Heart Disease

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

Phase I study of IM Injection of VEGF Producing MSC for the Treatment of Critical Limb Ischemia

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05423
ICOC Funds Committed: 
$76 861
Disease Focus: 
Heart Disease
oldStatus: 
Closed
Public Abstract: 
Critical limb ischemia (CLI) represents a significant unmet medical need without any approved medical therapies for patients who fail surgical or angioplasty procedures to restore blood flow to the lower leg. CLI affects 2 million people in the U.S. and is associated with an increased risk of leg amputation and death. Amputation rates in patients not suitable for surgery or angioplasty are reported to be up to 30-50% after 1 year. Patients who are not eligible for revascularization procedures are managed with palliative care, but would be candidates for the proposed phase I clinical trial. In an effort to combat CLI, prior and ongoing clinical trials that our group and others have conducted have evaluated direct injection of purified growth factors into the limb that has low blood flow. Some trials have tested plasmids that would produce the blood vessel growth factors for a short period of time. These therapies did show benefit in early stage clinical trials but were not significantly better than controls in Phase III (late stage) trials, probably due to the short duration of presence of the growth factors and their inability to spread to the areas most needed. Other clinical trials ongoing in our vascular center and others are testing the patient’s own stem cells, moved from the bone marrow to the damaged limb, and those studies are showing some benefit, although the final assessments are not yet completed. Stem cells can have benefit in limb ischemia because they can actively seek out areas of low oxygen and will produce some growth factors to try to encourage blood vessel growth. But in cases where the circulation needs very high levels of rescue, this strategy might not be enough. As an improved strategy we are combining the stem cell and growth factor approaches to make a more potent therapy. We have engineered human Mesenchymal Stem Cells (MSCs) to produce high levels of the strong angiogenic agent VEGF for this novel approach (MSC/VEGF). We and others have documented over the past 20+ years that MSC are capable of sustained expression of growth factors, migrate into the areas of lowest oxygen in the tissues after injection, and wrap around the damaged or tiny blood vessels to secrete their factors where they are needed most. These MSC/VEGF cells are highly potent, safe and effective in our preclinical studies. These human stem cells designed to produce VEGF as “paramedic delivery vehicles armed with growth factor to administer” rapidly restored blood flow to the limbs of rodents who had zero circulation in one leg. With funding that could be potentially obtained through the proposed application we will follow the detailed steps to move this candidate therapy into clinical trials, and will initiate and complete an early phase clinical trial to test safety and potential efficacy of this product that is designed to save limbs from amputation.
Statement of Benefit to California: 
Critical Limb Ischemia (CLI) represents a significant unmet medical need without any curative therapies in its end stages, after even the best revascularization attempts using sophisticated catheters, stents, and bypass surgeries have failed. CLI affects over 2 million people in the US and the prevalence is increasing due to the aging of our population and the diabetes epidemic. In 2007, the treatment of diabetes and its complications in the USA generated $116 billion in direct costs; at least 33% of these costs were linked to the treatment of ischemic foot ulcers, associated with CLI. Once a patient develops CLI in a limb, the risk of needing amputation of the other limb is 50% after 6 years, with devastating consequences. Treatment costs are immense and lives are significantly shortened by this morbid disease. The symptoms associated with this very severe form of lower extremity peripheral artery disease (PAD) are pain in the foot at rest, non- healing ulcers, limb/digital gangrene and delayed wound healing. The quality of life for those with CLI is extremely poor and reported to be similar to that of patients with end stage malignancy. Most patients with CLI will undergo repeat hospitalizations and surgical/endovascular procedures in an effort to preserve the limb, progress to immobility and need constant care. Unfortunately, the limb salvage efforts are often not effective enough, and despite multiple attempts at revascularization, the wounds still fail to heal. The final stage in 25% of cases is limb amputation, which is associated with a high mortality rate within 6 months. Amputation rates in patients not suitable for revascularization are reported to be up to 30-50% after 1 year. Fewer than half of all CLI patients achieve full mobility after an amputation and only one in four above-the-knee amputees will ever wear a prosthesis. Between 199– 1999, over 28,000 first time lower extremity bypass procedures were performed in California for CLI, and 29% of patients were admitted to the hospital for at least one subsequent bypass operation or revision procedure. The 5-year amputation free survival rate for this group of CLI patients from California was only 51.1%. The direct costs to California for the treatment of CLI and diabetic ischemic ulcers are substantial. The lost ability of no-option CLI patients to remain in the CA workforce, to support their families, and to pay taxes causes additional financial strain on the state’s economy. The goal of the proposed study is to develop and apply a safe and effective stem cell therapy to save limbs from amputation due to disorders of the vasculature that currently cannot be cured. The successful implementation of our planned therapies will significantly reduce the cost of healthcare in California and could bring people currently unable to work due to immobility back to the workforce and active lifestyles, with a significantly improved quality of life.
Progress Report: 
  • A) Pre-clinical: The remainder of the IND-enabling studies for the development candidate MSC/VEGF were designed in consultation with Biologics Consulting Group (BCG). The project will begin with the IND-enabling phase and transition through regulatory approvals and through the Phase I clinical trial. The project has a Preclinical unit under the leadership of co-PI Dr. Jan Nolta, and a Clinical unit under the leadership of PI Dr. John Laird. The two units are well integrated, since the team has been meeting frequently since 2008 to plan the testing of the current and prior development candidates. The team is currently performing a Phase I stem cell therapy to test a medical device, as the result of those interactions. During the planning phase we met weekly, and worked continually on the MSC/VEGF project.
  • Co-PI Jan Nolta, Ph.D. is Scientific Director of the UC Davis/CIRM GMP Facility. Dr. Nolta’s team is expert in translational applications of gene-modified MSC at the level of GLP. The Pre-Clinical team is performing all IND-enabling studies for MSC/VEGF, and will manufacture and qualify the MSC and MSC/VEGF products in the GMP facility at UC Davis that is directed by Dr. Bauer (CMC lead). These studies are ongoing and we have been advised by BCG consulting lead Andra Miller, who was formerly Gene Therapy Group Leader at the FDA, CBER, Division of Cell and Gene Therapies. BCG is assisting with preIND preparation, through the planning grant period funding for this project.
  • B) Clinical: The Clinical team is led by PI John Laird MD, Medical Director of the UCD Vascular Center, who is an internationally recognized leader in the field of peripheral vascular interventions. He is the PI for multicenter and multinational trials to evaluate novel treatments for peripheral arterial disease. He has led clinical trials investigating the use of FGF-1, Hif, and VEGF to treat claudication and CLI. Christy Pifer is the experienced Project Manager who will guide the entire process. She is the Vascular Center’s clinical trials manager and orchestrates accrual of patients to all trials, including one ongoing Phase I stem cell clinical trial and another pending, as well as a Phase III gene transfer clinical trial. Ms. Pifer has coordinated over 100 Phase I, II and III clinical trials over the past 12 years. The planning grant allowed Ms. Pifer to contribute significant amounts of time to conducting meetings and designing the clinical study with Dr. Laird and other Vascular Center faculty. We had weekly meetings with the clinical and translational team members to finalize the CIRM Disease Team Grant Application.
  • C) Consultant meetings conducted through the Planning Grant Mechanism:
  • - Paragon was chosen as our CRO for the proposed trial. We had on-site meetings and conference calls with Paragon during the planning phase.
  • - Our consultant Dr. Andy Balber, was a Founder, and for ten years served as the CSO of Aldagen, Inc. At Aldagen since 2000, he helped the Company establish and maintain a clinical program during which patients were treated with stem cell products under seven cleared INDs. Dr. Balber has assisted our team with preparation of the preIND application, and will assist with further dialog with the FDA. We met frequently through conference call and email, and he edited our Disease Teams Grant proposal.
  • - Andra Miller, Director, Cell and Gene Therapy, Biologics Consulting Group, Inc, is a consultant for the development of regulatory strategies to facilitate rapid development of our cell and gene based therap. She and her team are providing support for CMC submission, pre-IND, RAC and IND preparation, Phase I product development strategies and assessment of cGLP compliance. Dr. Miller was Gene Therapy Group Leader for the Division of Cellular and Gene Therapies, Office of Therapeutics of FDA's Center for Biologics Evaluation and Research, for ten years. We met through conference call and email during the Planning Grant period and she edited our Disease Teams grant application.
  • Partner PI group: Dr. Herrera from the Reina Sofia Hospital, Cordoba University, Andalucia, is our partner, identified through the planning grant phase. Her team is currently performing clinical trials of MSC injections for CLI using intra-arterial administration. Now, using the strong development candidate MSC/VEGF, the two teams will each embark upon parallel clinical trials in their respective countries, each capitalizing on their own team’s stem cell delivery strengths to patients at the same stage of no option CLI. The two teams will use similar inclusion and exclusion criteria and will work closely together, if funded, to develop Phase I trials that are highly similar except for the route of injection. We had Skype and conference call meetings with interpreters, and frequent email contact during the Planning Grant phase. This partnership would not have been possible without the CIRM Planning Award.

