Heart Disease

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

Human ES cell based therapy of heart failure without allogenic immune rejection

Funding Type: 
Early Translational III
Grant Number: 
TR3-05559
ICOC Funds Committed: 
$1 857 600
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Heart failure is a major and ever-growing health problem affecting an estimated 5.8 million Americans with about half a million new cases every year. There are limited therapeutic options for heart failure. Heart transplantation is effective but has limited impact due to scarcity of donor organs and eventual immune rejection even under chronic immune suppression. Therefore, there is a clear unmet medical need to develop new effective therapies to treat heart failure. Human ES cell based cell therapy could provide a cure for heart diseases by providing renewable source of human cardiomyocytes (CMs) to restore lost cardiomyocytes and cardiac functions. In support of this notion, hESC-derived cardiomyocytes (hESC-CMs) can repopulate lost cardiac muscle and improve heart function in preclinical animal models of advanced heart failure. However, one key bottleneck hindering such clinic development is that hESC-CMs will be rejected by allogenic immune system of the recipients, and the typical immunosuppressant regimen is especially toxic for patients with heart diseases and leads to increased risk of cancer and infection. To resolve this bottleneck, I propose to develop a novel approach to protect the hESC-CMs from allogenic immune system. If successful, our approach will not only greatly improve the feasibility of developing hESC-CMs to treat heart failure but also has broad application in other hESC-based cell therapies for which allogenic immune rejection remains a major hurdle.
Statement of Benefit to California: 
Heart disease is a leading cause of death and disability among Californians with an above average rate of mortality. It costs the State tremendous expenditure for the treatment and loss of productivity. There are limited therapeutic options for advanced heart diseases. In this context, heart transplantation is effective but limited by the shortage of donors. Therefore, there is clearly an urgent unmet medical need for new and effective therapies to treat heart failure. Human ES cell based cell therapy approach offers the unique potential to provide renewable source of cardiomyocytes to treat heart failure by restoring lost cardiomyocytes and cardiac function. However, one key bottleneck is that the allogenic hESC-derived cardiomyocytes will be immune rejected by recipients, and the typical immunosuppression regimen is especially toxic for fragile patients with heart diseases. In addition, chronic immune suppression greatly increases the risk of cancer and infection. Our proposed research is aimed to develop novel strategies to prevent allogenic immune rejection of hESC-derived cardiomyocytes without inducing systemic immune suppression. If successful, our approach will greatly facilitate the development of hESC-derived cardiomyocytes for treating heart disease and also has broad application in other hESC-based therapy for which allogenic immune rejection remains a bottleneck.
Progress Report: 
  • Heart failure affects an estimated 5.8 million Americans with about half a million new cases every year. It is also one of the leading causes of death and loss of productivity in California. There is a clear unmet medical need to develop new therapies to treat patients with heart failure. Human embryonic stem cells (hESCs) can undergo unlimited self-renewal and retain the pluripotency to differentiate into all cell types in the body. Therefore, as a renewable source of various cell types in the body, hESCs hold great promise for the cell replacement therapy of many human diseases. In this context, significant progress has been made in the differentiation of hESCs into functional cardiomyocytes (CMs), providing the potential of cell replacement therapy to cure heart diseases through the restoration of lost cardiac function. However, one key bottleneck hindering the clinic development of hESC-derived CMs is that hESC-derived CMs will be rejected by allogenic immune system of the recipients, and the typical immunosuppressant regimen can be highly toxic for patients with heart diseases. To resolve this bottleneck and improve the feasibility of the hESC-based therapy of heart failure, we developed and validated a novel approach to protect the hESC-derived CMs from the allogenic human immune system in vivo.
  • Heart failure is a major disease in California with limited therapeutic options. It costs the State tremendous expenditure in treatment and loss in productivity. While heart transplant is effective in treating the disease, this option is limited by the scarcity of heart donors and the modest graft survival rate (50%) ten years after transplantation. With their unlimited self-renewal capability and pluripotency to differentiate into all cell types in the body, human ES cells (hESCs) hold great promise for human cell therapy. Therefore, cell therapy approaches with hESC-derived CMs have the unique potential for a cure by restoring lost CMs and cardiac function. Despite significant progress in differentiating hESCs into CMs that are capable of partially restoring heart functions in myocardia infarction (MI) animal models, one key bottleneck remaining is that the allogenic hESC-derived CMs will be immune rejected by the recipients, and the typical immunosuppression regimen is especially toxic for patients with advanced heart diseases. By developing a novel approach to prevent allogenic immune rejection of hESC-derived CMs without the typical immunosuppression, we showed that genetically modified hESCs can be efficiently differentiated into cardiomyocytes, which exhibit characteristic electric physiological properties and are protected from allogenic immune rejection.

Engineering microscale tissue constructs from human pluripotent stem cells

Funding Type: 
Research Leadership 14
Grant Number: 
LA1_C14-08015
ICOC Funds Committed: 
$6 368 285
Disease Focus: 
Heart Disease
Neurological Disorders
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Directly Reprogrammed Cell
Public Abstract: 
Tissues derived from stem cells can serve multiple purposes to enhance biomedical therapies. Human tissues engineered from stem cells hold tremendous potential to serve as better substrates for the discovery and development of new drugs, accurately model development or disease progression, and one day ultimately be used directly to repair, restore and replace traumatically injured and chronically degenerative organs. However, realizing the full potential of stem cells for regenerative medicine applications will require the ability to produce constructs that not only resemble the structure of real tissues, but also recapitulate appropriate physiological functions. In addition, engineered tissues should behave similarly regardless of the varying source of cells, thus requiring robust, reproducible and scalable methods of biofabrication that can be achieved using a holistic systems engineering approach. The primary objective of this research proposal is to create models of cardiac and neural human tissues from stem cells that can be used for various purposes to improve the quality of human health.
Statement of Benefit to California: 
California has become internationally renowned as home to the world's most cutting-edge stem cell biology and a global leader of clinical translation and commercialization activities for stem cell technologies and therapies. California has become the focus of worldwide attention due in large part to the significant investment made by the citizens of the state to prioritize innovative stem cell research as a critical step in advancing future biomedical therapies that can significantly improve the quality of life for countless numbers of people suffering from traumatic injuries, congenital disorders and chronic degenerative diseases. At this stage, additional investment in integration of novel tissue engineering principles with fundamental stem cell research will enable the development of novel human tissue constructs that can be used to further the translational use of stem cell-derived tissues for regenerative medicine applications. This proposal would enable the recruitment of a leading biomedical engineer with significant tissue engineering experience to collaborate with leading cardiovascular and neural investigators. The expected result will be development of new approaches to engineer transplantable tissues from pluripotent stem cell sources leading to new regenerative therapies as well as an enhanced understanding of mechanisms regulating human tissue development.

