Fig. 3.

The stage-specific effects of substrate stiffness on pancreatic endoderm differentiation. Human iPSCs were differentiated on either soft (PCL) or stiff (PEKK) electrospun substrates to (a, b) mesendodermal, (c, d) posterior foregut, or (e, f) pancreatic endoderm lineage. a Gene expression of mesendodermal markers GSC and MIXL1 was significantly upregulated on stiff substrates as compared to soft substrates. b Immunofluorescent imaging and quantification of percent-positive cells showed that GSC protein expression was significantly higher on stiff substrates (green: GSC; blue: DAPI; scale bar = 100 μm). c Gene expression of posterior foregut markers HNF4A and FOXA2 was significantly upregulated on soft substrates as compared to stiff substrates. d Immunofluorescent imaging and quantification of percent-positive cells showed that FOXA2 protein expression was significantly higher for cells cultured on soft substrates (green: FOXA2; blue: DAPI; scale bar = 100 μm). e Gene expression of pancreatic endoderm markers NKX2.2 and NKX6.1 was significantly upregulated on soft substrates as compared to stiff substrates. f Immunofluorescent imaging and quantification of percent-positive cells showed that PDX1 protein expression was higher on soft substrates (green: PDX1; blue: DAPI; scale bar =100 μm). 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