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.

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.

Antibody tools to deplete or isolate teratogenic, cardiac, and blood stem cells from hESCs

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02060
ICOC Funds Committed: 
$1 869 487
Disease Focus: 
Blood Disorders
Heart Disease
Liver Disease
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
Purity is as important for cell-based therapies as it is for treatments based on small-molecule drugs or biologics. Pluripotent stem cells possess two properties: they are capable of self regeneration and they can differentiate to all different tissue types (i.e. muscle, brain, heart, etc.). Despite the promise of pluripotent stem cells as a tool for regenerative medicine, these cells cannot be directly transplanted into patients. In their undifferentiated state they harbor the potential to develop into tumors. Thus, tissue-specific stem cells as they exist in the body or as derived from pluripotent cells are the true targets of stem cell-based therapeutic research, and the cell types most likely to be used clinically. Existing protocols for the generation of these target cells involve large scale differentiation cultures of pluripotent cells that often produce a mixture of different cell types, only a small fraction of which may possess therapeutic potential. Furthermore, there remains the real danger that a small number of these cells remains undifferentiated and retains tumor-forming potential. The ideal pluripotent stem cell-based therapeutic would be a pure population of tissue specific stem cells, devoid of impurities such as undifferentiated or aberrantly-differentiated cells. We propose to develop antibody-based tools and protocols to purify therapeutic stem cells from heterogeneous cultures. We offer two general strategies to achieve this goal. The first is to develop antibodies and protocols to identify undifferentiated tumor-forming cells and remove them from cultures. The second strategy is to develop antibodies that can identify and isolate heart stem cells, and blood-forming stem cells capable of engraftment from cultures of pluripotent stem cells. The biological underpinning of our approach is that each cell type can be identified by a signature surface marker expression profile. Antibodies that are specific to cell surface markers can be used to identify and isolate stem cells using flow cytometry. We can detect and isolate rare tissue stem cells by using combinations of antibodies that correspond to the surface marker signature for the given tissue stem cell. We can then functionally characterize the potential of these cells for use in regenerative medicine. Our proposal aims to speed the clinical application of therapies derived from pluripotent cell products by reducing the risk of transplanting the wrong cell type - whether it is a tumor-causing residual pluripotent cell or a cell that is not native to the site of transplant - into patients. Antibodies, which exhibit exquisitely high sensitivity and specificity to target cellular populations, are the cornerstone of our proposal. The antibodies (and other technologies and reagents) identified and generated as a result of our experiments will greatly increase the safety of pluripotent stem cell-derived cellular therapies.
Statement of Benefit to California: 
Starting with human embryonic stem cells (hESC), which are capable of generating all cell types in the body, we aim to identify and isolate two tissue-specific stem cells – those that can make the heart and the blood – and remove cells that could cause tumors. Heart disease remains the leading cause of mortality and morbidity in the West. In California, 70,000 people die annually from cardiovascular diseases, and the cost exceeded $48 billion in 2006. Despite major advancement in treatments for patients with heart failure, which is mainly due to cellular loss upon myocardial injury, the mortality rate remains high. Similarly, diseases of the blood-forming system, e.g. leukemias, remain a major health problem in our state. hESC and induced pluripotent stem cells (collectively, pluripotent stem cells, or PSC) could provide an attractive therapeutic option to treat patients with damaged or defective organs. PCS can differentiate into, and may represent a major source of regenerating, cells for these organs. However, the major issues that delay the clinical translation of PSC derivatives include lack of purification technologies for heart- or blood-specific stem cells from PSC cultures and persistence of pluripotent cells that develop into teratomas. We propose to develop reagents that can prospectively identify and isolate heart and blood stem cells, and to test their functional benefit upon engraftment in mice. We will develop reagents to identify and remove residual PSC, which give rise to teratomas. These reagents will enable us to purify patient-specific stem cells, which lack cancer-initiating potential, to replenish defective or damaged tissue. The reagents generated in these studies can be patented forming an intellectual property portfolio shared by the state and the institutions where the research is carried out. The funds generated from the licensing of these technologies will provide revenue for the state, will help increase hiring of faculty and staff (many of whom will bring in other, out-of-state funds to support their research) and could be used to ameliorate the costs of clinical trials – the final step in translation of basic science research to clinical use. Only California businesses are likely to be able to license these reagents and to develop them into diagnostic and therapeutic entities; such businesses are at the heart of the CIRM strategy to enhance the California economy. Most importantly, this research will set the platform for future stem cell-based therapies. Because tissue stem cells are capable of lifelong self-renewal, stem cell therapies have the potential to be a single, curative treatment. Such therapies will address chronic diseases with no cure that cause considerable disability, leading to substantial medical expense. We expect that California hospitals and health care entities will be first in line for trials and therapies. Thus, California will benefit economically and it will help advance novel medical care.
Progress Report: 
  • Our program is focused on improving methods that can be used to purify stem cells so that they can be used safely and effectively for therapy. A significant limitation in translating laboratory discoveries into clinical practice remains our inability to separate specific stem cells that generate one type of desired tissue from a mixture of ‘pluripotent’ stem cells, which generate various types of tissue. An ideal transplant would then consist of only tissue-specific stem cells that retain a robust regenerative potential. Pluripotent cells, on the other hand, pose the risk, when transplanted, of generating normal tissue in the wrong location, abnormal tissue, or cancer. Thus, we have concentrated our efforts to devise strategies to either make pluripotent cells develop into desired tissue-specific stem cells or to separate these desired cells from a mixture of many types of cells.
  • Our approach to separating tissue-specific stem cells from mixed cultures is based on the theory that every type of cell has a very specific set of molecules on its surface that can act as a signature. Once this signature is known, antibodies (molecules that specifically bind to these surface markers) can be used to tag all the cells of a desired type and remove them from a mixed population. To improve stem cell therapy, our aim is to identify the signature markers on: (1) the stem cells that are pluripotent or especially likely to generate tumors; and (2) the tissue-specific stem cells. By then developing antibodies to the pluripotent or tumor-causing cells, we can exclude them from a group of cells to be transplanted. By developing antibodies to the tissue-specific stem cells, we can remove them from a mixture to select them for transplantation. For the second approach, we are particularly interested in targeting stem cells that develop into heart (cardiac) tissue and cells that develop into mature blood cells. As we develop ways to isolate the desired cells, we test them by transplanting them into animals and observing how they grow.
