Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells

Abstract

Human skin uses millions of hairs and glands distributed across the body surface to function as an external barrier, thermoregulator, and stimuli sensor. The large-scale generation of human skin with these appendages would be beneficial however is challenging. Here, we describe a detailed protocol for generating hair-bearing skin tissue entirely from a homogeneous population of human pluripotent stem cells (PSCs) in a three-dimensional in vitro culture system. Defined culture conditions are used over a two-week period to induce differentiation of PSCs to surface ectoderm and cranial neural crest cells, which give rise to the epidermis and dermis, respectively, in each organoid unit. After 60 days of incubation, the skin organoids produce hair follicles. By day ~130, the skin organoids reach full complexity and contain stratified skin layers, pigmented hair follicles, sebaceous glands, Merkel cells, and sensory neurons, recapitulating the cell composition and architecture of fetal skin tissue at week-18 of gestation. Skin organoids can be maintained in culture using this protocol for up to 150 days enabling the organoids to be used to investigate basic skin biology, model disease, and further the reconstruction or regeneration of skin tissue.