(453d) Directing Pluripotent Stem Cell Differentiation Using Hydrogel Microspheres

Pluripotent stem cells are a promising cell source for treatment of diseased or damaged tissues, including repairing the human heart.  Current pluripotent stem cell differentiation protocols generally focus on deriving cardiac tissue with high purity and function, yet few of these methods are highly scalable and also tightly control the cellular microenvironment.  Using a water-in-oil emulsion technique, this research has created a scalable and easily modifiable hydrogel microsphere system for pluripotent stem cell differentiation.  Microspheres supported the proliferation of the encapsulated pluripotent stem cells and subsequent cardiac differentiation. MESCs were successfully encapsulated into the PEG-fibrinogen microspheres and maintained for over 23 days.  mESCs proliferated and initiated formation of embryoid bodies (EBs) within the microspheres during the first week of differentiation.  It was found that proliferation and EB formation occurred more readily when cells were encapsulated at high densities (45 and 60 million cells/mL precursor solution) rather than low densities (below 15 million cells/mL precursor). Cell encapsulation concentration did not have a significant impact on initial mean sphere diameter. EBs typically degraded the microsphere and attached to the culture vessel within 10 days of differentiation; these attached tissues initiated spontaneous contraction as early as Day 11 of differentiation.  qPCR analysis of gene expression confirmed the down-regulation of pluripotent marker Oct4 and the up-regulation of Nkx2.5. Frequency of spontaneous contraction ranged from 1.5 to 2 Hz.