Metabolic-Glycoengineering-Enabled Molecularly Specific Acoustic Tweezing Cytometry for Targeted Mechanical Stimulation of Cell Surface Sialoglycans.

In this study, we developed a novel type of dibenzocyclooctyne (DBCO)-functionalized microbubbles (MBs) and validated their attachment to azide-labelled sialoglycans on human pluripotent stem cells (hPSCs) generated by metabolic glycoengineering (MGE). This enabled the application of mechanical forces to sialoglycans on hPSCs through molecularly specific acoustic tweezing cytometry (mATC), that is, displacing sialoglycan-anchored MBs using ultrasound (US). It was shown that subjected to the acoustic radiation forces of US pulses, sialoglycan-anchored MBs exhibited significantly larger displacements and faster, more complete recovery after each pulse than integrin-anchored MBs, indicating that sialoglycans are more stretchable and elastic than integrins on hPSCs in response to mechanical force. Furthermore, stimulating sialoglycans on hPSCs using mATC reduced stage-specific embryonic antigen-3 (SSEA-3) and GD3 expression but not OCT4 and SOX2 nuclear localization. Conversely, stimulating integrins decreased OCT4 nuclear localization but not SSEA-3 and GD3 expression, suggesting that mechanically stimulating sialoglycans and integrins initiated distinctive mechanoresponses during the early stages of hPSC differentiation. Taken together, these results demonstrated that MGE-enabled mATC uncovered not only different mechanical properties of sialoglycans on hPSCs and integrins but also their different mechanoregulatory impacts on hPSC differentiation, validating MGE-based mATC as a new, powerful tool for investigating the roles of glycans and other cell surface biomolecules in mechanotransduction.