Extracellular Matrix Bioscaffold Augmented with Human Stem Cells for Cardiovascular Repair

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.

Cardiovascular diseases remain the leading cause of death and disability in the United States. On average, an American suffers a coronary event every 34 seconds with one American dying of such an event every 1 minute and 23 seconds. Once the infarction has occurred, and even with optimal intervention, patients are prone to development of ischemic heart disease (IHD). Current therapies for IHD 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 novel potential cure for IHD. Numerous cell therapies are currently under investigation for myocardial repair; however success rates remain modest. Typically, cells are injected in suspension into either the systemic or coronary vasculature or directly into the ischemic myocardium. Outcomes have clearly demonstrated the safety of these cell based therapies but clinical improvements have been modest. Major limitations that have been identified include inconsistent cell delivery methods and poor long term donor cells survival and retention. Reliable methods for delivery of therapeutic cells are therefore critically needed in order to improve cell based cardiac regenerative therapies. Bioscaffolds have the potential to improve cell retention and localization to the site of injury. Porcine small intestinal submucosa extracellular matrix (SIS-ECM) is one such bioscaffold that may serve as a binding scaffold for MSCs. SIS-ECM itself has been found to exert a variety of beneficial pro-regenerative functions, hereunder modulation of the host chemotactic and immune response and release of large amounts of pro-angiogenic factors. Critically, SIS-ECM is already FDA approved for cardiac tissue repair after open heart surgery. MSCs are an ideal candidate for stem cell transplantation because of their ability to modulate the local tissue environment, induce local angiogenesis and recruit recipient effector populations. They also exhibit numerous pro-regenerative functions including immunomodulation by secreting a range of immunomodulatory factors and they are thus an ideal candidate for allogeneic transplantation. Furthermore, MSCs play a role in angiogenesis by secretion of angiogenic factors as well as chemotactic factors that facilitate recruitment of host stem and progenitor cells. The overall goal of this project then has been to generate an MSC seeded SIS-ECM device for the treatment of IHD. The hypothesis is that the combination of MSCs and SIS-ECM will result in a device with regenerative properties that exceed either component alone. We have in the course of the project developed a porcine model that mimics the hallmarks of the human IHD by injecting an embolizing agent into the coronary artery. We have tested the proposed device in this model and monitored functional improvement as compared to control animals and animals receiving cells or ECM alone. We have also verified that human and porcine MSCs are phenotypically and functionally equivalent and finally, we have explored the possible mechanisms underlying such a therapeutic benefit both in the lab and in a rat myocardial infarct model. With this final report, we consider this project to have been successfully executed and to have yielded significant new insight - not only into the utility of MSCs and SIS-ECM in the treatment of ischemic heart disease, but also in multiple areas of basic stem cell biology, animal modeling and translational research; all of which points far beyond the immediate scope of this project. We have completed the entire planned animal work and optimized all assays required for final analysis of the collected tissue and data. From the echocardiographic data, we expect to gain substantial insight into the effect of the different treatment options on the function of the ailing heart. We have also established new collaborations with colleagues who through sharing of tissue and data will enable us to analyze the hearts in far greater detail than first anticipated, thus substantially furthering our understanding of tissue regeneration. The data that will ultimately be derived from this huge body of work is, however, not at a stage of completion where we can offer any final conclusions regarding the main specific aims of the study. What can, however, be said with absolute certainty at this point is, that this project has contributed significant new insight into the area of regenerative medicine, large animal and translational research and beyond which could not have been achieved without the support of CIRM. Equally important, this work has laid the foundation for numerous future studies by both our team and by our colleagues at large that are certain to benefit heart disease patient in California and beyond.