  • Thus, the first goal of our program is to develop tools to isolate pluripotent stem cells, especially those that can generate tumors in transplant recipients. To this end, we tested an antibody aimed at a pluripotent cell marker (stage-specific embryonic antigen-5 [SSEA-5]) that we previously identified. We transplanted into animals a population of stem cells that either had the SSEA-5-expressing cells removed or did not have them removed. The animals that received the transplants lacking the SSEA-5-expressing cells developed smaller and fewer teratomas (tumors consisting of an abnormal mixture of many tissues). Approaching the problem from another angle, we analyzed teratomas in animals that had received stem cell transplants. We found SSEA-5 on a small group of cells we believe to be responsible for generating the entire tumor.
  • The second goal of the program is to develop methods to selectively culture cardiac stem cells or isolate them from mixed cultures. Thus, in the last year we tested procedures for culturing pluripotent stem cells under conditions that cause them to develop into cardiac stem cells. We also tested a combination of four markers that we hypothesized would tag cardiac stem cells for separation. When these cells were grown under the proper conditions, they began to ‘beat’ and had electrical activity similar to that seen in normal heart cells. When we transplanted the cells with the four markers into mice with normal or damaged hearts, they seemed to develop into mature heart cells. However, these (human) cells did not integrate with the native (mouse) heart cells, perhaps because of the species difference. So we varied the approach and transplanted the human heart stem cells into human heart tissue that had been previously implanted in mice. In this case, we found some evidence that the transplanted cells differentiated into mature heart cells and integrated with the surrounding human cells.
  • The third goal of our project is to culture stem cells that give rise only to blood cells and test them for transplantation. In the past year, we developed a new procedure for treating cultures of pluripotent stem cells so that they differentiate into specific stem cells that generate blood cells and blood vessels. We are now working to refine our understanding and methods so that we end up with a culture of differentiated stem cells that generates only blood cells and not vessels.
  • In summary, we have discovered markers and tested combinations of antibodies for these markers that may select unwanted cells for removal or wanted cells for inclusion in stem cell transplants. We have also begun to develop techniques for taking a group of stem cells that can generate many tissue types, and growing them under conditions that cause them to develop into tissue-specific stem cells that can be used safely for transplantation.
  • Our program is focused on improving methods to purify blood-forming and heart-forming stem cells so that they can be used safely and effectively for therapy. Current methods to identify and isolate blood-forming stem cells from bone marrow and blood are efficient. In addition, we found that if blood-forming stem cells are transplanted, they create in the recipient an immune system that will tolerate (i.e., not reject) organs, tissues, or other types of tissue stem cells (e.g. skin, brain, or heart) from the same donor. Many living or recently deceased donors often cannot contribute these stem cells, so we need, in the future, a single biological source of each of the different types of stem cells (e.g., blood and heart) to change the entire field of regenerative medicine. The ultimate reason to fund embryonic stem cell and other pluripotent stem cell research is to create safe banks of predefined pluripotent cells. Protocols to differentiate these cells to the appropriate blood-forming stem cells could then be used to induce tolerance of other tissue stem cells from the same embryonic stem cell line. However, existing protocols to differentiation stem cells often lead to pluripotent cells (cells that generate multiple types of tissue), which pose a risk of generating normal tissue in the wrong location, abnormal tissue, or cancers called teratomas. To address these problems, we have concentrated our efforts to devise strategies to (a) make pluripotent cells develop into desired tissue-specific stem cells, and (b) to separate these desired cells from all other cells, including teratoma-causing cells. In the first funding period of this grant, we succeeded in raising antibodies that identify and eliminate teratoma-causing cells.
  • In the past year, we identified surface markers of cells that can only give rise to heart tissue. First we studied the genes that were activated in these cells, further confirming that they would likely give rise to heart tissue. Using antibodies against these surface markers, we purified heart stem cells to a higher concentration than has been achieved by other purification methods. We showed that these heart stem cells can be transplanted such that they integrate into the human heart, but not mouse heart, and participate in strong and correctly timed beating.
  • In the embryo, a group of early stem cells in the developing heart give rise to (a) heart cells; (b) cells lining the inner walls of blood vessels; and (c) muscle cells surrounding blood vessels. We identified cell surface markers that could be used to separate each of these subsets from each other and from their common stem cell parents. Finally, we determined that a specific chemical in the body, fibroblast growth factor, increased the growth of a group of pluripotent stem cells that give rise to more specific stem cells that produce either blood cells or the lining of blood vessels. This chemical also prevented blood-forming stem cells from developing into specific blood cells.
  • In the very early embryo, pluripotent cells separate into three distinct categories called ‘germ layers’: the endoderm, mesoderm, and ectoderm. Each of these germ layers later gives rise to certain organs. Our studies of the precursors of mesoderm (the layer that generates the heart, blood vessels, blood, etc.) led us by exclusion to develop techniques to direct ES cell differentiation towards endoderm (the layer that gives rise to liver, pancreas, intestinal lining, etc.). A graduate student before performed most of this work before he joined in our effort to find ways to make functional blood forming stem cells from ES cells. He had identified a group of proteins that we could use to sequentially direct embryonic stem cells to develop almost exclusively into endoderm, then subsets of digestive tract cells, and finally liver stem cells. These liver stem cells derived from embryonic stem cells integrated into mouse livers and showed signs of normal liver tissue function (e.g., secretion of albumin, a major protein in the blood). Using the guidelines of the protocols that generated these liver stem cells, we have now turned our attention back to our goal of generating from mesoderm the predecessors of blood-forming stem cells, and, ultimately, blood-forming stem cells.
  • In summary, we have continued to discover signals that cause pluripotent stem cells (which can generate many types of tissue) to become tissue-specific stem cells that exclusively develop into only heart, blood cells, blood vessel lining cells, cells that line certain sections of the digestive tract, or liver cells. This work has also involved determining the distinguishing molecules on the surface of various cells that allow them to be isolated and nearly purified. These results bring us closer to being able to purify a desired type of stem cell to be transplanted safely to generate only a single type of tissue.

