Recent advances in pluripotent stem cell research have provided investigators with potent sources of cardiogenic cells. However, tissue engineering methodologies to assemble cardiac progenitors into aligned, 3-dimensional (3D) myocardial tissues capable of physiologically relevant electrical conduction and force generation are lacking. In this study, we introduced 3D cell alignment cues in a fibrin-based hydrogel matrix to engineer highly functional cardiac tissues from genetically purified mouse embryonic stem cell derived cardiomyocytes (CMs) and cardiovascular progenitors (CVPs). Procedures for CM and CVP derivation, purification, and functional differentiation in monolayer cultures were first optimized to yield robust intercellular coupling and maximize velocity of action potential propagation. A versatile soft-lithography technique was then applied to reproducibly fabricate engineered cardiac tissues with controllable size and 3D architecture. While purified CMs assembled into a functional 3D syncytium only when supplemented with supporting non-myocytes, purified CVPs differentiated into cardiomyocytes, smooth muscle, and endothelial cells, and autonomously supported the formation of functional cardiac tissues. After a total culture time similar to period of mouse embryonic development (21 days), the engineered cardiac tissues exhibited unprecedented levels of 3D organization and functional differentiation characteristic of native neonatal myocardium, including: 1) dense, uniformly aligned, highly differentiated and electromechanically coupled cardiomyocytes, 2) rapid action potential conduction with velocities between 22 and 25 cm/s, and 3) significant contractile forces of up to 2 mN. These results represent an important advancement in stem cell-based cardiac tissue engineering and provide the foundation for exploiting the exciting progress in pluripotent stem cell research in the future tissue engineering therapies for heart disease.

This protocol describes a cell/hydrogel molding method for precise and reproducible biomimetic fabrication of three-dimensional (3D) muscle tissue architectures in vitro. Using a high aspect ratio soft lithography technique, we fabricate polydimethylsiloxane (PDMS) molds containing arrays of mesoscopic posts with defined size, elongation and spacing. On cell/hydrogel molding, these posts serve to enhance the diffusion of nutrients to cells by introducing elliptical pores in the cell-laden hydrogels and to guide local 3D cell alignment by governing the spatial pattern of mechanical tension. Instead of ultraviolet or chemical cross-linking, this method utilizes natural hydrogel polymerization and topographically constrained cell-mediated gel compaction to create the desired 3D tissue structures. We apply this method to fabricate several square centimeter large, few hundred micron-thick bioartificial muscle tissues composed of viable, dense, uniformly aligned and highly differentiated cardiac or skeletal muscle fibers. The protocol takes 4–5 d to fabricate PDMS molds followed by 2 weeks of cell culture.

Glycosylation of therapeutic proteins has a profound impact on their safety and efficacy. Many factors shape the glycosylation of biotherapeutics, ranging from expression systems and cell culture processes to downstream purification strategies. Various analytical technologies have been developed to address questions concerning different aspects of glycosylation. Informatics tools are also crucial for a systematic understanding of the glycosylation processes. Hence, an integrated approach is required to harness glycosylation for the production of optimal and consistent glycoprotein-based therapeutic drugs. Here, we review the latest developments and challenges in glycosylation analysis and control in the context of bioprocessing monoclonal antibodies.

Heart attack remains the leading cause of death in both men and women worldwide. Stem cell-based therapies, including the use of engineered cardiac tissues, have the potential to treat the massive cell loss and pathological remodeling resulting from heart attack. Specifically, embryonic and induced pluripotent stem cells are a promising source for generation of therapeutically relevant numbers of functional cardiomyocytes and engineering of cardiac tissues in vitro. This review will describe methodologies for successful differentiation of pluripotent stem cells towards the cardiovascular cell lineages as they pertain to the field of cardiac tissue engineering. The emphasis will be placed on comparing the functional maturation in engineered cardiac tissues and developing heart and on methods to quantify cardiac electrical and mechanical function at different spatial scales.

The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors’ ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.