Human Induced Pluripotent Stem Cell Encapsulation Geometry Impacts Three-Dimensional Developing Human Engineered Cardiac Tissue Functionality.

Cardiac tissue engineering has been working to alleviate the immense burden of cardiovascular disease for several decades. To improve cardiac tissue homogeneity and cardiomyocyte (CM) maturation, in this study, we investigated altering initial encapsulation geometry in a three-dimensional (3D) direct cardiac differentiation platform. Traditional engineered cardiac tissue production utilizes predifferentiated CMs to produce 3D cardiac tissue and often involves various cell selection and exogenous stimulation methods to promote CM maturation. Starting tissue formation directly with human induced pluripotent stem cells (hiPSCs), rather than predifferentiated CMs, simplifies the engineered cardiac tissue formation process, making it more applicable for widespread implementation and scale-up. In this study, hiPSCs were encapsulated in poly (ethylene glycol)-fibrinogen in three tissue geometries (disc-shaped microislands, squares, and rectangles) and subjected to established cardiac differentiation protocols. Resulting 3D engineered cardiac tissues (3D-ECTs) from each geometry displayed similar CM populations (∼65%) and gene expression over time. Notably, rectangular tissues displayed less tissue heterogeneity and suggested more advanced features of maturing CMs, including myofibrillar alignment and Z-line formation. In addition, rectangular tissue showed significantly higher anisotropic contractile properties compared to square and microisland tissues (MI 0.28 ± 0.03, SQ 0.35 ± 0.05, RT 0.79 ± 0.04). This study demonstrates a straightforward method for simplifying and improving 3D-ECT production without the use of exogenous mechanical or electrical pacing and has the potential to be utilized in bioprinting and drug testing applications. Impact statement Current methods for improving cardiac maturation postdifferentiation remain tedious and complex. In this study, we examined the impact of initial encapsulation geometry on improvement of three-dimensional engineered cardiac tissue (3D-ECT) production and postdifferentiation maturation for three tissue geometries, including disc-shaped microislands, squares, and rectangles. Notably, rectangular 3D-ECTs displayed less tissue heterogeneity and more advanced features of maturing cardiomyocytes, including myofibrillar alignment, Z-line formation, and anisotropic contractile properties, compared to microisland and square tissues. This study demonstrates an initial human induced pluripotent stem cell-encapsulated rectangular tissue geometry can improve cardiac maturation, rather than implementing cell selection or tedious postdifferentiation manipulation, including exogenous mechanical and/or electrical pacing.

Engineered cardiac tissue microsphere production through direct differentiation of hydrogel-encapsulated human pluripotent stem cells.

Engineered cardiac tissues that can be directly produced from human induced pluripotent stem cells (hiPSCs) in scalable, suspension culture systems are needed to meet the demands of cardiac regenerative medicine. Here, we demonstrate successful production of functional cardiac tissue microspheres through direct differentiation of hydrogel encapsulated hiPSCs. To form the microspheres, hiPSCs were suspended within the photocrosslinkable biomaterial, PEG-fibrinogen (25 million cells/mL), and encapsulated at a rate of 420,000 cells/minute using a custom microfluidic system. Even at this high cell density and rapid production rate, high intra-batch and batch-to-batch reproducibility was achieved. Following microsphere formation, hiPSCs maintained high cell viability and continued to grow within and beyond the original PEG-fibrinogen matrix. These initially soft microspheres (<250 Pa) supported efficient cardiac differentiation; spontaneous contractions initiated by differentiation day 8, and the microspheres contained >75% cardiomyocytes (CMs). CMs responded appropriately to pharmacological stimuli and exhibited 1:1 capture up to 6.0 Hz when electrically paced. Over time, cells formed cell-cell junctions and aligned myofibril fibers; engineered cardiac microspheres were maintained in culture over 3 years. The capability to rapidly generate uniform cardiac microsphere tissues is critical for advancing downstream applications including biomanufacturing, multi-well plate drug screening, and injection-based regenerative therapies.

Direct Production of Human Cardiac Tissues by Pluripotent Stem Cell Encapsulation in Gelatin Methacryloyl.

Direct stem cell encapsulation and cardiac differentiation within supporting biomaterial scaffolds are critical for reproducible and scalable production of the functional human tissues needed in regenerative medicine and drug-testing applications. Producing cardiac tissues directly from pluripotent stem cells rather than assembling tissues using pre-differentiated cells can eliminate multiple cell-handling steps that otherwise limit the potential for process automation and production scale-up. Here we asked whether our process for forming 3D developing human engineered cardiac tissues using poly(ethylene glycol)-fibrinogen hydrogels can be extended to widely used and printable gelatin methacryloyl (GelMA) hydrogels. We demonstrate that low-density GelMA hydrogels can be formed rapidly using visible light (<1 min) and successfully employed to encapsulate human induced pluripotent stem cells while maintaining high cell viability. Resulting constructs had an initial stiffness of approximately 220 Pa, supported tissue growth and dynamic remodeling, and facilitated high-efficiency cardiac differentiation (>70%) to produce spontaneously contracting GelMA human engineered cardiac tissues (GEhECTs). GEhECTs initiated spontaneous contractions on day 8 of differentiation, with synchronicity, frequency, and velocity of contraction increasing over time, and displayed developmentally appropriate temporal changes in cardiac gene expression. GEhECT-dissociated cardiomyocytes displayed well-defined and aligned sarcomeres spaced at 1.85 ± 0.1 µm and responded appropriately to drug treatments, including the ß-adrenergic agonist isoproterenol and antagonist propranolol, as well as to outside pacing up to 3.0 Hz. Overall results demonstrate that GelMA is a suitable biomaterial for the production of developing cardiac tissues and has the potential to be employed in scale-up production and bioprinting of GEhECTs.