Heart Disease

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

Chemical Genetic Approach to Production of hESC-derived Cardiomyocytes

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00132
ICOC Funds Committed: 
$3 036 002
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Adult heart muscle cells retain negligible proliferative capacity and this underlies the inability of the heart to replace muscle cells that are lost to injury, such as infarct, and underlies progression to heart failure. To date, no stem cell therapiy has produced significant cardiomyocyte replacement. Instead, transplanted cells, if they persist at all, produce endothelial cells or fibroblasts and the ameliorating effects on heart function that have been reported have been achieved by improving contractility, perfusion or other processes that are impaired in the failing heart. This proposal is to develop specific reagents and ultimately drugs to stimulate regeneration. Our approach integrates advances in stem cell biology, high-throughput (HT) biology, informatics and proteomics to identify small molecules, proteins and signal transduction pathways that control heart muscle formation from human embryonic stem cells (hESCs). High throughput assays will be developed and implemented to identify genes, signaling proteins, and small molecules that that control important steps in the differentiation, proliferation, and maturation of cardiac cells. Computer modeling and informatics will be used to identify and validate the signaling pathways that direct these critical processes. The discovery of small molecules and pathways will lead to protocols for 1) efficient directed differentiation of cardiomyogenic precursors from hESCs for research into transplantation and toxicology, 2) biotech reagents to stimulate cardiomyocyte renewal through directed differentiation of hESCs, and 3) cellular targets or lead compounds to develop drugs that stimulate regeneration from endogenous cells.
Statement of Benefit to California: 
This proposal is a multidisciplinary collaboration among stem cell biologists, chemists, and engineers to address a critical problem that limits the widespread use of hESC for cardiology applications. Developing the multidisciplinary technology and overcoming the hurdles to application of hESCs to biotech and clinic will benefit California in many ways, including: Research to discover novel tools to stimulate heart muscle regeneration from hESCs is clinically important. Cardiovascular disease is the single largest cause of death in the U.S. and the assays we will develop and the reagents themselves will be useful tools to direct cardiomyocyte regeneration. This will speed the translation of hESCs to the clinic, specifically by stimulating production of cardiomyocytes and potentially by enhancing their integration and function after engraftment. Heart regeneration from hESCs probably uses similar cellular proteins and signaling pathways as regeneration of cardiomyocytes from other sources, thus, this research might be broadly applicable to heart muscle repair. Regeneration from endogenous cells remains controversial but these tools should be useful reagents to study and hopefully stimulate endogenous repair. Bringing the diverse people together (chemists, cell biologists, and engineers) to address a stem cell problem forges new links in the academic community that should be capable of opening new areas of research. These new areas of research will be a important legacy of the stem cell initiative and promises to invigorate academic research. The technology that we are developing applies the new discipline of chemical biology to stem cell biology, and the merger promises to spin off new areas of investigation and biotech products with the potential to benefit the practice of medicine and the local economy. Lastly, supporting the leading edge technology and the collaboration will build the California infrastructure of high throughput chemical library screening so that it can be focused on other areas of biomedical research, both stem cell and non-stem cell.
Progress Report: 
  • The goal of this project is to identify small molecules that stimulate cardiomyocyte differentiation from stem cells. The strategy is to use embryonic stem ESC)-derived progenitors to screen for compounds and then optimize their chemical properties to generate molecules that can be used as reagents and potentially as lead compounds to develop drugs to stimulate regeneration in patient hearts. During year 2, progress is reported in: 1) optimizing the biological and pharmaceutical properties of 4 chemically diverse compounds discovered in year 1; 2) patent application filed on these compounds; 3) identification of targets and biological mechanism of action of 2 of the 4 compounds; 4) 1 compound has been validated in hESCs; 5) pilot screening completed of a new stem cell screen to discover molecules that act on late stage progenitors similar to cells thought to exist in the adult heart; 5) new assays developed and screened for discovering modulators of the Wnt pathway that enhance cardiomyocyte production. Thus, there are a total of 8 chemically distinct compounds under study and additional assays have been developed that should bring additional compounds into the pipeline during year 3.
  • This progress report covers FY3 of the project to identify and characterize novel small molecule probes of cardiomyocyte differentiation from stem cells. During FY3, we characterized 11 novel chemical entities that promote cardiomyocyte differentiation. The small, drug-like molecules affect distinct steps in cardiomyocyte differentiation – 5 compounds promote formation of uncommitted cardiac progenitors, 2 stimulated committed cardiac precursors, while 2 compounds act later to stimulate differentiation into cardiomyocytes. Thus, these compounds are novel probes of stem cell differentiation. Some of the compounds are characterized to act upon particular cellular target proteins while the targets of other compounds are unknown. Of the latter class, candidate targets have been characterized by biochemical studies; one of which has been confirmed by RNA interference, yielding a new pathway in cardiac cell formation from stem cells. Three of the chemical series have been described in a patent application. Additional primary hits are being characterized.
  • For FY4, we will continue characterization of a novel compounds. Particular focus will be on 4 chemical entities that promote later stages of human stem cell cardiomyocyte differentiation and on characterizing and discovering additional candidates that act on late-stage differentiation. In addition, we will develop a new pathway screen for a cellular target involved in specifying cardiomyocyte progenitors that have recently been shown to form new myocytes in vivo. Our new compounds are valuable probes of the underlying mechanism(s) responsible for making cardiac cells from stem cells. Moreover, recent data has shown that endogenous stem cells that reside in the adult heart resemble progenitors in the hESC cultures, so certain of our compounds can be considered as targeting cellular proteins and signaling pathways that might be beneficial to stimulate endogenous regeneration. Towards this goal, we will optimize the drug-like properties of the compounds in anticipation of in vivo testing for regenerative potential.
  • This research led to the discovery of small molecules that promote the formation of heart muscle cells from human pluripotent stem cells. The project used high throughput screening technology and medicinal chemistry, similar to that used in pharmaceutical companies, to discover and optimize the molecules. The cellular processes targeted by the compounds were also investigated, and in several cases this research uncovered novel roles for key cellular proteins and signaling pathways, such as Wnt and TGFb signaling, in stem cell differentiation. The compounds will be useful as reagents for cardiomyocyte preparation from stem cells, and patent applications have been filed.

