Dataset Information

Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.

ABSTRACT: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide a promising source for cell therapy and drug screening. Several high-yield protocols exist for hESC-CM production; however, methods to significantly advance hESC-CM maturation are still lacking. Building on our previous experience with mouse ESC-CMs, we investigated the effects of 3-dimensional (3D) tissue-engineered culture environment and cardiomyocyte purity on structural and functional maturation of hESC-CMs. 2D monolayer and 3D fibrin-based cardiac patch cultures were generated using dissociated cells from differentiated Hes2 embryoid bodies containing varying percentage (48-90%) of CD172a (SIRPA)-positive cardiomyocytes. hESC-CMs within the patch were aligned uniformly by locally controlling the direction of passive tension. Compared to hESC-CMs in age (2 weeks) and purity (48-65%) matched 2D monolayers, hESC-CMs in 3D patches exhibited significantly higher conduction velocities (CVs), longer sarcomeres (2.09 ± 0.02 vs. 1.77 ± 0.01 ?m), and enhanced expression of genes involved in cardiac contractile function, including cTnT, ?MHC, CASQ2 and SERCA2. The CVs in cardiac patches increased with cardiomyocyte purity, reaching 25.1 cm/s in patches constructed with 90% hESC-CMs. Maximum contractile force amplitudes and active stresses of cardiac patches averaged to 3.0 ± 1.1 mN and 11.8 ± 4.5 mN/mm(2), respectively. Moreover, contractile force per input cardiomyocyte averaged to 5.7 ± 1.1 nN/cell and showed a negative correlation with hESC-CM purity. Finally, patches exhibited significant positive inotropy with isoproterenol administration (1.7 ± 0.3-fold force increase, EC50 = 95.1 nm). These results demonstrate highly advanced levels of hESC-CM maturation after 2 weeks of 3D cardiac patch culture and carry important implications for future drug development and cell therapy studies.

SUBMITTER: Zhang D

PROVIDER: S-EPMC3660435 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.

Zhang Donghui D Shadrin Ilya Y IY Lam Jason J Xian Hai-Qian HQ Snodgrass H Ralph HR Bursac Nenad N

Biomaterials 20130502 23

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide a promising source for cell therapy and drug screening. Several high-yield protocols exist for hESC-CM production; however, methods to significantly advance hESC-CM maturation are still lacking. Building on our previous experience with mouse ESC-CMs, we investigated the effects of 3-dimensional (3D) tissue-engineered culture environment and cardiomyocyte purity on structural and functional maturation of hESC-CMs. 2D monolayer an ...[more]

PMID: 23642535

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Project description:UnlabelledA tissue-engineered cardiac patch provides a method to deliver cardiomyoctes to the injured myocardium with high cell retention and large, controlled infarct coverage, enhancing the ability of cells to limit remodeling after infarction. The patch environment can also yield increased survival. In the present study, we sought to assess the efficacy of a cardiac patch made from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to engraft and limit left ventricular (LV) remodeling acutely after infarction. Cardiac patches were created from hiPSC-CMs and human pericytes (PCs) entrapped in a fibrin gel and implanted acutely onto athymic rat hearts. hiPSC-CMs not only remained viable after in vivo culture, but also increased in number by as much as twofold, consistent with colocalization of human nuclear antigen, cardiac troponin T, and Ki-67 staining. CM+PC patches led to reduced infarct sizes compared with myocardial infarction-only controls at week 4, and CM+PC patch recipient hearts exhibited greater fractional shortening over all groups at both 1 and 4 weeks after transplantation. However, a decline occurred in fractional shortening for all groups over 4 weeks, and LV thinning was not mitigated. CM+PC patches became vascularized in vivo, and microvessels were more abundant in the host myocardium border zone, suggesting a paracrine mechanism for the improved cardiac function. PCs in a PC-only control patch did not survive 4 weeks in vivo. Our results indicate that cardiac patches containing hiPSC-CMs engraft onto acute infarcts, and the hiPSC-CMs survive, proliferate, and contribute to a reduction in infarct size and improvements in cardiac function.SignificanceIn the present study, a cardiac patch was created from human induced pluripotent stem cell-derived cardiomyocytes and human pericytes entrapped in a fibrin gel, and it was transplanted onto infarcted rat myocardium. It was found that a patch that contained both cardiomyocytes and pericytes survived transplantation and resulted in improved cardiac function and a reduced infarct size compared with controls.

