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Methods In Molecular Biology
Ohyama, M;Kobayashi, T;
The epithelial (keratinocyte) stem cells locating at the bulge region of hair follicles have been reported to possess high proliferative capacity in vitro and multipotency to repopulate hair follicles, sebaceous glands, and the epidermis, indicating the importance of those cells for the clinical applications including gene therapy and regenerative medicine. However, most of previous investigations adopted rodent bulge cells. The biological properties of human and rodent bulge cells have been reported to be distinct. Accordingly, it is crucial to directly isolate and characterize human bulge cells. However, the supply of human hair follicles for investigative purposes may be extremely limited in some situation. Dogs share analogous hair loss disorders with humans. Recent investigations have uncovered the biological similarities between canine bulge cells, suggesting that canine bulge cells are valuable substitute for the biological characterization of human bulge cells. Here, we provided the protocols for the isolation and characterization of human and canine hair follicle stem cell-enriched keratinocytes. Manual microdissection still represents the most commonly used technique to enrich bulge cells from human and canine hair follicles. Positive selection using a cell surface marker, CD200, should enable further enrichment of human bulge cells. Confirmation of successful isolation and molecular and cellular biological characterization of bulge cells are possible by real-time PCR and flow cytometry analyses described here. Colony-forming assay enables not only the evaluation of in vitro proliferative capacity but also the subcloning of holoclone keratinocytes, putative keratinocyte stem cells. Finally, hair reconstitution assay is available for the assessment of multipotency in vivo and sets a basis for tissue engineering of hair follicles.