Embryonic Stem Cell-Derived Therapies Targeting Cardiac Ischemic Disease

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00124
ICOC Funds Committed: 
$2 524 617
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Cardiovascular disease (CVD) is the leading cause of death in the United States. Over one million Americans will suffer from a new or recurrent heart attacks this year and over 40 percent of those will die suddenly. In addition, about two-thirds of the patients develop congestive heart failure; and in people diagnosed with CHF, sudden cardiac death occurs at 6-9 times the general population rate. Heart transplantation remains the only viable solution for severely injured hearts; however, this treatment is limited by the availability of donor hearts. Therefore, alternative strategies to treat end stage heart failure and blocked blood vessels are needed. The objective of this proposal is to determine whether human embryonic stem (hES) cell can be used for repairing the heart. Our collaborator Advanced Cell Technology (ACT) has recently succeeded in identifying conditions for the reproducible isolation of hES cells which have the characteristics of cells which form blood vessels and heart muscle. This proposal will assess whether the hES cells can form new functional blood vessels and repair injured heart muscle in a rat model of heart attacks. Results from these studies will help develop new therapies for treating patients with heart attacks.
Statement of Benefit to California: 
Cardiovascular disease (CVD) is the leading cause of death in California and the United States. Over one million Americans will suffer from a new or recurrent myocardial infarction this year and over 40 percent of those will die suddenly. In addition, about two-thirds of myocardial infarction patients develop congestive heart failure. The 5-year mortality rate for CHF is about 50%, and in people diagnosed with CHF, sudden cardiac death occurs at 6-9 times the general population rate. Heart transplantation remains the only viable solution for severely injured hearts; however, this treatment is limited by the availability of donor hearts. It is estimated that health care costs for CVD is over 18 billion dollars a year. Additionally, the morbidity associated with CVD cost California and the nation billions of dollars a year. Therefore, alternative strategies to treat end stage heart failure and ischemia are needed. (Source: American Heart Association. Heart Disease and Stroke Facts, 2004, Dallas, TX: AHA 2004; American Heart Association. Heart Disease and Stroke Statistics-2006 Update, Dallas, TX: AHA 2006). The field of regenerative medicine is important to California and the nation. Advances in the technology to find cell based therapies will be revolutionary in their impact on patient care. Human embryonic stem (hES) cells have the potential to become all of the cells in the human body, and their unique properties give researchers the hope that from these primitive cells new therapies can result that may be available in time for the looming health care crisis. This project is focused on a pre-clinical application of a specific hES cell based therapy for myocardial regeneration and an antibody targeting technology to direct stem cells to injured organs. This project will benefit California in several ways including: 1) support for UC trainees, 2) potential of developing important clinical trials in CA based on results from this proposal, and 3) enhancement of the biotechnology industry in CA which would lead to the creation of new jobs in CA and an enhanced tax base.
Progress Report: 
  • Myocardial infarction can lead to death and disability with a 5-year death rate for congestive heart failure of 50%. It is estimated that cardiovascular disease is the leading cause of mortality and morbidity and is predicted to be the leading cause of death worldwide by 2020. Currently, heart transplantation is the only successful treatment for end-stage heart failure; however, the ability to provide this treatment is limited by the availability of donor hearts. Therefore, alternative therapies for both acute and chronic myocardial ischemia need to be developed.
  • Our results demonstrate that human embryonic stem cell (hESC)-derived hemangioblasts can create new blood vessels and improve blood flow in a rodent model of myocardial infarction. We demonstrated that adult stem cells (bone marrow CD34+ cells) can be successfully targeted to injured heart tissue, thus avoiding surgery or invasive catheter based therapies. The antibody technology can be used to target hESC-derived hemangioblasts specifically to injured heart tissue.
  • Further studies are needed to confirm our initial findings, determine whether the new blood vessel formation lead to an increase in heart function and safety studies. Studies are in progress to improve the efficiency and effectiveness of hESC-derived hemangioblasts to create new blood vessels. Additionally, investigations are underway to determine if immunosuppressive drugs will be necessary to increase survival of the hESC-derived hemangioblast. Our initial finding of hES-derived hemangioblasts inducing new blood vessel formation may eventually lead to the development of an unlimited and reliable cell source for renewing blood vessels and treating myocardial infarction.
  • Coronary artery disease (CAD) remains the leading cause of morbidity and mortality worldwide and is predicted to be the leading cause of death by 2020. In the US, it is estimated that cardiovascular disease affects 60 million patients costing the healthcare system approximately $186 billion annually. Approximately two-thirds of patients sustaining a myocardial infarction do not make a complete recovery and often are left with debilitating congestive heart failure. Despite the advances in medical treatment and interventional procedures to reduce mortality in patients with CAD, the number of patients with refractory myocardial ischemia and congestive heart failure is rapidly increasing. For end-stage heart failure, heart transplantation is the only successful treatment. However, the ability to provide this treatment is limited by the availability of donor hearts. Therefore, alternative therapies in the prevention and treatment of end-stage heart failure are needed.
  • Critical to any heart repair strategy is the need to provide vessels to allow for an adequate blood supply to nourish the heart. Our results demonstrate that human embryonic stem cell (hESC)-derived hemangioblasts can create new blood vessels and improve blood flow in a rodent model of myocardial infarction. Studies are in progress to improve the efficiency and effectiveness of hESC-derived hemangioblasts to create new blood vessels. Strategies to improve efficiency and effectiveness include the use of extracellular matrix proteins (components that make up the structural aspect of the heart) to increase the survival of the cells or the use of antibodies to direct and link the cells to the damaged heart muscle. Additionally, to decrease the risk of tumor formation from the hESC-derived hemangioblasts, the hESC-derived hemangioblasts are being cultured to form more mature endothelial cells (cells that mimic the bodies natural cells that produce blood vessels). These cells are being tested to determine whether they can effectively induce blood vessels in the heart. Our initial finding of hES-derived hemangioblasts inducing new blood vessel formation may eventually lead to the development of an unlimited and reliable cell source for renewing blood vessels and treating myocardial infarction.
  • Coronary artery disease (CAD) remains the leading cause of morbidity and mortality worldwide and is predicted to be the leading cause of death by 2020. In the US, it is estimated that cardiovascular disease affects 60 million patients costing the healthcare system approximately $186 billion annually. Approximately two-thirds of patients sustaining a myocardial infarction do not make a complete recovery and often are left with debilitating congestive heart failure. Despite the advances in medical treatment and interventional procedures to reduce mortality in patients with CAD, the number of patients with refractory myocardial ischemia and congestive heart failure is rapidly increasing. For end-stage heart failure, heart transplantation is the only successful treatment. However, the ability to provide this treatment is limited by the availability of donor hearts. Therefore, alternative therapies in the prevention and treatment of end-stage heart failure are needed.
  • Critical to any heart repair strategy is the need to provide vessels to allow for an adequate blood supply to nourish the heart. Our results demonstrate that human embryonic stem cell (hESC)-derived hemangioblasts can create new blood vessels and improve blood flow in a rodent model of myocardial infarction. Subsequent studies with hESC-derived endothelial progenitor cells decreased MI size and improved LV function in a mouse model of myocardial ischemia. Studies are in progress to improve the efficiency and effectiveness of hESC-derived endothelial progenitor cells to create new blood vessels.
  • Strategies to improve efficiency and effectiveness of stem cell therapy include the use of extracellular matrix proteins (components that make up the structural aspect of the heart) to increase the survival of the cells or the use of antibodies to direct and link the cells to the damaged heart muscle. We have demonstrated that antibodies can direct stem cells to injured myocardial tissue. Continued studies are in progress to perform studies needed for the submission of an IND. The development of peptide-modified scaffolds for the treatment of chronic heart failure has produced initial proof of concept studies that a tissue engineering approach for restoration of an injured heart is possible. Additionally, we have demonstrated that extracellular matrix derived peptides can recruit endogenous cardiac stem cells. Further development of a biopolymer scaffold for the treatment of chronic heart failure is in progress.
  • Coronary artery disease (CAD) remains the leading cause of morbidity and mortality worldwide and is predicted to be the leading cause of death by 2020. In the US, it is estimated that cardiovascular disease affects 60 million patients costing the healthcare system approximately $186 billion annually. Approximately two-thirds of patients sustaining a myocardial infarction do not make a complete recovery and often are left with debilitating congestive heart failure. Despite the advances in medical treatment and interventional procedures to reduce mortality in patients with CAD, the number of patients with refractory myocardial ischemia and congestive heart failure is rapidly increasing. For end-stage heart failure, heart transplantation is the only successful treatment. However, the ability to provide this treatment is limited by the availability of donor hearts. Therefore, alternative therapies in the prevention and treatment of end-stage heart failure are needed.
  • Critical to any heart repair strategy is the need to provide vessels to allow for an adequate blood supply to nourish the heart. Our results demonstrate that human embryonic stem cell (hESC)-derived hemangioblasts can create new blood vessels and improve blood flow in a rodent model of myocardial infarction. Subsequent studies with hESC-derived endothelial progenitor cells decreased MI size and improved LV function in a mouse model of myocardial ischemia. Studies are in progress to improve the efficiency and effectiveness of hESC-derived endothelial progenitor cells to create new blood vessels.
  • Strategies to improve efficiency and effectiveness of stem cell therapy include the use of extracellular matrix proteins (components that make up the structural aspect of the heart) to increase the survival of the cells or the use of antibodies to direct and link the cells to the damaged heart muscle. We have demonstrated that antibodies can direct stem cells to injured myocardial tissue. Continued studies are in progress to perform studies needed for the submission of an IND. The development of peptide-modified scaffolds for the treatment of chronic heart failure has produced initial proof of concept studies that a tissue engineering approach for restoration of an injured heart is possible. Additionally, we have demonstrated that extracellular matrix derived peptides can recruit endogenous cardiac stem cells. Further development of a biopolymer scaffold for the treatment of chronic heart failure is in progress.

