Integrin‐Targeted Cyclic Forces Accelerate Neural Tube‐Like Rosette Formation from Human Embryonic Stem Cells

Abstract

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...

Rapid responses of human pluripotent stem cells to cyclic mechanical strains applied to integrin by acoustic tweezing cytometry

“…Modulation of YAP activity is necessary for hPSCs to exit pluripotency and begin differentiation into specific lineages 21 , 29 , 44 , 45 . Our observation of decreased OCT4 in hPSCs after ATC application is consistent with our previous results 28 , 30 , indicating that the hPSCs were undergoing differentiation due to ATC application. However, YAP nucleocytoplasmic translocation in hPSCs after 30 min ATC treatment was observed in our previous study 28 rather than nuclear translocation observed in our current study.…”

“…Our data of rapid downregulation of OCT4 in hPSCs by ATC (as short as 1 min) show the early mechanoresponses of hPSCs, showing their unique mechanosensitivity to ATC stimulations and the likely role in the long-term effects on hPSCs after ATC treatments manifested as faster differentiation and neural rosette formation 28 , 30 .…”

“…While higher concentrations of bubbles might result in more efficient responses of hPSCs, we chose a concentration of bubbles that yielded a bubble:cell ratio of about 1:10 to minimize potential damages to hPSCs due to sonoporation 38 or the impact of cavitation as shown in our previous studies 24 , 38 , 39 . Importantly, our previous studies show hPSCs in a colony could respond globally to ATC applications, exhibiting increased CSK contractility and downregulation of pluripotency markers across the entire colony, even when not all cells are coated with microbubbles 28 , 30 . The global response of hPSC colonies likely results from mechanical and chemical interactions between adjacent hPSCs in the same colony 39 .…”

“…In particular, we have demonstrated that applications of ATC-mediated mechanical forces targeted to integrins of hPSCs for 30 min reduced expression of pluripotency-related transcription factors Octamer-binding transcription factor 4 (OCT4) and Nanog homeobox (NANOG) and induced epithelial-mesenchymal transition (EMT) immediately after ATC application 28 . Treatment with neural induction medium (NIM) following 30 min ATC application further resulted in increased expression of Paired Box 6 (PAX6), an early neuroectodermal (NE) differentiation marker, as early as 6 h, and neural rosette-like structure formation as early as 48 h 30 . These results show that ATC-facilitated NE differentiation of hPSCs occurred significantly faster than conventional NE differentiation protocols that rely on soluble factors only, which normally takes about at least 7 days to obtain PAX6 + neural rosette structures 31 , 32 .…”

“…Although we have previously shown that ATC-induced mechanoresponses in hPSCs depended on focal adhesion kinase (FAK) and CSK tension previously 28 , 30 , how integrin-targeted cyclic force/strain in ATC is transduced to elicit hPSC responses remains incompletely understood. Robust responses of hPSCs to ATC-mediated cyclic forces to integrins for only 30 min is intriguing and suggest exciting potentials of ATC to promote hPSC differentiation.…”