Organ systems play essential roles in the physiological functions required for homeostasis. A 3D integumentary organ system (3D-IOS) comprises the skin and skin appendages such as hair follicles and sebaceous glands. This protocol describes how to induce the differentiation of murine induced pluripotent stem (iPS) cells into a 3D-IOS. First, iPS cells are grown for 7 d under conditions that encourage the formation of embryoid bodies (EBs). The iPS cell–derived EBs are stimulated by Wnt10b one day before transplantation of multiple EBs in vivo (a method we describe as the clustering-dependent embryoid body (CDB) transplantation method). After a further 30 d, the transplanted EBs will have differentiated into a 3D-IOS containing mature hair follicles and sebaceous glands. These can be removed and transplanted into wounds in the skin of other mice. After transplantation of a 3D-IOS, the organ system shows full physiological function in vivo starting 14 d following transplant. Thus, this protocol enables a whole functional organ system to be generated from pluripotent stem cells.
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The datasets generated during the current study are available from the corresponding author upon reasonable request.
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We are grateful to R. Takagi, J. Ishimaru, and A. Sugawara for performing the experiments. This work was partially supported by a Grant-in-Aid for KIBAN (A) from the Ministry of Education, Culture, Sports, and Technology (no. 25242041).
This work was partially funded by Organ Technologies Inc. T.T. is a director at Organ Technologies Inc. This work was performed under an Invention Agreement between Tokyo University of Science, RIKEN, and Organ Technologies Inc. This work was partially supported by a collaboration grant from Organ Technologies Inc. to T.T.
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Key references using this protocol
Nakao, K. et al. Nat. Methods 4, 227–230 (2007): https://doi.org/10.1038/nmeth1012
Toyoshima, K. E. et al. Nat. Commun. 3, 784 (2012): https://doi.org/10.1038/ncomms1784
Takagi, R. et al. Sci. Adv. 2, e1500887 (2016): https://doi.org/10.1126/sciadv.1500887
Integrated supplementary information
Supplementary Figure 1 The occurrence of hair follicles and the epithelial classification types of CDB transplants.
(a) Number of hair follicles in the CDB transplants. The data are presented as the mean ± SEM of individual experiments; n = 13 (single iPS injection), n = 74 (CDB transplants without Wnt10b), and n = 7 (CDB transplants with Wnt10b). *P < 0.001 by Student’s t test. (b) The frequency of epithelial types in CDB transplants. Epithelial types in CDB transplants were classified based on cell morphology and number. The data are presented as the mean ± SEM of individual experiments; n = 5. b reproduced with permission from Takagi et al.22, American Association for the Advancement of Science.
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Toyoshima, Ke., Ogawa, M. & Tsuji, T. Regeneration of a bioengineered 3D integumentary organ system from iPS cells. Nat Protoc 14, 1323–1338 (2019). https://doi.org/10.1038/s41596-019-0124-z
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