TBX20 Improves Contractility and Mitochondrial Function During Direct Human Cardiac Reprogramming.

BACKGROUND: Direct cardiac reprogramming of fibroblasts into cardiomyocytes has emerged as a promising strategy to remuscularize injured myocardium. However, it is insufficient to generate functional induced cardiomyocytes from human fibroblasts using conventional reprogramming cocktails, and the underlying molecular mechanisms are not well studied. METHODS: To discover potential missing factors for human direct reprogramming, we performed transcriptomic comparison between human induced cardiomyocytes and functional cardiomyocytes. RESULTS: We identified TBX20 (T-box transcription factor 20) as the top cardiac gene that is unable to be activated by the MGT133 reprogramming cocktail (MEF2C, GATA4, TBX5, and miR-133). TBX20 is required for normal heart development and cardiac function in adult cardiomyocytes, yet its role in cardiac reprogramming remains undefined. We show that the addition of TBX20 to the MGT133 cocktail (MGT+TBX20) promotes cardiac reprogramming and activates genes associated with cardiac contractility, maturation, and ventricular heart. Human induced cardiomyocytes produced with MGT+TBX20 demonstrated more frequent beating, calcium oscillation, and higher energy metabolism as evidenced by increased mitochondria numbers and mitochondrial respiration. Mechanistically, comprehensive transcriptomic, chromatin occupancy, and epigenomic studies revealed that TBX20 colocalizes with MGT reprogramming factors at cardiac gene enhancers associated with heart contraction, promotes chromatin binding and co-occupancy of MGT factors at these loci, and synergizes with MGT for more robust activation of target gene transcription. CONCLUSIONS: TBX20 consolidates MGT cardiac reprogramming factors to activate cardiac enhancers to promote cardiac cell fate conversion. Human induced cardiomyocytes generated with TBX20 showed enhanced cardiac function in contractility and mitochondrial respiration.

Cardiomyocytes from CCND2-overexpressing human induced-pluripotent stem cells repopulate the myocardial scar in mice: A 6-month study.

BACKGROUND: Cardiomyocytes that have been differentiated from CCND2-overexpressing human induced-pluripotent stem cells (hiPSC-CCND2OE CMs) can proliferate when transplanted into mouse hearts after myocardial infarction (MI). However, it is unknown whether remuscularization can replace the thin LV scar and if the large muscle graft can electrophysiologically synchronize to the recipient myocardium. Our objectives are to evaluate the structural and functional potential of hiPSC-CCND2OE CMs in replacing the LV thin scar. METHODS: NOD/SCID mice were treated with hiPSC-CCND2OE CMs (i.e., the CCND2OE group), hiPSC-CCND2WT CMs (the CCND2WT group), or an equal volume of PBS immediately after experimentally-induced myocardial infarction. The treatments were administered to one site in the infarcted zone (IZ), two sites in the border zone (BZ), and a fourth group of animals underwent Sham surgery. RESULTS: Six months later, engrafted cells occupied >50% of the scarred region in CCND2OE animals, and exceeded the number of engrafted cells in CCND2WT animals by ~8-fold. Engrafted cells were also more common in the IZ than in the BZ for both cell-treatment groups. Measurements of cardiac function, infarct size, wall thickness, and cardiomyocyte hypertrophy were significantly improved in CCND2OE animals compared to animals from the CCND2WT or PBS-treatment groups. Measurements in the CCND2WT and PBS groups were similar, and markers for cell cycle activation and proliferation were significantly higher in hiPSC-CCND2OE CMs than in hiPSC-CCND2WT CMs. Optical mapping of action potential propagation indicated that the engrafted hiPSC-CCND2OE CMs were electrically coupled to each other and to the cells of the native myocardium. No evidence of tumor formation was observed in any animals. CONCLUSIONS: Six months after the transplantation, CCND2-overexpressing hiPSC-CMs proliferated and replaced >50% of the myocardial scar tissue. The large graft hiPSC-CCND2OE CMs also electrically integrated with the host myocardium, which was accompanied by a significant improvement in LV function.

Large Cardiac Muscle Patches Engineered From Human Induced-Pluripotent Stem Cell-Derived Cardiac Cells Improve Recovery From Myocardial Infarction in Swine.

