Application of time-lapse imaging, single-cell tracking, and competing risks analysis to characterise cardiac stem cell growth dynamics

Abstract

Heart failure due to damaged heart muscle is amongst the most common cause of death worldwide. Adult stem cells hold promise for their potential to repair damaged heart muscle after injury. Cardiac colony forming unit fibroblasts (cCFU-F) are a population of cardiac-resident mesenchymal stem cells hypothesised to be involved in remodelling the heart after injury. Basic characterisation of cCFU-F in vitro has been a key to understanding their function and behaviour. However, heterogeneity of cCFU-F cultures has limited the utility of traditional assays that rely on population-based snapshots as they obscure details of single-cell behaviour. Therefore, the aim of this thesis was to apply time-lapse imaging and single-cell tracking to characterise cCFU-F growth dynamics at the single-cell level. In achieving this aim this thesis identified a lack of unbiased methods for analysing cell tracking data, since most studies have excluded right-censored and competing cell fates. Therefore, competing risks and concordance analysis are introduced. The utlity of these methods was demonstrated by quantifying breast cancer cellresponses to chemotherapy and concordance in fate for granulocyte macrophage progenitors and their differentiated progeny. Time-lapse imaging, single-cell tracking and competing risks analysis were then applied to characterise the onset of heterogeneity within cCFU-F cultures during adaption to in vitro culture, as well as the role of platelet-derived growth factor receptor alpha (PDGFR-alpha) signalling in regulating cCFU-F fate (division, death, and self-renewal). Cell cycle entry and progression were heterogeneous though correlated with cCFU-F colony size. Manipulation of PDGFR-alpha signalling modulated cCFU-F self-renewal probability, particularly for freshly isolated cCFU-F. The In vitro characterisation of cCFU-F has also been limited by the use of serum-containing medium which is undefined. Therefore, a serum-free medium was developed using factorial design experiments to identify cytokines that stimulated cCFU-F self-renewal. cCFU-F isolated and expanded in serum-free medium supplemented with cytokines achieved a greater number of population doublings and rate of self-renewal, yet had a similar molecular phenotype and potency to cCFU-F expanded using serum. In conclusion this study describes methodological advances for characterising adult stem cells in vitro and a detailed analysis of cCFU-F growth dynamics at the single-cell level.