| Heart disease is the leading cause of death in the world and is likely to remain so in the foreseeable future. There is a great deal of optimism about cardiac regenerative therapies because recent advances in stem cell technology have made it possible to derive millions of cardiomyocytes, i.e., functional heart muscle cells, from human embryonic stem cells and induced pluripotent stem cells. These stem cell-derived cardiac cells also have enormous potential in drug discovery, toxicology studies and fundamental cardiac physiology. Cardiomyocytes (CMs) do not have well-established, specific surface markers, which could enable their separation from heterogeneous differentiation cultures using transient antibody labelling and traditional methods such as fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS). The next major challenge in realizing their potential is the development of separation methods that are compatible with clinical applications and can isolate cardiomyocytes and specific cardiac cell subpopulations in sufficient numbers, purity and quality after in vitro culture. This thesis presents a review of currently available cardiomyocyte separation strategies; develops a mathematical model to describe the novel labeling-free application of high gradient magnetic sorting for cardiomyocytes; reports the CM separation conducted; and finally avalidates a CM prosurvival preconditioning strategy to protect against an in vitro simulated ischemic/reperfusion injury such as one that occurs after cells are implantation in vivo. Neonatal rat and hESC-derived cardiomyocytes were collected at 94.8+/-3.3% and 93.6+/-2.8% (Day 20-25) purity from the separation respectively. Sodium nitrite/hypoxic preconditioned cells' viability increased 20% post hypoxic/reoxygenation injury compared to cells without preconditioning treatment. |