Modeling Myocardial Therapy with Human Embryonic Stem Cells

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00104
ICOC Funds Committed: 
$2 229 140
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Five million people in the U.S. suffer with heart failure, at a cost of $30 billion/year. Heart failure occurs when the heart is damaged and becomes unable to meet the demands placed on it. Unlike some tissues, heart muscle does not regenerate. Human embryonic stem cells grow and divide indefinitely while maintaining the potential to develop into many tissues of the body, including heart muscle. They provide an unprecedented opportunity to both study human heart muscle in culture in the laboratory, and advance cell-based therapy for damaged heart muscle. We have developed methods for identifying and isolating specific types of human embryonic stem cells, stimulating them to become human heart muscle cells, and delivering these into the hearts of mice that have had a heart attack. This research will identify those human embryonic stem cells that are best at repairing damaged heart muscle, thereby treating or avoiding heart failure.
Statement of Benefit to California: 
More than 90,000 people in California suffer with heart failure, at a cost of ~$540 million/year. Heart failure occurs when the heart is damaged and becomes unable to meet the demands placed on it. Unlike some tissues, heart muscle does not regenerate. This research will identify human embryonic stem cells that are able to repair damaged heart muscle, thereby treating or avoiding heart failure. The medical treatments developed as a result of these studies will not only benefit the health of Californians with heart failure, but also should result in significant savings in health care costs. This research will push the field of cardiovascular regenerative medicine forward despite the paucity of federal funds, and better prepare us to utilize these funds when they become available in the future.
Progress Report: 
  • Five million people in the U.S. suffer with heart failure, at a cost of $30 billion/year. Heart failure occurs when the heart is damaged and becomes unable to meet the demands placed on it. Unlike some tissues, heart muscle does not regenerate. Human embryonic stem cells grow and divide indefinitely while maintaining the potential to develop into many tissues of the body, including heart muscle. They provide an unprecedented opportunity to both study human heart muscle in culture in the laboratory, and advance cell-based therapy for damaged heart muscle. During the first year of CIRM support, we have developed methods for identifying and isolating specific types of human embryonic stem cells, and stimulating them to become human heart muscle cells. We are currently working to determine the best methods and timing for delivering these cells into the hearts of mice that have had a heart attack. This research will identify those human embryonic stem cells that are best at repairing damaged heart muscle, thereby treating or avoiding heart failure.
  • Five million people in the U.S. suffer with heart failure, at a cost of $30 billion/year. Heart failure occurs when the heart is damaged and becomes unable to meet the demands placed on it. Unlike some tissues, heart muscle does not regenerate. Human embryonic stem cells grow and divide indefinitely while maintaining the potential to develop into many tissues of the body, including heart muscle. They provide an unprecedented opportunity to both study human heart muscle in culture in the laboratory, and advance cell-based therapy for damaged heart muscle. During this year of CIRM support, we have developed methods for identifying and isolating specific types of human embryonic stem cells, and stimulating them to become human heart muscle cells. We are currently working to determine the best methods and timing for delivering these cells into the hearts of mice that have had a heart attack. This research will identify those human embryonic stem cells that are best at repairing damaged heart muscle, thereby treating or avoiding heart failure.
  • Five million people in the U.S. suffer with heart failure, at a cost of $30 billion/year. Heart failure occurs when the heart is damaged and becomes unable to meet the demands placed on it. Unlike some tissues, heart muscle does not regenerate. Human embryonic stem cells grow and divide indefinitely while maintaining the potential to develop into many tissues of the body, including heart muscle. They provide an unprecedented opportunity to both study human heart muscle in culture in the laboratory, and advance cell-based therapy for damaged heart muscle. During this year of CIRM support, we have developed methods for identifying and isolating specific types of human embryonic stem cells, and stimulating them to become human heart muscle cells. We are currently working to determine the best methods and timing for delivering these cells into the hearts of mice that have had a heart attack. This research will identify those human embryonic stem cells that are best at repairing damaged heart muscle, thereby treating or avoiding heart failure.

In Vivo Molecular Magnetic Resonance Imaging of Human Embryonic Stem Cells in Murine Model of Myocardial Infarction

