CHARACTERIZATION OF ADULT HUMAN BONE MARROW MESENCHYMAL STEM CELLS FOR EFFECTIVE MYOCARDIAL REPAIR GENEVIEVE TAN MEI YUN (B.Sc (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF SURGERY NATIONAL UNIVERSITY OF SINGAPORE 2009 Summary Cardiovascular disease is a prevalent cause of death in the world Cell transplantation therapy has recently been developed as an alternative therapy for cardiovascular disease However, current studies employing the use of undifferentiated bone marrow stem cells have resulted in variable clinical outcomes with modest efficacies Differentiating primitive adult bone marrow stem cells into a stable and committed cardiac (-like) phenotype ex vivo prior to transplantation into an injured myocardium may be more effective for the treatment of cardiovascular disease Fibrosis and ventricular remodeling following a myocardial infarction begins with elevated extracellular matrix (ECM) deposition, which stiffens the myocardium and inadvertently contributes to ventricular dysfunction Notwithstanding, ECMs reportedly influence critical cellular processes such as survival, proliferation and differentiation in many cell types via the engagement of specific integrins However, it is not well understood if ECMs exert a significant influence on the proliferation and cardiac transdifferentiation of primitive mesenchymal stem cells Additionally, the effect/s of myocardial fibrosis and post-infarct remodeling on stem cell differentiation in vivo is not well studied This project has specific objectives: To explore the role/s of extracellular matrices and their integrin partners on the cell fate and development of CLCs vs, MSCs in vitro and in vivo and To compare the relative therapeutic efficacies of the cell types Adult human bone marrow mesenchymal stem cells (MSCs) can differentiate into cardiomyocyte-like cells (CLCs) with the concomitant use of collagen V extracellular matrices and a simple non-toxic culture medium in vitro Importantly, this distinct cardiaclike phenotype is stable in prolonged cultures In contrast, MSCs exhibited a spontaneous but transient expression of cardiac-specific proteins Objective 1: Cell-ECM interactions are mediated via the engagement of integrins, which in turn activates a cascade of downstream intracellular signaling events that lead to the expression of multiple proteins among other biological processes CLCs demonstrated preferential interaction with specific collagen subtypes Specifically, Collagen -V, but not collagen -I, promoted the cellular adhesion and cardiac differentiation of MSC-derived CLCs More remarkably, collagen V matrices promoted the large-scale production of CLCs that was valuable for subsequent transplantation therapies Initial cellular adhesion to collagen V but not collagen I, was dependent on the 2 integrin but independent of the v3 and v integrins However, inhibition of v integrin, but not 21 integrin reduced gene expression levels of troponin T, sarcomeric -actin and RyR2 in CLCs cultured on collagen V ECM Importantly, the engraftment of CLCs within close proximity of collagen V-expressing myofibers promoted their integration into the cardiac syncytium More remarkably, CLCs demonstrated distinct striations that were indistinguishable from host cardiomyocytes in collagen V-enriched areas in the infarcted myocardium, while CLCs that engrafted in collagen I-enriched areas in the infarct borders did not Thus, collagens -I and -V may play pivotal roles in the cell fate development of CLCs in vivo, although it remains to be elucidated if the colocalization of CLCs with the collagen V-enriched, endomysial-lined myofibers correlates with a specific interaction between the endogenous ECM and the transplanted cells, that is reminiscent of their affinity in vitro It is also unclear if the localization of CLCs in the interstitial tissues enriched in collagens -I and -III prevented their integration into the host myofibrillar architecture Notwithstanding, significant improvements in cardiac function were observed in rats administered with low-dose CLC therapy despite low incidences of cell integration in the i host myocardium However, it is highly unlikely that such remarkable benefits were attributed to myocyte replacement Instead, the introduction of an exogenous supply of viable cells may possibly improve cardiac function by modulating the ECM architecture in vivo to retain a certain degree of pliancy in the post-infarcted myocardium, thus reducing overall tissue stiffness in the compromised myocardium Hence, CLCs may facilitate functional recovery by preserving tissue compliance in the peri-infarct borders, which in turn sustains the contractile efficiency for long-term functional recovery in the infarcted myocardium Objective 2: CLC-therapy was more effective when administered in higher doses as demonstrated by increasingly evident improvements in cardiac function Additionally, high- dose CLC therapy resulted in enhanced cell integration with the host myocardium Notwithstanding similar trends in functional improvements observed in both low- and high-dose cell therapy groups, the high-dose therapy groups were relatively better reflections of the direct cellular effects of cell- transplantation on the infarcted myocardium Correspondingly, cell-treated rats exhibited smaller cardiac volumes and LV internal diameters Cell therapy generally improves the injured myocardium by restraining progressive wall thinning and ventricular dilatation Thus, cell-therapy alleviated adverse remodeling effects, possibly by sustaining myocardial tissue compliance However, CLCs were more effective than MSCs in improving cardiac function as CLC-treated rats demonstrated persistently superior systolic activities with respect to control and in particular, MSC-treated rats Echocardiography assessments showed that highdose CLC-therapy mediated a significant 9.9 ± 12.1% improvement in LV fractional shortening (FS) as compared to a decrease of 14.4 ± 13.6% in control rats (p