Heart Disease

Coding Dimension ID: 
295
Coding Dimension path name: 
Heart Disease
Funding Type: 
Basic Biology III
Grant Number: 
RB3-05174
Investigator: 
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.
  • 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 this project, 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 last year, we have determined the epigenetic changes occurring and correlated those with 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 are revealing approaches to refine and improve human cardiac reprogramming and will aid in translation of this technology for human cardiac regenerative purposes.
Funding Type: 
New Faculty I
Grant Number: 
RN1-00566
Investigator: 
Type: 
PI
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.
Funding Type: 
New Faculty I
Grant Number: 
RN1-00562
Investigator: 
ICOC Funds Committed: 
$3 149 806
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Congenital and acquired defects of cardiac pacemakers are leading causes of morbidity and mortality in our society. Dysfunctions of the SA node and the lower conduction cells lead to a variety of complex arrhythmias that typically necessitate anti-arrhythmic therapy and implantation of devices. These treatments have significant limitations in their efficacy and risk-benefit ratio. Thus, it would be ideal to generate cell-based therapeutic approaches towards treating arrhythmias. Experimental data has provided compelling evidence that pacemaker and conduction cells of the heart separate early in development from the working myocardium and retain a relatively undifferentiated state. Prior cell-based approaches in regenerating myocardial damage in the heart have met limited success in part due to implantation of a diverse population of cells. This generally results in poor engraftment and undesirable outcomes. There is now evidence for resident conduction progenitor cells in myocardium that orchestrate the process of cell recruitment into the conduction tissue. In the current proposal we aim to identify the molecular events that lead to differentiation and formation of cardiac pacemaker cells. We will utilize the information obtained from the above experiments to generate cell based methods to treat cardiac arrhythmias. We aim to genetically manipulate the human embryonic stem cells so we can identify a selected population that is destined to become pacemaker cells. By replacing the cells responsible for normal beating of the heart, we hope to provide natural therapies for human conduction system disease
Statement of Benefit to California: 
The ultimate of goal of our proposal is identify a reliable mechanism for implementing a cell-based approach for treating human arrhythmias. Sudden cardiac death related to cardiac arrhythmia is a leading cause of morbidity and mortality in our society. The people of California have voted to implement new innovative ways of treating human disease by using human stem cells, the current project is in line with such wishes to create new therapeutic modalities towards treating heart disease.
Progress Report: 
  • Cardiovascular disease is a major source of morbidity and mortality in our society. In this case, cardiac arrhythmias are leading cause of sudden cardiac death. Therefore, it is empirical to identify the source and mechanisms of cardiac arrhythmias. The long-term objectives of our laboratory is identify the key molecules that are involved in differentiation and formation of cardiac conduction system. We utilize mouse as a model system to identify the molecular pathways leading the formation of cardiac conduction cells.
  • In the past year we have identified some of regulatory pathways that allows for the proper formation of cardiac conduction tissue. We are using mice that have specific mutations in the cells of cardiac conduction system to identify these special pathways. One such molecule that orchestrates the differentiation of cardiac conduction cells is Nkx2-5. We have determined that loss of this transcription factor is of significant detriment to the health of cardiac conduction and is the underlying factor in common arrhythmias. Our ultimate goal is to utilize the information obtained by our studies in mice, and apply them towards therapeutic functions in humans. To this end, we are trying to develop a mechanism to reprogram cardiac stem cells to behave like conduction system cells. Ultimately, this approach would be used towards stem cell therapy for cardiac arrhythmias.
  • A leading cause of heart related morbidity and mortality is cardiac rhythm disturbances. In fact sudden cardiac death is primarily due to abnormalities of cardiac electrical conduction abnormalities. At present, the therapeutic approaches to treatment of cardiac arrhythmias are limited to cardiac device including pacemakers and defibrillators. These devices are expensive and carry additional risks to the patients during after surgical implantation. Our overall goal is to identify the key regulatory pathways that lead to differentiation and formation of various cells type of cardiac conduction cells.
