Heart Disease

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

Generation and characterization of high-quality, footprint-free human induced pluripotent stem cell lines from 3,000 donors to investigate multigenic diseases

Funding Type: 
hiPSC Derivation
Grant Number: 
ID1-06557
ICOC Funds Committed: 
$16 000 000
Disease Focus: 
Developmental Disorders
Genetic Disorder
Heart Disease
Infectious Disease
Alzheimer's Disease
Neurological Disorders
Autism
Respiratory Disorders
Vision Loss
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Induced pluripotent stem cells (iPSCs) have the potential to differentiate to nearly any cells of the body, thereby providing a new paradigm for studying normal and aberrant biological networks in nearly all stages of development. Donor-specific iPSCs and differentiated cells made from them can be used for basic and applied research, for developing better disease models, and for regenerative medicine involving novel cell therapies and tissue engineering platforms. When iPSCs are derived from a disease-carrying donor; the iPSC-derived differentiated cells may show the same disease phenotype as the donor, producing a very valuable cell type as a disease model. To facilitate wider access to large numbers of iPSCs in order to develop cures for polygenic diseases, we will use a an episomal reprogramming system to produce 3 well-characterized iPSC lines from each of 3,000 selected donors. These donors may express traits related to Alzheimer’s disease, autism spectrum disorders, autoimmune diseases, cardiovascular diseases, cerebral palsy, diabetes, or respiratory diseases. The footprint-free iPSCs will be derived from donor peripheral blood or skin biopsies. iPSCs made by this method have been thoroughly tested, routinely grown at large scale, and differentiated to produce cardiomyocytes, neurons, hepatocytes, and endothelial cells. The 9,000 iPSC lines developed in this proposal will be made widely available to stem cell researchers studying these often intractable diseases.
Statement of Benefit to California: 
Induced pluripotent stem cells (iPSCs) offer great promise to the large number of Californians suffering from often intractable polygenic diseases such as Alzheimer’s disease, autism spectrum disorders, autoimmune and cardiovascular diseases, diabetes, and respiratory disease. iPSCs can be generated from numerous adult tissues, including blood or skin, in 4–5 weeks and then differentiated to almost any desired terminal cell type. When iPSCs are derived from a disease-carrying donor, the iPSC-derived differentiated cells may show the same disease phenotype as the donor. In these cases, the cells will be useful for understanding disease biology and for screening drug candidates, and California researchers will benefit from access to a large, genetically diverse iPSC bank. The goal of this project is to reprogram 3,000 tissue samples from patients who have been diagnosed with various complex diseases and from healthy controls. These tissue samples will be used to generate fully characterized, high-quality iPSC lines that will be banked and made readily available to researchers for basic and clinical research. These efforts will ultimately lead to better medicines and/or cellular therapies to treat afflicted Californians. As iPSC research progresses to commercial development and clinical applications, more and more California patients will benefit and a substantial number of new jobs will be created in the state.

Preclinical evaluation of human embryonic stem cell-derived cardiovascular progenitors

