Generation of human-induced pluripotent stem cell-derived adherent 3-dimensional skin hair-follicle organoids

Regenerating hair follicles (HFs) is a critical medical need for patients who suffer from serious hair loss. To generate equivalent hair-bearing skin systems that could mimic the complexity of native tissues with pluripotent stem cells, floating culture has been employed as a standard method; however, it is still necessary to improve limitations such as the heterogeneity of organoids and the difficulty of handling them, which increases during long-term culture. Here, we devise a floating-adherent combinatory culture system to establish skin HF organoids from human-induced pluripotent stem cells (hiPSCs). Specifically, embryoid bodies were generated in a free-floating environment, followed by the induction process, which occurred in adherent conditions. With our approach, hair germ-like buds were shown to protrude and extend faster. After 100 days of culture, mature cystic skin organoids stratified to form the epidermis, dermis, and outer root sheath, as evident from quantitative polymerase chain reaction and immunohistochemistry analysis. Dermal condensate cells (Sox2+, PDGFRα+, P75+), which are the precursors of HFs, were detected together with HF stem cells (NFATC1+, LGR5+), putative bulge stem cells (LHX2+, KRT15+) and melanocytes (PMEL+). Notably, our constructed HFs could recapitulate the sensory function of native tissues, as illustrated by the formation of a network of sensory neurons and Schwann cells connecting towards HF cells and epidermal progenitors. In summary, our results demonstrate a new protocol for the simplified and efficient induction of skin HFs from hiPSCs, thereby contributing to research on optimizing HF growth and investigating novel therapeutic strategies to treat alopecia.