Figure 4: EDICTS resolves stem cell phenotypes steered by graded extracellular mechanics.

H3K4K27me expression is responsive to minute changes in Young’s modulus. (A) PEGDM continuous Young’s modulus gradient hydrogels were fabricated by varying the dispensing rates of different PEGDM concentration solutions into a custom mold, yielding a final hydrogel “strip” measuring ~10 mm in width and ~55 mm in length. (B) hMSCs cultured on PEGDM continuous Young’s modulus gradient hydrogels were fixed and immunolabeled for H3K4me3 (green) and H3K27me3 (red) at early (72 hours) and late (2 weeks) time points along the entire length of the hydrogel, which ranged in Young’s modulus from 3.8 to 27 kPa. (C) Haralick texture descriptors were acquired and analyzed via PCA followed by k-means cluster analysis to determine centroids of clusters of cells cultured for a specified time and position on the gradient. Centroids of cells grown on the softest part of the gel (S1) were closest in proximity to the centroid of pre-differentiated adipogenic cells, indicating their similarity of H3K4K27me3 expression and subsequent lineage commitment. (D) Heat-map of normalized texture descriptor values reveals increasing (outlined in red) and decreasing (outlined in blue) trends that vary with decreasing hydrogel stiffness.