Differentiation of Mouse Embryonic Stem Cells Using PEG-Fibrinogen Hydrogel Microspheres

Abstract

Cardiac disease is the leading cause of death worldwide, and current treatments are either ineffective at significantly prolonging lifespan or are unavailable to the majority of patients in need due to a lack of supply. Application of engineered heart tissue is a promising method to repair damaged hearts, but there are many challenges to generate functional cardiac tissue which can integrate with damaged myocardium. We have developed a highly scalable method for producing cardiac tissue from mouse embryonic stem cells (mESCs) using poly(ethylene glycol)-diacrylate (PEGDA) based hydrogels with a conjugated fibrinogen protein functional motif. Hydrogels consisted of dispersed microsphere cell-carriers to be utilized as in vitro differentiation systems or as a possible injectable delivery vehicle. Cells were encapsulated within the hydrogel polymer precursor and microspheres were generated via water-in-oil emulsification. The generation of microspheres with reproducible diameter distributions within the range of roughly 70-200 µm was demonstrated. The microspheres acted as differentiation vehicles; as the cells proliferated and remodeled the surrounding matrix, they formed embryoid bodies which exhibited spontaneous contraction as early as 10 days into differentiation. Cell activity within the microsphere was primarily characterized by phase-contrast microscopy. Differentiation of encapsulated cells was verified using qPCR and immunohistochemical techniques. This method is the first to describe the proliferation and differentiation of encapsulated mouse pluripotent stem cells using a PEG-based hydrogel.