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Title: Towards the in vitro modelling of the cardiac channelopathies using human induced pluripotent stem cells
Author: Rajamohan, Divya
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2013
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LQTl (Long QT Syndrome 1) and CPVTl (Catacholaminergic polymorphic ventricular tachycardia-i) are two commonly prevalent cardiac channelopathies. Whilst LQTl accounts for ~45-54% of all genotyped cases of LQTS (Medeiros-Domingo et al., 2007), CPVTl is thought to be accountable for ~15% of all cases of sudden cardiac death (Priori et ai, 2002). Limitations to currently available models of these disorders include the inter-specific differences in cardiac electrophysiology associated with animal models, the lack of a multi- ion channel phenotype in recombinant cell models and the limited availability of human cardiac tissue (Rajamohan et ai, 2013). This necessitates the development of humanised hPSC-based models of LQTl and CPVTl that can: 1) help better model and understand their pathogenesis and 2) aid in the development of new strategies for their treatment. Skin fibroblasts were derived from 3x LQTl patients and lx CPVTl patient. These were reprogrammed into human induced pluripotent stem cells (hiPSCs) and subsequently differentiated into functional cardiomyocytes (hiPSC-CMs). Spontaneously beating cells were analysed, by multi-electrode array and patch clamp technologies, for their electrophysiology and response to pharmacology. Initial experiments have shown that, relative to controls, LQTlhiPSC- CMs exhibit prolonged action potential durations under basal conditions, and that CPVTl-afflicted hiPSC-CMs develop putative DADs on treatment with isoprenaline - electrophysiological hallmarks of LQT and CPVT, respectively. Further experiments are ongoing to confirm these observations and to demonstrate that these iPSC models are capable of responding appropriately to a range of clinically relevant pharmaceuticals. This will ascertain the suitability of these cells as humanised assays for the evaluation of new treatments of LQTl and CPVTl, respectively. This study also looked at deriving hiPSCs from a range of different tissue types in order to compare the feasibility and efficiencies of hiPSC generation from different cell sources and to give potential donors the option of making tissue donations in a manner that is less invasive and painful than a skin biopsy. In this regard, hiPSCs were successfully derived from skin, tooth and oral gingival samples. Finally, steps were taken to increase the feasibility of the use of hPSC-CMs as large-scale pharmaceutical screens. First, an improvement in the cardiac differentiation of hiPSCs was achieved by modulation of the BMP/Activin/Nodal cardiac signalling pathways. Next, in collaboration with engineers at Loughborough University, a novel MEA (multi-electrode array) II biochip was designed and fabricated with a view to allow cost-effective and high-throughput MEA-based assessment of hPSC-CM electrophysiology. Lastly, in collaboration with scientists at BioFocus (Cambridge), steps were taken to translate the patch clamp analysis of hPSC-CMs onto a commercially available robotic, planar patch platform - The PatchXpress (Molecular Devices). III
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available