Myocardium that has been acutely or chronically infarcted presents an unwelcoming environment for the survival of transplanted cardiomyocytes. The large amount of cell necrosis in acutely infarcted tissue is associated with significant quantities of inflammatory cells, reactive oxygen species and cytokines that are toxic to the grafted cell, while the scar tissue of an old infarction can be poorly receptive to the engraftment and integration of new cells. In both situations, the ischaemia associated with the non-vascularized cell grafts presents an immediate challenge to cell survival, and the positive outcome of blocking cell death and augmenting cell survival pathways speaks to the need to overcome the inhospitable ischaemic and inflammatory environment.
Modifying inflammation and immunogenicity
The inflammatory cytokine interleukin (IL)-1 is elevated in acutely infarcted myocardium, and mediates cardiac myocyte apoptosis and adverse remodelling . Skeletal myoblasts that express an IL-1 inhibitor had improved survival after transplantation into infarcted myocardium than control cells, by over sixfold at 3 weeks associated with improved left ventricular size and function and reduced fibrosis . Co-injection of skeletal myoblasts with superoxide dismutase into an acute infarct reduced levels of tumour necrosis factor-α, TGF-β, IL-1β, IL-6, GM-CSF, while improving cell survival .
In contrast to autotransplant studies with adult stem cells or circulating progenitor cells, allograft transplant of hESCs elicits an adaptive immunological response and compounds the problem of cells trying to survive in this inflammatory milieu . Given the inherent incompatibilities of xenotransplantation, animal studies evaluating hESC-derived cardiomyocyte grafts have used immunologically compromised hosts, such as athymic rats  or immunodeficient mice . In studies of allogenic donor skeletal myoblasts transplanted into the ischaemic hind limb of mice, pre-treatment of the host tissue with antibodies against CD4, CD8 and C3 complement increased survival of the graft . Treatment of host tissue with prednisone and cyclosporine has been used successfully to transplant hESC into immunocompetent mice . In clinical use, down-regulating host immunity prior and subsequent to transplant will be required until means of inducing tolerance (e.g. through haematopoietic chimerism)  or immune-evading grafts are generated. Several studies indicate that ESCs have low or undetectable levels of class I or II major histocompatibility antigens, but after differentiation, both class I and II antigens can be up-regulated by inflammatory cytokines . Thus, while immature ESC derivatives may have reduced immunogenicity compared with adult transplanted tissue, as they mature it seems likely that they will have the full immunogenicity of an adult cell . One encouraging possibility is that cell grafts, because of their relative simplicity compared with whole organs, may require less intense immunosuppression regimens than those used currently for organ transplantation.
Improving blood flow to infarcted myocardium has also been explored as a means to reduce the persistent ischaemia of the host tissue that threatens the survival of grafts. Pre-treating the host myocardium with adenovirus encoding VEGF 3 weeks prior to transplanting foetal cardiomyocytes led to increased capillary density in the infarct and higher rate of survival of the transplanted cells . Fibroblast growth factor (FGF) given 1 week prior to foetal cardiomyocyte cell grafting into infarcted hearts was associated with enhanced ventricular size and function, and greater distribution of transplanted cells throughout the scar area . The improvement in ventricular function in this study may be a result of angiogenesis, or a direct effect of the growth factors on the cells, but nevertheless has positive implications for maximizing cardiac regeneration. Another approach to improving blood supply is to co-transplant hESC-derived endothelial cells along with hESC-derived cardiomyocytes, to promote neovascularization of the graft .
Extracellular and bioengineering solutions
While direct intramyocardial injection of cells results in a greater retention of cells than intracoronary or intravenous methods, as much as 90% of the cells can be immediately lost from the injection site because of mechanical extrusion from the injection track and washout of cells into the circulation [6-8]. Furthermore, when cell attachments to extracellular matrix or other cells are lost, as in during transfer of cardiomyocytes from cell culture into the host myocardium, an apoptotic pathway is initiated, termed anoikis. The loss of adhesion-related survival signals will eventually lead to cell death unless those attachments are re-established. For these reasons, the use of biomaterials has been explored as a ways of mechanically increasing retention of cells and mimicking extracellular matrix, as well as provide a niche environment with a depot of pro-survival factors and drugs. These agents have generally been hydrogels composed of synthetic polymers or natural proteins [36-48].
