Single-cell benchmarking separates epidermal stratification from appendage programme maintenance in an air-liquid interface skin model.

Full-thickness skin equivalents (FTS) reproduce epidermal stratification, yet rarely generate hair follicles or glands. Defining the cellular features associated with this limitation could inform the engineering of appendage-bearing skin. Here we profiled mouse induced pluripotent stem cell-derived early epithelial progenitors (EEPs) and EEP-derived FTS by single-cell RNA sequencing, using developing mouse skin as a reference. FTS established a stratified epidermis with basal cell enrichment, but was accompanied by a marked reduction in appendage-primed cells (1.17% versus 8.69% in EEPs). Developmental mapping showed that FTS cells mapping to the hair follicle trajectory concentrated at early positions, whereas EEPs spanned a later and broader correspondence. Compared with matched developing skin states, FTS epithelium exhibited stronger injury-response and reactive transcriptional programmes. This epithelial reactivity coincided with an inferred dermal-epithelial signalling network with weaker BMP and noncanonical WNT inputs, no epithelial Hedgehog communication, and biased FGF and extracellular matrix interactions. Together, these findings link limited appendage formation in FTS to reduced appendage priming, a reactive interfollicular epidermal state, and a remodelled dermal signalling environment that diverges from native developmental niches.