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Optimization in the Identification, Selection and Induction of Maturation of Subtypes of Cardiomyocytes derived from Human Embryonic Stem Cells

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01143
Principle Investigator: 
Institution: 
Funds requested: 
$906 629
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
Cardiovascular diseases remain the major cause of death in the western world. Stem and progenitor cell-derived cardiomyocytes (SPC-CMs) hold great promise for the myocardial repair. However, most of SPC-CMs displayed heterogeneous and immature electrophysiological phenotypes with substantial automaticity. Implanting these electrically immature and inhomogeneous CMs to the hearts would be arrhythmogenic and deleterious. Further optimization in identification, selection and inducing maturation of subtypes of CMs from primitive SPC-CMs are paramount for developing a safe and effective cell-based therapy. Commonly used CM isolation techniques are microdissection, density sedimentation or promoter-driven, fluorescence-activated cell sorting (FACS). Microdissection and density sedimentation are labor intensive and lack of purity. Promoter-driven FACS may compromise cell viability and which promoter is proficient for selection remains unclear. We have established several antibiotics (Abx)-resistant human embryonic stem cell (hESC) lines conferred by lentiviral vectors under the control of various cardiac-specific promoters. With simple Abx treatment, we have easily isolated >95% pure hESC-CMs at various stages of differentiation from embryoid bodies (EBs). Using this Abx selection system, we also found that electrical maturation and differentiation of primitive hESC-CMs depended heavily on developmental cues from extracardiac cells in the EBs. This Abx selection system therefore could be used easily to purify CMs for mechanistic studies and future cell-based therapies. However, the subtype specification of atrial, ventricular and pacemaking CMs appears to occur at very early stages of differentiation because early EBs possess all three types of cells. Furthermore, various cardio-specific promoters have been shown to select preferentially certain subtypes of CMs. In order to use these promoters and Abx resistance to sub-select particular types of CMs at early stages of differentiation, we need to know the timing and sequence of expressions of various cardiac promoters during the EB development. For this later purpose, we will generate hESC lines expressing different colors of fluorescent proteins under the control of various cardiac-specific promoters respectively to determine the timing of expressions of these promoters in the EBs. Based on the sequence of expression, we will generate the Abx-resistant hESC lines under the control of these promoters to sub-select CMs. We will then study the EP properties of these sub-selected hESC-CMs and their interactions with extra-cardiac cells. The overall goal of this proposal is to establish an In Vitro system to track the sequence of expressions of various promoters in order to sub-select particular phenotypes of CMs by the Abx-resistance method. As a result, we will be able to optimize the selection and induction of a population of mature and homogeneous hESC-CMs for a safe and effective cell-based therapy.
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) 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 optimization in identification, selection and inducing maturation of subtypes of CMs from these primitive SPC-CMs are badly needed. Most frequently used isolation techniques are microdissection, density sedimentation or promoter-driven, fluorescence-activated cell sorting (FACS). Microdissection and density sedimentation are labor intensive and lack of purity. Promoter-driven FACS may compromise cell viability and which promoter is proficient for the cardiomyocyte selection remains to be determined. None of the laboratories in the world has success in developing an easy and efficient way to isolate the SPC-CMs. As a result, no method has been developed to induce the maturation of SPC-CMs. We already have the technology to efficiently isolate pure populations of human embryonic stem cell-derived CMs (hESC-CMs) from the embryoid bodies. The proposed research will further determine which type of promoter is best to properly sub-select a specific phenotype of hESC-CMs for future cell-based therapies in California. Most importantly, using this antibiotics-based selection method, we have started investigating the methods for inducing maturation of these sub-selected and primitive CMs. With both goals achieved, we will make California the first state to have a safe and effective cell-based therapy for myocardial repair with a mature and homogeneous population of hESC-CMs. None of stem cell-related research in California is devoted to optimize the selection, identification and induction of maturation of a specific phenotype of hESC-CMs in order to develop a safe cell-based therapy. The proposed research will be the first to achieve this goal proposed by CIRM Tools and Technologies Award. 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.
Review Summary: 
This application focuses on the development of new and improved techniques for deriving homogenous, mature, and functionally relevant cardiomyocyte populations from human embryonic stem cells (hESCs). The applicant proposes to generate transgenic hESC lines expressing a fluorescent protein under the control of promoters or enhancers which are associated with specific cardiomyocyte (CM) subtypes. The analysis of temporal expression and persistence of these reporters will allow identification of key steps and stages of CM subtype differentiation. This information, together with the generation of antibiotic-resistant hESC lines, will facilitate isolation of cardiomyocyte subtype populations. Finally, the resulting cells will be characterized by analysis of their electrophysiological properties, and the role of extra-cardiac cells in the differentiation and maturation process will be investigated. The reviewers agreed that this straightforward but unique effort could be of great impact, particularly in the field of cardiovascular stem cell research. This proposal directly addresses key roadblocks in this area, including the difficulty in deriving sufficient numbers of homogenous, pure, specific cardiomyocyte populations with mature functionalities. These studies could offer new insights as to what defines functional maturity as well as a powerful strategy for isolating and scaling up the production of these cells. In contrast to current methodologies, the proposed strategies are not labor intensive and would likely not compromise cell viability. Finally, while potentially useful for regenerative medicine, the resulting cells also could be extremely valuable for future drug screening efforts. In terms of feasibility, the reviewers were confident in the qualifications of the research team. The proposal was well written and conceived, and the applicant provided appropriate discussion of expected results, potential hurdles, and alternative methods that might become necessary. Furthermore, the preliminary data made a strong case for the overall feasibility of the selection scheme. Reviewers expressed some concern that the project was overly ambitious and that some plan details (e.g. the use of single promoter expression strategies and the heterogeneity of embryoid bodies) would prove problematic. However, the significant preliminary data and the overall simplicity and elegance of the approach, convinced reviewers of its potential to substantially impact the field. The investigators are very well qualified to pursue the objectives of this proposal. The principal investigator has an excellent track record. The collaborators bring exceptional expertise in all relevant areas, including stem cell biology, cardiac differentiation, antibiotic selection, electrophysiological profiling, and lentiviral gene targeting. The budget was judged to be fairly appropriate, although proposed travel expenses and consultant fees appeared somewhat excessive. Overall, this is a well written application that addresses several key roadblocks in stem cell biology. Despite some minor flaws, the reviewers were optimistic that this simple but elegant approach would prove useful and productive.
Conflicts: 

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