Single-cell sequencing reveals increased LAMB3+ basal keratinocytes and ZNF90+ fibroblasts in autologous cultured epithelium under serum- and feeder-free conditions

Abstract Autologous cultured epithelium grafting (ACEG) is a promising treatment for refractory vitiligo. Concerns for infections or immunological reactions caused by serum and feeder used in culture medium may limit the use for surgical interventions. Here, we cultured autologous epithelium under serum- and feeder-free (SFF) conditions and compared its safety and efficacy with epithelium cultured under serum- and feeder-dependent (SFD) conditions in patients with stable vitiligo. Then, single-cell RNA transcriptomics of SFF and SFD cultured epithelium and healthy skin were conducted. There were no significant differences in repigmentation between the SFF and the SFD conditioned grafting. Increased LAMB3 + basal keratinocytes and ZNF90 + fibroblasts were found in the SFF epithelial sheets. The LAMB3 + basal keratinocytes had active cellular metabolism and participated in extracellular matrix homeostasis. The ZNF90 + fibroblasts were more differentiated and implicated in collagen formation for cell adhesion. Both the LAMB3 + basal keratinocytes and the ZNF90 + fibroblasts were more involved in the interactions with melanocytes in the SFF epithelial sheets compared to the SFD epithelial sheets. Our findings support the LAMB3 + basal keratinocytes and the ZNF90 + fibroblasts as key factors behind the repigmentation in ACEG under SFF conditions. The study provides translational insights into ACEG repigmentation and potential therapeutic targets for vitiligo.