New Faculty II
$1 500 000
One of the obstacles to pharmaceutical and clinical applications of human embryonic stem cell (hESC)-derived cardiomyocytes is insufficient purity of these cells. Our proposed research to identify a pure population of hESC-derived ventricular cardiomyocytes will provide a solution to this obstacle: 1) hESC-derived cardiomyocytes could potentially be transplanted to replace heart muscle cells lost or damaged to cardiovascular disease such as heart attack, which is the leading cause of death in the united states and other developed countries. Present cardiac differentiation protocols induce a heterogeneous population of cardiomyocytes including ventricular, nodal and atrial subtypes, which co-exist with other undefined cell lineages and/or undifferentiated cells. Transplantation of undifferentiated cells poses the risk of tumorigenisis, while a mixed population of different subtypes may cause cardiac arrhythmias. A pure population of chamber-specific cells such as ventricular cardiomyocytes will provide safer testing of in vivo cell replacement therapy. 2) A pure population of hESC-derived ventricular cardiomyocytes could be used to develop in vitro cardiac disease models, such as cardiac hypertrophy, to assist in finding relevant cures. 3) Understanding of the molecular basis for cardiac differentiation, growth and maturation by studying the development of these cells may lead to novel approaches to regenerate damaged heart tissues by in situ induction of cardiomyocyte proliferation and/or differentiation of adult cardiac progenitor cells. 4) Pure populations of hESC-derived cardiomyocytes may provide accurate prediction of pharmaceutical drug cardiac safety. Pharmaceutical agents unintended for cardiac diseases may interfere with the normal function of ion channels on heart cells, leading to increased risks of cardiac arryhthmia and sudden death. Compared to current cell models used in cardiac toxicity test such as animal heart cells, these cells bear closer resemblance to human cardiac cells and therefore may offer greater sensitivity and specificity. In summary, our proposed research will greatly facilitate the development of applications using hESC-derived cardiomyocytes..
Statement of Benefit to California:
Our research goal is to use innovative strategies to identify a pure population of hESC-derived ventricular cardiomyocytes. The state of California and its citizens will benefit from the outcomes of this research through its roles in facilitating the development of cardiac disease therapy and improving cardiac toxicity test of pharmaceutical drugs. In addition, our research will engage both interested graduate and undergraduate students and help reach out to the broad base of students in our teaching-oriented institute, who might otherwise have no direct opportunity to learn about the significance and progress of stem cell research. This is important considering that as they become a main stream work force post graduation, they may have direct or indirect influence on the government's policy in related to stem cell research on a national level.
The focus of this proposal is to improve the derivation of cardiomyocytes (CM) from human embryonic stem cells (hESCs), with the aim of improving therapy in patients with heart failure. The principal investigator (PI) identifies two aspects in CM differentiation process that need improvement: (1) low efficiency of derivation; (2) low purity of CM preparations, including the fact that current differentiation methods yield heterogeneous populations of various CM subtypes. Three specific aims are proposed: (1) identification of a ventricular-specific CM promoter; (2) establishment of hESCs carrying a reporter gene under the control of this promoter and (3) using genetically engineered hESC lines with the appropriate phenotype to define cell surface markers of ventricular-specific CM. Genes that encode cell surface proteins uniquely or preferentially expressed in the differentiated CM cells will be used in combination with antibodies to the encoded proteins to refine means of purifying ventricular CM. Reviewers agreed that cardiomyocytes (CM) offer hope for therapeutic treatment of patients with heart disease/failure. If the proposed research is successful, identification, characterization, and generation of antibodies to surface markers of CM may have diagnostic and therapeutic utility. However, reviewers were concerned that the success of this proposal is predicated by the identification of CM markers. As is true for many cell subset surface markers, there is a high risk that a unique set of CM markers may not be found. Reviewers commented that specific aim 1 to identify a promoter region of a ventricular-specific gene appears to be unnecessary, and that this could be accomplished by using a knock-in hESC reporter line. Another weakness of the proposal lies in the fact that the PI does not propose to make clonal lines. This may confound the results with site-specific integration effects. It was pointed out that although much emphasis in the proposal is given to the production of unique CM–specific antibodies, preliminary data provided in the proposal seems to suggest that commercially available antibodies that specifically mark CM subsets are already available. Additional criticism on the proposal included the fact that these studies will evaluate only two hESC lines for derivation of CM. Given the published differences in differentiation capacities, reviewers felt two cell lines may not be enough. Similarly, reviewers felt that additional functional characterization of the CM, including animal studies, may be needed for full evaluation of the differentiation process. The PI is an assistant professor who has no other funding resources for research listed in the proposal, which was a cause for concern. The PI’s stem cell research experience is limited to a short postdoctoral fellowship in an established stem cell lab, and he/she has a modest publication record. He/She has listed two mentors, but the proposal does not include letters of support from these mentors. Reviewers were concerned that it is not clear from the application whether facilities exist at the PI’s institution to perform the state-of-the-art technologies described in the proposal, or whether the appropriate bioinformatics expertise is available to the applicant. It was also noted that the PI’s institution is mainly a teaching institution with almost no track record in the area of stem cells. In conclusion, reviewers found that the present application was interesting but weak and not well developed.