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06378
ICOC Funds Committed: 
$2 930 388
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Because the regenerative capacity of adult heart is limited, any substantial cell loss as a result of a heart attack is mostly irreversible and may lead to progressive heart failure. Human pluripotent stem cells can be differentiated to heart cells, but their properties when transplanted into an injured heart remain unresolved. We propose to perform preclinical evaluation for transplantation of pluripotent stem cell-derived cardiac cells into the injured heart of an appropriate animal model. However, an important issue that has limited the progress to clinical use is their fate upon transplantation; that is whether they are capable of integrating into their new environment or they will function in isolation at their own pace. As an analogy, the performance of a symphony can go into chaos if one member plays in isolation from all surrounding cues. Therefore, it is important to determine if the transplanted cells can beat in harmony with the rest of the heart and if these cells will provide functional benefit to the injured heart. We plan to isolate cardiac cells derived from human pluripotent stem cells, transplant them into the model’s injured heart, determine if they result in improvement of the heart function, and perform detailed electrophysiology studies to determine their integration into the host tissue. The success of the proposed project will set the platform for future clinical trails of stem cell therapy for heart disease.
Statement of Benefit to California: 
Heart disease remains the leading cause of mortality and morbidity in the US with an estimated annual cost of over $300 billion. In California alone, more than 70,000 people die every year from cardiovascular diseases. 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. Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) could provide an attractive therapeutic option to treat patients with damaged heart. We propose to isolate heart cells from hESCs and transplant them in an injured animal model's heart and study their fate. In the process, we will develop reagents that can be highly valuable for future research and clinical studies. The reagents generated in these studies can be patented forming an intellectual property portfolio shared by the state and the institution where the research is carried out. Most importantly, the research that is proposed in this application could lead to future stem cell-based therapies that would restore heart function after a heart attack. 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: 
  • Identification and isolation of pure cardiac cells derived from human pluripotent stem cells has proven to be a difficult task. We have designed a method to genetically engineer human embryonic stem cells (hESCs) to harbor a label that is expressed during sequential maturation of cardiac cells. This will allow us to prospectively isolate cardiac cells at different stages of development for further characterization and transplantation. Using this method, we have screened proteins that are expressed on the surface of cells as markers. Using antibodies against these surface markers allows for isolation of these cells using cell sorting techniques. Thus far, we have identified two surface markers that can be used to isolate early cardiac progenitors. Using these markers, we have enriched for cardiac cells from differentiating hESCs and have characterized their properties in the dish as well as in small animals. We plan to transplant these cells in large animal models and monitor their survival, expansion and their integration into the host myocardium. Molecular imaging techniques are used to track these cells upon transplantation.

Human Induced Pluripotent Stem Cell-Derived Cardiovascular Progenitor Cells for Cardiac Cell Therapy.

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06455
ICOC Funds Committed: 
$3 004 315
Disease Focus: 
Heart Disease
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Despite therapeutic advances, cardiovascular disease remains a leading cause of mortality and morbidity in California. Regenerative therapies that restore normal function after a heart attack would have an enormous societal and financial impact. Although very promising, regenerative cardiac cell therapy faces a number of challenges and technological hurdles. Human induced pluripotent stem cells (hiPSC) allow the potential to deliver patient specific, well-defined cardiac progenitor cells (CPC) for regenerative clinical therapies. We propose to translate recent advances in our lab into the development of a novel, well-defined hiPSC-derived CPC therapy. All protocols will be based on clinical-grade, FDA-approvable, animal product-free methods to facilitate preclinical testing in a large animal model. This application will attempt to translate these findings by: -Developing techniques and protocols utilizing human induced pluripotent stem cell-derived cardiac progenitor cells at yields adequate to conduct preclinical large animal studies. -Validation of therapeutic activity will be in small and large animal models of ischemic heart disease by demonstrating effectiveness of hiPSC-derived CPCs in regenerating damaged myocardium post myocardial infarction in small and large animal models. This developmental candidate and techniques described here, if shown to be a feasible alternative to current approaches, would offer a novel approach to the treatment of ischemic heart disease.
Statement of Benefit to California: 
Cardiovascular disease remains the leading cause of morbidity and mortality in California and the US costing the healthcare system greater than 300 billion dollars a year. Although current therapies slow progression of heart disease, there are few options to reverse or repair the damaged heart. The limited ability of the heart to regenerate following a heart attack results in loss of function and heart failure. Human clinical trials testing the efficacy of adult stem cell therapy to restore mechanical function after a heart attack, although promising, have had variable results with modest improvements. The discovery of human induced pluripotent stem cells offers a potentially unlimited renewable source for patient specific cardiac progenitor cells. However, practical application of pluripotent stem cells or their derivatives face a number of challenges and technological hurdles. We have demonstrated that cardiac progenitor cells, which are capable of differentiating into all cardiovascular cell types, are present during normal fetal development and can be isolated from human induced pluripotent stem cells. We propose to translate these findings into a large animal pre-clinical model and eventually to human clinical trials. This could lead to new therapies that would restore heart function after a heart attack preventing heart failure and death. This will have tremendous societal and financial benefits to patients in California and the US in treating heart failure.
Progress Report: 
  • Cardiovascular disease remains to be a major cause of morbidity and mortality in California and the United States. Despite the best medical therapies, none address the issue of irreversible myocardial tissue loss after a heart attack and thus there has been a great interest to develop approaches to induce regeneration. Our lab has focused on harvesting the full potential of patient specific induced pluripotent stem cells (iPSCs) to use to attempt to regenerate the damaged tissue. We believe that these iPSCs can be potentially used in the future to generate sufficient number of cells to be implanted in the damaged heart to regenerate the lost tissue post heart attack. Our lab has studied how these cardiac progenitors evolve in the developing heart and applied our finding to iPSCs to recapitulate the cardiac progenitors to ultimately use in clinical therapies. We have successfully derived these cardiac progenitors from patient derived iPSCs in a clinical grade fashion to ensure that we can apply same protocols in the future to clinical use if we are successful in demonstrating the efficacy of this therapy in our translational large animal studies that we will be conducting.