  • Our laboratories focuses on the molecular pathways that guide the formation of distinct cell types in the human heart. The proper formation of these cell types from a unique cardiac progenitor is an important, yet complex biological question that our laboratory is aiming to answer. In this regard, in the past year we have identified a unique molecular pathway by which a unique population of cardiac progenitor cells are added to heart and also participate in the formation and patterning of the cardiac pacemaker cells. We are using mouse models to study the formation of cardiac stem cells and also the mechanisms by which they acquire distinct identities. To this end, our mutant mouse models display abnormal formation of the SA node which is the primary site of cardiac beating. By studying the mutant mice generated by genetic manipulation of stem cells, we aim to further advance our knowledge of different forms of cardiac stem cell formation. During the past year we have made significant progress in elucidating the ways by which cardiac progenitor cells contribute the pacemaker cell formation and putting forth new paradigms for cardiac pacemaker stem cell formation.
  • Heart disease is a major cause of morbidity and mortality in our society. Congestive heart failure and cardiac arrhythmias are the most common mechanism by which heart disease leads to sudden cardiac death. Genetic studies in the general population have determined that susceptibility to cardiac arrhythmia and congestive heart failure is due to mutations in certain genes that guide cardiac development. Specifically, mutations in certain molecules called transcription factors are the leading mechanisms by which genetic defects lead to congenital heart defects and cardiac arrhythmias. Our laboratory studies the mechanism by which transcription factors and signaling molecules guide cardiac development and lead to selective formation of different cardiac cells. Our laboratory has pioneered work that has lead to the discovery of mutations that lead to cardiac arrhythmia and heart failure. In the past year, we have made steady progress in characterization of some of the key factors that guide cardiac cell development. To this end, we have identified a molecule called R-spondin-3 (Rspo3) that is critical for cardiac cell growth and probably survival. We have determined that Rspo3 functions to keep cardiac cell proliferating and loss of Rspo3 leads to thin cardiac muscle and heart failure. The mutation of Rspo3 in mouse leads to not only heart failure, but also leads to arrhythmias and valvlular heart disease. Therefore, Rspo3 functions in multiple aspect cardiac development and plays an essential role in proliferation of resident cardiac stem cells. Since, Rspo3 is known to function in a specific cardiac pathway called Wnt pathway, our hypothesis is that Rspo3 is a needed growth factor that is guiding cardiac stem cells towards growth and proliferation. We have submitted a manuscript about our work with Rspo3.
  • Our laboratory has also identified a molecule called OSR1 which plays a critical role in cardiac septation and development of conduction system. Mice that lack Osr1 have defects in atrial septation and show evidence of cardiac arrhythmias. We are in the process of submitting a manuscript that describes our results with OSR1. In summary, the generous funding by CIRM has helped us identify important new molecules with novel mechanisms critical in cardiac development.
  • Heart disease is a major cause of morbidity and mortality in our society. Congestive heart failure and cardiac arrhythmias are the most common mechanism by which heart disease leads to sudden cardiac death. Genetic studies in the general population have determined that susceptibility to cardiac arrhythmia and congestive heart failure is due to mutations in certain genes that guide cardiac development. Specifically, mutations in certain molecules called transcription factors are the leading mechanisms by which genetic defects lead to congenital heart defects and cardiac arrhythmias. Our laboratory studies the mechanism by which transcription factors and signaling molecules guide cardiac development and lead to selective formation of different cardiac cells. Our laboratory has pioneered work that has lead to the discovery of mutations that lead to cardiac arrhythmia and heart failure. In the past year, we have made steady progress in characterization of some of the key factors that guide cardiac cell development. To this end, we have identified a molecule called R-spondin-3 (Rspo3) that is critical for cardiac cell growth and probably survival. We have determined that Rspo3 functions to keep cardiac cell proliferating and loss of Rspo3 leads to thin cardiac muscle and heart failure. The mutation of Rspo3 in mouse leads to not only heart failure, but also leads to arrhythmias and valvlular heart disease. Therefore, Rspo3 functions in multiple aspect cardiac development and plays an essential role in proliferation of resident cardiac stem cells. Since, Rspo3 is known to function in a specific cardiac pathway called Wnt pathway, our hypothesis is that Rspo3 is a needed growth factor that is guiding cardiac stem cells towards growth and proliferation. We have submitted a manuscript about our work with Rspo3.
