Funding opportunities

Selection, maturation induction and enrichment of pacemaker cells from stem cells for generating biological pacemakers in clinical therapy

Funding Type: 
Early Translational I
Grant Number: 
TR1-01231
Funds requested: 
$5 110 827
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Optimal cardiac function depends on the properly coordinated cardiac conduction system, which includes sinoatrial node (SAN), atrioventricular node and His-Purkinje system. Genetic defects or postnatal damage of SAN cells result in impaired pulse generation (sinus node dysfunction, SND), accounting for >30% of permanent pacemaker placements in the US. However, electronic pacemakers have limited battery life and multiple associated risks. The ideal therapy for SND is to repair or replace the defective SAN by cellular or genetic approaches. Stem and progenitor cell-derived cardiomyocytes (SPC-CMs) hold great promise for generating biological pacemakers. However, three main bottlenecks need to be resolved before we could translate primitive SPC-CMs into a feasible biological pacemaker. First, we need to create methods to isolate SAN progenitor cells from primitive SPC-CMs to facilitate the development of pure pacemaker cells (PMCs) from SPCs. Second, most SPC-CMs display heterogeneous and immature electrophysiological (EP) phenotypes with variable automaticity. The molecular pathways of inducing EP maturation of primitive PMCs are largely unknown. Implanting these electrically immature and inhomogeneous CMs into hearts may carry arrhythmogenic risks. Third, methods to enrich and induce maturation of PMCs are needed for any feasible clinical application. Further development in selection, inducing maturation and enrichment of pacemaker cells from primitive SPC-CMs are needed in order to overcome bottlenecks of translating SPC-CMs into feasible biological pacemaker cells. Studying SAN development is a challenging task due to lack of specific SAN tracking markers or methods. Additionally, most mutant mice with defective SAN function are embryonic-lethal and cannot be studied further. We used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to investigate factors affecting the development of automaticity in embryoid bodies (EBs). We find that automaticity of primitive hESC-CMs evolves in the EB milieu from a simple intracellular Ca2+ based mechanism to a mature form of automaticity, involving sarcolemmal ion channels. We have developed an EP screen with a specific agent that can sub-select early PMCs from primitive SPC-CMs, as a result, facilitating the generation of pure PMCs. We also successfully generated induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CMs). Furthermore, in order to elucidate the molecular pathways of PMC development, we apply gene chip technologies and collaborate with other scientists to establish the developmental gene profiles of PMCs obtained from human adult hearts, fetal hearts, hESC-CMs and iPS-CMs. We will use automated microscopy with cell tracking technologies to screen small molecules to enrich early PMCs and to induce their maturation. The overall goal of this proposal is to purify, differentiate, and enrich pacemaker cells for cell-based therapy of intolerable slow heart rate from SND.
Statement of Benefit to California: 
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) display heterogeneous and immature electrophysiological (EP) phenotypes with substantial automaticity. Implanting these electrically immature and inhomogeneous CMs to hearts may carry arrhythmogenic and deleterious risks. Furthermore, genetic defects or postnatal damage of sinoatrial node (SAN) cells result in impaired pulse generation (sinus node dysfunction, SND), accounting for >30% of pacemaker placements in the US. However, electronic pacemakers have multiple associated risks. The ideal therapy for SND is to repair or replace the defective SAN by cellular or genetic approaches. SPC-CMs hold great promise for generating biological pacemakers. Several bottlenecks need to be resolved before we can translate SPC-CMs into a feasible biological pacemaker. We have successfully used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to investigate factors affecting the development of automaticity of early PMCs. We have developed an EP screen with a specific agent that can sub-select early pacemaker cells (PMCs) from primitive SPC-CMs, as a result, facilitating the generation of pure PMCs. With collaborations, we also successfully generated induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CMs). These two breakthroughs lead us a step closer toward generating patient-specific biological pacemaker cells for future clinical therapy. We further used gene chip technology to map the genetic makeup of PMCs in adult & fetal hearts, hESC-CMs and iPS-CMs in order to create a road map for inducing maturation of primitive PMCs. Finally, we will apply automated microscopy with cell tracking technologies to screen small molecules for enriching early PMCs for the gene chip analysis and for inducing the maturation of early PMCs. With aforementioned goals achieved, we will make California the first state to develop a patient-specific biological pacemaker with a mature and homogeneous population of PMCs. No otherstem cell-related research in California is devoted to optimize the selection, scale-up and induction of maturation of PMCs for achieving a safe cell-based therapy. The proposed research will be the first to achieve this goal indicated by CIRM translational I research awards. 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 SND.
