Characterizing the effect of substrate stiffness on neural stem cell differentiation
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Characterizing the effect of substrate stiffness on neural stem cell differentiation Colleen T. Curley1, Kristen Fanale1, and Sabrina S. Jedlicka1,2,3 1
Bioengineering Program, 111 Research Drive, Bethlehem, PA 18015, USA Materials Science and Engineering Department, 5 E. Packer Avenue, Bethlehem, PA 18015, USA 3 Center for Advanced Materials and Nanotechnology, 5 E. Packer Avenue, Bethlehem, PA 18015, USA 2
ABSTRACT Differentiated neurons (dorsal root ganglia and cortical neurons) have been shown to develop longer neurite extensions on softer materials than stiffer ones, but previous studies do not address the ability of neural stem cells to undergo differentiation as a result of material elasticity. In this study, we investigate neuronal differentiation of C17.2 neural stem cells due to growth on polyacrylamide gels of variable elastic moduli. Neurite growth, synapse formation, and mode of division (asymmetric vs. symmetric) were all assessed to characterize differentiation. C17.2 neural stem cells were seeded onto polyacrylamide gels coated with Type I collagen. The cells were then serum starved over a 14 day period, fixed, and analyzed for biochemical markers of differentiation. For division studies, time-lapse imaging of cells on various substrates was performed during serum withdrawal using the Nikon Biostation. Division events were analyzed using ImageJ to quantify sizes of resulting daughter. Data shows that C17.2 cell differentiation (as dictated by number and type of division events) is dependent upon substrate stiffness, with softer polyacrylamide surfaces (140 Pa) leading to increased populations of neurons and increased neurite length. Our data also indicates that the ability of neural stem cells to express synaptic proteins and develop synapses is dependent upon material elasticity. INTRODUCTION Factors contributing to cell fate consist of both chemical and mechanical cues from the microenvironment [1,2]. Recent studies show that the impact of the mechanical properties of the extracellular environment play an important role in regulating many cellular processes, including migration, proliferation, and differentiation [3-8]. Cell response to material elasticity seems to be cell-type specific and correlates to elasticity of a cell’s native tissue [8-11]. While previous studies have illustrated the importance of elasticity in regulating stem cell differentiation [7,8], the role of substrate mechanics in directing neuronal differentiation is not clear. In this study, we explore the effect of substrate stiffness on neurite length, synapse formation, and mode of division during differentiation. Neurite extension and synapse formation are crucial for the development of functional nervous tissue, while mode of division affects the composition of the adult cell population, with symmetric divisions giving rise to two cells with the same fate and asymmetric giving rise to daughters of differing developmental fate[12]. We utilize thin polyacrylamide gels coated with collagen as growth substrates, a widely used m
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