  • Our laboratory has also identified a molecule called OSR1 which plays a critical role in cardiac septation and development of conduction system. Mice that lack Osr1 have defects in atrial septation and show evidence of cardiac arrhythmias. We are in the process of submitting a manuscript that describes our results with OSR1. In summary, the generous funding by CIRM has helped us identify important new molecules with novel mechanisms critical in cardiac development.
  • The aims of the current proposal are to gain insight into the mechanisms of cardiac development as it relates to cardiac conduction system
  • and overall maturation of atria and ventricle. Our studies have identified a new key molecule that directs the maturation of cardiac cells. The secreted factor RSPO3 was found to have a significant role in the proper maturation of cardiac ventricles. We now aim to further identify the potential mechanisms by which RSPO3 Functions in the developmental maturation of the mammalian heart
Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01143
Investigator: 
Institution: 
Type: 
PI
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.
Funding Type: 
New Faculty II
Grant Number: 
RN2-00921
Investigator: 
Type: 
PI
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.
Funding Type: 
New Faculty II
Grant Number: 
RN2-00909
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$3 155 931
Disease Focus: 
Heart Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Coronary heart disease is the leading cause of death in the developed world. This disease results from atherosclerosis or fatty deposits in the vessel wall that causes blockage of coronary arteries. Blockage of these arteries cut off supplies of nutrients and oxygen to the heart muscle, causing heart attacks, heart failure or sudden death. To restore coronary blood supply, physicians use guide-wires to position an inflatable balloon at the blockage site of the artery, where the balloon is inflated to open up the artery. This procedure is called percutaneous transluminal coronary angioplasty or PTCA, which is usually accompanied by the placement of a metal tube (or stent) at the diseased site to maintain vessel opening. PTCA is the dominant procedure to restore blood flow in coronary arteries- in the United States alone nearly 1.3 million PTCA procedures were performed in 2004. However, as a response to PTCA-related vessel wall damage, cells from the vessel wall are activated to divide and grow into the vessel lumen, causing re-narrowing or restenosis of the artery. Restenosis of the vessel lumen is the major hurdle limiting the success of PTCA. It occurs in 20-50% of cases within six months of the initial PTCA procedure and requires repeated PTCA to open up the re-narrowed artery, leading to tremendous human and social expenses. Stents which contain drug inhibitors of cell growth (drug eluting stents, or DES) reduce restenosis; however, considerable concerns have emerged regarding the safety of DES due to an increased risk of sudden stent occlusion by platelet aggregates (or thrombosis). This sudden occlusion is caused by a concomitant drug inhibition of cells that cover the raw surface of metal stents to prevent platelet aggregation. This complication is frequently lethal, resulting in death or heart attack in 85% of cases. The safety concerns over DES have created an urgent need to define the mechanisms underlying the biology of restenosis. A population of cells resident in the vessel wall consists of progenitor cells that divide and grow into the vessel lumen when vessels are injured. The repair process mediated by these cells directly contributes to vessel restenosis. Our goal is to understand the biology of these stem cells in the repair of injured arteries- how vessel injury signals these cells to divide and invade the vessel lumen, what molecular effectors control the cellular responses, and how to intercept these signals and effectors to prevent vessel restenosis. This will provide a solid scientific basis for new therapeutic targets and strategies for vessel restenosis after PTCA. The proposal is a targeted response to CIRM New Faculty Awards II. It seeks to extend my research expertise into the field of stem cell biology related to clinically important vascular diseases. We are confident that our proposed studies will generate significant progress in this field, in both scientific knowledge and useful therapies.
