Murine and human pluripotent stem cell-derived cardiac bodies form contractile myocardial tissue in vitro.

AIMS We explored the use of highly purified murine and human pluripotent stem cell (PSC)-derived cardiomyocytes (CMs) to generate functional bioartificial cardiac tissue (BCT) and investigated the role of fibroblasts, ascorbic acid (AA), and mechanical stimuli on tissue formation, maturation, and functionality. METHODS AND RESULTS Murine and human embryonic/induced PSC-derived CMs were genetically enriched to generate three-dimensional CM aggregates, termed cardiac bodies (CBs). Addressing the critical limitation of major CM loss after single-cell dissociation, non-dissociated CBs were used for BCT generation, which resulted in a structurally and functionally homogenous syncytium. Continuous in situ characterization of BCTs, for 21 days, revealed that three critical factors cooperatively improve BCT formation and function: both (i) addition of fibroblasts and (ii) ascorbic acid supplementation support extracellular matrix remodelling and CB fusion, and (iii) increasing static stretch supports sarcomere alignment and CM coupling. All factors together considerably enhanced the contractility of murine and human BCTs, leading to a so far unparalleled active tension of 4.4 mN/mm(2) in human BCTs using optimized conditions. Finally, advanced protocols were implemented for the generation of human PSC-derived cardiac tissue using a defined animal-free matrix composition. CONCLUSION BCT with contractile forces comparable with native myocardium can be generated from enriched, PSC-derived CMs, based on a novel concept of tissue formation from non-dissociated cardiac cell aggregates. In combination with the successful generation of tissue using a defined animal-free matrix, this represents a major step towards clinical applicability of stem cell-based heart tissue for myocardial repair.

[1]  Hong Jiang,et al.  Creation of Engineered Cardiac Tissue In Vitro From Mouse Embryonic Stem Cells , 2006, Circulation.

[2]  P. Janmey,et al.  Tissue Cells Feel and Respond to the Stiffness of Their Substrate , 2005, Science.

[3]  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.

[4]  C. Mummery,et al.  Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells. , 2008, Differentiation; research in biological diversity.

[5]  R. James,et al.  Growth Induced by Incremental Static Stretch in Adult Rabbit Latissimus Dorsi Muscle , 2000, Experimental physiology.

[6]  E. Bettiol,et al.  Developmental Changes in Cardiomyocytes Differentiated from Human Embryonic Stem Cells: A Molecular and Electrophysiological Approach , 2007, Stem cells.

[7]  J. Mandl,et al.  Vitamin C: update on physiology and pharmacology , 2009, British journal of pharmacology.

[8]  K. Woodhouse,et al.  Fiber alignment and coculture with fibroblasts improves the differentiated phenotype of murine embryonic stem cell‐derived cardiomyocytes for cardiac tissue engineering , 2012, Biotechnology and bioengineering.

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

[10]  E. Olson,et al.  Transient Regenerative Potential of the Neonatal Mouse Heart , 2011, Science.

[11]  Larry A Taber,et al.  Regional epicardial strain in the embryonic chick heart during the early looping stages. , 2003, Journal of biomechanics.

[12]  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.

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

[14]  Andreas Hess,et al.  Cardiac Grafting of Engineered Heart Tissue in Syngenic Rats , 2002, Circulation.

[15]  Alexander Meissner,et al.  Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. , 2010, Cell stem cell.

[16]  Payam Akhyari,et al.  A novel miniaturized multimodal bioreactor for continuous in situ assessment of bioartificial cardiac tissue during stimulation and maturation. , 2011, Tissue engineering. Part C, Methods.

[17]  Wolfram-Hubertus Zimmermann,et al.  Optimizing Engineered Heart Tissue for Therapeutic Applications as Surrogate Heart Muscle , 2006, Circulation.

[18]  Thomas Eschenhagen,et al.  Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement , 2000, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[19]  Xiaojun Ma,et al.  Scalable Producing Embryoid Bodies by Rotary Cell Culture System and Constructing Engineered Cardiac Tissue with ES‐Derived Cardiomyocytes in Vitro , 2006, Biotechnology progress.

[20]  A. Haverich,et al.  Induced pluripotent stem cell (iPSC)-derived Flk-1 progenitor cells engraft, differentiate, and improve heart function in a mouse model of acute myocardial infarction. , 2011, European heart journal.

[21]  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.

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

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

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

[25]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[26]  Timothy J. Nelson,et al.  Repair of acute myocardial infarction by human stemness factors induced pluripotent stem cells. , 2009, Circulation.

[27]  P. Doevendans,et al.  Improvement of mouse cardiac function by hESC-derived cardiomyocytes correlates with vascularity but not graft size. , 2009, Stem cell research.

