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Regeneration of a bioengineered 3D integumentary organ system from iPS cells

Abstract

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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Fig. 1: Outline of the procedure for the generation of a functional 3D-IOS and other organ systems from 2D-cultured iPS cells.
Fig. 2: In vitro formation of iPS-derived EBs and preparation of clustering-dependent EBs.
Fig. 3: Transplantation of a CDB into the subrenal capsule of a SCID mouse and cystic epithelial tissue formation in the transplant.
Fig. 4: Structural and functional analysis of the bioengineered IOS.

Data availability

The datasets generated during the current study are available from the corresponding author upon reasonable request.

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Acknowledgements

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).

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Authors

Contributions

T.T. designed the protocol; K.T. and M.O. performed the experiments; and K.T., M.O., and T.T. wrote the protocol.

Corresponding author

Correspondence to Takashi Tsuji.

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Competing interests

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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Related links

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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