Extended Data Fig. 6: Long-term transcriptional changes in response to mechano-osmotic stimuli.

(a) Venn diagram and Z-score heatmap analyses of specific and overlapping genes from bulk RNA sequencing in 24 h upregulated in response to compression in basal medium or pluripotency medium when compared to their respective media conditions. Note that compression in basal medium leads to more genes being upregulated and the specific genes representing differentiation genes. Compression in pluripotency medium leads to upregulation of cytoskeletal and stress genes. (b) Gene set enrichment analyses of differentially expressed genes from bulk RNA sequencing in 24 h basal medium versus 30 min compression + 24 h recovery in basal medium (left panel) and 24 h basal medium versus 30 min hypertonic shock + 24 h recovery in basal medium (right panel). Note enrichment of gene sets involved in morphogenesis and metal ion homeostasis in both conditions. (c) Representative images of Pax6 staining and quantification of LaminB1-RFP hiPSCs exposed to 30 min basal medium (Control), 30 min basal medium + 10 min hypotonic shock (Hypotonic), 30 min compression (Compression) and 30 min compression +10 min hypotonic shock (Compression+Hypo) in basal medium followed by 96 h of spontaneous differentiation in basal medium. Note delayed differentiation in both shock conditions (scale bars 50 µm; n = 3 independent experiments with 33844 (Basal), 34959 (Hypo), 35860 (Comp), 27615 (Comp+Hypo) total cells/condition; ANOVA/Dunnett’s). (d) Schematic representation of experimental outline, representative images of Oct4 and Pax6 staining and quantification of cells exposed to 30 min compression or 30 min hypertonic shock in pluripotency medium for 96 h. Note delayed differentiation in both shock conditions (scale bars 75 µm; n = 3 independent experiments with 673 (control), 564 (Compression), 421, 832 (Hypertonic) total cells/condition; Kruskal-Wallis/Dunn’s, ns=not significant). Source numerical data are available in source data.

Source data