Search

Hair follicle stem cells (HFSCs) are considered one of the useful donor cell types for skin regenerative medicine owing to their robust proliferative capacity and multipotency. However, methods for easily and effectively obtaining HFSCs from a limited skin biopsy are still lacking. Here we report a novel approach for obtaining a subpopulation of HFSCs from a small skin sample from the rat tail, which uses the sebaceous glands (SGs) to capture the adjacent HFSCs. By means of organ culture, keratinocytes were expanded from the detached SGs, which also included adherent HFSCs from the hair follicle that could be passaged at the single-cell level. These SG-captured keratinocytes strongly expressed the basal layer markers K14, integrin α6, and p63; the bulge stem cell marker K15; and the upper isthmus stem cell marker Plet1. Furthermore, we reconstituted new epidermis, hair follicles, and SGs from the SG-captured keratinocytes using an easily operated, modified skin reconstitution assay based on silicone gel sheeting. This study suggests that the SGs could be an accessible capturer to harvest the adjacent HFSC subpopulation, particularly when the donor tissue is limited.

Hair follicle stem cells (HFSCs) are potentially useful for the treatment of skin injuries and diseases. To achieve clinical application, a prerequisite must be accomplished: harvesting enough HFSCs from limited skin biopsy. The commonly used sorting approach for isolating HFSCs, however, suffers from its intrinsic disadvantages, such as requirement of large-scale skin biopsy. Here, we report an efficient organ culture method to isolate and expand rat HFSCs from limited skin biopsy and these HFSCs could reconstitute the epidermis and the hair follicles (HFs). Seventy-three percent of cultured HFs formed hair follicle stem cell colonies from the bulge, and a single hair follicle provided all the HFSCs used in this research, demonstrating the high efficiency of this method. Quantitative RT-PCR and immunofluorescent staining results revealed that these stem cells obtained from the bulge highly expressed basal layer markers K14 and alpha-6 integrin, epithelial stem cell marker P63, and bulge stem cell marker K15. After long-term culture in vitro, GFP-labeled hair follicle stem cells formed new hair follicles, epidermis, and sebaceous glands following xenotransplantation into the back of nude mice. This study indicated that multipotent hair follicle stem cells could be efficiently harvested through organ culture from limited skin material—even a single hair follicle—and reconstitute hair follicles in vivo after long-term expansion culture, providing the basis for future clinical applications.