Funding opportunities

Inducing human embryonic stem cell differentiation into cardiomyocytes using extracellular matrix cues

Funding Type: 
New Faculty I
Grant Number: 
RN1-00531
Funds requested: 
$2 304 262
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Heart failure following a heart attack is the leading killer in the United States and is attributed to one out of every five deaths. Heart transplantation is a successful treatment, but there is a limited supply of donor organs and many patients die each year waiting for a heart. Since there is very limited regenerative capacity in the heart, researchers have investigated delivering adult cells in an attempt to replace damaged heart tissue, yet clinical success has not been achieved since these cell types do not match those in the heart. Cardiac muscle cells would be an ideal choice for cell transplantation, but adult cardiac cells do not replicate and thus a large supply cannot be grown. To overcome that limitation, we propose to develop a reliable method to direct human embryonic stem cells to form cardiac muscle cells using cues from the surface they are grown on. Human embryonic stem cells have the ability to become cardiac muscle cells, and are therefore an exciting possible cell source for transplantation to repair the heart after a heart attack. Prior to delivery, the cells must be directed to form cardiac muscle cells. A handful of studies have discovered soluble reagents that promote formation of cardiac muscle cells. Cell fate is not only determined by soluble factors, but by what the cells adhere to. Researchers know that cell adhesion mediates cell fate with adult cells, yet no studies have examined how the cell culture surface affects cardiac muscle cell formation from human embryonic stem cells. This proposal therefore aims to examine several surfaces to promote cardiac muscle cell formation, including the use of micro- and nanopatterned surfaces. With the knowledge gained from this research, cell transplantation for cardiac repair may become an available treatment for heart attacks in humans.
Statement of Benefit to California: 
Human embryonic stem cells have the power to become any cell in the body. Being able to direct them into becoming cardiac muscle cells could allow them to become a treatment for heart failure following a heart attack, which is the leading killer in California. Bioengineering experimental studies proposed here will help create methods to direct human embryonic stem cells into cardiac muscle cells. Researchers have already derived cardiac muscle cells from embryonic stem cells, but methods to enhance and control this process must be developed before the cells can be used in humans. In our approach, bioengineering techniques may be able to create the right environment for the development of cardiac muscle cells. Material cues and specialized surfaces will be used to influence and control embryonic stem cell fate. With success, this technology could provide a cell source for repairing damaged heart tissue. The lifetime risk of developing coronary heart disease after 40 is 49 percent for men and 32 percent for women. Moreover, approximately 38 percent of people who have a heart attack will die from it. It is estimated that this year approximately 700,000 Americans will have a new heart attack and an additional 500,000 will have a recurrent attack, with estimated direct and indirect costs of $151.6 billion. Considering the population of California is approximately 12% of the country, this equates to 84,000 new heart attacks and 60,000 recurrent attacks this year in the state, with an annual cost of $18.2 billion. Treatment using embryonic stem cell derived cardiac muscle cells could thus affect the lives of tens of thousands Californians each year. It would also reduce state health care costs associated with this disease.
Review Summary: 
SYNOPSIS: The proposal is to develop greater control of hESC cardiomyogenesis by using extracellular matrix (ECM) cues and soluble factors. The particular expertise is the patterning of proteins as either micropatterns or nano patterns in 2D or 3D. This plan has 4 aims with similar experiments in each with increasing complexity of patterned ECM proteins from homogeneous coating in 2D, to 3D to micro-patterned to nano-patterned. This is therefore a tissue engineering project with a strong biomedical engineering component. There are four aims that represent essentially the same experiment carried out in distinct ECM platforms. In each aim single ECM proteins in presence or absence of soluble inducers of cardiomyogenesis will be tested in triplicate on plated 24 hr embryoid bodies (EBs) of hESCs; combinations will be used if necessary. The ECM surfaces are: 1) A 2D matrix of collagen I, III, or IV, laminin, or fibronectin 2) A 3D matrix using porous scaffolds made with a hydrogel 3) A micro-patterned matrix generated via photo acid-based photolithography 4) A nano-patterned ECM surface generated by electron-beam cross-linking A key element of the project is that the ESCs will be infected by a lentiviral construct expressing GFP under the cardiac specific promoter (alpha-myosin heavy chain) as an easy read-out system. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: Improving and maximizing the efficiency of cardiomyocyte (CM) differentiation from hESC is an important and significant problem. ECM is recognized as being very important but it is a rather under-studied problem, compared to the role of soluble inducers. Understanding the ECM will likely also be useful for efficient cellular transplantation protocols. Research of hESC cultivation in various biomaterials (and hydrogels in particular) is growing, and there are published articles in the last year that are relevant to the proposed effort. The proposed work is significant, as it is focused on the type and spatial configuration of several proteins as a factor in cardiac differentiation of hESCs. The use of micropatterned substrates for cell culture is not entirely new, but there is a lot of innovation in applying the micro- and nano-patterns developed previously by the PI to hESC culture. The strengths of this proposal are the uniqueness of the expertise of Dr. Christman in both cardiomyocyte and protein patterning, the environment that fosters this study, and the need for more cardiomyocytes. The concept is well explained and the chemistry seems to be well in hand. The application of nanotechnology to manipulating in vitro differentiation of CM is a strength of the application. While interest in the bioengineering aspects and topic of the project was consistent, the reviewers felt that the proposal was not well-developed and was weakened by: the GFP reporter line is not yet validated, issues of cell biology that were less well explained, an oversimplified design, no clear rationale for the selection of culture