Regenerative therapies could be particularly beneficial for heart disease, which is the leading killer of adults in the U.S, and is responsible for the 5 million Americans with insufficient cardiac function. At the other end of the age spectrum, malformations of the heart involving abnormal cell lineage or morphogenetic decisions are the leading noninfectious cause of death in children. Unfortunately, since adult heart cells cannot multiply after birth, the heart has almost no regenerative capacity after injury or in response to malformations. Deciphering the secrets of heart formation might lead to novel approaches to repair or regenerate damaged heart muscle using embryonic stem cells (ESCs) and progenitor cells. Our research is focused on determining what causes ESCs to specialize into cells that belong to the mesodermal, or middle, layer of an embryo, which develops into blood, muscle, and bone, among other cells, with a specific focus on cues that stimulate cardiac and skeletal muscle formation. Small RNA molecules called microRNAs have emerged as an elegant and novel mechanism nature uses to titrate dosage of critical proteins by regulating the flow of genetic information as it is translated into proteins. microRNAs are active dynamically and specifically in developing cardiac and skeletal muscle during muscle formation. In mice and flies, microRNAs regulate the balance of muscle formation vs. expansion of progenitor cells. We have evidence that microRNAs can control mouse embryonic stem cells (mESCs) and can promote formation of mesoderm and inhibit formation of other cell types such as brain or gut cells. This may be true in human ESCs also. However, NIH-approved human ESC (hESC) lines are contaminated with mouse feeder cells, are difficult to disperse into single cells and do not grow robustly enough to generate homogeneous pools of genetically altered cells. This has made it difficult to generate homogenous population of cells that could be used for discovery and future potential therapeutic applications. The aims of this grant will use non-NIH approved lines to meet these objectives and are not fundable by the NIH. We hypothesize that specific microRNAs influence early mesoderm commitment and later steps of myogenic expansion or formation from hESCs by controlling other key regulatory events. To test this hypothesis, we propose three specific aims: 1) Determine if microRNAs can promote mesoderm formation and subsequent decisions of cardiac muscle proliferation or differentiation in hESCs; 2) Determine if specific microRNAs repress other lineages in hESCs; 3) Determine the mechanisms by which microRNAs regulate mesoderm commitment, muscle differentiation and proliferation. The tools and understanding developed here will ultimately be used to generate myocytes either directly or through subsequent screens for drugs targeted at the pathways discovered by the proposed work.
The work proposed here will reveal novel mechanisms to induce human embryonic stem cells (hESCs) to differentiate into cardiac and possibly skeletal muscle. The major focus of the aims is to more efficiently derive cardiac cells from hESCs and to understand the mechanisms by which this occurs. Novel pathways will lead to pharamcologic targets that are amenable to high throughput screening. This knowledge will lead to protocols that will allow efficient generation of cardiac muscle cells that could eventually be used for therapeutic purposes in individuals with heart disease. In the short-term, California will benefit from being at the forefront of technology and discovery in hESC biology and by remaining the epicenter of the most progressive basic and translational science. If we are successful in the long-term, California residents will benefit from novel therapies and potential commercialization of discoveries for heart disease, the number one cause of death in the U.S.
SYNOPSIS: This proposal seeks to define a role for miRNAs in cardiomyocyte differentiation. Preliminary data show that in mouse embryonic stem cells (mESCs), miR-1 and miR-133 favor mesoderm development and inhibit ectoderm and endoderm lineages. miR-1 promotes cardiac and skeletal muscle differentiation while miR-133 inhibits myogenesis. The research aims are: 1) Determine if miR-1 or miR-133 can promote mesoderm differentiation from hESCs and also impact the subsequent decision of proliferation versus cardiomyocyte differentiation; 2) Determine if miR-1 or miR-133 repress ectoderm or endoderm differentiation in hESCs; 3) Determine the mechanisms by which miR-1 and miR-133 regulate mesoderm commitment, myocyte differentiation and proliferation, and their potential interaction with the Notch and Wnt signaling pathways.
