Heart disease is the number one cause of death in the US. Heart muscle injured during a heart attack does not regenerate, and the resulting damage leads to heart failure, which inflicts almost 6 million people in the US alone. Recently, several studies have shown that direct injection of stem cell-derived heart cells may offer regenerative potential in the damaged heart. However, injected heart cells often lack the spatial and temporal organization required to create uniform tissue with synchronized beating, while rapid donor cell death poses another key limitation. For these reasons, we propose to transplant engineered heart muscle (EHM) that is spatially and temporally organized into a relevant large animal model. Our proposal addresses unique translational challenges pertaining to tissue engineered heart repair by scaling our established human induced pluripotent stem cell (iPSC) differentiation protocol to create one billion human and large animal model cardiomyocytes for each EHM, in order to meet clinical demands by: (1) adopting our established human EHM tissue engineering process to the large animal model; (2) defining conditions for EHM implantation; and (3) performing a pivotal feasibility, safety, and efficacy study in the large animal model with chronic heart failure. Our studies will establish long-term safety and efficacy of iPSC-EHM therapies in a clinically relevant large animal model, which will overcome a major unresolved bottleneck to the translation of stem cell therapies to humans.
Cardiovascular disease (CVD) affects more than 1.7 million Californians. The societal and financial costs are tremendous, with CVD accounting annually for an estimated $8 billion in California health care costs alone. Following a heart attack, the endogenous regenerative process is not sufficient to compensate for heart tissue death. Thus, using regenerative therapies with human stem cells to form engineered heart tissue is emerging as a promising therapeutic avenue. Engineered tissues are already being used in patients needing artificial blood vessels, bladders, and tracheas. Our multidisciplinary team proposes to create human engineered heart tissue (EHT) for treatment of post-attack heart failure in a clinically-enabling large animal model, and we are confident we will be able to move our potential therapy into preclinical human trials. Development of therapies 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, our research may provide an opportunity for California to benefit from royalties, patents, and licensing fees, which will create cutting-edge projects, attractive jobs, and innovative therapies that will generate millions of dollars in new tax revenues and opportunities in our state. Finally, our research could further advance the flourishing biotech industry in California, serving as a crucial engine to power California’s economic future.
Heart, stroke and other cardiovascular diseases are responsible for ~17 million deaths per year globally and this number is predicted to reach 23.3 million by 2030. Cardiovascular diseases impose a staggering annual cost of $300 billion on the U.S. health care system. Heart transplantation is the ultimate solution to end-stage heart failure. However, a major limitation in treating cardiac injury is the limited availability of donors; as a result, only a small fraction of patients will benefit from heart transplantation. Tissue engineering holds a great promise to create functional tissue constructs that can reestablish the structure and function of injured tissue with exciting success stories. However, many challenges regarding their development still remain. It is the goal of this project to develop a novel 3D bioprinting technology to fabricate cardiac tissues made from cell-laden hydrogels with engineered microvasculature. By integrating the advanced 3D bioprinting with stem cell technology, functional cardiac tissues will be created with biomimetic 3D microarchitecture and functional vasculature. This novel 3D-printed cardiac tissue will heal the damaged heart and improve its function to pave the way for a superior treatment option for the millions of cardiac patients in the U.S.
Heart disease and other cardiovascular diseases are the #1 killer in California and remain a leading cause of disability and death. A major limitation in treating cardiac injury is the failure of current therapies to induce myocardium regeneration. Due to the limited availability of donors, only a fraction of individuals who could benefit from heart transplantations actually receive them. One possible avenue for remedying this situation is to artificially engineer cardiac tissues. Tissue engineering techniques have been successfully applied to engineer many types of tissue; however, many challenges regarding their development still remain. This proposal aims to make an advance in tissue engineering by developing a novel 3D bioprinting technology to fabricate tissues made from cell-laden hydrogels with engineered microvasculature. The completion of this work will be a paradigm shift and a landmark achievement in efforts towards clinical treatments of vascularized cardiac tissue using stem cells. This advanced technology can also have a significant economical impact as heart diseases impose a staggering annual cost of $300 billion on the U.S. health care system. In addition, the development of the 3D bioprinting technology and advanced biomaterials will keep California and the U.S. as a whole in the leading position in this emerging field.
