Heart Disease

Coding Dimension ID: 
295
Coding Dimension path name: 
Heart Disease
Funding Type: 
Basic Biology III
Grant Number: 
RB3-05086
Investigator: 
ICOC Funds Committed: 
$1 181 306
Disease Focus: 
Heart Disease
Collaborative Funder: 
Germany
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 

Despite therapeutic advances, cardiovascular disease remains a leading cause of mortality and morbidity in both California and Europe. New insights into disease pathology, models to expedite in vitro testing and regenerative therapies would have an enormous societal and financial impact. Although very promising, practical application of pluripotent stem cells or their derivatives face a number of challenges and technological hurdles. For instance, recent reports have demonstrated that cardiac progenitor cells (CPCs), which are capable of differentiating into all three cardiovascular cell types, are present during normal fetal development and can be isolated from pluripotent stem cells. induced pluripotent stem cell (iPSC)-derived CPC therapy after a myocardial infarction would balance the need for an autologous, multipotent stem cell myocardial regeneration with the concerns of tumorigenicity using a more primitive stem cell. However, translating this discovery into a clinically useful therapy will require additional advances in our understanding of CPC biology and the factors that regulate their fate to develop optimized cell culture technology for CPC application in regenerative medicine.

Cardiac cell therapy with hiPSC-derived cells, will require reproducible production of large numbers of well-characterized cells under defined conditions in vitro. This is particularly true for the rare and difficult to culture intermediates, such as CPCs. Our preliminary data demonstrated that a CPC niche exists during cardiac development and that CPC expansion is regulated by factors found within the niche microenvironment including specific soluble factors and ECM signals. However, our current understanding of the cardiac niche and how this unique microenvironment influences CPC fate is quite limited. We believe that if large scale production of hiPSC-derived CPCs is ever to be successful, new 3D cell culture technologies to replicate the endogenous cardiac niche will be required. The goals of this proposal are to address current deficiencies in our understanding of the cardiac niche and its effects on CPC expansion and differentiation as well as utilize novel bioengineering approaches to fabricate synthetic niche environments in vitro. The development of advanced fully automated in vitro culture systems that reproduce key features of natural niche microenvironments and control proliferation and/or differentiation, are critically needed both for studying the role of the niche in CPC biology but also the advancement of the field of regenerative medicine.

Statement of Benefit to California: 

Heart disease, stroke and other cardiovascular diseases are the #1 killer in California. Despite medical advances, heart disease remains a leading cause of disability and death. Recent estimates of its cost to the U.S. healthcare system amounts to almost $300 billion dollars. Although current therapies slow the progression of heart disease, there are few, if any options, to reverse or repair damage. Thus, regenerative therapies that restore normal heart function would have an enormous societal and financial impact not only on Californians, but the U.S. more generally. The research that is proposed in this application could lead to new therapies that would restore heart function after and heart attack and prevent the development of heart failure and death. We will develop the techniques to expand and transplant human cardiac progenitor cells. Combining tissue engineering with human pluripotent stem cells will facilitate the creation of new cardiovascular therapies.

