Heart Disease

Coding Dimension ID: 
295
Coding Dimension path name: 
Heart Disease

Mechanism of heart regeneration by cardiosphere-derived cells

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-06215
ICOC Funds Committed: 
$1 367 604
Disease Focus: 
Heart Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
In the process of a heart attack, clots form suddenly on top of cholesterol-laden plaques, blocking blood flow to heart muscle. As a result, living heart tissue dies and is replaced by scar. The larger the scar, the higher the chance of premature death and disability following the heart attack. While conventional treatments aim to limit the initial injury (by promptly opening the clogged artery) and to prevent further damage (using various drugs), regenerative therapy for heart attacks seeks to regrow healthy heart muscle and to dissolve scar. To date, cell therapy with CDCs is the only intervention which has been shown to be clinically effective in regenerating the injured human heart. However, the cellular origin of the newly-formed heart muscle and the mechanisms underlying its generation remain unknown. The present grant seeks to understand those basic mechanisms in detail, relying upon state-of-the-art scientific methods and preclinical disease models. Our work to date suggests that much of the benefit is due to an indirect effect of transplanted CDCs to stimulate the proliferation of surrounding host heart cells. This represents a major, previously-unrecognized mechanism of cardiac regeneration in response to cell therapy. The proposed project will open up novel mechanistic insights which will hopefully enable us to boost the efficacy of stem cell-based treatments by bolstering the regeneration of injured heart muscle.
Statement of Benefit to California: 
Coronary artery disease is the predominant cause of premature death and disability in California. Clots form suddenly on top of cholesterol-laden plaques in the wall of a coronary artery, blocking blood flow to the heart muscle. This leads to a “heart attack”, in which living heart muscle dies and is replaced by scar. The larger the scar, the greater the chance of death and disability following the heart attack. While conventional treatments aim to limit the initial injury (by promptly opening the clogged artery) and to prevent further injury (using various drugs), regenerative therapy for heart attacks seeks to regrow healthy heart muscle and to dissolve scar. To date, cell therapy with CDCs is the only intervention that has been shown to be clinically effective in regenerating the injured human heart. However, the cellular origin of the newly-formed heart muscle and the mechanisms underlying its generation remain unknown. The present grant seeks to understand those basic mechanisms in detail, relying upon state-of-the-art scientific methods and preclinical disease models. The resulting insights will enable more rational development of novel therapeutic approaches, to the benefit of the public health of the citizens of California. Economic benefits may also accrue from licensing of new technology.
Progress Report: 
  • Key abbreviations:
  • CDCs: cardiosphere-derived cells
  • MI: myocardial infarction
  • The present award tests the hypothesis that CDCs promote regrowth of normal mammalian heart tissue through induction of adult cardiomyocyte cell cycle re-entry and proliferation (as occurs naturally in zebrafish and neonatal mice). Such a mechanism, if established, would challenge the dogma that terminally-differentiated adult cardiomyocytes cannot re-enter the cell cycle. We have employed an inducible cardiomyocyte-specific fate-mapping approach (to specifically mark resident myocytes and their progeny), coupled with novel methods of myocyte purification and rigorous quantification. We have also developed assays that enable us to exclude potential technical confounding factors. The use of bitransgenic mice is essential for our experimental design (as it enables fate mapping of resident myocytes in a mammalian model), while the use of mouse CDCs in our in vivo experiments (as opposed to human CDCs) enables us to avoid immunosuppression and its complications. To date, mouse, rat and pig models have proven to be reliable in predicting clinical effects of CDC therapy in humans, and results with human and mouse CDCs in comparable models (e.g., SCID mice for human CDCs versus wild-type mice for mouse CDCs) have not revealed any major mechanistic divergence. Our results demonstrate that induction of cardiomyocyte proliferation represents a major, previously-unrecognized mechanism of cardiac regeneration in response to cell therapy. One full-length publication describing these findings has appeared (K. Malliaras et al., EMBO Mol Med, 2013, 5:191-209), and another paper has been submitted. The work has already begun to open up novel mechanistic insights which will enable us to improve the efficacy of stem cell-based treatments and bolster cardiomyocyte repopulation of infarcted myocardium.

A new paradigm of lineage-specific reprogramming

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-06035
ICOC Funds Committed: 
$1 708 560
Disease Focus: 
Heart Disease
Stem Cell Use: 
Directly Reprogrammed Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Recently, we devised and reported a new regenerative medicine paradigm that entails temporal/transient overexpression of induced pluripotent stem cell based reprogramming factors in skin cells, leading to the rapid generation of “activated” cells, which can then be directed by specific growth factors and small molecules to “relax” back into various defined and homogenous tissue-specific precursor cell types (including nervous cells, heart cells, blood vessel cells, and pancreas and liver progenitor cells), which can be expanded and further differentiated into mature cells entirely distinct from fibroblasts. In this proposal, combined with small molecules that can functionally replace reprogramming transcription factors as well as substantially improve reprogramming efficiency and kinetics, we aim to further develop and mechanistically characterize chemically defined, non-integrating approaches (e.g., mRNA, miRNA, episomal plasmids and/or small molecule-based) to robustly and efficiently reprogram skin fibroblast cells into expandable heart precursor cells. Specifically, we will: determine if we can use non-integrating methods to destabilize human fibroblasts and facilitate their direct reprogramming into the heart precursor cells; characterize of heart cells generated by the direct programming methods, both in the tissue culture dish and in a mouse model of heart attack; and characterize newly identified reprogramming enhancing small molecules mechanistically.
Statement of Benefit to California: 
This study will develop and mechanistically characterize a new method of generating safe patient specific heart cells that could be useful in treating heart failure which afflicts millions of Californians and accounts for billions of dollars in healthcare spending annually. Additionally, the small molecules discovered in this study could be good candidates for future drug development as well as being broadly useful for other regenerative medicine applications. These advances could also be a platform for new personalized medicine/ cell banking businesses which could bring economic growth in addition to improving the health of Californians.
Progress Report: 
  • During the reporting period, we have made very significant progress toward the following research aims: (1) Using the Oct4-based reprogramming assay system established, we were able to screen for and identify small molecules that can replace the other three genes in the Cell-Activation and Signaling-Directed (CASD) lineage conversion paradigm for reprogramming fibroblasts into cardiac lineage. (2) Using in-depth assays, we have examined the process using lineage-tracing methods and characterized those Oct4/small molecules-reprogrammed cardiac cells in vitro. (3) Most importantly, we were able to identify a baseline condition that appears to reprogram human fibroblasts into cardiac cells using defined conditions.