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

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06455
ICOC Funds Committed: 
$3 179 315
Disease Focus: 
Heart Disease
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Despite therapeutic advances, cardiovascular disease remains a leading cause of mortality and morbidity in California. Regenerative therapies that restore normal function after a heart attack would have an enormous societal and financial impact. Although very promising, regenerative cardiac cell therapy faces a number of challenges and technological hurdles. Human induced pluripotent stem cells (hiPSC) allow the potential to deliver patient specific, well-defined cardiac progenitor cells (CPC) for regenerative clinical therapies. We propose to translate recent advances in our lab into the development of a novel, well-defined hiPSC-derived CPC therapy. All protocols will be based on clinical-grade, FDA-approvable, animal product-free methods to facilitate preclinical testing in a large animal model. This application will attempt to translate these findings by: -Developing techniques and protocols utilizing human induced pluripotent stem cell-derived cardiac progenitor cells at yields adequate to conduct preclinical large animal studies. -Validation of therapeutic activity will be in small and large animal models of ischemic heart disease by demonstrating effectiveness of hiPSC-derived CPCs in regenerating damaged myocardium post myocardial infarction in small and large animal models. This developmental candidate and techniques described here, if shown to be a feasible alternative to current approaches, would offer a novel approach to the treatment of ischemic heart disease.
Statement of Benefit to California: 
Cardiovascular disease remains the leading cause of morbidity and mortality in California and the US costing the healthcare system greater than 300 billion dollars a year. Although current therapies slow progression of heart disease, there are few options to reverse or repair the damaged heart. The limited ability of the heart to regenerate following a heart attack results in loss of function and heart failure. Human clinical trials testing the efficacy of adult stem cell therapy to restore mechanical function after a heart attack, although promising, have had variable results with modest improvements. The discovery of human induced pluripotent stem cells offers a potentially unlimited renewable source for patient specific cardiac progenitor cells. However, practical application of pluripotent stem cells or their derivatives face a number of challenges and technological hurdles. We have demonstrated that cardiac progenitor cells, which are capable of differentiating into all cardiovascular cell types, are present during normal fetal development and can be isolated from human induced pluripotent stem cells. We propose to translate these findings into a large animal pre-clinical model and eventually to human clinical trials. This could lead to new therapies that would restore heart function after a heart attack preventing heart failure and death. This will have tremendous societal and financial benefits to patients in California and the US in treating heart failure.
Progress Report: 
  • Cardiovascular disease remains to be a major cause of morbidity and mortality in California and the United States. Despite the best medical therapies, none address the issue of irreversible myocardial tissue loss after a heart attack and thus there has been a great interest to develop approaches to induce regeneration. Our lab has focused on harvesting the full potential of patient specific induced pluripotent stem cells (iPSCs) to use to attempt to regenerate the damaged tissue. We believe that these iPSCs can be potentially used in the future to generate sufficient number of cells to be implanted in the damaged heart to regenerate the lost tissue post heart attack. Our lab has studied how these cardiac progenitors evolve in the developing heart and applied our finding to iPSCs to recapitulate the cardiac progenitors to ultimately use in clinical therapies. We have successfully derived these cardiac progenitors from patient derived iPSCs in a clinical grade fashion to ensure that we can apply same protocols in the future to clinical use if we are successful in demonstrating the efficacy of this therapy in our translational large animal studies that we will be conducting.

Allogeneic Cardiac-Derived Stem Cells for Patients Following a Myocardial Infarction

Funding Type: 
Disease Team Therapy Development - Research
Grant Number: 
DR2A-05735
ICOC Funds Committed: 
$19 782 136
Disease Focus: 
Heart Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
The proposed research will demonstrate both safety and efficacy of a heart-derived stem cell product in patients who have experienced a heart attack either recently or in the past by conducting a mid-stage clinical trial. A prior early-stage trial showed that the product can repair damaged portions of the heart after a heart attack in ways that no commercial therapy currently can. Damaged areas turn irreversibly into scar tissue after the initial event, which can predispose a person to future events and lead to an ongoing worsening of general and heart health. Data from the early-stage trial suggest that treatment with the heart-derived cell product under development can turn scar tissue back into healthy heart muscle. The planned mid-stage trial will hopefully confirm that finding in a larger patient group and provide additional data to support the safety profile of the product. The product is manufactured using heart tissue obtained from a healthy donor and can be used in most other individuals. Its effect is thought to be long-lasting (months-years) although it is expected to be cleared from the body relatively quickly (weeks-months). Treatment is administered during a single brief procedure, requiring a local anesthetic and insertion of a tube (or catheter) into the heart. The overriding goal for the product is to prevent patients who have had a heart attack from deteriorating over time and developing heart failure, a condition which is defined by the heart’s inability to pump blood efficiently and one which affects millions of Americans. Successful completion of the proposed mid-stage trial would lead next to a final, confirmatory trial and then to the application process by which permission to market the product is obtained from the Food and Drug Administration. The end result could be an affordable stem cell therapy effective as part of a treatment regimen after a heart attack.
Statement of Benefit to California: 
The manufacturer of the heart-derived stem cell product under development is a California-based small company who currently employs 7 California residents. Five new local jobs will be created to support the proposed project. Three medical centers located in California will participate in the proposed mid-stage clinical trial. The trial will hopefully bring notoriety to both the company and the medical centers involved while at the same time provide a novel therapeutic option for the many citizens of California afflicted with heart disease. Recent statistics place California among the 50% of states with the highest death rates for heart disease. Therefore, a successfully developed cell product could have a meaningful impact on the home population. Furthermore, as manufacturing needs grow to accommodate the demands of early commercialization, the company anticipates generating 100+ new biotech jobs.
Progress Report: 
  • This project aims to demonstrate both safety and efficacy of a heart-derived cell product in patients who have experienced a heart attack either recently or in the past by conducting a mid-stage (Phase II) clinical trial. The cell product is manufactured using heart tissue obtained from a healthy donor and can be used in most other individuals. Its effect is thought to be long-lasting (months-years) although it is expected to be cleared from the body relatively quickly (weeks-months). Treatment is administered during a single brief procedure, requiring a local anesthetic and insertion of a tube (or catheter) into the heart. The overriding goal for the product is to prevent patients who have had a heart attack from deteriorating over time and developing heart failure, a condition which is defined by the heart’s inability to pump blood efficiently and one which affects millions of Americans. At the outset of the project, a Phase I trial was underway. By the close of the current reporting period, the Phase 1 trial had reached its main safety endpoint, and the Phase II trial was approved to proceed. Fourteen patients were treated with the heart-derived cell product as part of Phase I. The safety endpoint for the trial was pre-defined and took into consideration the following: inflammation in the heart accompanied by an immune response, death due to abnormal heart rhythms, sudden death, repeat heart attack, treatment for symptoms of heart failure, need for a heart assist device, and need for a heart transplant. Both an independent Data and Safety Monitoring Board (DSMB) and CIRM agreed that Phase I met its safety endpoint and that Phase II was approved to proceed. The Phase I participants continue to be monitored for safety and efficacy. Meanwhile, the manufacturing processes established to create cell products for use in Phase I, were employed to create cell products in anticipation of Phase II. A supply of products was readied for use in Phase II. Also in anticipation of Phase II, a number of clinical sites were readied for participation. Manufacturing data and trial status updates were also provided to the Food and Drug Administration (FDA).

