Mechanical forces play critical roles in influencing human embryonic stem cell (hESC) fate. However, it remains largely uncharacterized how local mechanical forces influence hESC behavior in vitro. Here, we used an ultrasound (US) technique, acoustic tweezing cytometry (ATC), to apply targeted cyclic subcellular forces to hESCs via integrin-bound microbubbles (MBs). We found that ATC-mediated cyclic forces applied for 30 min to hESCs near the edge of a colony induced immediate global responses throughout the colony, suggesting the importance of cell-cell connection in the mechanoresponsiveness of hESCs to ATC-applied forces. ATC application generated increased contractile force, enhanced calcium activity, as well as decreased expression of pluripotency transcription factors Oct4 and Nanog, leading to rapid initiation of hESC differentiation and characteristic epithelial-mesenchymal transition (EMT) events that depend on focal adhesion kinase (FAK) activation and cytoskeleton (CSK) tension. These results reveal a unique, rapid mechanoresponsiveness and community behavior of hESCs to integrin-targeted cyclic forces.

and cell replacement therapy. For example, recent demonstration that hESCs can be induced to differentiate and become motor neurons (MNs) offers unprecedented opportunity for studying MN development/function and developing cell-based therapies. [2] However, current MN differentiation protocols, based on soluble factors and small molecules that inhibit and/ or stimulate particular signaling pathways in defined culture conditions, not only are limited by low differentiation purity and yield, but also require prolonged cell culture that can take several weeks. [3] Mechanical forces are generated and transmitted across multiple scales, affecting cell fate during early embryonic development. [4] It has been increasingly recognized that besides chemical factors, biomechanical and topographical cues also play critical roles in differentiation and self-renewal of hESCs. [5] Thus new bioengineering tools and methods that leverage the intrinsic mechanosensitivity of hESCs may have the potential to improve hESC differentiation protocols. [6] While static mechanical factors such as substrate stiffness have been shown to mediate hESC behavior including their differentiation, [5] the effects of dynamic mechanical forces on hESCs have not been fully understood or exploited. This is due in part to the lack of appropriate techniques for applying dynamic forces to multiple cells in a high throughput fashion. Techniques such as atomic force microscopy (AFM) [7] and optical tweezer, although capable of applying subcellular dynamic forces, are limited to single cell analysis and often require expensive instrumentation. Magnetic twisting cytometry (MTC), [8] which uses functionalized magnetic microbeads attached to cells to apply forces to multiple cells, has been employed for microrheology and mechanobiology studies. However, solid microbeads are difficult to remove from cells, limiting post-MTC downstream assays and longitudinal studies that require continuous culture of cells devoid of exogenous materials.Acoustic tweezing cytometry (ATC) [9] is an ultrasound-based technique that utilizes ultrasound pulses to actuate encapsulated microbubbles (MBs) bound to integrin receptors to exert controlled forces to multiple cells simultaneously. MBs with stabilizing lipid or polymer shells (radius 1-3 µm) have been established as contrast agents for diagnostic ultrasound imaging [10] and exploited for drug/gene delivery applications. [11,12] Functionalization of MBs by decorating their shell Mechanical forces play important roles in human embryonic stem cell (hESC) differentiation. To investigate the impact of dynamic mechanical forces on neural induction of hESCs, this study employs acoustic tweezing cytometry (ATC) to apply cyclic forces/strains to hESCs by actuating integrin-bound microbubbles using ultrasound pulses. Accelerated neural induction of hESCs is demonstrated as the result of combined action of ATC and neural induction medium (NIM). Specifically, application of ATC for 30 min followed by culture in NIM upregulates neuroecd...

Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not Sox2. Translocations were directed by intracellular transmission of biophysical signals from cell surface integrins to nuclear CRM1 and were independent of exogenous soluble factors. On E-CADHERIN-coated substrates, presumably with minimal integrin engagement, mechanical strain-induced rapid nuclear-to-cytoplasmic translocation of the three transcription factors. These findings might provide fundamental insights into early developmental processes and may facilitate mechanotransductionmediated bioengineering approaches to influencing stem cell fate determination. Insight, innovation, integration How do mechanical stimuli, sensed through different types of cell adhesions, alter embryonic stem cell fate? This paper employs a stretchable device coated with different cell adhesion molecules to apply stretch to human embryonic stem cells in a manner that might mimic differentiation of cell lineages present at the blastocyst stage of development. The work reveals that stretch induces an unexpectedly rapid export of a subset of pluripotent transcription factors from the nucleus to the cytoplasm before significant decreases in overall levels. Interestingly, the subset of transcription factors exported differs between cells stretched on Matrigel-versus E-CADHERIN-coated devices. These results have physiological relevance to embryonic development where some cells experience mechanical forces predominantly through cell-cell contact while other cells also experience cell-ECM interactions leading to different cell-fate specification. These insights might enhance our understanding of early development and may also guide bioengineering approaches where stretching is used to accelerate or regulate the direction of differentiation.