Tissue-engineered skin substitutes preserve the potential of epithelial cells to differentiate into sebaceous glands

Summary: Abstract Body: Sebaceous glands maintain skin hydration by producing sebum, a lipid mixture secreted by sebocytes. To date, human tissue-engineered skin substitutes (TESs) have limited capacity to regenerate sebaceous glands. This study investigates the cell sources that promote sebaceous differentiation in TESs produced by the self-assembly method. Bilayered TESs were produced using epithelial cells and fibroblasts from scalp, foreskin, and breast tisssues. Epithelial cells were cultured on fibroblast sheets for 10 days at the air-liquid interface to promote maturation, then grafted onto athymic mice for 21 days. Biopsies were analyzed by histology, immunofluorescence, and transmission electron microscopy (TEM). Before grafting, sebocyte clusters were observed in TESs epidermis made with scalp epithelial cells. After grafting, glandular structures were seen only in these TESs, suggesting that in vitro clusters developed into sebaceous glands in vivo. TEM revealed lipid droplets and tonofilaments typical of sebaceous glands. Oil Red O staining showed an excretory duct connecting the structures to the skin surface, suggesting functionality. Immunofluorescence confirmed the presence of early and mature sebocytes (keratin 7, mucin-1) and the human origin of the sebaceous gland-like structures. Notably, the fibroblast origin did not affect this process. We developed a human model enabling the de novo formation of sebaceous-like glands in TESs and we highlighted the importance of epithelial stem cell origin for sebaceous differentiation. Future research will focus on identifying key molecular and cellular signals involved. These results provide new insights into sebaceous gland development in tissue-engineered substitutes, potentially enhancing their therapeutic use in treating burn patients. Bettina Cattier^{1, 2, 3}, Brice Magne^{1, 2, 3}, Marika Lemire-Rondeau^{1, 2, 3}, Amélie Morissette^{1, 2, 3}, Eric Philippe^{1, 2, 3}, Danielle Larouche^{1, 2, 3}, Lucie Germain^{1, 2, 3} 1. Centre de Recherche en Organogénèse Expérimentale de l’Université Laval/LOEX, Québec, QC, Canada. 2. Department of Surgery, Universite Laval Faculte de Medecine, Québec City, QC, Canada. 3. Regenerative Medicine Division, Centre de recherche du CHU de Québec-Université Laval, Québec, QC, Canada. Stem Cell Biology, Tissue Regeneration and Wound Healing