Year 2

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.