Letterhttps://doi.org/10.1038/s41586-018-0858-8Human blood vessel organoids as a model of diabetic vasculopathyreiner A. Wimmer 1 *, Alexandra Leopoldi 1 , Martin Aichinger 2 , Nikolaus Wick 3 , Brigitte Hantusch 3 , Maria Novatchkova 1 , Jasmin taubenschmid 1 , Monika Hämmerle 3 , Christopher esk 1 , Joshua A. Bagley 1 , Dominik Lindenhofer 1 , Guibin Chen 4 , Manfred Boehm 4 , Chukwuma A. Agu 1 , Fengtang Yang 5 , Beiyuan Fu 5 , Johannes Zuber 2 , Juergen A. Knoblich 1 , Dontscho Kerjaschki 3 & Josef M. Penninger 1,6 *The increasing prevalence of diabetes has resulted in a global epidemic 1 . Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells 2–4 . Diabetes also impairs the functions of endothelial cells 5 and disturbs the communication between endothelial cells and pericytes 6 . How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.Previous studies have used coculture techniques that combine induced pluripotent stem (iPS) cell-derived endothelial cells with pericytes 7,8 or early vascular cells 9,10 to establish vascular networks. With the aim to engineer entire human blood vessels, we devel-oped a multistep protocol to modulate mesoderm development and vascular specification 8,11–16 (Fig. 1a). Confocal imaging revealed the formation of complex, interconnected networks of CD31 + endothelial tubes (Fig. 1b). These self-organizing three-dimensional (3D) vascular networks showed proper localization of pericytes as defined by the molecular markers PDGFRβ, calponin 1 (Fig. 1c and Extended Data Fig. 1a) and α-smooth muscle actin (SMA) (data not shown). These vessel-like structures were enveloped by a basement membrane, which was visualized using immunostaining for collagen type IV (Extended Data Fig. 1a, b). Coculturing of purified, differentiated endothelial cells and pericytes resulted in tenuous endothelial networks that contained only a few pericyte interactions and were not covered by collagen type IV (Extended Data Fig. 1c). We reproducibly generated vascular net -works using the human embryonic stem cell line H9 as well as two additional iPS cell lines (Extended Data Fig. 1d).To standardize these microvasculatures, we developed 3D organoids in a 96-microwell format (Fig. 1a). These 1–2-mm vascular organoids formed 3D capillary networks that consisted of lumen-forming endothelial cells that were tightly associated with pericytes (Fig. 1d–f, Extended Data Fig. 1e and Supplementary Videos 1, 2). Electron microscopy confirmed the generation of a lumen, a basement mem-brane and typical tight junctions between endothelial cells (Extended Data Fig. 1f). We identified tip cells by CD31 + filopodia in vascular organoids (Extended Data Fig. 1g), indicative of newly forming vessels 17 . Vascular organoids were composed of PDGFRβ+ pericytes, CD31 + VE-cadherin + endothelium, CD90 + CD73 + CD44 + mesenchy-mal stem-like cells and CD45 + haematopoietic cells (Extended Data Fig. 2a). Gene expression profiling confirmed that CD31 + endothelial cells showed a typical endothelial signature, including expression of maturity markers such as von-Willebrand factor and VE-PTP (which is encoded by PTPRB), similar to primary human umbilical vein endothelial cells (Extended Data Fig. 2b). PDGFRβ+ cells displayed expression of typical pericyte markers, such as NG2 (which is encoded by CSPG4), SMA (which is encoded by ACTA2) and calponin 1 (which is encoded by CNN1), and clustered with primary human placental pericytes (Extended Data Fig. 2b, c). Endothelial cells in vascular orga-noids were positive for the lectin Ulex europaeus agglutinin I (UEA-I), showed uptake of acetylated low-density lipoprotein, expressed von Willebrand factor, generated Weibel–Palade bodies and responded to TNF by inducing ICAM-1 expression (Extended Data Fig. 2d–g), markers which are all indicative of functional maturity 13 .To investigate whether 3D organoids could form functional blood vessels in vivo 18 , we differentiated human iPS cells into vascular organoids in vitro and transplanted them under the kidney capsule of immunodeficient NOD/SCID/IL2Rγnull (NSG) mice. The human organoids reproducibly grew and survived (more than 95%) within the mice for more than 6 months (Fig. 1g). FITC–dextran and human- specific anti-CD31 antibody perfusion showed that human blood vessels had functionally connected to the mouse vasculature (Fig. 1h and Extended Data Fig. 3a, b). Quantitative magnetic resonance imag-ing (MRI) of perfusion rates and blood volumes showed well-vascu-larized and perfused transplants; moreover, the mean transit times and low vessel leakage confirmed normal organization and function of the human blood vessels (Extended Data Fig. 3c, d). Analyses of histological sections showed in vivo differentiation of the cells into arteries, arterioles, capillaries and venules 19 (Fig. 1i and Extended Data Fig. 3d–j). Transplantation of vascular organoids derived from human embryonic stem cells genetically tagged with blue fluorescent protein (BFP) or red fluorescent protein (RFP) confirmed the establishment of a vascular tree in mice that contained human endothelium; more than 90% of all pericytes covering the human endothelium were of human origin (Fig. 1j and Extended Data Fig. 3k).To assess microvascular changes induced by diabetes in humans, we examined the dermal skin microvasculature of normoglycaemic 1 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria. 2 Institute of Molecular Pathology (IMP), Vienna, Austria. 3 Clinical Department of Pathology, Medical University Vienna, Vienna, Austria. 4 Center for Molecular Medicine, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD, USA. 5 Wellcome Trust Sanger Institute, Hinxton, UK. 6 Present address: Life Science Institute, University of British Columbia, Vancouver, British Columbia, Canada. *e-mail: reiner.wimmer@imba.oeaw.ac.at; josef.penninger@imba.oeaw.ac.at2 4 J A N U A r Y 2 0 1 9 | V O L 5 6 5 | N A t U r e | 5 0 5© 2019 Springer Nature Limited. 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