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Generation of blood vessel organoids from human pluripotent stem cells

Abstract

Blood vessels are fundamental to animal life and have critical roles in many diseases, such as stroke, myocardial infarction and diabetes. The vasculature is formed by endothelial cells that line the vessel and are covered with mural cells, specifically pericytes in smaller vessels and vascular smooth muscle cells (vSMCs) in larger-diameter vessels. Both endothelial cells and mural cells are essential for proper blood vessel function and can be derived from human pluripotent stem cells (hPSCs). Here, we describe a protocol to generate self-organizing 3D human blood vessel organoids from hPSCs that exhibit morphological, functional and molecular features of human microvasculature. These organoids are differentiated via mesoderm induction of hPSC aggregates and subsequent differentiation into endothelial networks and pericytes in a 3D collagen I–Matrigel matrix. Blood vessels form within 2–3 weeks and can be further grown in scalable suspension culture. Importantly, in vitro–differentiated human blood vessel organoids transplanted into immunocompromised mice gain access to the mouse circulation and specify into functional arteries, arterioles and veins.

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Fig. 1: Schematic diagram of the time line for generating blood vessel organoids from hPSCs.
Fig. 2: Bright-field images of hPSC differentiation into vascular networks and blood vessel organoids.
Fig. 3: Characterization of vascular networks and blood vessel organoids differentiated from hPSCs.
Fig. 4: Characterization of blood vessel organoids transplanted into NSG mice.

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The data generated or analyzed during this study are included in this published article.

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Acknowledgements

We thank all members of our laboratories for constructive critiques and expert advice. We also thank M. Boehm (Center for Molecular Medicine, National Heart, Lung, and Blood Institute, National Institutes of Health) for sharing the iPSC line NC8 and A. Kavirayani, M. Zeba, T. Engelmaier, J. Klughofer and A. Piszczek for histology services. J.M.P. was supported by grants from IMBA, the Austrian Ministry of Sciences, the Austrian Academy of Sciences, an ERC grant and an Era of Hope Innovator award.

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Authors

Contributions

R.A.W., A.L. and M.A. performed experiments. R.A.W., D.K. and J.M.P. supervised the project. R.A.W. and J.M.P. wrote the manuscript.

Corresponding authors

Correspondence to Reiner A. Wimmer or Josef M. Penninger.

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Competing interests

A patent application related to this work has been filed. IMBA is in the process of applying for a patent application covering vascular organoid technology that lists R.A.W., D.K. and J.M.P. as inventors.

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Journal peer review information Nature Protocols thanks Andrew Baker and Valeria Orlova for their contributions to the peer review of this work.

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Wimmer, R.A. et al. Nature 565, 505–510 (2019): https://doi.org/10.1038/s41586-018-0858-8

Supplementary information

Supplementary Video 1

Dissection of vascular networks for blood vessel organoid production

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Wimmer, R.A., Leopoldi, A., Aichinger, M. et al. Generation of blood vessel organoids from human pluripotent stem cells. Nat Protoc 14, 3082–3100 (2019). https://doi.org/10.1038/s41596-019-0213-z

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