Department of Otolaryngology, Boston Children's Hospital, Boston, MA, USA.
F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, MA, USA.
Nat Protoc. 2022 May;17(5):1266-1305. doi: 10.1038/s41596-022-00681-y. Epub 2022 Mar 23.
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.
人体皮肤利用分布在体表的数百万根毛发和腺体作为外部屏障、体温调节器和刺激传感器发挥作用。大规模生成具有这些附属物的人类皮肤将是有益的，但具有挑战性。在这里，我们描述了一种详细的方案，即在三维体外培养系统中，从同质的人类多能干细胞群体中生成具有毛发的皮肤组织。在 2 周的时间内使用定义的培养条件诱导多能干细胞分化为表皮外胚层和颅神经嵴细胞，这两种细胞分别在每个类器官单位中产生表皮和真皮。孵育 60 天后，皮肤类器官产生毛囊。到第 130 天左右，皮肤类器官达到完全复杂的程度，包含分层的皮肤层、有色素的毛囊、皮脂腺、默克尔细胞和感觉神经元，再现了妊娠 18 周胎儿皮肤组织的细胞组成和结构。使用该方案，皮肤类器官可以在培养中维持长达 150 天，从而使类器官能够用于研究基本皮肤生物学、模拟疾病，并且进一步重建或再生皮肤组织。
