(521b) Matrix Remodeling Enhances the Differentiation Capacity of Neural Progenitor Cells in 3D Hydrogels

Authors: 
Madl, C. M., Stanford University
LeSavage, B. L., Stanford University
Dewi, R. E., Stanford University
Lampe, K., University of Virginia
Heilshorn, S. C., Stanford University
Neural progenitor cells (NPCs) are stem cells capable of differentiating into the major lineages of the central nervous system (neurons, astrocytes, and oligodendrocytes) and thus represent a promising cell source to repair damaged nervous tissue. However, expansion of therapeutically-relevant numbers of NPCs and their efficient differentiation into desired mature cell types remains a challenge. Materials-based strategies, including culture within three-dimensional (3D) hydrogels, have the potential to overcome these current limitations. An ideal material would enable both NPC expansion and subsequent differentiation within a single platform. We recently demonstrated that cell-mediated remodeling of 3D hydrogels was necessary to maintain the stem cell phenotype of NPCs during expansion, but the role of matrix remodeling on NPC differentiation and maturation remained unknown. To determine the effects of matrix remodeling on NPC differentiation, we designed an elastin-like protein (ELP) hydrogel system in which matrix stiffness and degradability can be co-varied independently of hydrogel swelling, microstructure, and nutrient diffusivity. Hydrogel stiffness was maintained in a neural-relevant range (E ~ 0.5-1.5 kPa) to permit differentiation into both neuronal and glial lineages, while matrix remodelability was tuned by varying the susceptibility of the hydrogels to proteolytic degradation. By culturing NPCs within these degradable, engineered-protein hydrogels, we identified that a critical amount of remodeling was necessary to enable NPC differentiation, even in highly degradable gels. NPCs cultured in highly degradable gels best retained expression of markers indicative of the multipotent stem cell state (nestin and Sox2), while also maintaining sustained metabolic activity throughout the culture duration. Successful differentiation into astrocytes and neurons was observed only if cells were permitted sufficient time to remodel the matrix prior to induction of differentiation. NPCs that were provided only one day of remodeling prior to induction failed to differentiate, whereas NPCs that were provided one week of remodeling robustly differentiated into neurons and astrocytes. The resulting neurons exhibited hallmarks of maturity, including expression of neurofilament proteins and altered calcium signaling in response to neurotransmitter treatment. Mechanistically, we demonstrated that maintenance of the multipotent stem cell state required the interaction of the transcriptional co-activator Yes-associated protein (YAP) with the TEAD-family of transcription factors. In contrast to numerous examples of biomaterial properties regulating YAP activity by altering the subcellular localization of YAP protein, we found that matrix remodeling modulated YAP expression at the mRNA level. Matrix remodeling enables cell-cell contact via N-cadherin, which activates β-catenin signaling and in turn drives expression of YAP to maintain NPC differentiation capacity. These results identify matrix degradation as an important but previously unrecognized regulator of NPC differentiation and maturation. Therefore, cell-remodelable hydrogels are an attractive platform to enable expansion of NPCs followed by differentiation of the cells into mature phenotypes for therapeutic use.