As ongoing CIRM-funded development of regenerative medicine (RM) progresses, the demand for increasing numbers of pluripotent stem cells and their differentiated derivatives has also increased. We have established a scalable suspension culture system for the production of large quantities of hESC for banking and to seed production of a number of regenerative medicine cell types, notably retinal pigmented epithelia, neural stem cells, dopaminergic neurons and cardiomyocytes, that support a number of CIRM and NIH-funded groups. In addition, we have adapted this system for the suspension production of several hESC derivative cell types, notably cardiomyocytes. While our system has provided unprecedented production capability for a number of cell products in pre-clinical and imminent clinical studies, it has proven impractical to scale up to the level that will be required for clinical trials for some hESC cell products, notably cardiomyocytes, due to high expected human doses. This project will resolve this scale-up challenge by adapting our suspension cell culture system, that is limited to 1-3L spinner culture flasks, to a more readily scalable and controllable suspension bioreactor system that utilizes “bags” capable of volumes up to 500L. Achieving this objective will remove a key barrier to progressing RM for cardiac applications as well as open the door to large clinical trials and commercialization of other regenerative medicine cell products in the years to come.
We have developed GMP-compliant suspension cell culture processes for scalable production of hPSC and derivatives. These processes have been invaluable in our support of CIRM- and NIH-funded regenerative medicine projects, including those with RPE, NSC, DA neurons and cardiomyocytes (CM), as well as for production of GMP banks of hPSC for various projects. Our GMP-compliant suspension culture CM production process has made pre-clinical animal studies and small early clinical trials practical. However, while our current CM system is readily transferred to other groups and is meeting current production requirements, the scale requirements for anticipated high dose clinical trials is beyond the practical limitation of our spinner flask-based system. hPSC and CM are sensitive to changes in shear encountered at every scale-up step and re-optimizing conditions at each step is prohibitively expensive. Our experience using bag-based bioreactors for non-hESC products suggests that scale-up in bags will be more controllable and predictable than spinners or stir-tanks reactors. It is also a readily transferred technology. We propose to adapt our suspension hPSC and CM processes to a bag system, optimize conditions at a small scale, then demonstrate scalability at a moderate scale. Success in this project will remove a key barrier to developing many regenerative medicine products, and in particular those where high human doses are anticipated, such as CM.
- Human embryonic stem cells (hESC) have the unique capabilities of unlimited replication and differentiation into multiple cell types. We can now generate induced pluripotent stem cells (iPSC) from adult tissues that possess hESC-like properties. Because of their unique features, both hESCs and iPSCs hold great potential for medical applications. Many different cell types have been produced from both hESCs and iPSCs to repair and/or replace diseased/ damaged tissues in treating conditions such as macular degeneration, heart disease, Parkinson’s disease, spinal cord injury and diabetes. This new type of therapeutic approach is called regenerative medicine (RM); we have already produced clinical grade retinal pigmented epithelial cells (RPEs) from hESC to support clinical trials for the treatment of macular degeneration.
- A crucial challenge for RM is the ability to generate large quantities of stem cell in excess of those produced by traditional 2 dimensional culture systems. We have established a 3-dimensional (3-D) suspension culture system using spinner flasks for large scale production of hESC and iPSC. In this system, hESC/iPSC form cell aggregates which increase in size during the cultivation period, thereby overcoming the bottleneck of 2 dimensional adherent culture. At CBG, hESCs/iPSCs have been produced in our suspension system and used to produce a number of various cell types for RM applications [RPEs, neural stem cells, dopaminergic neurons and cardiomyocytes (CMs)]. Importantly, we have successfully established a process to produce clinical grade CM in suspension. The same approach may also be applied to the production of other cell types.
- While the current suspension system has provided unprecedented production capability for a number of cell products in pre-clinical and clinical studies, it may not be practical to scale up to the level to fulfill the requirement for large clinical trials. To address this hurdle, we have proposed to develop a suspension culture process in the bag-based system to replace spinner flasks for large scale production of hESC/ iPSC and their differentiation. Based on our previous studies using the spinner flask system, we have demonstrated that the cell aggregates are very sensitive to shear force and the culture conditions need to be changed based on the size of each flask due to the differences in shear force caused by the geometry of the vessels. Shear force in the spinner flask system is generated by the rotating propeller inside the flask, while shear force generated by the bag-based system is created by a rocking platform. Based on other studies indicating that the wave bag produces more homogenous gentle shear force, we therefore hypothesize that the bag-based system would be superior to the spinner flask and other propeller-based systems. Specifically, the aim for this project is to resolve the scale-up challenges by adapting our current suspension cell culture system into the bag-based system (which can be more easily scaled up for large scale productions). By achieving this objective, the key barrier will be resolved for cardiac applications in RM and we will have opened the door for large clinical trials and commercialization of other cellular products in RM.
- The first year milestones for this project are to complete the establishment of the procedures for large scale production of hESC/iPSC in the bag-based culture system; the second year milestones include the establishment of the CM differentiation in this system. Here we report the completion of all first year milestones; the specific achievements are summarized below.