Funding Type: 
SEED Grant
Grant Number: 
RS1-00326
ICOC Funds Committed: 
$658 125
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Magnetic resonance imaging (MRI) has emerged as one of the predominant modalities to evaluate the effects of stem cells in restoring the injured myocardium. However, MRI does not enable assessment of a fundamental issue in cell therapy, survival of the transplanted cells. The transplanted human embryonic cells (hESC) must at the very least survive to restore the injured myocardium. This research proposal will address this specific challenge to image non-invasively both the survival of the transplanted hESC and the resultant restoration of the myocardium through sensitive detection of the molecular events indicating hESC survival and rapid imaging of myocardial function. In order to achieve this dual capability, there are 2 primary considerations: 1) amplification of molecular signals and 2) high spatial and temporal resolution imaging of the myocardium. No single imaging modality will fulfill all needs of non-invasive molecular imaging in the heart. Only an imaging modality that optimizes the 2 technical specifications will provide physiologically relevant meaning of the molecular signal of the transplanted hESC. The molecular signal will be useful if some correlation between hESC survival and functional restoration can be established. In order to address these critical issues, this proposal will describe efforts to implement molecular MRI to image both the survival of transplanted hESC and restoration of cardiac function using mouse model of myocardial infarction. This research proposes an integrated, multidisciplinary approach to converge innovative approaches in MRI and stem cell biology to address a fundamental yet very critical issue in cardiac restoration: survival of hESC following transplantation into the injured myocardium. This proposal combines novel molecular techniques with the high resolution capabilities of MRI. Upon conclusion of this research, an integrated MRI platform will be developed to allow dual evaluation of the survival of transplanted hESC and their effects on myocardial function. Maturation of this imaging technology will ultimately enable accurate assessment of the survival of hESC and restoration of recipient tissue in all human organs.
Statement of Benefit to California: 
Coronary artery disease (CAD) continues to be the leading cause of death in the United States. Recent advances in cardiovascular therapy have improved immediate survival following an acute myocardial infarction (MI). The persistence of high overall mortality of CAD despite improved treatment is due to a shift in the disease process. Studies have demonstrated a critical role of the infarcted myocardium in the development of congestive heart failure (CHF). The incidence of CHF is now reaching epidemic proportions. Today, there is higher number of deaths from patients developing CHF rather than those sustaining acute MI. CHF is the leading cause of hospital admissions resulting in approximately 300,000 deaths annually. There are nearly 5 million Americans who are suffering from this illness with 550,000 new cases reported each year. Over the last several decades, advances in biomedical technology provided significant improvement in morbidity and mortality. However, the average 5-year survival today still remains around a dismal 50%, creating a major public health concern. Heart transplantation is an established treatment for end-stage CHF. Yet, this definitive therapy is limited to only 2000 donor hearts per year. Thus, a strong mandate exists for an alternative therapeutic option. Human embryonic stem cells (hESC) have demonstrated the ability to differentiate into cardiac cells, representing a potential application of cell therapy to restore the injured myocardium. The public health impact of CHF in California is representative of the emerging trend seen across the United States. As the most populous State in the nation, CHF has resulted in equivalent burden to California’s health care cost, morbidity and mortality. The State of California stands to benefit tremendously with accurate MRI-guided monitoring of the therapeutic efficacy of hESC in an effort to advance the treatment for CHF.
Progress Report: 
  • Magnetic resonance imaging (MRI) has emerged as one of the predominant modalities to evaluate the effects of stem cells in restoring the injured heart. However, MRI does not enable assessment of a fundamental issue in cell therapy, survival of the transplanted cells. The transplanted human embryonic cells (hESCs) must at the very least survive to restore the injured heart. In order to address this issue, this research has conducted the fundamental work to develop a reporter gene as outlined in the proposal and developed a reliable system to evaluate the survival of the transplanted hESCs.
  • First, using a commercially available genetic construct, the reporter gene was designed to generate specific cell surface tags as a signal of cell survival. Molecular assays demonstrated proper characteristics of the reporter gene and the construct has been inserted into human embryonic kidney cells to demonstrate proof of concept. MRI signal was generated from these cells and this result has been validated by flow cytometry confirming the expression of cell surface tags by the reporter gene. Second, the metabolic effects of the contrast agent, iron-oxide, used to magnetically activate the antibodies have been evaluated. The results demonstrated that the iron-oxide has no toxic effects to the cell metabolism. Finally, preliminary MRI of the iron-oxide labeled hESC injected directly into the mouse heart was obtained.
  • Based on above results, the molecular signal was further refined to generate optical signal of cell survival as an additional validation tool. Robust molecular signal of hESC survival was generated following transplantation of the reporter gene transduced hESC into the mouse myocardium. During the no cost extenstion period, correlation between hESC survival and functional restoration of the injured heart will be assessed. Using MRI, cell survival and functional restoration of the heart will be imaged non-invasively in order to obtain longitudinal information regarding survival of transplanted hESC and restoration of heart function.
  • Magnetic resonance imaging (MRI) has emerged as one of the predominant modalities to evaluate the effects of stem cells in restoring the injured heart. However, MRI does not assess a fundamental issue in cell therapy, survival of the transplanted cells. The transplanted human embryonic cells (hESCs) must at the very least survive to restore the injured heart. In order to address this issue, this research has conducted the fundamental work to develop a reporter gene as outlined in the proposal and developed a reliable system to evaluate the survival and teratoma formation of the transplanted hESCs.
  • Using a commercially available genetic construct, the reporter gene was designed to generate specific cell surface tags as a signal of cell survival. Molecular assays demonstrated proper characteristics of the reporter gene and the construct has been inserted into human embryonic stem cells. MRI signal was generated from these cells and this result has been validated by flow cytometry confirming the expression of cell surface tags by the reporter gene in viable human embryonic stem cells. The viable cells expressing this reporter gene were transplanted into mouse heart and MRI signal was generated from the heart of a live mouse.
  • Based on the above results, the molecular signal was further refined to generate optical signal of cell survival as an additional validation tool. Robust molecular signal of hESC survival was generated following transplantation of the reporter gene transduced hESC into the mouse myocardium. During the no cost extenstion period, detection of hESC survival, proliferation, and early teratoma formation was studied. These biological properties of the transplanted hESCs were monitored accurately. This information will be used to correlate hESC survival/proliferation/teratoma formation with functional restoration of the injured heart.

Technology for hESC-Derived Cardiomyocyte Differentiation and Optimization of Graft-Host Integration in Adult Myocardium

Funding Type: 
SEED Grant
Grant Number: 
RS1-00242
ICOC Funds Committed: 
$634 287
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Stem cells therapies hold great promise in the treatment of cardiac diseases such as coronary heart disease or congestive heart failure. Thanks to their ability to transform into almost any kind of tissue, engrafted stem cells can potentially replace damaged heart tissues with healthy tissues, effectively restoring the heart’s original functions. While initial studies demonstrated the potential benefits of stem cell injection for repairing heart damage, they told researchers little about exactly how improvements were made to the heart and how the improvement might be enhanced. Also, there is concern that the stem cells could negatively impact some aspects of heart function and lead to disturbances of heart rhythm and future attacks. In light of this, we propose to develop a model to study the detailed interaction of stem cells and healthy heart tissue in the laboratory, where events within the cells and between the cells can be measured accurately and many experiments can be done to increase our understanding, without the use of human subjects. Specifically, we plan to focus on two main goals. The first goal is to develop a platform to better understand the gradual transition that stem cell lines make as they mature into heart cells, process known as differentiation. We will record the electrical activity arising from newly formed heart cells to determine when exactly they form and how the behave in response to electrical stimuli or drugs as they mature. This will tell us more about the behavior of the cells that could be injected into the heart so that we know how they will respond when they merge with the heart and when is the best time to introduce them. The second goal, building on the first one, is to observe how the stem cells make contact with the heart cells, including how they grow together mechanically and how they begin to communicate electrically as a repaired tissue. This will be carried out by growing the stem cells and heart cells separately and then allowing them to grow together, just as they would in the heart. Simultaneous recording of electrical activity at numerous locations in the culture will let us map the activity across the culture and evaluate the communication between heart cells (host) and stem cells (graft). Understanding the microscopic nature of integration of stem cells into healthy tissue will lead to a greater knowledge of what can happen when stem cells are injected into the heart and begin to replace the non-functional tissue and connect to healthy tissue. Insights gained with such model should lead to a better understanding of the repair process and highlight strategies for making stem cell-based therapies safer and more effective. This model will also allow testing and development of chemical or electrical manipulations that would increase the yield and reliability of the differentiation process, paving the way for the ultimate scale-up of stem cell therapies for clinical use.
Statement of Benefit to California: 
There is currently no cure for heart damage caused by heart attack, and stem cells offer a very promising solution to this problem that affects millions of Americans. We feel that addressing possible solutions to this pervasive problem is a very constructive and meaningful way to utilize some of the financial resources allocated for stem cell research in California. Within (and outside) the CIRM community, we also have the important goal of making currently unavailable electronic, microfabrication and signal processing technologies available in the form our proposed research platforms. With our planned outreach efforts, we will freely share our methods and equipment, hopefully enhancing the work of many other research groups. By using CIRM funds, we could make such systems available for use with non-registered (as well as registered) cell lines. The outcome of this research stands to impact not only citizens of California, but also the nation and the world. We aim to make considerable progress with research paid for by the citizens of California, demonstrating the degree to which we, as a people, are committed to solving problems in medicine and health care and improving the lives of others. This work will also benefit our State and taxpayers through the training of post-doctoral and graduate students with a clear mindset of leadership, creativity and compassion. Through publication and presentations at local, national and international forums, we hope to disseminate the knowledge gained and encourage further advances.
Progress Report: 
  • The success of cardiac cell grafts for repair of infarcts or congestive heart failure has been moderate to date. While graft cells may survive transplantation, their contribution to conduction and force generation is neither well-defined nor understood. Also, there is concern that the stem cells could negatively impact some aspects of heart function and lead to disturbances of heart rhythm. In light of this, we proposed to develop a model to study the detailed interaction of stem cells and healthy heart tissue in the laboratory, focusing on two main thrusts.
  • The first part of this project had seen the successful development of a platform to better understand the transition that stem cells make as they mature into heart cells, a process known as differentiation. Using arrays of microelectrodes, recording of electrical activity from maturing stem cells was demonstrated. Impact of electrical stimulation on the differentiation process had been probed. Investigation of the interaction between stem cells and heart cells had also been initiated. The second part focused mainly on the latter aspect – functional coupling of stem cells in the heart tissue. New analysis tools for the quantification of the conduction of the electrical activity across a heart tissue were developed. Studies with mixed co-cultures of cardiac cells and fibroblasts revealed a high sensitivity of the conduction properties to the presence of non-conductive cells (fibroblasts), and provide a model for assessing conduction in stem cell grafts of varying homogeneity. Co-cultures of heart cells (host) and stem cells (graft), first grown separately then allowed them to merge, highlighted issues of conduction mismatch at the interface between the host and graft tissue, as well as the dependence of this conduction on the maturity and purity of the grafts used. Most importantly, these studies demonstrated the value of the model developed under this grant for the investigation of electrical coupling and conduction in stem cell grafts, issues that are vital to the safe, effective and successful use of stem cell therapy.