Statement of Benefit to California: 
Coronary heart disease is the leading cause of death in California. This disease results from atherosclerosis or fatty deposits in the vessel wall that causes blockage of coronary arteries of the heart, causing heart attacks, heart failure or sudden death. Physicians use wires and balloons to open up the blocked artery (angioplasty) and a metal tube (stent) to keep the artery open and restore blood flow. Although effective, angioplasty and stenting cause some damages to the blood vessel, which leads to a recurrent blockage (or restenosis) of the vessel in 20-50% of patients within 6 months of the procedure. This vessel restenosis requires repeated angioplasties and stenting for restoration of blood flow. Given the large number of patients with coronary heart disease in California, the need for repeated surgical procedures has resulted in tremendous human, social and economic costs in our state. An attempt to reduce vessel restenosis is the placement of drug-eluting stents (or DES) in angioplastied vessels. Although drugs released from the stents reduce vessel restenosis, this approach creates a new and frequently fatal complication- sudden occlusion of the stented arteries. This complication is because drugs in the stents delay the repair of inner lining of the artery, whose function is to prevent platelet aggregation within the lumen of the artery. Sudden platelet aggregation (or thrombosis) within the vessel lumen causes instantaneous obstruction of the artery, leading to acute heart attacks or death. Thus, the safety concerns over DES have created an urgent need to define the mechanisms underlying the biology of restenosis. A population of cells present at the vessel wall possess stem cell characteristics. After vessel injury, these cells increase in number and turn into different kinds of cells, which then migrate from the vessel wall into the lumen, causing blockage of the vessel. Thus, understanding how these cells behave will inspire new ideas for treating recurrent vessel blockage or restenosis. We propose to study how and what molecular signals activate these cells when vessels are injured. Our goal is to provide a scientific strategy of intercepting these signals for the treatment of vessel restenosis. We believe that understanding the biology of vascular stem cells will lead to significant advances in the research and novel therapies of vessel injury and restenosis. Given the scope of this problem , an improved therapy of vessel restenosis will have a significant economic and social impact. We have proposed to use modern methods in genetics, cell biology, and molecular biology to attack the challenges of this project. At the same time, we will train a new generation of bright students and junior scientists in the areas of stem cell biology highly relevant to human disease. This ensures that an essential knowledge base will be preserved, passed on and expanded in California for the foreseeable future.
Progress Report: 
  • Coronary heart disease is the leading cause of death in the developed world. This disease results from atherosclerosis or fatty deposits in the vessel wall that causes blockage of coronary arteries. Blockage of these arteries cut off supplies of nutrients and oxygen to the heart muscle, causing heart attacks, heart failure or sudden death. To restore coronary blood supply, physicians use guide-wires to position an inflatable balloon at the blockage site of the artery, where the balloon is inflated to open up the artery. This procedure is called percutaneous transluminal coronary angioplasty or PTCA, which is usually accompanied by the placement of a metal tube (or stent) at the diseased site to maintain vessel opening. However, as a response to PTCA, cells from the vessel wall are mobilized to divide and grow into the vessel lumen, causing re-narrowing of the artery. Renarrowing of the vessel lumen is the major hurdle limiting the success of PTCA. Mental stents which contain drug inhibitors of cell growth (drug eluting stents, or DES) reduce re-narrowing; however, considerable concerns have emerged regarding the safety of DES due to an increased risk of sudden stent occlusion by platelet aggregates (or thrombosis). This sudden occlusion is caused by a concomitant drug inhibition of cells that cover the raw surface of metal stents to prevent platelet aggregation. This complication is frequently lethal, resulting in death or heart attack in 85% of cases. The safety concerns over DES have created an urgent need to define the mechanisms underlying the biology of vascular re-narrowing.
  • A population of cells resident in the vessel wall consists of stem cells that divide and grow into the vessel lumen when vessels are injured. The repair process mediated by these cells directly contributes to vessel re-narrowing. Our goal is to understand the biology of these stem cells in the repair of injured arteries- how vessel injury signals these cells to divide and invade the vessel lumen, what molecular effectors control the cellular responses, and how to intercept these signals and effectors to prevent vessel re-narrowing. This will provide a solid scientific basis for new therapeutic targets and strategies for vessel re-narrowing after PTCA.
  • In the past year, we have successfully developed in the laboratory a more efficient method of isolating the vessel wall stem cells (or adventitial stem cells) and growing these cells in test tubes. The ability to isolate and grow these stem cells has allowed us to study the effects of many biologically active molecules on these cells critical for vascular repair and re-narrowing. We are now using this method to study molecular pathways that can modify the biological behavior of the vessel wall stem cells. Furthermore, we have developed a different method of injuring the blood vessels to study how the vessel wall stem cells respond to different types of vessel injury. This method allows us to track the mobilization of vessel wall stem cells more precisely in the vascular repair process. We are using this method to study the activity of vessel wall stem cells following injury.