- 1. We have successfully adapted hESC to the bag-based system using gas permeable bags and determined agitation conditions (rocking speed and angles), cell density and passage interval, and the concentration of bFGF to be used for optimal results.
- 2. All process parameters were optimized and established for the production of hESC/iPSC in the bag-based system at both the 500 mL and l L scales.
- 3. The reliability of the bag-based system has been tested by passaging the cell culture repeatedly for 3-6 passages. The results have shown the reproducibility of the productions from the serial passages of hESC/iPSC using established parameters in the bag-based system and results met all the successful criteria.
- 4. We successfully performed a 3 L production in the bag-based system that proves the scale up potential of this system for hESC/iPSC. The results have shown 5-fold increases from two continuous passages of hESC with normal karyotype.
- 5. Cell dissociation procedure is a critical aspect of passaging the cells. We compared the use of GentleMACs (MACS, Mitenyi Biotec inc.) with the traditional manual dissociation process and the resultant viability and recovery rates are comparable between the two methods.
- This grant has enabled a plethora of activities in California Stem Cell Genomics. The Stanford Administrative Core for the Center of Excellence in Stem Cell Genomics (CESCG) has been established and is responsible for overseeing joint center activities, and the administration of center-initiated projects (CIP) 1 and 2, and several collaborative research projects (CRP). In the first year of the award the CESCG administration organized monthly telephone conference calls to share research progress and coordinate activities across the Center. On May 1, 2015 the CESCG held its first center-wide retreat in a one-day event at Clark Center on the campus of the Stanford Medical School. The two CIPs have made significant progress. CIP1 has generated a valuable resource of 38 induced pluripotent stem cell lines and established a reliable platform for high throughput derivation of human induced pluripotent stem cell-derived cardiomyocytes for use in downstream high throughput toxicity and drug pharmacology screening assays. CIP2 has completed the first human single cell brain analysis and is in the process of deriving a single cell pancreatic map. We have launched our collaborative research progress grant. Following on the receipt of applications in October 2014 and successful review in January 2015, the Administrative Core at Stanford has also issued subcontract awards for 3 CRPs managed by the CESCG from the Northern California site – two comprehensive project awards CRP-C2 to Daniel Geschwind of UCLA and CRP-C3 to Arnold Kriegstein of UCSF, and a regular project award CRP-R4 to Jeremy Sanford of UCSC. These activities will transform stem cell research in California and continue its preeminence in this area.
Cells in the body take up nutrients from their environment and metabolize them in a complex set of biochemical reactions to generate energy and replicate. Control of these processes is particularly important for heart cells, which need large amounts of energy to drive blood flow throughout the body. Not surprisingly, the nutritional requirements of heart cells are very different than those of stem cells. This proposal will investigate the metabolism of pluripotent stem cells and how this changes during differentiation to cardiac cells. We will determine which nutrients are important to make functional heart cells and use this information to optimize growth conditions for producing heart cells for regenerative medicine and basic biology applications. We accomplish this by feeding cells nutrients (sugar, fat) labeled with isotopes. As these labeled molecules are consumed, the isotopes are incorporated into different metabolites which we track using mass spectrometry. This advanced technique will allow us to see how sugars and fat are metabolized inside stem cells and cardiac cells obtained through differentiation. We will also study the electrical activity of these heart cells to ensure that adequate nutrients are provided for the generation of cells with optimal function. Ultimately, this project will lead to new methods for producing functional heart cells for regenerative medicine and may also lead to insights into how cardiac cells malfunction in heart disease.
Heart disease is one of the leading causes of death in California. As a result, much of the regenerative medicine community in the state and the many Californians suffering from heart failure are interested in obtaining functional heart cells from stem cells. Our work will identify the most important nutrients required to coax stem cell-derived heart cells to behave like true adult heart cells. This information will make more effective cell models for researchers and companies to study how this disease affects heart cell metabolism. Since enzymes are highly targetable with drugs, the basic scientific findings from our work will be of great interest to California biotechnology companies and can stimulate job growth in the state. Our findings will also provide insight into very specific types of genetic heart disease, and this work may lead to additional grants from federal funding sources, bringing about additional revenue and job growth in California. A better understanding of how different nutrients influence heart cell function may provide guidance into new treatment strategies for heart disease. Finally, this work will highlight the importance of diet, nutrition, and healthy heart function, providing useful information relating to public health.