Micro Platform for Controlled Cardiac Myocyte Differentiation

Funding Type: 
SEED Grant
Grant Number: 
RS1-00239-A
ICOC Funds Committed: 
$363 707
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Congestive heart failure, the inability of the heart to continue to pump effectively due to damage of its muscle cells, affects approximately 4.8 million Americans and is a leading cause of mortality. Causes of the irreversible damage to the cardiomyocytes that results in congestive heart failure include hypertension, heart attacks, and coronary disease. Because the cadiomyocytes in the adult heart tissue are terminally differentiated and thus cannot regenerate themselves, once they are damaged, they are irreversibly damaged. As a consequence, despite the advances in medical devices and pharmaceuticals, still more than 50% of congestive heart failure patients die within 5 years of initial diagnosis. The goal therefore must be to restore the heart cells’ functions. This is possible by transplanting fetal and neonatal cardiomyocytes which can then integrate into the host tissue. This approach has demonstrated success in improving heart function. However, the limited availability of fetal donors has prevented its adoption as a viable therapeutic approach. Embryonic stem cells can overcome this challenge as they proliferate continuously in vitro and can be furthermore stimulated to differentiate. Embryoid bodies are three-dimensional clusters of heterogenous stem cells, some of which contain cardiac myocytes, which demonstrate characteristic spontaneous contractions. Controlled and efficient differentiation of the stem cells into cardiomyocytes and an effective way to characterize/verify these cells is critical. Ensuring a pure population of cardiac myocytes is essential because otherwise there is a high-likelihood of tumor formation when transplanted. Previous studies have shown that a low percentage of all embryoid bodies spontaneously form cardiomyocytes. Our goal is to therefore develop a self-contained system to grow and controllably differentiate the human embryonic stem cells into cardiomyocytes in high-yields. Few studies have characterized the types of cardiac myocytes in the differentiating human EBs. Our strategy is to use electrical and chemical cues to induce the high-yield differentiation of stem cells into cardiomyocytes and to monitor this process over time both electrically and optically.
Statement of Benefit to California: 
Improvements in differentiating stem cells into homogenous populations of specific cell types are much needed for transplantation therapy in general—and for congestive heart failure patients in particular. The benefits associated with the development of this micro platform have even broader reaching implications beyond biomedical research. After this system is developed, it will serve as a first platform of its kind that can be later commercialized, which would help spur industry growth. To vitalize and enable high-tech/biotech companies to this {REDACTED} area {REDACTED}, engaging industry involvement to this area is necessary. Supporting such activities would furthermore foster the opportunity for student internships with industry and well as afford the students opportunities in entrepreneurship. Our institution is a Hispanic-serving undergraduate institute with almost 50% minority students. Such a proposed system is vital for promoting both the diversity and research culture {REDACTED} and will be leveraged extensively in outreach programs to encourage underrepresented minorities in science education and training. By actively reaching out to specific students who would particularly benefit from our proposed undergraduate internship program, we can attract at-risk students to engage them in research to promote their retention.

Micro Platform for Controlled Cardiac Myocyte Differentiation

Funding Type: 
SEED Grant
Grant Number: 
RS1-00239-B
ICOC Funds Committed: 
$363 707
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Congestive heart failure, the inability of the heart to continue to pump effectively due to damage of its muscle cells, affects approximately 4.8 million Americans and is a leading cause of mortality. Causes of the irreversible damage to the cardiomyocytes that results in congestive heart failure include hypertension, heart attacks, and coronary disease. Because the cadiomyocytes in the adult heart tissue are terminally differentiated and thus cannot regenerate themselves, once they are damaged, they are irreversibly damaged. As a consequence, despite the advances in medical devices and pharmaceuticals, still more than 50% of congestive heart failure patients die within 5 years of initial diagnosis. The goal therefore must be to restore the heart cells’ functions. This is possible by transplanting fetal and neonatal cardiomyocytes which can then integrate into the host tissue. This approach has demonstrated success in improving heart function. However, the limited availability of fetal donors has prevented its adoption as a viable therapeutic approach. Embryonic stem cells can overcome this challenge as they proliferate continuously in vitro and can be furthermore stimulated to differentiate. Embryoid bodies are three-dimensional clusters of heterogenous stem cells, some of which contain cardiac myocytes, which demonstrate characteristic spontaneous contractions. Controlled and efficient differentiation of the stem cells into cardiomyocytes and an effective way to characterize/verify these cells is critical. Ensuring a pure population of cardiac myocytes is essential because otherwise there is a high-likelihood of tumor formation when transplanted. Previous studies have shown that a low percentage of all embryoid bodies spontaneously form cardiomyocytes. Our goal is to therefore develop a self-contained system to grow and controllably differentiate the human embryonic stem cells into cardiomyocytes in high-yields. Few studies have characterized the types of cardiac myocytes in the differentiating human EBs. Our strategy is to use electrical and chemical cues to induce the high-yield differentiation of stem cells into cardiomyocytes and to monitor this process over time both electrically and optically.
Statement of Benefit to California: 
Improvements in differentiating stem cells into homogenous populations of specific cell types are much needed for transplantation therapy in general—and for congestive heart failure patients in particular. The benefits associated with the development of this micro platform have even broader reaching implications beyond biomedical research. After this system is developed, it will serve as a first platform of its kind that can be later commercialized, which would help spur industry growth. To vitalize and enable high-tech/biotech companies to this {REDACTED} area {REDACTED}, engaging industry involvement to this area is necessary. Supporting such activities would furthermore foster the opportunity for student internships with industry and well as afford the students opportunities in entrepreneurship. Our institution is a Hispanic-serving undergraduate institute with almost 50% minority students. Such a proposed system is vital for promoting both the diversity and research culture {REDACTED} and will be leveraged extensively in outreach programs to encourage underrepresented minorities in science education and training. By actively reaching out to specific students who would particularly benefit from our proposed undergraduate internship program, we can attract at-risk students to engage them in research to promote their retention.
Progress Report: 
  • This year, we have made quite some progress in developing the microtechnology platform. We have developed a new way to form and culture human embryonic stem cells into uniform embryoid bodies in a high throughput fashion. Instead of using the laborious ‘hanging drop method’ or the complicated ‘spinning flask method’, we have developed a way for researchers to easily pipette their cells into standard well plates and increase their throughput by almost 1000x. This is achieved by placing inserts with rounded-bottom microwells into standard well plates. Each one of these inserts that can fit into a standard 24 or 96 well plate can have up to 1000 wells and therefore can create 1000 embryoid bodies, all of uniform size. We can even create wells of various sizes such that we can induce embryoid bodies of predefined sizes and numbers of cells. Many recent publications have demonstrated that the initial size of the embryoid bodies affect differentiation. We have observed this as well. Moreover, this new platform allows researchers to perform real-time microscopy of the cells during this whole process.
  • In addition to developing this new chip, we have also electrically stimulated at different stages during differentiation. The different stages of differentiation include: 1) during embryoid body development 2) when transferred to gelatin coated dishes 3) after about a week on gelatin and 4) isolated beating areas. Electrical stimulation was accomplished using a C-PACE voltage pulsing device at a 1 Hz frequency, 4.5 V (2.5 V/cm), and a 1 ms duration. Unfortunately, none of the electrical stimulation yielded any exhibited increased expression of cardiac markers. Future studies will examine pacing of differentiated cardiac cells for synchronization and will employ more markers using a PCR super microarray.
  • We have also worked on custom software development that allows us to automatically identify and track individual cells within the microplatform.
  • There were a number of factors that caused some unexpected delays in scientific progress this year. Most notably, the PI Michelle Khine and her lab moved to a new university. Therefore, this took quite some time to take down and then re-establish the lab at its new location. Now at UC Irvine, she finally has the ideal infrastructure to make progress quickly on this project. This one year extension to finish this project is therefore much needed and greatly appreciated.
  • To uniformly control the differentiation of embryoid bodies (EBs), we have developed a very simple to use culture platform the create homogenous-sized EBs.
  • We have made quite some progress with the EB array culture plate development, described in detail in the last progress report. Since then, we have developed a way to 1) translate to a more transparent material with lower autofluorescence (cyclic olefin copolymer, COC) to be compatible with optical imaging (Figure 1, c) and then 2) mated the microwells to the bottom of 24 well plates for ease of handling. While we have not had success with applying electric fields to induce cardiomyocyte differentiation, we are now working with !) optimizing the EB size to yield the most cardiomyocytes and then 2)perfusing the EBs with soluble factors.

