Mohd Yusof, Nurul Ain Nasim
(2020)
Human pluripotent stem cells derived-cardiomyocytes (hPSC-CMs) to assess the impact of drug- and gene-induced changes on cell function.
PhD thesis, University of Nottingham.
Abstract
The majority of data available for drug predictive safety and disease/polymorphism modelling are based on results from genetic association studies or from models other than humans, which can lead to conflicting mechanistic explanations. This thesis considers whether human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) could complement existing assays to improve drug safety screening and disease/polymorphism modelling by assessing the impact of drug- and gene-induced changes on cell function.
To evaluate the suitability of hPSC-CMs for drug safety screening, changes in contractility and electrophysiology were quantitated. Protocols for the CellOPTIQ:hiPSC-CMs combination were established and unified via a drug ‘training’ set, allowing for the blinded evaluation of up to 27 drugs with known positive, negative or neutral inotropic effects. This demonstrated that the accuracy of CellOPTIQ:hiPSC-CMs configuration ranged from 50 to 60%, which was lower than might have been expected from a human cell-based system. Nevertheless, contraction and relaxation time appeared to be informative for positive inotropes, while contraction amplitude was informative for negative inotropes. Moreover, accuracy was further improved and increased to 75% (for CellOPTIQ) by refining the conditions that would reduce beat rate, increasing the signal to noise ration and enabling chronic long-term drug testing. The results show that hiPSC-CMs could be used to reduce socioeconomics cost and assist regulatory guidelines governing cardiac safety assessments.
To evaluate whether hPSC-CMs could be used to understand the gene-induced changes and influence of polymorphic variation in the genome on cell behaviour, the ADRB2 locus (encodes βeta2-adrenergic receptor; β2AR) was used as an exemplar. This is because the action of beta-blockers may be influenced by polymorphisms within the β2AR, particularly at amino acid positions 16 and 27, and there is a potential association with cardiovascular disorders such as heart failure.
To study β2AR polymorphism differences against a constant genetic background, CRISPR/Cas9 technology was used to genome-edit the HUES7 hESC line to produce 4 isogenic β2AR variants (single amino acid codes: GE, GQ, RE, RQ). The isogenic variants were able to retain their pluripotency and differentiation capabilities into hPSC-CMs whilst retaining responsiveness to β2AR pharmacology.
For the functional assessment, an initial phenotypic analysis was carried out by luciferase-based real-time cAMP (cyclic adenosine monophosphates) assay to estimate the differential response during acute and chronic agonist exposure to individual β2AR variants. It was found that RQ has the lower initial β2AR density, GQ has the highest receptor desensitisation, Arg16 was resistant to desensitisation and GQ tended towards being the most downregulated.
Since β2AR is regulated by multiple complex processes and their regulation has essential effects on signal transduction, it is expected that these isogenic models will help to understand the mechanisms that underlie ‘disease’ and drug- and gene-induced states. Moreover, the work shows how coupling hPSC-CMs with CRISPR/Cas9 technologies can be used to personalise diagnosis and identify new pathogenic variants for cardiovascular disease.
Thus, this thesis shows there is value in exploring the use of hPSC-CMs in assessing the impact of drug-induced changes on cell function, and provide a tool to gain mechanistic insights into how polymorphisms in the genome alter signalling cascades.
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