A Novel Engineered Niche to Explore the Vasculogenic Potential of Embryonic Stem Cells

Funding Type: 
New Faculty I
Grant Number: 
RN1-00566
ICOC Funds Committed: 
$2 108 683
Disease Focus: 
Heart Disease
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Cardiovascular diseases account for an estimated $330 billion in health care costs each year, afflict 61.8 million Americans, and will account for more than 1.5 million deaths in the U.S. this year alone. A number of these diseases are characterized by either insufficient blood vessel growth or damage to the existing vessels, resulting in inadequate nutrient and oxygen delivery to the tissues. The most common clinical example of this is a heart attack, or myocardial infarction, typically caused by blockage of a coronary artery. The resulting ischemia (reduced blood flow) induces irreversible damage to the heart, leaving behind a non-functional scar tissue. Efforts to restore blood flow to ischemic tissues have largely focused on the delivery of protein growth factors (called pro-angiogenic molecules) that stimulate new capillary growth. An alternative approach is to deliver an appropriate cell type that can either accelerate the recruitment of host vessels or can differentiate into a functional vasculature directly. While adult stem cells have shown promising potential with respect to the former, the potential of embryonic stem cells (ESCs) with respect to either of these two possibilities remains unclear. Therefore, this proposal seeks to: 1.) Utilize a novel, highly tunable, 3D engineered niche to investigate how changes in multiple instructive signals coordinately govern the differentiation of ESCs into capillary vessels; 2.) Exploit knowledge gained from basic studies using this model system to generate a purified population of ESC-derived endothelial progenitor cells (EPCs) and test their potential to repair ischemia in vivo. Specifically, in Aim 1, we propose to further develop and characterize our artificial engineered niche for fundamental studies on ESC fate decisions. Aim 2 will use this system to test two competing hypotheses, namely that: 1.) ESCs can facilitate capillary morphogenesis in an indirect manner, in much the same way as adult stem cells; or 2.) ESCs can be directed down an endothelial-specific lineage by manipulating one or more instructive signals. Finally, Aim 3 will utilize our engineered niche to generate a purified population of ESC-derived EPCs and then test their ability to enhance perfusion in an animal model. Successful completion of these proposed aims may transform the clinical use of stem cells for cardiovascular disease and other ischemic pathologies by enabling identification of those factors and conditions which promote vessel formation. The versatile artificial engineered niche developed here will also yield a new tool that could enormously benefit efforts to screen the combinatorial effects of promising therapeutic compounds. Completion of the planned studies will greatly facilitate the PI’s long-term goal of developing “instructive” biomaterials and strategies to direct tissue repair.
Statement of Benefit to California: 
Human embryonic stem cells (hESCs) are pluripotent stem cells that can theoretically give rise to every cell type in the human body. Their potential use for the treatment of human diseases has been heralded with great fanfare and even some controversy. However, their therapeutic potential has yet to be realized due to an incomplete fundamental understanding of the factors that govern their differentiation. This proposal describes studies intended to assess the ability of hESCs to develop into blood vessels; in particular, capillary networks that are responsible for the delivery of oxygen and essential nutrients to all tissues in the human body. This focus is motivated by the fact that cardiovascular disease accounts for an estimated $330 billion in health care costs each year, afflicts 61.8 million Americans, and will account for more than 1.5 million deaths in the United States this year alone. It is the number one killer in this country and in California. Since many cardiovascular diseases are characterized by either insufficient blood vessel growth or damage to the existing vessels, a therapy based on hESCs could have enormous benefit to the citizens of California, the United States, and the rest of the world. Therefore, this proposal has two primary goals. First, we seek to develop a novel technology to systematically investigate the influence of multiple instructive signals on the ability of hESCs to differentiate into capillary vessels. Second, we propose to exploit knowledge gained from the basic studies using this technology to generate a purified population of hESCs and test their potential to repair ischemia (lack of blood flow) in an animal model. Successfully achieving these goals will benefit the citizens of California in three significant ways. First, our efforts may help to transform the clinical use of stem cells, not only for cardiovascular disease but other diseases as well, by enabling identification of those factors and conditions which promote hESC differentiation. Second, the versatile technology developed here will yield a powerful new tool that could enormously benefit California’s biotechnology companies in their efforts to screen the combinatorial effects of promising therapeutic compounds. Third, we expect the proposed studies to directly benefit 8-10 researchers in training and indirectly trickle down to hundreds of undergraduate students [REDACTED] enrolled in courses taught by the PI. This final benefit may perhaps have the most significant long-term economic impact by training and inspiring future leaders to pursue research and development positions in California.

Optimization in the Identification, Selection and Induction of Maturation of Subtypes of Cardiomyocytes derived from Human Embryonic Stem Cells

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01143
ICOC Funds Committed: 
$906 629
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Cardiovascular diseases remain the major cause of death in the western world. Stem and progenitor cell-derived cardiomyocytes (SPC-CMs) hold great promise for the myocardial repair. However, most of SPC-CMs displayed heterogeneous and immature electrophysiological phenotypes with substantial automaticity. Implanting these electrically immature and inhomogeneous CMs to the hearts would be arrhythmogenic and deleterious. Further optimization in identification, selection and inducing maturation of subtypes of CMs from primitive SPC-CMs are paramount for developing a safe and effective cell-based therapy. Commonly used CM isolation techniques are microdissection, density sedimentation or promoter-driven, fluorescence-activated cell sorting (FACS). Microdissection and density sedimentation are labor intensive and lack of purity. Promoter-driven FACS may compromise cell viability and which promoter is proficient for selection remains unclear. We have established several antibiotics (Abx)-resistant human embryonic stem cell (hESC) lines conferred by lentiviral vectors under the control of various cardiac-specific promoters. With simple Abx treatment, we have easily isolated >95% pure hESC-CMs at various stages of differentiation from embryoid bodies (EBs). Using this Abx selection system, we also found that electrical maturation and differentiation of primitive hESC-CMs depended heavily on developmental cues from extracardiac cells in the EBs. This Abx selection system therefore could be used easily to purify CMs for mechanistic studies and future cell-based therapies. However, the subtype specification of atrial, ventricular and pacemaking CMs appears to occur at very early stages of differentiation because early EBs possess all three types of cells. Furthermore, various cardio-specific promoters have been shown to select preferentially certain subtypes of CMs. In order to use these promoters and Abx resistance to sub-select particular types of CMs at early stages of differentiation, we need to know the timing and sequence of expressions of various cardiac promoters during the EB development. For this later purpose, we will generate hESC lines expressing different colors of fluorescent proteins under the control of various cardiac-specific promoters respectively to determine the timing of expressions of these promoters in the EBs. Based on the sequence of expression, we will generate the Abx-resistant hESC lines under the control of these promoters to sub-select CMs. We will then study the EP properties of these sub-selected hESC-CMs and their interactions with extra-cardiac cells. The overall goal of this proposal is to establish an In Vitro system to track the sequence of expressions of various promoters in order to sub-select particular phenotypes of CMs by the Abx-resistance method. As a result, we will be able to optimize the selection and induction of a population of mature and homogeneous hESC-CMs for a safe and effective cell-based therapy.
Statement of Benefit to California: 
Cardiovascular diseases remain the major cause of death in the western world. Stem and progenitor cell (SPC)-based cell therapies in animal and human studies suggest promising therapeutic potentials. However, most SPC-derived cardiomyocytes (SPC-CMs) displayed heterogeneous and immature electrophysiological (EP) phenotypes with substantial automaticity. Implanting these electrically immature and inhomogeneous CMs to the hearts would be arrhythmogenic and deleterious. Further optimization in identification, selection and inducing maturation of subtypes of CMs from these primitive SPC-CMs are badly needed. Most frequently used isolation techniques are microdissection, density sedimentation or promoter-driven, fluorescence-activated cell sorting (FACS). Microdissection and density sedimentation are labor intensive and lack of purity. Promoter-driven FACS may compromise cell viability and which promoter is proficient for the cardiomyocyte selection remains to be determined. None of the laboratories in the world has success in developing an easy and efficient way to isolate the SPC-CMs. As a result, no method has been developed to induce the maturation of SPC-CMs. We already have the technology to efficiently isolate pure populations of human embryonic stem cell-derived CMs (hESC-CMs) from the embryoid bodies. The proposed research will further determine which type of promoter is best to properly sub-select a specific phenotype of hESC-CMs for future cell-based therapies in California. Most importantly, using this antibiotics-based selection method, we have started investigating the methods for inducing maturation of these sub-selected and primitive CMs. With both goals achieved, we will make California the first state to have a safe and effective cell-based therapy for myocardial repair with a mature and homogeneous population of hESC-CMs. None of stem cell-related research in California is devoted to optimize the selection, identification and induction of maturation of a specific phenotype of hESC-CMs in order to develop a safe cell-based therapy. The proposed research will be the first to achieve this goal proposed by CIRM Tools and Technologies Award. The success of this proposal will also make California the epicenter of the next generation of cell therapies and will benefit its citizens who have significant cardiovascular diseases.
Progress Report: 
  • The goal of our project is to develop methods to induce stem cells to differentiate into heart cells. Importantly, there are three major types of heart cells, which correspond to the ventricle (the major chambers that pump blood to the body), the atria (the smaller chambers that pump blood to the ventricles), and the nodes (these are the regions within the heart where the "pacemaker" cells are found, which control the heart rate). If we can produce pure populations of ventricular, atrial, or nodal cells, we can potentially use these cells for "replacement therapy" for patients which have had heart attacks or who have developed arrhythmias. During the first year of the research, we succeeded in producing cells that correspond to the ventricle. Furthermore, we have developed novel culturing techiques that improve the differentiation of the cells into atrial and nodal type myocytes, and the new strategies look very promising for the future research of this project.
  • The goal of our project is to develop methods to induce stem cells to differentiate into heart cells. Importantly, there are three major types of heart cells, which correspond to the ventricle (the major chambers that pump blood to the body), the atria (the smaller chambers that pump blood to the ventricles), and the nodes (these are the regions within the heart where the "pacemaker" cells are found, which control the heart rate). If we can produce pure populations of ventricular, atrial, or nodal cells, we can potentially use these cells for "replacement therapy" for patients which have had heart attacks or who have developed arrhythmias. During the first year of the research, we succeeded in producing cells that correspond to the ventricle. Furthermore, we have developed novel culturing techiques that improve the differentiation of the cells into atrial and nodal type myocytes, and the new strategies look very promising for the future research of this project.

