Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are a valuable resource for cardiac therapeutic development; however, generation of these cells in large numbers and high purity is a limitation in widespread adoption. Here, design of experiments (DOE) is used to investigate the cardiac differentiation space of three hiPSC lines when varying CHIR99027 concentration and cell seeding density, and a novel image analysis is developed to evaluate plate coverage when initiating differentiation. Metabolic selection via lactate purifies hiPSC-cardiomyocyte populations, and the bioenergetic phenotype and engineered tissue mechanics of purified and unpurified hiPSC-cardiomyocytes are compared. Findings demonstrate that when initiating differentiation one day after hiPSC plating, low (3 μM) Chiron and 72 x 103 cells/cm2 seeding density result in peak cardiac purity (50-90%) for all three hiPSC lines. Our results confirm that metabolic selection with lactate shifts hiPSC-cardiomyocyte metabolism towards oxidative phosphorylation, but this more "mature" metabolic phenotype does not by itself result in a more mature contractile phenotype in engineered cardiac tissues at one week of culture in 3D tissues. This study provides widely adaptable methods including novel image analysis code and parameters for refining hiPSC-cardiomyocyte differentiation and describes the practical implications of metabolic selection of cardiomyocytes for downstream tissue engineering applications

Human Cardiac Fibroblast Number and Activation State Modulate Electromechanical Function of hiPSC-Cardiomyocytes in Engineered Myocardium

Cardiac tissue engineering using hiPSC-derived cardiomyocytes is a promising avenue for cardiovascular regeneration, pharmaceutical drug development, cardiotoxicity evaluation, and disease modeling. Limitations to these applications still exist due in part to the need for more robust structural support, organization, and electromechanical function of engineered cardiac tissues. It is well accepted that heterotypic cellular interactions impact the phenotype of cardiomyocytes. The current study evaluates the functional effects of coculturing adult human cardiac fibroblasts (hCFs) in 3D engineered tissues on excitation and contraction with the goal of recapitulating healthy, nonarrhythmogenic myocardium in vitro. A small population (5% of total cell number) of hCFs in tissues improves tissue formation, material properties, and contractile function. However, two perturbations to the hCF population create disease-like phenotypes in engineered cardiac tissues. First, increasing the percentage of hCFs to 15% resulted in tissues with increased ectopic activity and spontaneous excitation rate. Second, hCFs undergo myofibroblast activation in traditional two-dimensional culture, and this altered phenotype ablated the functional benefits of hCFs when incorporated into engineered cardiac tissues. Taken together, the results of this study demonstrate that human cardiac fibroblast number and activation state modulate electromechanical function of hiPSC-cardiomyocytes and that a low percentage of quiescent hCFs are a valuable cell source to advance a healthy electromechanical response of engineered cardiac tissue for regenerative medicine applications

Wet-Spun Polycaprolactone Scaffolds Provide Customizable Anisotropic Viscoelastic Mechanics for Engineered Cardiac Tissues

Myocardial infarction is a leading cause of death worldwide and has severe consequences including irreversible damage to the myocardium, which can lead to heart failure. Cardiac tissue engineering aims to re-engineer the infarcted myocardium using tissues made from human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to regenerate heart muscle and restore contractile function via an implantable epicardial patch. The current limitations of this technology include both biomanufacturing challenges in maintaining tissue integrity during implantation and biological challenges in inducing cell alignment, maturation, and coordinated electromechanical function, which, when overcome, may be able to prevent adverse cardiac remodeling through mechanical support in the injured heart to facilitate regeneration. Polymer scaffolds serve to mechanically reinforce both engineered and host tissues. Here, we introduce a novel biodegradable, customizable scaffold composed of wet-spun polycaprolactone (PCL) microfibers to strengthen engineered tissues and provide an anisotropic mechanical environment to promote engineered tissue formation. We developed a wet-spinning process to produce consistent fibers which are then collected on an automated mandrel that precisely controls the angle of intersection of fibers and their spacing to generate mechanically anisotropic scaffolds. Through optimization of the wet-spinning process, we tuned the fiber diameter to 339 ± 31 µm and 105 ± 9 µm and achieved a high degree of fidelity in the fiber structure within the scaffold (fiber angle within 1.8° of prediction). Through degradation and mechanical testing, we demonstrate the ability to maintain scaffold mechanical integrity as well as tune the mechanical environment of the scaffold through structure (Young’s modulus of 120.8 ± 1.90 MPa for 0° scaffolds, 60.34 ± 11.41 MPa for 30° scaffolds, 73.59 ± 3.167 MPa for 60° scaffolds, and 49.31 ± 6.90 MPa for 90° scaffolds), while observing decreased hysteresis in angled vs. parallel scaffolds. Further, we embedded the fibrous PCL scaffolds in a collagen hydrogel mixed with hiPSC-CMs to form engineered cardiac tissue with high cell survival, tissue compaction, and active contractility of the hiPSC-CMs. Through this work, we develop and optimize a versatile biomanufacturing process to generate customizable PCL fibrous scaffolds which can be readily utilized to guide engineered tissue formation and function

Enhanced Electrical Integration of Engineered Human Myocardium via Intramyocardial versus Epicardial Delivery in Infarcted Rat Hearts.