  • Coronary heart disease is the leading cause of death in the developed world. This disease results from atherosclerosis or fatty deposits in the vessel wall that causes blockage of coronary arteries, causing shortage of blood supply with consequent heart attacks, sudden death, or heart failure. To restore coronary blood supply, physicians use guide-wires to position an inflatable balloon at the blockage site of the artery, where the balloon is inflated to open the artery. This angioplasty procedure is usually accompanied by the placement of a metal stent at the diseased site to maintain vessel opening. Such percutaneous coronary intervention (PCI) with angioplasty and stenting is the dominant procedure for opening obstructed coronary arteries. However, PCI activates a population of cells in the vessel wall to grow into the vessel lumen, causing re-narrowing of the artery. This vessel re-narrowing (restenosis) is the major hurdle limiting the success of PCI. Mental stents coated with drug inhibitors of cell growth (drug eluting stents, or DES) reduce re-narrowing; however, considerable concerns have emerged regarding the safety of DES due to an increased risk of sudden stent occlusion by platelet aggregates (or thrombosis) and the need for prolonged anti-platelet therapy, which poses bleeding risks especially to older patients or patients who need surgery. These concerns call for defining mechanisms that control re-narrowing of injured arteries.
  • A population of cells resident in the vessel wall consists of stem cells that are activated when vessels are injured. Activation of these cells directly contributes to vessel re-narrowing. Our goal is to understand how these cells are activated by vessel injury, how injury signals these cells to divide and invade the vessel lumen, what molecular effectors control the cellular responses, and how to intercept these signals and effectors to prevent vessel re-narrowing. In the past year, we successfully developed new methods for isolating and growing these vascular stem cells in test tubes. These new methods allowed us to determine how these stem cells turn into other types of vessel cells after injury and how they contribute to re-narrowing of injured vessels. We are using this method to define molecular pathways that control vessel wall stem cells to respond to vessel injury.
  • Coronary heart disease is a leading cause of morbidity and mortality. This disease results from blockage of coronary arteries that supply blood to the heart muscle. To restore blood supply, physicians use angioplasty to open the obstructed artery and apply stenting to maintain the arterial patency. Approximately 1.3 million angioplasty and stenting procedures are performed every year in the US to relieve coronary obstruction. However, these procedures activate a population of vascular cells to grow into the arterial lumen, causing re-narrowing of the artery. This re-narrowing (restenosis) is the major hurdle limiting the success of angioplasty and stenting. Mental stents coated with drug inhibitors of cell growth (drug eluting stents, or DES) reduce re-narrowing; however, considerable concerns have emerged regarding the safety of DES due to an increased risk of sudden stent occlusion by platelet aggregates (or thrombosis) and the need for prolonged anti-platelet therapy, which poses bleeding risks. These concerns call for defining mechanisms that control re-narrowing of injured arteries.
  • A population of stem cells resides in the arterial wall. These cells are activated when arteries are injured by mechanical stress such as angioplasty and stenting. Activation of these cells directly contributes to arterial re-narrowing. Our goal is to understand how these stem cells are activated by vessel injury, how injury signals these cells to divide and invade the vessel lumen, what molecular effectors control the cellular responses, and how to intercept these signals and effectors to prevent vessel re-narrowing. We developed new methods for isolating and growing these vascular stem cells in test tubes. In the past year, we successfully used these methods to determine how arterial injury or mechanical stress signals the stem cells to produce different types of cells which grow into the arterial lumen, causing narrowing of the artery. We are using these methods and also developing new methods to define molecular pathways that control the reaction of stem cells to arterial injury. This will help identify drug targets for therapeutic intervention.
