(A) Stacking five single EHTs (B) on a custom-made device facilitated EHT fusion and allowed contractions under auxotonic load resulting in synchronously contracting (C) multiloop EHTs ready for in vivo engraftment. (D) Six single-knot sutures served to fix multiloop EHTs on (E) the recipient hearts ((A-E), with permission from [7] ). (F) Photograph of an assembled electrical stimulation chamber developed by Radisic and colleagues for applying pulsatile electrical field stimuli to (G) cardiac cells seeded on scaffolds. (H) Representative transmission electron micrographs of stimulated and non-stimulated constructs after 8 days of cultivation, compared with neonatal rat ventricles. (I) Stimulated constructs and neonatal ventricles contained higher levels of β-Myosin heavy chain staining compared to non-stimulated constructs ((F-I), with permission from [8]). (J) Decellularized cadaveric rat heart. (K) H&E staining indicates no intact cells or nuclei with large vascular conduits maintained (black asterisks) Scale bar, 200 μm. (L) Immunofluorescence staining of cadaveric and SDS-decellularized rat heart thin sections showing the presence or absence, respectively, of DAPI-positive nuclei (purple), cardiac α-myosin heavy chain (green) and sarcomeric α-actin (red). Scale bars, 50 μm. (M) Recellularized whole rat heart at day 4 of perfusion culture, showing cross-sectional ring harvested for functional analysis (day 8, Upper Insert), and Masson’s trichrome staining of a ring thin section showing cells throughout the thickness of the wall (Lower Insert). Scale bar, 100 μm. Force generation in recellularized left ventricular rings after (N) 1-Hz and (O) 2-Hz electrical stimulation ((J-O), with permission from [20**]).

(A-E) Double transgenic mice (Isl1-dsRed and Nkx-2.5-eGFP) were used to create embryonic stem cells with a defined myogenic identity. (A) ESC-derived progenitor cells from the R+G+ population grown on micropatterned fibronectin lanes showed striated sarcomeric α-actinin staining (green) and little smooth muscle myosin heavy chain staining (red). (B) R+G- cells and (C) G+R- cells (D) Negative control exhibited less cardiac-like cells. (E) R+G+ progenitors were used to generate contractile muscular thin films (MTF) ((A-E) with permission from [55]). (F-I) Engineered heart tissues (EHTs) were used as surrogate tissues to evaluate integration of the injected ESC-derived cell populations. (F) Schematic depicting the EHT and injected cells. (G) Left Isochronal activation maps depict the speed of electrical impulses through the EHT, showing that ESC-derived cardiac progenitors improve conduction velocity and impulse propagation in EHTs. Right , Immunohistochemical staining of the injected EHT shows YFP+ injected cells (purple) and expression of key cardiac markers including Connexin-43 (Cx43, red) and cardiac troponin T (cTnT, green) as well as nuclear counterstaining (DAPI, blue). Conversely, impulse propagation is uniform but slower in EHTs injected with (H) neonatal cardiomyocytes (nCMs) and is even blocked by the addition of (I) cardiac fibroblasts (cFBs) ((F-I) with permission from [65*]).

(A) Tube formation after 7-day cultivation of endothelial cells on collagen scaffolds with covalently immobilized vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1). Images of PBS control group (scaffold with no growth factor) and VEGF+Ang1 group (scaffold with immobilized VEGF and Ang1) with live/dead staining (CFDA stains live cells green, PI stains dead cells red), hematoxylin and eosin staining, and von Willebrand factor staining. Sponges without cells are also shown. Random clusters of cells were found in PBS control group, compared to circular structures formed by elongated thin cells with nuclei in the peripheral position in the VEGF+Ang1 group ((A) with permission from [34]). (B-G) Implantation of collagen scaffolds with covalently immobilized VEGF to replace a full right ventricular free wall defect in rat hearts (B-D) Masson’s trichrome staining at 28 days after repair of right ventricular free wall defect shows higher patch thickness for patches with low dose VEGF (14.5 ± 1.4 ng VEGF) and high dose VEGF (97.2 ± 8.0 ng VEGF), compared to control patch with no VEGF. Arrows indicate thickness of the patch. (E-G) CD31 staining shows more CD31-positive vascular structures (indicated by arrows) for patch with high dose VEGF, compared to control low dose VEGF patch ((B-G) with permission from [36]). (H-L) Formation of realistic cardiac microstructure using micropatterned anisotropic slice cultures that replicate the natural fiber directions in ventricular cross sections. (H-J) The cells attached, spread and aligned to the micropatterned fibronectin lines and formed confluent cardiac fibers by Day 6. (K) A composite image of the entire micropatterned slice culture as well as (L) close-up images of four sections with the underlying fibronectin pattern (green, inset) is shown ((H-L) with permission from [44*]). (M-P) PDMS molds with arrays of (M) mesoscopic posts were used to fabricate three-dimensional muscle tissue architectures. (N) After 2 weeks of cultivation, tissue constructs were formed with densely aligned and striated cardiomyocytes, showing a high level of connexin-43 expression.. Directions of cell alignment in subregions (blue squares) were obtained to show more aligned cells in (O) PDMS molds with posts compared to (P) those without posts. (P,O) Inset shows the same image with F-actin stain (green) ((M-P) with permission from [47] ).