To improve the predictive capability of pre-clinical models and reduce the use of animal models in drug discovery and disease modelling, advanced in vitro models are being developed. These... Show moreTo improve the predictive capability of pre-clinical models and reduce the use of animal models in drug discovery and disease modelling, advanced in vitro models are being developed. These microphysiological systems (MPS) or “Organs-on-Chip” (OoC) are being developed to include all aspects of the human physiology to improve the in vitro cellular response. OoCs combined with differentiated human induced pluripotent stem cells (hiPSC) allow the use of cells with patient specific genotypes and aid the development of personalized and precision medicine.In this thesis, the development of tractable models of the vasculature is described. These models allow for the combination of hiPSC-derived vascular and tissue specific cells with haemodynamics to recapitulate essential stimuli of blood vessels. Show less
Ebola virus can trigger a release of pro-inflammatory cytokines with subsequent vascular leakage and impairment of clotting finally leading to multiorgan failure and shock after entering and... Show moreEbola virus can trigger a release of pro-inflammatory cytokines with subsequent vascular leakage and impairment of clotting finally leading to multiorgan failure and shock after entering and infecting patients. Ebola virus is known to directly target endothelial cells and macrophages, even without infecting them, through direct interactions with viral proteins. These interactions affect cellular mechanics and immune processes, which are tightly linked to other key cellular functions such as metabolism. However, research regarding metabolic activity of these cells upon viral exposure remains limited, hampering our understanding of its pathophysiology and progression. Therefore, in the present study, an untargeted cellular metabolomic approach was performed to investigate the metabolic alterations of primary human endothelial cells and M1 and M2 macrophages upon exposure to Ebola virus-like particles (VLP). The results show that Ebola VLP led to metabolic changes among endothelial, M1, and M2 cells. Differential metabolite abundance and perturbed signaling pathway analysis further identified specific metabolic features, mainly in fatty acid-, steroid-, and amino acid-related metabolism pathways for all the three cell types, in a host cell specific manner. Taken together, this work characterized for the first time the metabolic alternations of endothelial cells and two primary human macrophage subtypes after Ebola VLP exposure, and identified the potential metabolites and pathways differentially affected, highlighting the important role of those host cells in disease development and progression. Show less
A bicuspid aortic valve (BAV) is a congenital heart defect in which the heart valve between the left ventricle and the aorta consists of two valve flaps instead of the normal three (tricuspid... Show moreA bicuspid aortic valve (BAV) is a congenital heart defect in which the heart valve between the left ventricle and the aorta consists of two valve flaps instead of the normal three (tricuspid aortic valve – TAV). In a large proportion of people with a BAV, calcification of the heart valves or a widening of the aorta occurs early in life. It is not yet clear why patients with a BAV are prone to develop valvular calcification and aortic dilatation. The aim of the research described in this thesis is to study the pathogenesis of aortic valve calcification and aortic dilatation in BAV patients with a focus on the role of endothelial cells in these processes. With the research described in this thesis, we have shown that endothelial cells from BAV patients respond differently than cells from people with a TAV. In addition, the endothelial cell activation in the vascular wall of BAV patients is different and dependent on the blood flow. We found two good tissue culture methods to study heart valve calcification and used them to study the role of the protein FHL2 in this process. Show less
Abnormal vascular physiology and precipitating inflammatory pathways underlie many different diseases, including hemorrhage, stroke, vascular dementia and even cancer. Pluripotent stem cells (PSCs)... Show moreAbnormal vascular physiology and precipitating inflammatory pathways underlie many different diseases, including hemorrhage, stroke, vascular dementia and even cancer. Pluripotent stem cells (PSCs) can now be derived by reprogramming from any individual so that it is possible in principle to derive all somatic cells of the human body that would normally be difficult to access. In this thesis, I studied the derivation of myeloid cells from human induced pluripotent stem cells (hiPSCs) to model the inflammatory component of vascular disease and characterized the development path of hiPSC-derived endothelial cells (hiPSC-ECs) which form the vascular walls. Functional defects in either of these cell types can cause or exacerbate vascular disease. I then used these cell types to gain insight into the mechanisms underlying two genetic diseases: Hereditary Hemorrhagic Telangiectasia (HHT) which is caused by mutations in a gene called Endoglin expressed on cells of the vascular wall and inflammatory macrophages, and a vascular tumor called Pseudomyogenic hemangioendothelioma (PHE) in which endothelial cells are thought to be the tumor cell of origin. I developed new differentiation protocols to generate inflammatory cells from hiPSC, characterized these cells functionally and used Next-Generation Sequencing and bioinformatic analysis to gain insight into the molecular pathways controlling development of one particular type of endothelial cells from hiPSC and the underlying tumorigenic mechanisms of PHE. Show less
