(14b) Engineering Biomaterials to Recapitulate the Stem Cell Microenvironment

Despite the tremendous potential of stem cells to repair and replace damaged or diseased tissue, little progress has been made in translating stem cell-based therapies from the laboratory to the clinic. Several critical engineering challenges remain that have limited the clinical use of stem cells, including difficulties producing the large number of stem cells needed for therapeutic applications, the efficient and reproducible differentiation of stem cells into mature cell types of interest, delivery of cells to target tissues during transplantation, and maintenance of the viability and function of the cells post-transplantation. Materials science approaches stand to overcome these significant hurdles. By designing synthetic systems to mimic the native stem cell microenvironment, we can exert greater control over cell fate decisions, such as self-renewal and differentiation. In this talk, I will focus on my work using materials-based strategies to engineer platforms to expand neural progenitor cells (NPCs). NPCs are capable of differentiating into the major neural lineages of the central nervous system, and thus represent a promising cell source for therapies to treat numerous debilitating conditions, including spinal cord injury and neurodegenerative diseases. However, recent studies have highlighted the difficulties in maintaining the regenerative potential of cultured neural progenitor cells (NPCs) for use in clinical trials. I hypothesized that culturing NPCs within 3D biomimetic hydrogels would allow for significant stem cell expansion in vitro, while better maintaining the regenerative capacity of the cultured cells. While it has been well established that 2D material parameters, such as substrate stiffness, influence the differentiation of NPCs, previous 3D studies have not clearly determined which parameters are key to maintaining NPC stemness and regenerative capacity. I therefore designed a family of engineered elastin-like protein (ELP) hydrogels with simultaneous tuning of stiffness, degradability, and adhesive ligand concentration to probe the contributions of these parameters toward maintenance of NPC phenotype. NPCs were encapsulated in gels with stiffness spanning a physiologically-relevant range (E~0.5-50 kPa) and high, medium, or low degradability. Expression of the NPC stemness markers Nestin and Sox2 was positively correlated with matrix degradability, but no correlation was observed with matrix stiffness. Accordingly, NPCs cultured within high degradability gels exhibited increased proliferation and better maintained their differentiation capacity compared with cells cultured in low degradability gels. In contrast to previous results with 3D cultured mesenchymal stem cells, biochemical inhibition of cytoskeletal contractility and culture in hydrogels without cell-adhesive ligands did not decrease NPC stemness in high degradability gels, suggesting a mechanism independent of tension generation. However, blocking degradation by NPC-secreted proteases through biochemical inhibitors or shRNA-mediated knockdown resulted in a marked reduction in NPC stemness. Confocal microscopy revealed enhanced cell spreading and cell-cell contact in high degradability gels. Inhibiting cadherin-mediated contact with function blocking peptides decreased NPC stemness. Independently blocking matrix degradation and inhibiting cadherin contact both decreased β-catenin signaling, providing a mechanism by which matrix remodeling regulates NPC stemness. Culturing NPCs in two additional hydrogel systems (proteolytically-degradable poly(ethylene glycol) and physically-remodelable alginate) demonstrated the generality of our results, indicating that NPC stemness maintenance required matrix remodeling but was insensitive to matrix stiffness. These results have identified matrix remodeling as a previously unknown requirement for NPC stemness maintenance in 3D hydrogels, suggesting that biomaterials that exploit matrix remodeling may increase the regenerative potential of NPC therapies.