Generation of Human Induced Pluripotent Stem Cell-derived Planar Hair-bearing Skin Organoids Using an Air-Liquid Interface Culture System

Human induced pluripotent stem cell (hiPSC)-derived skin organoids (SOs) containing hair follicles provide a sophisticated in vitro model for investigating intercellular interactions within the skin microenvironment. Initially developed in an inverted cystic configuration, where the dermis is exposed to the environment and the epidermis faces inward toward the organoid center, SOs can subsequently be transferred to a planar configuration in an air-liquid interface (ALI) culture system, which more closely resembles human skin physiology. This manuscript presents a comprehensive protocol for generating and selecting cystic SOs that can be dissected into planar SOs and further developed in an ALI culture system. The protocol includes defined checkpoints for macroscopic qualitative assessment at critical stages of organoid development, details the technical steps required for flattening cystic SOs into planar skin, specifies optimal conditions for ALI culture, and demonstrates outcomes through molecular analysis. At Day 27, immunofluorescence staining with lineage-specific markers revealed a pluristratified pigmented epithelium with skin appendages, including hair follicles and sebaceous gland-like structures. This protocol provides a robust method for generating planar SOs with appendages, offering a physiologically relevant in vitro model. This system supports diverse applications in skin research and has already been successfully used to model pathogen infection, ultraviolet radiation-induced tissue damage, and drug responses.