Human blood vessel organoids as a model of diabetic vasculopathy

Authors:
Wimmer RA, Leopoldi A, Aichinger M, Wick N, Hantusch B, Novatchkova M, Taubenschmid J, Hämmerle M, Esk C, Bagley JA, Lindenhofer D, Chen G, Boehm M, Agu CA, Yang F, Fu B, Zuber J, Knoblich JA, Kerjaschki D, Penninger JM.
In:
Source: Nature
Publication Date: (2019)
Issue: 565 (7740): 505-510
Research Area:
Stem Cells
Gene Expression
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Platform:
4D-Nucleofector® X-Unit
Experiment

A mammalian expression vector expressing Cas9 from S. pyogenes with a 2A-Puro cassette42 (Addgene, 62988) was cut with BbSI (Thermo Fisher ER1011) shortly after the U6 promoter. Subsequently, the plasmid was religated introducing single-guide RNAs (sgRNAs) for either NOTCH3 or DLL4. The following primers were used for sgRNA annealing with lowercase sequences indicating overhang sites for insertion into the vector: NOTCH3 forward, caccgGCCACTATGTGAGAACCCCG; NOTCH3 reverse,
aaacCGGGGTTCTCACATAGTGGCc. DLL4 forward, caccgCAGGAGTTCATCAACGAGCG; DLL4 reverse, aaacCGCTCGTTGATGAACTCCTGc. sgRNA plasmids were verified by Sanger sequencing and used for electroporation of iPS cells (NC8) with the 4D-Nucleofector System (Lonza). In brief, 2 µg plasmid DNA was transfected using the P3 Primary Cell 4D-Nucleofector Kit. Transfected NC8 cells were seeded on Matrigel-coated six-well plates in Essential 8 medium (Gibco) containing 50 µM Y27632 (Calbiochem) and cultured for 24 h before puromycin treatment (0.2 µg ml-1) for 48 h. Remaining cells were cultivated until colony formation could be observed and single colonies were further expanded for genotyping using Sanger sequencing. Knockout cell lines were verified by western blot or immunofluorescence staining.

Abstract

The increasing prevalence of diabetes has resulted in a global epidemic1. 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 cells2-4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. 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.