Progress Report: 
  • Cardiovascular disease is the leading cause of morbidity and mortality in the United States. As humans lack the ability to regenerate myocardial tissue lost afte a heart attcak, there has been great focus on cardiovascualr regenerative therapies with the use of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). There has been increased attention towards developing tissue engineering as a method to standardize methods to differentiate human ESCs and iPSCs into cardiovascular progenitor cells (CPC) expand these progenitor cells in a standardized manor. We have focused on developing techniques to allow expansion of these CPCs into clinically relevany numbers by determining: 1. Conditions to optimize their derivation into clinically numbers using clinical grade techniques.
  • 2. Defininy and optimizing the extracellular matrxi to be used to maintain these CPCs in an undifferentiated state to allow their expansion to clinically required numbers. We studied the endogenous environment that these CPCs exist in fetal development and focused on the extracellular matrix proteins that help support these CPCs during development. By studying the array of proteins endogenously in developing heart we now will shift our focus on re-engineering this environment in-vitro to be able to mimic this growth to use this as a mean to grow and expand these progenitors for use clinically in the future. Currently we are deriving these CPCs from human ESC and iPSC and expanding them on different combinations of proteins as determined in the staining of the endogenous fetal environment. We hope to by the end of this porject determine the ideal conditions for derivation of these CPCs from iPSCs and the environmental cues needed for culturing these cells to allow for maximal yield for potential use in clinical regenerative therapies in the future.
  • Cardiovascular disease is the leading cause of morbidity and mortality in the United States. As humans lack the ability to regenerate myocardial tissue lost afte a heart attcak, there has been great focus on cardiovascualr regenerative therapies with the use of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). There has been increased attention towards developing tissue engineering as a method to standardize methods to differentiate human ESCs and iPSCs into cardiovascular progenitor cells (CPC) expand these progenitor cells in a standardized manor. We have focused on developing techniques to allow expansion of these CPCs into clinically relevany numbers by determining: 1. Conditions to optimize their derivation into clinically numbers using clinical grade techniques.
  • 2. Defininy and optimizing the extracellular matrxi to be used to maintain these CPCs in an undifferentiated state to allow their expansion to clinically required numbers. We studied the endogenous environment that these CPCs exist in fetal development and focused on the extracellular matrix proteins that help support these CPCs during development. By studying the array of proteins endogenously in developing heart we now will shift our focus on re-engineering this environment in-vitro to be able to mimic this growth to use this as a mean to grow and expand these progenitors for use clinically in the future. Currently we are deriving these CPCs from human ESC and iPSC and expanding them on different combinations of proteins as determined in the staining of the endogenous fetal environment. We hope to by the end of this porject determine the ideal conditions for derivation of these CPCs from iPSCs and the environmental cues needed for culturing these cells to allow for maximal yield for potential use in clinical regenerative therapies in the future.
Funding Type: 
Basic Biology III
Grant Number: 
RB3-05174
Investigator: 
Institution: 
Type: 
PI
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: 
Early Translational I
Grant Number: 
TR1-01249
Investigator: 
Name: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$6 762 954
Disease Focus: 
Bone or Cartilage Disease
Stroke
Neurological Disorders
Heart Disease
Neurological Disorders
Skin Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 

All adult tissues contain stem cells. Some tissues, like bone marrow and skin, harbor more adult stem cells; other tissues, like muscle, have fewer. When a tissue or organ is injured these stem cells possess a remarkable ability to divide and multiply. In the end, the ability of a tissue to repair itself seems to depend on how many stem cells reside in a particular tissue, and the state of those stem cells. For example, stress, disease, and aging all diminish the capacity of adult stem cells to self-renew and to proliferate, which in turn hinders tissue regeneration.

Our strategy is to commandeer the molecular machinery responsible for adult stem cell self-renewal and proliferation and by doing so, stimulate the endogenous program of tissue regeneration. This approach takes advantage of the solution that Nature itself developed for repairing damaged or diseased tissues, and controls adult stem cell proliferation in a localized, highly controlled fashion. This strategy circumvents the immunological, medical, and ethical hurdles that exist when exogenous stem cells are introduced into a human. When utilizing this strategy the goal of reaching clinical trials in human patients within 5 years becomes realistic.

Specifically, we will target the growing problem of neurologic, musculoskeletal, cardiovascular, and wound healing diseases by local delivery of a protein that promotes the body’s inherent ability to repair and regenerate tissues. We have evidence that this class of proteins, when delivered locally to an injury site, is able to stimulate adult tissue stem cells to grow and repair/replace the deficient tissue following injury. We have developed technologies to package the protein in a specialized manner that preserves its biological activity but simultaneously restricts its diffusion to unintended regions of the body. For example, when we treat a skeletal injury with this packaged protein we augment the natural ability to heal bone by 350%; and when this protein is delivered to the heart immediately after an infarction cardiac output is improved and complications related to scarring are reduced. This remarkable capacity to augment tissue healing is not limited to bones and the heart: the same powerful effect can be elicited in the brain, and skin injuries.

The disease targets of stroke, bone fractures, heart attacks, and skin wounds and ulcers represent an enormous health care burden now, but this burden is expected to skyrocket because our population is quickly aging. Thus, our proposal addresses a present and ongoing challenge to healthcare for the majority of Californians, with a novel therapeutic strategy that mimics the body’s inherent repair mechanisms.

