Single cell transcriptomics of human skin equivalent organoids

Several methods for generating human skin equivalent (HSE) organoid cultures are regularly used to study skin biology and test pharmaceuticals, however few studies have thoroughly characterized these systems. To fill this gap, we used single cell-RNA sequencing to compare the cellular states of in vitro HSEs generated from distinct culture methods, HSEs xenografted onto mice, and in vivo epidermis. By combining differential gene expression, pseudotime analyses, splicing kinetics, and spatial localization, we reconstructed HSE keratinocyte differentiation trajectories that recapitulated known in vivo epidermal differentiation pathways and show that HSEs contain many of the major in vivo cellular states. However, HSEs also develop several unique keratinocyte states, an expanded basal stem cell program, and disrupted terminal differentiation. In addition, cell-cell communication modeling showed the presence of EMT-associated signaling pathways not normally active in homeostatic skin and we show that EGF supplementation influences the EMT signature. Lastly, xenografted HSEs at early timepoints post-transplantation significantly rescued many of the observed in vitro deficits, while undergoing a hypoxic response that drove an alternative differentiation lineage. This study highlights the strengths and limitations of organoid cultures and identifies areas for potential innovation.