Characterization and Engineering of the Cardiac Stem Cell Niche

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

Prospective isolation of hESC-derived hematopoietic and cardiomyocyte stem cells

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00354
ICOC Funds Committed: 
$2 636 900
Disease Focus: 
Blood Disorders
Heart Disease
Immune Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The capacity of human embryonic stem cells (hESCs) to perpetuate themselves indefinitely in culture and to differentiate to all cell types of the body has lead to numerous studies that aim to isolate therapeutically relevant cells for the benefit of patients, and also to study how genetic diseases develop. However, hESCs can cause tumors called teratomas when placed in the body and therefore, we need to separate potentially beneficial cells from hazardous hESCs. Thus, potential therapeutics cannot advance until the development of methodologies that eliminate undifferentiated cells and enrich tissue stem cells. In our proposal we hope to define the cell surface markers that are differentially expressed by committed hESC-derived stem cells and others that are expressed by teratogenic hESCs. To do this we will carry out a large screen of cell subsets that form during differentiation using a collection of unique reagents called monoclonal antibodies, many already obtained or made by us, to define the cell-surface markers that are expressed by teratogenic cells and others that detect valuable tissue stem cells. This collection, after filing for IP protection, would be available for CIRM investigators in California. We were the first to isolate mouse and human adult blood-forming stem cells, human brain stem cells, and mouse muscle stem cells, all by antibody mediated cell-sorting approaches. Antibody mediated identification of cell subsets that arise during early hESC differentiation will allow separation and characterization of defined subpopulations; we would isolate cells that are committed to the earliest lineage known to form multiple cell types in the body including bone, blood, heart and muscle. These cells would be induced to differentiate further to the blood forming and heart muscle forming lineages. Enriched, and eventually purified hESC-derived blood-forming stem cells and heart muscle stem cells will be tested for their potential capacity to engraft and improve function in animal models. Blood stem cells will be transplanted into immunodeficient mice to test their capacity to give rise to all blood cell types; and heart muscle stem cells will be transferred to mouse hearts that had an artificial coronary artery blockage, a model for heart attack damage. Finally, we will test the capacity of blood stem cell transplantation to induce transplantation tolerance towards heart muscle stem cells from the same donor cell line. Transplantation tolerance in this case means that the heart cells would be accepted as ‘self’ by the mouse that had it’s unrelated donor immune system replaced wholly or in part by blood forming stem cells from the same hESC line that gave rise to the transplantable heart stem cells, and therefore would not be rejected by it’s own immune system. This procedure would allow transplantation of beneficial tissues such as heart, insulin-producing cells, etc., without the use of immunosuppressive drugs.
Statement of Benefit to California: 
The principle objective of this proposal is to develop reagents which, in combinations, can identify and isolate tissue-regenerating stem cells derived from hESC lines. The undifferentiated hESCs are dangerous for transplantation into humans, as they cause tumors. We propose to prepare reagents that identify and can be used to delete or prospectively isolate these tumor-causing undifferentiated hESCs. HESC-derived tissue stem cells have the potential to regenerate damaged tissues and organs, and don’t cause tumors. We propose to develop reagents that can be used to identify and prospectively isolate pure human blood-forming stem cells derived from hESCs, and separately other reagents that can be used to identify and prospectively isolate pure heart-forming stem or progenitor cells. These “decontaminated” hESC-derived tissue stem cells may eventually be used to treat human tissue degenerative diseases. These reagents could also be used to isolate the same cells from somatic cell nuclear transfer (SCNT)-derived pluripotent stem cell lines from patients with genetic diseases. This procedure would enable us to analyze the effects of the genetic abnormalities on blood stem and progenitor cells in patients with genetic blood and immune system disorders, and on heart stem and progenitor cells in patients with heart disorders. The antibodies and stem cells (hESCs, tissue regenerating, etc) that will be isolated from patients with specific diseases will be invaluable tools that can be used to create model(s) for understanding the diseases and their progression. In addition, the antibodies and the stem cells generated in these studies are entities that could be patented or