AbstractThe purpose of this PhD project was to validate induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) as a model system for studying alterations at the cellular level that are seen in cardiomyopathy in situ. Initial work involved acquiring iPSC reprogramming, iPSC culture and cardiac differentiation techniques in collaboration with The University of Göttingen. Healthy control iPSC-CMs were validated for their pluripotency and then characterised for their immaturity on the protein level through immunofluorescence. Long term culture, 3D culture and/or high-density plating were used to promote cardiomyocyte maturation, with successes including the presence of M-protein, and decreased expression of embryonic heart (EH-) myomesin, α-smooth muscle actin and vimentin. Engineered heart tissues (EHTs) were established as a model for use in this study as part of an effort to include them for both the analysis of maturation of iPSC-CMs and for functional comparisons between iPSC-CM disease models.
iPSC-CM cardiomyopathy cell lines were generated from a healthy control cell line in order to develop disease models with isogenic controls. A missense point mutation, D94A, was introduced into myosin regulatory light chain 2 (MYL2) using CRISPR/Cas9 technology, in order to generate a dilated cardiomyopathy (DCM) model. Similarly, a missense point mutation, A13T, was separately introduced into MYL2 to generate a hypertrophic cardiomyopathy (HCM) model. These edited iPSCs were differentiated in vitro into cardiomyocytes to allow their cytoarchitecture to be explored using techniques such as immunocytochemistry followed by confocal microscopy. Preliminary data indicated a potential increase in NRAP expression and decrease in β-catenin expression in the DCM line when compared with the isogenic control. Additionally, a DCM patient derived iPSC-CM line harbouring a mutation in RBM20 was explored, with findings including sarcomere irregularity and altered cell-cell contacts.
In conclusion, the present study generated iPSC-CMs with a degree of maturation as a result of 3D culture, long-term culture and/or high-density plating. Several known DCM related phenotypes were further validated using both transgenic mouse hearts and iPSC-CM DCM models. Cell lines for DCM and HCM as a result of mutations in MYL2 encoding the regulatory light chain (RLC) were generated. These cell lines can be used for establishing markers and potentially identifying disease specific therapeutics, as well as enabling a greater understanding of MYL2 mutations in cardiomyopathies.
|Date of Award||1 Aug 2021|
|Supervisor||Elisabeth Ehler (Supervisor) & Katrin Streckfuss-Bömeke (Supervisor)|