Introduction: An estimated 1 billion cardiomyocytes are killed during a myocardial infarction (MI), leading to MI being the primary etiology of heart failure (HF). Due to loss of cardiac contractility in HF, ongoing work in the field of cardiac regeneration is aimed at developing strategies to restore both cell loss and functional decline post-MI. Delivery of cardiomyocytes derived from human embryonic or human induced pluripotent stem cells (hiPSC-CMs) via engineered tissue or intramuscular injection has emerged as a promising means to stabilize cardiac function. Delivery of CMs through epicardially-implanted engineered cardiac tissues (ECTs) is especially advantageous as increased CM maturity, improved engraftment, and increased mechanical support of the ventricular wall have been reported as compared to intramuscular CM injection, and new data suggests lower incidence of arrhythmia. We have identified novel approaches to biomanufacturing for clinical translation of ECTs with respect to scaling up implant size and CM density; quantify the impact on formation and electromechanical function of the ECTs; and demonstrate engraftment in a large animal model of MI. Through this study, novel challenges have emerged and are leading us into the next generation of ECTs for heart regeneration.
Materials and
Methods: Differentiation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) uses biphasic control of Wnt signaling and is followed by proliferative expansion of hiPSC-CMs and lactate-based selection. Cardiac purity by flow cytometry analysis of cardiac troponin T (cTnT) expression ensures >70% purity for experiments. To fabricate ECTs, hiPSC-CMs are mixed with 5% human primary ventricular fibroblasts and collagen-1 hydrogel for gelation in polydimethylsiloxane (PDMS) molds customized for surface area, volume, and shape to accommodate increasing hiPSC-CM number and density. Nondestructive, longitudinal (daily) brightfield optical microscopy images and videos of ECTs allow for assessment of formation and compaction. Mechanical analysis of the passive (elastic modulus) and active properties (contractile twitch stress generation) of the ECTs is achieved using a custom micromechanical tensile apparatus (Aurora Scientific, Aurora, Canada) after 7 days of in vitro culture. Electrophysiological function is assessed by optical mapping of voltage and calcium kinetics in ECTs to quantify conduction velocity and arrhythmia risk. Finally, a scaled up ECT containing 1 billion hiPSC-CMs is carefully implanted on a pig model of chronic myocardial ischemia and assessed after 4 weeks. Immunofluorescent imaging confirms engraftment and is used to assess maturation of implanted hiPSC-CMs in ECTs. New directions use computational and experimental approaches to identify novel tissue scaffold support systems with polycaprolactone (PCL) for surgical handling during implantation of ECTs with the added benefit of mechanical support to the ventricular wall.
Results, Conclusions, and Discussions: ECTs exhibited hiPSC-CM dose-dependent responses in structure and mechanics where high-density ECTs show reduced compaction (Fig 1A) and active stress generation (Fig 1B). Scaled up and cell-dense macro-ECTs were able to follow point stimulation pacing with no circular reentrant patterns that would suggest arrhythmogenesis (Fig 1C). Finally, we successfully fabricated mega-ECTs at clinical scale containing 1 billion hiPSC-CMs for implantation in a swine model of chronic myocardial ischemia (Fig 1D-E), successfully demonstrating the technical feasibility of biomanufacturing, surgical implantation, engraftment, and maturation of implanted hiPSC-CMs (Fig 1F-G). Through this iterative engineering process, we define the impact of manufacturing variables on ECT formation and function as well as identify challenges that must be overcome to successfully accelerate ECT clinical translation. The most pressing issue related to new biomanufacturing approaches is the handling of ECTs, as the stiffness of high-density ECTs is well below that of other products used in surgical applications. We are addressing this challenge with novel architectures of synthetic scaffolds to create composites while preserving hiPSC-CM viability and function. Computational models suggest that anisotropy promotes functional recovery of the heart, and thus aligned fibrous PCL scaffolds are being integrated with ECTs for evaluation of tissue mechanics and heart regeneration post-MI. In conclusion, our work to deliver 1 billion hiPSC-CMs to the infarcted heart has led us into exciting new areas of engineering design and biomanufacturing to create the next generation of ECTs for heart regeneration.
Acknowledgements (Optional): NIH P20GM103652 pilot project award with Co-I Frank Sellke. [1] Dwyer et al. Bioengineering (Basel). 2023 May 13;10(5):587.