The most widely used hydrogels have properties resembling extracellular matrix that can provide adhesive peptides to maintain survival signalling of transplanted hESCs . In vitro, hESCs cultured and transplanted in collagen have reduced apoptosis . Furthermore, collagen patches provide a three-dimensional framework into which transplanted embryonic stem cells can align . Matrigel is a gelatinous biologic mixture that has also been used successfully in delivering hESC, which, when combined with pro-survival factors, has been associated with improved survival and engraftment of hESC cardiomyocytes into infarcted tissue . The mixture itself manifests angiogenic properties when injected into infarcted myocardium and may promote survival through multiple pathways .
Fibrin glue is generated by mixing fibrinogen and thrombin and has been successfully used in the injection track to prevent extrusion of skeletal myoblasts transplanted into a myocardial infarction, resulting in a greater number of cells in the target site, smaller scar and increased arteriole density . Bioengineered hydrogels can be designed to be pH- or temperature-sensitive, hence they take the form of an injectable liquid for cell suspension, which converts into a biodegradable gel with adhesive properties within heart tissue . A chitosan hydrogel with such properties has been used to deliver embryonic stem cells in a rat myocardial infarction model . In vitro, cells survived, proliferated and aggregated, and following injection into the infarct, the chitosan formed a temporary scaffold that improved cell retention and survival, resulting in a significantly larger graft compared with direct cell injections alone. A study performed with Rat H9c2 neonatal heart cells injected into infarcted myocardium with a mixture of collagen gel and Matrigel had a threefold increase in survival compared with direct cell injections or either vehicle alone (Fig. 2) . Hyaluronic acid–based gels are also appealing for co-injection, as this glycosaminoglycan is a component of the naturally occurring extracellular matrix found within connective tissues. It has been shown to improve wall thickness and angiogenesis in infarcted hearts [46, 47, 49, 50]. A combination of hyaluronic acid, to improve cell retention and survival, covalently linked to thiolated collagen, to aid cell attachment, demonstrated a marked improvement in retention of cardiosphere-derived cells in a mouse infarct model. This was associated with reduction in apoptosis, increased angiogenesis and with an improvement in left-ventricular function .
The hydrogels themselves may have ameliorative effects on ischaemic myocardium by providing structural support to the heart and promoting angiogenesis. Cell-free fibrin glue injected into infarcted myocardium prevented scar expansion and wall thinning compared with control injections , and induced microvessel formation within the infarct . Increased density of arterioles and capillaries has also been reported at the site of alginate  and hyaluronic acid [46, 50] injections, as well as decreased host cell apoptosis in these regions, leading investigators to hypothesize that these hydrogels recruit pro-angiogenic cells while favourably modulating the inflammatory microenvironment .
Anti-apoptotic, angiogenic and anti-inflammatory factors can be added to the hydrogels, providing a depot of pro-survival factors for controlled release. The use of VEGF- and FGF-loaded hydrogels promotes increased capillary ingrowth and angiogenesis when added to stem cell transplants [38, 40]. Other investigators have demonstrated increased stem cell homing and myocardial repair associated with injected hydrogels providing sustained levels of SDF-1  and erythropoietin .
Injection of a foreign substance, particularly a synthetic material, however, may not always be beneficial. Hyaluronic acid has also been shown to impair IGF-1 signalling , which may lead to greater apoptosis. Investigators have shown that a hydrogel composite of oligopolyethylene glycol injected with mouse ESCs increased graft size and reduced infarct size in a rat myocardial infarction model more than cells injected alone . On the other hand, a study of synthetic hydrogels containing FGF achieved sustained high levels of the growth factor, but also showed extensive inflammation at the interface of the tissue and gel . The presence of foreign material producing inflammation could potentially disrupt electrical integration of the cells or generate circuits for arrhythmias in these sites. Extracellular matrix–based components such as hyaluronic acid and collagen may prove superior in this regard and deserve further investigation.
Other novel methods for improving cell retention have been described. One such technique uses superparamagnetic microspheres to magnetize cardiac-derived cells that are then localized and retained with a magnet superimposed over target tissue .