protected by copyright, forming an intellectual property portfolio shared by the state and the state institutions wherein the research was carried out. The funds generated from the licensing of these technologies will help pay back the state, will help support increasing faculty and staff (many of whom bring in other, out of state funds for their research), and could be used to ameliorate the costs of clinical trials. Only California businesses are likely to be able to license these antibodies and cells, to develop them into diagnostic and therapeutic entities; such businesses are the heart of the CIRM strategy to enhance the California economy. Most importantly, however, is that this research will lead to tissue stem cell therapies. Such therapies will address chronic diseases that cause considerable disability and misery, currently have no cure, and therefore lead to huge medical expenses. Because tissue stem cells renew themselves for life, stem cell therapies are one-time therapies with curative intent. We expect that California hospitals and health care entities will be first in line for trials and therapies, and for CIRM to negotiate discounts on such therapies for California taxpayers, thus California will benefit both economically and with advanced novel medical care.
Progress Report: 
  • The objectives of our proposal are the isolations of blood-forming and heart-forming stem cells from human embryonic stem cell (hESCs) cultures, and the generation of monoclonal antibodies (mAbs) that eliminate residual teratogenic cells from transplantable populations of differentiated hESCs. For isolation of progenitors, we hypothesized that precursors derived from hESCs could be identified and isolated using mAbs that label unique combinations of lineage-specific cell surface molecules. We used hundreds of defined mAbs, generated hundreds of novel anti-hESC mAbs, and used these to isolate and characterize dozens of hESC-derived populations. We discovered four precursor types from early stages of differentiating cells, each expressing genes indicative of commitment to either embryonic or extraembryonic tissues. Together, these progenitors are candidates to give rise to meso-endodermal lineages (heart, blood, pancreas, etc), and yolk sac, umbilical cord and placental tissues, respectively. Importantly, we have found that cells of the meso-endodermal population give rise to beating cardiomyocytes. We are currently enriching cardiomyocyte precursors from this population using cardiac-specific genetic markers, and are assaying the putative progenitors using electrophysiological assays and by transplantation into animal hearts (a test for restoration of heart function). In addition, we established in vitro conditions that effectively promote hESC-differentiation towards the hematopoietic (blood) lineages and isolated populations that resemble hematopoietic stem cells (HSCs) in both surface phenotype as well as lineage potentials, as determined by assays in vitro. We have generated hESC-lines that express the anti-apoptotic gene BCL2, and have found that these cells produce significantly greater amounts of hematopoietic and cardiac cells, because of their increased survival during culturing and sorting. We are currently isolating hematopoietic precursors from BCL2-hESCs and will test their ability to engraft in immunodeficient mice, to examine the capacity of hESC-derived HSCs to regenerate the blood system. Finally, we have utilized the novel mAbs that we prepared against undifferentiated hESCs, to deplete residual teratogenic cells from differentiated cultures that were transplanted into animal models. We discovered that following depletion teratoma rarely formed, and we expect to determine a final cocktail of mAbs for removal of teratogenic cells from transplantation products this year.
  • The main objective of our proposal is to isolate therapeutic stem cells and progenitors from human embryonic stem cells (hESCs) that give rise to blood and heart cells. Our approach involves isolation of differentiated precursor subset of cells using monoclonal antibodies (mAbs) and cell sorting instruments, and subsequent characterization of their respective hematopoietic and cardiomyogenic potential in culture as well as following engraftment into mouse models of disease. In addition, we aim to develop mAbs that specifically bind to undifferentiated hESCs for removal of residual teratoma-initiating cells from therapeutic cell preparations, to ensure transplantation safety.