BACKGROUND: Here, we generated human cardiac muscle patches (hCMPs) of clinically relevant dimensions (4 cm × 2 cm × 1.25 mm) by suspending cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human induced-pluripotent stem cells in a fibrin scaffold and then culturing the construct on a dynamic (rocking) platform. METHODS: In vitro assessments of hCMPs suggest maturation in response to dynamic culture stimulation. In vivo assessments were conducted in a porcine model of myocardial infarction (MI). Animal groups included: MI hearts treated with 2 hCMPs (MI+hCMP, n=13), MI hearts treated with 2 cell-free open fibrin patches (n=14), or MI hearts with neither experimental patch (n=15); a fourth group of animals underwent sham surgery (Sham, n=8). Cardiac function and infarct size were evaluated by MRI, arrhythmia incidence by implanted loop recorders, and the engraftment rate by calculation of quantitative polymerase chain reaction measurements of expression of the human Y chromosome. Additional studies examined the myocardial protein expression profile changes and potential mechanisms of action that related to exosomes from the cell patch. RESULTS: The hCMPs began to beat synchronously within 1 day of fabrication, and after 7 days of dynamic culture stimulation, in vitro assessments indicated the mechanisms related to the improvements in electronic mechanical coupling, calcium-handling, and force generation, suggesting a maturation process during the dynamic culture. The engraftment rate was 10.9±1.8% at 4 weeks after the transplantation. The hCMP transplantation was associated with significant improvements in left ventricular function, infarct size, myocardial wall stress, myocardial hypertrophy, and reduced apoptosis in the periscar boarder zone myocardium. hCMP transplantation also reversed some MI-associated changes in sarcomeric regulatory protein phosphorylation. The exosomes released from the hCMP appeared to have cytoprotective properties that improved cardiomyocyte survival. CONCLUSIONS: We have fabricated a clinically relevant size of hCMP with trilineage cardiac cells derived from human induced-pluripotent stem cells. The hCMP matures in vitro during 7 days of dynamic culture. Transplantation of this type of hCMP results in significantly reduced infarct size and improvements in cardiac function that are associated with reduction in left ventricular wall stress. The hCMP treatment is not associated with significant changes in arrhythmogenicity.

Myocardial Tissue Engineering With Cells Derived From Human-Induced Pluripotent Stem Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold.

RATIONALE: Conventional 3-dimensional (3D) printing techniques cannot produce structures of the size at which individual cells interact. OBJECTIVE: Here, we used multiphoton-excited 3D printing to generate a native-like extracellular matrix scaffold with submicron resolution and then seeded the scaffold with cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human-induced pluripotent stem cells to generate a human-induced pluripotent stem cell-derived cardiac muscle patch (hCMP), which was subsequently evaluated in a murine model of myocardial infarction. METHODS AND RESULTS: The scaffold was seeded with ≈50 000 human-induced pluripotent stem cell-derived cardiomyocytes, smooth muscle cells, and endothelial cells (in a 2:1:1 ratio) to generate the hCMP, which began generating calcium transients and beating synchronously within 1 day of seeding; the speeds of contraction and relaxation and the peak amplitudes of the calcium transients increased significantly over the next 7 days. When tested in mice with surgically induced myocardial infarction, measurements of cardiac function, infarct size, apoptosis, both vascular and arteriole density, and cell proliferation at week 4 after treatment were significantly better in animals treated with the hCMPs than in animals treated with cell-free scaffolds, and the rate of cell engraftment in hCMP-treated animals was 24.5% at week 1 and 11.2% at week 4. CONCLUSIONS: Thus, the novel multiphoton-excited 3D printing technique produces extracellular matrix-based scaffolds with exceptional resolution and fidelity, and hCMPs fabricated with these scaffolds may significantly improve recovery from ischemic myocardial injury.

The stress kinase JNK regulates gap junction Cx43 gene expression and promotes atrial fibrillation in the aged heart.

