In this study, a detailed analysis of the outgrowth of primary keratinocytes from plucked human hair follicles was conducted. Plucked hair follicles offer an easily accessible and non-invasive method as a primary cell source for adult somatic keratinocytes, providing a simple starting material for induced pluripotent stem cell (iPSC) reprogramming. In this study, we laid our focus on the precise examination of timing and location of the first keratinocyte outgrowth after plucking, as well as the morphological changes that occur during cultivation. Our results show that the region of the hair follicle, from which the initial outgrowth occurs, is crucial for successful cultivation. Additionally, first appearing protrusions and first visible cells show the same specific marker expression as the intact outer root sheath. We therefore conclude that initially appearing keratinocytes arise from the basal layer of the outer root sheath. To improve the whole process, the protocol was adjusted to reduce the time until successful outgrowth. These optimizations are particularly relevant for developing a standardized protocol that works efficiently across all individuals, including patients with difficult-to-culture hair follicles. The accelerated cell harvesting could prove valuable in future applications in regenerative medicine, especially for patients where culture times have previously been too long.
Keywords: Cytokeratins; Keratinocytes; Keratins; Keratocysts; Outer root sheath (ORS); Plucked hair follicle; iPSCs.
© 2025 Klingenstein et al.