IMPACT AND SIGNIFICANCE: This proposal has both scientific and clinical significance, related to its potential for discovering new pathways that govern the fate of hESC-derived cardiac progenitors and their differentiated progeny. This proposal seeks to characterize the role of a subset of microRNAs, two miRNAs (miR-133 and miR-1) in cardiomyocyte differentiation in hESC. The PI has shown that these miRNAs play potentially important roles in cardiogenesis in other model systems. Thus, this work could contribute to the understanding of microRNA regulation in hESC in general. The real impact and significance of this research is using novel approaches to generate cells which can repair or regenerate damaged heart muscle, possibly using hESC. It should be noted that this is a highly controversial field and a significant number of other attempts to repopulate damaged heart muscle and show functional changes has been met with much controversy and skepticism. This proposal is reasonably innovative as the PI seeks to manipulate well-known signal transduction pathways in cardiomycytes and hESCs using miRNA, a very fashionable and potentially powerful approach. The real innovation is the integration of superb expertise in cardiobiology, signal transduction and miRNAs as effectors of hESC function.
QUALITY OF THE RESEARCH PLAN: This is a very dense, well written proposal from a seasoned investigator who is an expert in cardiac development. The PI has used multiple tools to make major insights into signaling processes, mostly using the mouse model. The PI has recently identified two miRNAs which are expressed in both cardiac and skeletal muscle and which appear to influence distinctly different signaling pathways which regulate differentiation and proliferation of myoblasts. The hypothesis is that these miRNAs can be used to manipulate hESC to generate myocytes for drug screening or directly for therapeutic use.
The experimental design proposed is very straightforward and appears to be simply transferring everything that the investigator has already done in mouse embryonic cells and cardiomyocytes to hESCs. The PI will over express the miRNAs, determine lineage markers and look at Notch and Wnt signaling pathways. The PI already has voluminous data showing that all of these function in murine cells but there is also preliminary data in drosophila, and some compelling data in H9 hESC. The PI has little experience in human ES cell manipulation but has formed appropriate collaborations. Thus, the proposal will allow a seasoned molecular cardiologist to enter into the hESC field.
That notwithstanding, the hypothesis that the miRNAs might, very specifically, allow expansion of cardiomyocyte progenitors is fraught with difficulties. As indicated by the PI, each miRNA likely has 30 – 50 different targets in the cell and thus the hypothesis that there will be specificity in directing lineage specificity of hESC differentiation is difficult to swallow. Moreover, the PI has already shown that these miRNAs already affect the Wnt and Notch signaling pathways, which are almost ubiquitous. Also, the exact targets of these miRNAs in these pathways are as yet undefined even in the mouse system. Thus, the premise that one might be able to use the manipulation of miRNAs to generate specific types of cardiac lineages is difficult to come to terms with. Moreover, the expression patterns shown in the preliminary data for both miRNAs are quite diverse, although not ubiquitous.
In summary, it is reasonable that the investigator will learn whether these miRNAs can promote mesoderm differentiation and suppress other pathways but whether or not these miRNAs could be that specific for cardiomyocyte differentiation for eventual therapeutic use is highly unclear. This is another example of a seasoned investigator working on a specific developmental pathway and organogenesis model taking this opportunity to apply insights in model systems to the hESC system.
STRENGTHS: The PI is one of the international leaders in the field of cardiovascular developmental biology and molecular cardiology. He has historic productivity in uncovering mechanisms of cardiomyocyte development and differentiation and is experienced in the relatively new (but very hot) field of microRNA regulation. The PI has an excellent track record; a team that is uniquely suited to the work; and is in a superb scientific environment that includes hESC expertise. Finally, there is the potential to recruit a superb investigator into the hESC field.
A major strength of the proposal is the preliminary data, primarily in other system(s), which suggest that the proposed research is entirely feasible and likely to yield valuable information. CIRM has seen many microRNA grants, most of which were very superficial. This proposal, because of the background work, aims to dig much deeper into important regulatory issues. The work on suppression of non-mesodermal lineages seems of more general interest than even the work on cardiomyocyte differentiation for the community as a whole.
WEAKNESSES: The PI has limited experience in hESC but this is not regarded as a major concern, given his track record; a team that is uniquely suited to the work; and a superb scientific environment that includes hESC expertise.