BACKGROUND: The stress kinase c-jun N-terminal kinase (JNK) is critical in the pathogenesis of cardiac diseases associated with an increased incidence of atrial fibrillation (AF), the most common arrhythmia in the elderly. We recently discovered that JNK activation is linked to the loss of gap junction connexin43 (Cx43) and enhanced atrial arrhythmogenicity. However, direct evidence for JNK-mediated impairment of intercellular coupling (cell-cell communication) in the intact aged atrium is lacking, as is evidence for whether and how JNK suppresses Cx43 in the aged human atrium. METHODS AND RESULTS: JNK activity in human atrial samples is correlated with both reduced Cx43 expression and increasing age. Using a unique technique of optical mapping space constant measurement, we found that impaired intercellular coupling and reduced Cx43 were linked to enhanced activation of JNK in intact aged rabbit atria. These JNK-associated alterations were further confirmed in naturally JNK activated aged mice and in cardiac-specific inducible MKK7D (JNK upstream activator) young mice. Moreover, JNK inhibition, using either JNK specific inhibitors in aged wild-type (WT) mice and JNK activator anisomycin-treated young WT mice or JNK1/2 dominant-negative mice with genetically inhibited cardiac JNK activity, completely eliminated these functional abnormalities. Furthermore, we discovered for the first time that long-term JNK activation downregulates Cx43 expression via c-jun suppressed transcriptional activity of the Cx43 gene promoter. CONCLUSION: Our results demonstrate that JNK is a critical regulator of Cx43 expression, and that augmented JNK activation in aged atria downregulates Cx43 to impair cell-cell communication and promote the development of AF. JNK inhibition may represent a promising therapeutic approach to prevent or treat AF in the elderly.

Spheroids of cardiomyocytes derived from human-induced pluripotent stem cells improve recovery from myocardial injury in mice.

The microenvironment of native heart tissue may be better replicated when cardiomyocytes are cultured in three-dimensional clusters (i.e., spheroids) than in monolayers or as individual cells. Thus, we differentiated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs) and allowed them to form spheroids and spheroid fusions that were characterized in vitro and evaluated in mice after experimentally induced myocardial infarction (MI). Synchronized contractions were observed within 24 h of spheroid formation, and optical mapping experiments confirmed the presence of both Ca2+ transients and propagating action potentials. In spheroid fusions, the intraspheroid conduction velocity was 7.0 ± 3.8 cm/s on days 1- 2 after formation, whereas the conduction velocity between spheroids increased significantly ( P = 0.003) from 0.8 ± 1.1 cm/s on days 1- 2 to 3.3 ± 1.4 cm/s on day 7. For the murine MI model, five-spheroid fusions (200,000 hiPSC-CMs/spheroid) were embedded in a fibrin patch and the patch was transplanted over the site of infarction. Later (4 wk), echocardiographic measurements of left ventricular ejection fraction and fractional shortening were significantly greater in patch-treated animals than in animals that recovered without the patch, and the engraftment rate was 25.6% or 30% when evaluated histologically or via bioluminescence imaging, respectively. The exosomes released from the spheroid patch seemed to increase cardiac function. In conclusion, our results established the feasibility of using hiPSC-CM spheroids and spheroid fusions for cardiac tissue engineering, and, when fibrin patches containing hiPSC-CM spheroid fusions were evaluated in a murine MI model, the engraftment rate was much higher than the rates we have achieved via the direct intramyocardial injection. NEW & NOTEWORTHY Spheroids fuse in culture to produce structures with uniformly distributed cells. Furthermore, human cardiac lineage-induced pluripotent stem cells in cardiomyocytes in adjacent fused spheroids became electromechanically coupled as the fusions matured in vitro, and when the spheroids were combined with a biological matrix and administered as a patch over the infarcted region of mouse hearts, the engraftment rate exceeded 25%, and the treatment was associated with significant improvements in cardiac function via a paracrine mechanism, where exosomes released from the spheroid patch.

Hemodynamic Stimulation Using the Biomimetic Cardiac Tissue Model (BCTM) Enhances Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell (hiPSC)-derived cardio-myocytes (hiPSC-CMs) hold great promise for cardiovascular disease modeling and regenerative medicine. However, these cells are both structurally and functionally -immature, primarily due to their differentiation into cardiomyocytes occurring under static culture which only reproduces biomolecular cues and ignores the dynamic hemo-dynamic cues that shape early and late heart development during cardiogenesis. To evaluate the effects of hemodynamic stimuli on hiPSC-CM maturation, we used the biomimetic cardiac tissue model to reproduce the hemodynamics and pressure/volume changes associated with heart development. Following 7 days of gradually increasing stimulation, we show that hemodynamic loading results in (a) enhanced alignment of the cells and extracellular matrix, (b) significant increases in genes associated with physiological hypertrophy, (c) noticeable changes in sarcomeric organization and potential changes to cellular metabolism, and (d) a significant increase in fractional shortening, suggestive of a positive force frequency response. These findings suggest that culture of hiPSC-CMs under conditions that accurately reproduce hemodynamic cues results in structural orga-nization and molecular signaling consistent with organ growth and functional maturation.