Statement of Benefit to California: 

Californians represent 1 in 7 Americans, and make up the single largest healthcare market in the United States. The diseases and injuries that affect Californians affect the rest of the US, and the world. For example, stroke is the third leading cause of death, with more than 700,000 people affected every year. It is a leading cause of serious long-term disability, with an estimated 5.4 million stroke survivors currently alive today. Symptoms of musculoskeletal disease are the number two most cited reasons for visit to a physician. Musculoskeletal disease is the leading cause of work-related and physical disability in the United States, with arthritis being the leading chronic condition reported by the elderly. In adults over the age of 70, 40% suffer from osteoarthritis of the knee and of these nearly 80% have limitation of movement. By 2030, nearly 67 million US adults will be diagnosed with arthritis. Cardiovascular disease is the leading cause of death, and is a major cause of disability worldwide. The annual socioeconomic burden posed by cardiovascular disease is estimated to exceed $400 billion annually and remains a major cause of health disparities and rising health care costs. Skin wounds from burns, trauma, or surgery, and chronic wounds associated with diabetes or pressure ulcer, exact a staggering toll on our healthcare system: Burns alone affect 1.25M Americans each year, and the economic global burden of these injuries approaches $50B/yr. In California alone, the annual healthcare expenditures for stroke, skeletal repair, heart attacks, and skin wound healing are staggering and exceed 700,000 cases, 3.5M hospital days, and $34B.

We have developed a novel, protein-based therapeutic platform to accelerate and enhance tissue regeneration through activation of adult stem cells. This technology takes advantage of a powerful stem cell factor that is essential for the development and repair of most of the body’s tissues. We have generated the first stable, biologically active recombinant Wnt pathway agonist, and showed that this protein has the ability to activate adult stem cells after tissue injury. Thus, our developmental candidate leverages the body’s natural response to injury. We have generated exciting preclinical results in a variety of animals models including stroke, skeletal repair, heart attack, and skin wounding. If successful, this early translational award would have enormous benefits for the citizens of California and beyond.