Induction of Pluripotent Stem Cell-Derived Pacemaking Cells

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-05764
ICOC Funds Committed: 
$1 334 880
Disease Focus: 
Heart Disease
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Currently, over 350,000 patients per year with abnormal heart rhythm receive electronic pacemakers to restore their normal heart beat. Electronic pacemakers do not respond to the need for changing heart rate in situations such as exercise and have limited battery life, which can be resolved with biopacemakers. In this proposed project, we will examine methods that improve the generation of pacemaking cells from human induced pluripotent stem cells as candidates for biopacemaker.
Statement of Benefit to California: 
This proposal aims to generate pacemaking cells through facilitated differentiation from human induced pluripotent stem cells that may serve as biopacemakers. Over 350,000 patients a year in the U.S. require the implantation of an electronic pacemaker to restore their heart rhythm, with more than 3 million patients that are dependent on this device. At the cost of $58K per pacemaker implantation, the healthcare burden is greater than $20 billion a year. However, the cost associated with these electronic devices does not end with surgery for implantation. These devices require a battery change every 5 to 10 years that involve another surgical procedure. With California being the most populated state, this can be very costly to the Californians. It also does not give the patients the quality of life by having to endure repeated surgeries. The possibility of biopacemaker that requires no future battery replacements and other advantages such as physiological adaptation to the active state of the patient make biopacemakers a truly desirable alternative to electronic devices. Moreover, one in 20,000 infants or preemies with congenital sinoatrial node dysfunction are also inappropriate candidates to receive electronic pacemakers because they are physically too small and require a proportional increase in the length of pacing leads with their significant growth rate. Therefore, there is a great need for biopacemakers that may overcome the deficiencies of electronic devices.
Progress Report: 
  • This goal of this project is to improve the yield of pacemaking cells from human induced pluripotent stem cells (hiPSCs) that can be used to engineer biopacemaker. We have demonstrated that manipulation of the membrane potential of hiPSCs using small molecules can upregulate genes of the desired cell type progressing to the pacemaking cells at all differentiation stages. In the differentiation stage to mesodermal cells, treated hiPSCs exhibit a membrane potential that is further down the differentiation path than untreated control. This source was this change was examined.

Epigenetic regulation of human cardiac differentiation

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-05901
ICOC Funds Committed: 
$1 708 560
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
Each cell type in our body has its own identity. This identity allows a heart cell to contract repetitively, and a brain cell to conduct nerve impulses. Each cell type gains its identity by turning on or off thousands of genes that together give the cell its identity. Understanding how these sets of genes are regulated together as a cell gains its identity is important to be able to generate new cells in disease. For example, after a heart attack, heart muscle dies, leaving scar tissue and a poorly functioning heart. It would be very useful to be able to make new heart muscle by introducing the right set of instructions into other cells in the heart, and turn them into new heart muscle cells. One way that many genes are turned on or off together is by a cellular mechanism called epigenetic regulation. This global regulation coordinates thousands of genes. We plan to understand the epigenetic regulatory mechanisms that give a human heart muscle cell its identity. Understanding their epigenetic blueprint of cardiac muscle cells will help develop strategies for cardiac regeneration, and for a deeper understanding of how cells in our body acquire their individual identities and function.
Statement of Benefit to California: 
This research will benefit the state of California and its citizens by helping develop new approaches to cardiac regeneration that will be more efficient than current approaches, and amenable to drug-based approaches. In addition, the knowledge acquired in these studies will be important not only for heart disease, but for any other disease where reprogramming to regenerate new cells is desirable. The mechanisms revealed by this research will also lead to new understanding of the basis for congenital heart defects, which affect several thousand Californian children every year, and for which we understand very little.
Progress Report: 
  • We have made considerable progress on this project, which is aimed at understanding how genes are controlled during the conversion of human stem cells into heart cells. We have been able to use advanced techniques that allow us to make millions of human heart cells in a dish from "Induced Pluripotent Stem Cells" (known as iPS cells), which are cells derived from skin cells that have properties of embryonic stem cells. We are now using genome engineering techniques to insert a mutation that is associated with human congenital heart defects. We are now starting to map the chromatin marks that will tell us how heart genes are turned on, while genes belonging to other cell types are kept off. This "blueprint" of a heart cell will help us understand how to make better heart cells to repair injured hearts, and will allow us to model human congenital heart disease in a human experimental system.

Studying Arrhythmogenic Right Ventricular Dysplasia with patient-specific iPS cells

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-06276
ICOC Funds Committed: 
$1 582 606
Disease Focus: 
Heart Disease
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Most heart conditions leading to sudden death or impaired pumping heart functions in the young people (<35 years old) are the results of genetic mutations inherited from parents. It is very difficult to find curative therapy for these inherited heart diseases due to late diagnosis and lack of understanding in how genetic mutations cause these diseases. Using versatile stem cells derived from patients’ skin cells with genetic mutations in cell-cell junctional proteins, we have made a significant breakthrough and successfully modeled one of these inherited heart diseases within a few months in cell cultures. These disease-specific stem cells can give rise to heart cells, which allow us to discover novel abnormalities in heart energy consumption that causes dysfunction and death of these diseased heart cells. Currently, there is no disease-slowing therapy to these inherited heart diseases except implanting a shocking device to prevent sudden death. We propose here to generate more patient-specific stem cell lines in a dish from skin cells of patients with similar clinical presentations but with different mutations. With these new patient-specific stem cell lines, we will be able to understand more about the malfunctioned networks and elucidate common disease-causing mechanisms as well as to develop better and safer therapies for treating these diseases. We will also test our new therapeutic agents in a mouse model for their efficacy and safety before applying to human patients.
Statement of Benefit to California: 
Heart conditions leading to sudden death or impaired pumping functions in the young people (<35 years old) frequently are the results of genetic mutations inherited from parents. Currently, there is no disease-slowing therapy to these diseases. It is difficult to find curative therapy for these diseases due to late diagnosis. Many cell culture and animal models of human inherited heart diseases have been established but with significant limitation in their application to invent novel therapy for human patients. Recent progress in cellular reprogramming of skin cells to patient-specific induced pluripotent stem cells (iPSCs) enables modeling human genetic disorders in cell cultures. We have successfully modeled one of the inherited heart diseases within a few months in cell cultures using iPSCs derived from patients’ skin cells with genetic mutations in cell-cell junctional proteins. Heart cells derived from these disease-specific iPSCs enable us to discover novel disease-causing abnormalities and develop new therapeutic strategies. We plan to generate more iPSCs with the same disease to find common pathogenic pathways, identify new therapeutic strategies and conduct preclinical testing in a mouse model of this disease. Successful accomplishment of proposed research will make California the epicenter of heart disease modeling in vitro, which very likely will lead to human clinical trials and benefit its young citizens who have inherited heart diseases.
Progress Report: 
  • Most heart conditions leading to sudden death or impaired cardiac pumping functions in the young people (<35 years old) are the results of genetic mutations inherited from parents. It is very difficult to find curative therapy for these inherited heart diseases due to late diagnosis and lack of understanding in how genetic mutations cause these diseases. One of these inherited heart diseases is named arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). The signature features of sick ARVD/C hearts are progressive heart muscle loss and their replacement by fat and scare tissues, which can lead to lethal irregular heart rhythms and/or heart failure. We have made a significant breakthrough and successfully modeled the sick ARVD/C heart muscles within two months in cell cultures using versatile stem cells derived from ARVD/C patients’ skin cells with genetic mutations in one of the desmosomal (a specific type of cell-cell junctions in hearts) proteins, named plakophilin-2. These disease-specific stem cells can give rise to heart cells, which allow us to discover specific abnormalities in heart energy consumption of ARVD/C heart muscles that causes dysfunction and death of these diseased heart cells. In the Year 1 of this grant support, we have made and characterized additional stem cells lines from ARVD/C patients with different desmosomal mutations. We are in the process to determine if heart muscles derived from these new ARVD/C patient-specific stem cells have common disease-causing mechanisms as we had published. We found two proposed therapeutic agents are ineffective in suppressing ARVD/C disease in culture but we have identified one potential drug that suppressed the loss of ARVD/C heart cells in culture. We also started to establish a known ARVD/C mouse model for future preclinical drug testing.

