WOFAPS 2025 8th World Congress of Pediatric Surgery

Yanghonghong Fei, Vincent CH Lui, Kenneth KY Wong

Department of Surgery, The University of Hong Kong

Objective: Pediatric burn injuries frequently result in scarring that impairs normal growth and development. Current reconstructive techniques, except full-thickness skin grafts, cannot restore sweat glands, severely compromising patients' thermoregulatory capacity. While induced pluripotent stem cell (iPSC) technology enables the creation of personalized cell banks and has successfully generated hair follicles in skin organoids, sweat gland development still relies on primary cells, limiting its clinical application. Here, we aim to develop an epidermal organoid system derived from mouse iPSCs with dual potential to generate both interfollicular epidermis (IFE) and functional sweat glands.

Methods: Mouse iPSCs were maintained on MEF feeder layers through 15 passages before using. Following Lee et al.'s protocol, cells were cultured in suspension until day 8, then dissociated and embedded in matrigel with specialized epidermal organoid growth medium. Third-passage iPSC-derived epidermal organoids (iEOs) were characterized using immunofluorescence (IF) and single cell RNA sequencing.

Results: Within the same culture system, iEOs developed into two distinct morphologies: predominantly spherical structures with characteristic keratin cores representing stratified epidermal differentiation, and less frequent budding structures with irregular outgrowths forming glandular aggregates, observed at approximately a 3:1 ratio. IF staining confirmed the presence of basal keratinocyte marker Krt5 in all iEOs. Spherical iEOs expressed suprabasal marker Krt10, while glandular iEOs demonstrated positive staining for AQP5, SMA, and K19, indicating sweat gland differentiation. Single-cell RNA sequencing validated the presence of distinct cell populations corresponding to interfollicular epidermis, sweat glands, and hair follicles, with sweat gland populations expressing both ductal (Scnn1a) and secretory (AQP5) markers.

Conclusion: We demonstrate successful generation of dual-potential mouse iPSC-derived epidermal organoids that develop both stratified epidermis and functional sweat glands. These organoids express characteristic molecular markers of native tissue, representing a potential standardized, scalable source for functional skin reconstruction with thermoregulatory capacity.