Chris Madl1 Bauer LeSavage1 Sarah Heilshorn1

1, Stanford University, Stanford, California, United States

While stem cells and their progeny have significant therapeutic promise, the difficulty and cost of expanding a large number of high-quality stem cells remains a significant barrier to widespread clinical use. Recently, 3D hydrogels have been proposed as in vitro culture platforms for the expansion of stem cell populations to overcome the space limitations of 2D culture and to mimic critical aspects of the native stem cell niche. Here we explore the material properties required to maintain the stemness of neural progenitor cells (NPCs). Using three different material platforms, we demonstrate that NPCs must be able to adaptively remodel the 3D material in order to maintain their stemness during proliferation. Mechanistically, material adaptation allows NPCs to establish cell-cell contacts that initiate beta-catenin signaling that drives expression of stem cell genes. Material adaptation could be achieved either through use of on-demand proteolytic degradation by cell-expressed enzymes or through use of reversible crosslinks that can be remodeled over time to alter the polymeric network structure. Interestingly, we also present data demonstrating that 3D matrix stiffness does not correlate with the maintenance of NPC stemness over a broad range of matrix mechanical properties (E~0.5-50 kPa). It is well-established that matrix stiffness modulates stemness in strongly contractile stem cells, including mesenchymal stem cells and muscle satellite cells, but the impact of stiffness on stemness maintenance in less contractile stem cells such as NPCs is not well known. These results highlight that different material requirements may exist for the expansion of different stem cell types. Our results have identified matrix remodeling as a previously unknown requirement for maintenance of NPC stemness in 3D hydrogels and suggest that adaptable biomaterials will be useful for expansion of therapeutically relevant numbers of NPCs.