  • We have made substantial advancement towards achieving these goals. First, we discovered that the initial differentiation of hESCs occurs through only 4-5 different progenitor types, of which one is destined to give rise to heart lineages. We purified this population using three novel cell surface markers, and found a significant enrichment of cardiomyocyte clones in colony formation assays that we developed. This subset also expressed particularly high levels of cardiac genes and was receptive to further differentiation into beating cardiomyocytes or vascular endothelial cells. When transplanted into immunodeficient mice these progenitors differentiated into ventricular myocytes and vascular endothelial cells. In the coming year we will perform transplantation experiments to evaluate whether they improve the functional outcome of heart infarction in hearts of mice. Second, we have optimized cell culture conditions and cell surface markers to sort hematopoietic progenitors derived from hESCs. We have also begun to transplant these populations into immunodeficient mouse recipients to identify blood-reconstituting hematopoietic populations. Third, we identified 5 commercial and 1 custom mAbs that are specific to human pluripotent cells (hESCs and induced pluripotent cells). We are currently testing the capacity of combinations of 3 pluripotency surface markers to remove all teratoma-initiating cells from transplanted differentiated cell populations. In summary, we expect provide functional validation of the blood and heart precursor populations that we identified from hESCs by the end term of this grant.
  • The main objective of our proposal is to isolate therapeutic stem and progenitor cells derived from human embryonic stem cells (hESCs) that can give rise to blood and heart cells. Our approach involves developing differentiation protocols to drive hematopoietic (blood) and cardiac (heart) development of hESCs, then to identify and isolate stem/progenitor cells using monoclonal antibodies (mAbs) specific to surface markers expressed on blood and heart stem/progenitor cells, and finally to characterize their functional properties in vitro and in vivo. In addition, we sought to develop mAbs that specifically bind to undifferentiated hESCs for removal of residual teratoma (tumor)-initiating cells from therapeutic preparations, to ensure transplantation safety.
  • We have made substantial progress toward achieving these goals. First, we discovered that the initial differentiation of hESCs occurs through only 4-5 different progenitor types, of which one is destined to give rise to heart lineages. We purified this population using four novel cell surface markers (ROR2, PDGFRα, KDR, and CD13), and found a significant enrichment of cardiomyocyte clones in colony formation assays that we developed. This subset also expressed particularly high levels of cardiac genes and was receptive to further differentiation into beating cardiomyocytes or vascular endothelial cells. When transplanted into immunodeficient mice these progenitors differentiated into ventricular myocytes and vascular endothelial cells. We have also successfully developed a human fetal heart xenograft model to test hESC-derived cardiomyocyte stem/progenitor cells in human heart tissue for engraftment and function.
  • Second, we have optimized cell culture conditions and cell surface markers to sort hematopoietic progenitors derived from hESCs. In doing so, we have mapped the earliest stages of hematopoietic specification and commitment from a bipotent hematoendothelial precursor. Our culture conditions drive robust hematopoietic differentiation in vitro but these hESC-derived hematopoietic progenitors do not achieve hematopoietic engraftment when transplanted in mouse models. Furthermore, we overexpressed the anti-apoptotic protein BCL2 in hESCs, and discovered a significant improvement in viability upon single cell sorting, embryoid body formation, and in cultures lacking serum replacement. Moving forward, we feel the survival advantages exhibited by this BCL2-expressing hESC line will improve our chances of engrafting hESC-derived hematopoietic stem/progenitor cells.
  • Third, we identified a cocktail of 5 commercial and 1 novel mAbs that enable specific identification of human pluripotent cells (hESCs and induced pluripotent cells). We have found combinations of 3 pluripotency surface markers that can remove all teratoma-initiating cells from differentiated hESC and induced pluripotent stem cell (iPSC) populations prior to transplant. While these combinations can vary depending on the differentiation culture, we have generated a simple, easy-to-follow protocol to remove all teratogenic cells from large-scale differentiation cultures.
  • In summary, we accomplished most of the goals stated in our original proposal. We successfully achieved cardiac engraftment of an hESC-derived cardiomyocyte progenitor using a novel human heart model of engraftment. While we unfortunately did not attain hematopoietic engraftment of hESC-derived cells, we are exploring a strategy to address this. Our research has led to four manuscripts: one on the protective effects of BCL2 expression on hESC viability and pluripotency (published in PNAS, 2011), another describing markers of pluripotency and their use in depleting teratogenic potential in differentiated PSCs (accepted for publication in Nature Biotechnology), and two submitted manuscripts, one describing a novel xenograft assay to test PSC-derived cardiomyocytes for functional engraftment and the other describing the earliest fate decisions downstream of a PSC.

