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

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 μm), and enhanced expression of genes involved in cardiac contractile function, including cTnT, αMHC, 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.

[1]  J. Lüdemann,et al.  Shortening versus isometric contractions in isolated human failing and non-failing left ventricular myocardium: dependency of external work and force on muscle length, heart rate and inotropic stimulation. , 1998, Cardiovascular research.

[2]  Gordon Keller,et al.  Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. , 2011, Cell stem cell.

[3]  Masayuki Yamato,et al.  Electrical coupling of cardiomyocyte sheets occurs rapidly via functional gap junction formation. , 2006, Biomaterials.

[4]  Thomas Rau,et al.  Human Engineered Heart Tissue as a Versatile Tool in Basic Research and Preclinical Toxicology , 2011, PloS one.

[5]  G. Fraedrich,et al.  Positive and negative inotropic effects of DL-sotalol and D-sotalol in failing and nonfailing human myocardium under physiological experimental conditions. , 1995, Circulation.

[6]  Stefan Wagner,et al.  Murine and human pluripotent stem cell-derived cardiac bodies form contractile myocardial tissue in vitro. , 2013, European heart journal.

[7]  J. Itskovitz‐Eldor,et al.  Functional Properties of Human Embryonic Stem Cell–Derived Cardiomyocytes: Intracellular Ca2+ Handling and the Role of Sarcoplasmic Reticulum in the Contraction , 2006, Stem cells.

[8]  Kumaraswamy Nanthakumar,et al.  Interrogating functional integration between injected pluripotent stem cell-derived cells and surrogate cardiac tissue , 2009, Proceedings of the National Academy of Sciences.

[9]  Christine L Mummery,et al.  Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. , 2012, Circulation research.

[10]  Sean P. Palecek,et al.  Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions , 2012, Nature Protocols.

[11]  Doris A Taylor,et al.  Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart , 2008, Nature Medicine.

[12]  I. Efimov,et al.  Application of blebbistatin as an excitation-contraction uncoupler for electrophysiologic study of rat and rabbit hearts. , 2007, Heart rhythm.

[13]  K. Bendixen,et al.  Physiological function and transplantation of scaffold-free and vascularized human cardiac muscle tissue , 2009, Proceedings of the National Academy of Sciences.

[14]  Jing Liu,et al.  Functional Sarcoplasmic Reticulum for Calcium Handling of Human Embryonic Stem Cell‐Derived Cardiomyocytes: Insights for Driven Maturation , 2007 .

[15]  Shulamit Levenberg,et al.  Tissue Engineering of Vascularized Cardiac Muscle From Human Embryonic Stem Cells , 2007, Circulation research.

[16]  James A Thomson,et al.  Human Embryonic Stem Cells Develop Into Multiple Types of Cardiac Myocytes: Action Potential Characterization , 2003, Circulation research.

[17]  Gordon Keller,et al.  SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells , 2011, Nature Biotechnology.

[18]  Nenad Bursac,et al.  The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle. , 2011, Biomaterials.

[19]  G. Lyons,et al.  Extracellular Matrix Promotes Highly Efficient Cardiac Differentiation of Human Pluripotent Stem Cells: The Matrix Sandwich Method , 2012, Circulation research.

[20]  G. Vunjak‐Novakovic,et al.  Hybrid Gel Composed of Native Heart Matrix and Collagen Induces Cardiac Differentiation of Human Embryonic Stem Cells without Supplemental Growth Factors , 2011, Journal of cardiovascular translational research.

[21]  Charles E. Murry,et al.  Growth of Engineered Human Myocardium With Mechanical Loading and Vascular Coculture , 2011, Circulation research.

[22]  W. Bloch,et al.  Contractile properties of early human embryonic stem cell-derived cardiomyocytes: beta-adrenergic stimulation induces positive chronotropy and lusitropy but not inotropy. , 2012, Stem cells and development.

[23]  Nenad Bursac,et al.  Mesoscopic hydrogel molding to control the 3D geometry of bioartificial muscle tissues , 2009, Nature Protocols.

[24]  L. Gepstein,et al.  A combined cell therapy and in-situ tissue-engineering approach for myocardial repair. , 2011, Biomaterials.

[25]  Raphael Rubin,et al.  Rubin's Pathology: Clinicopathologic Foundations of Medicine. , 2011 .

[26]  Sean P. Palecek,et al.  Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling , 2012, Proceedings of the National Academy of Sciences.

