Research on hair follicle neogenesis using cultured follicular cells remains constrained by the rapid cellular senescence of primary cells, including hair follicle keratinocytes (HFKs), significant inter-donor variability, and the progressive loss of hair-inductive potential (trichogenicity). To overcome these bottlenecks, we established immortalized human HFK lines via the co-transfection of SV40 large T antigen (SV40T) and human telomerase reverse transcriptase (hTERT). Among the generated clones, the Kyungpook National University Keratinocyte-8 (KNUK-8) line exhibited superior proliferative capacity and was selected for comprehensive characterization. Notably, although the karyotype underwent certain modifications during the immortalization process, KNUK-8 demonstrated no tumorigenic potential in vivo. Furthermore, KNUK-8 preserved its genetic identity with parental cells and maintained specific HFK marker expression. In chamber hair reconstitution assays, KNUK-8 combined with newborn mouse dermal cells generated robust and mature hair follicles comparable to primary HFKs for up to 12 weeks, whereas dermal cells or keratinocytes alone failed to induce neogenesis. Crucially, this trichogenic potential was reproducibly maintained even at extended passages (passages 35 and 45). To our knowledge, KNUK-8 represents the first immortalized human HFK line that reliably retains long-term trichogenic potential in vivo, offering a highly valuable standard resource for hair regeneration research.
