Engineering Hydrogels in vitro with ECM Components for Stem Cell Differentiation and Implantation in vivo After Stroke
Although strokes are the leading cause of adult disability, the leading treatment after initial intervention is rest and physical therapy. Thus, there exists a current lack of therapy to promote recovery of damaged tissue in the brain. While stem and progenitor cell transplantation have shown promise in promoting recovery, critical obstacles include the low survival rate post-transplantation and the lack of differentiation in the cells that do survive. An alternative approach to direct cell transplantation is to deliver encapsulated cells in a hydrogel matrix. This hydrogel matrix should be designed to "gel" within the brain to create a minimally invasive surgery. It should also 1) protect the cells from the harsh environment that exist post-stroke, 2) direct the differentiation of encapsulated cells and 3) not cause additional damage to brain tissue by increasing immune response. The research presented in this dissertation can be broadly split into two categories: in vivo hydrogel/neural progenitor cell transplantation studies and in vitro hydrogel development for directing encapsulated cell behavior.
To test the hypothesis that delivering neural progenitor cells in a hydrogel matrix with an optimized transplantation process can improve transplanted cell viability in the stroke cavity, we began by studying the brain's immune response to different hydrogel formulations. These experiments showed that our hyaluronic acid hydrogel system did not cause an increased inflammatory response when it had mechanics similar to those of the brain (~300 Pa). Next, we optimized the injection process by studying the effect of needle gauge, infusion speed and cell concentration on cell viability. We found that too high of an infusion speed and/or cell concentration can significantly decrease viability. Next we injected neural progenitor cells derived from induced pluripotent stem cells into the stroke cavity of immune compromised mice using the optimized injection protocol with and without a hydrogel. Although we did not see a significant difference in transplanted cell viability, the hydrogel did promote more neuronal differentiation from the transplanted cells.
Concurrently, the hyaluronic acid hydrogel was developed in vitro via experiments aimed at finding additional ways to control and direct cell behavior. One approach studied was the incorporation of additional adhesion ligands (RGD, YIGSR, IKVAV). A design of experiments (DOE) methodology was taken to systematically modulate the three ligands to determine their individual and combinatorial effects on neural progenitor cell survival and differentiation. Three DOE setups were used to optimize the concentrations of the three ligands for encapsulated cell survival. We also found that this optimal concentration increased progenitor cell differentiation when compared to hydrogels with equimolar signal distribution and cells plated on traditional two-dimensional surfaces. A second approach to directing cell behavior was developed by modulating ligand presentation throughout the hydrogel. This was done by pre-reacting RGD to specific portions of the hyaluronic acid polymer prior to hydrogel formation. We found that the clustering of the adhesion peptide modulates cell spreading and integrin expression. Additionally, there is an optimal degree of clustering for cell spreading that applies across different RGD concentrations. Finally we looked at a novel way to crosslink hydrogels for stem cell encapsulation. Oxime click chemistry was able to efficiently form crosslinked polymer networks without any toxic effects.