Development and characterization of human epidermal organoids and their application to model keratinocyte disorders with a focus on Darier disease

An organoid is a collection of organ-specific cells that develops from stem cells or progenitor cells and recapitulates the structure and function of the organ. Human epidermal organoids (HEOs) are derived from the epidermis and offer several advantages over 2D keratinocyte cultures. They exhibit an in vivo-like organization and replicate the physiology of the epidermis. Darier disease (DD) is a dominantly inherited disorder characterized by skin plaques. DD features include acantholysis and abnormal keratinization. These histological characteristics result from mutations in the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum calcium-ATPase (SERCA2), leading to desmosome malformation. Currently, no effective in vitro models exist for studying DD.
We established culture conditions and protocols to develop a reliable method for generating human epidermal organoids. Then, we utilized this model to create and characterize an in vitro 3D model of DD. HEOs were generated from keratinocytes and characterized through immunofluorescence, immunohistochemistry, and qPCR. They expressed epidermal genes indicative of stratification and differentiation (collagen 17, keratin-1, -5, -10, -14, and -15, filaggrin, and transglutaminase-1 and -3).
Darier organoids were generated using four distinct approaches: 1) treating HEOs with thapsigargin (TG) to induce DD-like features (tgHEOs), 2) generating HEOs from keratinocytes isolated from Darier patients (ddHEOs), 3) creating HEOs from keratinocytes with ATP2A2 knockdown via siRNA (srHEOs), and 4) producing HEOs from CRISPR- modified keratinocytes with ATP2A2 mutations (ntHEOs). In TG-treated HEOs, HEOs derived from Darier patients, and N/TERT Darier organoids desmosomal proteins displayed abnormal cytoplasmic localization instead of being expressed at the cell membrane, replicating a crucial aspect of DD. Furthermore, keratins in Darier organoids were mislocalized in the nuclear and perinuclear regions.
HEOs reproduce the complexity and architecture of the human epidermis. They can also be derived from patients with DD, enabling the generation of in vitro models of skin diseases. This makes HEOs essential for pathophysiology research, drug development, and personalized medicine for epidermal disorders.