A new paradigm of lineage-specific reprogramming

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

Epigenetic regulation of human cardiac differentiation

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

Extracellular Matrix Bioscaffold Augmented with Human Stem Cells for Cardiovascular Repair

Funding Type: 
Early Translational III
Grant Number: 
TR3-05626
ICOC Funds Committed: 
$4 939 140
Disease Focus: 
Heart Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
An estimated 16.3 million Americans suffer from coronary heart disease. Every 25 seconds, someone has a coronary event and every minute, someone dies from one. Treatment for coronary heart disease has improved greatly in recent years, yet 1 in 6 deaths in the US in 2007 was still caused by this terrible disease. Stem cells have been used as an supplemental form of treatment but they have been most effective for patients treated immediately after their first heart attack. Unfortunately, stem cell therapy for chronic heart disease and heart failure has been less successful. With current delivery methods for stem cells into the heart, most are washed away quickly, whereas our device will hold them in the area that needs repair. With this project we are testing a novel approach to improve the benefits of stem cell therapy for patients suffering from chronic heart disease. By applying a type of bone marrow stem cells known to enhance tissue repair (mesenchymal stem cells) to a biological scaffold, we hope to greatly amplify the beneficial properties of both the stem cells and the biological scaffold. This device will be implanted onto an appropriate preclinical model that have been treated so as to mirror the chronic heart disease seen in humans. We predict that this novel device will heal the damaged heart and improve its function to pave the way for a superior treatment option for the thousands of Americans for whom the unlikely prospect of a heart transplant is currently the only hope.
Statement of Benefit to California: 
Heart disease is the number one cause of death and disability in California and in the US as a whole. An estimated 16.3 million Americans over the age of 20 suffer from coronary heart disease (CHD) with an estimated associated cost of $177.5 billion and CHD accounted for 1 in 6 deaths in the US in 2007. Advances in treatment have decreased early mortality but consequently lead to an increase in the incidences of heart failure (HF). Patients with HF have a 50 percent readmission rate within six months, which is a heavy cost both in terms of quality of life and finances. The high cost of caring for patients with HF results primarily from frequent hospital readmissions for exacerbations. The need for efficient treatment strategies that address the underlying cause, massive loss of functional myocardium, is yet to be met. We believe that present project proposal, development of a combined mesenchymal stem cell and extra cellular matrix scaffold device, will lead to improved standards of care for patients suffering from chronic myocardial infarction who are thus at risk of developing HF. By not only retarding disease progression but by actually restoring cardiac function, we believe that the proposed project will have a tremendous impact on both the cost of care as well as the quality of life for large groups of Californians and patients worldwide for whom the improbable prospect of heart transplantation is the only curative treatment option available.
Progress Report: 
  • Heart disease is a major cause of death and disability in the US, accounting for 1 in every 4 deaths and costing more than 100 billion annually. While significant improvements have been made towards treating and managing heart disease, we are still not able to effectively return the heart to a healthy state and cure the patients. With our project we have set out to develop a novel strategy for not only halting the disease progression but to reverse the devastating effect on the function of the heart. By combining bone marrow mesenchymal stromal cells with a biological scaffold material, we hope to create a patch for the heart that will support and regenerate the diseased tissue to the point where the patient will be relieved of the burden of their disease and have a markedly improve quality of life. We have in the past year made significant advances toward establishing an animal disease model in which we can study novel ways of treating heart disease. We have in the same time isolated and characterized cells that reside in the bone marrow and that have the potential to heal the diseased tissue by improving blood flow, minimize scarring and generally promoting recovery of the heart function. We have studies these cells under when grown under different conditions and found their ability to mediate tissue regeneration to be highly dependent on their local environments. We are currently trying to identify the optimal combination of cells and microenvironment that may achieve maximal regenerative effect in our disease model and ultimately help our patient combat their heart disease.