[27]  Peter Kohl,et al.  Simultaneous Voltage and Calcium Mapping of Genetically Purified Human Induced Pluripotent Stem Cell–Derived Cardiac Myocyte Monolayers , 2012, Circulation research.

[28]  G Arnold,et al.  Evidence for functional relevance of an enhanced expression of the Na(+)-Ca2+ exchanger in failing human myocardium. , 1996, Circulation.

[29]  Jürgen Hescheler,et al.  Organotypic slice culture from human adult ventricular myocardium. , 2012, Cardiovascular research.

[30]  N. Alpert,et al.  Energetics of isometric force development in control and volume-overload human myocardium. Comparison with animal species. , 1991, Circulation research.

[31]  G. Hasenfuss,et al.  Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts. , 2013, European heart journal.

[32]  N. Alpert,et al.  Altered Myocardial Force‐Frequency Relation in Human Heart Failure , 1992, Circulation.

[33]  C. Moravec,et al.  Human cardiac myosin heavy chain isoforms in fetal and failing adult atria and ventricles. , 2001, American journal of physiology. Heart and circulatory physiology.

[34]  M. Suematsu,et al.  Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. , 2013, Cell stem cell.

[35]  R. Passier,et al.  NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes , 2011, Nature Methods.

[36]  Kam W Leong,et al.  Pluripotent stem cell-derived cardiac tissue patch with advanced structure and function. , 2011, Biomaterials.

[37]  J. Schiller,et al.  Calcium Handling in Embryonic Stem Cell–Derived Cardiac Myocytes , 2006, Annals of the New York Academy of Sciences.

[38]  N. Bursac,et al.  Cardiac fibroblast paracrine factors alter impulse conduction and ion channel expression of neonatal rat cardiomyocytes. , 2009, Cardiovascular research.

[39]  Nenad Bursac,et al.  Local tissue geometry determines contractile force generation of engineered muscle networks. , 2012, Tissue engineering. Part A.

[40]  C. Hodonsky,et al.  Variable optimization for the formation of three-dimensional self-organized heart muscle , 2009, In Vitro Cellular & Developmental Biology - Animal.

[41]  D. Allen,et al.  Effects of Acidosis on Ventricular Muscle From Adult and Neonatal Rats , 1988, Circulation research.

[42]  R. Passier,et al.  Differentiation of human embryonic stem cells to cardiomyocytes by coculture with endoderm in serum-free medium. , 2007, Current protocols in stem cell biology.

[43]  Thomas Boudou,et al.  A microfabricated platform to measure and manipulate the mechanics of engineered cardiac microtissues. , 2012, Tissue engineering. Part A.

[44]  M. Valderrábano,et al.  Influence of anisotropic conduction properties in the propagation of the cardiac action potential. , 2007, Progress in biophysics and molecular biology.

[45]  R J Cohen,et al.  Cardiac muscle tissue engineering: toward an in vitro model for electrophysiological studies. , 1999, American journal of physiology. Heart and circulatory physiology.

[46]  Norio Nakatsuji,et al.  A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions. , 2012, Cell reports.

[47]  Andreas Hess,et al.  Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts , 2006, Nature Medicine.

[48]  Jackie Schiller,et al.  Calcium Handling in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes , 2011, PloS one.

[49]  Justin S. Weinbaum,et al.  Cell-induced alignment augments twitch force in fibrin gel-based engineered myocardium via gap junction modification. , 2009, Tissue engineering. Part A.

[50]  Ofer Binah,et al.  Functional and developmental properties of human embryonic stem cells-derived cardiomyocytes. , 2007, Journal of electrocardiology.

[51]  Ronald A. Li,et al.  Human pluripotent stem cell-based approaches for myocardial repair: from the electrophysiological perspective. , 2011, Molecular pharmaceutics.

[52]  Yoram Rudy,et al.  Simulation of the Undiseased Human Cardiac Ventricular Action Potential: Model Formulation and Experimental Validation , 2011, PLoS Comput. Biol..

[53]  B. Nadal-Ginard,et al.  Expression of the cardiac ventricular alpha- and beta-myosin heavy chain genes is developmentally and hormonally regulated. , 1984, The Journal of biological chemistry.

[54]  E. Sasaki,et al.  Nongenetic method for purifying stem cell–derived cardiomyocytes , 2010, Nature Methods.