The spatial lipidome of the human epidermis and epidermal models

The structure and barrier function of the human skin are closely linked to its lipid composition, especially in the epidermis, the outermost part in contact with the environment. The differentiation process of living epidermal keratinocytes into corneocytes, and the subsequent formation of the epidermal permeability barrier are accompanied by major changes in the lipids contained in and secreted by keratinocytes. Conventional lipidomic methods can characterize overall lipid composition but provide limited information on how lipid species are spatially organized within epidermal tissue. Here, we used high-resolution MALDI FTICR mass spectrometry imaging to study and compare the spatial lipidome in native human skin and a skin equivalent model with a fully stratified epidermis. Across both ionization polarities, we annotated 509 lipid-related m/z features; 452 were detected in native human skin and 214 in skin equivalents. 157 were detected in both native and reconstructed skin, corresponding to more than 70% overlap and indicating that the model reproduces a substantial lipid core relevant to further analyses. Native skin showed greater overall lipid diversity, consistent with its higher cellular and structural complexity. However, representative ion images showed similar distribution of phospholipids, barrier-associated lipids, and selected sphingolipid-related features across the stratum corneum and epidermis of both native and reconstructed skins. In particular, ceramide-, hexosylceramide-, ceramide phosphate-, and sphingomyelin-related signals followed comparable spatial ordering, with minor shifts in the skin equivalent that may reflect differences in epidermal differentiation. Heatmap-based clustering of annotated lipid-related m/z features further revealed specific lipid profiles by epidermal layers and could therefore be used as unbiased regions of interest (ROI) for further analysis. These data show that skin equivalent models replicate the major aspects of lipid composition and distribution in native human epidermis, providing a practical and biologically relevant model for spatial lipidomics methods and a deeper understanding of lipids’ contribution to the structure and function of the human epidermis.