Heart Repair with Human Tissue Engineered Myocardium

Funding Type: 
Early Translational III
Grant Number: 
TR3-05556
ICOC Funds Committed: 
$4 766 231
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Heart disease is the number one cause of morbidity and mortality in the US. With an estimated 1.5 million new or recurrent myocardial infarctions, the total economic burden on our health care system is enormous. Although conventional pharmacotherapy and surgical interventions often improve cardiac function and quality of life, many patients continue to develop refractory symptoms. Thus, the development of new therapies is urgently needed. “Tissue engineering” can be broadly defined as the application of novel bioengineering methods to understand complex structure-function relationships in normal or pathological conditions and the development of biological substitutes to restore, maintain, or improve function. It is different from “cell therapy”, which is designed to improve the function of an injured tissue by simply injecting suspensions of isolated cells into the injury site. To date, two main limitations of cell therapy are (1) acute donor cell death due to unfavorable seeding environment and (2) the lack of suitable cell type that genuinely resembles human cardiac cells. Our proposal seeks to use engineered tissue patches seeded with human embryonic stem cell-derived cardiomyocytes for treatment of ischemic heart disease in small and large animal models. It represents a significant development of novel techniques to address both of the main limitations of cell therapy, and will provide a new catalyst for the entire field of stem cell-based tissue engineering.
Statement of Benefit to California: 
Patients with end-stage heart failure have a 2-year survival rate of 25% by conventional medical therapy. Not commonly known to the public is that this dismal survival rate is actually worse when compared to patients with AIDS, liver cirrhosis, or stroke. Following a heart failure, the endogenous repair process is not sufficient to compensate for cardiomyocyte death. Thus, novel therapies with stem cells in combination with supportive scaffolds to form engineered cardiac tissue grafts is emerging as a promising therapeutic avenue. Engineered tissues have now been used to make new bladders for patients needing cystoplasty, bioarticial heart patches seeded with bone marrow cells, and more recently new trachea for patient with late stage tracheal cancer. Our multi-disciplinary team intends to push the therapeutic envelop by developing human tissue engineered myocardium for treatment of post-myocardial infarction heart failure. We will first test our engineered cardiac tissue in small and large animal models. We will perform extensive quality control measures to define morphological, molecular, and functional properties. At the end of 3 years, we are confident we will be able to derive a lead candidate that can move into IND-enabling preclinical development. These discoveries will benefit the millions of patients with heart failure in California and globally.
Progress Report: 
  • Despite advances in medical and device therapies, patients with end-stage heart failure have a survival rate of only 25% during the first 2 years following their diagnosis. Heart failure typically follows from damage induced by severe myocardial infarction (MI; heart attack). After a severe MI, the human heart may lose up to 1 billion heart muscle cells (cardiomyocytes). For most of these patients, heart transplantation is the only useful therapy, but there is a severe shortage of donor hearts. Recently, left ventricular assist devices (LVADs) have become available to take over the pumping function of the crucial left ventricle chamber of the heart. These devices were originally used as “bridge to transplant” (a temporary measure to keep patients alive until a new heart became available); recently some patients have received LVADs as “destination therapies” (permanent substitutes for transplanted hearts). The problems associated with these mechanical implants, however, include increased risk of stroke (blood clots that form due to the devices) and infection (the LVADs are powered from batteries that are carried outside the body and require wires to pierce the skin).
  • We are working to develop cardiac regenerative medicine using Engineered Heart Muscle (EHM). We are using human embryonic stem cells (hESCs) because they can be grown in very large quantities and, with the appropriate methods, can be triggered to differentiate into the cardiomyocytes, fibroblasts and smooth muscle that are lost after MI. Because these cells can be produced in essentially unlimited quantities, we could theoretically treat a very large number of patients who currently have no options.
  • During the first year of this project, we have a) established methods for producing the multi-billion quantities of hESC-derived cells needed to address this problem; b) developed methods to freeze and ship these cells to our collaborator in Germany for EHM assembly, and c) used these cells to generate 2 different forms of EHMs to compare their survival and function both in vitro (composition, force generated) and in vivo (after transplantation into rats that have been given MIs). We are now refining the EHM design with the goal of moving forward to testing them in animals with more human-like hearts (based on size and heart rate); this step will be essential to evaluate their safety and function before any clinical trial.