VEGF signaling in adventitial stem cells in vascular physiology and disease

Funding Type: 
New Faculty II
Grant Number: 
RN2-00909
ICOC Funds Committed: 
$3 155 931
Disease Focus: 
Heart Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Coronary heart disease is the leading cause of death in the developed world. This disease results from atherosclerosis or fatty deposits in the vessel wall that causes blockage of coronary arteries. Blockage of these arteries cut off supplies of nutrients and oxygen to the heart muscle, causing heart attacks, heart failure or sudden death. To restore coronary blood supply, physicians use guide-wires to position an inflatable balloon at the blockage site of the artery, where the balloon is inflated to open up the artery. This procedure is called percutaneous transluminal coronary angioplasty or PTCA, which is usually accompanied by the placement of a metal tube (or stent) at the diseased site to maintain vessel opening. PTCA is the dominant procedure to restore blood flow in coronary arteries- in the United States alone nearly 1.3 million PTCA procedures were performed in 2004. However, as a response to PTCA-related vessel wall damage, cells from the vessel wall are activated to divide and grow into the vessel lumen, causing re-narrowing or restenosis of the artery. Restenosis of the vessel lumen is the major hurdle limiting the success of PTCA. It occurs in 20-50% of cases within six months of the initial PTCA procedure and requires repeated PTCA to open up the re-narrowed artery, leading to tremendous human and social expenses. Stents which contain drug inhibitors of cell growth (drug eluting stents, or DES) reduce restenosis; however, considerable concerns have emerged regarding the safety of DES due to an increased risk of sudden stent occlusion by platelet aggregates (or thrombosis). This sudden occlusion is caused by a concomitant drug inhibition of cells that cover the raw surface of metal stents to prevent platelet aggregation. This complication is frequently lethal, resulting in death or heart attack in 85% of cases. The safety concerns over DES have created an urgent need to define the mechanisms underlying the biology of restenosis. A population of cells resident in the vessel wall consists of progenitor cells that divide and grow into the vessel lumen when vessels are injured. The repair process mediated by these cells directly contributes to vessel restenosis. Our goal is to understand the biology of these stem cells in the repair of injured arteries- how vessel injury signals these cells to divide and invade the vessel lumen, what molecular effectors control the cellular responses, and how to intercept these signals and effectors to prevent vessel restenosis. This will provide a solid scientific basis for new therapeutic targets and strategies for vessel restenosis after PTCA. The proposal is a targeted response to CIRM New Faculty Awards II. It seeks to extend my research expertise into the field of stem cell biology related to clinically important vascular diseases. We are confident that our proposed studies will generate significant progress in this field, in both scientific knowledge and useful therapies.
Statement of Benefit to California: 
Coronary heart disease is the leading cause of death in California. This disease results from atherosclerosis or fatty deposits in the vessel wall that causes blockage of coronary arteries of the heart, causing heart attacks, heart failure or sudden death. Physicians use wires and balloons to open up the blocked artery (angioplasty) and a metal tube (stent) to keep the artery open and restore blood flow. Although effective, angioplasty and stenting cause some damages to the blood vessel, which leads to a recurrent blockage (or restenosis) of the vessel in 20-50% of patients within 6 months of the procedure. This vessel restenosis requires repeated angioplasties and stenting for restoration of blood flow. Given the large number of patients with coronary heart disease in California, the need for repeated surgical procedures has resulted in tremendous human, social and economic costs in our state. An attempt to reduce vessel restenosis is the placement of drug-eluting stents (or DES) in angioplastied vessels. Although drugs released from the stents reduce vessel restenosis, this approach creates a new and frequently fatal complication- sudden occlusion of the stented arteries. This complication is because drugs in the stents delay the repair of inner lining of the artery, whose function is to prevent platelet aggregation within the lumen of the artery. Sudden platelet aggregation (or thrombosis) within the vessel lumen causes instantaneous obstruction of the artery, leading to acute heart attacks or death. Thus, the safety concerns over DES have created an urgent need to define the mechanisms underlying the biology of restenosis. A population of cells present at the vessel wall possess stem cell characteristics. After vessel injury, these cells increase in number and turn into different kinds of cells, which then migrate from the vessel wall into the lumen, causing blockage of the vessel. Thus, understanding how these cells behave will inspire new ideas for treating recurrent vessel blockage or restenosis. We propose to study how and what molecular signals activate these cells when vessels are injured. Our goal is to provide a scientific strategy of intercepting these signals for the treatment of vessel restenosis. We believe that understanding the biology of vascular stem cells will lead to significant advances in the research and novel therapies of vessel injury and restenosis. Given the scope of this problem , an improved therapy of vessel restenosis will have a significant economic and social impact. We have proposed to use modern methods in genetics, cell biology, and molecular biology to attack the challenges of this project. At the same time, we will train a new generation of bright students and junior scientists in the areas of stem cell biology highly relevant to human disease. This ensures that an essential knowledge base will be preserved, passed on and expanded in California for the foreseeable future.
Progress Report: 
  • Coronary heart disease is the leading cause of death in the developed world. This disease results from atherosclerosis or fatty deposits in the vessel wall that causes blockage of coronary arteries. Blockage of these arteries cut off supplies of nutrients and oxygen to the heart muscle, causing heart attacks, heart failure or sudden death. To restore coronary blood supply, physicians use guide-wires to position an inflatable balloon at the blockage site of the artery, where the balloon is inflated to open up the artery. This procedure is called percutaneous transluminal coronary angioplasty or PTCA, which is usually accompanied by the placement of a metal tube (or stent) at the diseased site to maintain vessel opening. However, as a response to PTCA, cells from the vessel wall are mobilized to divide and grow into the vessel lumen, causing re-narrowing of the artery. Renarrowing of the vessel lumen is the major hurdle limiting the success of PTCA. Mental stents which contain drug inhibitors of cell growth (drug eluting stents, or DES) reduce re-narrowing; however, considerable concerns have emerged regarding the safety of DES due to an increased risk of sudden stent occlusion by platelet aggregates (or thrombosis). This sudden occlusion is caused by a concomitant drug inhibition of cells that cover the raw surface of metal stents to prevent platelet aggregation. This complication is frequently lethal, resulting in death or heart attack in 85% of cases. The safety concerns over DES have created an urgent need to define the mechanisms underlying the biology of vascular re-narrowing.
  • A population of cells resident in the vessel wall consists of stem cells that divide and grow into the vessel lumen when vessels are injured. The repair process mediated by these cells directly contributes to vessel re-narrowing. Our goal is to understand the biology of these stem cells in the repair of injured arteries- how vessel injury signals these cells to divide and invade the vessel lumen, what molecular effectors control the cellular responses, and how to intercept these signals and effectors to prevent vessel re-narrowing. This will provide a solid scientific basis for new therapeutic targets and strategies for vessel re-narrowing after PTCA.
  • In the past year, we have successfully developed in the laboratory a more efficient method of isolating the vessel wall stem cells (or adventitial stem cells) and growing these cells in test tubes. The ability to isolate and grow these stem cells has allowed us to study the effects of many biologically active molecules on these cells critical for vascular repair and re-narrowing. We are now using this method to study molecular pathways that can modify the biological behavior of the vessel wall stem cells. Furthermore, we have developed a different method of injuring the blood vessels to study how the vessel wall stem cells respond to different types of vessel injury. This method allows us to track the mobilization of vessel wall stem cells more precisely in the vascular repair process. We are using this method to study the activity of vessel wall stem cells following injury.
