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, but is challenging. Here, we describe a detailed protocol for generating hair-bearing skin tissue entirely from a homogeneous population of human pluripotent stem cells in a three-dimensional in vitro culture system. Defined culture conditions are used over a 2-week period to induce differentiation of pluripotent stem cells to surface ectoderm and cranial neural crest cells, which give rise to the epidermis and dermis, respectively, in each organoid unit. After 60 d 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 d, enabling the organoids to be used to investigate basic skin biology, model disease and, further, reconstruct or regenerate skin tissue.

Differentiation of pluripotent stem cells for modeling human skin development and potential applications

“…Because the neural crest develops into cranial dermis, the described hair follicles resembled facial beard and ear hair. The work provided proof of concept of the potential of iPSCs to regenerate skin appendages, and underscores the capability of such systems to test adequate morphogens in an orderly manner to derive skin from different locations in the human body ( Lee et al, 2020 ; Sun et al, 2021 ; Lee et al, 2022 ). Ebner-Peking et al (2021) reported the assembly of skin spheroid organoids containing vasculature by culturing together human iPSCs-derived keratinocytes, fibroblasts, and endothelial cells.…”

Hair Follicle Morphogenesis During Embryogenesis, Neogenesis, and Organogenesis

“…Hair follicle organoids are also successfully made with mouse and human-derived induced pluripotent stem cells (Figure 2C) (Lee et al, 2018;Lee et al, 2020). The hair follicle organoids mimic the actual hair follicle development process and matured hair follicles in the organoid can make the hair shafts functional (Lee et al, 2022). Interestingly, in a spherical organoid, hair follicles grow outward and hair shafts grow inward.…”

“…Unlike the WIHN, melanocytes are present and can produce pigmented hair. Although adipocytes, sensory neurons, and Schwann cells are present, the organoids still lack other cell populations, including sweat glands, blood vessels, arrector pili muscle (rarely observed), and immune cells (Lee et al, 2022). Therefore, advances in protocols will be needed to generate fully mature skin organoids comprising entire niche components.…”

In vitro models for investigating itch

“…After several weeks, sebaceous glands and hair follicle formation occurred, as well as neural crest-like cells, showing the potential for eventual sensory neuron manipulation to create a whole skin itch model ( Lee et al, 2018 ). With even more physiological relevance to the human system, the same researchers later used hiPSCs to successfully create similar self-assembled and fully stratified skin organoids including hair follicle formation ( Lee J. et al, 2020 , 2022 ; Ramovs et al, 2022 ). In another highly complementary organoid system, hESC-SNs were introduced to endothelial cells and proved essential for vascular formation ( Kannan et al, 2021 ).…”