Biomimetic Cardiac Tissue Model Enables the Adaption of Human Induced Pluripotent Stem Cell Cardiomyocytes to Physiological Hemodynamic Loads.

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) provide a human source of cardiomyocytes for use in cardiovascular research and regenerative medicine. However, attempts to use these cells in vivo have resulted in drastic cell death caused by mechanical, metabolic, and/or exogenous factors. To explore this issue, we designed a Biomimetic Cardiac Tissue Model (BCTM) where various parameters associated with heart function including heart rate, peak-systolic pressure, end-diastolic pressure and volume, end-systolic pressure and volume, and ratio of systole to diastole can all be precisely manipulated to apply hemodynamic loading to culture cells. Using the BCTM, two causes of low survivability in current cardiac stem cell therapies, mechanical and metabolic, were explored. iPSC-CMs were subject to physiologically relevant mechanical loading (50 mmHg systolic, 10% biaxial stretch) in either a low- or high-serum environment and mechanical loads were applied either immediately or gradually. Results confirm that iPSC-CMs subject to mechanical loading in low-serum conditions experienced widespread cell death. The rate of application of stress also played an important role in adaptability to mechanical loading. Under high-serum conditions, iPSC-CMs subject to gradual imposition of stress were comparable to iPSC-CMs maintained in static culture when evaluated in terms of cell viability, sarcomeric structure, action potentials and conduction velocities. In contrast, iPSC-CMs that were immediately exposed to mechanical loading had significantly lower cell viability, destruction of sarcomeres, smaller action potentials, and lower conduction velocities. We report that iPSC-CMs survival under physiologically relevant hemodynamic stress requires gradual imposition of mechanical loads in a nutrient-rich environment.

The role of dye affinity in optical measurements of Cai(2+) transients in cardiac muscle.

Previous experiments in cultures of neonatal rat myocytes demonstrated that the shape of Cai(2+) transients measured using high-affinity Ca(2+)-sensitive dyes may be misrepresented. The purpose of this study was to examine the role of dye affinity in Cai(2+) measurements in intact adult cardiac tissue by comparing optical recordings obtained with high- and low-affinity dyes. Experiments were carried out in porcine left ventricular (LV) wedge preparations stained locally by intramural injection via microcapillaries (diameter = 150 µm) with a low-affinity Ca(2+)-sensitive dye Fluo-4FF or Fluo-2LA (nominal Kd, ~7-10 µmol/l), high-affinity dye Rhod-2 (Kd = 0.57 µmol/l), and Fluo-4 or Fluo-2MA (Kd, ~0.4 µmol/l); in addition, tissue was stained with transmembrane potential (Vm)-sensitive dye RH-237. Optical recordings of Vm and Cai(2+) were made using optical fibers (diameter = 325 µm) glued with the microcapillaries. The durations of Cai(2+) transients measured at 50% level of recovery (CaD50) using high-affinity Fluo-4/Fluo-2MA dyes were up to ~81% longer than those measured with low-affinity Fluo-4FF/Fluo-2LA at long pacing cycle lengths (CL). In Fluo-4/Fluo-2MA measurements at long CLs, Cai(2+) transients often (~50% of cases) exhibited slow upstroke rise and extended plateau. In Rhod-2 measurements, CaD50 was moderately longer (up to ~35%) than in Fluo-4FF recordings, but Cai(2+) transient shapes were similar. In all series of measurements, mean action potential duration values were not significantly different (P > 0.05). The delays between Vm and Cai(2+) upstrokes were comparable for low- and high-affinity dyes (P > 0.05). In conclusion, measurements of Cai(2+) transient in ventricular myocardium are strongly affected by the affinity of Ca(2+) dyes. The high-affinity dyes may overestimate the duration and alter the shape of Cai(2+) transients.

