Year 3

Pluripotent stem cells (PSCs) harbor several attractive features for regenerative medicine: they are capable of self-renewal and have the capacity to differentiate to the tissue lineage of all three germ layers. The overall objective of this grant is to develop new technologies that can facilitate generation of cardiovascular progenitors that upon transplantation are capable to integrate into the host tissue and function as part of the normal myocardium with no adverse effect.
Several clinical trials of cell-based therapy have generated enthusiasm about the potential of adult stem cells to treat heart disease. However, no study has yet confirmed the delivery of a pure population of stem cells capable of robust regeneration of the injured myocardium. Furthermore, the electrical properties of the viable engrafted cells remain unknown. While adult stem cells have yet failed to convincingly regenerate myocardial tissue, human embryonic stem cells (hESCs) have proven to be a potential and unlimited source for cardiomyocyte regeneration. Most attempts to isolate transplantable cells from hESCs have aimed to isolate mature cells. Mature cardiomyocytes have passed the stage of self-renewal and may pose problems due to lack of proper integration. Cardiovascular progenitors, on the other hand, could adapt to the microenvironment for optimal integration into the host tissue and reside there for the lifetime of the patient.
We have employed gene editing technology to generate new hESC lines, in which fluorescent reporter proteins are expressed only in cardiac cells. Hence, upon differentiation of hESCs towards cardiac lineage, a distinct fluorescent color is expressed sequentially at each stage of cardiovascular development. Our data shows that cell isolation by mechanical cell sorting exerts harmful effect on the cells, compromising their viability. Therefore, we have developed a novel method where cardiac cells of interest harbor an antibiotic resistant gene. Therefore, treatment of these cells with an antibiotic results in selection of the desired heart cells while eliminating all unwanted contaminant cells. We have isolated these cells and have performed detailed analysis to fully characterize their developmental potential. We have performed global gene expression analysis to identify novel biomarkers unique to specific cardiac populations.
We have started pre-clinical studies of transplanting cardiovascular progenitors (at different stages of development) and mature cardiomyocytes in animal models. We have shown engraftment of these cells into the host heart, albeit a very low efficiency. Furthermore, we have shown that transplantation of immature cardiovascular progenitors may generate cardiomyocytes in addition to supporting cells such as vascular endothelial cells and fibroblasts. These results highlight the potential benefit of progenitor cells to generate other cell types that contribute to heart regeneration. A major challenge to clinical translation of stem cells for heart regeneration is the lack of data on the integration of the transplanted cells into the host heart. . It is possible that the transplanted cells fail to physiologically couple with the host tissue, or they may modify the substrate such that a pro-arrhythmic focus is created. As an analogy, consider the performance of a great symphony orchestra that is interrupted by several members playing in isolation. As such, grafted cells in the heart can also be an ectopic source of activities, promoting arrhythmic events. These are critical issues that need to be addressed in detail in the right animal models before any clinical application can be pursued. We are in the process of developing imaging modalities to investigate the extent of structural and functional integration of the transplanted cells into the host heart.