Specification of Ventricular Myocyte and Pacemaker Lineages Utilizing Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00198
ICOC Funds Committed: 
$609 999
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Heart failure is a leading cause of mortality in California and the United States. Currently, there are no “cures” for heart failure.Other life threatening forms of heart disease include dysfunction of cardiac pacemaker cells, necessitating implantation of mechanical pacemakers. Although mechanical pacemakers can be efficacious, there are potential associated problems, including infection, limited battery half-life, and lack of responsiveness to normal biological cues. Our research with human embryonic stem cells will be aimed at developing therapies for heart failure, and cardiac pacemaker dysfunction. In each of these disease settings, one might effect a “cure” by replacing worn out or dysfunctional cardiac cells with new ones. In the case of heart failure, the cells that need to be replaced are heart muscle cells, which do the majority of the work in the heart. In the case of pacemaker dysfunction, the cells that need to be replaced are pacemaker cells, a highly specialized type of heart muscle cell. To replace these cells, we need to find cells that can become heart muscle or cardiac pacemaker cells, understand how to generate fairly large numbers of them, and how to persuade them to become either heart muscle or cardiac pacemaker cells. Potential cardiac progenitor cells may come from a number of different sources, either from patients themselves, or from extrinsic sources. Regardless of the source,we need to define factors which will make the cells multiply and will make them become the cell type that we need for repair. The biology of human heart cells is likely to be distinctive from that of heart cells from other animals. For example, a human heart has to function for multiple decades, unlike hearts of other animals who live in general for shorter periods of time. The size, required function, and rhythm of the human heart are also distinct from that of other animals. For these reasons, for repair of human heart, it is important to study human cardiac progenitors and to define pathways required to grow them and to differentiate them utilizing human cells as a model experimental system. Our proposed research will utilize human embryonic stem cells as a source of cardiac progenitors. As human embryonic stem cells can turn into many different kinds of cells, we will create special lines of human embryonic stem cells that will become fluorescent when they adopt the cardiac progenitor, heart muscle, or pacemaker state. These lines will then be treated with a large number of small molecules to find small molecules which amplify cells the number of fluorescent cells in each of these states. The small molecules activate known biochemical pathways, so we can then use the small molecules themselves, or activate identified pathways to achieve the goal of obtaining sufficient numbers of specific cardiac cell types for cardiac therapy.
Statement of Benefit to California: 
More Californians die each year of cardiovascular disease than from the next four leading causes of death combined. Californians continue to die or be disabled as a direct result of cardiovascular disease. Although advances in medical treatment have improved post-infarct survival, heart failure is an increasingly abundant manifestation of cardiovascular disease. A secondary complication of heart failure, and other cardiac diseases, is cardiac pacemaker dysfunction, a potentially fatal condition which is currently ameliorated by mechanical pacemakers. However, mechanical pacemakers have many associated complications,particularly for pediatric patients. For both heart failure and pacemaker dysfunction, replacement of heart muscle cells or biological pacemaker cells offers the hope of improving upon current medical practice. Our research is aimed toward developing new therapies which will allow for the replacement of these critical cell types in diseased heart.
Progress Report: 
  • In the current reporting period, we have worked on the generation of human embryonic stem cell derived marker cell lines for different steps of cardiomyocyte differentiation. The cell lines are designed to express fluorescent proteins under the control of gene promoters that mark cardiac progenitor cells, cardiomyocytes, or cardiac conduction system cells.
  • We tested several HUES cell lines for this purpose and chose cell lines that can differentiate into cardiomyocytes efficiently but are easy to expand and appear stable over several passages in culture. We generated several BAC transgenic cell lines that specifically express green fluorescent protein in cardiomyocytes. Further cell lines are being generated. The cell lines will be used in high-throughput screens to identify molecules and mechanisms that direct the efficient in vitro differentiation into different cardiac cells.

Development of Neuro-Coupled Human Embryonic Stem Cell-Derived Cardiac Pacemaker Cells.

