Stimulation of Stem Cell Survival, Proliferation and Differentiation for Endogenous Cardiac Repair

Abstract

Heart failure as a consequence of myocardial infarction (MI) remains the leading cause of morbidity and mortality worldwide, taking more lives than all cancers combined. Since the loss of functional cardiomyocytes underpins the pathophysiology of MI and subsequent development of heart failure, stem cell therapy has been widely investigated as a new therapeutic strategy. The success of a cardiac stem cell therapy largely depends on the selection of the appropriate stem cell type. Furthermore, the molecular mechanisms involved in cardiac repair, but also in progenitor cell proliferation and myocardial differentiation need to be further elucidated. Finally, the hostile microenvironment of the infarct, characterized by inflammation, ischemia and fibrosis needs to be tackled in order to improve stem cell survival, integration and differentiation. Cardiac stem cells isolated based on an elevated aldehyde dehydrogenase (ALDH) activity, called cardiac atrial appendage stem cells (CASCs), are a promising candidate for myocardial regeneration. In the first part of this study, we demonstrated that CASC proliferation is not unlimited and accompanied by a minor but significant reduction in absolute telomere length due to the lack of telomerase activity. However, despite a decrease in the proliferative percentage of CASCs during culture, clinically relevant cell numbers were generated, equaling ranges used in previous clinical trials with cardiac stem cells. Furthermore, CASCs preserved their biological properties during culture, including their antigenic expression profile, ALDH expression and more importantly their myocardial differentiation potential as demonstrated by the sarcomeric organization of cardiac troponin T and I. Finally, CASCs were also successfully expanded in human platelet plasma supernatant while maintaining their biological properties, which is an important step towards the clinical application of CASCs. In a second part of this study, we demonstrated that within the adult heart, CASCs are predominantly present in the atrial appendages and more abundant in the right than in the left atrial appendage. We showed that they express multiple early cardiac differentiation markers such as NKX2.5, GATA4, TBX5 and TBX18 and are committed towards myocardial differentiation as demonstrated by the expression of TNNT2 and MYL2. These results also suggest a possible heterogeneous embryonic origin of CASCs since these early cardiac differentiation markers are expressed in distinctive cardiac progenitor cell populations during cardiac development. Besides, the presence of several Frizzled receptors on CASCs suggested a role of Wnt signaling in self-renewal, proliferation and differentiation of the CASCs. However, despite an active role of Wnt signaling in CASCs as shown by the increase in total and active β-catenin levels, Wnt activation did not affect CASC proliferation or self-renewal. Furthermore, Wnt inhibition upregulated early cardiac markers without inducing mature myocardial differentiation. So, although Wnt signaling is functional in CASCs and has been described to play a crucial role in cardiogenesis and differentiation of pluripotent stem cells towards cardiac lineages, it has only limited effects on CASC proliferation and differentiation in vitro. In a third part of this study, we aimed to improve CASC survival under oxygen deprived conditions since the microenvironment of the infarct area is characterized by ischemia, inflammation and fibrosis and the survival of transplanted cells will most likely be negatively affected by the low oxygen levels in the targeted area. Here we showed that the declined CASC viability associated with hypoxia but not anoxia could be partly recovered by treating them with conditioned medium of mesenchymal stem cells (CM-MSC). The observed increase in CASC survival was also accompanied by an increase in CASC proliferation as shown by an increase in the number of Ki67 positive cells cultured in CM-MSC under hypoxic conditions. This paracrine effect was not mediated via VEGF or PDGF and the CM-MSC protection of CASCs against hypoxia induced cell death occurred in an Akt independent manner. Instead, CM-MSC treatment of CASCs upregulated catalase expression levels under hypoxic conditions. Finally, we developed an experimental approach to study cardiac fibrosis in an in vitro setting. Cardiac fibrosis does not only lead to the development of heart failure but is also an important limiting factor in the development of a successful cardiac regeneration therapy since CASCs that end up in the fibrotic tissue are not able to make contact with functional cardiomyocytes and therefore do not differentiate towards cardiomyocytes. This experimental approach can now be used to study the effect TGF-β1 and TGF-β3 on cardiac fibroblasts and to investigate whether the process of fetal wound healing can be mimicked in the heart. In contrast to adult wound healing, fetal wound healing is not accompanied by scar tissue formation, partly mediated by the low TGF-β1 to TGF-β3 ratios and the absence of TGF-β1 induced collagen deposition. In conclusion, the results described in this thesis provide important molecular and cellular insights in CASC proliferation, differentiation and survival. However, further research is essential to fully understand the underlying mechanisms and responsible factors which will improve CASC therapy for patients with heart failure by targeting CASC biology and the hostile microenvironment after an infarction.