  • Coronary heart disease, the major cause of morbidity and mortality in our society, results from blockage of the coronary arteries that supply blood to the heart muscle. Blockage of the coronary arteries causes heart attack. Angioplasty and stenting are used to open the obstructed coronary artery and maintain the arterial patency. ~1.3 million angioplasty and stenting procedures are performed in the US every year to treat coronary artery disease. However, these procedures activate a population of vascular cells to grow into the arterial lumen, causing re-narrowing of the artery. This re-narrowing (restenosis) is the major hurdle limiting the success of angioplasty and stenting. Mental stents coated with drug inhibitors of cell growth (drug eluting stents, or DES) reduce re-narrowing; however, considerable concerns have emerged regarding the safety of DES due to an increased risk of sudden stent occlusion by platelet aggregates (or thrombosis) and the need for prolonged anti-platelet therapy, which poses bleeding risks. Defining the mechanisms that control re-narrowing of injured arteries is therefore important for treating coronary artery disease.
  • The arterial wall contains a population of stem cells. These stem cells are activated when arteries are injured by mechanical stress such as angioplasty and stenting. Activation of these cells directly contributes to arterial re-narrowing. Our goal is to understand how these stem cells are activated by vessel injury, how injury signals these cells to divide and invade the vessel lumen, what molecular effectors control the cellular responses, and how to intercept these signals and effectors to prevent vessel re-narrowing. We developed new methods for isolating and growing these vascular stem cells in test tubes, and we have successfully used these methods to determine how arterial injury or mechanical stress signals the stem cells to produce different types of cells which grow into the arterial lumen, causing narrowing of the artery. In the past year, we developed new genetic tools to further understand the mechanism of vascular injury and repair. We are using the new genetic tool to define molecular and cellular pathways that control the reaction of stem cells to arterial injury.
  • Blockage of coronary arteries that supply blood to the heart muscle is the major cause of morbidity and mortality in our society. Angioplasty and stenting are used to open the obstructed coronary artery and maintain the arterial patency. In US, ~1.3 million angioplasty and stenting procedures are performed every year to treat coronary artery disease. Although effective in restoring the blood flow, these procedures activate a population of vascular cells resident in the arterial wall to grow into the vesslel lumen, causing re-narrowing (restenosis) of the treated artery months or years later. This arterial re-narrowing is a major hurdle limiting the success of angioplasty and stenting. Mental stents coated with drug inhibitors of cell growth (drug eluting stents, or DES) reduce re-narrowing; however, the safety of DES has raised considerable concerns due to an increased risk of sudden stent occlusion by platelet aggregates (or thrombosis) as well as the need for prolonged anti-platelet therapy, which poses bleeding risks, especially in the elderly population. It is therefore important to define the underlying mechanisms of re-narrowing of injured arteries in order to design new therapies for coronary artery disease.
  • A population of stem cells resides in the arterial wall. These stem cells are activated when arteries are injured by angioplasty and stenting. Once activated, these cells grow and differentiate into cells that invade the vascular luman and contribute to arterial re-narrowing. We developed new genetic tools to further understand the mechanism of vascular injury and repair. We are using the new genetic tool to define molecular and cellular pathways that control the reaction of stem cells to arterial injury. The goal is to understand how these stem cells are activated by vessel injury, how injury signals these cells to divide and invade the vessel lumen, what molecular effectors control the cellular responses, and how to intercept these signals and effectors to prevent vessel re-narrowing.
Funding Type: 
New Faculty II
Grant Number: 
RN2-00903
Investigator: 
Type: 
PI
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.
