M5 isolation method preserves higher trichogenic identity and proliferative capacity of human dermal papilla cells

Human dermal papilla cells (hDPCs) play a critical role in hair follicle regeneration. However, their clinical application is limited by inefficient isolation procedures and the progressive loss of trichogenic potential during in vitro expansion. This study aimed to establish an optimized isolation strategy to balance scalability with the preservation of functional identity. Five isolation methods (M1–M5) were systematically evaluated based on attachment efficiency, proliferation kinetics, and manufacturability. The molecular characteristics of hDPCs derived via the five different methods were examined using gene expression profiling and signaling pathway analysis. Concurrently, functional competence was assessed using three-dimensional (3D) in vitro hair follicle-like constructs and an in vivo mouse hair regeneration model. Among the tested methods, the M5 approach demonstrated superior performance, notably achieving high attachment efficiency and reduced processing time. The M5-derived hDPCs exhibited significantly enhanced proliferative capacity while maintaining elevated population doubling levels through passage 4. Molecular analyses revealed sustained expression of key hair-inductive markers, including LEF1, AXIN2, and Noggin, accompanied by activation of the Wnt/β-catenin and TGF-β/SMAD signaling pathways. In vivo, the M5-derived hDPCs effectively induced anagen-phase hair follicles and robust hair growth. Furthermore, the M5-derived hDPCs demonstrated physiological responsiveness comparable to those achieved by pharmacological modulation with minoxidil and dihydrotestosterone, confirming retention of cellular plasticity. Collectively, the optimized M5 isolation strategy provides a robust and standardized platform for hDPC isolation, preserving biological potency while supporting scalable manufacturing for regenerative applications in alopecia.