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Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant

Abstract

Generation of functional and vascularized organs from human induced pluripotent stem cells (iPSCs) will facilitate our understanding of human developmental biology and disease modeling, hopefully offering a drug-screening platform and providing novel therapies against end-stage organ failure. Here we describe a protocol for the in vitro generation of a 3D liver bud from human iPSC cultures and the monitoring of further hepatic maturation after transplantation at various ectopic sites. iPSC-derived specified hepatic cells are dissociated and suspended with endothelial cells and mesenchymal stem cells. These mixed cells are then plated onto a presolidified matrix, and they form a 3D spherical tissue mass termed a liver bud (iPSC-LB) in 1–2 d. To facilitate additional maturation, 4-d-old iPSC-LBs are transplanted in the immunodeficient mouse. Live imaging has identified functional blood perfusion into the preformed human vascular networks. Functional analyses show the appearance of multiple hepatic functions in a chronological manner in vivo.

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Figure 1: Protocol for generating a vascularized and functional human liver from iPSC–derived organ bud transplants.
Figure 2: Generation of 3D liver buds from iPSCs in vitro by recapitulating early organogenetic interactions.
Figure 3: Ectopic transplantation of human iPSC-derived liver buds at various sites.
Figure 4: Intravital visualization of vascularized and functional human liver formation from iPSC-derived liver buds inside the cranium.
Figure 5: Functional characterization of human iPSC-LB transplants at various ectopic sites based on in vivo albumin (ALB) production.

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Acknowledgements

We thank F. Kawamata, A. Takahashi, S. Funayama, N. Hijikata and N. Sasaki for kindly providing technical support, and Y.-W. Zheng, Y. Ueno and all of the members of our laboratory for help with several comments. We express our great thanks to Y. Sato, SAIKOU, Inc. for the illustrations for the protocols (http://www.medicalillustration.jp/). This work was supported by Grants-in-Aid of the Ministry of Education, Culture, Sports, Science, and Technology of Japan to T.T. (nos. 24106510, 24689052), N.K. (no. 22390260) and H.T. (nos. 21249071, 25253079). This work was also supported by grants to H.T. from the Research Center Network for Realization of Regenerative Medicine and the Strategic Promotion of Innovative Research and Development program (S-innovation, no. 62890004) of the JST; by a Specified Research Grant of the Takeda Science Foundation and a grant from the Japan IDDM network to H.T.; and by a grant of the Yokohama Foundation for Advanced Medical Science to T.T. K. Eto and H. Nakauchi (Institute of Medical Science, University of Tokyo) provided TkDA3 iPSCs.

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Authors

Contributions

T.T. conceived, designed and conducted the experiments, analyzed the data and wrote the paper. R.-R.Z., H.K., M.K., E.Y. and M.E. performed experiments and wrote the paper. N.K., K.S. and H.T. supervised the project.

Corresponding authors

Correspondence to Takanori Takebe or Hideki Taniguchi.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Time-lapse imaging of human iPSC-LB formation process. (MOV 1112 kb)

Supplementary Video 2

Procedures for preparing a transparency cranial window. (MOV 4004 kb)

Supplementary Video 3

Procedures for transplantation under a cranial window. (MOV 3363 kb)

Supplementary Video 4

Procedures for transplantation under subrenal capsule. (MOV 4079 kb)

Supplementary Video 5

Procedures for transplantation onto distal mesentery. (MOV 2337 kb)

Supplementary Video 6

Procedures for transplantation onto proximal mesentery. (MOV 3414 kb)

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Takebe, T., Zhang, RR., Koike, H. et al. Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant. Nat Protoc 9, 396–409 (2014). https://doi.org/10.1038/nprot.2014.020

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