Funding opportunities

Directed differentiation of hES cells into the heart valve lineage

Funding Type: 
SEED Grant
Grant Number: 
RS1-00469
Funds requested: 
$675 000
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Tissue engineering is an approach that promises a limitless supply of replacement tissues and organ to treat a wide variety of diseases. These approaches have worked somewhat with artificial skin but not for cardiovascular tissues. Because adult cardiovascular tissues heal poorly, defects in cardiovascular tissues need to be replaced with synthetic materials or tissues taken from cadavers. For heart valves, cadaveric tissues are in short supply and synthetic devices have long-term complications. In children, neither approach works well and these patients face repeat surgery every few years. The common approach for heart valve tissue engineering is to harvest the patients own cells, seed them on degradable substrates and hope that the cells somehow regenerate the substrate before it all dissolves away. This has not worked for heart valves. The main limitation appears to be the absence of cells that are capable of generating a mature structurally complex heart valve matrix. Indeed, adult heart valves have limited potential for self repair and the valve cells appear to generate only disorganized scar tissue. We believe that he field of heart valve tissue engineering can benefit greatly from the availability of heart-valve cells with characteristics similar to those cells that produce valve leaflet tissue during embryonic development. These cells can be harvested in very small numbers from embryos. For medical applications, millions of these cells are needed, however. Growing large numbers of these cells from these few harvested cells is impossible, because during amplification in culture, cells are know to change their characteristic features. After many divisions, these cells no longer possess the desirable features. We believe that human embryonic stem cells can be use for heart valve tissue engineering. Stem cells can be grown in very large numbers and then made to differentiate into the particular cell line with appropriate growth factors. Discovering what these growth factors are is the main objective of this work. These growth factors will be discovered by culturing human embryonic stem cells inside developing mouse hearts, right next to the developing heart valve tissues. This will be done first inside the whole heart that has been taken from the embryo and grown on a culture dish, and later by isolated portions of the developing valve tissue. If we can induce the stem cells to differentiate into the valve cell type, we can then sample the surrounding fluids for the growth factors that have been secreted by the embryonic tissues. Once identified, these growth factors can be use directly on stem cells grown simply in culture dishes. This can be done in very large numbers. We expect that such properly differentiated cells will behave in a more constructive way to remodel a matrix into one that is morphologically closer to living valvular tissue.
Statement of Benefit to California: 
Californians suffer from cardiac diseases no differently than the rest of the citizens in the US. Valvular heart disease is one such condition and affects over 150,000 individuals in the US per year. It is a slow, debilitating process during which the cardiac valves become stenosed or incompetent. Unless the diseased valve is repaired or replaced, this disease eventually results in cardiopulmonary failure and death. Indeed, many patients are misdiagnosed, fail to receive treatment and become so compromised that surgical correction becomes impossible. The most widely used treatment is the implantation of artificial heart valves – either mechanical or tissue-based devices. Mechanical valves are typically constructed from pyrolytic carbon and tissue valves are fabricated from glutaraldehyde-tanned bovine pericardium or whole porcine aortic valves. These devices, however, are imperfect. Mechanical valves are rigid, require chronic anticoagulant therapy, and can fail suddenly and catastrophically. The chronic anticoagulation is associated with cumulative morbidity and mortality with rates as high as 5% per year, essentially guaranteeing some undesirable event within 20 years. Although animal tissue valves or bioprostheses do not require anticoagulation, they have poor long-term durability and eventually fail through calcification and rupture of the valve cusps. For children, none of the solutions described above are satisfactory. Children grow and the best possible solution for these patients is a living valve that can grow with the patient and support cardiac function for decades into the future. Tissue engineering technologies offer the promise of an unlimited supply of organs and tissues to treat a wide variety of injuries and diseases. Like many new technologies, however, tissue engineering has promised more than it has delivered. In the cardiovascular field, in particular, tissue engineering has not made great inroads into product lines occupied by conventional synthetic devices. Part of the problem has been that cardiovascular tissues are more complex than initially envisioned and have a capacity for self-repair that is far less effective than tissue engineering principles demand. Stem cell research, and the harnessing of stem cell technologies, will thus eventually produce devices that are living, rather than inert, and last the life of the patient, rather than a decade or two. California is well-positioned to capitalize on this technology, since two of the three main heart valve manufacturers reside in Santa Anna. Almost all of the new start-up companies making innovative valve technologies are also based in California. Californian is the hub of the world heart valve industry. Advancing this field into stem cell applications is thus best done in California, where the established industry base can translate these technologies into commercial products, and make them available for the treatment of patients in California, and worldwide.
Review Summary: 
The PI is a biomedical engineer in cardiothoracic surgery and is interested in developing approaches to generate heart valve tissues from hES cells. In general, seeding cells onto biodegradable surfaces to generate bioprosthetic valves has not worked. The goal here is to identify signals that will transform cells to the correct phenotype needed for valve tissue formation. The PI will test if placing ES cells in association with cushion tissue is sufficient to transform them into a valvular interstitial cell, and if so, experiments are proposed to identify the relevant morphogen(s). Aim1. Inject diI-labelled human ES cells into E13.5 cushions of cultured mouse hearts. Co-staining of injected cells with SM-actin and MHC will be used as indication of transformation. The PI will not need to culture ES cells, but rather they will be obtained in batches from Dr. Pera. Aim 2. ESC will be co-cultured with isolated valvular tissues derived from E13.5 mouse hearts. Transwell membranes SIGNIFICANCE AND INNOVATION: The problem being addressed is significant and clinically relevant. Based on limitations of available donor tissues and poor progress in developing prosthetic valves, advances in generating useful tissues would be important. The generation of valvular tissue de novo could have importance from a clinical standpoint. In fact there have been reports recently of the ability to generate valvular tissue from amniotic fluid cells. This study will examine whether human ES cells can be converted to valvular tissue upon direct injection into endocardial cushion tissue. STRENGTHS: 1) Intriguing question 2) Preliminary data with amniotic fluid cells WEAKNESSES: Reviewer one: The major problem with the project is alluded to by the applicant: the likelihood that multiple distinct signals are needed to progress to the normal phenotype. ESCs injected into myocardium form teratomas rather than myocardium, and it seems likely the same thing will happen here. The ES will need to be instructed along a mesoderm to mesenchymal fate before they will be receptive to further instruction from the cushion tissue. Another issue is the general lack of definition for a valvular interstitial cell. Will expression of SM actin be sufficient to generate an appropriate functional phenotype? A further complication is that valve formation is a morphological process not just a differentiation program, and this seems unlikely to be recapitulated, other than perhaps in the first Aim. Finally, the ability to purify proteins from CM of these limited cultures seems very unlikely. Reviewer two: 1) Studies will not discriminate between differentiation and fusion 2) Clinical utility somewhat questionable given propensity for forming teratomas would still exist 3) A "flyer", "home run" type experimental design, either works or does not. High likelihood of a negative results and a positive results would still be difficult to interpret. DISCUSSION: There was no further discussion following the comments of the reviewers.
Conflicts: 

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