Technology for hESC-Derived Cardiomyocyte Differentiation and Optimization of Graft-Host Integration in Adult Myocardium
Recommended if funds allow
Stem cells therapies hold great promise in the treatment of cardiac diseases such as coronary heart disease or congestive heart failure. Thanks to their ability to transform into almost any kind of tissue, engrafted stem cells can potentially replace damaged heart tissues with healthy tissues, effectively restoring the heart’s original functions. While initial studies demonstrated the potential benefits of stem cell injection for repairing heart damage, they told researchers little about exactly how improvements were made to the heart and how the improvement might be enhanced. Also, there is concern that the stem cells could negatively impact some aspects of heart function and lead to disturbances of heart rhythm and future attacks. In light of this, we propose to develop a model to study the detailed interaction of stem cells and healthy heart tissue in the laboratory, where events within the cells and between the cells can be measured accurately and many experiments can be done to increase our understanding, without the use of human subjects. Specifically, we plan to focus on two main goals. The first goal is to develop a platform to better understand the gradual transition that stem cell lines make as they mature into heart cells, process known as differentiation. We will record the electrical activity arising from newly formed heart cells to determine when exactly they form and how the behave in response to electrical stimuli or drugs as they mature. This will tell us more about the behavior of the cells that could be injected into the heart so that we know how they will respond when they merge with the heart and when is the best time to introduce them. The second goal, building on the first one, is to observe how the stem cells make contact with the heart cells, including how they grow together mechanically and how they begin to communicate electrically as a repaired tissue. This will be carried out by growing the stem cells and heart cells separately and then allowing them to grow together, just as they would in the heart. Simultaneous recording of electrical activity at numerous locations in the culture will let us map the activity across the culture and evaluate the communication between heart cells (host) and stem cells (graft). Understanding the microscopic nature of integration of stem cells into healthy tissue will lead to a greater knowledge of what can happen when stem cells are injected into the heart and begin to replace the non-functional tissue and connect to healthy tissue. Insights gained with such model should lead to a better understanding of the repair process and highlight strategies for making stem cell-based therapies safer and more effective. This model will also allow testing and development of chemical or electrical manipulations that would increase the yield and reliability of the differentiation process, paving the way for the ultimate scale-up of stem cell therapies for clinical use.
Statement of Benefit to California:
There is currently no cure for heart damage caused by heart attack, and stem cells offer a very promising solution to this problem that affects millions of Americans. We feel that addressing possible solutions to this pervasive problem is a very constructive and meaningful way to utilize some of the financial resources allocated for stem cell research in California. Within (and outside) the CIRM community, we also have the important goal of making currently unavailable electronic, microfabrication and signal processing technologies available in the form our proposed research platforms. With our planned outreach efforts, we will freely share our methods and equipment, hopefully enhancing the work of many other research groups. By using CIRM funds, we could make such systems available for use with non-registered (as well as registered) cell lines. The outcome of this research stands to impact not only citizens of California, but also the nation and the world. We aim to make considerable progress with research paid for by the citizens of California, demonstrating the degree to which we, as a people, are committed to solving problems in medicine and health care and improving the lives of others. This work will also benefit our State and taxpayers through the training of post-doctoral and graduate students with a clear mindset of leadership, creativity and compassion. Through publication and presentations at local, national and international forums, we hope to disseminate the knowledge gained and encourage further advances.
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