Xufeng Xue

Acoustic tweezing cytometry enhances osteogenesis of human mesenchymal stem cells through cytoskeletal contractility and YAP activation

Human mesenchymal stem cells (hMSCs) have great potential for cell-based therapies for treating degenerative bone diseases. It is known that mechanical cues in the cell microenvironment play an important role in regulating osteogenic (bone) differentiation of hMSCs. However, mechanoregulation of lineage commitment of hMSCs in conventional two-dimensional (2D) monocultures or bioengineered three-dimensional (3D) tissue constructs remains suboptimal due to complex biomaterial design criteria for hMSC culture. In this study, we demonstrate the feasibility of a novel cell mechanics and mechanobiology tool, acoustic tweezing cytometry (ATC), for mechanical stimulation of hMSCs. ATC utilizes ultrasound (US) pulses to actuate functionalized lipid microbubbles (MBs) which are covalently attached to hMSCs via integrin binding to exert forces to the cells. ATC stimulation increases cytoskeletal contractility of hMSCs regardless of the cell area. Furthermore, ATC application rescues osteogenic differentiation of hMSCs in culture conditions that are intrinsically repressive for hMSC osteogenesis (e.g., soft cell culture surfaces). ATC application activates transcriptional regulator YAP to enhance hMSC osteogenesis. Our data further show that F-actin, myosin II, and RhoA/ROCK signaling functions upstream of YAP activity in mediating ATC-stimulated hMSC osteogenesis. With the capability of applying controlled dynamic mechanical stimuli to cells, ATC provides a powerful tool for mechanoregulation of stem cell behaviors in tissue engineering and regenerative medicine applications.

Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells

Classic embryological studies have successfully applied genetics and cell biology principles to understand embryonic development. However, it remains unresolved how mechanics, as an integral driver of development, is involved in controlling tissue-scale cell fate patterning. Here we report a micropatterned human pluripotent stem (hPS)-cell-based neuroectoderm developmental model, in which pre-patterned geometrical confinement induces emergent patterning of neuroepithelial and neural plate border cells, mimicking neuroectoderm regionalization during early neurulation in vivo. In this hPS-cell-based neuroectoderm patterning model, two tissue-scale morphogenetic signals—cell shape and cytoskeletal contractile force—instruct neuroepithelial/neural plate border patterning via BMP-SMAD signalling. We further show that ectopic mechanical activation and exogenous BMP signalling modulation are sufficient to perturb neuroepithelial/neural plate border patterning. This study provides a useful microengineered, hPS-cell-based model with which to understand the biomechanical principles that guide neuroectoderm patterning and hence to study neural development and disease.Mechanical cues play critical roles in embryonic development. A micropatterned neuroectoderm developmental model based on human pluripotent stem cells now reveals how morophogenetic signals such as cell shape and contractility regulate neural tissue development.

Acoustic Tweezing Cytometry Induces Rapid Initiation of Human Embryonic Stem Cell Differentiation

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.

A systems mechanobiology model to predict cardiac reprogramming outcomes on different biomaterials

During normal development, the extracellular matrix (ECM) regulates cell fate mechanically and biochemically. However, the ECMs influence on lineage reprogramming, a process by which a cells developmental cycle is reversed to attain a progenitor-like cell state followed by subsequent differentiation into a desired cell phenotype, is unknown. Using a material mimetic of the ECM, here we show that ligand identity, ligand density, and substrate modulus modulate indirect cardiac reprogramming efficiency, but were not individually correlated with phenotypic outcomes in a predictive manner. Alternatively, we developed a data-driven model using partial least squares regression to relate short-term cell states, defined by quantitative mechanosensitive responses to different material environments, with long-term changes in phenotype. This model was validated by accurately predicting the reprogramming outcomes on a different material platform. Collectively, these findings suggest a means to rapidly screen candidate biomaterials that support reprogramming with high efficiency, without subjecting cells to the entire reprogramming process.

Carbon Nanotube Strain Sensor Based Hemoretractometer for Blood Coagulation Testing

Coagulation monitoring is essential for perioperative care and thrombosis treatment. However, existing assays for coagulation monitoring have limitations such as a large footprint and complex setup. In this work, we developed a miniaturized device for point-of-care blood coagulation testing by measuring dynamic clot retraction force development during blood clotting. In this device, a blood drop was localized between a protrusion and a flexible force-sensing beam to measure clot retraction force. The beam was featured with micropillar arrays to assist the deposition of carbon nanotube films, which served as a strain sensor to achieve label-free electrical readout of clot retraction force in real time. We characterized mechanical and electrical properties of the force-sensing beam and optimized its design. We further demonstrated that this blood coagulation monitoring device could obtain results that were consistent with those using an imaging method and that the device was capable of differentiating blood samples with different coagulation profiles. Owing to its low fabrication cost, small size, and low consumption of blood samples, the blood coagulation testing device using carbon nanotube strain sensors holds great potential as a point-of-care tool for future coagulation monitoring.

Capillary assisted deposition of carbon nanotube film for strain sensing

Advances in stretchable electronics offer the possibility of developing skin-like motion sensors. Carbon nanotubes (CNTs), owing to their superior electrical properties, have great potential for applications in such sensors. In this paper, we report a method for deposition and patterning of CNTs on soft, elastic polydimethylsiloxane (PDMS) substrates using capillary action. Micropillar arrays were generated on PDMS surfaces before treatment with plasma to render them hydrophilic. Capillary force enabled by the micropillar array spreads CNT solution evenly on PDMS surfaces. Solvent evaporation leaves a uniform deposition and patterning of CNTs on PDMS surfaces. We studied the effect of the CNT concentration and micropillar gap size on CNT coating uniformity, film conductivity, and piezoresistivity. Leveraging the piezoresistivity of deposited CNT films, we further designed and characterized a device for the contraction force measurement. Our capillary assisted deposition method of CNT films showed great application potential in fabrication of flexible CNT thin films for strain sensing.

Emerging Roles of YAP/TAZ in Mechanobiology

Understanding mechanotransduction is a major goal in the field of mechanobiology. YAP, and its paralog TAZ, are transcription coactivators at the core of the canonical Hippo signaling pathway. Recent studies have identified YAP/TAZ as both mechano-sensors and -transducers that respond to multiple extracellular mechanical signals and relay them to downstream transcriptional signals to regulate cell functions. In this chapter, we discuss how different types of mechanical cues, including the actin cytoskeleton, substrate rigidity, and external mechanical forces, mediate YAP/TAZ activities. We also discuss some possible mechanosensitive molecular machineries that function upstream of YAP/TAZ to control their mechanotransductive properties.