Cardiac muscle patches containing four types of cardiac cells derived from human pluripotent stem cells improve recovery from cardiac injury in mice.

AIMS: We have shown that human cardiac muscle patches (hCMPs) containing three different types of cardiac cells-cardiomyocytes (CMs), smooth muscle cells (SMCs), and endothelial cells (ECs), all of which were differentiated from human pluripotent stem cells (hPSCs)-significantly improved cardiac function, infarct size, and hypertrophy in a pig model of myocardial infarction (MI). However, hPSC-derived CMs (hPSC-CMs) are phenotypically immature, which may lead to arrhythmogenic concerns; thus, since hPSC-derived cardiac fibroblasts (hPSC-CFs) appear to enhance the maturity of hPSC-CMs, we compared hCMPs containing hPSC-CMs, -SMCs, -ECs, and -CFs (4TCC-hCMPs) with a second hCMP construct that lacked hPSC-CFs but was otherwise identical [hCMP containing hPSC-CMs, -AECs, and -SMCs (3TCC-hCMPs)]. METHODS AND RESULTS: hCMPs were generated in a fibrin scaffold. MI was induced in severe combined immunodeficiency (SCID) mice through permanent coronary artery (left anterior descending) ligation, followed by treatment with cardiac muscle patches. Animal groups included: MI heart treated with 3TCC-hCMP; with 4TCC-hCMP; MI heart treated with no patch (MI group) and sham group. Cardiac function was evaluated using echocardiography, and cell engraftment rate and infarct size were evaluated histologically at 4 weeks after patch transplantation. The results from experiments in cultured hCMPs demonstrate that the inclusion of cardiac fibroblast in 4TCC-hCMPs had (i) better organized sarcomeres; (ii) abundant structural, metabolic, and ion-channel markers of CM maturation; and (iii) greater conduction velocities (31 ± 3.23 cm/s, P < 0.005) and action-potential durations (APD50 = 365 ms ± 2.649, P < 0.0001; APD = 408 ms ± 2.757, P < 0.0001) than those (velocity and APD time) in 3TCC-hCMPs. Furthermore, 4TCC-hCMPs transplantation resulted in better cardiac function [ejection fraction (EF) = 49.18% ± 0.86, P < 0.05], reduced infarct size (22.72% ± 0.98, P < 0.05), and better engraftment (15.99% ± 1.56, P < 0.05) when compared with 3TCC-hCMPs (EF = 41.55 ± 0.92%, infarct size = 39.23 ± 4.28%, and engraftment = 8.56 ± 1.79%, respectively). CONCLUSION: Collectively, these observations suggest that the inclusion of hPSC-CFs during hCMP manufacture promotes hPSC-CM maturation and increases the potency of implanted hCMPs for improving cardiac recovery in mice model of MI.

Transmural heterogeneity and remodeling of ventricular excitation-contraction coupling in human heart failure.

BACKGROUND: Excitation-contraction (EC) coupling is altered in end-stage heart failure. However, spatial heterogeneity of this remodeling has not been established at the tissue level in failing human heart. The objective of this article was to study functional remodeling of excitation-contraction coupling and calcium handling in failing and nonfailing human hearts. METHODS AND RESULTS: We simultaneously optically mapped action potentials and calcium transients in coronary perfused left ventricular wedge preparations from nonfailing (n=6) and failing (n=5) human hearts. Our major findings are the following. First, calcium transient duration minus action potential duration was longer at subendocardium in failing compared with nonfailing hearts during bradycardia (40 bpm). Second, the transmural gradient of calcium transient duration was significantly smaller in failing hearts compared with nonfailing hearts at fast pacing rates (100 bpm). Third, calcium transient in failing hearts had a flattened plateau at the midmyocardium and exhibited a 2-component slow rise at the subendocardium in 3 failing hearts. Fourth, calcium transient relaxation was slower at the subendocardium than at the subepicardium in both groups. Protein expression of sarcoplasmic reticulum Ca(2+)-ATPase 2a was lower at the subendocardium than the subepicardium in both nonfailing and failing hearts. Sarcoplasmic reticulum Ca(2+)-ATPase 2a protein expression at subendocardium was lower in hearts with ischemic cardiomyopathy compared with those with nonischemic cardiomyopathy. CONCLUSIONS: For the first time, we present direct experimental evidence of transmural heterogeneity of excitation-contraction coupling and calcium handling in human hearts. End-stage heart failure is associated with the heterogeneous remodeling of excitation-contraction coupling and calcium handling.

