[Culture and identification of human induced pluripotent stem cell-derived skin organoids]

Objective: To establish and identify a culture system for skin organoids based on human induced pluripotent stem cells (hiPSCs). Methods: This study was a basic research with a self-controlled pre-post design. Human primary skin fibroblasts (Fbs) were obtained from discarded hair follicle tissue of a 45-year-old male patient undergoing hair transplant surgery at Tongji Medical College of Huazhong University of Science and Technology. Primary skin Fbs were induced into hiPSCs, which were then cultured to form three-dimensional aggregates. Then the induced differentiation was performed as followings. On differentiation day 0 (the day of differentiation initiation), the three-dimensional aggregates were transferred to ultra-low attachment culture plate and cultured in E6 medium supplemented with Matrigel, transforming growth factor-β type Ⅰ receptor inhibitor of SB431542, basic fibroblast growth factor (bFGF), and bone morphogenetic protein-4 to induce non-neural ectoderm formation. On differentiation day 3, one-quarter volume of E6 medium supplemented with bone morphogenetic protein signaling pathway inhibitor of LDN193189 and bFGF was added to the original medium to continue culturing and to induce the formation of cranial neural crest cells. On differentiation day 6, culture continued with approximately 3/5 volume of plain E6 medium added to the original medium. Half-medium changes were performed on differentiation days 8 and 10 (once each), followed by continued culture. On differentiation day 12, cell clumps were seeded into new ultra-low attachment culture plate to continue culturing, and epidermal self-assembly was induced using organoid maturation medium supplemented with Matrigel. On differentiation day 15, a half-medium change was performed, and culture was continued. On differentiation day 18, the original medium was replaced with organoid maturation medium supplemented with α-melanocyte-stimulating hormone to continue culturing. Starting from differentiation day 21, half-medium changes were performed every 3 days, and culture continued. After differentiation initiation, the stage-specific morphological characteristics of hiPSC differentiation into skin organoids were observed daily. Immunofluorescence staining was performed to assess skin organoids on different days of differentiation: on day 12 for the presence of mesenchymal cells, on day 20 for epidermal terminal differentiation, on day 35 for mesenchymal and epithelial structure formation as well as hair follicle stem cell and basal layer keratinocyte related characteristics, on day 55 for dermal papilla formation, on day 75 for hair germ-like structure formation, and on day 90 for organoid proliferative activity. Fluorescence probe staining was performed on differentiation day 110 to detect hair follicle formation, as well as lipid deposition and sebaceous gland-like structure formation. Results: On differentiation day 0, hiPSCs formed three-dimensional aggregates with clear boundaries and relatively uniform size. On differentiation day 3, ectoderm-like structures appeared on the surface of the aggregates, accompanied by non-epithelial-like cells beginning to migrate outward. During differentiation days 6 to 8, mesenchymal cells and neuroglial-like cells gradually increased. During differentiation days 12 to 18, organoids with spatial heterogeneity gradually formed within the aggregates, exhibiting preliminary epidermal-dermal-like bilayer structures. On differentiation day 60, hair germ-like structures were observed. During differentiation days 80 to 130, more mature hair germ-like structures and hair follicle-like structures gradually emerged. Immunofluorescence detection showed that on differentiation day 12, early mesenchymal cells were observed around the skin organoids. On differentiation day 20, keratinocytes emerged in the outer layer of the skin organoids. On differentiation day 35, dermal-like cells emerged in the skin organoids, forming distinct spatial compartments with epithelial-like structures, accompanied by the presence of basal layer-like cell populations. On differentiation day 55, dermal papilla-like cell populations were detected and were spatially adjacent to epidermal regions. On differentiation day 75, thickened epithelial-like structures growing inward were observed in localized areas of the skin organoids, along with cell aggregation forming hair germ-like structures; dermal papilla-like cell populations were detected in regions adjacent to the hair germ and were spatially adjacent to epithelial structures. On differentiation day 90, proliferating cell enrichment was observed in the hair germ region. Fluorescence probe staining showed that by differentiation day 110, more mature hair follicle-like structures, hair shaft-like protrusions, lipid deposition, and sebaceous gland-like structures were observed in the skin organoids. Conclusions: By stage-wise modulation of key signaling pathways, we successfully established a hiPSC-based a culture system for skin organoids. The induced differentiation process of this system highly mimics the in vivo developmental program of skin and hair follicles, resulting in the reconstitution of a complex in vitro skin model with hair follicle-like structures.