and distinctive morphological features that, together, facilitate the identification of human epithelial HF stem cell in their natural in situ context. This is complemented with the demonstration that, despite the fact that s1 integrin expression is not a useful marker for human epithelial HF stem cells, s1 integrin-mediated signalling still affects physiological key functions of their immediate cell progeny and may be pharmacologically targeted for stimulating human hair growth.