BackgroundDevelopmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.Methodology/Principal FindingsHere we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and α-myosin heavy chain (αMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.Conclusion/SignificanceThe protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.

In vitro prevascularization of bone grafts with endothelial progenitor cells (EPCs) is a promising strategy to improveimplant survival. In this study we show bone formation in constructs that contain multipotent stromal cells (MSCs) and EPCs. Early and late EPCs from peripheral blood and bone marrow of adult goats were characterized for differentiation markers and functional responses. EPCs from peripheral blood are more proliferative than bone-marrow-derived EPCs, express higher numbers of endothelial markers for longer periods of time, and form more intricate networks. We demonstrate that EPCs derived from peripheral blood contribute to osteogenic differentiation by MSCs in vitro, and that MSCs support the proliferation of EPCs and stabilize the formed cellular networks. In vivo, EPCs from peripheral blood assemble into early blood vessel networks, which are more pronounced in the presence of MSCs. These results show that the EPCs isolated from peripheral blood are suitable for prevascularization strategies, and that coseeding of EPCs and MSCs is favorable for bone formation after 6 weeks.

Cell adhesion molecules are ubiquitously expressed proteins playing a central role in controlling cell migration, proliferation, survival, and apoptosis. Besides their key function in physiological maintenance of tissue integrity, adhesion molecules play an eminent role in various pathological processes. In cardiovascular disorders, cell adhesion molecules are particularly involved in atherogenesis and atherosclerotic plaque progression. They also play a critical role in myocardial infarction and reperfusion damage and a minor role in valvular stenosis and cardiomyopathy. Their common denominator: An increased expression of adhesion molecules involved in leukocyte extravasation and accumulation. Leukocyte extravasation is a multistep process, mediated by several cell adhesion molecules including selectins (P-, E- and L-), integrins and members of the Ig superfamily (ICAM-1, VCAM-1). These molecules can be targeted for imaging purposes (e.g. to identify atherosclerotic plaques) or can serve as biomarkers for plaque destabilization. Furthermore, cell adhesion molecules can serve as drugable targets to prevent leukocyte extravasation where warranted to decrease inflammatory tissue damage (e.g. reperfusion injury). Current techniques involve blocking of binding sites, targeted drug delivery using liposomes and polymeric particles as carriers or imaging of inflammation sites using labeled cells or antibodies. This review focuses on the role of cell adhesion molecules in cardiovascular disease and the use of targeting adhesion molecules for imaging purposes and local drug delivery in cardiovascular medicine.

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