Funding Type: 
New Cell Lines
Grant Number: 
RL1-00662
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$1 424 412
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Closed
Public Abstract: 
The field of regenerative medicine revolves around the capacity of a subset of cells, called stem cells, to become the mature tissues of the adult human body. By studying stem cells, we hope to develop methods and reagents for treating disease. For instance, we hope to develop methods for making stem cells become cardiovascular cells in the lab which could then be used to rapidly screen large numbers drugs that may be used to treat cardiovascular disease. In another example, if we are able to create bone in the lab from stem cells, we may be able to help treat people with catastrophic skeletal injuries such as wounded soldiers. Until recently, the most flexible type of stem cell known was the embryonic stem cell. Embryonic stem cells are pluripotent, meaning they can give rise to all of the adult tissues. In contrast, stem cells found in the adult are considered only multipotent, in that they can only become a limited number of mature cells. For example, bone marrow stem cells can give rise to all of the components of the blood, but cannot make nerves for a spinal chord. Breakthroughs in the past couple of months have indicated that it is possible to "reprogram" adult skin cells and make them become pluripotent, like stem cells from an embryo. These new kind of cells ares called "induced pluripotent cells" or iPS cells for short. This has lead to great excitement within the scientific community because it raises the possibility that we may use this technology to rapidly create pluripotent stem cells from a large host of human diseases using skin from affected individuals. However, whether the new iPS cells made from skin cells and embryonic stem cells are functionally the same in all applications remains to be seen. Our lab is in the unique position to test this hypothesis. We have derived several normal embryonic stem cell lines and are in the process of deriving iPS cells from normal skin. Furthermore, we are fortunate enough to have begun deriving a new embryonic stem cell line harboring an inherited mutation that results in severe cardiovascular and bone disease that affects more than 7,500 Californians. What's more, one of our collaborators has over the past ten years assembled a cell bank of more that 50 unique adult skin cell lines with the same inherited disease. Therefore, for our proposal, we will make new normal and disease specific iPS and embryonic stem cell lines. We will use these new stem cell lines to test whether the iPS and embryonic stem cells are truly functionally the same, by comparing them after we make them become cardiovascular and bone cells. This work will allow us to advance the field of regenerative medicine on two fronts. 1. We will perform an important comparison of iPS and embryonic stem cell lines. 2. We will compare the disease specific cells with normal cells which will help us better understand cardiovascular and bone disease and pave the way for the development of new therapies.
Statement of Benefit to California: 
Our proposal compares normal and disease specific pluripotent stem cells derived from embryonic and adult skin sources. This proposal will benefit the state of California and its citizens in several specific ways. First, the specific inherited disease we are studying affects approximately one in every 5,000 people worldwide. That translates into over 7,500 Californians and over 60,000 men, women and children of every race and ethnic group in the United States. By examining the characteristics of the disease specific lines, we hope to better understand the mechanisms of the disease and create assays for screening new drugs that can be used to treat people with the disease. Second, this disease is one of a broad class of cardiovascular disease, called thoracic aortic disease. An estimated 3,700 Californians are treated for thoracic aortic disease every year. Our findings may provide insight into the mechanisms underlying these diseases and other cardiovascular diseases. Third, this disease also results in skeletal defects. By studying the mechanisms of the skeletal defects, we will better understand the mechanisms of bone development, which will lead to improved applications of stem cell therapies for individuals with bone injury and disease. Finally, by providing detailed comparisons of iPS and embryonic stem cells, our work will have important ramifications for the future direction of the entire field of stem cell research and regenerative medicine.
Progress Report: 
  • During the past year, we have used the funds from this grant to derive a new embryonic stem cell line with an inherited mutation that results in a severe cardiovascular and bone disease called Marfan syndrome that affects more than 7,500 Californians. In addition, using adult skin cell lines with the same inherited disease, we have made significant progress deriving iPS cells with Marfan syndrome. During the next year we also hope to expand our studies by recruiting patients with a disease very similar to Marfan syndrome called Loeys-Dietz syndrome, to donate skin biopsies so that we can make iPS cells to study that disease as well. Using these new stem cell lines, we are testing whether the iPS and embryonic stem cells are truly functionally the same, by comparing them after we make them become cardiovascular and bone cells.
  • One of the biggest challenges in stem cell biology is figuring out how to make the stem cells become the adult cells we want to study and not some other random adult cells. Over the past year, we have made great strides in turning our stem cells into the cell types most severely affected in people with Marfan syndrome, namely bone and cardiovascular cells. What is most exciting to us is that even with these preliminary studies, it looks like we might be seeing differences between the stem cells with Marfan syndrome and normal stem cells after they are coaxed into become the bone and cardiovascular cells. These results are still very preliminary though, and we need to take great care during the next year to rigorously repeat our experiments before we can be certain of those results. If we can reproduce the differences, these differences may be the basis for screening for new drugs to treat people with Marfan syndrome or lead to a better understanding as to what exactly is the sequence of cellular events that leads to the patient’s symptoms. What’s more, by studying how to efficiently make bone and cardiovascular cells from human embryonic stem cells and iPS cells in the dish, we hope to provide important data that could be beneficial in a wide variety of applications such as tissue engineering or cellular replacement therapies using bone or blood vessels.