[28]  W. Claycomb,et al.  Effect of Mechanical Loading on Three-Dimensional Cultures of Embryonic Stem Cell-Derived Cardiomyocytes , 2008 .

[29]  Hideki Uosaki,et al.  Directed and Systematic Differentiation of Cardiovascular Cells From Mouse Induced Pluripotent Stem Cells , 2008, Circulation.

[30]  Xuan Yuan,et al.  A Universal System for Highly Efficient Cardiac Differentiation of Human Induced Pluripotent Stem Cells That Eliminates Interline Variability , 2011, PloS one.

[31]  N. Bursac,et al.  Implantation of Mouse Embryonic Stem Cell-Derived Cardiac Progenitor Cells Preserves Function of Infarcted Murine Hearts , 2010, PloS one.

[32]  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.

[33]  B. Fleischmann,et al.  Developmental changes in contractility and sarcomeric proteins from the early embryonic to the adult stage in the mouse heart , 2003, The Journal of physiology.

[34]  篠原 隆司,et al.  Induction of pluripotent stem cell cells from germ cells , 2012 .

[35]  Stefan Wagner,et al.  Generation of induced pluripotent stem cells from human cord blood. , 2009, Cell stem cell.

[36]  Lars S. Maier,et al.  Generation of Functional Murine Cardiac Myocytes From Induced Pluripotent Stem Cells , 2008, Circulation.

[37]  Wolfgang A. Linke,et al.  Terminal Differentiation, Advanced Organotypic Maturation, and Modeling of Hypertrophic Growth in Engineered Heart Tissue , 2011, Circulation research.

[38]  Stanley Nattel,et al.  Regional and tissue specific transcript signatures of ion channel genes in the non‐diseased human heart , 2007, The Journal of physiology.

[39]  P. Doevendans,et al.  Cardiomyocyte cell cycle activation improves cardiac function after myocardial infarction. , 2008, Cardiovascular research.

[40]  T. Eschenhagen.  The beat goes on: human heart muscle from pluripotent stem cells. , 2011, Circulation research.

[41]  A. Kirschning,et al.  Fully defined in situ cross-linkable alginate and hyaluronic acid hydrogels for myocardial tissue engineering. , 2013, Biomaterials.

[42]  Liu Wang,et al.  Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells , 2011, Cell Research.

[43]  K. Plath,et al.  Reprogrammed Mouse Fibroblasts Differentiate into Cells of the Cardiovascular and Hematopoietic Lineages , 2008, Stem cells.

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

[45]  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.

[46]  Rafael Beyar,et al.  Transplantation of human embryonic stem cell-derived cardiomyocytes improves myocardial performance in infarcted rat hearts. , 2007, Journal of the American College of Cardiology.

[47]  Takahiro Ishiwata,et al.  Developmental Changes in Ventricular Diastolic Function Correlate With Changes in Ventricular Myoarchitecture in Normal Mouse Embryos , 2003, Circulation research.

[48]  Steven P Jones,et al.  Functional Integration of Electrically Active Cardiac Derivatives From Genetically Engineered Human Embryonic Stem Cells With Quiescent Recipient Ventricular Cardiomyocytes: Insights Into the Development of Cell-Based Pacemakers , 2005, Circulation.

[49]  Peter W Zandstra,et al.  Creation of mouse embryonic stem cell-derived cardiac cell sheets. , 2011, Biomaterials.

[50]  J. Itskovitz‐Eldor,et al.  Generation and Characterization of Functional Cardiomyocytes from Rhesus Monkey Embryonic Stem Cells , 2006, Stem cells.

[51]  Lior Gepstein,et al.  Derivation and cardiomyocyte differentiation of induced pluripotent stem cells from heart failure patients. , 2013, European heart journal.

[52]  B. Fleischmann,et al.  Fibroblasts facilitate the engraftment of embryonic stem cell-derived cardiomyocytes on three-dimensional collagen matrices and aggregation in hanging drops. , 2010, Stem cells and development.

[53]  Lila R Collins,et al.  Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts , 2007, Nature Biotechnology.

[54]  Takashi Aoi,et al.  Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts , 2008, Nature Biotechnology.

[55]  R. Jaenisch,et al.  Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases , 2009, Nature Biotechnology.

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

[57]  Karl-Ludwig Laugwitz,et al.  Patient-specific induced pluripotent stem-cell models for long-QT syndrome. , 2010, New England Journal of Medicine.

[58]  K. McCreath,et al.  Mitochondrial Reactive Oxygen Species Mediate Cardiomyocyte Formation from Embryonic Stem Cells in High Glucose , 2010, Stem cells.

[59]  L Gepstein,et al.  Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. , 2001, The Journal of clinical investigation.

[60]  Milica Radisic,et al.  Challenges in cardiac tissue engineering. , 2010, Tissue engineering. Part B, Reviews.