Elucidating Molecular Basis of Hypertrophic Cardiomyopathy with Human Induced Pluripotent Stem Cells

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05129
Investigator: 
ICOC Funds Committed: 
$1 425 600
Disease Focus: 
Heart Disease
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people, including trained athletes, and is the most common inherited heart defect. Until now, studies in humans with HCM have been limited by a variety of factors, including variable environmental stimuli which may differ between individuals (e.g., diet, exercise, and lifestyle), the relative difficulty in obtaining human cardiac samples, and inadequate methods of maintaining human heart tissue in cell culture systems. Cellular reprogramming methods that enable derivation of human induced pluripotent stem cells (hiPSCs) from adult cells, which can then be differentiated into cardiomyocytes (hiPSC-CMs), are a revolutionary tool for creating disease-specific cell lines that may lead to effective targeted therapies. In this proposal, we will derive hiPSC-CMs from patients with HCM and healthy controls, then perform a battery of functional and molecular tests to determine the presence of cardiomyopathic disease and associated abnormal molecular programs. With these preliminary studies, we believe hiPSC-CMs with HCM phenotype will dramatically enhance the ability to perform future high-throughput drug screens, evaluate gene and cell therapies, and assess novel electrophysiologic interventions for potential new therapies of HCM. Because HCM is not a rare disease but rather the leading cause of inherited heart defects, we believe the findings here should have broad clinical and scientific impact toward understanding the molecular and cellular basis of HCM.
Statement of Benefit to California: 
Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people and is the most common inherited heart defect. In this study, we will generate hiPSC-derived cardiomyocytes from patients with HCM, then perform a number of functional, molecular, bioinformatic, and imaging analyses to determine the extent and nature of cardiomyopathic disease. We believe hiPSC-CMs with HCM phenotype will dramatically enhance the ability to perform future high-throughput drug screens, evaluate gene and cell therapies, and assess electrophysiologic interventions for potential novel therapies of HCM. The experiments outlined are pertinent and central to the overall mission of CIRM, which seeks to explore the use of stem cell platforms to yield novel mechanistic insights into the molecular and cellular basis of disease. Because HCM is not an orphan disease, but rather the leading cause of sudden cardiac death in young people, we believe the research findings will benefit the state of California and its citizens.
Progress Report: 
  • Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people, including trained athletes, and is the most common inherited heart defect. In this proposal, we will generate human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with HCM. The specific aims are as follow:
  • Specific Aim 1: Generate iPSCs from patients with HCM and healthy controls.
  • Specific Aim 2: Determine the extent of disease by performing molecular and functional analyses of hiPSC-CMs.
  • Specific Aim 3: Rescue the molecular and functional phenotypes using zinc finger nuclease (ZFN) technology.
  • Over the past year, we have now derived iPSCs from a 10-patient family cohort with the MYH7 mutation. Established iPSC lines from all subjects were differentiated into cardiomyocyte lineages (iPSC-CMs) using standard 3D EB differentiation protocols. We found hypertrophic iPSC-CMs exhibited features of HCM such as cellular enlargement and multi-nucleation beginning in the sixth week following induction of cardiac differentiation. We also found hypertrophic iPSC-CMs demonstrated other hallmarks of HCM including expression of atrial natriuretic factor (ANF), elevation of β-myosin/α-myosin ratio, calcineurin activation, and nuclear translocation of nuclear factor of activated T-cells (NFAT) as detected by immunostaining. Blockade of calcineurin-NFAT interaction in HCM iPSC-CMs by cyclosporin A (CsA) and FK506 reduced hypertrophy by over 40%. In the absence of inhibition, NFAT-activated mediators of hypertrophy such as GATA4 and MEF2C were found to be significantly upregulated in HCM iPSC-CMs beginning day 40 post-induction of cardiac differentiation, but not prior to this point. Taken together, these results indicate that calcineurin-NFAT signaling plays a central role in the development of the HCM phenotype as caused by the Arg663His mutation.
  • Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people, including trained athletes, and is the most common
  • inherited heart defect. In this proposal, we will generate and characterize human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with HCM. The
  • specific aims are as follow:
  • Specific Aim 1: Generate iPSCs from patients with HCM and healthy controls.
  • Specific Aim 2: Determine the extent of disease by performing molecular and functional analyses of hiPSC-CMs.
  • Specific Aim 3: Rescue the molecular and functional phenotypes using zinc finger nuclease (ZFN) technology.
  • Over the past year, we have characterized the pathological phenotypes from iPSCs derived from a 10-patient family cohort with the MYH7 mutation.
  • We've differentiated all stablished iPSC lines from all subjects into cardiomyocyte using a modified protocol from that published by Palacek in PNAS 2011. This protocol increased the yield of cardiomyocytes significantly to consistently greater than 70% beating cardiomyocytes. We then tested the electrophysiological properties of iPSC-CMs from control and patients with HCM and found that both control and patient iPSC-CM display atrial, ventricular and nodal-like electrical waveforms by whole cell patch clamping. However, by day 30, a large subfraction (~40%) of the HCM iPSC-CM exhibit arrhythmic waveforms including delayed after-depolarizations (DADs) compared with control (~5.1%). In addition we found that treatment of HCM hiPSC-CM with positive inotropic agents (beta-adrenergic agonist - isoproterenal) for 5 days caused an earlier increase in cell size by 1.7 fold as compared to controls and significant increase in irregular calcium transients. Furthermore, we found that HCM iPSC-CMs exhibited frequent arrhythmia due to their increased intracellular calcium level by 30% at baseline. These HCM iPSC-CM also exhibited decreased calcium release by the sarcoplasmic reticulum. These findings emphasize the role of irregular calcium recycling in the pathogenesis of HCM. To confirm that the regulation of myocyte calcium is the key to HCM pathogenesis, we treated several lines from multiple HCM patients with calcium channel blocker (verapamil/diltiazem) and found that this treatment significantly ameliorated all aspects of the HCM phenotype including myocyte hypertrophy, calcium handling abnormalities, and arrhythmia. These finding supports the use of calcium channel blockers in patients with HCM and encourages further clinical studies in HCM patients using these agents.
  • Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people, including trained athletes, and is the most common
  • inherited heart defect. In this proposal, we will generate human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with HCM. The
  • specific aims are as follow:
  • Specific Aim 1: Generate iPSCs from patients with HCM and healthy controls.
  • Specific Aim 2: Determine the extent of disease by performing molecular and functional analyses of hiPSC-CMs.
  • Specific Aim 3: Rescue the molecular and functional phenotypes using zinc finger nuclease (ZFN) technology.
  • Over the past year, we have characterized iPSC-CMs from a 10-patient family cohort with the MYH7 mutation using standard 3D EB differentiation protocols.
  • We found normal and hypertrophic iPSC-CMs were predictive as in vitro model for arrhythmia screening using microelectroarrays and single cell patch-clamping
  • analysis. For example, we found that administration of catecholamine drug norepinephrine causes the formation of torsade de point which is a lethan arrhythmia.
  • This recapitulates the phenotype in patients with HCM receiving catecholamine drugs. We also found increase in torsade formation when the iPSC-CMs are treated
  • with hERG blockers that are also known to cause increases in arrhythmia in HCM patients. We believe the use of hiPSC-CM from healthy individuals and patients with
  • genetic heart disease can help predict the potential arrhythmic risk in existing or new drug agents that are undergoing FDA evaluation.
  • We have also generated HCM mutations in lines of normal iPSC to determine whether these mutant lines will exhibit HCM phenotype. This would satisfy the Koch's postulate
  • with regards to the role of the mutant DNA sequence on HCM manifestation. We found, using TALEN and piggyBac transposon technologies that genome edited can be generated
  • to carry R663H mutation in the MYH7 gene and that these genome edited iPSC-CM recapitulated the HCM phenotype associated with the R663H mutation such as sarcomere
  • disassembly and intracellular calcium abnormalities as well as contractile arrhythmias. We have also corrected mutant HCM human iPSC from patients with MYH7 R663H mutation
  • and show that these corrected iPSC-CM exhibit normal sarcomeric phenotype with smaller cell size and reduced calcium transient irregularities.

