A cardiac patch from aligned microvessel and cardiomyocyte patches.

Cardiac tissue engineering aims to produce replacement tissue patches in the lab to replace or treat infarcted myocardium. However, current patches lack preformed microvascularization and are therefore limited in thickness and force production. In this study, we sought to assess whether a bilayer patch composed of a layer made from human induced pluripotent stem cell-derived cardiomyocytes and a microvessel layer composed of self-assembled human blood outgrowth endothelial cells and pericytes was capable of engrafting on the epicardial surface of a nude rat infarct model and becoming perfused by the host 4 weeks after acute implantation. The bilayer configuration was found to increase the twitch force production, improve human induced pluripotent stem cell-derived cardiomyocyte survival and maturation, and increase patent microvessel lumens compared with time-matched single layer controls after 2 weeks of in vitro culture. Upon implantation, the patch microvessels sprouted into the cardiomyocyte layer of the patch and inosculated with the host vasculature as evidenced by species-specific perfusion labels and erythrocyte staining. Our results demonstrate that the added microvessel layer of a bilayer patch substantially improves in vitro functionality and that the bilayer patch is capable of engraftment with rapid microvessel inosculation on injured myocardium. The bilayer format will allow for scaling up in size through the addition of layers to obtain thicker tissues generating greater force in the future.

Inosculation and perfusion of pre-vascularized tissue patches containing aligned human microvessels after myocardial infarction.

A major goal of tissue engineering is the creation of pre-vascularized tissues that have a high density of organized microvessels that can be rapidly perfused following implantation. This is especially critical for highly metabolic tissues like myocardium, where a thick myocardial engineered tissue would require rapid perfusion within the first several days to survive transplantation. In the present work, tissue patches containing human microvessels that were either randomly oriented or aligned were placed acutely on rat hearts post-infarction and for each case it was determined whether rapid inosculation could occur and perfusion of the patch could be maintained for 6 days in an infarct environment. Patches containing self-assembled microvessels were formed by co-entrapment of human blood outgrowth endothelial cells and human pericytes in fibrin gel. Cell-induced gel contraction was mechanically-constrained resulting in samples with high densities of microvessels that were either randomly oriented (with 420 ± 140 lumens/mm(2)) or uniaxially aligned (with 940 ± 240 lumens/mm(2)) at the time of implantation. These patches were sutured onto the epicardial surface of the hearts of athymic rats following permanent ligation of the left anterior descending artery. In both aligned and randomly oriented microvessel patches, inosculation occurred and perfusion of the transplanted human microvessels was maintained, proving the in vivo vascularization potential of these engineered tissues. No difference was found in the number of human microvessels that were perfused in the randomly oriented (111 ± 75 perfused lumens/mm(2)) and aligned (173 ± 97 perfused lumens/mm(2)) patches. Our results demonstrate that tissue patches containing a high density of either aligned or randomly oriented human pre-formed microvessels achieve rapid perfusion in the myocardial infarct environment - a necessary first-step toward the creation of a thick, perfusable heart patch.

Tissue Contraction Force Microscopy for Optimization of Engineered Cardiac Tissue.

We developed a high-throughput screening assay that allows for relative comparison of the twitch force of millimeter-scale gel-based cardiac tissues. This assay is based on principles taken from traction force microscopy and uses fluorescent microspheres embedded in a soft polydimethylsiloxane (PDMS) substrate. A gel-forming cell suspension is simply pipetted onto the PDMS to form hemispherical cardiac tissue samples. Recordings of the fluorescent bead movement during tissue pacing are used to determine the maximum distance that the tissue can displace the elastic PDMS substrate. In this study, fibrin gel hemispheres containing human induced pluripotent stem cell-derived cardiomyocytes were formed on the PDMS and allowed to culture for 9 days. Bead displacement values were measured and compared to direct force measurements to validate the utility of the system. The amplitude of bead displacement correlated with direct force measurements, and the twitch force generated by the tissues was the same in 2 and 4 mg/mL fibrin gels, even though the 2 mg/mL samples visually appear more contractile if the assessment were made on free-floating samples. These results demonstrate the usefulness of this assay as a screening tool that allows for rapid sample preparation, data collection, and analysis in a simple and cost-effective platform.

Directed Fusion of Mesenchymal Stem Cells with Cardiomyocytes via VSV-G Facilitates Stem Cell Programming.

Mesenchymal stem cells (MSCs) spontaneously fuse with somatic cells in vivo, albeit rarely, and the fusion products are capable of tissue-specific function (mature trait) or proliferation (immature trait), depending on the microenvironment. That stem cells can be programmed, or somatic cells reprogrammed, in this fashion suggests that stem cell fusion holds promise as a therapeutic approach for the repair of damaged tissues, especially tissues not readily capable of functional regeneration, such as the myocardium. In an attempt to increase the frequency of stem cell fusion and, in so doing, increase the potential for cardiac tissue repair, we expressed the fusogen of the vesicular stomatitis virus (VSV-G) in human MSCs. We found VSV-G expressing MSCs (vMSCs) fused with cardiomyocytes (CMs) and these fusion products adopted a CM-like phenotype and morphology in vitro. In vivo, vMSCs delivered to damaged mouse myocardium via a collagen patch were able to home to the myocardium and fuse to cells within the infarct and peri-infarct region of the myocardium. This study provides a basis for the investigation of the biological impact of fusion of stem cells with CMs in vivo and illustrates how viral fusion proteins might better enable such studies.