- Growing up we have all heard the phrase, “You are what you eat.” Just like our bodies, stem cells require large quantities of fuel for energy and growth. The same is true for heart cells that continually drive blood flow. Therefore, understanding how stem cells and cardiac cells they generate consume and use different foods is important for characterizing their clinical potential. This grant aims to study how different nutritional fuels influence stem cell and cardiomyocyte (heart cell) growth, differentiation, and function. Using advanced methods that allow us to track how sugar (carbs), protein, and fat are consumed and produced by stem cells, we have identified key nutritional factors that affect stem cell performance and function. Surprisingly, most advanced stem cell media are lacking in several important factors. This deficiency negatively impacts stem cell metabolism in a number of ways, causing increased nutrient consumption, decreased respiration, and oxidative stress. Using this information we have developed improved stem cell growth conditions that mitigates these effects. Finally, we have compared the metabolism of stem cell-derived heart cells to parental stem cells, identifying key differences that will serve as benchmarks to functionally validate the performance and “maturity” of cardiac cells.
This project uses patient hiPSC-derived cardiomyocytes to develop a safe and effective drug to treat a serious heart health condition. This research and product development will provide a novel method for a human genetic heart disorder characterized by long delay (long Q-T interval) between heart beats caused by mutations in the Na+ channel α subunit. Certain patients are genetically predisposed to a potentially fatal arrhythmogenic response to existing drugs to treat LQT3 since the drugs have off-target effects on other important ion channels in cardiomyocytes. We will use patient-derived hiPSC-cardiomyocytes to develop a safer drug (development candidate, DC) that will retain efficacy against the "leaky" Na+-channel yet minimize off-target effects in particular against the K+ hERG channel that can be responsible for the existing drug’s pro-arrhythmic effect. Since this problem is thought to occur severely in patients with the common KCHN2 variant, K897T (~33% of the white population), removing the off-target liability addresses a serious unmet clinical need. Futher, since we propose to modify an existing drug (i.e., do drug rescue), the path from patient-specific hiPSCs to clinic might be easier than for a completely new chemical entity. Lastly, an appealing aspect is that the hiPSCs were derived from a child to test his therapy, & we aim to produce a better drug for his treatment. Our goal is to complete development of the DC and initiate IND-enabling in vivo studies.
In the US, an estimated 850,000 adults are hospitalized for arrhythmias each year, making arrhythmias one of the top five causes of healthcare expenditures in the US with a direct cost of more than $40 billion annually for diagnosis, treatment & rehabilitation. The State of California has approximately 12% of the US population which translates to 102,000 individuals hospitalized every year for arrhythmias. Another 30,000 Californians die of sudden arrhythmic death syndrome every year. Arrhythmias are very common in older adults and because the population of California is aging, research to address this issue is important for human health and the State economy. Most serious arrhythmias affect people older than 60. This is because older adults are more likely to have heart disease & other health problems that can lead to arrhythmias. Older adults also tend to be more sensitive to the side effects of medicines, some of which can cause arrhythmias. Some medicines used to treat arrhythmias can even cause arrhythmias as a side effect. In the US, atrial fibrillation (a common type of arrhythmia that can cause problems) affects millions of people & the number is rising. Accordingly, the same problem is present in California. Thus, successful completion of this work will not only provide citizens of California much needed advances in cardiovascular health technology & improvement in health care but an improved heart drug. This will provide high paying jobs & significant tax revenue.
- The project objective is to design, synthesize and test a sodium-channel inhibitor analog that selectively inhibits the sodium channel and not the potassium channel in patient-derived IPSCs. The strategy is to first work out the approach with wild-type human IPSCs in advance of the patient-derived cells. The status is that the milestones for Year 1 have largely been accomplished. The achievements for this reporting period include nearly locking down the IPSC protocol, developing ultra high throughput kinetic analysis of human cardiomyocytes, developing an enantioselective synthesis of sodium-channel inhibitors and analogs and identifying from a pool of only 49 compounds, a promising sodium-channel inhibitor that provides insight into selective sodium channel inhibition.
- We have been successful with a cardiovascular drug re-purposing. The parent drug possesses significant adverse off-target properties and pharmacological liabilities. We have synthesized new drug candidates that showed improved potency compared to the parent drug molecule. The new compounds showed greater than 50-100-fold improvement in the on-target versus off-target effects compared to the currently used drug treatment. We have found that minor chemical changes to the position of key substituents on the molecule had a great impact on potency and off-target effects. This improvement in potency for the on-target effects of the molecule may lead to lower doses and/or greater therapeutic efficacy of the new drug candidate compared to the currently used drug.
- We have nominated a small subset as lead compounds and advanced these compounds into pre-clinical testing that included chemical and metabolic stability studies. The results from chemical stability studies showed the lead drug candidates to be stable with a half-life of degradation of greater than 30 days. We have tested one of the lead compounds in liver microsomes to monitor potential hepatic metabolism of the compound. In addition, we studied the lead compounds in an in vitro measure of cytotoxicity and found the compounds are sufficiently non-toxic to move forward. The results showed one lead compound to have sufficient stability against metabolic enzymes of the liver to move the molecule forward into more advanced in vivo pre-clinical studies, including safety studies and efficacy studies.
