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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.

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Fig. 1: Schematics comparing in vivo and in vitro skin organogenesis.
Fig. 2: Illustration of day-by-day differentiation protocol and representative checkpoint images.
Fig. 3: Images from optimal timepoints to check differentiation and characterization of the resulting skin organoids structure.
Fig. 4: qCe3D whole-mount immunostaining.
Fig. 5: Morphologies of developing skin organoids during differentiation.
Fig. 6: Morphological differences of skin organoids incubated with differing BMP4 concentrations.
Fig. 7: Representative phase-contrast images of recommended cell seeding density and cell confluency.

Data availability

Examples of results obtained are included in the figures.

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Acknowledgements

This work was supported by the Ralph W. and Grace M. Showalter Trust (K.R.K.), the Indiana CTSI (core pilot grant UL1 TR001108 to K.R.K.), the Indiana Center for Biomedical Innovation (Technology Enhancement Grant to K.R.K.) and the NIH (grants R01AR075018 and R01DC017461 to K.R.K.). Cell lines associated with this study were stored in a facility constructed with support from the NIH (grant C06 RR020128-01). We thank M. Steinhart, C. Nist-Lund, A. Sheets, B. Cooper, S. Heller, B. Woodruff, M. Rendl, P. Rompolas, D. Spandau, J. Foley, R. Rotting, M. Haniffa, M. Boniotto, S. Sahu, S. Sharan, K. Andrykovich, R. Jaenisch, L. Biggs and S. Wickström for constructive feedback and technical assistance during the development of this method.

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Contributions

J.L. and K.R.K. conceived the study and wrote the manuscript. W.H.v.d.V. performed whole-mount immunostaining. W.H.v.d.V., S.A.S. and C.D. performed imaging. J.L., W.H.v.d.V., S.A.S., C.D., J.K., A.P.L. and K.R.K. contributed to figure making, writing and manuscript editing. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Jiyoon Lee or Karl R. Koehler.

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Competing interests

J.L. and K.R.K., with the Indiana University Research and Technology Corporation, have submitted a patent application covering the entire skin organoid induction method (WO2017070506A1). The other authors declare no competing interests.

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Nature Protocols thanks Ming Xing Lei, Yunfang Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references used in this protocol

Lee, J. et al. Nature 582, 399–404 (2020): https://doi.org/10.1038/s41586-020-2352-3

Lee, J. et al. Cell Rep. 22, 242–254 (2018): https://doi.org/10.1016/j.celrep.2017.12.007

Koehler, K. R. et al. Nat. Biotechnol. 35, 583–589 (2017): https://doi.org/10.1038/nbt.3840

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Lee, J., van der Valk, W.H., Serdy, S.A. et al. Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells. Nat Protoc 17, 1266–1305 (2022). https://doi.org/10.1038/s41596-022-00681-y

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