Perez-Aso, M. | bioRxiv Science

bioRxiv2025

Enhanced Differentiation Potential of Pigmented Human Epidermal Equivalents

Melanocyte-keratinocyte interactions are vital for regulating melanogenesis and maintaining epidermal homeostasis. However, most 3D human skin equivalents lack melanocytes, limiting their relevance for pigmentation studies. To address this, we utilized a pigmented human epidermal equivalent (PmtHEE) that incorporates melanocytes into the epidermis. Using single-cell RNA sequencing (scRNA-seq), we characterized PmtHEE and compared it with neonatal foreskin epidermis (FsEpi) and a fibroblast-containing human skin equivalent model (FibHSE). PmtHEE showed a higher proportion of differentiated cells and model-specific cell state transition paths that reflect in vivo trajectories. We further uncovered a shared L1CAM-EZR-driven external signaling network using exSigNet in FsEpi and PmtHEE that mediates melanocyte-to-keratinocyte communication and supports epidermal differentiation. Altogether, PmtHEE provides a distinct and physiologically relevant model of pigmented skin, with enhanced differentiation potential compared to conventional in vitro systems.

cell biology↗

bioRxiv2024

Characterization of Reconstructed Human Epidermis in a Chemically-defined, Animal Origin-free Cell Culture System

The Reconstructed Human Epidermis (RHE) model derived from epidermal keratinocytes offers an ethical and scientifically valid alternative to animal experimentation, particularly in cutaneous toxicology and dermatological research, where elimination of animal cruelty is of paramount importance. Moreover, safer cell- and tissue-based cutaneous therapies are also possible, where the removal of animal-originated products from the RHE culture media lessens the risk of adverse clinical events. Thus, we compared chemically-define animal origin-free (cdAOF) supplements and the historically utilized supplement (HKGS), which contains growth factors and bovine pituitary extract. Herein we present the first extended characterization of RHE using cdAOF culture systems with newborn, adult, and immortalized N/TERT keratinocytes. Culture of RHE in the cdAOF media produced histological features that were nearly identical to that produced using HKGS, with the exception that the basal keratinocytes were less cylindrical, and with some immunolocalization of involucrin in the basal layer. Additionally, increased mRNA expression of several inflammatory-proliferative markers was observed in the cdAOF RHEs as well. Importantly, in RHEs cultured in cdAOF media, expression and immunolocalization of other expected markers of keratinization, as well as monitoring of barrier function revealed results equal or close to those observed in RHE cultured in HKGS.

cell biology↗