Characterisation of cardiosphere derived cells :investigating hypoxic pre-conditioning on pro-angiogenic properties and tracking the cardiac fibroblast component

Abstract

Coronary heart disease is still the leading cause of death in the UK, despite significant advances in clinical treatments. Stem cell transplantation has the potential to improve cardiac function and patient outcome, but optimal cell types, cell preparation methods and cell delivery routes are yet to be established. The heart contains a small population of progenitor cells that, in culture, contribute to spontaneously formed spheroids known as cardiospheres (Csphs). Following further culture, Csphs give rise to cardiosphere derived cells (CDCs). Both Csphs and CDCs show paracrine benefit including neovascularisation in myocardial ischaemia, leading to improvement in heart function. The aims of this project were to use mouse models to (i) investigate the effect of hypoxic preconditioning on the pro-angiogenic potential of CDCs and (ii) characterise the contribution of cardiac fibroblasts (CFs) to CDCs. I used Col1a2-CreERT;Rosa26-STOP-YFP mice to track YFP-expressing CFs in myocardial tissue and in CDC culture. Co-staining experiments showed only partial overlap of YFP with other CF markers (vimentin and Fsp1) in heart tissue, which may be due to the heterogeneity of CFs and/or incomplete activation of YFP in CFs. I showed that CF-derived cells exist in all stages of CDC culture, and a small subset of these cells also expressed the stem cell markers Sca-1 or cKit, suggesting CF derived cells may contribute to the progenitor cell population. My results showed that preconditioning CDCs with 3%O2 enhances cell outgrowth from heart explants and promotes expression of stem cell and pro-angiogenic markers. I then assessed the pro-angiogenic potential of CDCs in vivo using a sub-dermal matrigel plug assay and showed that CDCs alone have limited pro-angiogenic potential. However, 3%O2 preconditioning of CDCs significantly enhances this process. Further research will increase our understanding of CDC-mediated angiogenesis and improve clinical therapies for MI patients.