Antibody tools to deplete or isolate teratogenic, cardiac, and blood stem cells from hESCs

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02060
ICOC Funds Committed: 
$1 869 487
Disease Focus: 
Blood Disorders
Heart Disease
Liver Disease
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
Purity is as important for cell-based therapies as it is for treatments based on small-molecule drugs or biologics. Pluripotent stem cells possess two properties: they are capable of self regeneration and they can differentiate to all different tissue types (i.e. muscle, brain, heart, etc.). Despite the promise of pluripotent stem cells as a tool for regenerative medicine, these cells cannot be directly transplanted into patients. In their undifferentiated state they harbor the potential to develop into tumors. Thus, tissue-specific stem cells as they exist in the body or as derived from pluripotent cells are the true targets of stem cell-based therapeutic research, and the cell types most likely to be used clinically. Existing protocols for the generation of these target cells involve large scale differentiation cultures of pluripotent cells that often produce a mixture of different cell types, only a small fraction of which may possess therapeutic potential. Furthermore, there remains the real danger that a small number of these cells remains undifferentiated and retains tumor-forming potential. The ideal pluripotent stem cell-based therapeutic would be a pure population of tissue specific stem cells, devoid of impurities such as undifferentiated or aberrantly-differentiated cells. We propose to develop antibody-based tools and protocols to purify therapeutic stem cells from heterogeneous cultures. We offer two general strategies to achieve this goal. The first is to develop antibodies and protocols to identify undifferentiated tumor-forming cells and remove them from cultures. The second strategy is to develop antibodies that can identify and isolate heart stem cells, and blood-forming stem cells capable of engraftment from cultures of pluripotent stem cells. The biological underpinning of our approach is that each cell type can be identified by a signature surface marker expression profile. Antibodies that are specific to cell surface markers can be used to identify and isolate stem cells using flow cytometry. We can detect and isolate rare tissue stem cells by using combinations of antibodies that correspond to the surface marker signature for the given tissue stem cell. We can then functionally characterize the potential of these cells for use in regenerative medicine. Our proposal aims to speed the clinical application of therapies derived from pluripotent cell products by reducing the risk of transplanting the wrong cell type - whether it is a tumor-causing residual pluripotent cell or a cell that is not native to the site of transplant - into patients. Antibodies, which exhibit exquisitely high sensitivity and specificity to target cellular populations, are the cornerstone of our proposal. The antibodies (and other technologies and reagents) identified and generated as a result of our experiments will greatly increase the safety of pluripotent stem cell-derived cellular therapies.
Statement of Benefit to California: 
Starting with human embryonic stem cells (hESC), which are capable of generating all cell types in the body, we aim to identify and isolate two tissue-specific stem cells – those that can make the heart and the blood – and remove cells that could cause tumors. Heart disease remains the leading cause of mortality and morbidity in the West. In California, 70,000 people die annually from cardiovascular diseases, and the cost exceeded $48 billion in 2006. Despite major advancement in treatments for patients with heart failure, which is mainly due to cellular loss upon myocardial injury, the mortality rate remains high. Similarly, diseases of the blood-forming system, e.g. leukemias, remain a major health problem in our state. hESC and induced pluripotent stem cells (collectively, pluripotent stem cells, or PSC) could provide an attractive therapeutic option to treat patients with damaged or defective organs. PCS can differentiate into, and may represent a major source of regenerating, cells for these organs. However, the major issues that delay the clinical translation of PSC derivatives include lack of purification technologies for heart- or blood-specific stem cells from PSC cultures and persistence of pluripotent cells that develop into teratomas. We propose to develop reagents that can prospectively identify and isolate heart and blood stem cells, and to test their functional benefit upon engraftment in mice. We will develop reagents to identify and remove residual PSC, which give rise to teratomas. These reagents will enable us to purify patient-specific stem cells, which lack cancer-initiating potential, to replenish defective or damaged tissue. The reagents generated in these studies can be patented forming an intellectual property portfolio shared by the state and the institutions where the research is carried out. The funds generated from the licensing of these technologies will provide revenue for the state, will help increase hiring of faculty and staff (many of whom will bring in other, out-of-state funds to support their research) and could be used to ameliorate the costs of clinical trials – the final step in translation of basic science research to clinical use. Only California businesses are likely to be able to license these reagents and to develop them into diagnostic and therapeutic entities; such businesses are at the heart of the CIRM strategy to enhance the California economy. Most importantly, this research will set the platform for future stem cell-based therapies. Because tissue stem cells are capable of lifelong self-renewal, stem cell therapies have the potential to be a single, curative treatment. Such therapies will address chronic diseases with no cure that cause considerable disability, leading to substantial medical expense. We expect that California hospitals and health care entities will be first in line for trials and therapies. Thus, California will benefit economically and it will help advance novel medical care.
Progress Report: 
  • Our program is focused on improving methods that can be used to purify stem cells so that they can be used safely and effectively for therapy. A significant limitation in translating laboratory discoveries into clinical practice remains our inability to separate specific stem cells that generate one type of desired tissue from a mixture of ‘pluripotent’ stem cells, which generate various types of tissue. An ideal transplant would then consist of only tissue-specific stem cells that retain a robust regenerative potential. Pluripotent cells, on the other hand, pose the risk, when transplanted, of generating normal tissue in the wrong location, abnormal tissue, or cancer. Thus, we have concentrated our efforts to devise strategies to either make pluripotent cells develop into desired tissue-specific stem cells or to separate these desired cells from a mixture of many types of cells.
