Funding opportunities
Basic Mechanisms Underlying Human Cardiac Cell-Cell Junction Differentiation in Human iPSC
Funding Type:
Basic Biology II
Grant Number:
RB2-01608
Funds Committed:
$1,361,721
Funding Recommendations:
Not recommended
Public Abstract:
Heart disease is the number one cause of death and disability in California and in the United States. Especially devastating is Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC), an inherited form of heart disease associated with a high frequency of arrhythmias and sudden cardiac death in young people. It is a common heart disease in young athletes, who despite their appearance of health are struck down by this type of heart disease. Understanding this disease is key. Even though it is an inherited disease early detection is hindered because parents carrying the genetic code have highly variable clinical symptoms, making ARVC and catastrophic cardiac events very hard to predict and avoid. There is some evidence that this kind of heart disease is caused by mistakes in the genetic code essential for holding the mechanical integrity of heart muscle cells together or cell junctions. What is missing is an understanding of the basic biology of these heart muscle cell junctions in humans and appropriate human model systems to study their biology and dynamics in the absence and presence of disease. The goal of the studies proposed here is to understand the basic biology of how the human heart muscle cell junctions differentiate and mature and what happens when normal development goes wrong. We will do this by deriving and studying heart muscle cells from normal populations derived from human induced pluripotent stem cells (iPS) and heart muscle cells derived from human iPS from people we know are carrying mistakes in the genetic code for cell junction components. Human iPS are a unique population of stem cells from our own tissues, such as skin, that have the same genetic information as the rest of our bodies. Therefore, human iPS from people who carry the ARVC heart disease mistakes can be used in our laboratory to provide a true human model of that disease. Recent advances in stem cell biology have highlighted the unique potential of human iPS that could be used in the future as a source of cells for cell-based therapies for heart disease. However, before there can be any clinical application of these approaches, we need a detailed fundamental understanding of the basic biology and differentiation/maturation of these human iPS into the heart muscle cells. In this proposal we also propose to determine whether the microenvironment can influence human heart muscle cell junction differentiation/maturation in the absence and presence of disease in the human iPS model system, so that we might be able to obtain high yielding cultures of fully mature functional cells, a future goal for realizing stem cell medicine for heart disease. Knowledge gained from these studies will advance the field by providing us with model systems to study the biology of human cardiac muscle cell junction differentiation/maturation, which are crucially important in understanding human cell-cell junction diseases such as ARVC.
Statement of Benefit to California:
Heart disease is the number one cause of death and disability within the United States and the rates are calculated to be even higher for citizens of the State of California when compared to the rest of the nation. These diseases place tremendous financial burdens on the people and communities of California, which highlights the need to find therapies to alleviate these growing burdens. Especially devastating is Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC), an inherited form of heart disease associated with a high frequency of arrhythmias and sudden cardiac death in young people. Even though it is an inherited disease early detection is hindered because parents carrying the genetic code have highly variable clinical symptoms, making ARVC and catastrophic cardiac events very hard to predict and avoid. There is some evidence that this kind of heart disease is caused by mistakes in the genetic code essential for holding the mechanical integrity of heart muscle cells together or cell junctions. What is missing is an understanding of the basic biology of these heart muscle cell junctions in humans and appropriate human model systems to study their biology and dynamics in the absence and presence of disease. The goal of the studies proposed here is to understand the basic biology of how the human heart muscle cell junctions differentiate and mature and what happens when normal development goes wrong using human induced pluripotent stem cells (iPS) as a model system. Recent important advances in stem cell biology have highlighted the unique potential of human iPS, which is an endogenous population of stem cells, which can be derived from our own tissues, such as skin, that could be used in the future as a source of cells for patient-specific cell-based therapies for heart disease. However, before there can be any clinical application of these approaches, a fundamental understanding of the basic biology and maturation/differentiation of these unique stem cells into heart muscle cells and muscle cell-cell junctions will be required. Our proposal meets this goal and tries to identify avenues to advance stem cell medicine for heart disease by developing model systems to systematically characterize and manipulate the maturation and differentiation of human iPS into fully mature heart muscle cells by studying the basic biology behind how heart muscle cell junctions differentiate and mature. Since various forms of human heart diseases, including ARVC, have underlying defects in muscle cell junctions, this knowledge will be crucially important in unraveling the biological defects burdening Californians with these forms of heart disease alongside advancing the application of stem cell based approaches as therapies for heart disease, such as ARVC, for citizens of the State of California.
Review Summary:
EXECUTIVE SUMMARY
The goal of this proposal is to study the differentiation of cardiomyocytes from human induced pluripotent stem cells (iPSCs) and their subsequent functional maturation. The applicant proposes to derive iPSCs from patients with Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) as well as healthy controls. Experiments will focus on the establishment of cell-cell junctions between cardiomyocytes, called desmosomes, which are required for normal cardiac function and disrupted in ARVC. The applicant proposes two Specific Aims. First, the applicant proposes to characterize the development and function of cell-cell junctions in cardiomyocytes derived from normal iPSCs and test the effects of several extracellular matrix (ECM) culture substrates. In Aim 2, the applicant proposes to derive iPSCs from ARVC patients and compare their differentiation and maturation into cardiomyocytes with those derived from healthy iPSC controls.
Reviewers agreed that this proposal addresses a major unsolved problem in stem cell biology: the inability to differentiate pluripotent stem cells into cardiomyocytes that are mature and form functional cell-cell junctions. Since several human heart diseases, including ARVC, have underlying defects in these junctions, results generated from this proposal could have therapeutic implications. Reviewers did find a potentially significant flaw in the rationale for the proposed approach, however. The applicant hypothesizes that ECM cues present in a native, fully-developed heart (of porcine origin) will direct iPSC-derived cardiomyocyte differentiation and maturation. However, reviewers cautioned that this approach may not be as effective as using a less mature matrix, similar to one that would be present during early stages of cardiac development when the heart undergoes its most rapid period of growth and differentiation. Furthermore, the applicant describes the complexity of the cardiac-derived matrix as a benefit, but it may also be a weakness because it will be impossible to thoroughly characterize the biochemical components, their relative amounts, and the batch-to-batch variation of such matrices. Without this characterization it will be difficult to establish mechanistic links between ECM composition and cardiomyocyte differentiation. Supplemental data from recent mass spectrometry analysis indicate that the applicant is attempting to address this concern, but it will likely take significant time and effort to definitively resolve these issues.
Reviewers described the research plan as well organized and clearly written but not particularly ambitious; they found the two Aims redundant in their experimental design. In addition, they noted that the applicant does not propose the most sophisticated methods for characterizing the electrophysiological properties of cardiomyocytes. Reviewers found the preliminary data to be generally supportive of the proposed approaches. However, they cited the lack of data demonstrating iPSC differentiation into cardiomyocytes as a weakness, since differences in the differentiation potential between human embryonic stem cells and iPSC, and amongst iPSC lines derived from different donors, may exist. They did appreciate that the applicant has previously developed a mouse model of ARVC and should be able to compare the results from this study to in vivo mouse studies. While viewing this as an asset, reviewers nonetheless cautioned that species differences in cardiac physiology might inherently limit the impact of what could be derived from this information.
The reviewers found the principal investigator (PI) and research team to be clear strengths of the proposal. They praised the excellent collaborators, who bring complementary expertise to the project. In general, reviewers found the research team well qualified to conduct the proposed studies.
Overall, while reviewers appreciated that this proposal addresses an important question, they raised questions about the scientific rationale and experimental design that reduced their enthusiasm.
Conflicts:
- Ali Brivanlou