Review Summary: 
Optimal cardiac function depends on properly coordinated electrical conduction and subsequent myocardial contraction. Sinoatrial node (SAN) dysfunction accounts for greater than 30% of permanent pacemaker placements in the US. This proposal intends to repair defective SAN using pacemaker cells (PMC) derived from human pluripotent stem cells (hPSC). The applicant addresses three major bottlenecks to the generation of such a biological pacemaker: (1) the need for specific markers and methods to isolate pacemaker progenitor cells from primitive SPC-CMs (stem and progenitor cell-derived cardiomyocytes), (2) definition of the largely unknown molecular pathways regulating PMC or SAN development and maturation; (3) development of methods to enrich PMCs and to induce their maturation for clinical application. The applicant will approach these bottlenecks as follows. Early PMC derived from hPSC will be identified by resistance to an inhibitor that selectively blocks beating of non-pacemaker cell types. This will be used to characterize the electrophysiological phenotypes of early iPSC derived pacemakers and to develop an automated system for screening such phenotypes. To identify potential pathways that may regulate PMC differentiation and maturation, the applicant will perform comprehensive molecular profiling of adult vs. fetal working cardiac myocytes and pacemaker cells. Finally, to generate mature PMC at high purity, the applicant proposes to develop an automated system to enrich for PMC and to conduct a chemical screen for compounds that increase PMC maturation. While this proposal addresses significant bottlenecks to the advancement of PMC into the clinic, reviewers questioned the impact. They noted that well-tolerated, reliable electronic pacemakers with sophisticated mechanisms to adjust heart rates exist and considered it difficult to justify the development of this experimental therapy without a clear unmet medical need. Reviewers noted that stem cell derived cardiac myocytes have already been shown to have automaticity and to function as a biological pacemaker but did note that significant advances made since 2004 were not addressed in the scientific rationale. Despite the team’s successful record advancing cell selection technology, the reviewers had issues with the application and preliminary data that dampened confidence in a successful outcome. Reviewers had difficulty reading the dense, difficult to navigate application and found aspects of the research design unclear. They noted that the comprehensive molecular profiling at selected stages of cardiomyocyte development will produce a significant body of data, the interpretation of which and the specific application to the development of biological pacemakers was unclear. Specifically, cardiac cellular heterogeneity and motility during development could confound interpretation of the heart matrix molecular expression profiles. They noted that the impact of processing PMC upon viability was not addressed and that the method will have little utility if it negatively impacts PMC viability. They were unclear how the screening program will build upon findings from previous, similar screens performed by the collaborator’s group. In addition, reviewers expressed particular frustration with the preliminary data. The small size of the figures hampered interpretation of preliminary data and the data itself were not adequate to justify conclusions. The reviewers considered the principal Investigator (PI) as well as the members of the assembled team to have expertise in the areas of the proposed research. Reviewers considered strength of the proposal to be the strong team of collaborators. Reviewers considered the combined resources and environments of the applicant and collaborators as enabling for the research. However, they did raise concerns about integration and communication among the investigators and considered there to be poor justification for a team of the proposed size. They found the budget justification to be lacking in detail for a budget of the proposed size. They also noted a number of inconsistencies in the budget. In summary, the reviewers did not have a lot of enthusiasm for this proposal. They found the research plan unclear, the preliminary data unconvincing and the medical need to be reasonably well met by existing treatments.
Conflicts: 

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