Engineering three dimensional cardiospheres from pluripotent stem cells

Abstract

Previously, we have shown that generating 3D cardiospheres from pre-differentiated cardiomyocytes derived from human pluripotent stem cells has enriched and matured the cardiomyocyte populations compared to the 2D controls. However, current limitations with cardiomyocytes include highly variable efficiency resulting from differentiation protocols, and improvement in purification is necessary. Additionally, the differentiaton yields an immature phenotype resembling that of embryonic cardiomyocytes, not mature adult cardiomyocytes. In order for cell-based cardiac therapies to realize their clinical potential, methods to enrich and mature the cardiomyocyte populations are required. This project identifes which time point during the pre-differentiation process we can create cardiospheres with the highest yield of mature cardiomyocytes using the fewest number of steps, creating a faster protocol. The project forms cardiospheres at four different time points during the differentiation process. Time point one is equivalent to day zero of the protocol, where the cells are still pluripotent. Time point two is equivalent to day five of the protocol, after mesoderm differentiation is achieved. Time point three is equivalent to day nine of the protocol, when spontaneous beating is first noticed. Time point four is equivalent to day 14 of the protocol, when the cells show robust beating. At each of these time points 3D aggregates will be formed and will continue to be cultured for a total of 21 days before being evaluated. Initial results indicate the cardiospheres formed on day 5 are more mature than those formed in the control. Further experiments this semester will concrete the results.