Critical to the long term success of the CIRM iPSC Initiative of generating and ensuring the availability of high quality disease-specific human IPSC lines is the establishment and successful operation of a biorepository with proven methods for quality control, safe storage and capabilities for worldwide distribution of high quality, highly-characterized iPSCs. Specifically the biorepository will be responsible for receipt, expansion, quality characterization, safe storage and distribution of human pluripotent stem cells generated by the CIRM stem cell initiative. This biobanking resource will ensure the availability of the highest quality hiPSC resources for researchers to use in disease modeling, target discovery and drug discovery and development for prevalent, genetically complex diseases.
The generation of induced pluripotent stem cells (iPSCs) from patients and subsequently, the ability to differentiate these iPSCs into disease-relevant cell types holds great promise in facilitating the “disease-in-a-dish” approach for studying our understanding of the pathological mechanisms of human disease. iPSCs have already proven to be a useful model for several monogenic diseases such as Parkinson’s, Fragile X Syndrome, Schizophrenia, Spinal Muscular Atrophy, and inherited metabolic diseases such as 1-antitrypsin deficiency, familial hypercholesterolemia, and glycogen storage disease. In addition, the differentiated cells obtained from iPSCs represent a renewable, disease-relevant cell model for high-throughput drug screening and toxicology/safety assessment which will ultimately lead to the successful development of new therapeutic agents. iPSCs also hold great hope for advancing the use of live cells as therapies for correcting the physiological manifestations caused by disease or injury.
- The California Institute for Regenerative Medicine (CIRM) Human Pluripotent Stem Cell Biorepository is operated by the Coriell Institute for Medical Research and is a critical component of the CIRM Human Stem Cell Initiative. The overall goal of this initiative is to generate, for world-wide use by non-profit and for-profit entities, high quality, disease-specific induced pluripotent stem cells (iPSCs). These cells are derived from existing tissues such as blood or skin, and are genetically manipulated in the laboratory to change into cells that resemble embryonic stem cells. iPSCs can be grown indefinitely in the Petri dish and have the remarkable capability to be converted into most of the major cell types in the body including neurons, heart cells, and liver cells. This ability makes iPSCs an exceptional resource for disease modeling as well as for drug screening. The expectation is that these cells will be a major benefit to the process for understanding prevalent, genetically complex diseases and in developing innovative therapeutics.
- The Coriell CIRM iPSC Biorepository, located at the Buck Institute for Research on Aging in Novato, CA, is funded through a competitive grant award to Coriell from CIRM and is managed by Mr. Matt Self under the supervision of the Program Director, Dr. Steven Madore, Director of Molecular Biology at Coriell. The Biorepository will receive biospecimens consisting of peripheral blood mononuclear cells (PBMCs) and skin biopsies obtained from donors recruited by seven Tissue Collector grant awardees. These biospecimens will serve as the starting material for iPSC derivation by Cellular Dynamics, Inc (CDI). Under a contractual agreement with Coriell, CDI will expand each iPSC line to generate sufficient aliquots of high quality cryopreserved cells for distribution via the Coriell on-line catalogue. Aliquots of frozen cell lines and iPSCs will be stored in liquid nitrogen vapor in storage units at the Buck Institute with back-up aliquots stored in a safe off-site location.
- Renovation and construction of the Biorepository began at the Buck Institute in late January. The Biorepository Manger was hired March 1 and after installation of cryogenic storage vessels and alarm validation, the first biospecimens were received on April 30, 2014. Additionally, Coriell has developed a Clinical Information Management System (CIMS) for storing all clinical and demographic data associated with enrolled subjects. Tissue Collectors utilize CIMS via a web interface to upload and edit the subject demographic and clinical information that will ultimately be made available, along with the iPSCs, via Coriell’s on-line catalogue
- As of November 1 specimens representing a total of 725 unique individuals have been received at the Biorepository. These samples include PBMCs obtained from 550 unique individuals, skin biopsies from 72 unique individuals, and 103 primary dermal fibroblast cultures previously prepared in the laboratories of the CIRM Tissue Collectors. A total of 280 biospecimen samples have been delivered to CDI for the purpose of iPSC derivation. The Biorepository is anticipating delivery of the first batches of iPSCs for distribution in early 2015. These lines, along with the associated clinical data, will become available to scientists via the on-line Coriell catalogue. The CIRM Coriell iPSC Biorepository will ensure safe long-term storage and distribution of high quality iPSCs.