Intramural optical mapping of V(m) and Ca(i)2+ during long-duration ventricular fibrillation in canine hearts.

Intramural gradients of intracellular Ca(2+) (Ca(i)(2+)) Ca(i)(2+) handling, Ca(i)(2+) oscillations, and Ca(i)(2+) transient (CaT) alternans may be important in long-duration ventricular fibrillation (LDVF). However, previous studies of Ca(i)(2+) handling have been limited to recordings from the heart surface during short-duration ventricular fibrillation. To examine whether abnormalities of intramural Ca(i)(2+) handling contribute to LDVF, we measured membrane voltage (V(m)) and Ca(i)(2+) during pacing and LDVF in six perfused canine hearts using five eight-fiber optrodes. Measurements were grouped into epicardial, midwall, and endocardial layers. We found that during pacing at 350-ms cycle length, CaT duration was slightly longer (by ≃10%) in endocardial layers than in epicardial layers, whereas action potential duration (APD) exhibited no difference. Rapid pacing at 150-ms cycle length caused alternans in both APD (APD-ALT) and CaT amplitude (CaA-ALT) without significant transmural differences. For 93% of optrode recordings, CaA-ALT was transmurally concordant, whereas APD-ALT was either concordant (36%) or discordant (54%), suggesting that APD-ALT was not caused by CaA-ALT. During LDVF, V(m) and Ca(i)(2+) progressively desynchronized when not every action potential was followed by a CaT. Such desynchronization developed faster in the epicardium than in the other layers. In addition, CaT duration strongly increased (by ∼240% at 5 min of LDVF), whereas APD shortened (by ∼17%). CaT rises always followed V(m) upstrokes during pacing and LDVF. In conclusion, the fact that V(m) upstrokes always preceded CaTs indicates that spontaneous Ca(i)(2+) oscillations in the working myocardium were not likely the reason for LDVF maintenance. Strong V(m)-Ca(i)(2+) desynchronization and the occurrence of long CaTs during LDVF indicate severely impaired Ca(i)(2+) handling and may potentially contribute to LDVF maintenance.

Mechanisms of defibrillation.

Electrical shock has been the one effective treatment for ventricular fibrillation for several decades. With the advancement of electrical and optical mapping techniques, histology, and computer modeling, the mechanisms responsible for defibrillation are now coming to light. In this review, we discuss recent work that demonstrates the various mechanisms responsible for defibrillation. On the cellular level, membrane depolarization and electroporation affect defibrillation outcome. Cell bundles and collagenous septae are secondary sources and cause virtual electrodes at sites far from shocking electrodes. On the whole-heart level, shock field gradient and critical points determine whether a shock is successful or whether reentry causes initiation and continuation of fibrillation.

Bioreactor Suspension Culture: Differentiation and Production of Cardiomyocyte Spheroids From Human Induced Pluripotent Stem Cells.

Human induced-pluripotent stem cells (hiPSCs) can be efficiently differentiated into cardiomyocytes (hiPSC-CMs) via the GiWi method, which uses small-molecule inhibitors of glycogen synthase kinase (GSK) and tankyrase to first activate and then suppress Wnt signaling. However, this method is typically conducted in 6-well culture plates with two-dimensional (2D) cell sheets, and consequently, cannot be easily scaled to produce the large numbers of hiPSC-CMs needed for clinical applications. Cell suspensions are more suitable than 2D systems for commercial biomanufacturing, and suspended hiPSCs form free-floating aggregates (i.e., spheroids) that can also be differentiated into hiPSC-CMs. Here, we introduce a protocol for differentiating suspensions of hiPSC spheroids into cardiomyocytes that is based on the GiWi method. After optimization based on cardiac troponin T staining, the purity of hiPSC-CMs differentiated via our novel protocol exceeded 98% with yields of about 1.5 million hiPSC-CMs/mL and less between-batch purity variability than hiPSC-CMs produced in 2D cultures; furthermore, the culture volume could be increased ∼10-fold to 30 mL with no need for re-optimization, which suggests that this method can serve as a framework for large-scale hiPSC-CM production.

