Investigation of adipose-derived stem cells for applications in cardiac regeneration: cell source characterization and the effect of cardiac ECM on cardiomyogenesis
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Cardiovascular disease is the leading cause of mortality in the world and stem cell therapy has shown promise for its treatment. Focusing on adipose-derived stem cells (ASCs), numerous aspects still need to be investigated, including depot- and donor- dependent differences in ASC characteristics and the cardiomyogenic potential of these cells within the complex microenvironment of the cardiac extracellular matrix (ECM). In the first part of my thesis, a standardized comparative study was conducted between donor-matched subcutaneous and omentum fat ASCs, as well as donor-matched pericardial fat and thymic remnant ASCs. This study highlighted variability in the yield, viability, immunophenotype, clonogenic potential, doubling time, and adipogenic and osteogenic potential of the ASC populations. More specifically, ASCs isolated from both intrathoracic depots had a significantly higher proportion of CD34+ cells at passage 2. Furthermore, ASCs from subcutaneous and pericardial adipose tissue demonstrated enhanced adipogenic capacity, whereas ASCs isolated from the omentum displayed the highest levels of osteogenic markers in culture. Through cell culture analysis under hypoxic conditions (5% O2), oxygen tension was shown to be a key mediator of colony forming efficiency and osteogenesis for all depots. In the second stage, non-crosslinked 3-D porous foams derived from decellularized porcine cardiac ECM were developed. Mincing and milling processing methods were explored during scaffold fabrication and the architecture, ECM composition and physical properties of the foams were characterized. The minced foams demonstrated enhanced mechanical properties, improved long-term stability under simulated culturing conditions and better conservation of ECM constituents. The final stage of experimental work focused on investigating the cardiomyogenic differentiation of pericardial fat ASCs on minced cardiac foams, collagen I gels and tissue culture polystyrene using modified cardiomyogenic medium (MCM) or 5-azacytidine stimulation. Results showed that MCM was more effective than 5-azacytidine in stimulating cardiac gene and protein expression. Furthermore, enhanced differentiation was observed with both treatments on the cardiac foams, suggesting a synergistic effect of the cardiac ECM. Further, the cardiac foams were observed to provide an inductive microenvironment for the ASCs under control medium culture conditions, inducing cardiomyogenic gene and protein expression in the absence of external differentiation factors.