(567e) Pluripotent Stem Cell Colony Formation and Cardiogenesis within PEG-Fibrinogen Microspheres

Heart disease is the greatest single cause of death worldwide, and treatments currently rely on drug therapies that are limited in their capacity to restore or improve function of damaged heart tissue.  Cardiomyocytes (CM) are highly specialized electrically active cells which contribute to the heart’s ability to contract, but are irreversibly damaged during ischemic events and do not regenerate.  Because adult CMs are difficult to expand in vitro while maintaining their specialized phenotype, it is desired to engineer cardiac tissue derived from pluripotent stem cells. The development of functional engineered heart tissue can greatly benefit heart disease patients by doubling as a source for cell therapy and also serving as a robust drug screening platform.  However, because of the current nascent understanding of biochemical and biophysical cues that influence stem cell differentiation fate, homogeneous differentiation of CMs has yet to be accomplished.  Encapsulation of stem cell colonies using hydrogel biomaterials allows for uniform temporal and spatial presentation of biochemical cues, as well as providing the cells with structural support to facilitate their differentiation.  A common in vitro method of mesodermal differentiation begins with the hanging drop method of embryoid body (EB) formation, which produces multicellular EBs of relatively uniform size, but provides limited control over the microenvironment because of diffusion gradients.  This study examines the utility of a unique mouse embryonic stem cell (mESC) encapsulation system using poly(ethylene glycol) (PEG) hydrogel-based microsphere scaffolds as differentiation vehicles.  PEG hydrogels are a promising synthetic biomaterial due to their high biocompatibility, high propensity for functional modification, and in situ crosslinking capability.  This study has utilized a PEG diacrylate-fibrinogen conjugate as the microsphere material.  By using a material containing both natural and synthetic components, the cells are provided with a natural substrate for adhesion and biological recognition while also being exposed to a material with tunable mechanical characteristics.  Briefly, mouse embryonic stem cells (mESCs) were suspended within the copolymer solution.  Encapsulation and sphere formation was achieved via single-emulsion method, where the cell-copolymer solution was mixed with mineral oil and vortexed.  Spheres were simultaneously photo-crosslinked by exposure to a metal halide lamp source using the photoinitiators Irgacure 2959 and 2-2-dimethoxy-2-phenyl acetophenone.  The resulting cell-laden microspheres were cultured in suspension, allowing for colony formation and differentiation as well as matrix remodeling.  Cell viability was assessed via fluorescent microscopy and Live/Dead viability staining.  Differentiation was functionally assessed by monitoring cell beating, and quantitatively assessed via immunostaining and gene expression analysis for cardiac markers and germ layer markers.  The effects of microsphere size, mechanical characteristics, material, and processing methods on stem cell differentiation were assessed.  This study focused on understanding EB formation within PEG-fibrinogen microspheres and developing a correlation between microsphere size and differentiation capacity.