Absence of full-length dystrophin impairs normal maturation and contraction of cardiomyocytes derived from human-induced pluripotent stem cells.

AIMS: Heart failure invariably affects patients with various forms of muscular dystrophy (MD), but the onset and molecular sequelae of altered structure and function resulting from full-length dystrophin (Dp427) deficiency in MD heart tissue are poorly understood. To better understand the role of dystrophin in cardiomyocyte development and the earliest phase of Duchenne muscular dystrophy (DMD) cardiomyopathy, we studied human cardiomyocytes differentiated from induced pluripotent stem cells (hiPSC-CMs) obtained from the urine of a DMD patient. METHODS AND RESULTS: The contractile properties of patient-specific hiPSC-CMs, with no detectable dystrophin (DMD-CMs with a deletion of exon 50), were compared to CMs containing a CRISPR-Cas9 mediated deletion of a single G base at position 263 of the dystrophin gene (c.263delG-CMs) isogenic to the parental line of hiPSC-CMs from a healthy individual. We hypothesized that the absence of a dystrophin-actin linkage would adversely affect myofibril and cardiomyocyte structure and function. Cardiomyocyte maturation was driven by culturing long-term (80-100 days) on a nanopatterned surface, which resulted in hiPSC-CMs with adult-like dimensions and aligned myofibrils. CONCLUSIONS: Our data demonstrate that lack of Dp427 results in reduced myofibril contractile tension, slower relaxation kinetics, and to Ca2+ handling abnormalities, similar to DMD cells, suggesting either retarded or altered maturation of cardiomyocyte structures associated with these functions. This study offers new insights into the functional consequences of Dp427 deficiency at an early stage of cardiomyocyte development in both patient-derived and CRISPR-generated models of dystrophin deficiency.

Mechanical Stress Conditioning and Electrical Stimulation Promote Contractility and Force Maturation of Induced Pluripotent Stem Cell-Derived Human Cardiac Tissue.

BACKGROUND: Tissue engineering enables the generation of functional human cardiac tissue with cells derived in vitro in combination with biocompatible materials. Human-induced pluripotent stem cell-derived cardiomyocytes provide a cell source for cardiac tissue engineering; however, their immaturity limits their potential applications. Here we sought to study the effect of mechanical conditioning and electric pacing on the maturation of human-induced pluripotent stem cell-derived cardiac tissues. METHODS: Cardiomyocytes derived from human-induced pluripotent stem cells were used to generate collagen-based bioengineered human cardiac tissue. Engineered tissue constructs were subjected to different mechanical stress and electric pacing conditions. RESULTS: The engineered human myocardium exhibits Frank-Starling-type force-length relationships. After 2 weeks of static stress conditioning, the engineered myocardium demonstrated increases in contractility (0.63±0.10 mN/mm2 vs 0.055±0.009 mN/mm2 for no stress), tensile stiffness, construct alignment, and cell size. Stress conditioning also increased SERCA2 (Sarco/Endoplasmic Reticulum Calcium ATPase 2) expression, which correlated with a less negative force-frequency relationship. When electric pacing was combined with static stress conditioning, the tissues showed an additional increase in force production (1.34±0.19 mN/mm2), with no change in construct alignment or cell size, suggesting maturation of excitation-contraction coupling. Supporting this notion, we found expression of RYR2 (Ryanodine Receptor 2) and SERCA2 further increased by combined static stress and electric stimulation. CONCLUSIONS: These studies demonstrate that electric pacing and mechanical stimulation promote maturation of the structural, mechanical, and force generation properties of human-induced pluripotent stem cell-derived cardiac tissues.

Isolation and Mechanical Measurements of Myofibrils from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Tension production and contractile properties are poorly characterized aspects of excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Previous approaches have been limited due to the small size and structural immaturity of early-stage hiPSC-CMs. We developed a substrate nanopatterning approach to produce hiPSC-CMs in culture with adult-like dimensions, T-tubule-like structures, and aligned myofibrils. We then isolated myofibrils from hiPSC-CMs and measured the tension and kinetics of activation and relaxation using a custom-built apparatus with fast solution switching. The contractile properties and ultrastructure of myofibrils more closely resembled human fetal myofibrils of similar gestational age than adult preparations. We also demonstrated the ability to study the development of contractile dysfunction of myofibrils from a patient-derived hiPSC-CM cell line carrying the familial cardiomyopathy MYH7 mutation (E848G). These methods can bring new insights to understanding cardiomyocyte maturation and developmental mechanical dysfunction of hiPSC-CMs with cardiomyopathic mutations.

Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

An improved cryosection method for polyethylene glycol hydrogels used in tissue engineering.

The high water content of hydrogels allows these materials to closely mimic the native biological extracellular conditions, but it also makes difficult the histological preparation of hydrogel-based bioengineered tissue. Paraffin-embedding techniques require dehydration of hydrogels, resulting in substantial collapse and deformation, whereas cryosectioning is hampered by the formation of ice crystals within the hydrogel material. Here, we sought to develop a method to obtain good-quality cryosections for the microscopic evaluation of hydrogel-based tissue-engineered constructs, using polyethylene glycol (PEG) as a test hydrogel. Conventional sucrose solutions, which dehydrate cells while leaving extracellular water in place, produce a hydrogel block that is brittle and difficult to section. We therefore replaced sucrose with multiple protein-based and nonprotein-based solutions as cryoprotectants. Our analysis demonstrated that overnight incubation in bovine serum albumin (BSA), fetal bovine serum (FBS), polyvinyl alcohol (PVA), optimum cutting temperature (OCT) compound, and Fisher HistoPrep frozen tissue-embedding media work well to improve the cryosectioning of hydrogels. The protein-based solutions give background staining with routine hematoxylin and eosin, but the use of nonprotein-based solutions PVA and OCT reduces this background by 50%. These methods preserve the tissue architecture and cellular details with both in vitro PEG constructs and in constructs that have been implanted in vivo. This simple hydrogel cryosectioning technique improves the methodology for creation of good-quality histological sections from hydrogels in multiple applications.

Lack of thrombospondin-2 reduces fibrosis and increases vascularity around cardiac cell grafts.

BACKGROUND: Fibrosis around cardiac cell injections represents an obstacle to graft integration in cell-based cardiac repair. Thrombospondin-2 (TSP-2) is a pro-fibrotic, anti-angiogenic matricellular protein and an attractive target for therapeutic knockdown to improve cardiac graft integration and survival. METHODS: We used a TSP-2 knockout (KO) mouse in conjunction with a fetal murine cardiomyocyte grafting model to evaluate the effects of a lack of TSP-2 on fibrosis, vascular density, and graft size in the heart. RESULTS: Two weeks after grafting in the uninjured heart, fibrosis area was reduced 4.5-fold in TSP-2 KO mice, and the thickness of the peri-graft scar capsule was reduced sevenfold compared to wild-type (WT). Endothelial cell density in the peri-graft region increased 2.5-fold in the absence of TSP-2, and cardiomyocyte graft size increased by 46% in TSP-2 KO hearts. CONCLUSIONS: TSP-2 is a key regulator of fibrosis and angiogenesis following cell grafting in the heart, and its absence promotes better graft integration, vascularization, and survival. SUMMARY: Fibrosis around cardiac cell injections impairs graft integration in cell-based cardiac repair. TSP-2 is a pro-fibrotic, anti-angiogenic matricellular protein. Using a TSP-2-knockout mouse model and cardiac cell transplantation, we found significantly reduced fibrosis and increased endothelial cell density in the peri-graft region. Thus, TSP-2 is an attractive target for therapeutic knockdown to improve cardiac graft integration and survival.

Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts.

Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts, but two critical issues related to their electrophysiological behaviour in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear whether these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea-pig model to show that hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia. To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically encoded calcium sensor, GCaMP3 (refs 4, 5). By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host­graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.

Growth of engineered human myocardium with mechanical loading and vascular coculture.