Direct Cardiac Reprogramming for Heart Regeneration

Funding Type: 
Early Translational III
Grant Number: 
TR3-05593
ICOC Funds Committed: 
$6 319 110
Disease Focus: 
Heart Disease
Stem Cell Use: 
Directly Reprogrammed Cell
oldStatus: 
Active
Public Abstract: 
Heart disease is a leading cause of mortality. The underlying pathology is typically loss of heart muscle cells that leads to heart failure. Because heart muscle has little or no regenerative capacity after birth, current therapeutic approaches are limited for the over 5 million Americans who suffer from heart failure. Our recent findings regarding direct reprogramming of a type of structural cell of the heart, called fibroblasts, into cardiac muscle-like cells using just three genes offers a novel approach to achieving cardiac regeneration. 50% of cells in the human heart are cardiac fibroblasts, providing a potential source of new heart muscle cells for regenerative therapy. We simulated a heart attack in mice by blocking the coronary artery, and have been able to reprogram existing mouse cardiac fibroblasts in to new muscle by delivering the 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 of cells in the intact organ was more complete than in cells in a dish. We now propose to develop the optimal gene therapy approach to introduce cardiac reprogramming genes into the heart, to establish the optimal delivery approach to administer virus encoding cardiac reprogramming factors that results in improvement in cardiac function in a preclinical model of cardiac injury, and to establish the safety profile of in vivo cardiac reprogramming in a preclinical model.
Statement of Benefit to California: 
This research will benefit the state of California and its citizens by helping develop a new therapeutic approach to cardiac regeneration. Heart disease is a leading cause of death in adults and children in California, but there is no current treatment that can promote cardiac regeneration. This proposal will lay the groundwork for a clinical trial that could result in generation of new heart muscle cells from within the heart. If successful, there is potential economic benefit in terms of productive lives saved and in the commercialization of this technology.
Progress Report: 
  • Heart disease is a leading cause of mortality. The underlying pathology is typically loss of heart muscle cells that leads to heart failure. Because heart muscle has little or no regenerative capacity after birth, current therapeutic approaches are limited for the over 5 million Americans who suffer from heart failure. Our recent findings regarding direct reprogramming of a type of structural cell of the heart, called fibroblasts, into cardiac muscle-like cells using just three genes offers a novel approach to achieving cardiac regeneration. 50% of cells in the human heart are cardiac fibroblasts, providing a potential source of new heart muscle cells for regenerative therapy. We simulated a heart attack in mice by blocking the coronary artery, and have been able to reprogram existing mouse cardiac fibroblasts into new muscle by delivering the 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 of cells in the intact organ was more complete than in cells in a dish. We now identified a combination of factors that reprogram human and pig cardiac fibroblasts and are optimizing a gene therapy approach to introduce cardiac reprogramming genes into the heart of pigs. In a pig model of cardiac injury, these factors were able to convert non-muscle cells into new muscle in the area of injury. We also found a viral vector that can preferentially infect the fibroblasts compare to the muscle cells. We are now in a position to test for functional improvement in pigs.

Pages

Subscribe to RSS - Heart Disease

© 2013 California Institute for Regenerative Medicine