- The California Institute for Regenerative Medicine (CIRM) Human Pluripotent Stem Cell Biorepository is operated by the Coriell Institute for Medical Research and is a critical component of the CIRM Human Stem Cell Initiative. The overall goal of this initiative is to generate, for world-wide use by non-profit and for-profit entities, high quality, disease-specific induced pluripotent stem cells (iPSCs). These cells are derived from human tissues such as blood or skin, and are genetically manipulated in the laboratory to change into cells that resemble embryonic stem cells. iPSCs can be grown indefinitely in the laboratory and have the remarkable capability to be converted into most of the major cell types in the body including neurons, heart and liver cells, etc. This ability makes iPSCs an exceptional resource for disease modeling as well as for therapy target identification and drug screening. The expectation is that these cells will be a major benefit to the process for understanding prevalent, genetically complex diseases and in developing innovative therapeutics.
- The Coriell CIRM hPSC Biorepository, located at the Buck Institute for Research on Aging in Novato, CA, is funded through a competitive grant award to Coriell from CIRM. The Biorepository receives biospecimens consisting of peripheral blood mononuclear cells (PBMCs) and skin biopsies obtained from donors recruited by seven California-based Tissue Collectors funded by CIRM. These biospecimens serve as the starting material for iPSC derivation by Cellular Dynamics, Inc (CDI). Under a contractual agreement with Coriell, CDI expands iPSC clones to generate sufficient aliquots of high quality cryopreserved cells for distribution via Coriell's on-line CIRM Collection Catalog. Aliquots of frozen cell lines and iPSCs are stored in liquid nitrogen vapor in storage units at the Buck Institute with back-up aliquots stored in a Coriell off-site safe storage location.
- Coriell has established a Biorepository facility at the Buck Institute and has also developed a Clinical Information Management System (CIMS) for storing all clinical and demographic data associated with enrolled subjects. Tissue Collectors utilize CIMS via a web interface to upload and edit the subject demographic and clinical information that will ultimately be made available, along with the iPSCs, via Coriell’s on-line catalog.
- The CIRM hPSC Biorepository released in September 2015 the first 285 iPSCs for distribution. These lines, along with the associated clinical data, are available to scientists via the on-line Coriell catalog. The CIRM hPSC Biorepository will ensure safe long-term storage and distribution of high quality iPSCs and their associated demographic, clinical and genomic data.
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.
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.
- First year progress on grant ID1-06557, " Generation and Characterization of High-Quality, Footprint-Free Human Induced Pluripotent Stem Cell (iPSC) Lines From 3000 Donors to Investigate Multigenic Disease" has met all agreed-upon milestones. In particular, Cellular Dynamics International (CDI) has taken lease to approximately 5000 square feet of lab space at the Buck Institute for Research on Aging in Novato, CA. The majority of this space is located within the new CIRM-funded Stem Cell Research Building at the Buck Institute and was extensively reconfigured to meet the specific needs of this grant. All equipment, including tissue culture safety cabinets and incubators, liquid-handling robotics, and QC instrumentation have been installed and qualified. A total of 16 scientists have been hired and trained (13 in Production and 3 in Quality) and more than 20 Standard Operating Procedures (SOPs) have been developed and approved specifically for this project. These SOPs serve to govern the daily activities of the Production and Quality staff and help ensure consistency and quality throughout the iPSC derivation and characterization process. In addition, a Laboratory Information Management System (LIMS) had to be developed to handle the large amount of data generated by this project and to track all samples from start to finish. The first and most important phase of this LIMS project has been completed; additional functionalities will likely be added to the LIMS during the next year, but completion of phase 1 will allow us to enter full production mode on schedule in the first quarter of year 2. Procedures for the shipping, infectious disease testing, and processing of donor samples were successfully implemented with the seven Tissue Collectors. To date, over 700 samples have been received from these Tissue Collectors and derivation of the first 50 patient-derived iPSC lines has been completed on schedule. These cells have been banked in the Coriell BioRepository, also located at the Buck Institute. The first Distribution Banks will be available for commercial release during year 2.
- The goal of CIRM Grant ID1-06557 is to generate high quality induced pluripotent stem cells (iPSC) from blood and skin samples from 3000 donors, many of whom suffer from untreatable medical conditions, and place them in a Repository accessible to scientists around the world. Many common diseases have a complex or unknown origin that makes it difficult to develop effective treatments. iPSCs have the great advantage that they can be generated from adults suffering from a known disease and then converted ("differentiated") into any cell type in the body. For example, iPSC lines from patients with heart disease can be converted into heart cells, iPSC lines from patients with Alzheimer's disease can be converted to brain cell, and iPSC lines from patients with pulmonary fibrosis can be converted into cells of the lung. Comparison of these "patient-derived" iPSC lines with those from healthy donors can help to illuminate the underlying cause of disease and also to serve as a system for discovering drugs to treat the disease in question. Our lab has been tasked with converting control and patient blood or skin cells, collected by scientists throughout the state of California, into high quality iPSCs. In the past year, 1100 samples have been converted to iPSCs and passed quality control. These lines are being used to produce large Distribution Banks that will be made available to scientists globally.