substrates, limited readouts, and lack of adequate preliminary data. The reporter cells will be provided by Dr. Mercola (Burnham), but it is not clear that these cells are currently available or will be stable. Even if daily monitoring cannot be done on a basis by GFP, one set of each will be examined after 14 days culture by immunostaining for cardiac markers (both fetal and mature), by qRT-PCR and by calcium flux for functional assessment. Beyond the initial experiments, the PI should consider the use of low oxygen for favoring cardiomyocyte differentiation. Additionally one wonders if after optimizing the conditions in Aim 1, it would be advantageous to limit the number of conditions in the subsequent aims. The rationale for using 24 hr EBs was not explained, although this is presumably so that they will flatten down and interact with the matrix. But there might be benefit in dissociating later stage EBs, or inducing for a period of time before plating out on the ECM. There is a focus on inducers such as RA, DMSO, cyclosporine, etc. and not on the soluble inducers that are likely more relevant to the natural pathway such as dkk, FGF, BMPs, etc. The assays are relatively limited, for example ignoring contamination of CMs with other derivatives, such as smooth muscle (SM) or endothelium. The focus on the engineering and not the biology is expected given the background of the PI, but the biology really is limited. The commitment to focus on these limited ECM components might be necessary for the purpose of feasibility. However, another perhaps stronger approach would be to consider ECM that is relevant during embryogenesis at the time of CM specification. This “philosophy” has proven valid for considering relevant soluble factors (growth factors rather than DMSO, for example) and could be applied to this project very nicely. The rationale for the selection and design of patterned materials is not provided, and it is not clear why the specific patterns are expected to promote cardiogenesis. More importantly, the considerations are limited on the chemical composition of protein and the pattern geometry, while other properties of the attachment substrate such as mechanical properties (critically important for electrically excitable, contractile cells) are not included. This oversimplified design clearly reflects the lack of experience with stem cells. The existing information in the literature on cardiac differentiation of stem cells (animal as well as human) that would provide guidance to the proposed work does not seem to be consulted. Likewise, the readouts are really limited to the molecular factors. Cardiac differentiation is more than just the expression of certain cardiac markers. It is surprising that the experimental plans do not include studies of phenotypic stability of the cells, and none of the known functional assays. Overall, the experimental design is not developed to sufficient depth and detail, and it is not clear if the stated objectives will be met. Preliminary data are extensive and solid, but do not establish the feasibility of the proposed approach. There are no studies with hESCs, not even the testing of the general biocompatibility of the planned substrates for culture with hESCs. Given that the PI has no published experience with hESCs, preliminary studies are essential for the project of this scale. Also, it is surprising that there are no advisors or Co-Is included to support cell culture work. Finally, the budget for supplies is excessive. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Christman is a newly hired Asst. Professor (2007) in the UCSD Dept. of Bioengineering. She is highly trained in the field of bioengineering with a relevant BS from Northwestern, PhD from Berkeley, and PD training in nanotechnology from UCLA. She has relevant experience during graduate studies in the delivery of polymers to ischemic myocardium. The PI established a strong publication record as a graduate student and post doc, in biomaterials for treatment of myocardial infarction and in nanotechnology of patterning proteins (12 peer reviewed articles, 4 first author papers in the last two years). She just started her faculty position, has no publications or funding, so it is not possible to evaluate her success as an independent investigator, but her prior publication record indicates a good potential for future development. Dr. Christman presents a logical and ambitious plan to develop a career in tissue engineering and to translate her results to the clinic. Her strengths are clearly in the chemistry and nanotechnology, and she has arranged for mentorship in the biology. She has no current experience in ES or hESC research. The career development plan has strong emphasis on stem cell research. The investigator provides reasonable metrics for success, including teaching the next generation of stem cell biologists. She would clearly benefit from this award in terms of affiliation with the stem cell groups and biologists. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The institutional letter is very strong as is the institution’s commitment to stem cell research. The school has provided Dr. Christman with a solid startup package and support for 2 graduate students, with good access to both bio engineering and cell culture facilities. There are designated mentors including the department Chair, Andrew McCulloch, and two strong investigators outside of the Dept., Sylvia Evans in Pharmacology and Mark Mercola at the Burnham. This outside mentorship of strong biologists will be a very important component for success at bridging the disciplines. Dr. McCulloch provides a strong letter detailing the plans for the school to build in stem cell biology and particularly to bridge disciplines for example via an Engineering in Medicine initiative, and affinity groups across the LaJolla region. The school was funded with a CIRM infrastructure grant. A human ES cell core facility for NIH and Melton lines is established and this is in the process of expansion. Overall there is a very strong environment and clear commitment to building strength in both the department and school. DISCUSSION: The discussants agreed that the proposal has a strong bioengineering component, with an investigator well-trained in biomaterials and nanotechnology. This strength of the application could not off-set the fact that throughout the proposal, the discussion of the biology was rudimentary, and overall, weak. For example, only a single time point is to be analyzed (24 hour embryoid bodies), and without justification for choosing that time point; there is no assessment of contamination by other cell types; endogenous pathways are not being investigated (only artificial inducers are part of the plan); and no functional or phenotypic data is being analyzed.
Conflicts: 

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