Layer-By-Layer Fabrication of Large and Thick Human Cardiac Muscle Patch Constructs With Superior Electrophysiological Properties.

Engineered cardiac tissues fabricated from human induced pluripotent stem cells (hiPSCs) show promise for ameliorating damage from myocardial infarction, while also restoring function to the damaged left ventricular (LV) myocardium. For these constructs to reach their clinical potential, they need to be of a clinically relevant volume and thickness, and capable of generating synchronous and forceful contraction to assist the pumping action of the recipient heart. Design prerequisites include a structure thickness sufficient to produce a beneficial contractile force, prevascularization to overcome diffusion limitations and sufficient structural development to allow for maximal cell communication. Previous attempts to meet these prerequisites have been hindered by lack of oxygen and nutrient transport due to diffusion limits (100-200 µm) resulting in necrosis. This study employs a layer-by-layer (LbL) fabrication method to produce cardiac tissue constructs that meet these design prerequisites and mimic normal myocardium in form and function. Thick (>2 mm) cardiac tissues created from hiPSC-derived cardiomyocytes, -endothelial cells (ECs) and -fibroblasts (FBs) were assessed, in vitro, over a 4-week period for viability (<6% necrotic cells), cell morphology and functionality. Functional performance assessment showed enhanced t-tubule network development, gap junction communication as well as previously unseen, physiologically relevant conduction velocities (CVs) (>30 cm/s). These results demonstrate that LbL fabrication can be utilized successfully to create prevascularized, functional cardiac tissue constructs from hiPSCs for potential therapeutic applications.

Fabrication and characterization of a thick, viable bi-layered stem cell-derived surrogate for future myocardial tissue regeneration.

Cardiac tissue surrogates show promise for restoring mechanical and electrical function in infarcted left ventricular (LV) myocardium. For these cardiac surrogates to be usefulin vivo, they are required to support synchronous and forceful contraction over the infarcted region. These design requirements necessitate a thickness sufficient to produce a useful contractile force, an area large enough to cover an infarcted region, and prevascularization to overcome diffusion limitations. Attempts to meet these requirements have been hampered by diffusion limits of oxygen and nutrients (100-200 µm) leading to necrotic regions. This study demonstrates a novel layer-by-layer (LbL) fabrication method used to produce tissue surrogates that meet these requirements and mimic normal myocardium in form and function. Thick (1.5-2 mm) LbL cardiac tissues created from human induced pluripotent stem cell-derived cardiomyocytes and endothelial cells were assessed,in vitro, over a 4-week period for viability (<5.6 ± 1.4% nectrotic cells), cell morphology, viscoelastic properties and functionality. Viscoelastic properties of the cardiac surrogates were determined via stress relaxation response modeling and compared to native murine LV tissue. Viscoelastic characterization showed that the generalized Maxwell model of order 4 described the samples well (0.7

N-cadherin overexpression enhances the reparative potency of human-induced pluripotent stem cell-derived cardiac myocytes in infarcted mouse hearts.

AIMS: In regenerative medicine, cellular cardiomyoplasty is one of the promising options for treating myocardial infarction (MI); however, the efficacy of such treatment has shown to be limited due to poor survival and/or functional integration of implanted cells. Within the heart, the adhesion between cardiac myocytes (CMs) is mediated by N-cadherin (CDH2) and is critical for the heart to function as an electromechanical syncytium. In this study, we have investigated whether the reparative potency of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) can be enhanced through CDH2 overexpression. METHODS AND RESULTS: CDH2-hiPSC-CMs and control wild-type (WT)-hiPSC-CMs were cultured in myogenic differentiation medium for 28 days. Using a mouse MI model, the cell survival/engraftment rate, infarct size, and cardiac functions were evaluated post-MI, at Day 7 or Day 28. In vitro, conduction velocities were significantly greater in CDH2-hiPSC-CMs than in WT-hiPSC-CMs. While, in vivo, measurements of cardiac functions: left ventricular (LV) ejection fraction, reduction in infarct size, and the cell engraftment rate were significantly higher in CDH2-hiPSC-CMs treated MI group than in WT-hiPSC-CMs treated MI group. Mechanistically, paracrine activation of ERK signal transduction pathway by CDH2-hiPSC-CMs, significantly induced neo-vasculogenesis, resulting in a higher survival of implanted cells. CONCLUSION: Collectively, these data suggest that CDH2 overexpression enhances not only the survival/engraftment of cultured CDH2-hiPSC-CMs, but also the functional integration of these cells, consequently, the augmentation of the reparative properties of implanted CDH2-hiPSC-CMs in the failing hearts.