  • Coronary heart disease is the leading cause of death in the developed world. This disease results from atherosclerosis or fatty deposits in the vessel wall that causes blockage of coronary arteries, causing shortage of blood supply with consequent heart attacks, sudden death, or heart failure. To restore coronary blood supply, physicians use guide-wires to position an inflatable balloon at the blockage site of the artery, where the balloon is inflated to open the artery. This angioplasty procedure is usually accompanied by the placement of a metal stent at the diseased site to maintain vessel opening. Such percutaneous coronary intervention (PCI) with angioplasty and stenting is the dominant procedure for opening obstructed coronary arteries. However, PCI activates a population of cells in the vessel wall to grow into the vessel lumen, causing re-narrowing of the artery. This vessel re-narrowing (restenosis) is the major hurdle limiting the success of PCI. Mental stents coated with drug inhibitors of cell growth (drug eluting stents, or DES) reduce re-narrowing; however, considerable concerns have emerged regarding the safety of DES due to an increased risk of sudden stent occlusion by platelet aggregates (or thrombosis) and the need for prolonged anti-platelet therapy, which poses bleeding risks especially to older patients or patients who need surgery. These concerns call for defining mechanisms that control re-narrowing of injured arteries.
  • A population of cells resident in the vessel wall consists of stem cells that are activated when vessels are injured. Activation of these cells directly contributes to vessel re-narrowing. Our goal is to understand how these cells are activated by vessel injury, how injury signals these cells to divide and invade the vessel lumen, what molecular effectors control the cellular responses, and how to intercept these signals and effectors to prevent vessel re-narrowing. In the past year, we successfully developed new methods for isolating and growing these vascular stem cells in test tubes. These new methods allowed us to determine how these stem cells turn into other types of vessel cells after injury and how they contribute to re-narrowing of injured vessels. We are using this method to define molecular pathways that control vessel wall stem cells to respond to vessel injury.
  • Coronary heart disease is a leading cause of morbidity and mortality. This disease results from blockage of coronary arteries that supply blood to the heart muscle. To restore blood supply, physicians use angioplasty to open the obstructed artery and apply stenting to maintain the arterial patency. Approximately 1.3 million angioplasty and stenting procedures are performed every year in the US to relieve coronary obstruction. However, these procedures activate a population of vascular cells to grow into the arterial lumen, causing re-narrowing of the artery. This re-narrowing (restenosis) is the major hurdle limiting the success of angioplasty and stenting. Mental stents coated with drug inhibitors of cell growth (drug eluting stents, or DES) reduce re-narrowing; however, considerable concerns have emerged regarding the safety of DES due to an increased risk of sudden stent occlusion by platelet aggregates (or thrombosis) and the need for prolonged anti-platelet therapy, which poses bleeding risks. These concerns call for defining mechanisms that control re-narrowing of injured arteries.
  • A population of stem cells resides in the arterial wall. These cells are activated when arteries are injured by mechanical stress such as angioplasty and stenting. Activation of these cells directly contributes to arterial re-narrowing. Our goal is to understand how these stem cells are activated by vessel injury, how injury signals these cells to divide and invade the vessel lumen, what molecular effectors control the cellular responses, and how to intercept these signals and effectors to prevent vessel re-narrowing. We developed new methods for isolating and growing these vascular stem cells in test tubes. In the past year, we successfully used these methods to determine how arterial injury or mechanical stress signals the stem cells to produce different types of cells which grow into the arterial lumen, causing narrowing of the artery. We are using these methods and also developing new methods to define molecular pathways that control the reaction of stem cells to arterial injury. This will help identify drug targets for therapeutic intervention.
  • Coronary heart disease, the major cause of morbidity and mortality in our society, results from blockage of the coronary arteries that supply blood to the heart muscle. Blockage of the coronary arteries causes heart attack. Angioplasty and stenting are used to open the obstructed coronary artery and maintain the arterial patency. ~1.3 million angioplasty and stenting procedures are performed in the US every year to treat coronary artery disease. However, these procedures activate a population of vascular cells to grow into the arterial lumen, causing re-narrowing of the artery. This re-narrowing (restenosis) is the major hurdle limiting the success of angioplasty and stenting. Mental stents coated with drug inhibitors of cell growth (drug eluting stents, or DES) reduce re-narrowing; however, considerable concerns have emerged regarding the safety of DES due to an increased risk of sudden stent occlusion by platelet aggregates (or thrombosis) and the need for prolonged anti-platelet therapy, which poses bleeding risks. Defining the mechanisms that control re-narrowing of injured arteries is therefore important for treating coronary artery disease.
  • The arterial wall contains a population of stem cells. These stem cells are activated when arteries are injured by mechanical stress such as angioplasty and stenting. Activation of these cells directly contributes to arterial re-narrowing. Our goal is to understand how these stem cells are activated by vessel injury, how injury signals these cells to divide and invade the vessel lumen, what molecular effectors control the cellular responses, and how to intercept these signals and effectors to prevent vessel re-narrowing. We developed new methods for isolating and growing these vascular stem cells in test tubes, and we have successfully used these methods to determine how arterial injury or mechanical stress signals the stem cells to produce different types of cells which grow into the arterial lumen, causing narrowing of the artery. In the past year, we developed new genetic tools to further understand the mechanism of vascular injury and repair. We are using the new genetic tool to define molecular and cellular pathways that control the reaction of stem cells to arterial injury.
  • Blockage of coronary arteries that supply blood to the heart muscle is the major cause of morbidity and mortality in our society. Angioplasty and stenting are used to open the obstructed coronary artery and maintain the arterial patency. In US, ~1.3 million angioplasty and stenting procedures are performed every year to treat coronary artery disease. Although effective in restoring the blood flow, these procedures activate a population of vascular cells resident in the arterial wall to grow into the vesslel lumen, causing re-narrowing (restenosis) of the treated artery months or years later. This arterial re-narrowing is a major hurdle limiting the success of angioplasty and stenting. Mental stents coated with drug inhibitors of cell growth (drug eluting stents, or DES) reduce re-narrowing; however, the safety of DES has raised considerable concerns due to an increased risk of sudden stent occlusion by platelet aggregates (or thrombosis) as well as the need for prolonged anti-platelet therapy, which poses bleeding risks, especially in the elderly population. It is therefore important to define the underlying mechanisms of re-narrowing of injured arteries in order to design new therapies for coronary artery disease.
  • A population of stem cells resides in the arterial wall. These stem cells are activated when arteries are injured by angioplasty and stenting. Once activated, these cells grow and differentiate into cells that invade the vascular luman and contribute to arterial re-narrowing. We developed new genetic tools to further understand the mechanism of vascular injury and repair. We are using the new genetic tool to define molecular and cellular pathways that control the reaction of stem cells to arterial injury. The goal is to understand how these stem cells are activated by vessel injury, how injury signals these cells to divide and invade the vessel lumen, what molecular effectors control the cellular responses, and how to intercept these signals and effectors to prevent vessel re-narrowing.