RATIONALE: The developing heart requires both mechanical load and vascularization to reach its proper size, yet the regulation of human heart growth by these processes is poorly understood. OBJECTIVE: We seek to elucidate the responses of immature human myocardium to mechanical load and vascularization using tissue engineering approaches. METHODS AND RESULTS: Using human embryonic stem cell and human induced pluripotent stem cell-derived cardiomyocytes in a 3-dimensional collagen matrix, we show that uniaxial mechanical stress conditioning promotes 2-fold increases in cardiomyocyte and matrix fiber alignment and enhances myofibrillogenesis and sarcomeric banding. Furthermore, cyclic stress conditioning markedly increases cardiomyocyte hypertrophy (2.2-fold) and proliferation rates (21%) versus unconditioned constructs. Addition of endothelial cells enhances cardiomyocyte proliferation under all stress conditions (14% to 19%), and addition of stromal supporting cells enhances formation of vessel-like structures by ≈10-fold. Furthermore, these optimized human cardiac tissue constructs generate Starling curves, increasing their active force in response to increased resting length. When transplanted onto hearts of athymic rats, the human myocardium survives and forms grafts closely apposed to host myocardium. The grafts contain human microvessels that are perfused by the host coronary circulation. CONCLUSIONS: Our results indicate that both mechanical load and vascular cell coculture control cardiomyocyte proliferation, and that mechanical load further controls the hypertrophy and architecture of engineered human myocardium. Such constructs may be useful for studying human cardiac development as well as for regenerative therapy.

Developing vasculature and stroma in engineered human myocardium.

We recently developed a scaffold-free patch of human myocardium with human embryonic stem cell-derived cardiomyocytes and showed that stromal and endothelial cells form vascular networks in vitro and improve cardiomyocyte engraftment. Here, we hypothesize that stromal cells regulate the angiogenic phenotype by modulating the extracellular matrix (ECM). Human marrow stromal cells (hMSCs) support the greatest degree of endothelial cell organization, at 1.3- to 2.4-fold higher than other stromal cells tested. Stromal cells produce abundant ECM components in patches, including fibrillar collagen, hyaluronan, and versican. We identified two clonal hMSC lines that supported endothelial networks poorly and robustly. Interestingly, the pro-angiogenic hMSCs express high levels of versican, a chondroitin sulfate proteglycan that modulates angiogenesis and wound healing, whereas poorly angiogenic hMSCs produce little versican. When transplanted onto uninjured athymic rat hearts, patches with proangiogenic hMSCs develop ~ 50-fold more human vessels and form anastomoses with the host circulation, resulting in chimeric vessels containing erythrocytes. Thus, stromal cells play a key role in supporting vascularization of engineered human myocardium. Different stromal cell types vary widely in their proangiogenic ability, likely due in part to differences in ECM synthesis. Comparison of these cells defines an in vitro predictive platform for studying vascular development.

Scaffold-free human cardiac tissue patch created from embryonic stem cells.

Progress in cardiac tissue engineering has been limited by (1) unfavorable cell and host responses to biomaterial scaffolds, (2) lack of suitable human cardiomyocyte sources, and (3) lack of fabrication techniques for scalable production of engineered tissue constructs. Here we report a novel and scalable method to generate scaffold-free human cardiac tissue patches. Human embryonic stem cells were differentiated to cardiomyocytes using activin A and BMP4 and placed into suspension on a rotating orbital shaker. Cells aggregated to form macroscopic disc-shaped patches of beating tissue after 2 days. Patch diameter was directly proportional to input cell number (approximately 11 mm with 12 million cells), and patches were 300-600 mum thick. Cardiomyocytes were concentrated around the patch edges and exhibited increased purity and maturation with time, comprising approximately 80% of total cells after 11 days. Noncardiac cell elements, primarily epithelium, were present at day 2 but were diminished markedly at later time points. Cardiomyocyte proliferation occurred throughout the patches at day 2 but declined by day 8. Patches exhibited automaticity and synchronous calcium transients, indicating electromechanical coupling. These novel scaffold-free human myocardial patches address critical challenges related to human cell sourcing and tissue fabrication that previously inhibited progress in cardiac tissue engineering.

Chromatin remodeling during mouse and human embryonic stem cell differentiation.