Von Willebrand disease (VWD) is the most common inherited bleeding disorder. Patients mainly develop mucocutaneous bleeding, like bruises, epistaxis and menorrhagia. The more severely affected... Show moreVon Willebrand disease (VWD) is the most common inherited bleeding disorder. Patients mainly develop mucocutaneous bleeding, like bruises, epistaxis and menorrhagia. The more severely affected patients may also develop joint bleeding, or bleeding from the gastrointestinal tract. Also, trauma, surgery or dental procedures may lead to critical bleeding events. VWD-related bleeding are caused by defects in von Willebrand factor (VWF), a large multimeric protein that is produced by endothelial cells and megakaryocytes. Most VWD patients develop the disease because of dominant-negative mutations in VWF. In this thesis we investigated whether inhibition of production of mutant VWF with limited inhibition of wildtype VWF positively affects the function of VWF and improves VWD phenotypes. We used small interfering RNAs (siRNAs) to selectively inhibit production of mutant VWF. These siRNAs were tested in several models for VWD. We indeed prove that siRNAs can distinguish a mutant and wildtype VWF allele in vitro in heterologous cell systems, ex vivo in patient-derived endothelial cells, and in vivo in a VWD mouse model. We also show in these disease models that we can improve several VWD phenotypes. These results are promising for further development of allele-specific siRNAs as a new treatment strategy for VWD. Show less
Koning, M.; Berg, C.W. van den; Rabelink, T.J. 2019
Kidney organoids can be generated from human pluripotent stem cells (PSCs) using protocols that resemble the embryonic development of the kidney. The renal structures thus generated offer great... Show moreKidney organoids can be generated from human pluripotent stem cells (PSCs) using protocols that resemble the embryonic development of the kidney. The renal structures thus generated offer great potential for disease modeling, drug screening, and possibly future therapeutic application. At the same time, use of these PSC-derived organoids is hampered by lack of maturation and off-target differentiation. Here, we review the main protocols for the generation of kidney organoids from human-induced PSCs, discussing their advantages and limitations. In particular, we will focus on the vascularization of the kidney organoids, which appears to be one of the critical factors to achieve maturation and functionality of the organoids. Show less
Aims High-fat diet-induced obesity (DIO) is amajor contributor to type II diabetes and micro-andmacro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of... Show moreAims High-fat diet-induced obesity (DIO) is amajor contributor to type II diabetes and micro-andmacro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of induced pluripotent stemcell-derived endothelial cells (iPSC-ECs) fromobese individuals could potentially limit their therapeutic efficacy forPVD. The aimof this studywas to compare the function of iPSC-ECs from normal and DIO mice using comprehensive in vitro and in vivo assays.Methods and results Six-week-old C57Bl/6 micewere fed with a normal or high-fat diet. At 24weeks, iPSCs were generated fromtail tip fibroblasts and differentiated into iPSC-ECs using a directed monolayerapproach. In vitro functional analysis revealed that iPSCECs from DIO mice had significantly decreased capacity to form capillary-like networks, diminished migration, and lower proliferation. Microarray and ELISA confirmed elevated apoptotic, inflammatory, and oxidative stress pathways in DIO iPSC-ECs. Following hindlimb ischaemia, mice receiving intramuscular injections of DIO iPSC-ECs had significantly decreased reperfusion compared with mice injected with control healthy iPSC-ECs. Hindlimb sections revealed increased muscle atrophy and presence of inflammatory cells in mice receiving DIO iPSC-ECs. When pravastatin was co-administered to mice receiving DIO iPSC-ECs, a significant increase in reperfusionwas observed; however, this beneficial effect was blunted by co-administration of the nitric oxide synthase inhibitor, Nv-nitro-L-arginine methyl ester.Conclusion This is the first study to provide evidence that iPSC-ECs from DIO mice exhibit signs of endothelial dysfunction and have suboptimal efficacy following transplantation in a hindlimb ischaemia model. These findings may have important implications for future treatment of PVD using iPSC-ECs in the obese population. Show less
Egorova, A.D.; Heiden, K. van der; Poelmann, R.E.; Hierck, B.P. 2012