Elucidating Molecular Basis of Hypertrophic Cardiomyopathy with Human Induced Pluripotent Stem Cells

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05129
Investigator: 
ICOC Funds Committed: 
$1 425 600
Disease Focus: 
Heart Disease
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people, including trained athletes, and is the most common inherited heart defect. Until now, studies in humans with HCM have been limited by a variety of factors, including variable environmental stimuli which may differ between individuals (e.g., diet, exercise, and lifestyle), the relative difficulty in obtaining human cardiac samples, and inadequate methods of maintaining human heart tissue in cell culture systems. Cellular reprogramming methods that enable derivation of human induced pluripotent stem cells (hiPSCs) from adult cells, which can then be differentiated into cardiomyocytes (hiPSC-CMs), are a revolutionary tool for creating disease-specific cell lines that may lead to effective targeted therapies. In this proposal, we will derive hiPSC-CMs from patients with HCM and healthy controls, then perform a battery of functional and molecular tests to determine the presence of cardiomyopathic disease and associated abnormal molecular programs. With these preliminary studies, we believe hiPSC-CMs with HCM phenotype will dramatically enhance the ability to perform future high-throughput drug screens, evaluate gene and cell therapies, and assess novel electrophysiologic interventions for potential new therapies of HCM. Because HCM is not a rare disease but rather the leading cause of inherited heart defects, we believe the findings here should have broad clinical and scientific impact toward understanding the molecular and cellular basis of HCM.
Statement of Benefit to California: 
Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people and is the most common inherited heart defect. In this study, we will generate hiPSC-derived cardiomyocytes from patients with HCM, then perform a number of functional, molecular, bioinformatic, and imaging analyses to determine the extent and nature of cardiomyopathic disease. We believe hiPSC-CMs with HCM phenotype will dramatically enhance the ability to perform future high-throughput drug screens, evaluate gene and cell therapies, and assess electrophysiologic interventions for potential novel therapies of HCM. The experiments outlined are pertinent and central to the overall mission of CIRM, which seeks to explore the use of stem cell platforms to yield novel mechanistic insights into the molecular and cellular basis of disease. Because HCM is not an orphan disease, but rather the leading cause of sudden cardiac death in young people, we believe the research findings will benefit the state of California and its citizens.
Progress Report: 
  • Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people, including trained athletes, and is the most common inherited heart defect. In this proposal, we will generate human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with HCM. The specific aims are as follow:
  • Specific Aim 1: Generate iPSCs from patients with HCM and healthy controls.
  • Specific Aim 2: Determine the extent of disease by performing molecular and functional analyses of hiPSC-CMs.
  • Specific Aim 3: Rescue the molecular and functional phenotypes using zinc finger nuclease (ZFN) technology.
  • Over the past year, we have now derived iPSCs from a 10-patient family cohort with the MYH7 mutation. Established iPSC lines from all subjects were differentiated into cardiomyocyte lineages (iPSC-CMs) using standard 3D EB differentiation protocols. We found hypertrophic iPSC-CMs exhibited features of HCM such as cellular enlargement and multi-nucleation beginning in the sixth week following induction of cardiac differentiation. We also found hypertrophic iPSC-CMs demonstrated other hallmarks of HCM including expression of atrial natriuretic factor (ANF), elevation of β-myosin/α-myosin ratio, calcineurin activation, and nuclear translocation of nuclear factor of activated T-cells (NFAT) as detected by immunostaining. Blockade of calcineurin-NFAT interaction in HCM iPSC-CMs by cyclosporin A (CsA) and FK506 reduced hypertrophy by over 40%. In the absence of inhibition, NFAT-activated mediators of hypertrophy such as GATA4 and MEF2C were found to be significantly upregulated in HCM iPSC-CMs beginning day 40 post-induction of cardiac differentiation, but not prior to this point. Taken together, these results indicate that calcineurin-NFAT signaling plays a central role in the development of the HCM phenotype as caused by the Arg663His mutation.
  • Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people, including trained athletes, and is the most common
  • inherited heart defect. In this proposal, we will generate and characterize human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with HCM. The
  • specific aims are as follow:
  • Specific Aim 1: Generate iPSCs from patients with HCM and healthy controls.
  • Specific Aim 2: Determine the extent of disease by performing molecular and functional analyses of hiPSC-CMs.
  • Specific Aim 3: Rescue the molecular and functional phenotypes using zinc finger nuclease (ZFN) technology.
  • Over the past year, we have characterized the pathological phenotypes from iPSCs derived from a 10-patient family cohort with the MYH7 mutation.
  • We've differentiated all stablished iPSC lines from all subjects into cardiomyocyte using a modified protocol from that published by Palacek in PNAS 2011. This protocol increased the yield of cardiomyocytes significantly to consistently greater than 70% beating cardiomyocytes. We then tested the electrophysiological properties of iPSC-CMs from control and patients with HCM and found that both control and patient iPSC-CM display atrial, ventricular and nodal-like electrical waveforms by whole cell patch clamping. However, by day 30, a large subfraction (~40%) of the HCM iPSC-CM exhibit arrhythmic waveforms including delayed after-depolarizations (DADs) compared with control (~5.1%). In addition we found that treatment of HCM hiPSC-CM with positive inotropic agents (beta-adrenergic agonist - isoproterenal) for 5 days caused an earlier increase in cell size by 1.7 fold as compared to controls and significant increase in irregular calcium transients. Furthermore, we found that HCM iPSC-CMs exhibited frequent arrhythmia due to their increased intracellular calcium level by 30% at baseline. These HCM iPSC-CM also exhibited decreased calcium release by the sarcoplasmic reticulum. These findings emphasize the role of irregular calcium recycling in the pathogenesis of HCM. To confirm that the regulation of myocyte calcium is the key to HCM pathogenesis, we treated several lines from multiple HCM patients with calcium channel blocker (verapamil/diltiazem) and found that this treatment significantly ameliorated all aspects of the HCM phenotype including myocyte hypertrophy, calcium handling abnormalities, and arrhythmia. These finding supports the use of calcium channel blockers in patients with HCM and encourages further clinical studies in HCM patients using these agents.