Building Cardiac Tissue from Stem Cells and Natural Matrices

Funding Type: 
New Faculty II
Grant Number: 
RN2-00921
ICOC Funds Committed: 
$1 706 255
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Congestive heart failure afflicts 4.8 million people, with 400,000 new cases each year. Myocardial infarction (MI), also known as a "heart attack", leads to a loss of cardiac tissue and impairment of left ventricular function. Because the heart does not contain a significant number of multiplying stem, precursor, or reserve cells, it is unable to effectively heal itself after injury and the heart tissue eventually becomes scar tissue. The subsequent changes in the workload of the heart may, if the scar is large enough, deteriorate further leading to congestive heart failure. Many stem cell strategies are being explored for the regeneration of heart tissue, however; full cardiac tissue repair will only become possible when two critical areas of tissue regeneration are addressed: 1) the generation of a sustainable, purified source of functional cardiac progenitors and 2) employment of cell delivery methods leading to functional integration with host tissue. This proposal will explore both of these 2 critical areas towards the development of a living cardiac patch material that will enable the regeneration of scarred hearts.
Statement of Benefit to California: 
The research proposed in expected to result in new techniques and methodology for the differentiation of stem cell-derived cardiomyocytes and delivery methods optimal for therapeutic repair of scarred heart tissue after a heart attack. The citizens of California could benefit from this research in three ways. The most significant impact would be in the potential potential for new medical therapies to treat a large medical problem. The second benefit is in the potential for these technologies to bring new usiness ventures to the state of California. The third benefit is the stem cell training of the students and postdocs involved in this study.
Progress Report: 
  • The proposed project aims to develop cardiac tissue for enhancing the regeneration of damaged heart. The progress in the first year involved generation of cardiac cells from stem cells, developing fabrication techniques for stem cell differentiation, and exporing cell interactions with various biodegradable materials.
  • Progress towards developing heart tissue for repairing damaged/diseaesed hearts includes stem cell differentiation towards cells that make up heart tissue and blood vessels, optimization of methods for cell expansion and cell-cell integration to generate functional tissues, and preliminary investigations of delivery materials fabrication.
  • We have optimized cardiac cell numbers from embyronic stem cells and generated a cardiac patch for delivery of these cardiac cells into damaged myocardium.
  • The aims for this study are to 1) develop methods for generating highly efficient numbers of cardiovascular cells from stem cells, and then 2) develop methods for packaging the cells into tissue-like implantable materials for repair of dead tissue following a heart attack. The final aim 3) was to examine the repair/restorative ability of the developed product in a damaged animal heart.
  • This year (4th year of the grant) was very productive. We have highly efficient methods for generating both heart (70% purity) and blood vessel cells (90% purity) and have developed a sophisticated design for packaging these into heart tissue-like materials. The animal studies are underway and initial data is promising.
  • The aim of this research proposal was to develop cardiac tissue for heart repair. Aim 1 focused on the generation of cardiac cells from stem cells. Aim 2 looks at biomaterials and patterning for building the complex multicellular integrated tissue. Aim 3 examined the ability of these tissues to repair a damaged heart. During this last year of the grant, we have successfully generate large numbers of cardiac cells from stem cells and have generated "sheets" of these cardiac cells. The animal studies on the cell injections and material injection show some success in the repair of heart tissue, but expect that the fully integrated heart tissue, once implanted, will be superior to cells or material alone.

Induction of cardiogenesis in pluripotent cells via chromatin remodeling factors

Funding Type: 
New Faculty II
Grant Number: 
RN2-00903
ICOC Funds Committed: 
$2 847 600
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Heart disease is one of the biggest killers in the civilized world, and as populations age, this trend will increase dramatically. Currently the only way to treat failing hearts is with expensive and relatively ineffective drugs, or by heart transplantation. Ideally, we would like to be able to regenerate sick or dead heart tissue. The best strategy would be to make new heart cells that match the patients' cells (to avoid rejection), and inject them into diseased heart so that they could regenerate the sick heart.Unfortunately, current strategies that are planned to do so are ineffectual. We wish to attempt to generate heart cells from human embryonic stem cells, or skin-derived "induced pluripotent cells" by "reprogramming" the stem cells into heart cells. This would be accomplished by turning on heart genes that normally are off in stem cells and seeing if this turns stem cells into heart cells. If this approach is successful, these newly generated stem cells could be used for regenerative therapies in the future.
Statement of Benefit to California: 
Heart disease is the leading killer of adults in the Western world. Hundreds of thousands of people in the US die of heart failure of sudden cardiac death each year. Largely, this is because inadequate therapies exist for the repair or treatment of the diseased heart. Our goal is to develop a means to efficiently convert pluripotent stem cells, including induced pluripotent cells (iPS cells) into new heart cells that could be used therapeutically to help regenerate healthy heart tissue. The results of our studies will help develop new technology that is likely to contribute to the California biotechnology industry. Our studies will develop technologies that can be used by biotechnology companies and researchers who wish to develop regenerative medicine therapies in a clinical setting. We are working closely with California companies to develop new microscopes, assay devices, and analytical software that could be the basis for new product lines or new businesses. If therapies do come to fruition, we anticipate that California medical centers will be leading the way. The most important contribution of this study will be to improve the health of Californians. Heart disease is a major cause of mortality and morbidity, resulting in billions of dollars in health care costs and lost days at work. Our goal is to contribute research that would ultimately improve the quality of life and increase productivity for millions of people who suffer from heart disease.
Progress Report: 
  • We hypothesized that human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPS cells, which are derived from skin or other adult cells) can be efficiently reprogrammed to become heart cells using a combination of factors that includes proteins that unwind DNA. To test this hypothesis, we proposed three specific aims. For each we have achieved significant progress. In progress toward our first aim, we have been able to enhance cardiac differentiation of mouse iPS cells by 20%, and have devised strategies to increase this success rate. Our second aim was directed at understanding how important the chromatin remodeling factor, Baf60c, was in the induction of heart cells from pluripotent cells. We have made significant progress in this regard, mostly in developing the complex genetic tools required to investigate this important question. The third aim was to understand how Baf60c and its collaborating factors work to enhance heart cell formation. Again, we have had considerable success in early experiments that indicate that we will be able to address these questions fully in the remaining years of the granting period. Overall, our first year of funding has allowed us to move rapidly forward in understanding how to propel a stem cell toward becoming a heart cell; these results will be important to understand how heart cells are made in the body, and how their genesis can be harnessed using the power of stem cells.
  • We have been studying ways to understand how heart cells form from stem cells, and how we could help make the process more efficient, to generate new heart cells for patients with damaged hearts due to heart attacks. We have focused on the finding that cellular machines that unwind DNA from chromosomes, so-called chromatin remodeling factors, are important for turning on heart genes. To date we have been generating the important biological tools required for these studies. These include stem cells in which some of these chromatin genes have been inactivated, as well as DNA constructions that will be inserted into embryonic stem cells to attempt to induce them to become heart cells. In parallel we have been working towards using these factors to transform other types of cells, such as skin cells, into cardiomyocytes; in collaboration with our colleagues we have made significant progress towards this goal, and are now investigating the importance of the chromatin remodeling complexes in this process. Our progress has been excellent, and we are confident that we are making great strides towards regenerative medicine in the context of heart disease.
  • We have been studying ways to understand how heart cells form from stem cells, and how we could help make the process more efficient, to generate new heart cells for patients with damaged hearts due to heart attacks. We have focused on the finding that cellular machines that unwind DNA from chromosomes, so-called chromatin remodeling factors, are important for turning on heart genes. To date we have been generating the important biological tools required for these studies. These include stem cells in which some of these chromatin genes have been inactivated, as well as DNA constructions that will be inserted into embryonic stem cells to attempt to induce them to become heart cells. In parallel we have been working towards using these factors to transform other types of cells, such as skin cells, into cardiomyocytes; in collaboration with our colleagues we have made significant progress towards this goal, and are now investigating the importance of the chromatin remodeling complexes in this process. Our progress has been excellent, and we are confident that we are making great strides towards regenerative medicine in the context of heart disease.
  • In the last year, we have made significant progress on this project, which aims to understand how heart cells can be produced from pluripotent cells. We have been able to understand the gene program that is controlled by a so-called chromatin remodeling protein, a protein that unwinds DNA to allow genes to be turned on. This protein, called Baf60c, turns on many of the genes that give a heart cell its basic functions, like beating. We have also created stem cell -based tools that will allow us in the final year of this project to identify the partner proteins that allow Baf60c to function, and where in our genome Baf60c turns genes on.
  • During the tenure of this award, we have made some exciting discoveries about how genes are regulated during the process of heart cell formation from embryonic stem cells. In particular, we focused our efforts on a group of proteins that regulate other genes using a process called chromatin remodeling. We discovered that one such chromatin remodeling protein is required for genes that are specific to the heart to be turned on in heart cells. We also discovered new proteins that are also important for the formation of the heart. In studying these chromatin remodeling proteins in an embryonic stem cell system, we identified how these proteins turn on the "right" set of genes in the earliest stages of commitment of stem cells to heart cell progenitors. Finally, we identified the nature of the group of proteins that work together as part of chromatin remodeling "complexes", which for the first time tells us how these proteins assemble together to regulate heart genes. These results have paved the way for studies aimed at creating new heart cells, and have opened up some exciting new possibilities to improve this process.

