Scalable Production of Human Cardiac Tissues through hiPSC Encapsulation in Gelatin Methacryloyl

Biomaterials are widely used in cardiac tissue engineering to provide the three-dimensional (3D) structure and physiological cues required to closely mimic the native tissue environment. Engineered cardiac tissues have the potential to revolutionize our ability to understand and treat heart disease. However, progress toward commercial production of engineered cardiac tissues has been limited by the prohibitively complex process of assembly, which typically involves the use of multiple pre-differentiated cell types and numerous cell handling steps. We have previously shown that using poly (ethylene glycol)-fibrinogen (PEG-Fb) and our one-step cell handling approach provides the necessary structural and biological cues to support human induced pluripotent stem cell (hiPSC) differentiation into maturing engineered cardiac tissues with aligned sarcomeres and evidence of T-tubule presence. Our transformational approach produces cardiac tissue directly from hydrogel encapsulated human induced pluripotent stem cells, eliminating the need to pre-differentiate, dissociate and then re-assemble cardiac cells. Here we sought to determine if this one cell handling step approach for direct 3D cardiac tissue production could be translated for use with the printable material gelatin methacryloyl (GelMa) to directly produce 3D GelMa human engineered cardiac tissues (GEhECTs).

Methods:

Low density GelMa with a methacrylation percentage of approximately 22% was synthesized and hiPSCs were encapsulated by photocrosslinking using visible light while maintaining high cell viability. After 3 days in culture, differentiation was initiated by adding small molcules CHIR and IWP-2 at respective time points. Videos were taken at Days 14, 17 and 40 to analyze the frequency and velocity of contraction. Cardiac gene expression was measured using qPCR at day 10, 20, and 70. Staining was performed using F-Actin, alpha-sarcomeric actinin(aSA), and connexin 43 (Cx43) to show morphological changes in the cells over time with cardiac markers presenting at late time points. Lastly, response to pacing and drug treatment was assessed through MEA recordings of dissociated GEhECT cardiomyocytes.

Results:

GelMA constructs supported hiPSC cardiac differentiation forming GEhECTs with spontaneous contraction initiating on Day 8. Frequency, velocity, and synchronicity of contraction increased with time. GEhECTs had high differentiation efficiency with approximately 70% of cells positive for the cardiac marker cTnT based on flow cytometry. Temporal changes in gene expression followed expected trends for cardiac differentiation with expression of cardiac genes, connexin 43 and β-myosin heavy chain, increasing over time. Cell morphology in the GEhECTS changed over the time course ofdifferentiation; round stem cell colonies prior to differentiation became elongated thin muscle cells, as visualized by F-actin staining. aSA and Cx43 staining displayed well aligned sarcomeres with functional cardiac gap junctions. Lastly, dissociated cardiomyocytes responded appropriately pharmacologic stimulation with the β-adrenergic agonist isoproterenol and antagonist propranolol, as well as exogenous pacing up to 3 Hz.

Conclusions:

Overall, results demonstrate that GelMa encapsulated hiPSCs can be directly differentiated into maturing 3D GEhECTs, eliminating the need to use pre-differentiated CMs for engineered cardiac tissue formation. Production of GEhECTs has the potential to be scaled-up for use in studying drug toxicity and cardiac developmental biology and for disease modeling.