  • Marfan Syndrome (MFS) is a genetic disorder that affects more than 7,500 Californians. Patients develop severe complications, affecting several parts of the body (eyes, limbs, aorta). During the last two years, we have used the funds from this grant to develop new cell lines aimed at studying MFS in a dish. These cell lines, are called pluripotent stem cells, and have been generated from: (i) an embryo that was donated for research and was known to have inherited the MFS disease (these cell lines are named human embryonic stem cells (hESCs)); and (ii) from skin biopsies of adult patients (these cell lines are named induced pluripotent stem cells (iPSCs)). These stem cell lines allow us to study MFS by differentiating the cells to adult cells (mainly bone and cardiovascular cells) and not other random adult cells. Using these new stem cell lines, we can test whether hESCs and iPSCs are functionally the same, by comparing them after we make them become cardiovascular and bone cells. We have observed that when the cells form bone or muscle cells, the stem cells with MFS are different and do not behave the same as those made with normal stem cells. We also started to use reagents that can force MFS cells to resemble and behave like normal bone cells. This is called “rescuing the disease phenotype”. For the first time, we are close to describing a stem cell-based technology not only to understand the mechanism(s) of the MFS but also to develop a screen for new drugs to treat people with MFS. However, we still need to confirm our results by repeating the experiments. Our results are very promising for understanding the bone issues in MFS, but continued efforts are also required to understand the cardiovascular issue. It is important to point out that the most important health risk associated with the disease is an aortic aneurysm that, if untreated, leads to death around 35 years old. In conclusion, we are continuing to generate data that will provide the foundation for improving our knowledge of the disease, and also will potentially assist us in developing new therapies for improving MFS patient lives.
  • The field of regenerative medicine revolves around the capacity of a subset of cells, called stem cells, to become the mature tissues of the adult human body. By studying stem cells, we hope to develop methods for treating a wide variety of diseases. For instance, we hope to develop methods for making stem cells become cardiovascular cells in the lab, which could then be used to rapidly screen large numbers of drugs that may be used to treat cardiovascular disease. We are also trying to create skeletal tissue from stem cells so that we may be able to help treat people with catastrophic skeletal injuries such as wounded soldiers.
  • Until recently, the most flexible type of stem cell known was the embryonic stem cell. Embryonic stem cells are pluripotent, meaning they can give rise to all cell types in the body. In contrast, stem cells found in the adult are considered only multipotent, in that they can only become a limited number of mature cells. Breakthroughs in the past five years have indicated that it is possible to "reprogram" adult skin cells and make them become pluripotent, like stem cells from an embryo. These new kinds of cells are called "induced pluripotent cells" or iPS cells. This has lead to great excitement within the scientific community because it raises the possibility that we may use this technology to rapidly create pluripotent stem cells from a large host of human diseases using easy to obtain tissue like skin and fat from affected individuals.
  • Our laboratory is in the unique position to test this hypothesis. We have derived several normal embryonic stem cell lines and iPS cells from normal skin. Furthermore, we have derived a new embryonic stem cell line and induced pluripotent stem cells from fibroblasts harboring an inherited mutation that results in severe cardiovascular and bone disease that affects more than 7,500 Californians, called Marfan's Syndrome.
  • We have created stem cells lines, both embryonic and induced pluripotent stem cells from cells having this disease. We have compared these cells to normal embryonic and induced pluripotent stem cells to examine exactly what makes these diseased cells behave in a way to have impaired bone formation. In addition, we have completed the differentiation, banking and full characterization of vascular cells derived from Marfan's Syndrome embryonic stem cells and Marfan’s syndrome induced pluripotent stem cells. We have seen that the cells with Marfan’s syndrome have a particular signaling pathway that has functional disregulation compared to normal, healthy cells. We have been able to explore how this disease process manipulates this pathway to cause this specific disease. Through this kind of modeling, we can use these cells to screen for treatment as well as model the disease in a way to manipulate the specific pathways this disease impacts to hopefully bring clinical treatments to patients who suffer from this disease.
Funding Type: 
New Cell Lines
Grant Number: 
RL1-00639
Investigator: 
Type: 
PI
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.
Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00354
Investigator: 
Institution: 
Type: 
PI
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.

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