Complexity is not necessarily expected from monogenic diseases but for many cardiovascular diseases (CVD), simple genotype-phenotype relationship may be far from reality. As millions of people... Show moreComplexity is not necessarily expected from monogenic diseases but for many cardiovascular diseases (CVD), simple genotype-phenotype relationship may be far from reality. As millions of people globally die of CVD, it is important to find models to study CVD that recapitulate the conditions as manifest in humans, most importantly for these cases of unexpected complexity. For these, simple gene mutation or deletion in mice has often failed. Combining human induced pluripotent stem cell (hiPSC) with genetic editing technologies is providing new opportunities to bridge the gap, with many hPSC-CM models now showing promising results for testing drugs, discovering molecular pathways associated with disease and other types of (gene) therapies. The work in this thesis contributes to this area of research. Show less
Inserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease... Show moreInserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR-Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation) by (1) inserting various combinations of fluorescent reporters into hiPSCs to assess the excitation-contraction coupling cascade in derivative cardiomyocytes and (2) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorders into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically matched panels of hiPSC lines efficiently and cost effectively. Show less
Brink, L. van den; Brandao, K.O.; Yiangou, L.; Blanch Asensio, A.; Mol, M.P.H.; Mummery, C.L.; ... ; Davis, R.P. 2021
While rare mutations in ion channel genes are primarily responsible for inherited cardiac arrhythmias, common genetic variants are also an important contributor to the clinical heterogeneity... Show moreWhile rare mutations in ion channel genes are primarily responsible for inherited cardiac arrhythmias, common genetic variants are also an important contributor to the clinical heterogeneity observed among mutation carriers. The common single nucleotide polymorphism (SNP) KCNH2-K897T is associated with QT interval duration, but its influence on the disease phenotype in patients with long QT syndrome type 2 (LQT2) remains unclear. Human induced pluripotent stem cells (hiPSCs), coupled with advances in gene editing technologies, are proving an invaluable tool for modeling cardiac genetic diseases and identifying variants responsible for variability in disease expressivity. In this study, we have used isogenic hiPSC-derived cardiomyocytes (hiPSC-CMs) to establish the functional consequences of having the KCNH2-K897T SNP in cis- or trans-orientation with LQT2-causing missense variants either within the pore-loop domain (KCNH2A561T/WT) or tail region (KCNH2N996I/WT) of the potassium ion channel, human ether-a-go-go-related gene (hERG). When KCNH2-K897T was on the same allele (cis) as the primary mutation, the hERG channel in hiPSC-CMs exhibited faster activation and deactivation kinetics compared to their trans-oriented counterparts. Consistent with this, hiPSC-CMs with KCNH2-K897T in cis orientation had longer action and field potential durations. Furthermore, there was an increased occurrence of arrhythmic events upon pharmacological blocking of hERG. Collectively, these results indicate that the common polymorphism KCNH2-K897T differs in its influence on LQT2-causing KCNH2 mutations depending on whether it is present in cis or trans. This study corroborates hiPSC-CMs as a powerful platform to investigate the modifying effects of common genetic variants on inherited cardiac arrhythmias and aids in unraveling their contribution to the variable expressivity of these diseases. Show less
Brandao, K.O.; Brink, L. van den; Miller, D.C.; Grandela, C.; Meer, B.J. van; Mol, M.P.H.; ... ; Davis, R.P. 2020
Mutations in KCNH2 can lead to long QTsyndrome type 2. Variable disease manifestation observed with this channelopathy is associated with the location and type of mutation within the protein,... Show moreMutations in KCNH2 can lead to long QTsyndrome type 2. Variable disease manifestation observed with this channelopathy is associated with the location and type of mutation within the protein, complicating efforts to predict patient risk. Here, we demonstrated phenotypic differences in cardiomyocytes derived from isogenic human induced pluripotent stem cells (hiPSC-CMs) genetically edited to harbor mutations either within the pore or tail region of the ion channel. Electrophysiological analysis confirmed that the mutations prolonged repolarization of the hiPSC-CMs, with differences between the mutations evident in monolayer cultures. Blocking the hERG channel revealed that the pore-loop mutation conferred greater susceptibility to arrhythmic events. These findings showed that subtle phenotypic differences related to KCNH2 mutations could be captured by hiPSC-CMs under genetically matched conditions. Moreover, the results support hiPSC-CMs as strong candidates for evaluating the underlying severity of individual KCNH2 mutations in humans, which could facilitate patient risk stratification. Show less
Brink, L. van den; Brandao, K.O.; Yiangou, L.; Mol, M.P.H.; Grandela, C.; Mummery, C.L.; ... ; Davis, R.P. 2020
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a powerful platform for in vitro modelling of cardiac diseases, safety pharmacology and drug screening. All... Show moreHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a powerful platform for in vitro modelling of cardiac diseases, safety pharmacology and drug screening. All these applications require large quantities of well-characterised and standardised batches of hiPSC-CMs. Cryopreservation of hiPSC-CMs without affecting their biochemical or biophysical phenotype is essential for facilitating this, but ideally requires the cells being unchanged by the freeze-thaw procedure. We therefore compared the in vitro functional and molecular characteristics of fresh and cryopreserved hiPSC-CMs generated from multiple independent hiPSC lines. While the frozen hiPSC-CMs exhibited poorer replating than their freshly-derived counterparts, there was no difference in the proportion of cardiomyocytes retrieved from the mixed population when this was factored in, although for several lines a higher percentage of ventricular-like hiPSC-CMs were recovered following cryopreservation. Furthermore, cryopreserved hiPSC-CMs from one line exhibited longer action potential durations. These results provide evidence that cryopreservation does not compromise the in vitro molecular, physiological and mechanical properties of hiPSC-CMs, though can lead to an enrichment in ventricular myocytes. It also validates this procedure for storing hiPSC-CMs, thereby allowing the same batch of hiPSC-CMs to be used for multiple applications and evaluations. Show less
