Optimal cardiac function depends on the properly coordinated cardiac conduction system (CCS). The CCS is a group of specialized cells responsible for generating cardiac rhythm and conducting these signals efficiently to working myocardium. This specialized CCS includes the sinoatrial node, atrioventricular node and His-Purkinje system. These specialized pacemaking /conducting cells have different properties from the surrounding myocytes responsible for the contractile force. Genetic defects or postnatal damage by diseases or aging processes of these cells would result in impaired pulse generation (sinus node dysfunction, SND) or propagation (heart block). Implantation of an electronic cardiac pacemaker is necessary for intolerant bradycardia to restore cardiac rhythm. However, the electronic implantable pacemaker has multiple associated risks (e.g. infections) and requires frequent generator changes due to limited battery life. Sinus node dysfunction is a generalized abnormality of cardiac impulse formation and accounts for >30 percent of permanent pacemaker placements in the US. A perfect therapy to SND will be to repair or replace the defective sinus node by cellular or genetic approaches. Many recent studies have demonstrated, in a proof-of-concept style, of generating a biological pacemaker by implanting various types of progenitor or stem cells into ventricular myocardium to form a pulse-generating focus. However, a perfect biological pacemaker will require good connections with the intrinsic neuronal network for proper physiological responses. Elucidation of the factors controlling the evolution of pacemaker cells and their interaction with the peripheral neuronal precursor cells (neural crest cells, NCCs) will be paramount for creating an adaptive biological pacemaker. The NCCs have been shown to be contiguous with the developing conduction system in embryonic hearts of humans. However, the influence and interaction of the NCCs with the developing cardiac pacemaker cells remains unclear. In addition, there is no simple marker for identifying the pacemaker cells and the electrophysiological (EP) recording is the only physiological method to trace the evolution of cardiac pacemaker cells from human embryonic stem cells (hESCs). We have successfully obtained the EP properties of early hESC-derived cardiomyocytes. We propose here an in vitro co-culture system to study fate of the pacemaker cells evolved from hESCs and to investigate the influence of NCCs on the early, cardiac committed myocytes derived from hESCs. Such a study will provide insight in the development of pacemaker cells and in the mechanisms of early neuro-cardiac interaction. Results from the proposed study may suggest strategies for developing efficient and neuro-coupled cardiac pacemakers from ESCs. These neuro-coupled biological pacemaker cells may one day used clinically to replace the need for implanting an electronic pacemaker for the treatment of intolerant bradycardia.
Optimal cardiac function depends on the properly coordinated cardiac conduction system. Genetic defects or postnatal damage by diseases or aging processes of these pacemaker cells would result in impaired pulse generation (sinus node dysfunction) or propagation (heart block). The implantation of an electronic cardiac pacemaker is necessary for intolerant bradycardia to restore physiologic cardiac rhythm. However, the electronic implantable pacemaker has multiple associated risks (e.g. infections and thrombosis) and requires frequent generator changes due to limited battery life. Sinus node dysfunction (SND) is a generalized abnormality of cardiac impulse formation and accounts for 30-50 percent of permanent pacemaker placements in the US. A perfect therapy to SND will be to repair or replace the defective sinus node by cellular or genetic approaches. Most of the research work on developing biological pacemakers are performed in Columbia University at New York City, Johns Hopkins University at Baltimore, and Technion-Israel Institute of Technology at Haifa, Israel. All of their approaches produced short-lived and non-responsive biological pacemakers to physiological demands. None of human stem cell-related research in California is devoted to this highly promising field of developing biological pacemakers. The proposed research here will elucidate the factors controlling the evolution of pacemaker cells and their interaction with the peripheral neuronal precursor cells (neural crest cells). Such a study will provide insight in the development of pacemaker cells and in the mechanisms of early neuro-cardiac interaction. These factors then can be used to generate better neuro-coupled biological pacemaker cells in California. These neuro-coupled biological pacemaker cells may one day be used clinically to replace the need for implanting an electronic pacemaker for the treatment of intolerant bradycardia. Creating the neuro-coupled, adaptive biological pacemakers will make California the epicenter of the next generation of pacemaker therapy, and will benefit its citizens who have intolerant cardiac bradycardia.
