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

Nature Protocols (2019) | Download Citation

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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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Data availability

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

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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

References

  1. 1.

    Takeichi, M. Self-organization of animal tissues: cadherin-mediated processes. Dev. Cell 21, 24–26 (2011).

  2. 2.

    Sasai, Y. Next-generation regenerative medicine: organogenesis from stem cells in 3D culture. Cell Stem Cell 12, 520–530 (2013).

  3. 3.

    Sasai, Y. Cytosystems dynamics in self-organization of tissue architecture. Nature 493, 318–326 (2013).

  4. 4.

    Pispa, J. & Thesleff, I. Mechanisms of ectodermal organogenesis. Dev. Biol. 262, 195–205 (2003).

  5. 5.

    Lu, C. & Fuchs, E. Sweat gland progenitors in development, homeostasis, and wound repair. Cold Spring Harb. Perspect. Med. 4, a015222 (2014).

  6. 6.

    Jiang, T. X. et al. Integument pattern formation involves genetic and epigenetic controls: feather arrays simulated by digital hormone models. Int. J. Dev. Biol. 48, 117–135 (2004).

  7. 7.

    Schneider, M. R., Schmidt-Ullrich, R. & Paus, R. The hair follicle as a dynamic miniorgan. Curr. Biol. 19, R132–R142 (2009).

  8. 8.

    Fuchs, E. Scratching the surface of skin development. Nature 445, 834–842 (2007).

  9. 9.

    Lim, X. & Nusse, R. Wnt signaling in skin development, homeostasis, and disease. Cold Spring Harb. Perspect. Biol. 5, a008029 (2013).

  10. 10.

    Sun, B. K., Siprashvili, Z. & Khavari, P. A. Advances in skin grafting and treatment of cutaneous wounds. Science 346, 941–945 (2014).

  11. 11.

    Sun, T. T. & Green, H. Differentiation of the epidermal keratinocyte in cell culture: formation of the cornified envelope. Cell 9, 511–521 (1976).

  12. 12.

    Atala, A. Human stem cell-derived retinal cells for macular diseases. Lancet 385, 487–488 (2015).

  13. 13.

    Sharpe, P. T. & Young, C. S. Test-tube teeth. Sci. Am. 293, 34–41 (2005).

  14. 14.

    Broutier, L. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat. Protoc. 11, 1724–1743 (2016).

  15. 15.

    Rookmaaker, M. B., Schutgens, F., Verhaar, M. C. & Clevers, H. Development and application of human adult stem or progenitor cell organoids. Nat. Rev. Nephrol. 11, 546–554 (2015).

  16. 16.

    Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 (2016).

  17. 17.

    Nakao, K. et al. The development of a bioengineered organ germ method. Nat. Methods 4, 227–230 (2007).

  18. 18.

    Ikeda, E. et al. Fully functional bioengineered tooth replacement as an organ replacement therapy. Proc. Natl. Acad. Sci. USA 106, 13475–13480 (2009).

  19. 19.

    Toyoshima, K. E. et al. Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches. Nat. Commun. 3, 784 (2012).

  20. 20.

    Ogawa, M. et al. Functional salivary gland regeneration by transplantation of a bioengineered organ germ. Nat. Commun. 4, 2498 (2013).

  21. 21.

    Hirayama, M. et al. Functional lacrimal gland regeneration by transplantation of a bioengineered organ germ. Nat. Commun. 4, 2497 (2013).

  22. 22.

    Takagi, R. et al. Bioengineering a 3D integumentary organ system from iPS cells using an in vivo transplantation model. Sci. Adv. 2, e1500887 (2016).

  23. 23.

    Cohen, D. E. & Melton, D. Turning straw into gold: directing cell fate for regenerative medicine. Nat. Rev. Genet. 12, 243–252 (2011).

  24. 24.

    Walck-Shannon, E. & Hardin, J. Cell intercalation from top to bottom. Nat. Rev. Mol. Cell Biol. 15, 34–48 (2014).

  25. 25.

    Eiraku, M. et al. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3, 519–532 (2008).

  26. 26.

    Nakano, T. et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 10, 771–785 (2012).

  27. 27.

    Koehler, K. R., Mikosz, A. M., Molosh, A. I., Patel, D. & Hashino, E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature 500, 217–221 (2013).

  28. 28.

    Suga, H. et al. Self-formation of functional adenohypophysis in three-dimensional culture. Nature 480, 57–62 (2011).

  29. 29.

    Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

  30. 30.

    Takasato, M. et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526, 564–568 (2015).

  31. 31.

    Sharmin, S. et al. Human induced pluripotent stem cell-derived podocytes mature into vascularized glomeruli upon experimental transplantation. J. Am. Soc. Nephrol. 27, 1778–1791 (2016).

  32. 32.

    Nishizawa, M. et al. Epigenetic variation between human induced pluripotent stem cell lines is an indicator of differentiation capacity. Cell Stem Cell. 19, 341–354 (2016).

  33. 33.

    Ozone, C. et al. Functional anterior pituitary generated in self-organizing culture of human embryonic stem cells. Nat. Commun. 7, 10351 (2016).

  34. 34.

    Egusa, H. et al. Gingival fibroblasts as a promising source of induced pluripotent stem cells. PLoS ONE 5, e12743 (2010).

  35. 35.

    Schad, C. R. et al. Application of fluorescent in situ hybridization with X and Y chromosome specific probes to buccal smear analysis. Am. J. Med. Genet. 66, 187–192 (1996).

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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).

Author information

Affiliations

  1. Laboratory for Organ Regeneration, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan

    • Koh-ei Toyoshima
    • , Miho Ogawa
    •  & Takashi Tsuji
  2. Organ Technologies Inc., Tokyo, Japan

    • Koh-ei Toyoshima
    • , Miho Ogawa
    •  & Takashi Tsuji

Authors

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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.

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.

Corresponding author

Correspondence to Takashi Tsuji.

Integrated supplementary information

  1. 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.

Supplementary information

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DOI

https://doi.org/10.1038/s41596-019-0124-z

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