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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References
Carmeliet, P. Angiogenesis in health and disease. Nat. Med. 9, 653–660 (2003).
Park, C. A hierarchical order of factors in the generation of FLK1- and SCL-expressing hematopoietic and endothelial progenitors from embryonic stem cells. Development 131, 2749–2762 (2004).
Faloon, P. et al. Basic fibroblast growth factor positively regulates hematopoietic development. Development 127, 1931–1941 (2000).
Ferguson, J. E., Kelley, R. W. & Patterson, C. Mechanisms of endothelial differentiation in embryonic vasculogenesis. Arterioscler. Thromb. Vasc. Biol. 25, 2246–2256 (2005).
Potente, M., Gerhardt, H. & Carmeliet, P. Basic and therapeutic aspects of angiogenesis. Cell 146, 873–887 (2011).
Gebala, V., Collins, R., Geudens, I., Phng, L. K. & Gerhardt, H. Blood flow drives lumen formation by inverse membrane blebbing during angiogenesis in vivo. Nat. Cell Biol. 18, 443–450 (2016).
Charpentier, M. S. & Conlon, F. L. Cellular and molecular mechanisms underlying blood vessel lumen formation. BioEssays 36, 251–259 (2014).
Simons, M., Gordon, E. & Claesson-Welsh, L. Mechanisms and regulation of endothelial VEGF receptor signalling. Nat. Rev. Mol. Cell Biol. 17, 611–625 (2016).
Armulik, A., Genové, G. & Betsholtz, C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev. Cell 21, 193–215 (2011).
Yamashita, J. et al. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408, 92–96 (2000).
Gerhardt, H. et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J. Cell Biol. 161, 1163–1177 (2003).
Stratman, A. N. & Davis, G. E. Endothelial cell-pericyte interactions stimulate basement membrane matrix assembly: influence on vascular tube remodeling, maturation, and stabilization. Microsc. Microanal. 18, 68–80 (2012).
Lindahl, P., Johansson, B. R., Levéen, P. & Betsholtz, C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 277, 242–245 (1997).
Davis, G. E., Norden, P. R. & Bowers, S. L. K. Molecular control of capillary morphogenesis and maturation by recognition and remodeling of the extracellular matrix: functional roles of endothelial cells and pericytes in health and disease. Connect. Tissue Res. 56, 392–402 (2015).
Wimmer, R. A. et al. Human blood vessel organoids as a model of diabetic vasculopathy. Nature 565, 505–510 (2019).
Adams, W. J. et al. Functional vascular endothelium derived from human induced pluripotent stem cells. Stem Cell Rep. 1, 105–113 (2013).
Rufaihah, A. J. et al. Human induced pluripotent stem cell-derived endothelial cells exhibit functional heterogeneity. Am. J. Transl. Res. 5, 21–35 (2013).
Kusuma, S. et al. Self-organized vascular networks from human pluripotent stem cells in a synthetic matrix. Proc. Natl. Acad. Sci. USA 110, 12601–12606 (2013).
Orlova, V. V. et al. Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells. Nat. Protoc. 9, 1514–1531 (2014).
Orlova, V. V. et al. Functionality of endothelial cells and pericytes from human pluripotent stem cells demonstrated in cultured vascular plexus and zebrafish xenografts. Arterioscler. Thromb. Vasc. Biol. 34, 177–186 (2014).
Lian, X. et al. Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of WNT signaling. Stem Cell Rep. 3, 804–816 (2014).
Cheung, C., Bernardo, A. S., Trotter, M. W. B., Pedersen, R. A. & Sinha, S. Generation of human vascular smooth muscle subtypes provides insight into embryological origing-dependent disease susceptibility. Nat. Biotechnol. 30, 165–173 (2012).
Bajpai, V. K., Mistriotis, P., Loh, Y. H., Daley, G. Q. & Andreadis, S. T. Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates. Cardiovasc. Res 96, 391–400 (2012).
Trotter, M. W. B., Cheung, C., Pedersen, R. A., Sinha, S. & Bernardo, A. S. Generation of human vascular smooth muscle subtypes provides insight into embryological origin–dependent disease susceptibility. Nat. Biotechnol. 30, 165–173 (2012).
Ren, X. et al. Engineering pulmonary vasculature in decellularized rat and human lungs. Nat. Biotechnol. 33, 1097–1102 (2015).
Samuel, R. et al. Generation of functionally competent and durable engineered blood vessels from human induced pluripotent stem cells. Proc. Natl. Acad. Sci. USA 110, 12774–12779 (2013).
James, D. et al. Expansion and maintenance of human embryonic stem cell-derived endothelial cells by TGFΒ inhibition is Id1 dependent. Nat. Biotechnol. 28, 161–166 (2010).
Patsch, C. et al. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nat. Cell Biol. 17, 994–1003 (2015).
Samuel, R., Duda, D. G., Fukumura, D. & Jain, R. K. Vascular diseases await translation of blood vessels engineered from stem cells. Sci. Transl. Med. 7, 309rv6 (2015).
Hockemeyer, D. & Jaenisch, R. Induced pluripotent stem cells meet genome editing. Cell Stem Cell 18, 573–586 (2016).
Potente, M. & Mäkinen, T. Vascular heterogeneity and specialization in development and disease. Nat. Rev. Mol. Cell Biol. 18, 477–494 (2017).
Homan, K. A. et al. Flow-enhanced vascularization and maturation of kidney organoids in vitro. Nat. Methods 16, 255–262 (2019).
Augustin, H. G. & Koh, G. Y. Organotypic vasculature: from descriptive heterogeneity to functional pathophysiology. Science 357, eaal2379 (2017).
Ungrin, M. D., Joshi, C., Nica, A., Bauwens, C. & Zandstra, P. W. Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates. PLoS ONE 3, e1565 (2008).
Huttala, O. et al. Human vascular model with defined stimulation medium—a characterization study. ALTEX 32, 125–136 (2015).
Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564 (2016).
Takebe, T. et al. Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant. Nat. Protoc. 9, 396–409 (2014).
Beers, J. et al. Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat. Protoc. 7, 2029–2040 (2012).
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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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.
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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
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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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DOI: https://doi.org/10.1038/s41596-019-0213-z
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