Cutaneous nestin+ cells are of substantial interest in regenerative medicine. However, the location of nestin+ cells in situ remains controversial. We therefore sought to determine their location in female human scalp skin, using stringently controlled immunohistochemical techniques, Western blot analysis, and in situ hybridization and complementing those techniques with relative and quantitative reverse transcriptase-PCR of enzymatically digested or laser-capture microdissected human hair follicle (HF) compartments. We show here that the immunoreactivity (IR) patterns obtained with anti-nestin antibodies are highly dependent on the tissue-fixation and immunohistochemical methods used. NESTIN mRNA could not be detected within HF-associated epithelial cells in situ or in RNA extracts of the microdissected HF epithelium. Instead, NESTIN transcripts were found only in intramesenchymal skin compartments. Individual cells showing both, specific nestin IR and NESTIN mRNA were detectable in the connective-tissue sheaths of human HFs, sebaceous and sweat glands. Moreover, stimulation of organ-cultured human scalp skin with the adipokine leptin increased the number of nestin+ cells in these intramesenchymal skin locations, whereas no specific nestin IR could be induced by leptin within the HF epithelium, including the bulge. Therefore, nestin expression at the gene and protein levels in human scalp skin is restricted to the periappendage mesenchyme and can be stimulated by leptin.
