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Hair follicle stem cells are pluripotent and have a self-renewal capacity and multi-differentiation potential in vitro. As hair follicle stem cells can be easily sampled from the skin and hair of clinical patients at a considerable quantity, these cells have potential applications in wound repair and skin tissue engineering. Effective approaches for the in vitro culture and amplification of mouse hair follicle stem cells, as well as the in vitro osteogenic differentiation potential and cell source when obtaining mouse-separated cells were examined. Serial subculture was performed in different culture systems. Cell source was detected based on the relevant surface markers derived from mouse hair follicles at the gene and protein levels, and the differential potential was determined. The proliferative ability of hair follicle-derived stem cells obtained from mouse embryonic fibroblast (MEF)/keratinocyte serum-free medium (KSF)-conditioned medium was the highest among all culture systems. The induced group had a stronger osteogenic differentiation potential compared with the non-induced group, indicating that the cells obtained from MEF/KSF-conditioned medium were cells derived from the hair follicle dermal papilla. Therefore, the strong osteogenic differentiation potential of the hair follicle-derived mesenchymal stem cells was screened with MEF/KSF-conditioned culture medium following amplification, and biological characteristics similar to those of hair follicle dermal papilla cells were observed. [ABSTRACT FROM AUTHOR]