Embryonic stem cell (ESC) differentiation is an excellent model to study chromatin changes at developmentally regulated loci. Differentiating mouse and human ESCs increase genome-wide acetylation (euchromatic) and tri-methylation (heterochromatic) of lysine 9 on histone H3. The Oct4 locus is euchromatic when expressed in undifferentiated ESCs and heterochromatic after differentiation. Brachyury T, a mesoderm-specific transcription factor, is not yet expressed in undifferentiated cells, where its locus has "bivalent" tri-methyl lysine 4 and lysine 27 modifications. During directed differentiation to pre-cardiac mesoderm, the activated brachyury locus has high levels of tri-methyl lysine 4 (euchromatin), switching to heterochromatin after gene silencing. Thus, ESC differentiation is accompanied by genome-wide commitment to euchromatin or heterochromatin. Undifferentiated hESCs bivalently modify the brachyury locus, activate it to euchromatin during mesoderm induction, and subsequently repress it to heterochromatin, demonstrating, to our knowledge, the first analysis of chromatin dynamics at a locus essential for mesoderm and endoderm differentiation.

Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts.

Cardiomyocytes derived from human embryonic stem (hES) cells potentially offer large numbers of cells to facilitate repair of the infarcted heart. However, this approach has been limited by inefficient differentiation of hES cells into cardiomyocytes, insufficient purity of cardiomyocyte preparations and poor survival of hES cell-derived myocytes after transplantation. Seeking to overcome these challenges, we generated highly purified human cardiomyocytes using a readily scalable system for directed differentiation that relies on activin A and BMP4. We then identified a cocktail of pro-survival factors that limits cardiomyocyte death after transplantation. These techniques enabled consistent formation of myocardial grafts in the infarcted rat heart. The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function after myocardial infarction compared with controls receiving noncardiac hES cell derivatives or vehicle. The ability of hES cell-derived cardiomyocytes to partially remuscularize myocardial infarcts and attenuate heart failure encourages their study under conditions that closely match human disease.

Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response.

Embryonic stem (ES) cells are promising for cardiac repair, but directing their differentiation toward cardiomyocytes remains challenging. We investigated whether the heart guides ES cells toward cardiomyocytes in vivo and whether allogeneic ES cells were immunologically tolerated. Undifferentiated mouse ES cells consistently formed cardiac teratomas in nude or immunocompetent syngeneic mice. Cardiac teratomas contained no more cardiomyocytes than hind-limb teratomas, suggesting lack of guided differentiation. ES cells also formed teratomas in infarcted hearts, indicating injury-related signals did not direct cardiac differentiation. Allogeneic ES cells also caused cardiac teratomas, but these were immunologically rejected after several weeks, in association with increased inflammation and up-regulation of class I and II histocompatibility antigens. Fusion between ES cells and cardiomyocytes occurred in vivo, but was rare. Infarct autofluorescence was identified as an artifact that might be mistaken for enhanced GFP expression and true regeneration. Hence, undifferentiated ES cells were not guided toward a cardiomyocyte fate in either normal or infarcted hearts, and there was no evidence for allogeneic immune tolerance of ES cell derivatives. Successful cardiac repair strategies involving ES cells will need to control cardiac differentiation, avoid introducing undifferentiated cells, and will likely require immune modulation to avoid rejection.

Formation of human myocardium in the rat heart from human embryonic stem cells.

Human embryonic stem cells (hESCs) offer the opportunity to replenish cells lost in the postinfarct heart. We explored whether human myocardium could be generated in rat hearts by injecting differentiated cardiac-enriched hESC progeny into the left ventricular wall of athymic rats. Although initial grafts were predominantly epithelial, noncardiac elements were lost over time, and grafts consisted predominantly of cardiomyocytes by 4 weeks. No teratomatous elements were observed. Engrafted cardiomyocytes were glycogen-rich and expressed expected cardiac markers including beta-myosin heavy chain, myosin light chain 2v, and atrial natriuretic factor. Heat-shock treatment improved graft size approximately threefold. The cardiac implants exhibited substantial angiogenesis, both recipient and graft derived. Importantly, there was greater proliferation in human cardiomyocytes than previously seen in rodent-derived cardiomyocytes: 14.4% of graft cardiomyocytes expressed the proliferation marker Ki-67, and 2.7% incorporated the thymidine analog BrdU 4 weeks after transplantation. This proliferation was associated with a sevenfold increase in graft size over the 4-week interval. Thus, hESCs can form human myocardium in the rat heart, permitting studies of human myocardial development and physiology and supporting the feasibility of their use in myocardial repair.