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InterScience or from the author. Regeneration or repair of the damaged myocardium requires different strategies including engineered constructs for more efficient cell delivery. This study was undertaken to examine the potential of a new nanostructured elastomer to deliver embryonic stem cell-derived cardiomyocytes (ESC-CM) to an infarcted area of the myocardium. Engineered materials were biocompatible, mechanically stable, and elastomeric nanocomposites serving as substrates for delivery of ESC-CM and as a left ventricular support device in myocardial regeneration strategies. Materials investigated were soft and strong poly(aliphatic/aromatic-ester) multiblock thermoplastic elastomers with poly(ethylene terephthalate) (PET) hard segments and dimerized fatty acid, i.e., dilinoleic acid (DLA) soft segments, respectively, with and without addition of 0.2 wt% TiO2 nanoparticles to form nanocomposites. The PET/ DLA-TiO2 nanocomposite exhibited over 8MPa tensile strength and 900% elongation at break. Addition of TiO2 nanoparticles significantly altered surface roughness and enhanced adhesion and spreading of ESC-CM derived from mouse and human embryonic stem cells. The newly developed materials did not affect the functional activity of spontaneously beating hESC-CM, as demonstrated by unaltered rate of their beating, and the cells continued to demonstrate contractile activity on the materials for more than two months in culture (the longest time tested). Quantitative proliferation and survival assays using fibroblasts confirmed the ability of the new materials to support cells as well as or better than the present commercial-type thermoplastic elastomer analog. The results indicate that PET/ DLA and PET/DLA-TiO2 are promising candidates for the manufacture of engineered patches to deliver ESC-CMs.

Treatment of human disease with human embryonic stem cell (hESC)-derived cells is now close to reality, but little is known of their responses to physiological and pathological insult. The ability of cells to respond via activation of Toll like receptors (TLR) is critical in innate immune sensing in most tissues, but also extends to more general danger sensing, e.g. of oxidative stress, in cardiomyocytes. We used biomarker release and gene-array analysis to compare responses in hESC before and after differentiation, and to those in primary human endothelial cells. The presence of cardiomyocytes and endothelial cells was confirmed in differentiated cultures by immunostaining, FACS-sorting and, for cardiomyocytes, beating activity. Undifferentiated hESC did not respond with CXCL8 release to Gram positive or Gram negative bacteria, or a range of PAMPs (pathogen associated molecular patterns) for TLRs 1-9 (apart from flagellin, an activator of TLR5). Surprisingly, lack of TLR-dependent responses was maintained over 4 months of differentiation of hESC, in cultures which included cardiomyocytes and endothelial cells. In contrast, primary cultures of human aortic endothelial cells (HAEC) demonstrated responses to a broad range of PAMPs. Expression of downstream TLR signalling pathways was demonstrated in hESC, and IL-1beta, TNFalpha and INFgamma, which bypass the TLRs, stimulated CXCL8 release. NFkappaB pathway expression was also present in hESC and NFkappaB was able to translocate to the nucleus. Low expression levels of TLRs were detected in hESC, especially TLRs 1 and 4, explaining the lack of response of hESC to the main TLR signals. TLR5 levels were similar between differentiated hESC and HAEC, and siRNA knockdown of TLR5 abolished the response to flagellin. These findings have potential implications for survival and function of grafted hESC-derived cells.