The process of new blood vessel formation is essential for repair of damaged or diseased tissues and organs. Regeneration of the heart is an important goal in modern medicine. Ideally, new therapies should give patients long-term normalization of the heart. Embryonic stem cells could be induced to form heart muscle cells and cells that line the blood vessels known as endothelial cells. Here we propose to use a natural hormone-like protein known as leptin, to stimulate embryonic stem cells to form cardiac muscle and endothelial cells. In our laboratory, we have done early experiments using organized stem cell clusters known as embryoid bodies treated with leptin. Our early studies showed that leptin can induce the formation of primitive blood vessels and contractile heart muscle cells. Based on our findings, we propose that leptin is important to stimulate human embryonic stem cells to form heart muscle and endothelial cells. Furthermore, we also think that the presence of primitive blood vessels in embryoid bodies can facilitate or be required for the formation of mature cardiac muscle cells. It is possible that blood vessel formation in response to leptin (or similar proteins) supplies a blood vessel network providing an appropriate environment where stem cells can then become heart muscle cells. Therefore, we propose three objectives: (I) To characterize the effect of leptin treatment on embryoid bodies cultures; (II)To optimize the conditions under which leptin (and possibly other related substances) induce formation of cells from the cardiovascular system thus increasing the number of endothelial and heart muscle cells in the cultures; and (III)To test the function of the endothelial and heart muscle cells formed in the cultures by using them in an experimental animal model of heart attack (myocardial infarction). We will also study the molecular aspects of the response to leptin. In addition, we will use specific markers to ascertain the endothelial and cardiac muscle character of the cells formed. Finally, we will separate the endothelial and heart muscle cells from the cultures and then use them to study if heart muscle cells benefit from the presence of endothelial cells. These cells will also be given to mice of the myocardial infarction model to prove their repair potential in the injured heart. To see if leptin can always induce formation of endothelial and heart muscle cells, we will use four different human embryonic stem cell lines, including a new cell line. The use of a new line has advantages to meet the strict regulatory requirements of the Food and Drug Administration (FDA) for therapeutic use in humans. Knowledge derived from the proposed studies will contribute to understand how human embryonic stem cells become mature functional cells. Moreover, these studies may result in optimized experimental conditions leading to the large scale production of heart muscle and endothelial cells for therapeutic applications.
Statement of Benefit to California:
Heart disease is the number one cause of death in California and in the United States. In California alone, heart disease accounts for 39% of all deaths and it is a major cause of disability and poor quality of life. The economic impact of heart disease is astonishing. In California, the health care costs and loss of productivity due to heart disease is approximately $30 billion per year. The yearly cost of coronary heart disease alone is at least $15 billion in the State. These expenditures account for one tenth of the total cost burden in the entire nation. In addition, type II diabetes and obesity are well-known risk factors for coronary heart disease. These two diseases are increasing at alarming rates every year and consequently contributing to the rising incidence of heart disease. The people of California made an important choice by voting to pass Proposition 71. Embryonic stem cells are likely to be unique tools in the treatment of genetic and neurological diseases. However, their potential use for effective therapeutic repair and regeneration of the heart could be life-saving for millions of individuals suffering from heart disease. In this regard, our research proposal seeks not only to advance our current understanding of the coordinate development of a complex tissue from stem cells, but most importantly, to focus on the translational aspect of this knowledge inasmuch as it provides a means to solve a major medical challenge, namely the repair of injured or damaged heart tissue with cells derived from human embryonic stem cells. In the long term, effective therapies to repair the myocardium, coupled with a comprehensive package of preventive health policies instituted statewide, would undoubtedly translate into significant savings to the State. The magnitude of such savings in heart disease costs alone is likely to surpass or at least return the investment made by California when Proposition 71 was passed.
SYNOPSIS: The project seeks to study the differentiation from embryoid bodies (EBs) of endothelial cells (EC) and cardiomyocytes (CM), with a focus on the role of leptin in enhancing this process. Leptin is a cytokine with angiogenic activity and the Principal Investigator (PI) made the observation that EB cultures treated with leptin have increased numbers of beating CM clusters. The hypothesis is that leptin might induce vascular networks that provide a niche for CM development.
Aim 1 will define the effect of leptin on the EB cultures. Markers and microarrays will be analyzed and STAT3 signaling evaluated. It appears that the PI can show an approximate 2x increase in numbers of beating clusters at day 13. It is not clear why microarray experiments will be useful.
Aim 2 will optimize the procedure, although it is not clear exactly how this differs from Aim 1. Additional variables will be tested including other cytokines, media, O2, temperature, etc.
In Aim 3 the cells will be tested functionally. Purified cells will be put into infracted animals and hearts analyzed for
INNOVATION & SIGNIFICANCE: This proposal focuses on the utility of human embryonic stem cells (hESCs) as a tool for coordinated differentiation of cardiomyocyte and endothelial co-cultures. Most present systems for use of ESC in myocardial regeneration approaches utilize either one or the other of these lineages, so the dual differentiation concept is a novel one. Myocardial regeneration remains an elusive goal, so the proposal has underlying merit in this regard. Advances in stimulating cardiovascular fates from hESCs will be important. This project derives from an observation made from an NIH supplement, and might lead to improved induction protocols for EC and/or CM.
STRENGTHS: The PI has expertise in neo-vascularization in wound response. The expertise for studying animal models is apparent. Other strengths include: (1)Innovative use of leptin-directed stem cell differentiation; (2)Good environment; (3) Important clinical problem.
WEAKNESSES: The project is not well described and it is unclear how much new information will be learned other than perhaps confirming a modest enhancement of CM from EB cultures. How specific is this to CM? Were total cell numbers normalized? In the animal models, assuming similar numbers of cells are used, it needs to be explained why these would behave differently than purified cells that develop naturally in EBs. Experiments in the first two aims are essentially a series of different culture conditions without any apparent rationale or priority. Experiments in the last aim on animals are also not described with clarity (mouse or rat?) and appear to be premature even at this pilot stage.
Other concerns include:(1) The preliminary data are pretty sketchy. Data are presented in only a semi-quantitative fashion. Quantitative approaches to sort various cell types are available and would allow a much better discrimination of the effects of leptin in culture. (2) No effect to ascertain electromechanical coupling of cardiomyocytes—a key step if they are to function in vivo.(3)The collaborative team seems only loosely pulled together—the microarray studies are dependent on participation of Dr. Srivastava, who doesn’t seem to have committed to the project. It is unclear how the infarction experiments will be completed—will these be done by the PI (who doesn’t seem to have the equipment for the studies) or in the collaborator’s lab? Infarction requires survival surgery in SCID mice, which is a challenge and the procedures must be done in a more sterile environment than is typical for mouse surgeries. The logistics do not seem to be well considered.
DISCUSSION: There was no further discussion following the reviewers' comments