SYNOPSIS: This proposal will explore the potential isolation of specialized cardiac cells from human embryonic stem cells (hESCs) that are responsible for normal cardiac conduction. Disorders of such cells in humans lead to problems with cardiac conduction and frequent intervention with electronic pacemakers. Isolation of such specialized cells from hESCs could provide an alternative therapeutic approach. In the first Specific Aim, the applicant will attempt to isolate pacemaking cells from hESCs and identify these through their electrophysiologic properties. In the second aim, the PI will co-culture hESC-derived cardiomyocytes with neural crest cells to attempt to maintain the pacemaking activities of these cells. Patch clamp and multi-electrode array will be used to study these cells.
SIGNIFICANCE AND INNOVATION: This proposal will lay the initial groundwork for deriving pacemaker cells from hESC. This is an important goal as there would be value in creating models of human cardiovascular disease that result from defects and degeneration of conduction system cells. Relatively little is known about the pathways which drive pacemaker cell formation and regulation at a cellular level, in the mouse or in the human context. Also, a longer term goal of creating biological pacemaker tissue could be particularly valuable in congenital forms of heart block, where repeated implantation of pacemakers has inherent complications. This is an interesting proposal which clearly has great clinical significance, and the applicant has proposed some highly innovative strategies to explore the questions asked.
STRENGTHS: This is an important area of ESC research with likely clinical significance. The proposal has several strengths, including: 1) the importance of the problem and relevance to physiological, developmental, and clinical areas of cardiovascular medicine and science; 2) the strength of the well-qualified, young PI, who is a physician scientist that has a command of the technological requirements of the proposal, and a deep clinical understanding of the physiological aspects as well; 3) an excellent academic background and good collaborative interactions with investigators that have a thorough command of the protocols for culturing human ES cells and their differentiation; and 4) preliminary studies with mouse ES cell derived pacemaker cells.
WEAKNESSES: This proposal has a few weaknesses. First, the pathways that drive the formation of pacemaker cells are not well established at present. Second, molecular markers that would allow the unequivocal, selective sorting of pacemaker cells would enhance the proposal. Analogies with markers that have been shown to be useful for mouse could be employed, such as HCN channels, connexins, etc.. Manipulation of human ES cells to stably carry BACs to drive the expression of reporters under the control of the native locus of the genes that encode these markers could be valuable. Third, the importance of the interactions of neural crest cells and pacemaker cells is not clear at present and could be minimal; however, there is a strong chance of a modulatory role, akin to innervation of skeletal muscle.
DISCUSSION: This is a risky project utilizing highly innovative strategies. The enthusiasm is strong since this is a young field that needs new blood, and this well qualified young investigator already has preliminary data. The main weakness cited by reviewers is the potential difficulty in differentiation. The derivation efficiency of pacemaker cells is such an unknown that they may be too difficult to generate. However, despite this risk, there is not much known about pacemaker cells and there is a high likelihood that this would be a productive investigation. Also, while the phenotypic characterization at the single cell level is very convincing, it could be improved by using molecular markers. The use of electrophysiology in the phenotypic analysis is a concern because it is not high throughput.
Reviewers had several suggestions for the applicant:
1) Develop an approach to find molecular markers of the progenitors destined to become pacemaker cells.
2) Test candidates that might serve as molecular markers based on studies that have already been performed in mouse model systems.
3) Focus on the use of the cells as a model system, as opposed to tissue engineering at this early stage.
There was a brief discussion about whether this work could be done using the presidential lines. One discussant felt that this could be done and therefore this work would be NIH fundable, but another discussant felt that this was not an issue because the presidential lines have their own inherent problems (e.g., problems with growth properties, karyotype, etc.)