Molecular Mechanisms Underlying Human Cardiac Cell Junction Maturation and Disease Using Human iPSC

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05103
ICOC Funds Committed: 
$1 341 955
Disease Focus: 
Heart Disease
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
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, including young athletes, who despite their appearance of health are struck down by this type of heart disease. Even though it is inherited, early detection is hindered because people carrying the genetic code have highly variable clinical symptoms, making ARVC and catastrophic cardiac events very hard to predict and avoid. Evidence suggests that this 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 dynamics in heart disease, which is important since other heart diseases also share some of these same heart cell defects. Our goal is to understand the basic biology of how human heart muscle cell junctions mature and what happens in disease, by studying ARVC. Human iPS cells 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. Thus, hiPS from people who carry the ARVC heart disease mistakes can be used in our laboratory to provide a true human model of that disease. We will generate heart muscle cells from hiPS from normal and ARVC donors that carry mistakes in the genetic code for cell junction components. We have identified new pathways that may be important causes of ARVC, thus we will also use our hiPS lines, to confirm whether these new pathways are truly important in human ARVC disease progression and if our approaches reverse disease progression. Characterization of our hiPS derived heart cells can also be exploited for translational medicine to predict an individual's heart cell response to drug treatment and provides a promising platform to identify new drugs for heart diseases, such as ARVC, which are currently lacking in the field. Recent advances in stem cell biology have highlighted the unique potential of hiPS to be used in the future as a source of cells for cell-based therapies for heart disease. However, prior to clinical application, a detailed understanding of the basic biology and maturation of these hiPS into heart muscle cells is required. Our studies seek to advance our understanding of how cell-cell junctions mature in hiPS and highlight tools that influence the microenvironment of the hiPS in a dish, to accelerate this process. This knowledge can also be exploited in regenerative medicine to achieve proper electromechanical integration of cardiac stem cells when using stem cells for heart repair, to improve longterm successful clinical outcomes of cardiac stem cell therapies.
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 an urgency to understand the underlying molecular basis of heart diseases as well as find more effective therapies to alleviate these growing burdens. Our goal is to improve heart health and quality of life of Californians by generating human stem cell models from people with an especially devastating form of genetic heart disease that affects young people and results in sudden cardiac death, to improve our molecular and medical understanding of how cardiac cells go wrong in the early stages of heart disease in humans. We will also test current drugs used to treat heart disease and new candidate pathways, that we have uncovered, to determine if and how they reverse and intervene with these defects. We believe that our model systems have tremendous potential in being used to diagnose, test an individual's heart cell's response to drug treatment, as well as predict severity of symptoms in heart diseases at an early stage, to monitor drug treatment strategies for the heart. We believe our studies also have a direct impact on regenerative medicine as a therapy for Californians suffering from heart disease, since data from our studies can identify ways to improve cardiac stem cell integration into the diseased heart when used for repair, as a way to improve long-term successful clinical outcomes of cardiac stem cell therapies. We also believe that our development of multiple human heart disease stem cells lines with unique genetic characteristics could be of tremendous value to biotechnology companies and academic researchers interested in large scale drug screening strategies to identify more effective compounds to rescue defects and treat Californians with heart disease, as well as provide important economic revenue and resources to California, which is stimulated by the development of businesses interested in developing these therapies further.
Progress Report: 
  • 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, including young athletes, who despite their appearance of health are struck down by this type of heart disease. Even though it is inherited, early detection is hindered because people carrying the genetic code have highly variable clinical symptoms, making ARVC and catastrophic cardiac events very hard to predict and avoid. Evidence suggests that this 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 dynamics in heart disease, which is important since other heart diseases also share some of these same heart cell defects. Our goal is to understand the basic biology of how human heart muscle cell junctions mature and what happens in disease, by studying ARVC. Human iPS cells 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. Thus, hiPS from people who carry the ARVC heart disease mistakes can be used in our laboratory to provide a true human model of that disease. During the first year of our grant, we have enrolled sufficient numbers of normal and ARVC donors into our study. We have collected skin biopsy tissues from donors as means to generate hiPS cells. Our results show that hiPS cell lines can be efficiently generated from both normal and ARVC donors and we have extensively characterized their profiles, such that we know they are bona fide stem cell lines and can be used as a model system to dissect defects in cardiac cell junction biology between these various different hiPS lines. We have also developed efficient and robust methodologies to generate heart muscle cells from hiPS from normal and ARVC donors that carry mistakes in the genetic code for cell junction components and are now in the midst of characterizing their molecular, genetic, biochemical and functional profiles to identify features in these cells that are unique for ARVC. Through our previous studies, we identified new pathways that may be important causes of ARVC, thus we will also use our hiPS lines, to confirm whether these new pathways are truly important in human ARVC disease progression and if our approaches reverse disease progression. Towards this goal, we have generated novel tools to increase and decrease a component of this pathway in order to test these approaches and have preliminary data to show that these tools are efficient in altering levels of this component in heart muscle cells, which we are now applying towards understanding these pathways in hiPS derived heart muscle cells and reversing defects in heart muscle cells from ARVC hiPS derived lines. Based on our progress, we have met all of the milestones stated in our grant proposal and in some cases, surpassed some milestones. We believe progress over the next year, will allow us to define some of the key cellular defects in ARVC and advance our understanding of how cell-cell junctions mature in hiPS and highlight tools that influence the microenvironment of the hiPS in a dish, to accelerate this process.
  • Overall, we have been able to achieve the milestones proposed for Year 2 of the grant. We have generated a panel of control and ARVC hiPSC lines using integration-free based methods. We provide evidence of our method to generate robust numbers of hiPSC-derived cardiac cells that express desmosomal cell-cell junction proteins. We show ARVC lines that display disease symptom-specific features (adipogenic or arrhythmic), which phenocopy the striking and differential symptoms found in respective individual ARVC-patients as tools to study human ARVC. We also uncover desmosomal defects in hiPSC-derived cardiac muscle cells that underlie the disease features found in ARVC cells. We have also published two reviews in the field of cell-cell junctional remodeling and stem cell approaches that helps to further our understanding of this field in cardiomyocytes, that is relevant to human disease and our research using hiPS.
  • Overall, we have been able to complete the milestones proposed for our grant. We have generated a unique panel of control and ARVC hiPSC lines using integration-free methods. We provide evidence of our method to generate robust numbers of hiPSC derived cardiac cells that express key components of the cardiac muscle cell-cell junction include mechanical junctions and electrical junctions. We show that our ARVC hiPSC lines display disease symptom-specific features (adipogenic and arrhythmic), which phenocopy the striking and differential diagnosis observed in our ARVC donor hearts and provide a platform to study the varying disease features underlying ARVC. We uncover novel and classic molecular and ultrastructural defects underlying the arrhythmogenic defects in our ARVC hiPSC lines that mimic the gradation in disease severity observed in ARVC donor hearts. We exploit conventional ARVC drugs to determine their impact on arrhythmogenic behavior and reversibility of phenotypes in our cells. We have published 4 articles in the field of cell-cell junction remodeling, protein turnover and stem cell approaches that further our understanding of this field in cardiac muscle cells as well as filed a provisional patent application on the use of a novel drug discovery system for fat arrhythmogenic disorders that exploit the genetic diversity and clinical features observed in our ARVC lines.