Cardiac tissue engineering is a promising approach to provide large-scale tissues for transplantation to regenerate the heart after ischemic injury, however, integration with the host myocardium will be required to achieve electromechanical benefits. To test the ability of engineered heart tissues to electrically integrate with the host, 10 million human embryonic stem cell (hESC)-derived cardiomyocytes were used to form either scaffold-free tissue patches implanted on the epicardium or micro-tissue particles (~1000 cells/particle) delivered by intramyocardial injection into the left ventricular wall of the ischemia/reperfusion injured athymic rat heart. Results were compared to intramyocardial injection of 10 million dispersed hESC-cardiomyocytes. Graft size was not significantly different between treatment groups and correlated inversely with infarct size. After implantation on the epicardial surface, hESC-cardiac tissue patches were electromechanically active, but they beat slowly and were not electrically coupled to the host at 4 weeks based on ex vivo fluorescent imaging of their graft-autonomous GCaMP3 calcium reporter. Histologically, scar tissue physically separated the patch graft and host myocardium. In contrast, following intramyocardial injection of micro-tissue particles and suspended cardiomyocytes, 100% of the grafts detected by fluorescent GCaMP3 imaging were electrically coupled to the host heart at spontaneous rate and could follow host pacing up to a maximum of 300-390 beats per minute (5-6.5 Hz). Gap junctions between intramyocardial graft and host tissue were identified histologically. The extensive coupling and rapid response rate of the human myocardial grafts after intramyocardial delivery suggest electrophysiological adaptation of hESC-derived cardiomyocytes to the rat heart's pacemaking activity. These data support the use of the rat model for studying electromechanical integration of human cardiomyocytes, and they identify lack of electrical integration as a challenge to overcome in tissue engineered patches

Human grafts contain striated cardiomyocytes.

<p>Confocal fluorescent imaging of engrafted hESC-cardiomyocytes at 4 weeks indicate that all three engraftment methods produce grafts that have high cardiac purity, as indicated by a double-positive stain for α-actinin (red) and GFP (green) with nuclear DAPI (blue), in representative images of all grafts. All grafts demonstrate sarcomere striations at higher magnification (far right). Scar tissue appears as the dark band separating the patch graft and host myocardium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131446#pone.0131446.g003" target="_blank">Fig 3</a> for picrosirius red labeled scar, black arrowhead) and this extends to the edges of the patch (outside the field of view; not shown). White arrowhead in patch image points to GFP⁺/α-actinin^- non-cardiac cells. g, graft; s, scar, h, host. Scale bar = 500 μm and 50 μm (far right column).</p

Cardiomyocyte engraftment in injured rat hearts at 4 weeks.

<p>(A) Human cardiac grafts are identified by immunohistochemistry for GFP (brown, DAB) that binds to the GCaMP3 protein for the cell, micro-tissue particle (MTP), and patch groups. Hematoxalyin nuclear counterstain (blue). (B) Infarct scar area quantified by picrosirius red-positive area shows no difference between groups, normalized to left ventricular (LV) area at 4 weeks. (C) Graft size measured by GFP-positive graft area is normalized to LV area and is equivalent between groups (n = 8/group). (D) GFP-positive graft size declines with larger scar size for the cell grafts identified by histological analysis using Pearson correlation analysis (see text), but is weak and not significant in the micro-tissue particle or patch groups. (E) Micro-tissue particles (GFP, green) engrafted in the infarct region and lateral border within the septum of a rat heart are shown at 4 weeks stained for GFP (green) with DAPI (blue) nuclear stain. Topically applied dye (yellow-orange) marks the location of where an intramyocardial graft was detected by <i>ex vivo</i> imaging (arrow head). (F) Analysis of graft distribution in the heart indicates that cells and micro-tissue particles engraft in the scar, border zone, and healthy myocardium with equal distribution, while patch implants are found on the epicardium.</p

Excitation response of stimulated hESC-cardiomyocytes in 2D culture <i>in vitro</i>.

<p>Spontaneous contraction rate was higher at 2 weeks with pacing at 1 and 6 Hz (*P < 0.05). Similarly, excitation threshold (ET) was significantly lower after 2 weeks of 1 and 6 Hz electrical pacing compared to unstimulated control. However, maximum capture rate (MCR) was not different among groups at any time point and all significant differences are lost after 4 weeks in culture.</p

Formation of hESC-derived cardiac engineered tissues.

<p>(A) hESCs are differentiated into cardiomyocytes with high efficiency as characterized by flow cytometry analysis. An example cell population (85.5% single cell population, left) shows 81% expression of cardiac troponin T (cTnT, PE fluorescence, right) relative to isotype control (not shown). (B) Micro-tissue particles (MTPs) are formed by seeding approximately 1000 cells per microwell (left) and are easily removed from molds by a gentle media wash (right). (C) <i>In vitro</i> characterization of MTPs indicates highly defined particle diameter based on cell input number. (D) Cardiac engineered tissues have high cardiac purity as indicated by β-myosin heavy chain (β-MHC; brown, DAB) in MTPs (top) and cardiac patches (bottom). (E) Cardiac purity by β-MHC staining shows increasing purity with culture time. * P < 0.05 versus Day 1.</p

Summary of hESC-cardiomyocyte engraftment at 4 weeks by histology and <i>ex vivo</i> fluorescent imaging.

<p>No significant differences exist between treatment groups. MCR, maximum capture rate; NA, not applicable.</p

Assessment of human myocardial graft coupling to host heart by GCaMP3 <i>ex vivo</i> fluorescent imaging 4 weeks after implantation.

<p>(A) All micro-tissue particle and cell injection graft regions detected by <i>ex vivo</i> imaging are electrically coupled to the host, while no patch grafts were coupled to the host. Fluorescence signal of a GCaMP3 graft region vs. time is synchronized with the host electrocardiogram (ECG) for cell grafts at spontaneous rate (B; note that coupling persists during premature beats) and with 6 Hz stimulation (C) and for micro-tissue particle grafts at spontaneous rate (D) and 6 Hz stimulation (E). Epicardial patch grafts are easily detected during imaging but are uncoupled at a slower spontaneous rate (F).</p