Induced Pluripotent Stem Cells for Cardiovascular Diagnostics

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00639
ICOC Funds Committed: 
$1 708 560
Disease Focus: 
Heart Disease
Toxicity
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Our objective is to use induced pluripotent stem (iPS) cell technology to produce a cell-based test for long QT syndrome (LQTS), a major form of sudden cardiac death. Nearly 500,000 people in the US die of sudden cardiac death each year. LQTS can be triggered by drug exposure or stresses. Drug-induced LQTS is the single most common reason for drugs to be withdrawn from clinical trials, causing major setbacks to drug discovery efforts and exposing people to dangerous drugs. In most cases, the mechanism of drug-induced LQTS is unknown. However, there are genetic forms of LQTS that should allow us to make iPS cell–derived heart cells that have the key features of LQTS. Despite the critical need, current tests for drug-induced LQTS are far from perfect. As a result, potentially unsafe drugs enter clinical trials, endangering people and wasting millions of dollars in research funds. When drugs causing LQTS such as terfenadine (Seldane) enter the market, millions of people are put at serious risk. Unfortunately it is very difficult to know when a drug will cause LQTS, since most people who develop LQTS have no known genetic risk factors. The standard tests for LQTS use animal models or hamster cells that express human heart genes at high levels. Unfortunately, cardiac physiology in animal models (rabbits and dogs) differs from that in humans, and hamster cells lack many key features of human heart cells. Human embryonic stem cells (hESCs) can be differentiated into heart cells, but we do not know the culture conditions that would make the assay most similar to LQTS in a living person. These problems could be solved if we had a method to grow human heart cells from people with genetic LQTS mutations, so that we know the exact test conditions that would reflect the human disease. This test would be much more accurate than currently available tests and would help enable the development of safer human pharmaceuticals. Our long-term goal is to develop a panel of iPS cell lines that better represent the genetic diversity of the human population. Susceptibility to LQTS varies, and most people who have life-threatening LQTS have no known genetic risk factors. We will characterize iPS cells that have well-defined mutations that have clinically proven responses to drugs that cause LQTS. These iPS cell lines will be used to refine testing conditions. To validate the iPS cell–based test, the results will be directly compared to the responses in people. These studies will provide the foundation for an expanded panel of iPS cell lines from people with other genetic mutations and from people who have no genetically defined risk factor but still have potentially fatal drug-induced LQTS. This growing panel of iPS cell lines should allow for testing drugs for LQTS more effectively and accurately than any current test.
Statement of Benefit to California: 
Heart disease is the leading killer of adults in the Western world. Nearly 500,000 people in the US die of sudden cardiac death each year. Our goal is to develop a cell-based test to screen for drugs that can cause sudden cardiac death. Drug-induced cardiac side effects are the most common reason for withdrawal of drugs from clinical trials, causing major setbacks to drug discovery efforts. Therefore our test we will improve the safety of pharmaceuticals. Our test will also reduce the change that a drug in development will fail during clinical trials, thereby decreasing the financial risk for pharmaceutical companies. The results of our studies will help develop new technology that is likely to contribute to the California biotechnology industry. Our studies will develop multiple lines of iPS cells with unique genetic characteristics. These cell lines could be valuable for biotechnology companies and researchers who are screening for drug compounds. We are working closely with California companies to develop new microscopes, assay devices, and analytical software that could be the basis for new product lines or new businesses. If therapies do come to fruition, we anticipate that California medical centers will be leading the way. The most important contribution of this study will be to improve the health of Californians. Heart disease is a major cause of mortality and morbidity, resulting in billions of dollars in health care costs and lost days at work. Our goal is to contribute research that would ultimately improve the quality of life and increase productivity for millions of people who suffer from heart disease.
Progress Report: 
  • Nearly 500,000 people in the US die of sudden cardiac death each year, and long QT syndrome (LQTS) is a major form of sudden cardiac death. LQTS can be triggered by drug exposure or stresses. Drug-induced LQTS is the single most common reason for drugs to be withdrawn from clinical trials, causing major setbacks to drug discovery efforts and exposing people to dangerous drugs. In most cases, the mechanism of drug-induced LQTS is unknown. However, there are genetic forms of LQTS that should allow us to make iPS cell–derived heart cells that have the key features of LQTS. Our objective is to produce a cell-based test for LQTS with induced pluripotent stem (iPS) cell technology, which allows adult cells to be “reprogrammed” to be stem cell–like cells.
  • Despite the critical need, current tests for drug-induced LQTS are far from perfect. As a result, potentially unsafe drugs enter clinical trials, endangering people and wasting millions of dollars in research funds. When drugs that cause LQTS, such as terfenadine (Seldane), enter the market, millions of people are put at serious risk. Unfortunately, it is very difficult to know when a drug will cause LQTS, since most people who develop LQTS have no known genetic risk factors. The standard tests for LQTS use animal models or hamster cells that express human heart genes at high levels. Unfortunately, cardiac physiology in animal models (rabbits and dogs) differs from that in humans, and hamster cells lack many key features of human heart cells. Human embryonic stem cells (hESCs) can be differentiated into heart cells, but we do not know the culture conditions that would make the assay most similar to LQTS in a living person. These problems could be solved if we had a method to grow human heart cells from people with genetic LQTS mutations, so that we know the exact test conditions that would reflect the human disease. This test would be much more accurate than currently available tests and would help enable the development of safer human pharmaceuticals.
  • Our long-term goal is to develop a panel of iPS cell lines that better represent the genetic diversity of the human population. Susceptibility to LQTS varies, and most people who have life-threatening LQTS have no known genetic risk factors. We will characterize iPS cells with well-defined mutations that have clinically proven responses to drugs that cause LQTS. These iPS cell lines will be used to refine testing conditions. To validate the iPS cell–based test, the results will be directly compared to the responses in people. These studies will provide the foundation for an expanded panel of iPS cell lines from people with other genetic mutations and from people who have no genetically defined risk factor but still have potentially fatal drug-induced LQTS. This growing panel of iPS cell lines should allow for testing drugs for LQTS more effectively and accurately than any current test.
  • To meet these goals, we made a series of iPS cells that harbor different LQTS mutations. These iPS cells differentiate into beating cardiomyocytes. We are now evaluating these LQTS cell lines in cellular assays. We are hopeful that our studies will meet or exceed all the aims of our original proposal.
  • Nearly 500,000 people in the US die of sudden cardiac death each year, and long QT syndrome (LQTS) is a major form of sudden cardiac death. LQTS can be triggered by drug exposure or stresses. Drug-induced LQTS is the single most common reason for drugs to be withdrawn from clinical trials, causing major setbacks to drug discovery efforts and exposing people to dangerous drugs. In most cases, the mechanism of drug-induced LQTS is unknown. However, there are genetic forms of LQTS that should allow us to make iPS cell–derived heart cells that have the key features of LQTS. Our objective is to produce a cell-based test for LQTS with induced pluripotent stem (iPS) cell technology, which allows adult cells to be “reprogrammed” to be stem cell–like cells.
  • Despite the critical need, current tests for drug-induced LQTS are far from perfect. As a result, potentially unsafe drugs enter clinical trials, endangering people and wasting millions of dollars in research funds. When drugs that cause LQTS, such as terfenadine (Seldane), enter the market, millions of people are put at serious risk. Unfortunately, it is very difficult to know when a drug will cause LQTS, since most people who develop LQTS have no known genetic risk factors. The standard tests for LQTS use animal models or hamster cells that express human heart genes at high levels. Unfortunately, cardiac physiology in animal models (rabbits and dogs) differs from that in humans, and hamster cells lack many key features of human heart cells. Human embryonic stem cells (hESCs) can be differentiated into heart cells, but we do not know the culture conditions that would make the assay most similar to LQTS in a living person. These problems could be solved if we had a method to grow human heart cells from people with genetic LQTS mutations, so that we know the exact test conditions that would reflect the human disease. This test would be much more accurate than currently available tests and would help enable the development of safer human pharmaceuticals.
  • Our long-term goal is to develop a panel of iPS cell lines that better represent the genetic diversity of the human population. Susceptibility to LQTS varies, and most people who have life-threatening LQTS have no known genetic risk factors. We will characterize iPS cells with well-defined mutations that have clinically proven responses to drugs that cause LQTS. These iPS cell lines will be used to refine testing conditions. To validate the iPS cell–based test, the results will be directly compared to the responses in people. These studies will provide the foundation for an expanded panel of iPS cell lines from people with other genetic mutations and from people who have no genetically defined risk factor but still have potentially fatal drug-induced LQTS. This growing panel of iPS cell lines should allow for testing drugs for LQTS more effectively and accurately than any current test.
  • To meet these goals, we have made a series of iPS cells that harbor different LQTS mutations. These iPS cells differentiate into beating cardiomyocytes. We are now evaluating these LQTS cell lines in cellular assays. We are hopeful that our studies will meet or exceed all the aims of our original proposal.
  • Cardiac arrhythmias are a major cause of morbidity and mortality. Yet we lack appropriate human tissue models to develop new therapies of this deadly disease. Despite the importance of this disease, the current in vitro models utilize overexpressed channels in fibroblasts that do not accurately recapitulate human cardiac myocytes. With our CIRM funding, we greatly improved our in vitro models by using cardiomyocytes derived from human induced pluripotent stem cells (iPS cells) from donors who harbor cardiac arrhythmia mutations. We enrolled a series of research subjects with genetic forms of LQTS. All participants in our study signed a consent form that was approved by the UCSF human subjects committee. We found that iPS cell–derived cardiomyocytes developed disease-related phenotypes in vitro that could be readily demonstrated by electrophysiological techniques. Such measurements enabled the pharmacological characterization of underlying mechanisms of disease and may point to potential novel therapies. The CIRM funding has allowed our laboratory develop new methods for human disease modeling in iPS cell–derived tissues. This project served as a critical catalyst for human disease research that would otherwise be impossible.

