An optimized protocol for generating appendage-bearing skin organoids from human-induced pluripotent stem cells
Open accessAbstract
Organoid generation from pluripotent stem cells is a cutting-edge technique that has created new possibilities for modelling human organs in vitro, as well as opening avenues for regenerative medicine. Here, we present a protocol for generating skin organoids (SKOs) from human-induced pluripotent stem cells (hiPSCs) via direct embryoid body formation. This method provides a consistent start point for hiPSC differentiation, resulting in SKOs with complex skin architecture and appendages (e.g. hair follicles, sebaceous glands, etc.) across hiPSC lines from two different somatic sources.
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hiPSC-derived skin organoids have hair follicles, pigmentation, dermal stratification, appendage patterning, and innervation.
“Recent advances in human induced pluripotent stem cell (hiPSC) technology have enabled the generation of skin organoids (SKO) with hair follicles, pigmentation, dermal stratification, appendage patterning, and innervation.[ 25 , 26 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ]”
From Coordinated Development of Immune Cell Populations in Vascularized Skin Organoids from Human Induced Pluripotent Stem Cells (2025) · cites this paper as [32]
The Introduction and the Methods figure 4 caption describe hiPSC-derived skin organoids with hair follicles, other appendages, hair placodes, and neurites. Troubleshooting, Problem 4 says follicles can develop pigmentation. The copy describes epidermal layers and an epidermis–dermis arrangement, but does not establish dermal stratification or a defined pattern of appendages; pigmentation is described as conditional. Page numbers were not available in our copy. (LLM, conf=0.86)
Experimental — accuracy not yet measuredH&E staining demonstrated formation of a thick epithelium, hair follicles, and dermal papillae within the dermis in VSKOs, consistent with previous reports on SKOs.
“Furthermore, hematoxylin and eosin staining demonstrated the formation of a thick epithelium, hair follicles, and dermal papillae within the dermis, consistent with previous reports on SKOs (Figure 7a).[ 32 ]”
From Coordinated Development of Immune Cell Populations in Vascularized Skin Organoids from Human Induced Pluripotent Stem Cells (2025) · cites this paper as [32]
Previous human SKO models were devoid of vascularization and immune cells.
“Previous human SKO models were devoid of vascularization and immune cells.[ 25 , 26 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 59 ]”
From Coordinated Development of Immune Cell Populations in Vascularized Skin Organoids from Human Induced Pluripotent Stem Cells (2025) · cites this paper as [32]
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Hair-bearing human skin generated entirely from pluripotent stem cells
Lee, J., Rabbani, C. C., Gao, H., Steinhart, M. R., Woodruff, B. M., Pflum, Z. E., Kim, A., Heller, S., Liu, Y., Shipchandler, T. Z., et al. (2020) · Nature · doi:10.1038/s41586-020-2352-3 · Cited by 487 · see citing works
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Research works printed but not linked: 7
- [1]McKnight G, Shah J, Hargest R. Physiology of the skin. Surgery (Oxford) 2022; 40: 8– 12. 10.1016/j.mpsur.2021.11.005 · DOI
- [2]Hosseini M, Koehler KR, Shafiee A. Biofabrication of human skin with its appendages. Adv Healthc Mater 2022; 11: e2201626. 10.1002/adhm.202201626 36063498 PMC11469047 · DOI · Paper page
- [3]Itoh M, Umegaki-Arao N, Guo Z et al Generation of 3D skin equivalents fully reconstituted from human induced pluripotent stem cells (iPSCs). PLoS One 2013; 8: e77673. 24147053 PMC3795682
- [4]Black AF, Bouez C, Perrier E et al Optimization and characterization of an engineered human skin equivalent. Tissue Eng 2005; 11: 723– 33. 15998214
- [6]Lee J, van der Valk WH, Serdy SA et al Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells. Nat Protoc 2022; 17: 1266– 305. 10.1038/s41596-022-00681-y 35322210 PMC10461778 · DOI · Paper page
- [7]Shafiee A, Sun J, Ahmed IA et al Development of physiologically relevant skin organoids from human induced pluripotent stem cells. Small 2023; e2304879. 10.1002/smll.202304879 38044307 · DOI · Paper page
- [8]Cakir B, Xiang Y, Tanaka Y et al Engineering of human brain organoids with a functional vascular-like system. Nat Methods 2019; 16: 1169– 75. 10.1038/s41592-019-0586-5 31591580 PMC6918722 · DOI
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The empowering influence of air-liquid interface culture on skin organoid hair follicle development
Sun, J., Ahmed, I., Brown, J., Khosrotehrani, K., & Shafiee, A. (2025) · Burns & Trauma · doi:10.1093/burnst/tkae070 · Cited by 19 · see citing works
Self-citationMostina, M., Sun, J., Sim, S. L., Ahmed, I. A., Souza‐Fonesca‐Guimaraes, F., Wolvetang, E. J., Brown, J., Kumari, S., Khosrotehrani, K., & Shafiee, A. (2025) · Advanced Healthcare Materials · doi:10.1002/adhm.202502108 · Cited by 17 · see citing works
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- Authors
- Ahmed, I., Sun, J., Brown, J., Khosrotehrani, K., & Shafiee, A.
- Affiliations
- The University of Queensland (AU)
- Queensland Health (AU)
- Corresponding author
- Khosrotehrani, Kiarash
- Shafiee, Abbas
- Venue
- Biology Methods and Protocols
- Publisher
- Oxford University Press (OUP)
- Published
- 2024-01-01
- Volume
- 9
- Issue
- 1
- Pages
- bpae019-bpae019
- ISSN
- 2396-8923 (ISSN-L)
- PMC
- PMC11009018
- OpenAlex
- W4394762730
- Cited by
- 16
- References
- 1
- Citations by year
- 2026: 7 · 2025: 6 · 2024: 3
- FWCI
- 2.18
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Biology Methods and Protocols (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- Metro North Hospital and Health Service
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Health Sciences › Medicine › Urology › Hair Growth and Disorders
- Health Sciences › Medicine › Genetics › Mesenchymal stem cell research
- Keywords
- skin organoids, human induced pluripotent stem cells, hair follicle, sebaceous glands, skin differentiation, regenerative medicine
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