Fig. 2

The stage-specific effects of substrate stiffness on motor neuron differentiation. Human iPSCs were differentiated on either soft (PCL) or stiff (PEKK) electrospun substrates to (a, b) ectodermal, (c, d) neural progenitor, or (e, f) motor neuron lineage. a Gene expression of ectodermal markers FGF5 and PAX6 was significantly upregulated on soft substrates as compared to stiff substrates. b Immunofluorescent imaging and quantification of percent-positive cells showed that PAX6 protein expression was significantly higher on soft substrates after ectodermal induction (green: PAX6; blue: DAPI; scale bar = 100 μm). c Gene expression of neural progenitor markers NCAM1 and NES was significantly upregulated on stiff substrates as compared to soft substrates. d Immunofluorescent imaging and quantification of percent-positive cells showed that NESTIN protein expression was higher on stiff substrates (green: NESTIN; blue: DAPI; scale bar = 100 μm). e Gene expression of motor neuron markers NEUROG2 and ISL1 was significantly upregulated on stiff substrates as compared to soft substrates. f Immunofluorescent imaging and quantification of percent-positive cells showed that HB9 protein expression was higher on stiff substrates (green: HB9; blue: DAPI; scale bar = 100 μm). Gene expression was normalized to that of cells from the preceding stage of differentiation cultured on TCPS (fold change = 1). The dashed line represents the average fold change of differentiated cells on TCPS. ^# p < 0.05, ^## p < 0.01, versus TCPS differentiated controls. *p < 0.05, **p < 0.01, between substrates