Acoustic Tweezing Cytometry (ATC) on Dissociated Human Embryonic Stem Cells (HESCS)

Abstract

Human embryonic stem cells (hESCs) have wide potential use in regenerative medicine and cell therapies. However, large-scale cell expansion is limited as hESCs undergo differentiation and apoptosis after single-cell dissociation. Currently, Rho-associated kinase (ROCK) inhibitors are used in hESC culture to maintain cell survival, yet with the risk of long-term negative effects on hESCs. In previous work, we have shown acoustic tweezing cytometry (ATC), an ultrasound-based subcellular mechanical stimulation technique, can significantly improve survival rate of dissociated hESCs while maintaining their pluripotency without the presence of ROCK inhibitor Y-27632. Herein, we further investigated the impact of ATC on dissociated hESCs and the mechanisms underlying the enhancement of hESC survival by ATC. To determine optimal ATC treatment conditions, we exposed hESCs to ATC treatments with different acoustic pressure levels (i.e., 0.05 - 0.25 MPa) and exposure frequencies, and assayed their effects on hESC survival. To explore the role of E-cadherin and integrin mediated signaling pathways in hESC survival, we used microbubbles targeted to E-cadherin and integrin on the cells in ATC application, and evaluated the effects on hESCs in terms of survival rate, cell area, colony size, and stemness. In addition, we monitored cell intracellular cytoskeleton contractility using a fluorescence-labeled micropost system in the initial cell spreading stage, and compared the contractility changes over time under various ATC stimulation conditions. In summary, results from this study will help us to develop an effective treatment scheme using ATC to enhance dissociated hESC survival and reveal the insights about mechanotransductive mechanisms involving E-cadherin and integrin-mediated adhesion signaling for hESC survival and pluripotency maintenance.