Effect of Young's modulus on neural differentiation of human induced pluripotent stem cell derived neural stem cells

摘要

Introduction: The Young's modulus of culture substrates has been shown to have a significant effect on the lineage choice and differentiation state of stem cells. Typically, these studies have examined a mechanical range over several orders of magnitude using discrete samples to identify optimal mechanical regions for the differentiation of specific lineages. However, a more detailed examination of the region of interest for a specific lineage, such as neurons, could further improve the optimization of biomaterial mechanics for specific applications. In this study the optimal range for neural differentiation of human induced pluripotent stem cell (hiPSC) derived neural stem cells (NSC) will be examined using poly ethylene glycol dimethacrylate (PEGDM) hydrogels containing a continuous gradient in Young's Modulus. Material and Methods: 50 mm×10 mm × 1 mm hydrogel gradients were fabricated by dispensing 10% and 55% 10 kDa PEGDM solutions containing 0.1% Matrigel through two syringe pumps running in inverse linear ramping profiles ranging from 0 mL/h to 52 mL/h, respectively, over 75s into a custom mold, followed by photopolymerization with 2.3 mJ/cm2 for 7 min. After swelling in PBS, one 9.6-mm disc was punched out every 10 mm along each gradient. Discs were then evaluated for swelling ratio, mesh size and mechanical properties. For cellular experiments, the discs were placed in the wells of 48-well plates, seeded with ND2.0 hiPSC derived NCS and cultured with serum-free neurs differentiation media for 14 days with medium exchanges every other day. Cell-seeded constructs were stained for neuron-specific β3-tubulin (TUJ1) and length of neurite extension was measured. Cultured cells on constructs were harvested for analyses of gene expression. Statistical significance was determined using one-way or two-way ANOVA in conjunction with the paired t-test with a p-value of ＜ 0.05. Results and Discussion: The Young's modulus of gradient samples ranged from 0.5 kPA to 3.5 kPA. Gene expression of TUJ1 (immature neuron marker) in cells cultures cultured on hydrogels decreases with increasing hydrogel Young's modulus (Figure 1). The gene expression of PAX6 (neural stem cell marker), MAP2 (mature neuron marker), GFAP (astrocyte marker) and Oligo2 (oligodendrocyte progenitor marker) were not affected by Young's modulus changes in this range and oligodendrocyte marker, PDGFRa, was not detected over the time course. Maximal neurite extension was observed in cells cultured on hydrogels with a Young's modulus of 0.91 kPa (Figure 2). Conclusion: Hydrogel Young's modulus effects hiPSC derived NSC gene expression of TUJ1 and neurite extension, but does not have a significant effect on the gene expression of other tested neural lineage markers. Modulation of matrix mechanics for neural differentiation is an important matrix property requiring futher optimization and study to fully understand its complex effect on cellular differentiation.