Characterization and Engineering of the Cardiac Stem Cell Niche

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05086
ICOC Funds Committed: 
$1 181 306
Disease Focus: 
Heart Disease
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Despite therapeutic advances, cardiovascular disease remains a leading cause of mortality and morbidity in both California and Europe. New insights into disease pathology, models to expedite in vitro testing and regenerative therapies would have an enormous societal and financial impact. Although very promising, practical application of pluripotent stem cells or their derivatives face a number of challenges and technological hurdles. For instance, recent reports have demonstrated that cardiac progenitor cells (CPCs), which are capable of differentiating into all three cardiovascular cell types, are present during normal fetal development and can be isolated from pluripotent stem cells. induced pluripotent stem cell (iPSC)-derived CPC therapy after a myocardial infarction would balance the need for an autologous, multipotent stem cell myocardial regeneration with the concerns of tumorigenicity using a more primitive stem cell. However, translating this discovery into a clinically useful therapy will require additional advances in our understanding of CPC biology and the factors that regulate their fate to develop optimized cell culture technology for CPC application in regenerative medicine. Cardiac cell therapy with hiPSC-derived cells, will require reproducible production of large numbers of well-characterized cells under defined conditions in vitro. This is particularly true for the rare and difficult to culture intermediates, such as CPCs. Our preliminary data demonstrated that a CPC niche exists during cardiac development and that CPC expansion is regulated by factors found within the niche microenvironment including specific soluble factors and ECM signals. However, our current understanding of the cardiac niche and how this unique microenvironment influences CPC fate is quite limited. We believe that if large scale production of hiPSC-derived CPCs is ever to be successful, new 3D cell culture technologies to replicate the endogenous cardiac niche will be required. The goals of this proposal are to address current deficiencies in our understanding of the cardiac niche and its effects on CPC expansion and differentiation as well as utilize novel bioengineering approaches to fabricate synthetic niche environments in vitro. The development of advanced fully automated in vitro culture systems that reproduce key features of natural niche microenvironments and control proliferation and/or differentiation, are critically needed both for studying the role of the niche in CPC biology but also the advancement of the field of regenerative medicine.
Statement of Benefit to California: 
Heart disease, stroke and other cardiovascular diseases are the #1 killer in California. Despite medical advances, heart disease remains a leading cause of disability and death. Recent estimates of its cost to the U.S. healthcare system amounts to almost $300 billion dollars. Although current therapies slow the progression of heart disease, there are few, if any options, to reverse or repair damage. Thus, regenerative therapies that restore normal heart function would have an enormous societal and financial impact not only on Californians, but the U.S. more generally. The research that is proposed in this application could lead to new therapies that would restore heart function after and heart attack and prevent the development of heart failure and death. We will develop the techniques to expand and transplant human cardiac progenitor cells. Combining tissue engineering with human pluripotent stem cells will facilitate the creation of new cardiovascular therapies.
Progress Report: 
  • Cardiovascular disease is the leading cause of morbidity and mortality in the United States. As humans lack the ability to regenerate myocardial tissue lost afte a heart attcak, there has been great focus on cardiovascualr regenerative therapies with the use of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). There has been increased attention towards developing tissue engineering as a method to standardize methods to differentiate human ESCs and iPSCs into cardiovascular progenitor cells (CPC) expand these progenitor cells in a standardized manor. We have focused on developing techniques to allow expansion of these CPCs into clinically relevany numbers by determining: 1. Conditions to optimize their derivation into clinically numbers using clinical grade techniques.
  • 2. Defininy and optimizing the extracellular matrxi to be used to maintain these CPCs in an undifferentiated state to allow their expansion to clinically required numbers. We studied the endogenous environment that these CPCs exist in fetal development and focused on the extracellular matrix proteins that help support these CPCs during development. By studying the array of proteins endogenously in developing heart we now will shift our focus on re-engineering this environment in-vitro to be able to mimic this growth to use this as a mean to grow and expand these progenitors for use clinically in the future. Currently we are deriving these CPCs from human ESC and iPSC and expanding them on different combinations of proteins as determined in the staining of the endogenous fetal environment. We hope to by the end of this porject determine the ideal conditions for derivation of these CPCs from iPSCs and the environmental cues needed for culturing these cells to allow for maximal yield for potential use in clinical regenerative therapies in the future.
  • Cardiovascular disease is the leading cause of morbidity and mortality in the United States. As humans lack the ability to regenerate myocardial tissue lost afte a heart attcak, there has been great focus on cardiovascualr regenerative therapies with the use of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). There has been increased attention towards developing tissue engineering as a method to standardize methods to differentiate human ESCs and iPSCs into cardiovascular progenitor cells (CPC) expand these progenitor cells in a standardized manor. We have focused on developing techniques to allow expansion of these CPCs into clinically relevany numbers by determining: 1. Conditions to optimize their derivation into clinically numbers using clinical grade techniques.
  • 2. Defininy and optimizing the extracellular matrxi to be used to maintain these CPCs in an undifferentiated state to allow their expansion to clinically required numbers. We studied the endogenous environment that these CPCs exist in fetal development and focused on the extracellular matrix proteins that help support these CPCs during development. By studying the array of proteins endogenously in developing heart we now will shift our focus on re-engineering this environment in-vitro to be able to mimic this growth to use this as a mean to grow and expand these progenitors for use clinically in the future. Currently we are deriving these CPCs from human ESC and iPSC and expanding them on different combinations of proteins as determined in the staining of the endogenous fetal environment. We hope to by the end of this porject determine the ideal conditions for derivation of these CPCs from iPSCs and the environmental cues needed for culturing these cells to allow for maximal yield for potential use in clinical regenerative therapies in the future.

