PMC

Patches actively contract in response to electrical stimulation. Patches were electrically stimulated at frequencies of 0.5–5 Hz, and contraction was measured by using video edge detection. (A) A representative cardiomyocyte-only patch trace and (B) expanded views of select frequencies of this trace show a patch that captured 0.5- to 3-Hz but not 4- or 5-Hz stimuli. (C) Patches captured stimuli less than or equal to 2 Hz but exhibited reduced capacity to keep pace with frequencies of 3–5 Hz.

Prevascularization of patches. (A) Representative images of patches created by using human cardiomyocytes, HUVECs, and/or MEFs cultured in huEB medium for 8 days are shown. Human endothelial cells stained for human CD31 (Left) and human cardiomyocytes stained for β-MHC (Right) are shown. Patches derived from cardiomyocytes only (Top) contained only rare CD31-positive cells. Patches derived from cardiomyocytes and HUVECs (Middle) were characterized by clumps of necrotic, CD31-positive debris, predominantly at the patch centers, and patches containing cardiomyocytes, HUVECs, and MEFs (Bottom) exhibited CD31-positive endothelial cell networks morphologically resembling a vascular plexus. (B) Patches comprising all three cell types contained significantly more vessel structures than patches comprising only cardiomyocytes or cardiomyocytes and HUVECs in both huEB and RPMI-B27 culture media. *, P < 0.05. Higher-magnification images of patches containing cardiomyocytes, HUVECs, and MEFs (C) or cardiomyocytes, hESC-derived endothelial cells, and MEFs (D) show that these patches contained human CD31-positive elongated vessel structures and lumens that morphologically resemble blood vessels.

Cardio-HUVEC-MEF patches are stiffer than patches composed of cardiomyocytes only. (A) Passive mechanical properties of patches were analyzed by stretching patch strips incrementally by using a square-wave length stretch protocol (Lower) and then measuring force (Upper). (B) Peak force elicited in response to increasing length increments for a representative cardio-HUVEC-MEF and cardio-only patch strip is shown. The slope of the force–strain line for a given patch strip denotes the stiffness of that strip. (C) The average stiffness of cardio-HUVEC-MEF patches was ≈4-fold greater than cardio-only patches. *, P < 0.05. (D–F) Patch sections were stained by using Sirius red (collagen) and fast green (other tissue elements). Representative cardio-only (D) and cardio-HUVEC-MEF (E) patches are shown. Cardio-HUVEC-MEF patches had greater than 5-fold collagen per unit area than cardio-only or cardio-HUVEC patches (F).

Cardio-HUVEC-MEF patches form vascularized human grafts after implantation in skeletal muscle. Cardio-only or cardio-HUVEC-MEF patches were implanted in the gluteus superficialis muscle of nude rats for 1 week. Graft sections were immunostained by using antibodies against β-MHC, human CD31, and Ter-119 to identify human cardiomyocytes, human endothelial cells, and red blood cells, respectively. (A) Representative images of β-MHC-positive human cardiomyocyte grafts from animals implanted with patches comprising cardiomyocytes only (Left inset magnified 2×) or cardiomyocytes, HUVECs, and MEFs (Right). (B) Higher-magnification images of β-MHC-positive and human CD31-positive graft areas from a cardio-HUVEC-MEF patch implant showed that human cardiomyocytes remained small and structurally disorganized (Left) and that human CD31-positive cells formed vessel-like lumens containing Ter-119-positive red blood cells (Middle and Right). (C) β-MHC-positive cardiomyocyte graft area was ≈11-fold larger in animals that received cardio-HUVEC-MEF patches. *, P < 0.05. (D) Additionally, grafts in animals that received cardio-HUVEC-MEF patches had ≈12-fold more human CD31-positive vessel lumens than those that received cardio-only patches. *, P < 0.05.

Cardiac tissue patches form human cardiac muscle and integrated human microvessels in rodent hearts. Cardio-HUVEC-MEF or cardio-HUVEC-NHDF patches were implanted onto nude rat hearts for 1 week. (A) Gross examination of the heart immediately after sacrifice demonstrated that patches (arrow) attached with sutures were firmly adhered to the heart. (B) Patches had significant β-MHC-positive human cardiac muscle tissue (brown immunostaining; representative cardio-HUVEC-MEF patch). (C) A higher-magnification image of the graft from B shows that β-MHC-positive cardiomyocytes were relatively small and had immature sarcomeric organization, and that (D) grafts contained Nkx2.5-positive (pink nuclei) cardiac progenitor cells that had not yet matured to express β-MHC (green). CD31-positive endothelial cells in animals implanted with cardio-HUVEC-MEF (E) or cardio-HUVEC-NHDF (F and G) patches frequently formed vessel-like lumens that contained leukocytes (arrows in E) and Ter-119-positive red blood cells (G), indicating that grafted human vessels had connected with the host vasculature.