Heart failure is a very common and chronic condition defined by an inability of the heart to pump blood effectively. Over half of cases of heart failure are caused by a condition called dilated cardiomyopathy, which involves dilation of the heart cavity and weakening of the muscle. Importantly, many cases of this disease do not have a known cause and are called “idiopathic” (i.e., physicians do not know why). Over the past 2 decades, doctors and scientists started realizing the disease can cluster in families, leading them to think there is a genetic cause to the disease. This resulted in discovering multiple genes that cause this disease. Nonetheless, the majority of cases of dilated hearts that cluster in families do not have a known genetic cause. Now scientists can turn blood and skin cells into heart cells by genetically manipulating them and creating engineered stem cells called “induced pluripotent stem cells” or iPSCs. This approach enables the scientists to study what chemical or genetic changes are happening to cause the problem. Also because these cells behave similar to the cells in the heart, scientists can now test new medicines on these cells first before trying them in patients. Here we aim to collect tissue from 800 patients without a known cause for their dilated hearts (and 200 control individuals) to help accelerate our understanding of this debilitating disease and hopefully offer new and better treatments.
Heart failure is a significant health burden in California with rising hospitalization and death rates in the state. We have a very limited understanding of the disease and so far the existing treatments only slow down the disease and the changes that happen rather than target the root cause. By studying a subgroup of the dilated cardiomyopathy patients who have no identified cause, we can work on identifying genetic causes of the disease, some of the biology happening inside the heart cell, and provide new treatments that can prevent the disease from happening or progressing. Improving the outcome of this debilitating disease and providing new treatments will go a long way to helping a large group of Californians lead healthier and longer lives. There are estimates that the US economy loses $10 billion (not counting medical costs), because heart failure patients are unable to work. Hence new knowledge and developments gained from this research can go a long way to ameliorating that cost. Finally, heart failure is the most common chronic disease patients in California are hospitalized for. This research targets over half of those admissions. If this research is able to cut the hospitalization rate even by 1%, this would translate to millions of dollars in savings to the state. Continuing to invest in innovation will make our state a hotbed for the biotechnology industry, which in turn advances the state’s economic and educational status.
- Heart failure is a leading cause of morbidity and mortality in California and the Western world with a significant economic burden due to the disease. Over half of heart failure cases are due to dilated cardiomyopathy, a disorder of progressive ventricular dilation and decreased contractility. However, after ischemic cardiomyopathy, the majority of familial cases of dilated cardiomyopathy are unknown or "idiopathic", suggesting a polygenic etiology with a complex genetic-environmental interaction. Traditionally, studying this disorder has been impaired by inability to access cardiac tissue and the limitation of mouse models in recapitulating the disorder. Thus, we propose using human induced pluripotent stem cells (iPSCs) to study idiopathic familial dilated cardiomyopathy (IFDC). We propose collecting tissue from individuals identified with the disorder In summary, this proposal represents a unique
- opportunity to improve our understanding of idiopathic familial dilated cardiomyopathy (which remains largely a mystery), identifying novel genetic causes (rendering many of these patients no longer “idiopathic), and proposing new therapeutic targets.
- Heart failure is a leading cause of mortality and morbidity in California and Western world. Over half of heart failure causes are due to dilated cardiomyopathy, a disorder of progressive ventricular dilatation and decreased contractility. However, the mortality rate for heart failure has been stagnant over the past few years nationally and in California. Hence new knowledge and developments gained from this research can go a long way to ameliorating the burden caused by this disorder. Our goal here is to collect tissues from patients with heart failure so that induced pluripotent stem cells (iPSCs) can be generated to understand their disease mechanism.
Because the regenerative capacity of adult heart is limited, any substantial cell loss as a result of a heart attack is mostly irreversible and may lead to progressive heart failure. Human pluripotent stem cells can be differentiated to heart cells, but their properties when transplanted into an injured heart remain unresolved. We propose to perform preclinical evaluation for transplantation of pluripotent stem cell-derived cardiac cells into the injured heart of an appropriate animal model. However, an important issue that has limited the progress to clinical use is their fate upon transplantation; that is whether they are capable of integrating into their new environment or they will function in isolation at their own pace. As an analogy, the performance of a symphony can go into chaos if one member plays in isolation from all surrounding cues. Therefore, it is important to determine if the transplanted cells can beat in harmony with the rest of the heart and if these cells will provide functional benefit to the injured heart. We plan to isolate cardiac cells derived from human pluripotent stem cells, transplant them into the model’s injured heart, determine if they result in improvement of the heart function, and perform detailed electrophysiology studies to determine their integration into the host tissue. The success of the proposed project will set the platform for future clinical trails of stem cell therapy for heart disease.