Progress Report: 
  • In the first year of CIRM funding our objectives were to optimize the activity of the Wnt protein for use in the body and then to test, in a variety of injury models, the effects of this lipid-packaged form of Wnt. We have made considerable progress on both of these fronts. For example, in Roel Nusse and Jill Helms’ groups, we have been able to generate large amounts of the mouse form of Wnt3a protein and package it into liposomal vesicles, which can then be used by all investigators in their studies of injury and repair. Also, Roel Nusse succeeded in generating human Wnt3a protein. This is a major accomplishment since our ultimate goal is to develop this regenerative medicine tool for use in humans. In Jill Helms’ lab we made steady progress in standardizing the activity of the liposomal Wnt3a formulation, and this is critically important for all subsequent studies that will compare the efficacy of this treatment across multiple injury repair scenarios.
  • Each group began testing the effects of liposomal Wnt3a treatment for their particular application. For example, in Theo Palmer’s group, the investigators tested how liposomal Wnt3a affected cells in the brain following a stroke. We previously found that Wnt3A promotes the growth of neural stem cells in a petri dish and we are now trying to determine if delivery of Wnt3A can enhance the activity of endogenous stem cells in the brain and improve the level of recovery following stroke. Research in the first year examined toxicity of a liposome formulation used to deliver Wnt3a and we found it to be well tolerated after injection into the brains of mice. We also find that liposomal Wnt3a can promote the production of new neurons following stroke. The ongoing research involves experiments to determine if these changes in stem cell activity are accompanied by improved neurological function. In Jill Helms’ group, the investigators tested how liposomal Wnt3a affected cells in a bone injury site. We made a significant discovery this year, by demonstrating that liposomal Wnt3a stimulates the proliferation of skeletal progenitor cells and accelerates their differentiation into osteoblasts (published in Science Translational Medicine 2010). We also started testing liposomal Wnt3a for safety and toxicity issues, both of which are important prerequisites for use of liposomal Wnt3a in humans. Following a heart attack (i.e., myocardial infarction) we found that endogenous Wnt signaling peaks between post-infarct day 5-7. We also found that small aggregates of cardiac cells called cardiospheres respond to Wnt in a dose-responsive manner. In skin wounds, we tested the effect of boosting Wnt signaling during skin wound healing. We found that the injection of Wnt liposomes into wounds enhanced the regeneration of hair follicles, which would otherwise not regenerate and make a scar instead. The speed and strength of wound closure are now being measured.
  • In aggregate, our work on this project continues to move forward with a number of great successes, and encouraging data to support our hypothesis that augmenting Wnt signaling following tissue injury will provide beneficial effects.
  • In the second year of CIRM funding our objectives were to optimize packaging of the developmental candidate, Wnt3a protein, and then to continue to test its efficacy to enhance tissue healing. We continue to make considerable progress on the stated objectives. In Roel Nusse’s laboratory, human Wnt3a protein is now being produced using an FDA-approved cell line, and Jill Helms’ lab the protein is effectively packaged into lipid particles that delay degradation of the protein when it is introduced into the body.
  • Each group has continued to test the effects of liposomal Wnt3a treatment for their particular application. In Theo Palmer’s group we have studied how liposomal Wnt3a affects neurogenesis following stroke. We now know that liposomal Wnt3a transiently stimulates neural progenitor cell proliferation. We don’t see any functional improvement after stroke, though, which is our primary objective.
  • In Jill Helms’ group we’ve now shown that liposomal Wnt3a enhances fracture healing and osseointegration of dental and orthopedic implants and now we demonstrate that liposomal Wnt3a also can improve the bone-forming capacity of bone marrow grafts, especially when they are taken from aged animals.
  • We’ve also tested the ability of liposomal Wnt3a to improve heart function after a heart attack (i.e., myocardial infarction). Small aggregates of cardiac progenitor cells called cardiospheres proliferate to Wnt3a in a dose-responsive manner, and we see an initial improvement in cardiac function after treatment of cells with liposomal Wnt3a. the long-term improvements, however, are not significant and this remains our ultimate goal. In skin wounds, we tested the effect of boosting Wnt signaling during wound healing. We found that the injection of liposomal Wnt3a into wounds enhanced the regeneration of hair follicles, which would otherwise not regenerate and make a scar instead. The speed of wound closure is also enhanced in regions of the skin where there are hair follicles.
  • In aggregate, our work continues to move forward with a number of critical successes, and encouraging data to support our hypothesis that augmenting Wnt signaling following tissue injury will provide beneficial effects.
  • Every adult tissue harbors stem cells. Some tissues, like bone marrow and skin, have more adult stem cells and other tissues, like muscle or brain, have fewer. When a tissue is injured, these stem cells divide and multiply but only to a limited extent. In the end, the ability of a tissue to repair itself seems to depend on how many stem cells reside in a particular tissue, and the state of those stem cells. For example, stress, disease, and aging all diminish the capacity of adult stem cells to respond to injury, which in turn hinders tissue healing. One of the great unmet challenges for regenerative medicine is to devise ways to increase the numbers of these “endogenous” stem cells, and revive their ability to self-renew and proliferate.
  • The scientific basis for our work rests upon our demonstration that a naturally occurring stem cell growth factor, Wnt3a, can be packaged and delivered in such a way that it is robustly stimulates stem cells within an injured tissue to divide and self-renew. This, in turn, leads to unprecedented tissue healing in a wide array of bone injuries especially in aged animals. As California’s population ages, the cost to treat such skeletal injuries in the elderly will skyrocket. Thus, our work addresses a present and ongoing challenge to healthcare for the majority of Californians and the world, and we do it by mimicking the body’s natural response to injury and repair.
