Funding opportunities

Technology for hESC-Derived Cardiomyocyte Differentiation and Optimization of Graft-Host Integration in Adult Myocardium

Funding Type: 
SEED Grant
Grant Number: 
Principle Investigator: 
Funds requested: 
$634 287
Funding Recommendations: 
Recommended if funds allow
Grant approved: 
Public Abstract: 
Statement of Benefit to California: 
Review Summary: 
SYNOPSIS: The goal of the proposed research is to develop microelectronic cell-monitoring technologies that would provide a better understanding of cardiomyocyte differentiation from ES cells, identify optimal stages of differentiation for cell-transplantation therapy, and perhaps facilitate directed in vitro differentiation strategies. In the first aim, the applicant will grow human embryoid bodies on novel microelectrode arrays to follow the development of extracellular action potential signals as a measure of the formation of this property crucial for effectiveness in vivo. This aim would also test whether electrophysiologic stimulation from the array could drive this and other aspects of myocyte differentiation in a deterministic manner. Aim 2 will develop a co-culture technique to map the electrical and physical integration process as an assay to predict the potential for functional connections of transplanted cells in vivo. INNOVATION AND SIGNIFICANCE: From a strategic perspective, this proposed research addresses important issues regarding the suitability of cells for cardiac myocyte transplantation; that is, how to optimize the functional integration of differentiated stem cells into the tissue of a damaged organ. Understanding the temporal acquisition of electrophysiologic functions and the nature of those functions could help refine the process of producing beating cells and develop pharmacologic strategies to drive differentiation in vitro. This application will use microelectrode arrays to monitor the acquisition of electrophysiological properties by hES cells stimulated to differentiate into cardiac myocytes. This approach will allow continuous analysis of differentiation over an extended period of time. Whether cardiomyocyte differentiation can be entrained by electrophysiologic stimulation is a novel idea that deserves a test. If successful, it could complement or possibly supplant emerging pharmacologic protocols. The novel co-culture approach of Aim 2 will attempt to model the injection of hES cells into the mature but infracted myocardium as a way to monitor physical and electrical integration with host tissue. If successful, this may provide the means to answer a number of important open questions regarding functional integration of mycocyte grafts into host cardiac tissue. STRENGTHS: This is a bold and innovative experimental strategy that addresses important questions still outstanding in the field of cell-based therapy of cardiac muscle. The problem of optimizing functional integration of cells derived by stem cell differentiation is of crucial importance to all plans for cell-based transplantation therapies. The applicants have developed an innovative two chamber culture system to monitor the interconnection of cells at tissue junctions that mimic engraftment. The ability to test for functional integration with a “host” tissue environment, under defined conditions, is a significant strength of the application. The principal investigator and his team have extensive experience and proven expertise in biosensor design with applications to cardiomyocyte biology needed for this project. Extensive preliminary results demonstrate the function of the microsensor and the success of the co-culturing technique. Dr. Joseph Wu will provide the necessary background in stem cell and cardiomyocyte technologies and experience with the engraftment of cardiomyocytes from hESC cultures in a mouse myocardial infarction model that uses noninvasive imaging to monitor the persistence of engrafted cells. Preliminary results include the ability to grow undifferentiated WiCell H9 hES cells and derive beating embryoid bodies after differentiation. WEAKNESSES: If the goal is to provide large numbers of cardiomyocytes, driving them toward terminal differentiation may inhibit their ability to incorporate into the myocardium. Because the use of embryoid bodies would be an inefficient means to mass produce a single cell type, the use of BMP4, activin and perhaps other morphogens to direct cardiomyocyte differentiation of more homogeneous cultures of hESCs without embryoid body formation may be a more useful strategy and still amenable to the planned experimental approaches. The proposal is almost entirely focused on discussing the technology of measuring the onset and development of electrical signaling. The proposal would benefit from a strategy correlating the appearance of molecular markers of differentiation with electrophysiologic maturation to provide a common language with developmental biologists. The proposal lacks a clear discussion of metrics that might be used to compare the success of differentiation (aim 1) or integration (aim 2) from one experiment to the next. Also, there is insufficient mention of positive control experiments using primary cardiac myocytes against which the success or failure of hES-derived cells could be evaluated. The applicant raises the possibility that electrical stimulation may aid in promoting differentiation into cardic-type cells, but does not present any evidence in support of the approach and does not propose optimizing stimulation parameters or any other experiments to maximize the extent of cardiac differentiation. The initial statement of Specific Aims highlights the utility of this experimental paradigm for testing pharmacologic and physiologic manipulations that would drive differentiation – but no such tests are described within the proposal. DISCUSSION: There was some discussion of the applicant's publication record with one discussant noting that the applicant has not published much since 2001 and another discussant noting that what has published has been in prominent bio-engineering journals. There was also some discussion as to the rationale for conducting these studies with human rather than mouse ESC given that is not clear that our knowledge is at the stage for human ESC studies; human studies may be more compelling given ultimate clinical goal

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