  • Our approach to separating tissue-specific stem cells from mixed cultures is based on the theory that every type of cell has a very specific set of molecules on its surface that can act as a signature. Once this signature is known, antibodies (molecules that specifically bind to these surface markers) can be used to tag all the cells of a desired type and remove them from a mixed population. To improve stem cell therapy, our aim is to identify the signature markers on: (1) the stem cells that are pluripotent or especially likely to generate tumors; and (2) the tissue-specific stem cells. By then developing antibodies to the pluripotent or tumor-causing cells, we can exclude them from a group of cells to be transplanted. By developing antibodies to the tissue-specific stem cells, we can remove them from a mixture to select them for transplantation. For the second approach, we are particularly interested in targeting stem cells that develop into heart (cardiac) tissue and cells that develop into mature blood cells. As we develop ways to isolate the desired cells, we test them by transplanting them into animals and observing how they grow.
  • Thus, the first goal of our program is to develop tools to isolate pluripotent stem cells, especially those that can generate tumors in transplant recipients. To this end, we tested an antibody aimed at a pluripotent cell marker (stage-specific embryonic antigen-5 [SSEA-5]) that we previously identified. We transplanted into animals a population of stem cells that either had the SSEA-5-expressing cells removed or did not have them removed. The animals that received the transplants lacking the SSEA-5-expressing cells developed smaller and fewer teratomas (tumors consisting of an abnormal mixture of many tissues). Approaching the problem from another angle, we analyzed teratomas in animals that had received stem cell transplants. We found SSEA-5 on a small group of cells we believe to be responsible for generating the entire tumor.
  • The second goal of the program is to develop methods to selectively culture cardiac stem cells or isolate them from mixed cultures. Thus, in the last year we tested procedures for culturing pluripotent stem cells under conditions that cause them to develop into cardiac stem cells. We also tested a combination of four markers that we hypothesized would tag cardiac stem cells for separation. When these cells were grown under the proper conditions, they began to ‘beat’ and had electrical activity similar to that seen in normal heart cells. When we transplanted the cells with the four markers into mice with normal or damaged hearts, they seemed to develop into mature heart cells. However, these (human) cells did not integrate with the native (mouse) heart cells, perhaps because of the species difference. So we varied the approach and transplanted the human heart stem cells into human heart tissue that had been previously implanted in mice. In this case, we found some evidence that the transplanted cells differentiated into mature heart cells and integrated with the surrounding human cells.
  • The third goal of our project is to culture stem cells that give rise only to blood cells and test them for transplantation. In the past year, we developed a new procedure for treating cultures of pluripotent stem cells so that they differentiate into specific stem cells that generate blood cells and blood vessels. We are now working to refine our understanding and methods so that we end up with a culture of differentiated stem cells that generates only blood cells and not vessels.
  • In summary, we have discovered markers and tested combinations of antibodies for these markers that may select unwanted cells for removal or wanted cells for inclusion in stem cell transplants. We have also begun to develop techniques for taking a group of stem cells that can generate many tissue types, and growing them under conditions that cause them to develop into tissue-specific stem cells that can be used safely for transplantation.
  • Our program is focused on improving methods to purify blood-forming and heart-forming stem cells so that they can be used safely and effectively for therapy. Current methods to identify and isolate blood-forming stem cells from bone marrow and blood are efficient. In addition, we found that if blood-forming stem cells are transplanted, they create in the recipient an immune system that will tolerate (i.e., not reject) organs, tissues, or other types of tissue stem cells (e.g. skin, brain, or heart) from the same donor. Many living or recently deceased donors often cannot contribute these stem cells, so we need, in the future, a single biological source of each of the different types of stem cells (e.g., blood and heart) to change the entire field of regenerative medicine. The ultimate reason to fund embryonic stem cell and other pluripotent stem cell research is to create safe banks of predefined pluripotent cells. Protocols to differentiate these cells to the appropriate blood-forming stem cells could then be used to induce tolerance of other tissue stem cells from the same embryonic stem cell line. However, existing protocols to differentiation stem cells often lead to pluripotent cells (cells that generate multiple types of tissue), which pose a risk of generating normal tissue in the wrong location, abnormal tissue, or cancers called teratomas. To address these problems, we have concentrated our efforts to devise strategies to (a) make pluripotent cells develop into desired tissue-specific stem cells, and (b) to separate these desired cells from all other cells, including teratoma-causing cells. In the first funding period of this grant, we succeeded in raising antibodies that identify and eliminate teratoma-causing cells.
  • In the past year, we identified surface markers of cells that can only give rise to heart tissue. First we studied the genes that were activated in these cells, further confirming that they would likely give rise to heart tissue. Using antibodies against these surface markers, we purified heart stem cells to a higher concentration than has been achieved by other purification methods. We showed that these heart stem cells can be transplanted such that they integrate into the human heart, but not mouse heart, and participate in strong and correctly timed beating.
  • In the embryo, a group of early stem cells in the developing heart give rise to (a) heart cells; (b) cells lining the inner walls of blood vessels; and (c) muscle cells surrounding blood vessels. We identified cell surface markers that could be used to separate each of these subsets from each other and from their common stem cell parents. Finally, we determined that a specific chemical in the body, fibroblast growth factor, increased the growth of a group of pluripotent stem cells that give rise to more specific stem cells that produce either blood cells or the lining of blood vessels. This chemical also prevented blood-forming stem cells from developing into specific blood cells.
