Central to this thesis was the use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to model a rare form of mitochondrial cardiomyopathy. To model this disease required the... Show moreCentral to this thesis was the use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to model a rare form of mitochondrial cardiomyopathy. To model this disease required the design of new methodologies, improving upon the current limitations of hiPSC-CMs as model systems, notably the variability and the immature state of the resulting cardiomyocytes. The diseases we were interested in manifest soon after birth. At the outset of the studies, it was unclear whether a phenotype would be evident in our standard immature 2D cultures, or whether more complex models would be required to capture more salient features of the condition. Our focus was on the rare mitochondrial disease Combined Oxidative Phosphorylation Deficiency, caused by mutations in the gene alanyl-tRNA synthetase 2. We refined our emerging 3D cardiac microtissue protocol to improve robustness and reproducibility and reduce cost by basing differentiation on small molecules rather than growth factors without altering the functionality of hiPSC-CMs. We also demonstrated that integrating pH and O2 sensors in a micro-physiological chip was possible for the assessment of metabolic parameters under microfluidic flow. The overall goal of this thesis was to provide additional tools that would have utility in studying mitochondrial and other cardiac diseases. Show less
Cardiac arrhythmias are a common cause of sudden death worldwide. However, despite decades of thorough investigation the underlying biophysical mechanisms of cardiac arrhythmias are still... Show moreCardiac arrhythmias are a common cause of sudden death worldwide. However, despite decades of thorough investigation the underlying biophysical mechanisms of cardiac arrhythmias are still insufficiently understood due to incomplete theories and the lack of precise spatiotemporal control in experiments. In the last decade, the problem of insufficient spatiotemporal control has started to be tackled by means of a new technique, called optogenetics. This technique employs expression of light-activated proteins, which are activated or deactivated in time and space by switching on/off light (in the near-ultraviolet to near-infrared wavelength range) in specific patterns thus realizing fully biological spatiotemporal control. However, with a few notable exceptions, cardiac optogenetic studies have only confirmed previously known mechanisms and yielded no or little novel mechanistic insights. In this thesis, to fill this gap, we combined nonlinear dynamics theory, numerical simulations and optogenetic experiments with unique spatiotemporal control to theoretically predict and demonstrate novel arrhythmogenic phenomena in cardiac tissue. Thanks to the robustness of the optogenetics methods and generality of the applied theories and computations, this thesis uncovered novel mechanisms for the biophysics of cardiac tissue that are applicable to the functioning of excitable systems in general. Show less
Windt, L.M.; Wiendels, M.; Dostanic, M.; Bellin, M.; Sarro, P.M.; Mastrangeli, M.; ... ; Meer, B.J. van 2023
Human heart tissues grown as three-dimensional spheroids and consisting of different cardiac cell types derived from pluripotent stem cells (hiPSCs) recapitulate aspects of human physiology better... Show moreHuman heart tissues grown as three-dimensional spheroids and consisting of different cardiac cell types derived from pluripotent stem cells (hiPSCs) recapitulate aspects of human physiology better than standard two- dimensional models in vitro. They typically consist of less than 5000 cells and are used to measure contraction kinetics although not contraction force. By contrast, engineered heart tissues (EHTs) formed around two flexible pillars, can measure contraction force but conventional EHTs often require between 0.5 and 2 million cells. This makes large-scale screening of many EHTs costly. Our goals here were (i) to create a physiologically relevant model that required fewer cells than standard EHTs making them less expensive, and (ii) to ensure that this miniaturized model retained correct functionality. We demonstrated that fully functional EHTs could be generated from physiologically relevant combinations of hiPSC-derived cardiomyocytes (70%), cardiac fibro-blasts (15%) and cardiac endothelial cells (15%), using as few as 1.6 ×104 cells. Our results showed that these EHTs were viable and functional up to 14 days after formation. The EHTs could be electrically paced in the frequency range between 0.6 and 3 Hz, with the optimum between 0.6 and 2 Hz. This was consistent across three downscaled EHT sizes tested. These findings suggest that miniaturized EHTs could represent a cost-effective microphysiological system for disease modelling and examining drug responses particularly in secondary screens for drug discovery. Show less
Studying diseases or effects of new drugs on the human body is challenging, not least because of the lack of proper testing models that recapitulate human physiology. Most research is done using... Show moreStudying diseases or effects of new drugs on the human body is challenging, not least because of the lack of proper testing models that recapitulate human physiology. Most research is done using animal models but these often show differences with humans in disease manifestation and responses to drugs. For example, drugs that had no effect on the hearts of animals later turned out to cause lethal arrhythmias in some humans. This – and the ethical issues around animal testing – is why, as in this thesis, there is increasing interest on making human heart models based on pluripotent stem cells (hPSCs). Nowadays small numbers of cells can be collected from a patient (e.g. from blood, urine or skin), reprogrammed to hiPSCs, and then differentiated to heart muscle cells (cardiomyocytes). Since the genetics of the patient are maintained during reprogramming, the phenotype of a genetic disease affecting cardiac function can also be captured. The focus of this thesis has been developing methods to measure these cardiac phenotypes robustly and with sufficient complexity to reflects drug responses and disease of the heart. Our results supported the notion that hPSC models will become human avatars and accurate measurement models able to recapitulate essential human-specific processes. Show less
