(616b) Hydrogels for Controlling Neural Stem Cell Fate Through Intracellular Redox State

Neurological injuries and diseases often result in an inflammatory environment hostile toward regeneration. This causes an acute release of reactive oxygen species (ROS) that can result in a secondary insult further impairing neural function. This inflammatory environment is also toxic toward transplanted cells, a key stumbling block toward stem cell therapy, as less than 20% of transplanted cells survive past two weeks. Even at sub-lethal levels, an increase in extracellular ROS can change the intracellular redox state and direct stem cells down a differentiation rather than self-renewal pathway. Poly(ethylene glycol) (PEG)-based hydrogels have been used to culture neural stem cells (NSCs) which survive and proliferate. Here we focus on the effects of the hydrolytically degradable poly(lactic acid)-b-PEG-b-poly(lactic acid) dimethacrylate block copolymer (PEG-PLA) as a means to influence encapsulated NSC survival and function.

We show that as PEG-PLA hydrogels degrade they release lactic acid, which scavenges free radicals and rescues NSCs from free radical-induced death in controlled monolayer cultures and 3D hydrogels. Monolayer cultures of NSCs were utilized to determine the concentration dependent ability of lactic acid to scavenge specific radical species, to improve NSC viability in the presence of ROS, and to increase NSC proliferation. We systematically synthesized a family of PEG-PLA hydrogels ranging from 0-100% degradable content to quantify the release of lactic acid into the medium and its neuroprotective capabilities. While keeping the initial (< 24 hours) mechanical properties constant, the degradable macromer content had an immediate (30 minutes) and prolonged (7 days) effect on intracellular redox state indicating fewer intracellular ROS. Cells encapsulated in hydrogels with increasing degradable content were more viable and more proliferative, with the 100% degradable material supporting a three-fold increase in metabolic ATP content and a more than two-fold increase in DNA content compared to the 0% degradable material after 7 days. Importantly, this increased survival and proliferation was correlated with an increase in the expression of neuronal and neural stem cell genes (beta-tubulin and nestin, respectively), indicating that they maintain NSC stemness and promote selective neuronal rather than astrocytic differentiation. In these highly degradable 3D hydrogels, we further showed that NSCs had greater concentrations of total glutathione, with a greater fraction of it found as reduced glutathione (GSH), a potent intracellular radical scavenger.

By using lactic-acid releasing hydrogels for in vitro cell culture, we can maintain NSC stemness and promote proliferation. These studies provide a fundamental understanding of the mechanisms governing NSC protection and maintenance by PEG-PLA hydrogels, which may inform future antioxidant drug delivery and biomaterial design. Furthermore, these hydrogels may be beneficial for cell transplantation therapy, where injured host cells as well as transplanted cells may be stimulated to survive via ROS-scavenging properties of the hydrogel.