  • In the very early embryo, pluripotent cells separate into three distinct categories called ‘germ layers’: the endoderm, mesoderm, and ectoderm. Each of these germ layers later gives rise to certain organs. Our studies of the precursors of mesoderm (the layer that generates the heart, blood vessels, blood, etc.) led us by exclusion to develop techniques to direct ES cell differentiation towards endoderm (the layer that gives rise to liver, pancreas, intestinal lining, etc.). A graduate student before performed most of this work before he joined in our effort to find ways to make functional blood forming stem cells from ES cells. He had identified a group of proteins that we could use to sequentially direct embryonic stem cells to develop almost exclusively into endoderm, then subsets of digestive tract cells, and finally liver stem cells. These liver stem cells derived from embryonic stem cells integrated into mouse livers and showed signs of normal liver tissue function (e.g., secretion of albumin, a major protein in the blood). Using the guidelines of the protocols that generated these liver stem cells, we have now turned our attention back to our goal of generating from mesoderm the predecessors of blood-forming stem cells, and, ultimately, blood-forming stem cells.
  • In summary, we have continued to discover signals that cause pluripotent stem cells (which can generate many types of tissue) to become tissue-specific stem cells that exclusively develop into only heart, blood cells, blood vessel lining cells, cells that line certain sections of the digestive tract, or liver cells. This work has also involved determining the distinguishing molecules on the surface of various cells that allow them to be isolated and nearly purified. These results bring us closer to being able to purify a desired type of stem cell to be transplanted safely to generate only a single type of tissue.
  • The main focus of our program is to improve methods to generate pure populations of tissue-specific stem cells that form only heart tissue or blood. Such tissue-specific stem cells are necessary for developing safe and effective therapies. If injected into patients with heart damage, heart-forming stem cells might be used to regenerate healthy heart tissue. Blood-forming stem cells are capable of regenerating the blood-forming system after cancer therapy and replacing a defective blood forming-system. We showed that blood-forming stem cells from a given donor induce in the recipient permanent transplant tolerance of all organs, tissues, or other tissue stem cells from the same donor. Therefore, having a single biological source of each of the different types of stem cells (e.g., blood and heart) would revolutionize regenerative medicine.
  • Our projects involve generating tissue-specific stem cells from pluripotent stem cells (PSCs), the latter of which are stem cells that can form all tissues of the body. PSCs (which include embryonic stem cells and induced pluripotent stem cells) can turn into all types of more specialized cells in a process known as “differentiation.” Because PSCs can be grown to very large numbers, differentiating PSCs into tissue-specific stem cells could lead to banks of defined tissue stem cells for transplantation into patients—the ultimate reason to conduct PSC research.
  • However, current methods to differentiate PSCs often generate mixtures of various cell types that are unsafe for injection into patients. Therefore, generating a pure population of a desired cell type from PSC is pivotal for regenerative medicine—purity is a key concern for cell therapy as it is with medications.
  • We have invented technologies to purify desired types of cells from complex cell populations, allowing us to precisely isolate a pure population of tissue-specific stem cells from differentiating PSCs for cell therapy. For instance, in our work on heart-forming cells, we developed labels for cells that progressively give rise to heart cells. We used these labeled cells to clarify the natural, stepwise, differentiation process that leads from PSCs to heart-forming stem cells, and finally to different cells within the heart. Exploiting these technologies to isolate desired cell types, we have completed the first step of turning human PSCs into heart-forming stem cells. In the laboratory, when we transplanted these heart-forming stem cells into a human heart, they integrated with the surrounding tissue and participated in correctly timed beating. In the future we hope to deliver heart-forming stem cells into the damaged heart to regenerate healthy tissue.
  • We have also attempted to turn PSCs into blood-forming stem cells by understanding the complex process of blood formation in the early embryo. As mentioned above, if blood-forming stem cells are transplanted into patients, they create in the recipient an immune system that will tolerate (i.e., not reject) other tissues and types of tissue stem cells (e.g., for skin or heart) from the same donor. Thus, turning PSCs into blood-forming stem cells will provide the basis for all regenerative medicine, whereby the blood-forming stem cells and the needed other tissue stem cells can be generated from the same pluripotent cell line and be transplanted together.
  • In parallel studies to those above, we have turned PSCs into liver-forming stem cells. In the embryo, the liver emerges from a cell type known as endoderm, whereas the blood and heart emerge from a different cell type known as mesoderm. We learned that PSCs could only be steered to form endoderm (and subsequently, liver) by diverting them away from the path that leads to mesoderm. Through this approach, we could turn human PSCs into endoderm cells (at >99% purity) and then into liver-forming stem cells that, when injected into the mouse liver, gave rise to human liver cells. This could be of therapeutic importance for human patients with liver damage.
  • Finally, we have developed methods to deplete PSCs from mixtures of cells differentiated from PSCs, because residual PSCs in these mixtures can form tumors (known as teratomas). These methods should increase the safety of PSC-derived tissue stem cell therapy.
  • In summary, we have developed methods to turn PSCs to tissue-specific stem cells that exclusively develop into only heart, blood cells, or liver cells. This work has involved determining the distinguishing molecules on the surface of various cells that allow them to be isolated and nearly purified. These results bring us closer to being able to purify a desired type of stem cell to be transplanted safely to generate only a single type of tissue.