Project description:Complex heterocellular interactions between cardiomyocytes and fibroblasts in the heart involve their bidirectional signaling via cell-cell contacts, paracrine factors, and extracellular matrix (ECM). These interactions vary with heart development and pathology leading to changes in cardiac structure and function. Whether cardiac fibroblasts of different ages interact differentially with cardiomyocytes to distinctly impact their function remains unknown. Here, we explored the direct structural and functional effects of fetal and adult cardiac fibroblasts on cardiomyocytes using a tissue-engineered 3D co-culture system. We show that the age of cardiac fibroblasts is a strong determinant of the structure, function, and molecular properties of co-cultured tissues. In particular, in vitro expanded adult, but not fetal, cardiac fibroblasts significantly deteriorated electrical and mechanical function of the co-cultured cardiomyocytes, as evidenced by slower action potential conduction, prolonged action potential duration, weaker contractions, higher tissue stiffness, and reduced calcium transient amplitude. This functional deficit was associated with structural and molecular signatures of pathological remodeling including fibroblast proliferation, interstitial collagen deposition, and upregulation of pro-fibrotic markers. Our studies imply critical roles of the age of supporting cells in engineering functional cardiac tissues and provide a new physiologically relevant in vitro platform to investigate influence of heterocellular interactions on cardiomyocyte function, development, and disease.Previous studies have shown that cardiomyocytes and fibroblasts in the heart interact through direct contacts, paracrine factors, and matrix-mediated crosstalk. However, whether cardiac fibroblasts of different ages distinctly impact cardiomyocyte function remains elusive. We employed a tissue-engineered hydrogel-based co-culture system to study interactions of cardiomyocytes with fetal or adult cardiac fibroblasts. We show that the age of cardiac fibroblasts is a strong determinant of the structure, function, and molecular properties of engineered cardiac tissues and that key features of fibrotic myocardium are replicated by supplementing cardiomyocytes with expanded adult but not fetal fibroblasts. These findings relate to implantation of stem cell-derived cardiomyocytes in adult myocardium and warrant further studies of how age and source of non-myocytes impact cardiac function and maturation.

Project description:BackgroundVentricular septal perforation and left ventricular aneurysm are examples of potentially fatal complications of myocardial infarction. While various artificial materials are used in the repair of these issues, the possibility of associated infection and calcification is non-negligible. Cell-seeded biodegradable tissue-engineered patches may be a potential solution. This study evaluated the feasibility of a new left ventricular patch rat model to study neotissue formation in biodegradable cardiac patches.MethodsHuman induced pluripotent stem cell-derived cardiac progenitor cells (hiPS-CPCs) were cultured onto biodegradable patches composed of polyglycolic acid and a 50:50 poly (l-lactide-co-?-caprolactone) copolymer for one week. After culturing, patches were implanted into left ventricular walls of male athymic rats. Unseeded controls were also used (n = 10/group). Heart conditions were followed by echocardiography and patches were subsequently explanted at 1, 2, 6, and 9 months post-implantation for histological evaluation.ResultThroughout the study, no patches ruptured demonstrating the ability to withstand the high pressure left ventricular system. One month after transplantation, the seeded patch did not stain positive for human nuclei. However, many new blood vessels formed within patches with significantly greater vessels in the seeded group at the 6 month time point. Echocardiography showed no significant difference in left ventricular contraction rate between the two groups. Calcification was found inside patches after 6 months, but there was no significant difference between groups.ConclusionWe have developed a surgical method to implant a bioabsorbable scaffold into the left ventricular environment of rats with a high survival rate. Seeded hiPS-CPCs did not differentiate into cardiomyocytes, but the greater number of new blood vessels in seeded patches suggests the presence of cell seeding early in the remodeling process might provide a prolonged effect on neotissue formation. This experiment will contribute to the development of a treatment model for left ventricular failure using iPS cells in the future.

Project description:Functional cardiac tissue engineering holds promise as a candidate therapy for myocardial infarction and heart failure. Generation of "strong-contracting and fast-conducting" cardiac tissue patches capable of electromechanical coupling with host myocardium could allow efficient improvement of heart function without increased arrhythmogenic risks. Towards that goal, we engineered highly functional 1?cm?×?1?cm cardiac tissue patches made of neonatal rat ventricular cells which after 2 weeks of culture exhibited force of contraction of 18.0?±?1.4?mN, conduction velocity (CV) of 32.3?±?1.8?cm/s, and sustained chronic activation when paced at rates as high as 8.7?±?0.8?Hz. Patches transduced with genetically-encoded calcium indicator (GCaMP6) were implanted onto adult rat ventricles and after 4-6 weeks assessed for action potential conduction and electrical integration by two-camera optical mapping of GCaMP6-reported Ca2+ transients in the patch and RH237-reported action potentials in the recipient heart. Of the 13 implanted patches, 11 (85%) engrafted, maintained structural integrity, and conducted action potentials with average CVs and Ca2+ transient durations comparable to those before implantation. Despite preserved graft electrical properties, no anterograde or retrograde conduction could be induced between the patch and host cardiomyocytes, indicating lack of electrical integration. Electrical properties of the underlying myocardium were not changed by the engrafted patch. From immunostaining analyses, implanted patches were highly vascularized and expressed abundant electromechanical junctions, but remained separated from the epicardium by a non-myocyte layer. In summary, our studies demonstrate generation of highly functional cardiac tissue patches that can robustly engraft on the epicardial surface, vascularize, and maintain electrical function, but do not couple with host tissue. The lack of graft-host electrical integration is therefore a critical obstacle to development of efficient tissue engineering therapies for heart repair.

Dataset Information

Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.

Publications

Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.

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