Preconditioning stem cells for cardiac tissue engineering

© 2010 Dr. Samantha Licy StubbsStem cells represent a promising tool for cell and tissue therapies due to their propensity to differentiate as well as their anti-apoptotic, pro-angiogenic and tissue remodeling properties. In practice however, the successful implementation of these cells into cell and tissue therapies for treating the complications of myocardial infarction has been limited by poor cell survival following implantation. Preconditioning stem cells prior to implantation to the ischaemic myocardium has been shown to improve implanted cell survival as well as improve functional outcomes. Preconditioning cells for tissue engineering however, remains largely unexplored. In this work, I aimed to explore how adipose-derived stem cells (ASC) can be protected against the hypoxic and ischaemic environments that occur in tissue engineering. I also aimed to determine whether preconditioning improves the survival of implanted stem cells in an in vivo tissue engineering environment. In establishing a model of ASC death in vitro, we found that ASC were resistant to severe hypoxia for up to 72 h but not to ischaemia. After just 24 h ischaemia, ASC morphology was altered consistent with cellular apoptosis. Concomitant with altered morphology, ischaemia significantly decreased cell viability compared with normoxia as measured by cell counting and MTT-based absorbance assays. Furthermore, compared with normoxia, ischaemia significantly increased cellular injury and apoptosis as measured by lactate dehydrogenase release into the medium and caspase 3/7 activity respectively. In investigating the susceptibility of other cell types important for cardiac tissue engineering to hypoxia or ischaemia-induced cell death, we found that endothelial cells and cardiomyocyte-like cells derived from ASC were much more susceptible to cellular injury and apoptosis during severe hypoxia alone. Our exploration of protection protocols showed that preconditioning ASC with hypoxia for 24 h protected them against ischaemia-induced cell death in vitro. Preconditioned ASC expressed significantly increased levels of hypoxia inducible factor-1α (HIF-1α) protein and its downstream target gene, vascular endothelial growth factor-A (VEGF-A) mRNA. This corresponded with a significant increase in hypoxia-mediated VEGF-A secretion into the culture medium. Phospho-Akt was also increased in preconditioned ASC compared with non-preconditioned cells. The protective effects of hypoxic preconditioning were abolished by a neutralising antibody against VEGF-A and the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 demonstrating the importance of VEGF and Akt in ASC survival. Interestingly, the protective paracrine effects of ASC were also potentiated by hypoxic preconditioning. We found that conditioned medium from hypoxic preconditioned ASC significantly decreased hypoxia-induced endothelial cell death compared with conditioned medium from non-preconditioned ASC. Hypoxic preconditioned ASC also promoted angiogenesis to a greater extent than non-preconditioned ASC. The addition of VEGF to conditioned medium from non-preconditioned ASC showed that this effect was mediated through the increase in ASC VEGF-A secretion under hypoxic conditions. Neither autocrine preconditioning protocols nor the protective paracrine effects of hypoxic preconditioned ASC on cardiomyogenic ASC were investigated however due to unreliable differentiation protocols. Finally, we investigated ASC death following implantation into an in vivo tissue engineering chamber. Immunohistochemisty revealed that ASC implanted into an in vivo tissue engineering chamber were able to survive the first two days following implantation. ASC nuclear morphology was regular and did not display any hallmarks of apoptosis such as chromatin condensation. Furthermore, we were unable to detect any cleaved caspase-3-positive ASC within the constructs at this time point suggesting that ASC were not apoptotic and had survived implantation through to two days. ASC were distributed throughout the chamber, remained in the growth factor-reduced Matrigel™, and tended to aggregate into clusters. Moreover, HIF-1α was present in these cells suggesting that within this environment, ASC were hypoxic and that HIF-1α may have contributed to their survival in these conditions. In collating this data, it appears that ASC are able to survive severe hypoxia both in vitro and in vivo, at least in part via a HIF-1α/VEGF-A/PI3K-Akt-mediated mechanism, and exert protective effects, which were potentiated following hypoxic preconditioning of ASC, on other cell types. Therefore, rather than requiring protection in in vivo tissue engineering environments themselves, it appears that hypoxic ASC may have an important role in supporting the survival of endothelial cells and possibly stem cell-derived cardiomyocytes in such environments for support and stabilisation of tissue growth