Engineering a Cardiovascular Tissue Graft from Human Embryonic Stem Cells

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00151
ICOC Funds Committed: 
$2 618 704
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Cardiovascular disease (CVD) affects more than 71 million Americans and 1.7 million Californians. Recently, engineered cardiovascular tissue grafts, or “patches”, including one made from mouse embryonic stem cells (ESC), have shown promising results as a future therapy for CVD. Our overall goal is to extend these recent results to human ESC as follows. Aim 1: Apply mechanical stretch and electrical pacemaker-like stimulation to hESC-derived heart cells in order to make them stronger and beat at the same time. Current methods to turn hESC into heart cells do not result in the organization required to generate enough strength to support a weak heart and to avoid irregular heart beats. We will use specially engineered devices to apply mechanical stretch and electrical pacemaker-like stimulation to hESC-derived heart cells in order to strengthen them and make them beat in unison. Aim 2: Engineer a cardiovascular patch from hESC-derived heart cells in order to make a potential new therapy for heart disease. Recently, heart cells from mouse ESC, supporting structures called scaffolds, and mechanical stretch have successfully been combined to engineer a cardiovascular patch. We will combine the hESC-derived heart cells from Aim 1, scaffolds, and the same stretch and pacemaker-like stimulation as in Aim 1 to engineer a cardiovascular patch. In addition, we will add a specialized substance called VEGF to our patch so that, potentially, a blood supply will form around it after it is implanted on a diseased heart. We believe a blood supply will be necessary to keep our patch healthy, and in turn, this will allow our patch to help a damaged heart pump better. Aim 3: Assess whether our patch can remain healthy and also strengthen the heart of a rat after it has undergone a heart attack. We will first implant our cardiovascular patch in the rat aorta, the main blood vessel that supplies blood to the body, to test whether the patch remains healthy and whether it can contract and beat on its own. We will first use the aortic position because we feel it will allow us to assess the inherent function of the patch, thus facilitating our efforts to improve its design. After testing in the aortic position, we will implant the patch over damaged heart tissue in a rat that has undergone an experimentally created heart attack. Over a period of several weeks, we will use novel imaging techniques, ultrasonography, echocardiography, and electrocardiography to non-invasively test whether the patch remains healthy and whether the patch helps the damaged heart pump better. We believe the above aims will address questions relevant to hESC-based cardiovascular therapies and will provide vital information needed for safe and effective future clinical translation. As we will evaluate both federally and non-federally approved cell lines, and thus unlikely to receive federal funding, we will need to rely on the support provided by CIRM to carry out our objectives.
Statement of Benefit to California: 
Cardiovascular disease (CVD) affects more than 1.7 million Californians and 71 million Americans. The societal and financial impacts are tremendous, with CVD accounting annually for an estimated $8 billion in CA and nearly $400 billion in US health care costs. In the case of chronic illness such as CVD, the state and national health care systems may not be able to meet the needs of patients or control spiraling costs, unless the focus of therapy switches away from maintenance and toward cures. Fortunately, the passage of Proposition 71 in 2004, and the subsequent creation of the California Institute for Regenerative Medicine (CIRM), has created the funding needed to advance human embryonic stem cell (hESC) research that could lead to curative therapies that would benefit both millions of Californians and Americans. Recently, engineered cardiovascular tissue grafts, made from rat neonatal cardiomyocytes (CM) and cardiomyocytes derived from mouse ESC, have shown promising results as a future therapy for CVD. The overall goal of our proposed research is to extend these recent studies to hESC and engineer a hESC-CM based cardiovascular tissue graft as a regenerative therapy for CVD. We believe the objectives of our research will benefit the people and the state of California by addressing questions relevant to hESC-based cardiovascular regenerative therapies and will provide vital information needed for safe and efficacious future clinical translation. Development of cures for diseases such as CVD could potentially improve the California health care system by reducing the long-term health care cost burden on California. In addition, the results of our research may provide an opportunity for California to benefit from royalties, patents, and licensing fees and benefit the California economy by creating projects, jobs, and therapies that will generate millions of dollars in new tax revenues in our state. Finally, stem cell research such as ours could further advance the biotech industry in California, serving as an engine for California’s economic future. We have assembled a multidisciplinary team of experienced investigators to attack the objectives of our proposed research. At the same time, we will train and mentor a new generation of bright students and junior scientists in the areas of hESC biology, regenerative medicine, and technology development. This ensures that an essential knowledge base will be preserved and passed on to both investigators and patients within and beyond California.
Progress Report: 
  • Specific Aim 1: To electromechanically condition hESC-derived cardiomyocytes.
  • Progress: Over the past year, we have designed and constructed a computer controlled integrated stretch system and electrical pacing system for applying mechanical and electrical stimulation. This system was used in conjunction with a stretchable microelectrode array (sMEA) and shown to successfully support, stretch, and pace primary murine cardiomyocytes (CM). We also have developed a strain array device for cell culture that effectively interfaces the desirable properties of high-throughput microscale fluidic devices with macroscale user-friendly features. One challenge we have encountered with our sMEAs is maintaining electrical continuity of electrodes as cells are stretched. As an alternative to traditional electrical stimulation we have created a system that optically induces electrical activity in hESC-CM. We are now able to optically and non-invasively pace cardiomyocytes differentiated from our modified hESC line.