General aim of this thesis, entitled Guide to the heart, was to explore the generation of multiple human pluripotent stem cell (hPSC)-derived cardiac subtypes and their application for selective... Show moreGeneral aim of this thesis, entitled Guide to the heart, was to explore the generation of multiple human pluripotent stem cell (hPSC)-derived cardiac subtypes and their application for selective pharmacology, understanding human cardiac development and cardiac repair.Approaches for the differentiation of hPSCs to cardiomyocytes (CMs) followed by purification from heterogeneous cultures are described. To generate subtype specific CMs, a protocol for the derivation and characterization of hPSC-derived CMs with atrial identity was developed. HPSC-derived atrial CMs have proven successful as pre-clinical pharmacological tool. The development of a human atrial reporter by CRISPR/Cas9-mediated knockin of red fluorescent mCherry into the genomic locus of atrial-enriched COUP-TFII allowed the selection of atrial and ventricular CMs. In addition, we evaluated the importance of COUP-TFII for atrial differentiation of hPSC and identified that COUP-TFII is dispensable for atrial differentiation of hPSCs. Importantly, hPSC-derived cardiac progenitors (CPCs) alleviated ventricular remodeling and fibrosis after transplantation to the heart in an acute myocardial infarction model in mice. This thesis ends with a review of the native cardiac environment during development, as well as the adult heart in health and disease. This was used to describe current knowledge regarding extracellular matrix preferences for engineering cardiac tissues from hPSC-CMs. Show less
Cell transplantation studies have shown that injection of progenitor cells can improve cardiac function after myocardial infarction (MI). Transplantation of human cardiac progenitor cells (hCPCs)... Show moreCell transplantation studies have shown that injection of progenitor cells can improve cardiac function after myocardial infarction (MI). Transplantation of human cardiac progenitor cells (hCPCs) results in an increased ejection fraction, but survival and integration are low. Therefore, paracrine factors including extracellular vesicles (EVs) are likely to contribute to the beneficial effects. We investigated the contribution of EVs by transplanting hCPCs with reduced EV secretion. Interestingly, these hCPCs were unable to reduce infarct size post-MI. Moreover, injection of hCPC-EVs did significantly reduce infarct size. Analysis of EV uptake showed cardiomyocytes and endothelial cells primarily positive and a higher Ki67 expression in these cell types. Yes-associated protein (YAP), a proliferation marker associated with Ki67, was also increased in the entire infarcted area. In summary, our data suggest that EV secretion is the driving force behind the short-term beneficial effect of hCPC transplantation on cardiac recovery after MI. Show less
The focus of this thesis is about cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs). hPSC-CMs are an established model used to study cardiac diseases as well as a good tool for... Show moreThe focus of this thesis is about cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs). hPSC-CMs are an established model used to study cardiac diseases as well as a good tool for drug efficacy and cardiotoxicity studies. The core aspect of the thesis is about hPSC-CMs derived from patients carrying genetic mutations that result in a cardiac arrhythmia known as Brugada syndrome. With these cells we manage to model the disease in vitro at the cellular level and assess the impact of specific drugs (readthrough-promoting) in an attempt to provide a valuable clinical perspective. The use of novel genetic engineering techniques (CRISPR-Cas9) helped us to further understand the link between the genotype of the patients and the phenotype of the disease. Furthermore the thesis contains our research attempts to improve hPSC-CMs so that they resemble more to adult human cardiomyocytes, since this would increase the validity of this model system and help us understand the nature of even more cardiac diseases. Show less
The research described in this thesis focuses on the use of both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to understand cardiac lineage development and disease. To... Show moreThe research described in this thesis focuses on the use of both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to understand cardiac lineage development and disease. To investigate the possibility of studying inherited cardiac diseases, we compared pluripotent stem cell-derived cardiomyocytes by investigating both a mouse and human model of a complex cardiac overlap syndrome caused by a mutation in the gene SCN5A. We demonstrated that both ESC- and iPSC-derived cardiomyocytes can recapitulate the characteristics of the disease. Furthermore this thesis describes a detailed protocol to differentiate human pluripotent stem cells to cardiomyocytes that was applied in the study comparing hESC- and hiPSC- derived cardiomyocytes at several time points during cardiac differentiation. We targeted fluorescent marker GFP to one allele of NKX2-5 in a human iPSC line that now matched a similar human ESC reporter line previously generated in the laboratory. This offered the opportunity to obtain cardiomyocytes and their precursors at different time points during the differentiation and determine the true degree of similarity between both pluripotent stem cell sources. Additionally the same hESC- and hiPSC-derived cardiomyocytes were compared to a unique set of foetal heart samples. Show less