Funding Type: 
SEED Grant
Grant Number: 
RS1-00171
ICOC Funds Committed: 
$744 639
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Optimal cardiac function depends on the properly coordinated cardiac conduction system (CCS). The CCS is a group of specialized cells responsible for generating cardiac rhythm and conducting these signals efficiently to working myocardium. This specialized CCS includes the sinoatrial node, atrioventricular node and His-Purkinje system. These specialized pacemaking /conducting cells have different properties from the surrounding myocytes responsible for the contractile force. Genetic defects or postnatal damage by diseases or aging processes of these cells would result in impaired pulse generation (sinus node dysfunction, SND) or propagation (heart block). Implantation of an electronic cardiac pacemaker is necessary for intolerant bradycardia to restore cardiac rhythm. However, the electronic implantable pacemaker has multiple associated risks (e.g. infections) and requires frequent generator changes due to limited battery life. Sinus node dysfunction is a generalized abnormality of cardiac impulse formation and accounts for >30 percent of permanent pacemaker placements in the US. A perfect therapy to SND will be to repair or replace the defective sinus node by cellular or genetic approaches. Many recent studies have demonstrated, in a proof-of-concept style, of generating a biological pacemaker by implanting various types of progenitor or stem cells into ventricular myocardium to form a pulse-generating focus. However, a perfect biological pacemaker will require good connections with the intrinsic neuronal network for proper physiological responses. Elucidation of the factors controlling the evolution of pacemaker cells and their interaction with the peripheral neuronal precursor cells (neural crest cells, NCCs) will be paramount for creating an adaptive biological pacemaker. The NCCs have been shown to be contiguous with the developing conduction system in embryonic hearts of humans. However, the influence and interaction of the NCCs with the developing cardiac pacemaker cells remains unclear. In addition, there is no simple marker for identifying the pacemaker cells and the electrophysiological (EP) recording is the only physiological method to trace the evolution of cardiac pacemaker cells from human embryonic stem cells (hESCs). We have successfully obtained the EP properties of early hESC-derived cardiomyocytes. We propose here an in vitro co-culture system to study fate of the pacemaker cells evolved from hESCs and to investigate the influence of NCCs on the early, cardiac committed myocytes derived from hESCs. Such a study will provide insight in the development of pacemaker cells and in the mechanisms of early neuro-cardiac interaction. Results from the proposed study may suggest strategies for developing efficient and neuro-coupled cardiac pacemakers from ESCs. These neuro-coupled biological pacemaker cells may one day used clinically to replace the need for implanting an electronic pacemaker for the treatment of intolerant bradycardia.
Statement of Benefit to California: 
Optimal cardiac function depends on the properly coordinated cardiac conduction system. Genetic defects or postnatal damage by diseases or aging processes of these pacemaker cells would result in impaired pulse generation (sinus node dysfunction) or propagation (heart block). The implantation of an electronic cardiac pacemaker is necessary for intolerant bradycardia to restore physiologic cardiac rhythm. However, the electronic implantable pacemaker has multiple associated risks (e.g. infections and thrombosis) and requires frequent generator changes due to limited battery life. Sinus node dysfunction (SND) is a generalized abnormality of cardiac impulse formation and accounts for 30-50 percent of permanent pacemaker placements in the US. A perfect therapy to SND will be to repair or replace the defective sinus node by cellular or genetic approaches. Most of the research work on developing biological pacemakers are performed in Columbia University at New York City, Johns Hopkins University at Baltimore, and Technion-Israel Institute of Technology at Haifa, Israel. All of their approaches produced short-lived and non-responsive biological pacemakers to physiological demands. None of human stem cell-related research in California is devoted to this highly promising field of developing biological pacemakers. The proposed research here will elucidate the factors controlling the evolution of pacemaker cells and their interaction with the peripheral neuronal precursor cells (neural crest cells). Such a study will provide insight in the development of pacemaker cells and in the mechanisms of early neuro-cardiac interaction. These factors then can be used to generate better neuro-coupled biological pacemaker cells in California. These neuro-coupled biological pacemaker cells may one day be used clinically to replace the need for implanting an electronic pacemaker for the treatment of intolerant bradycardia. Creating the neuro-coupled, adaptive biological pacemakers will make California the epicenter of the next generation of pacemaker therapy, and will benefit its citizens who have intolerant cardiac bradycardia.
Progress Report: 
  • Cardiovascular diseases remain the major cause of death in the US. Human Stem and progenitor cell-derived cardiomyocytes (SPC-CMs) hold great promise for myocardial repairs. Recent progress in cellular reprogramming of various somatic cell types into induced pluripotent stem cells opened the door for developing patient-specific, cell-based therapies. However, most SPC-CMs displayed heterogeneous and immature electrophysiological (EP) phenotypes with uncontrollable automaticity. Implanting these electrically immature and inhomogeneous CMs to the hearts would likely be arrhythmogenic and deleterious. Furthermore, as CMs mature, they undergo changes in automaticity and electrical properties. We used human embryonic stem cell-derived CMs (hESC-CMs) as the model system to study the development and maturation of CMs in the embryoid body (EB) environment. Elucidating molecular pathways governing EP maturation of early hESC-CMs in EBs would enable engineered microenvironment to create functional pacemaker cells or electrophysiologically compatible hESC-CMs for cell replacement therapies. We have established antibiotic (Abx)-resistant hESC lines conferred by lentiviral vectors under the control of a cardiac-specific promoter. With simple Abx treatment, we easily isolated >95% pure hESC-CMs at various stages of differentiation from EBs. In the first year of this grant support and using the Abx selection system, we found that hESC-CMs isolated at early stages of differentiation without further contacts with non-cardiomyocytes (non-CMs) depicted arrested electrical maturation. The intracellular Ca2+-mediated automaticity developed very early and contributed to dominant automaticity throughout hESC-CM differentiation regardless of the presence or absence of non-CMs. In contrast, sarcolemmal ion channels evolved later upon further differentiation within EBs and their maturation required the interaction with non-CMs. In the second year, we further developed an add-back co-culture system to enable adding non-CMs back to early isolated hESC-CMs, which rescued the arrest of EP maturation. We also developed techniques to isolate pure subsets of non-CMs, such as neural crest and endothelial cells, from hESC-derived EBs, which exerted influences on maturation of specific subsets of ion channel populations respectively. Therefore, our study showed for the first time that non-CMs exert significant influences on the EP maturation of hESC-CMs during differentiation. Knowledge of this study will allow us to improve functional maturation of primitive hESC-CMs or to create neuro-coupled pacemaker cells before cell transplantation.
  • Cardiovascular diseases remain the major cause of death in the US. Human Stem and progenitor cell-derived cardiomyocytes (SPC-CMs) hold great promise for myocardial repairs. Recent progress in cellular reprogramming of various somatic cell types into induced pluripotent stem cells opened the door for developing patient-specific, cell-based therapies. However, most SPC-CMs displayed heterogeneous and immature electrophysiological (EP) phenotypes with uncontrollable automaticity. Implanting these electrically immature and inhomogeneous CMs to the hearts would likely be arrhythmogenic and deleterious. Furthermore, as CMs mature, they undergo changes in automaticity and electrical properties. We used human embryonic stem cell-derived CMs (hESC-CMs) as the model system to study the development and EP maturation of CMs in the embryoid body (EB) environment. Elucidating molecular pathways governing EP maturation of early hESC-CMs in EBs would enable engineered microenvironment to create functional pacemaker cells or electrophysiologically compatible hESC-CMs for cell replacement therapies. We have established antibiotic (Abx)-resistant hESC lines conferred by lentiviral vectors under the control of a cardiac-specific promoter. With simple Abx treatment, we easily isolated >95% pure hESC-CMs at various stages of differentiation from EBs. In the first year of this grant support and using the Abx selection system, we found that hESC-CMs isolated at early stages of differentiation without further contacts with non-cardiomyocytes (non-CMs) depicted arrested electrical maturation. The intracellular Ca2+-mediated automaticity developed very early and contributed to dominant automaticity throughout hESC-CM differentiation regardless of the presence or absence of non-CMs. In contrast, sarcolemmal ion channels evolved later upon further differentiation within EBs and their maturation required the interaction with non-CMs. In the second year, we further developed an add-back co-culture system to enable adding non-CMs back to early isolated hESC-CMs, which rescued the arrest of EP maturation. In the third no-cost extension year, we further successfully established the cocultures of human neural crest cell (NCC)-derivatives and early-purified hESC-CMs. We found that peripheral neurons derived from human NCCs exerted strong influences on the development of a specific subset of ion channel populations during early hESC-CM differentiation. Therefore, our study showed for the first time that non-CMs, especially neurons derived from NCCs, exert significant influences on the EP maturation of hESC-CMs during differentiation. Knowledge of this study will allow us to improve functional maturation of primitive hESC-CMs or to create neuro-coupled pacemaker cells before cell transplantation.