  • Specific Aim 2: To engineer a hESC-CM based cardiovascular tissue graft.
  • Progress: From our first attempt at engineering a cardiovascular tissue graft as we reported in Year 1, we learned that our grafts would require large populatoins of relatively pure hESC-CMs. As a result, we concentrated our efforts over the past year in developing a more efficient differentiation method for producing larger yields and quantities of hESC-CM. Our method produces hESC-CM in a directed manner under feeder-free and serum-free conditions by controlling multiple cardiomyogenic developmental pathways. Also, in a collaborative effort, we are engineering a novel method for sorting cardiomyocytes. In order to promote improved viability of hESC-CM in our tissue grafts, co-transplantation with hESC-derived endothelial cells (hESC-EC), as opposed to VEGF alone, will likely be needed as shown recently by others. Over the past year, we have shown that we can produce hESC-EC and that their survival in the heart is enhanced by activation of acetylcholine receptors that lead to activation of pro-survival and anti-apoptosis pathways. Finally, in order to control spatial orientation of hESC-CM within our tissue grafts, we have demonstrated on-demand micropatterning of matrix proteins for cell localization and stem cell fate determination. We have illustrated the utility of a cantilever-based nano-contact printing technology for cellular patterning, mESC renewal, and mESC fate specification. We are currently extending our results to undifferentiated hESC and hESC-CM.
  • Specific Aim 3: To assess tissue graft viability and function in a small animal model.
  • Progress: Over the past year, we created hESC-CM based tissue grafts in linear form. In order to quantify the loss of cardiac function between healthy and diseased hearts, we have recently developed a novel in vitro hybrid experimental/computational system to measure active force generation in ventricular slices of rodent hearts. Quantification of the loss of cardiac function will guide us in determining the numbers of hESC-CM needed for producing grafts with varying force generating capacity. Finally, as outlined in our original proposal, we will first implant our tissue grafts in rat aortas as a novel test-bed to assess the graft’s inherent function while minimizing the confounding effects of underlying cardiac contractions. Over the past year we have successfully implanted decellularized aortic patches in rat aortas and are currently working on adding hESC-CM and hESC-EC to the patches to assess their viability and function.
  • In summary, in the second year of our project we have made strong progress on all three of our specific aims. Based on our current results, we anticipate we will continue to make significant progress in engineering a robust and functional cardiovascular tissue graft.
  • Specific Aim 1: To electromechanically condition hESC-derived cardiomyocyte(CM).
  • Progress: Over the past year, we tested, validated, and published an integrated strain and electrical pacing system that we designed and constructed. As mentioned in our previous reports, one challenge we encountered with our electromechanical devices is maintaining electrical continuity of electrodes as cells are stretched. As an alternative to traditional electrical stimulation, with collaborators at Stanford, we have created a system that optically induces electrical activity in hESC-CM by introducing light activated channelrhodopsin-2 (ChR2), a cationic channel, into undifferentiated hESC. In our initial manuscript we have also demonstrated the effects of light stimulation on a whole heart computational model in which we have virtually injected light-responsive hESC-CM in various areas of the simulated heart.
  • Specific Aim 2: To engineer a hESC-CM based cardiovascular tissue graft.
  • Progress: From our first attempt at engineering a cardiovascular tissue graft as we reported in Years 1, 2, and 3 we learned that our grafts would require large populations of relatively pure hESC-CMs. As a result, we’ve continued our efforts in developing a more efficient differentiation method for producing larger yields and quantities of hESC-CM. Our method produces hESC-CM and iPSC-CM in a directed manner under feeder-free, serum-free, and monolayer conditions by controlling TGF-beta/Activin, BMP, Wnt, and FGF pathways. We have used our differentiation protocols to contribute cardiomyocytes to our collaborators, which has resulted in one published manuscript and two submitted publications. Also, with our collaborator at UC Berkeley, we have engineered a novel method for identifying CMs based on their electrical signals and have reported our technology in one accepted manuscript and one under review.
  • Specific Aim 3: To assess tissue graft viability and function in a small animal model.
  • Progress: Over the past two years, we created hESC-CM based tissue grafts in linear and circular forms and our now creating grafts that can be optically controlled (see Aim 1 above). As described in our last progress report, in order to quantify the loss of cardiac function between healthy and diseased hearts, we have reported a novel in vitro hybrid experimental/computational system to measure active force generation in healthy ventricular slices of rodent hearts. Quantification of the loss of cardiac function will guide us in determining the numbers of hESC-CM needed for producing grafts with varying force generating capacity. We have also modeled eccentric and concentric cardiac growth through sarcomerogenesis in order to give us insight into how we might terminally mature our hESC-CM grafts. Finally, we have differentiated hESC into CM for one of our collaborators at Stanford and have performed detailed calcium imaging to show engraftment of hESC-CM with human heart tissue. This has given us great insight into how 3D tissue grafts might integrate with human heart tissue.
  • In summary, in the fourth year of our project we made good progress on all three of our specific aims. Based on our current results, we anticipate we will continue to make significant progress in engineering a robust and functional cardiovascular tissue graft as we originally proposed and we will continue our efforts. Undoubtedly, with the support of the CIRM grant over the past four years, we have made great strides towards creating a 3D tissue graft and believe we will demonstrate functional integration, not only with rodent hearts, but with human tissue, all within the coming year.

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