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

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

Human Embryonic Stem Cell-Derived Cardiomyocytes for Patients with End Stage Heart Failure

Funding Type: 
Disease Team Therapy Development - Research
Grant Number: 
DR2A-05394
ICOC Funds Committed: 
$19 999 899
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Patients with end-stage heart failure have a 2-year survival rate of only 50% with conventional medical therapy. This dismal survival rate is actually significantly worse than patients with AIDS, liver cirrhosis, stroke, and other comparable debilitating diseases. Currently available therapies for end stage heart failure include drug and device therapies, as well as heart transplantation. While drug and device therapies have proven effective at reducing symptoms, hospitalizations and deaths due to heart failure, new approaches are clearly required to improve this low survival rate. Organ transplantation is highly effective at increasing patient survival, but is severely limited in its potential for broad-based application by the very low number of hearts that are available for transplantation each year. Stem cell therapy may be a promising strategy for improving heart failure patient outcomes by transplanting cells rather than a whole heart. Several studies have convincingly shown that human embryonic stem cells can be differentiated into heart muscle cells (cardiomyocytes) and that these cells can be used to improve cardiac function following a heart attack. The key objective of this CIRM Disease Team Therapy proposal is to perform the series of activities necessary to obtain FDA approval to initiate clinical testing of human embryonic stem cell-derived cardiomyocytes in end stage heart failure patients.
Statement of Benefit to California: 
Coronary artery disease (CAD) is the number one cause of mortality and morbidity in the US. The American Heart Association has estimated that 5.7 million Americans currently suffer from heart failure, and that another 670,000 patients develop this disease annually. Cardiovascular disease has been estimated to result in an estimated $286 billion in direct and indirect costs in the US annually (NHLBI, 2010). As the most populous state in the nation, California bears a substantial fraction of the social and economic costs of this devastating disease. In recent years, stem cell therapy has emerged as a promising candidate for treating ischemic heart disease. Research by our group and others has demonstrated that human embryonic stem cells (hESCs) can be differentiated to cardiomyocytes using robust, scalable, and cGMP-compliant manufacturing processes, and that hESC-derived cardiomyocytes (hESC-CMs) can improve cardiac function in relevant preclinical animal models. In this proposal, we seek to perform the series of manufacturing, product characterization, nonclinical testing, clinical protocol development, and regulatory activities necessary to enable filing of an IND for hESC-CMs within four years. These IND development activities will be in support of a Phase 1 clinical trial to test hESC-CMs in heart failure patients for the first time. If successful, this program will both pave the way for a promising new therapy to treat Californians with heart failure numbering in the hundreds of thousands, and will further enhance California’s continuing prominence as a leader in the promising field of stem cell research and therapeutics.
Progress Report: 
  • Patients with end-stage heart failure (ESHF), which can result from heart attacks, have a 2-year survival rate of 50% with conventional medical therapy. Unlike cells of other organs, the billions of cardiomyocytes lost due to damage or disease do not regenerate. Recently, implantable mechanical pumps that take over the function of the failing left ventricle (left ventricular assist devices; LVADs) have been used to prolong the lives of heart failure patients. However, these devices carry an increased risk of stroke. The only current bona fide cure for ESHF is heart transplantation, but the shortage of donor organs and the risks associated with life-long use of powerful immunosuppressive drugs limit the number of patients that can be helped.
  • Human embryonic stem cells (hESCs) have the unique properties of being able to grow without limit and to be converted into all the cell types of the body, including cardiomyocytes. Our project seeks to find ways to treat patients by replacing their lost cardiomyocytes with healthy ones derived from hESC. The ultimate goal of this 4 year project is to evaluate the feasibility, safety, and efficacy of this approach in both small and large animal models of heart disease and to use this data to initiate a clinical trial to test the therapy in patients.
  • In our first year, we developed methods for producing essentially unlimited quantities of cardiomyocytes from hESCs using a process that is compatible both with clinical needs and large-scale industrial cell production. We have also developed models of heart disease in both rats and pigs, and have begun transplanting the stem cell-derived cardiomyocytes into the rat model. We have demonstrated that stem cell-derived cardiomyocytes can engraft in this animal model and we are testing their effects on the pumping function of the heart, the growth of replacement blood vessels lost during a heart attack, and the size of the scar that typically forms after injury. In the next several years, we will continue to evaluate the safety and function of these cells and will start to transplant in our large animal model of heart disease, which will enable us to test these cells in a heart with very similar characteristics to humans, delivered in a minimally invasive way that would be ideal for clinical use.

Extracellular Matrix Bioscaffold Augmented with Human Stem Cells for Cardiovascular Repair

