Abstract
Recent large-scale GWA meta-analyses identified more than 240 risk loci associated with coronary artery disease (CAD) on a genome-wide significance level. Pathway analyses confirmed established pathomechanisms contributing to the progression of CAD like lipid metabolism, inflammation, extracellular matrix remodelling, or neoangiogenesis. Human induced pluripotent stem cells (iPSCs) offer a great source to derive 3D models, so-called organoids, allowing a more physiological environment to be analyzed in vitro . We aim to establish vascular organoids from iPSCs as 3D model of angiogenesis to investigate the role of CAD- and angiogenesis-associated risk genes like ADAMTS7 , ARNTL , and TBX20 .Four iPSC lines (UCSD18i-3-6, female, healthy; WC028i-5807-6, female, healthy; UCSD167i-99-1, female, healthy; UCSD179i-27-1, male, healthy; all from skin fibroblasts) will be differentiated into vascular organoids following the protocol published by Wimmer et al (2019). The first iPSC lines were differentiated to estimate the optimal seeding density for each cell line, and organoids were characterized. During embedding of aggregates for vascular sprouting organoids will be used for angiogenesis assay, and sprout length and number will be analysed. Different inhibitors and stimulators of angiogenesis can easily be applied proving that vascular organoids can be used as angiogenesis model. Using siRNA we will then knock-down (KD) above-mentioned risk genes, assessing their effect on angiogenesis. Additionally, cells will be edited using CRISPR/Cas to knockout the same risk genes.The first organoids showed expression of pericyte (PC) and endothelial cell (EC) markers on RNA level, while smooth muscle cell markers were not expressed. These results were confirmed in immunofluorescence stainings in whole organoids. Additionally, we performed cryosections which we also stained with immunofluorescence, showing an even distribution of cells. Staining of the cryosections further confirmed expression of PC- and EC markers. In conclusion, we successfully established iPSC-derived vascular organoids from iPSCs. We have now a 3D cell model in our hand to study GWAS candidate genes and their role in angiogenesis in the context of CAD.
Published Version
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