Cardiovascular diseases remain the major cause of death in the western world. Stem and progenitor cell-derived cardiomyocytes (SPC-CMs) hold great promise for myocardial repairs. However, most SPC-CMs displayed heterogeneous and immature electrophysiological (EP) phenotypes with variable automaticity. Implanting these electrically immature and inhomogeneous CMs into hearts might carry arrhythmogenic risks. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide a model system to study the development of cardiomyocytes (CMs), in part because they are an immature population of CMs that could continue to mature in the embryoid body (EB) environment. Elucidating cellular factors and molecular pathways governing electrical maturation of early hESC-CMs would enable engineering microenvironment to create electrophysiologically compatible hESC-CMs for a safe, cell-based therapy of cardiovascular diseases.
Many temporal and regional cues from neighboring extra-cardiac cells or non-CMs direct the specification and maturation of CMs during normal cardiac development. How these regional and temporal cues influence EP maturation of primitive CMs in EBs remains to be explored. Without understanding the mechanism of EP maturation of SPC-CMs, the fate of SPC-CMs after cell transplantation is unpredictable and will remain a challenging hurdle for producing a clinically safe, cell-based therapy. Using hESC-CMs and an antibiotic-selection system to isolate hESC-CMs (>95% purity), we found that non-CMs in EBs induced electrical maturation and ion channel expression of primitive hESC-CMs during differentiation. A novel add-back (co-culture) method was also established to enable an engineered microenvironment for controlled EP maturation of primitive CMs. With these established methods and results, we studied the role of endothelial cells (ECs) and their molecular pathways in inducing EP maturation of primitive hESC-CMs since ECs have been shown to improve survival and development of early CMs. We found that ECs significant influenced the expression of specific types of ion channels of early hESC-CMs via paracrine pathways. In this grant application, we will first use calcium imaging and electrophysiological recording methods to characterize the EP phenotypes and maturation of hESC-CMs in EBs after blocking or activating several EC-related paracrine pathways. We will then apply information obtained from hESC-CM experiments to induce EP maturation of CMs derived from induced pluripotent stem cells. Our proposed study potentially will provide significant insights in directed ion channel maturation of primitive SPC-CMs and in improving the safety of current cell-based therapies in hearts.
Cardiovascular diseases remain the major cause of death in the western world. Stem and progenitor cell (SPC)-based cell therapies in animal and human studies suggest promising therapeutic potentials. However, most SPC-derived cardiomyocytes (SPC-CMs) displayed heterogeneous and immature electrophysiological (EP) phenotypes with substantial automaticity. Implanting these electrically immature and inhomogeneous CMs to the hearts would be arrhythmogenic and deleterious. Further understanding mechanisms of EP maturation of primitive SPC-CMs and creating methods of maturation induction of primitive SPC-CMs are badly needed so that a safe, cell-based myocardial repair could be achieved without arrhythmogenic risks. We have, for the first time, provided evidence that non-CMs in embryoid bodies (EBs) induced EP maturation of primitive human embryonic stem cell-derived CMs (hESC-CMs) during differentiation. Only a very small numbers of laboratories in the world have made progress in understanding the molecular pathways and cellular factors that control EP maturation of primitive hESC-CMs. As a result, no method has been developed to create directed EP maturation of SPC-CMs in vitro or in vivo. We have successfully developed the technology to efficiently isolate pure populations of hESC-CMs from EBs and established the co-culture method to promote EP maturation of early hESC-CMs by non-CMs. The proposed research will further determine the mechanisms by which endothelial cells affect ion channel expression and EP maturation of primitive hESC-CMs. Most importantly, using our established methods, we have started investigating strategies for inducing maturation of induced pluripotent stem cell-derived CMs (iPSC-CMs). With both goals achieved, we will make California the first state to produce a safe and patient-specific cell-based therapy for myocardial repair with an electrophysiologically mature population of iPSC-CMs. Currently, none of stem cell-related research in California or other states is devoted to induce EP maturation of primitive hESC-CMs or iPSC-CMs so that a clinically safe, cell-based therapy could be achieved. The proposed research will provide insights of mechanisms of inducing EP maturation of primitive SPC-CMs, which will enable future directed myogenesis with proper EP maturation from primitive hESC-CMs or iPSC-CMs for safe cell-based therapies in California. The success of this proposal will also make California the epicenter of the next generation of cell therapies and will benefit its citizens who have significant cardiovascular diseases.
The goal of this proposal is to explore the cellular factors and molecular pathways that control the electrophysiological maturation of human embryonic stem cell- or induced pluripotent stem cell-derived cardiomyocytes (hESC-CMs or iPSC-CMs). The applicant predicts that insights gained from these studies could lead to improvements in the derivation of mature and functionally authentic cell populations that may ultimately be useful for treating cardiovascular damage and disease. Aims 1 and 2 will use calcium imaging and electrophysiological recording methods to characterize the electrophysiological (EP) phenotypes and maturation of hESC-CMs in embryoid bodies (EBs) after blocking or activating specific endothelial cell (EC)-related paracrine pathways. In Aim 3, the applicant will apply information obtained from hESC-CM experiments to induce EP maturation in iPSC-CMs.
Reviewers agreed that the proposal addresses a timely and important topic. Cardiovascular disease represents a major and increasingly prevalent health concern. If successful, the insights gained from this study could enable safer and more authentic cardiomyocytes to be developed, a promising potential therapeutic approach for the repair of diseased or damaged hearts. The reviewers also appreciated the mechanistic focus, the emphasis on electrophysiological characterization and the innovative co-culture system that is the hallmark of this work.
The reviewers agreed that the research plan, though dense, was logical, provided adequate milestones and included solid and convincing body of preliminary data. However, the density and small size of the figures made it difficult to assess the merits and technical details such as the ratio of the various cell types, were not clearly stated or readily apparent. Furthermore, the reviewers thought that too much of the proposal had been dedicated to describing methodology in lieu of a clear presentation of the experimental rationale, plan and expected outcomes as well as a discussion of alternative plans and potential limitations of the proposed methodologies. The experimental design was short on specifics, leaving the reviewers to question, for example, the number and source of individual lines to be analyzed and how line-to-line variation will impact the findings. The heterogeneity of the non-CMs fraction of the co-culture studies was also a source of concern, as the introduction of variability into the experimental system could complicate subsequent analysis and interpretation of results. One reviewer was uncertain of the extent to which the in vitro read outs would be predictive of the in vivo situation. Finally, some reviewers were disappointed that previous reports from the literature on additional soluble factors that were shown to enhance differentiation and survival of hESC-CMs had not been discussed in the context of this work. Reviewers suggested that more focus on the experimental plan including specifically addressing issues related to heterogeneity and variability, would have overcome many of their reservations and significantly enhanced their impression of feasibility.
Reviewers praised the principal investigator’s expertise and track record and commended the level of effort proposed for this endeavor. The named collaborators were acknowledged for their extensive expertise and viewed as considerable assets to the team.
Overall, reviewers appreciated the focus of this proposal on electrophysiological characterization and acknowledged the strength of the research team. However, their enthusiasm was diminished by the proposal’s disproportionate emphasis on methodology as well as number of unanswered questions relating to feasibility.
A motion was made to move this application into Tier 1, Recommended for Funding. There was discussion as to whether the proposed plan would lead to findings that could be directly translated. The motion failed. Those voting in favor of the motion constituted the requisite 35% for a minority report but they chose not to make one.
- Ali Brivanlou