Funding Type: 
Early Translational III
Grant Number: 
TR3-05626
ICOC Funds Committed: 
$4 939 140
Disease Focus: 
Heart Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
An estimated 16.3 million Americans suffer from coronary heart disease. Every 25 seconds, someone has a coronary event and every minute, someone dies from one. Treatment for coronary heart disease has improved greatly in recent years, yet 1 in 6 deaths in the US in 2007 was still caused by this terrible disease. Stem cells have been used as an supplemental form of treatment but they have been most effective for patients treated immediately after their first heart attack. Unfortunately, stem cell therapy for chronic heart disease and heart failure has been less successful. With current delivery methods for stem cells into the heart, most are washed away quickly, whereas our device will hold them in the area that needs repair. With this project we are testing a novel approach to improve the benefits of stem cell therapy for patients suffering from chronic heart disease. By applying a type of bone marrow stem cells known to enhance tissue repair (mesenchymal stem cells) to a biological scaffold, we hope to greatly amplify the beneficial properties of both the stem cells and the biological scaffold. This device will be implanted onto an appropriate preclinical model that have been treated so as to mirror the chronic heart disease seen in humans. We predict that this novel device will heal the damaged heart and improve its function to pave the way for a superior treatment option for the thousands of Americans for whom the unlikely prospect of a heart transplant is currently the only hope.
Statement of Benefit to California: 
Heart disease is the number one cause of death and disability in California and in the US as a whole. An estimated 16.3 million Americans over the age of 20 suffer from coronary heart disease (CHD) with an estimated associated cost of $177.5 billion and CHD accounted for 1 in 6 deaths in the US in 2007. Advances in treatment have decreased early mortality but consequently lead to an increase in the incidences of heart failure (HF). Patients with HF have a 50 percent readmission rate within six months, which is a heavy cost both in terms of quality of life and finances. The high cost of caring for patients with HF results primarily from frequent hospital readmissions for exacerbations. The need for efficient treatment strategies that address the underlying cause, massive loss of functional myocardium, is yet to be met. We believe that present project proposal, development of a combined mesenchymal stem cell and extra cellular matrix scaffold device, will lead to improved standards of care for patients suffering from chronic myocardial infarction who are thus at risk of developing HF. By not only retarding disease progression but by actually restoring cardiac function, we believe that the proposed project will have a tremendous impact on both the cost of care as well as the quality of life for large groups of Californians and patients worldwide for whom the improbable prospect of heart transplantation is the only curative treatment option available.
Progress Report: 
  • Heart disease is a major cause of death and disability in the US, accounting for 1 in every 4 deaths and costing more than 100 billion annually. While significant improvements have been made towards treating and managing heart disease, we are still not able to effectively return the heart to a healthy state and cure the patients. With our project we have set out to develop a novel strategy for not only halting the disease progression but to reverse the devastating effect on the function of the heart. By combining bone marrow mesenchymal stromal cells with a biological scaffold material, we hope to create a patch for the heart that will support and regenerate the diseased tissue to the point where the patient will be relieved of the burden of their disease and have a markedly improve quality of life. We have in the past year made significant advances toward establishing an animal disease model in which we can study novel ways of treating heart disease. We have in the same time isolated and characterized cells that reside in the bone marrow and that have the potential to heal the diseased tissue by improving blood flow, minimize scarring and generally promoting recovery of the heart function. We have studies these cells under when grown under different conditions and found their ability to mediate tissue regeneration to be highly dependent on their local environments. We are currently trying to identify the optimal combination of cells and microenvironment that may achieve maximal regenerative effect in our disease model and ultimately help our patient combat their heart disease.
  • Cardiovascular diseases remain the leading cause of death and disability in the United States. Even with optimal intervention, patients that suffer from an initial coronary event are prone to development of ischemic heart disease (IHD). Current therapies for IHD such medication, percutaneous coronary intervention, anticoagulants, and coronary artery bypass grafting are incapable of rescuing necrotic tissue and recovering normal cardiac function. The only current curative therapy is heart transplantation; however donor organ supply is severely limited and the vast majority of patients die from congestive heart failure while on the transplant waiting list.
  • Cellular therapies are being explored as a potential cure for IHD. In the majority of these trials, cells are injected in suspension into either vasculature or directly into the ischemic myocardium. Clinical outcomes have clearly demonstrated the safety of these cell based therapies. However, clinical improvements have been modest at best, ostensibly due to poor long term donor cells survival and retention.
  • Mesenchymal stem cells (MSCs) are an attractive allogeneic stem cell source for cardiac regenerative therapies. MSCs are considered to be immunoprivileged in that they modulate and evade the host immune microenvironment, thus making them ideal candidates for allogeneic transplantation. MSCs also facilitate regeneration by secreting angiogenic and chemotactic factors that facilitate new blood vessel formation and recruitment of host stem and progenitor cells.
  • Porcine small intestinal submucosa extracellular matrix (SIS-ECM) is a bioscaffold produced from the small intestine of pigs. It has been found to exert a variety of beneficial pro-regenerative functions, hereunder modulating the chemotactic and immune response and releasing large amounts of pro-angiogenic factors. SIS-ECM is ideal in surgical applications as a replacement for synthetic materials in that it facilitates site specific regeneration and resorbs into native tissue without a need for later removal.
  • The overall goal of this project is to generate a MSC seeded SIS-ECM device for the treatment of IHD. The hypothesis is that the combination of MSCs and SIS-ECM will produce a device with regenerative properties that exceed either component alone. We will with this project develop a porcine myocardial infarct (MI) model that mimics the hallmarks of the human disease. We will then test the proposed device in this model and monitor functional improvement as compared to control animals and animals receiving cells or SIS-ECM alone. We will also verify in vitro that human and porcine MSCs are phenotypically and functionally equivalent to confirm that the results obtained in our porcine model are relevant for the human setting with a high probability. Finally, we will explore mechanisms of action in vitro in relevant assay and in vivo in rat myocardial infarct models.
  • Major accomplishments in this reporting period:
  • 1. We successfully established a reproducible porcine chronic MI model (CMI) and an acute myocardial infarct (AMI) model. We tested two routes of delivery, epicardial patch and intramyocardial injection. We also optimized orientation and seeding density of the device as well as telemetry implantation in a non-injury porcine sternotomy model. We conclude that the CMI model is well suited for the upcoming studies where we will transplantation our device as an epicardial patch with the MSC seeded side facing the epicardium and seeded below maximal capacity to be the favored approach.
  • 2. We found that MSCs from human and porcine bone marrow samples can readily be isolated, expanded and banked using identical methodology. We created master cell banks from three donors for each species. We additionally generated working cell banks of eGFP and Luciferase overexpressing MSCs for both species. We furthermore confirmed, again using identical methodology that both human and porcine MSCs are analogous with respect to tri-lineage potential, cells surface marker expression and karyotype. Moreover, these major MSC hallmarks are not altered in response to seeding onto SIS-ECM. Finally, we are completing similar studies for rat MSCs
  • 3. We have confirmed that human and porcine MSCs are analogous in the expression pattern of angiogenic factors. We also found that the migratory effect of culture supernatants from human or porcine MSCs seeded onto plastic or SIS-ECM is comparable. Additionally, we found that secretion levels of inflammatory cytokines and in vitro tube formation from culture supernatant was comparable for human MSCs seeded on plastic or SIS-ECM. We furthermore established an AMI model in both immune competent and immune deficient rats. Using these models we have demonstrated significant disease modifying effects of the rat DC analogue as compared to SIS-ECM or MSCs alone. Finally we found improved cell retention at the site of implant for our human DC in the immune deficient SCID rat AMI model.

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.

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