Heart Disease

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

Enhancing healing via Wnt-protein mediated activation of endogenous stem cells

Funding Type: 
Early Translational I
Grant Number: 
TR1-01249
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.

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

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

Building Cardiac Tissue from Stem Cells and Natural Matrices

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

VEGF signaling in adventitial stem cells in vascular physiology and disease

Funding Type: 
New Faculty II
Grant Number: 
RN2-00909
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.

Induction of cardiogenesis in pluripotent cells via chromatin remodeling factors

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

Prospective isolation of hESC-derived hematopoietic and cardiomyocyte stem cells

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

Engineering a Cardiovascular Tissue Graft from Human Embryonic Stem Cells

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

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.

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