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

Nat Protoc. 2022 May;17(5):1266-1305. doi: 10.1038/s41596-022-00681-y.

Jiyoon Lee ¹ ² ³ ⁴ ⁵, Wouter H van der Valk ^# ⁶ ⁷ ⁸ ⁹, Sara A Serdy ^# ⁷, CiCi Deakin ^# ⁷ ¹⁰, Jin Kim ⁶ ⁷ ¹¹ ⁸, Anh Phuong Le ⁶ ⁷ ¹¹ ⁸, Karl R Koehler ¹² ¹³ ¹⁴ ¹⁵

Affiliations

1 Department of Otolaryngology, Boston Children's Hospital, Boston, MA, USA. [email protected].
2 F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, MA, USA. [email protected].
3 Department of Plastic and Oral Surgery, Boston Children's Hospital, Boston, MA, USA. [email protected].
4 Department of Surgery, Harvard Medical School, Boston, MA, USA. [email protected].
5 Department of Otolaryngology-Head and Neck Surgery, Harvard Medical School, Boston, MA, USA. [email protected].
6 Department of Otolaryngology, Boston Children's Hospital, Boston, MA, USA.
7 F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, MA, USA.
8 Department of Otolaryngology-Head and Neck Surgery, Harvard Medical School, Boston, MA, USA.
9 Department of Otorhinolaryngology and Head & Neck Surgery, Leiden University Medical Center, Leiden, the Netherlands.
10 Department of Biological Engineering, Wentworth Institute of Technology, Boston, MA, USA.
11 Department of Plastic and Oral Surgery, Boston Children's Hospital, Boston, MA, USA.
12 Department of Otolaryngology, Boston Children's Hospital, Boston, MA, USA. [email protected].
13 F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, MA, USA. [email protected].
14 Department of Plastic and Oral Surgery, Boston Children's Hospital, Boston, MA, USA. [email protected].
15 Department of Otolaryngology-Head and Neck Surgery, Harvard Medical School, Boston, MA, USA. [email protected].
# Contributed equally.

PMID: 35322210 DOI: 10.1038/s41596-022-00681-y

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.

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