  • To our knowledge, there is no existing technology that displays such effectiveness, or that holds such potential for the stem cell-based treatment of skeletal injuries, as does a L-Wnt3a strategy. Because this approach directly activates the body’s own stem cells, it avoids many of the pitfalls associated with the introduction of foreign stem cells or virally reprogrammed autologous stem cells into the human body. In summary, our data show that L-Wnt3a constitutes a viable therapeutic approach for the treatment of skeletal injuries, especially those in individuals with diminished healing potential.
  • This progress report covers the period between Sep 01 2012through Aug 31 2013, and summarizes the work accomplished under ET funding TR1-01249. Under this award we developed a Wnt protein-based platform for activating a patient’s own stem cells for the purpose of tissue regeneration.
  • At the beginning of our grant period we generated research grade human WNT3A protein in quantities sufficient for all our discovery experiments. We then tested the ability of this WNT protein therapeutic to improve the healing response in animal models of stroke, heart attack, skin wounding, and bone fracture. These experimental models recapitulated some of the most prevalent and debilitating human diseases that collectively, affect millions of Californians.
  • At the end of year 2, we assembled an external review panel to select the promising clinical indication. The scientific advisory board unanimously selected skeletal repair as the leading indication. The WNT protein is notoriously difficult to purify; consequently in year 3 we developed new methods to streamline the purification of WNT proteins, and the packaging of the WNT protein into liposomal vesicles that stabilized the protein for in vivo use.
  • In years 3 and 4 we continued to accrue strong scientific evidence in both large and small animal models that a WNT protein therapeutic accelerates bone regeneration in critical size bony non-unions, in fractures, and in cases of implant osseointegration. In this last year of funding, we clarified and characterized the mechanism of action of the WNT protein, by showing that it activates endogenous stem cells, which in turn leads to faster healing of a range of different skeletal defects.
  • In this last year we also identified a therapeutic dose range for the WNT protein, and developed a route and method of delivery that was simultaneously effective and yet limited the body’s exposure to this potent stem cell factor. We initiated preliminary safety studies to identify potential risks, and compared the effects of WNT treatment with other commercially available bone growth factors. In sum, we succeeded in moving our early translational candidate from exploratory studies to validation, and are now ready to enter into the IND-enabling phase of therapeutic candidate development.
  • This progress report covers the period between Sep 01 2013 through April 30 2014, and summarizes the work accomplished under ET funding TR101249. Under this award we developed a Wnt protein-based platform for activating a patient’s own stem cells for purposes of tissue regeneration.
  • At the beginning of our grant period we generated research grade human WNT3A protein in quantities sufficient for all our discovery experiments. We then tested the ability of this WNT protein therapeutic to improve the healing response in animal models of stroke, heart attack, skin wounding, and bone fracture. These experimental models recapitulated some of the most prevalent and debilitating human diseases that collectively, affect millions of Californians. At the conclusion of Year 2 an external review panel was assembled and charged with the selection of a single lead indication for further development. The scientific advisory board unanimously selected skeletal repair as the lead indication.
  • In year 3 we accrued addition scientific evidence, using both large and small animal models, demonstrating that a WNT protein therapeutic accelerated bone healing. Also, we developed new methods to streamline the purification of WNT proteins, and improved our method of packaging of the WNT protein into liposomal vesicles (e.g., L-WNT3A) for in vivo use.
  • In year 4 we clarified the mechanism of action of L-WNT3A, by demonstrating that it activates endogenous stem cells and therefore leads to accelerated bone healing. We also continued our development studies, by identifying a therapeutic dose range for L-WNT3A, as well as a route and method of delivery that is both effective and safe. We initiated preliminary safety studies to identify potential risks, and compared the effects of L-WNT3A with other, commercially available bone growth factors.
  • In year 5 we initiated two new preclinical studies aimed at demonstrating the disease-modifying activity of L-WNT3A in spinal fusion and osteonecrosis. These two new indications were chosen by a CIRM review panel because they represent an unmet need in California and the nation. We also initiated development of a scalable manufacturing and formulation process for both the WNT3A protein and L-WNT3A formulation. These two milestones were emphasized by the CIRM review panel to represent major challenges to commercialization of L-WNT3A; consequently, accomplishment of these milestones is a critical yardstick by which progress towards an IND filing can be assessed.
  • With regards to objective 1, we employed established animal models of spinal fusion and osteonecrosis to demonstrate the disease-mitigating activity of our Developmental Candidate, autograftWNT.
  • With regards to objective 2, the mechanism of L-WNT3A action has been demonstrated in ex vivo, non-GLP pharmacology studies.
  • With regards to objective 3, we have completed characterization of the non-GLP substance, WNT3A drug product, and the drug product, L-WNT3A. Methods for reproducible and scaleable research grade production of the drug substance WNT3A, and the drug product, L-WNT3A, have been developed (see below for details). A serum free process has been achieved.
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: 
Institution: 
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.

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