Heart disease remains the leading cause of mortality and morbidity in the US with an estimated annual cost of over $300 billion. In California alone, more than 70,000 people die every year from cardiovascular diseases. 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. Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) could provide an attractive therapeutic option to treat patients with damaged heart. We propose to isolate heart cells from hESCs and transplant them in an injured animal model's heart and study their fate. In the process, we will develop reagents that can be highly valuable for future research and clinical studies. The reagents generated in these studies can be patented forming an intellectual property portfolio shared by the state and the institution where the research is carried out. Most importantly, the research that is proposed in this application could lead to future stem cell-based therapies that would restore heart function after a heart attack. 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.
- Identification and isolation of pure cardiac cells derived from human pluripotent stem cells has proven to be a difficult task. We have designed a method to genetically engineer human embryonic stem cells (hESCs) to harbor a label that is expressed during sequential maturation of cardiac cells. This will allow us to prospectively isolate cardiac cells at different stages of development for further characterization and transplantation. Using this method, we have screened proteins that are expressed on the surface of cells as markers. Using antibodies against these surface markers allows for isolation of these cells using cell sorting techniques. Thus far, we have identified two surface markers that can be used to isolate early cardiac progenitors. Using these markers, we have enriched for cardiac cells from differentiating hESCs and have characterized their properties in the dish as well as in small animals. We plan to transplant these cells in large animal models and monitor their survival, expansion and their integration into the host myocardium. Molecular imaging techniques are used to track these cells upon transplantation.
- Pluripotent stem cells (PSCs) harbor several attractive features for regenerative medicine: they are capable of self-renewal and have the capacity to differentiate to the tissue lineage of all three germ layers. The overall objective of this grant is to develop new technologies that can facilitate generation of cardiovascular progenitors that upon transplantation are capable to integrate into the host tissue and function as part of the normal myocardium with no adverse effect.
- Several clinical trials of cell-based therapy have generated enthusiasm about the potential of adult stem cells to treat heart disease. However, no study has yet confirmed the delivery of a pure population of stem cells capable of robust regeneration of the injured myocardium. Furthermore, the electrical properties of the viable engrafted cells remain unknown. While adult stem cells have yet failed to convincingly regenerate myocardial tissue, human embryonic stem cells (hESCs) have proven to be a potential and unlimited source for cardiomyocyte regeneration. Most attempts to isolate transplantable cells from hESCs have aimed to isolate mature cells. Mature cardiomyocytes have passed the stage of self-renewal and may pose problems due to lack of proper integration. Cardiovascular progenitors, on the other hand, could adapt to the microenvironment for optimal integration into the host tissue and reside there for the lifetime of the patient.
- We have employed gene editing technology to generate new hESC lines, in which fluorescent reporter proteins are expressed only in cardiac cells. Hence, upon differentiation of hESCs towards cardiac lineage, a distinct fluorescent color is expressed sequentially at each stage of cardiovascular development. We have isolated these cells and have performed detailed analysis to fully characterize their developmental potential. We have performed global gene expression analysis to identify novel biomarkers unique to specific cardiac populations.
- An addition, we have transplanted cardiovascular progenitors (at different stages of development) and mature cardiomyocytes in animal models. We have shown engraftment of these cells into the host heart, albeit a very low efficiency. Furthermore, we have shown that transplantation of immature cardiovascular progenitors may generate cardiomyocytes in addition to supporting cells such as vascular endothelial cells and fibroblasts. These results highlight the potential benefit of progenitor cells to generate other cell types that contribute to heart regeneration.
- A major challenge to clinical translation of stem cells for heart regeneration is the lack of data on the integration of the transplanted cells into the host heart. . It is possible that the transplanted cells fail to physiologically couple with the host tissue, or they may modify the substrate such that a pro-arrhythmic focus is created. As an analogy, consider the performance of a great symphony orchestra that is interrupted by several members playing in isolation. As such, grafted cells in the heart can also be an ectopic source of activities, promoting arrhythmic events. These are critical issues that need to be addressed in detail in the right animal models before any clinical application can be pursued. We plan to investigate the extent of structural and functional integration of the transplanted cells into the host heart.