Correction: Maturation of three-dimensional, hiPSC-derived cardiomyocyte spheroids utilizing cyclic, uniaxial stretch and electrical stimulation.

[This corrects the article DOI: 10.1371/journal.pone.0219442.].

Maturation of three-dimensional, hiPSC-derived cardiomyocyte spheroids utilizing cyclic, uniaxial stretch and electrical stimulation.

Functional myocardium derived from human induced pluripotent stem cells (hiPSCs) can be impactful for cardiac disease modeling, drug testing, and the repair of injured myocardium. However, when hiPSCs are differentiated into cardiomyocytes, they do not possess characteristics of mature myocytes which limits their application in these endeavors. We hypothesized that mechanical and electrical stimuli would enhance the maturation of hiPSC-derived cardiomyocyte (hiPSC-CM) spheroids on both a structural and functional level, potentially leading to a better model for drug testing as well as cell therapy. Spheroids were generated with hiPSC-CM. For inducing mechanical stimulation, they were placed in a custom-made device with PDMS channels and exposed to cyclic, uniaxial stretch. Spheroids were electrically stimulated in the C-Pace EP from IONOptix for 7 days. Following the stimulations, the spheroids were then analyzed for cardiomyocyte maturation. Both stimulated groups of spheroids possessed enhanced transcript and protein expressions for key maturation markers, such as cTnI, MLC2v, and MLC2a, along with improved ultrastructure of the hiPSC-CMs in both groups with enhanced Z-band/Z-body formation, fibril alignment, and fiber number. Optical mapping showed that spheroids exposed to electrical stimulation were able to capture signals at increasing rates of pacing up to 4 Hz, which failed in unstimulated spheroids. Our results clearly indicate that a significantly improved myocyte maturation can be achieved by culturing iPSC-CMs as spheroids and exposing them to cyclic, uniaxial stretch and electrical stimulation.

Shock-induced changes of Ca(i)2+ and Vm in myocyte cultures and computer model: Dependence on the timing of shock application.

OBJECTIVES: Responses of Ca(i)2+ to electrical shocks are believed to be important in defibrillation but measurements of shock-induced Ca(i)2+ changes during different phases of the action potential (AP) are lacking. The effects of shocks on Ca(i)2+ and Vm were investigated in geometrically defined cell cultures and in a computer model. METHODS: Uniform-field shocks (E = 10.4+/-0.9 V/cm) were applied 15-300 ms after AP upstroke in strands of cultured neonatal rat myocytes. Optical mapping was used to measure shock-induced Ca(i)2+ and Vm changes. A rat ionic model was used to elucidate ionic mechanisms of Ca(i)2+ responses. RESULTS: In experiments and simulations, shocks applied with short delays (15-40 ms) caused a transient decrease of Ca(i)2+ at sites of both DeltaV(+)m and DeltaV(-)m. Simulations indicated that the Ca(i)2+ decrease at DeltaV(+)m sites was caused by reversed outward flow of L-type Ca2+ current (I(CaL)), while the Ca(i)2+ decrease at DeltaV(-)m sites was due to the NaCa exchanger (NCX). At intermediate delays (40-150 ms), shocks caused a Ca(i)2+ decrease at sites of DeltaV(-)m and an increase at sites of DeltaV(+)m. Simulations indicated that the Ca(i)2+ increase at DeltaV(+)m sites was caused by transient reactivation of I(CaL) combined with a reverse-mode operation of NCX. Shocks applied at long delays (150-300 ms) caused a Ca(i)2+ increase at DeltaV(+)m and no change at DeltaV(-)m sites. CONCLUSION: Effects of shocks on Ca(i)2+ depend on the timing of shock application. Shocks applied during the early AP cause a transient Ca(i)2+ decrease, while later in AP shocks induce a Ca(i)2+ increase at sites of DeltaV(+)m. Shock-induced Ca(i)2+ changes in different AP phases are primarily determined by combination of I(CaL) and NCX.