This thesis describes research about the differentiation of human stem cells into cardiomyocytes (heart cells). During the differentiation process the stem cells become contractile myocytes... Show more This thesis describes research about the differentiation of human stem cells into cardiomyocytes (heart cells). During the differentiation process the stem cells become contractile myocytes that resemble the native heart cells. Nevertheless, the phenotype of these cardiomyocytes is comparable to a second trimester fetal heart cells. These thesis describes different approaches to induce maturity into the stem cell derived cardiomyocytes, such as changing the substrate stiffness, cell patterning and small molecule based medium. The maturation level achieved unveiled the contractile phenotype on cardiomyocytes derived from patients with hypertrophic cardiomyopathy. The end of this thesis describes a novel method to culture and expand multipotent cardiac progenitor cells. Show less
Cardiovascular disease is responsible for 17 million deaths globally each year. Research aimed at understanding the form and function of this vital organ will be key to improving patient care.... Show moreCardiovascular disease is responsible for 17 million deaths globally each year. Research aimed at understanding the form and function of this vital organ will be key to improving patient care. Although animal models such as rat, rabbit and dog have proven to be valuable to study heart physiology, there are a number of important species-specific differences, for e.g. heart rate, when compared with the human heart. In the recent years, research on cardiomyocytes(CMs) derived from human pluripotent stem cells has demonstrated that they resemble native human CMs and hence, make excellent models to study heart development and disease in vitro.In this dissertation,we present studies that describe the use of hPSC-CMs for pharamacological testing and for modeling inherited arrhythmogenic disorders.Combined with other novel technologies in the fields of genetic medicine, tissue engineering,and genome editing, hPSC-CM models will be valuable for 1) understanding lineage decisions determining CM specification, 2) unraveling molecular basis of disease, 3) translational applications such as target/drug discovery, diagnostic medicine and developing effective treatment strategies. Show less
It is critical to gain knowledge in the underlying mechanisms that control human cardiovascular developm ent, which helps us to understand the onset of congenital cardiovascular diseases, and to... Show moreIt is critical to gain knowledge in the underlying mechanisms that control human cardiovascular developm ent, which helps us to understand the onset of congenital cardiovascular diseases, and to develop optimal culture methods for efficient in vitro cardiomyocyte differentiation from hPSCs, which are of interest for final translational applications including screening and efficacy assays for disease modelling, drug discovery and development, personalized medicine, and perhaps the regeneration of cardiovascular tissues for therapeutic purposes. In this thesis, we show how genetic manipulation of human pluripotent stem cells (hPSCs), resulting in the genomic integration of a fluorescent protein encoding sequence at the locus of a key cardiac transcription factor, allows us to visualize and isolate early pre-cardiac progenitors subpopulations, and to study the molecular mechanisms involved in their further differentiation to cells of the cardiac lineage, including smooth muscle cells, endothelial cells, and cardiomyocyte subtypes. Show less
Kosmidis, G.; Bellin, M.; Ribeiro, M.C.; Meer, B. van; Ward-van Oostwaard, D.; Passier, R.; ... ; Casini, S. 2015
Since the first reports of human induced pluripotent stem cells (hiPSC), the field of pluripotent stem cell (PSC) research has grown in leap and bounds, particularly in the area of (cardiac)... Show moreSince the first reports of human induced pluripotent stem cells (hiPSC), the field of pluripotent stem cell (PSC) research has grown in leap and bounds, particularly in the area of (cardiac) disease modeling. This is in part because it is fairly easy to produce cardiomyocytes from hPSC and also there are now more assays available which can determine phenotypic behavior. This thesis describes and discusses improvements to reprogramming technology as well as its use in cardiac development and disease modeling Show less
To treat various cardiac diseases, modification of gene expression for the purpose of increased or decreased expression of a particular gene, is regarded as a potential therapy. As a vehicle to... Show moreTo treat various cardiac diseases, modification of gene expression for the purpose of increased or decreased expression of a particular gene, is regarded as a potential therapy. As a vehicle to introduce the gene of choice into the heart cell, virus vectors have given the most promising results. This thesis describes studies that are undertaken to investigate how virus vectors may be used to efficiently target cardiac cells and what the effects of certain genetic interventions are on the (patho)physiology of heart cells. We used lentivirus vectors to study the effects of integrin stimulation in neonatal rat cardiomyocytes on the uptake of macromolecules by these cells and through which pathway integrin stimulation leads to cardiac hypertrophy. Furthermore, the role of gap junctional coupling in the development of arrhythmias and in the cardiac differentiation of mesenchymal stem cells was investigated by modulating the expression of connexin 43 using lentivirus vectors. As adeno-associated virus vectors in particular have shown great potential as vector system to target the heart, we aimed to develop AAV vectors that may be used to specifically target either the cardiomyocytes or fibroblasts in the heart. Show less