The study appears to be more oriented toward microRNAs per se, than on key steps in hESC-based systems for cardiogenesis. The study of these microRNAs in mouse suggests the possibility that these microRNAs may not necessarily have a direct pivotal role in the control of the formation and fate of ES cell derived cardiac progenitors. Also, the multiple downstream targets of these microRNAs may make it difficult to pinpoint downstream specific pathways that relate to key steps in progenitor formation, renewal, or differentiation. The PI has developed a program to predict targets of microRNAs yet he does not tell us whether his targets mentioned throughout the application come up in this analysis. There was also a lack of justification for expanding these studies already well defined in mESCs to hESCs.
The expression of microRNA targets at different stages of differentiation are clearly important. In the preliminary data of the proposal, results and reports from skeletal muscle and cardiac muscle are intertwined (which is somewhat confusing). But this brings up the issue that, though the genes that determine commitment and terminal differentiation in these two lineages have similar overlap, they also use distinct family members. For example, mef2 is cited several times in the proposal, but the mef2 family members used to enhance myogenic bHLH function during cardiac differentiation are different from those used in skeletal muscle differentiation. There are methods available to enrich for skeletal myoblasts from hESC (independent of cardiomyocytes) and this may be a way to sort out the tissue-specific effects of the microRNAs vis-à-vis the different myogenic regulators.
Another weakness of the proposal is that over-expression of the microRNAs is the approach and is not informed by attempts at reducing expression of the microRNAs (even transiently). Another problem in the preliminary data that carries over to the research plan is the assumption that nestin uniquely marks neural precursors. Nestin is also expressed in other progenitor populations including some in mesoderm.
The microRNAs of most interest might be present in the undifferentiated cardiac progenitors versus those expressed in the already differentiated myocytes. Approaches to allow the isolation of an enriched population of human ES cell derived cardiac progenitors, followed by an examination of which subsets of microRNAs are found in these populations could be valuable. Stable expression in human ES cells of a GFP reporter that has been integrated into the NKX2.5 locus in a recombineered BAC, might allow FACS isolation of this population in differentiated human ES cells and could be valuable here.
A question arises about the efficiency of cardiomyocyte generation in embroid bodies from hESC in this lab. They cite 2-4% of cells adopt a cardiac lineage, whereas other applications in response to this RFA cite 10-25%. Are there issues of handling that should be discussed among CA investigators? Throughout the proposal the PI states that statistical analysis will be done, but does not say what tests will be used. Other important quantities are avoided. In the first aim, clones with varying levels of miRNA expression will be used. How many clones? Finally there is a lack of any in vivo model for determining if miRNA driven hESCs or mESCs can actually repopulate cardiac tissue and contribute to function.
DISCUSSION: This proposal is from a "card carrying cardiologist" and one of the top cardiovascular developmental biologists in the world who has made many major contributions. The PI is relatively new to the miRNA field but has already made important contributions; the miRNA field is also really "hot" now. The PI is new to the stem cell field as highlighted by the number of mistakes (eg the lack of distinction between cardiogenesis and myogenesis) in the application.
The proposal addresses miRNAs and cardiomyocyte development; the PI claims if specific miRNAs are expressed in ESC then differentiation is directed to cardiomyocytes. Generating functional cardiomyocytes that can engraft is an important clinical problem; there is controversy as to whether getting the cells means that they will engraft. The transgenic mice data were impressive. In contrast to other miRNA applications seen by the reviewers, this one really drills down. It is clear that there are multiple targets for any given miRNA therefore that leads to issues. The PI needs to give more thought to how specific miRNAs are likely to be functioning in dictating cell fate - they are not likely to be molecular switches. A reviewer thinks it unlikely that PI's underlying hypothesis will be proven correct but thinks it important to promote entry into the field of a young, world class investigator with a doable project that will generate valuable information. This is a unique situation. The application is not perfect, indicating that the investigator is on a learning curve, but there is a lot of promise and this would be an opportunity to recruit a really superb CV scientist into the field.
PROGRAMMATIC DISCUSSION: A proposal was made to recommend this application for funding. During the discussion it was noted that the PI is a very gifted physician scientist who would be desirable to bring into the hESC field. Also the PI is working in a new area – miRNA - that is "hot". The proposal was beautifully written and compared to other applications focused on miRNA, the research plan was well developed and had depth. All reviewers of the application endorsed the proposal to move to the ‘recommend for funding’ group; the motion was passed by the SMRFWG.