Pan-neuronal maturation but not neuronal subtype differentiation of adult neural stem cells is mechanosensitive

Abstract

Most past studies of the biophysical regulation of stem cell differentiation have focused on initial lineage commitment or proximal differentiation events. It would be valuable to understand whether biophysical inputs also influence distal endpoints more closely associated with physiological function, such as subtype specification in neuronal differentiation. To explore this question, we cultured adult neural stem cells (NSCs) on variable stiffness ECMs under conditions that promote neuronal fate commitment for extended time periods to allow neuronal subtype differentiation. We find that ECM stiffness does not modulate the expression of NeuroD1 and TrkA/B/C or the percentages of pan-neuronal, GABAergic, or glutamatergic neuronal subtypes. Interestingly, however, an ECM stiffness of 700 Pa maximizes expression of pan-neuronal markers. These results suggest that a wide range of stiffnesses fully permit pan-neuronal NSC differentiation, that an intermediate stiffness optimizes expression of pan-neuronal genes, and that stiffness does not impact commitment to particular neuronal subtypes.

Figure 1. Neuronal gene expression but not the generation of neurons is optimal on an intermediate stiffness near that of brain tissue.

Neural stem cells were cultured on polyacrylamide gels of different stiffnesses for 6 days. (A) TUJ1 gene expression normalized to ribosomal 18S, measured by quantitative reverse transcription polymerase chain reaction, is highest near 700 Pa. (B) The percentage of TUJ1+ neurons is invariant with substrate stiffness. (C) Immunofluorescence images corresponding to (B). TUJ1 is red, DAPI is blue. Error bars are 95% confidence intervals, n = 3. Means of multiple groups were compared by analysis of variance, Tukey-Kramer post hoc (ANOVA-TK), points with distinct group letters are statistically different with p < 0.05.

Figure 2. Neuronal subtype specification is not sensitive to substrate stiffness.

Neural stem cells were differentiated for 11 days on polyacrylamide gels of different stiffnesses and immunostained for TUJ1 (green), GABA (red), or VGlut1 (red), which are markers of neurons, GABAergic neurons, or glutamatergic neurons, respectively. DAPI is shown as blue. Inset images are outlined in white. Error bars are 95% confidence intervals, n = 6. Means compared by analysis of variance, Tukey-Kramer post hoc (ANOVA-TK), p > 0.05 for all comparisons.

Figure 3. Only specific neuronal genes are optimally expressed on intermediate substrate stiffnesses.

Neural stem cells were cultured on polyacrylamide gels of different stiffnesses for the indicated number of days. Gene expression, normalized to ribosomal 18S, was measured by quantitative reverse transcription polymerase chain reaction. Error bars are 95% confidence intervals, n = 3. Means compared by analysis of variance, Tukey-Kramer post hoc (ANOVA-TK), comparison bars represent p < 0.05. Note log-scale y-axes.