From basic biology to engineered therapies: the keratinocyte stem cell playbook

Keratinocyte stem cells (KSCs) are the principal drivers of epidermal renewal, barrier maintenance, and wound repair. Their ability to alternate between self-renewal and differentiation is orchestrated by tightly integrated extrinsic and intrinsic programs that ensure tissue stability while enabling rapid regeneration after injury. This review synthesizes current understanding of KSC homeostasis through a unified framework of three interdependent “fate locks”—the identity switch (ΔNp63 ↔ Notch/IRF6-KLF4/GRHL3/OVOL), the cell-cycle lock (E2F/MYC ↔ p21/p27-RB), and the mechanotransduction lock (YAP/TAZ ↔ Hippo/LATS). We summarize how niche-derived cues—integrins/ECM, EGFR, Wnt, Notch, Ca²⁺/CaSR, and TGF-β—interface with intrinsic timers such as asymmetric division, DNMT1-UHRF1-mediated epigenetic memory, the DNA-damage response, proteostasis/autophagy, and redox signaling to steer keratinocyte fate. Building on this biological foundation, we categorize current methods for isolation and xeno-free expansion of primary human keratinocytes, emphasizing advances in defined media, feeder-free substrates, and biomimetic culture surfaces. We further review 3D and organotypic models, hydrogel-based delivery systems, and the growing portfolio of keratinocyte-derived clinical products used in wound healing. Finally, we highlight emerging applications extending beyond cutaneous repair—including immunomodulation, pigment restoration, ocular and mucosal regeneration, and acellular exosome-based therapeutics.