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Human embryonic stem cells (hESCs) can differentiate to three germ layers within biochemical and biomechanical niches. The complicated mechanical environments in vivo could have diverse effects on the fate decision and biological functions of hESCs. To globally screen mechanosensitive molecules, three typical types of mechanical stimuli, i.e., tensile stretch, shear flow, and mechanical compression, were applied in respective parameter sets of loading pattern, amplitude, frequency, and/or duration, and then, iTRAQ proteomics test was used for identifying and quantifying differentially expressed proteins in hESCs. Bioinformatics analysis identified 37, 41, and 23 proteins under stretch pattern, frequency, and duration, 13, 18, and 41 proteins under shear pattern, amplitude, and duration, and 4, 0, and 183 proteins under compression amplitude, frequency, and duration, respectively, where distinct parameters yielded the differentially weighted preferences under each stimulus. Ten mechanosensitive proteins were commonly shared between two of three mechanical stimuli, together with numerous proteins identified under single stimulus. More importantly, functional GSEA and WGCNA analyses elaborated the variations of the screened proteins with loading parameters. Common functions in protein synthesis and modification were identified among three stimuli, and specific functions were observed in skin development under stretch alone. In conclusion, mechanomics analysis is indispensable to map actual mechanosensitive proteins under physiologically mimicking mechanical environment, and sheds light on understanding the core hub proteins in mechanobiology.

Background: Distinct mechanical stimuli are known to manipulate the behaviors of embryonic stem cells (ESCs). Fundamental rationale of how ESCs respond to mechanical forces and the potential biological effects remain elusive. Here we conducted the mechanobiological study for hESCs upon mechanomics analysis to unravel typical mechanosensitive processes on hESC-specific fluid shear., Methods: hESC line H1 was subjected to systematically varied shear flow, and mechanosensitive proteins were obtained by mass spectrometry (MS) analysis. Then, function enrichment analysis was performed to identify the enriched gene sets. Under a steady shear flow of 1.1 Pa for 24 h, protein expressions were further detected using western blotting (WB), quantitative real-time PCR (qPCR), and immunofluorescence (IF) staining. Meanwhile, the cells were treated with 200 nM trichostatin (TSA) for 1 h as positive control to test chromatin decondensation. Actin, DNA, and RNA were then visualized with TRITC-labeled phalloidin, Hoechst 33342, and SYTO® RNASelect™ green fluorescent cell stain (Life Technologies), respectively. In addition, cell stiffness was determined with atomic force microscopy (AFM) and annexin V-PE was used to determine the apoptosis with a flow cytometer (FCM)., Results: Typical mechanosensitive proteins were unraveled upon mechanomics analysis under fluid shear related to hESCs in vivo. Functional analyses revealed significant alterations in histone acetylation, nuclear size, and cytoskeleton for hESC under shear flow. Shear flow was able to induce H2B acetylation and nuclear spreading by CFL2/F-actin cytoskeletal reorganization. The resulting chromatin decondensation and a larger nucleus readily accommodate signaling molecules and transcription factors., Conclusions: Shear flow regulated chromatin dynamics in hESCs via cytoskeleton and nucleus alterations and consolidated their primed state.