Mechanisms of Direct Cardiac Reprogramming

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05174
ICOC Funds Committed: 
$1 708 560
Disease Focus: 
Heart Disease
oldStatus: 
Active
Public Abstract: 
Heart disease is a leading cause of adult and childhood mortality. The underlying pathology is typically loss of heart muscle cells that leads to heart failure, or improper development of specialized cardiac muscle cells called cardiomyocytes during embryonic development that leads to congenital heart malformations. Because cardiomyocytes have little or no regenerative capacity after birth, current therapeutic approaches are limited for the over 5 million Americans who suffer from heart failure. Embryonic stem cells possess clear potential for regenerating heart tissue, but efficiency of cardiac differentiation, risk of tumor formation, and issues of cellular rejection must be overcome. Our recent findings regarding direct reprogramming of a type of structural cell of the heart or skin called fibroblasts into cardiomyocyte-like cells using just three genes offer a potential alternative approach to achieving cardiac regeneration. The human heart is composed of muscle cells, blood vessel cells, and fibroblasts, with the fibroblasts comprising over 50% of all cardiac cells. The large population of cardiac fibroblasts that exists within the heart is a potential source of new heart muscle cells for regenerative therapy if it were possible to directly reprogram the resident fibroblasts into muscle cells. We simulated a heart attack in mice by blocking the coronary artery, and have been able to reprogram existing mouse cardiac fibroblasts after this simulated heart attack by delivering three genes into the heart. We found a significant reduction in scar size and an improvement in cardiac function that persists after injury. The reprogramming process starts quickly but is progressive over several weeks; however, how this actually occurs is unknown. Because this finding represents a new approach that could have clinical benefit, we propose to reveal the mechanism by which fibroblast cells become reprogrammed into heart muscle cells, which will be critical to refine the process for therapeutic use. We will do this by analyzing the changes in how the genome is interpreted and expressed at a genome-wide level at different time points during the process of fibroblast to muscle conversion, which represents the fundamental process that leads to reprogramming. The findings from this proposal will reveal approaches to refine and improve human cardiac reprogramming and will aid in translation of this technology for human cardiac regenerative purposes.
Statement of Benefit to California: 
This research will benefit the state of California and its citizens by helping develop a new approach to cardiac regeneration that would have a lower risk of tumor formation and cellular rejection. In addition, the approach could remove some of the hurdles of cell-based therapy including delivery challenges and incorporation challenges. The mechanisms revealed by this research will enable refinement of the method that could potentially then be used to treat the hundreds of thousands of Californians with heart failure.
Progress Report: 
  • Heart disease is a leading cause of adult and childhood mortality. The underlying pathology is typically loss of heart muscle cells that leads to heart failure, or improper development of specialized cardiac muscle cells called cardiomyocytes during embryonic development that leads to congenital heart malformations. Because cardiomyocytes have little or no regenerative capacity after birth, current therapeutic approaches are limited for the over 5 million Americans who suffer from heart failure. Embryonic stem cells possess clear potential for regenerating heart tissue, but efficiency of cardiac differentiation, risk of tumor formation, and issues of cellular rejection must be overcome.
  • Our recent findings regarding direct reprogramming of a type of structural cell of the heart or skin called fibroblasts into cardiac muscle-like cells using just three genes offer a potential route to achieve cardiac regeneration after cardiac injury. The large population of cardiac fibroblasts that exists within the heart is a potential source of new heart muscle cells for regenerative therapy if it were possible to directly reprogram the resident fibroblasts into muscle cells. In the last year, we simulated a heart attack in mice by blocking the coronary artery, and have been able to reprogram existing mouse cardiac fibroblasts after this simulated heart attack by delivering three genes into the heart. We found a significant reduction in scar size and an improvement in cardiac function that persists after injury. The reprogramming process starts quickly but is progressive over several weeks; however, how this actually occurs is unknown. Because this finding represents a new approach that could have clinical benefit, we are investigating the mechanism by which fibroblast cells become reprogrammed into heart muscle cells, which will be critical to refine the process for therapeutic use. During the last year, we have analyzed the changes in how the genome is interpreted and expressed at a genome-wide level at different time points during the process of fibroblast to muscle conversion, which represents the fundamental process that leads to reprogramming. We have also generated many reagents that will allow us to identify how the reprogramming factors interact with DNA to alter the interpretation. These reagents will be used in the coming year to more thoroughly investigate the epigenetic changes that induce changes in interpretation of the DNA, leading to the cardiac muscle phenotype. The findings from this proposal will reveal approaches to refine and improve human cardiac reprogramming and will aid in translation of this technology for human cardiac regenerative purposes.
  • Heart disease is a leading cause of adult and childhood mortality. The underlying pathology is typically loss of heart muscle cells that leads to heart failure, or improper development of specialized cardiac muscle cells called cardiomyocytes during embryonic development that leads to congenital heart malformations. Because cardiomyocytes have little or no regenerative capacity after birth, current therapeutic approaches are limited for the over 5 million Americans who suffer from heart failure. Embryonic stem cells possess clear potential for regenerating heart tissue, but efficiency of cardiac differentiation, risk of tumor formation, and issues of cellular rejection must be overcome.
  • Our recent findings regarding direct reprogramming of a type of structural cell of the heart or skin called fibroblasts into cardiac muscle-like cells using just three genes offer a potential route to achieve cardiac regeneration after cardiac injury. The large population of cardiac fibroblasts that exists within the heart is a potential source of new heart muscle cells for regenerative therapy if it were possible to directly reprogram the resident fibroblasts into muscle cells. We have simulated a heart attack in mice by blocking the coronary artery, and have been able to reprogram existing mouse cardiac fibroblasts after this simulated heart attack by delivering three genes into the heart. We found a significant reduction in scar size and an improvement in cardiac function that persists after injury. The reprogramming process starts quickly but is progressive over several weeks; however, how this actually occurs is unknown. Because this finding represents a new approach that could have clinical benefit, we are investigating the mechanism by which fibroblast cells become reprogrammed into heart muscle cells, which will be critical to refine the process for therapeutic use. During the last year, we have analyzed the changes in how the genome is interpreted and expressed at a genome-wide level at different time points during the process of fibroblast to muscle conversion, which represents the fundamental process that leads to reprogramming. We have mapped the dynamic and sequential changes that are occurring on the DNA during reprogramming of cells. In the coming year, we will be integrating data from studies of epigenetic changes, DNA-binding of reprogramming factors, and the resulting alterations in activation or repression of genes that are responsible for changing a fibroblast into a cardiac muscle cell. The findings from this proposal will reveal approaches to refine and improve human cardiac reprogramming and will aid in translation of this technology for human cardiac regenerative purposes.

Pages

Subscribe to RSS - Heart Disease

© 2013 California Institute for Regenerative Medicine