Discovering Potent Molecules with Human ESCs to Treat Heart Disease

Funding Type: 
SEED Grant
Grant Number: 
RS1-00169
ICOC Funds Committed: 
$714 654
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
This work is directly relevant to human embryonic stem cell (hESC) research because it brings new ideas about novel compounds to affect cardiomyogenesis. The work addresses an urgent need to develop new agents to treat cardiovascular disease. We will develop potent and selective drug-like molecules as cardiomyocyte differentiation agents. Heart disease is the leading cause of mortality and decline in the quality of life in the developed world. The ability of hESCs to form cardiomyocytes has spawned hope that these cells may be used to replace damaged myocardium. Despite their ability to form cardiomyocytes, efficient and controlled cardiomyogenesis in ESC cultures has not been achieved due to the unavailability of differentiation agents and an incomplete understanding of the pathways that regulate cardiac development. Success has been achieved in developing a robust and dependable high-throughput assay to study the effects of small molecules on cardiomyocyte differentiation. Powerful cell-based assays were developed and provided readouts that led to high-content results because multiple signals were probed. The assay is capable of capturing fast or long-acting biology because of the time-course readouts. Cell-based assays are superior to molecular screens because the cell-based assay delivers active compounds or “hits” that are permeable and non-cytotoxic. Moreover, refined “hits” can be used as probes to reveal novel signaling pathways and proteins that control differentiation, in a process termed chemical biology. By taking advantage of knowledge of the current “hits” we will rapidly synthesize novel drug-like compounds in a low-risk approach to. The “hits” will be refined and improved through an efficient synthetic process we use in our lab called “Dynamic Medicinal Chemistry”. Even after miniaturization and automation, screening is still expensive. A key to improve the screening process is to use pharmacologically active, drug-like compounds to provide rich target-relevant information. Intelligently designing libraries for screening by incorporating drug-like features into “lead” library design will improve the attrition rate and lead to more pharmacologically relevant compounds for future studies. This proposal is directly responsive to the California Institute for Regenerative Medicine SEED Grant Program because it provides for developing and testing new agents of use in cardiomyoenesis of hESCs. Importantly, it brings new investigators and a collaborative approach to the stem cell field. The agents discovered and developed may hold great promise as the groundwork for future medications development for a new class of damaged myocardium replacement agents. The theoretical rationale for the work is the use of high-content screening coupled with drug-like new agent discovery approaches. The work will also be of use in the elucidation of key biochemical targets and novel signaling pathways important in hESC cardiomyogenesis.
Statement of Benefit to California: 
In 2002, in the State of California, approximately 697,000 adult Californians died from heart disease. The cost as measured by loss of lifelong earnings was more than $79 billion. Setting aside the pain and suffering, the economic impact of cardiovascular disease to the State of California is staggering. Despite recent advances in cardiovascular medications development, new approaches and novel drug-like compounds are urgently needed to treat cardiovascular disease in California and elsewhere. The poor prognosis for heart disease for Californians underscores the critical need to develop alternative therapeutic strategies. The demonstrated ability of human embryonic stem cells (hESCs) to form cardiomyocytes has spawned widespread hope that these cells may be used as a source to replace damaged myocardium in humans. Despite their ability to form cardiomyocytes, efficient and controlled cardiomyogenesis in hESC culture has not been achieved due to the unavailability of differentiation agents and also because of an incomplete understanding of the pathways that regulate cardiac cell development. Using a high-throughput whole cell assay with image analysis, we have identified four small molecules that promote cardiomyogenesis in human ESCs. This proposal is directly responsive to the California Institute for Regenerative Medicine SEED Grant Program because it provides for developing and testing new agents of use in cardiomyogenesis of hESCs. It also brings new investigators and new collaborative approaches to the field. The promising agents discovered already constitute an excellent starting point and further refinement and development of these compounds may hold great promise as the groundwork for future medications development for a new class of damaged myocardium differentiation agents. The theoretical rationale for the work is the use of high-content screening coupled with drug-like new agent discovery approaches. The work will be of use in the elucidation of key biochemical targets and novel signaling pathways important in hESC cardiomyogenesis. The compounds discovered in our whole hESC-based assays thus far are not potent enough to be developed as drug candidates. But these compounds hold great promise as agents that could be refined further into drug leads. If the leads become drugs, promise of a new class of medication to treat cardiovascular disease may become a reality. Such drugs would decrease cardiovascular disease and decrease health care costs in California. This will likely have a significant economic impact to the State of California. The proposed work represents essential translational research required for new drug development.
Progress Report: 
  • The original goals of the proposal were to apply medicinal chemistry to generate more potent and drug-like analogs of small molecules that stimulate differentiation of cardiomyocytes from embryonic stem cell (ESC) and potentially other progenitor cell types found in adult human heart. During the grant period, we over-achieved each Aim and provided large numbers of drug-like small molecules for cardiomyocyte differentiation studies. In addition, other related information was gained that has considerably expanded our understanding related to developing regenerative medicines.
  • 1. Synthetic Chemistry: From an initial screen of thousands of compounds, six 'hits' were identified. Almost 1300 compounds were synthesized as analogs of these “hits” with the goal of generating more effective novel compounds as possible therapeutics for heart disease.
  • 2. Assay development and screening: Novel synthetic chemical analogs were studied in cell-based assays to evaluate potency of stimulating cardiac cell development relative to the starting 'hit' compounds. The biological data contributed to structure activity relationship (SAR) studies, and provided valuable information about parts of the molecules important for cardiomyocyte stem cell differentiation and for other important pharmaceutical properties. The iterative feedback from the biological testing helped to guide the next generation designs of new and ever more effective compounds.
  • 3. Chemical optimization. Focused structure activity relationship (SAR) studies for 4 chemical series from the ESC cardiogenesis differentiation screen were done. SAR for 2 additional chemical classes was done but those agents proved less potent. In addition to SAR, considerable information was obtained leading to improved solubility and membrane permeability of compounds in development, which became a focus of the chemical optimizations.
  • In summary, the work has already led to one or more promising drug-like compounds ready for efficacy testing in animal models and thus, efforts have greatly accelerated the timeline of getting compounds to human patients.
  • The original goals of the proposal were to apply medicinal chemistry to generate more potent and drug-like analogs of small molecules that stimulate differentiation of cardiomyocytes from embryonic stem cells (ESCs) and potentially other progenitor cell types found in adult human heart. During the grant period, we over-achieved each Aim and provided large numbers of drug-like small molecules for cardiomyocyte differentiation studies. In addition, other related information was gained that has considerably expanded our understanding related to developing regenerative medicines.
  • 1. Synthetic Chemistry: From an initial screen of thousands of compounds, six ‘hits’ were identified. Almost 1400 compounds were synthesized as analogs of these “hits” with the goal of generating more effective novel compounds as possible therapeutics for heart disease.
  • 2. Assay development and screening: Novel synthetic chemical analogs were studied in cell-based assays to evaluate potency of stimulating cardiac cell development relative to the starting ‘hit’ compounds. The biological data contributed to structure activity relationship (SAR) studies, and provided valuable information about parts of the molecules important for cardiomyocyte stem cell differentiation and for other important pharmaceutical properties. The iterative feedback from the biological testing helped to guide the next generation design of new and ever more effective compounds.
  • 3. Chemical optimization. Focused structure activity relationship (SAR) studies for 4 chemical series from the ESC cardiogenesis differentiation screen were done. SAR for 2 additional chemical classes was done but those agents proved less potent. In addition to SAR, considerable information was obtained leading to improved solubility and membrane permeability of compounds in development, which became a focus of the chemical optimizations. The most potent compounds increased stem cell differentiation to cardiomyocytes 5-10 fold. The compounds were non-toxic, reasonably tractable to make, stable and were water-soluble and hence relatively easy to handle.
  • 4. A number of biological signaling pathways were identified as affiliated with cardiomyocyte differentiation. One such pathway also is involved in anti-cancer activities. Thus, our efforts in identifying cardiomyocyte differentiation agents led us to study novel biology associated with cancer. One “hit” of this signaling pathway was chosen to do synthetic chemistry and “hit” to lead refinement. Approximately 100 compounds were synthesized and tested for inhibition of this signaling pathway.
  • In summary, the work has already led to a number of promising drug-like compounds ready for efficacy testing in animal models and thus, efforts have greatly accelerated the timeline of getting compounds to human patients. A total of 1500 compounds were synthesized to optimize the potency and properties of cardiomyocyte differentiation agents. The most potent stimulated production of human cardiomyocytes 5-10-fold compared to vehicle-stimulated cells.

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