Stem Cell Research
Human induced-pluripotent stem cells (iPSCs) can be generated from a large amount of adult somatic cells, reducing the risk for patients during surgical interventions for the isolation of primary cells. Human iPSCs further allow the generation of various cell types of the human body from an almost unfailing cell source. Additionally, ethical concerns are comparatively low, as patients/probands can be asked for permission.
The Stem Cell Research working group focus on the functional investigation of CAD-associated risk genes in stem cell models.
Additional projects in this group will be started soon, such as differentiation of iPSCs into vascular endothelial cells, and vascular organoids, which are 3D cell models of the human vasculature that allow interaction studies of all cell types located in the vessel.
Wilko Ziegler, MD candidate
Projects
Coronary artery disease (CAD) is the major cause of deaths worldwide. Many factors contribute to the development of CAD, such as lack of exercise, smoking, diabetes, malnutrition as well as genetics. Until today 163 CAD risk genes or loci have been identified in genome-wide association studies (GWASs) to be associated with CAD. Even though they all are associated with CAD they are involved in different pathways or mechanisms contributing to the progression of the disease including lipid metabolism, blood pressure, mitosis & proliferation, neovascularization or neoangiogenesis, NO-signaling, vascular remodeling, gene regulation, or inflammation. However, a subset of loci could not be assigned to a specific pathway or mechanism yet.Most commonly human cell lines, human primary cells or animal models are used to achieve data in cardiovascular research. Nevertheless, none of the before-mentioned models is an ideal model to investigate genetic causes of CAD in humans. Cell lines and primary cells are hard to obtain, and quickly lose their typical phenotypic behaviour. Results obtained in mice cannot be transferred to the human system as is common knowledge today. Therefore, human induced pluripotent stem cells (iPSCs) offer a great alternative as they overcome various problems that occur with primary cells. Stem cell derived 3D models, so-called organoids can be generated, allowing a more physiological situation that can be analyzed in vitro. Vascular organoids have first been described in 2019 by Rainer Wimmer and colleagues from iPSCs as well as embryonic stem cells. The described vascular organoids were made up of endothelial cells (ECs) as well as pericytes (PCs), connected to the blood system of mice after implantation and were perfused. We will use this model and further improve it to abstain from the use of animal contents, and to include vascular smooth muscle cells (VSMCs). We want to establish and improve this model. We will then use the model to investigate the role of CAD-associated genes like VEGFA and SMAD in angiogenesis or cell-cell communication.
Funded by the University of Lübeck 2022/2023
CAD and its complication atherosclerosis lead to a high number of deaths worldwide. Atherosclerosis, which is clogging of the vessels, is often accompanied by calcification besides lipid deposition and immune cell response. Calcium and phosphate is deposited within the atherosclerotic plaque and the smooth muscle cell layer in the vessel.
The CAD risk locus 9p21 has also been associated with vascular calcification. We used iPSCs of a healthy (nonrisk, NR) donor and a risk patient (R), containing the risk SNP, and differentiate them into VSMCs. Starting iPSCs as well as VSMCs are checked for cell typic characteristics, like morphology and gene expression, and behaviour. Cells of the NR and the R cell line do not show differences in cell type specific characteristics. However, cell behaviour is changed in the VSMCs of the R cell line.
In order to induce calcification in iPSC-derived VSMCs we apply a cocktail of calcium, phosphate and other crucial factors for 1 week. Calcification can be identified by staining, showing how much calcium and phosphate was deposited in the cells.
The 9p21 risk locus turned out to enhance calcification in iPSC-derived VSMCs of the R patient. This leads to the conclusion that 9p21 might be a driving factor for vascular calcification.
The functional basis for this aspect of the disease remains unclear and needs to be investigated.
Dilated cardiomyopathy (DCM) is a leading cause of death especially in the Western world. DCM is characterized by wall thinning and enlargement of the left heart ventricle leading to less effective pumping of the blood through the body. The disease is caused by several factors: life style factors such as diet and activity but also genetic risk factors play a major role in the development of the disease. Up to date >50 genes are identified to play a role in the development of dilated cardiomyopathy in patients. One of the genes identified is Nexilin (Nexn). So far Nexilin has been described as a structural protein at the Z-disk and numerous mutations have been described in patients with dilated and hypertrophic cardiomyopathy.
We aim to understand the role of Nexilin in the development of dilated cardiomyopathy better. We are working with human induced pluripotent stem cells (hiPSC) and hiPSC-derived cardiomyocytes with and without expression of Nexilin to elucidate its function during cardiomyocyte differentiation and afterwards in cardiomyocytes. Knocking out of Nexilin in the cells is done by using CRISPR/Cas9, a well known method to specifically knock out a gene of interest. Characterization of the cells is done at several time points throughout differentiation process and at DNA, RNA and protein level.
Finally, we hope to understand the role of Nexilin during development of DCM better to be able to design better diagnosis and ultimately better treatment tools.