Yubing Sun

Forcing Stem Cells to Behave: A Biophysical Perspective of the Cellular Microenvironment

Physical factors in the local cellular microenvironment, including cell shape and geometry, matrix mechanics, external mechanical forces, and nanotopographical features of the extracellular matrix, can all have strong influences on regulating stem cell fate. Stem cells sense and respond to these insoluble biophysical signals through integrin-mediated adhesions and the force balance between intracellular cytoskeletal contractility and the resistant forces originated from the extracellular matrix. Importantly, these mechanotransduction processes can couple with many other potent growth-factor-mediated signaling pathways to regulate stem cell fate. Different bioengineering tools and microscale/nanoscale devices have been successfully developed to engineer the physical aspects of the cellular microenvironment for stem cells, and these tools and devices have proven extremely powerful for identifying the extrinsic physical factors and their downstream intracellular signaling pathways that control stem cell functions.

Nanotopography Influences Adhesion, Spreading, and Self-Renewal of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have great potentials for future cell-based therapeutics. However, their mechanosensitivity to biophysical signals from the cellular microenvironment is not well characterized. Here we introduced an effective microfabrication strategy for accurate control and patterning of nanoroughness on glass surfaces. Our results demonstrated that nanotopography could provide a potent regulatory signal over different hESC behaviors, including cell morphology, adhesion, proliferation, clonal expansion, and self-renewal. Our results indicated that topological sensing of hESCs might include feedback regulation involving mechanosensory integrin-mediated cell-matrix adhesion, myosin II, and E-cadherin. Our results also demonstrated that cellular responses to nanotopography were cell-type specific, and as such, we could generate a spatially segregated coculture system for hESCs and NIH/3T3 fibroblasts using patterned nanorough glass surfaces.

Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells

Our understanding of the intrinsic mechanosensitive properties of human pluripotent stem cells (hPSCs), in particular the effects that the physical microenvironment has on their differentiation, remains elusive1. Here, we show that neural induction and caudalization of hPSCs can be accelerated by using a synthetic microengineered substrate system consisting of poly(dimethylsiloxane) micropost arrays (PMAs) with tunable mechanical rigidities. The purity and yield of functional motor neurons (MNs) derived from hPSCs within 23 days of culture using soft PMAs were improved more than 4- and 10-fold, respectively, compared to coverslips or rigid PMAs. Mechanistic studies revealed a multi-targeted mechanotransductive process involving Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo-YAP activities and actomyosin cytoskeleton integrity and contractility. Our findings suggest that substrate rigidity is an important biophysical cue influencing neural induction and subtype specification, and that microengineered substrates can thus serve as a promising platform for large-scale culture of hPSCs.

Mechanics Regulates Fate Decisions of Human Embryonic Stem Cells

Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function.

Elastomeric microposts integrated into microfluidics for flow-mediated endothelial mechanotransduction analysis

Mechanotransduction is known as the cellular mechanism converting insoluble biophysical signals in the local cellular microenvironment (e.g. matrix rigidity, external mechanical forces, and fluid shear) into intracellular signalling to regulate cellular behaviours. While microfluidic technologies support a precise and independent control of soluble factors in the cellular microenvironment (e.g. growth factors, nutrients, and dissolved gases), the regulation of insoluble biophysical signals in microfluidics, especially matrix rigidity and adhesive pattern, has not yet been achieved. Here we reported an integrated soft lithography-compatible microfluidic methodology that could enable independent controls and modulations of fluid shear, substrate rigidity, and adhesive pattern in a microfluidic environment, by integrating micromolded elastomeric micropost arrays and microcontact printing with microfluidics. The geometry of the elastomeric micropost array could be regulated to mediate substrate rigidity and adhesive pattern, and further the elastomeric microposts could be utilized as force sensors to map live-cell subcellular contractile forces. To illustrate the general application of our methodology, we investigated the flow-mediated endothelial mechanotransduction process and examined specifically the involvement of subcellular contractile forces in the morphological realignment process of endothelial cells under a sustained directional fluid shear. Our results showed that the cytoskeletal contractile forces of endothelial cells were spatiotemporally regulated and coordinated to facilitate their morphology elongation process along the direction of flow. Together, our study provided an integrated microfluidic strategy to modulate the in vitro cellular microenvironment with both defined soluble and insoluble signals, and we demonstrated its application to investigate quantitatively the involvement of cytoskeletal contractile forces in the flow-mediated mechanotransduction process of endothelial cells.

Acoustic tweezing cytometry for live-cell subcellular modulation of intracellular cytoskeleton contractility

Mechanical forces are critical to modulate cell spreading, contractility, gene expression, and even stem cell differentiation. Yet, existing tools that can apply controllable subcellular forces to a large number of single cells simultaneously are still limited. Here we report a novel ultrasound tweezing cytometry utilizing ultrasound pulses to actuate functionalized lipid microbubbles covalently attached to single live cells to exert mechanical forces in the pN - nN range. Ultrasonic excitation of microbubbles could elicit a rapid and sustained reactive intracellular cytoskeleton contractile force increase in different adherent mechanosensitive cells. Further, ultrasound-mediated intracellular cytoskeleton contractility enhancement was dose-dependent and required an intact actin cytoskeleton as well as RhoA/ROCK signaling. Our results demonstrated the great potential of ultrasound tweezing cytometry technique using functionalized microbubbles as an actuatable, biocompatible, and multifunctional agent for biomechanical stimulations of cells.

Synthesis of Necklace-like Magnetic Nanorings

Necklace-like magnetite and maghemite nanorings, composed of magnetic nanoparticles (NPs) with average size about 40 nm, have been prepared via a solvothermal process in a colloidal solution by a self-assembly process. The composition, phase, and morphology of these nanorings have been characterized by X-ray diffraction, X-ray absorption, and transmission electron microscopy. In this paper, we discuss the influence of reaction conditions on the formation of nanorings structure including the amount of PVP in starting materials, reaction time, and temperature. On the basis of experimental observation, we supposed that magnetite NPs may first assemble into chains by magnetic dipole-dipole interactions. These dipolar chains, which are metastable structures relative to necklace-like nanorings, then produced the rings. So, the stability of chains may determine the yield, size, and morphologies of necklace-like nanorings.

Mechanobiology: a new frontier for human pluripotent stem cells

Research on human pluripotent stem cells (hPSCs) has expanded rapidly over the last two decades, owing to the promises of hPSCs for applications in regenerative medicine, disease modeling, and developmental biology studies. While most studies of hPSCs have so far focused on identifying extrinsic soluble factors, intracellular signaling pathways, and transcriptional networks that are involved in regulating hPSC self-renewal and differentiation, a few promising studies have emerged in recent years to reveal some unique mechano-sensitive and -responsive properties of hPSCs and the effect of the physical aspects of the local cellular microenvironment on regulating hPSC behaviors. This Frontier Review is to highlight these recent studies of mechanobiology in hPSCs and to discuss the impact of advancing our understanding of mechanoregulation of hPSC behaviors on improving survival, self-renewal and differentiation of hPSCs using well-controlled synthetic micro/nanoscale cell culture tools.

Microfabricated nanotopological surfaces for study of adhesion-dependent cell mechanosensitivity.

Cells exhibit high sensitivity and diverse responses to the intrinsic nanotopography of the extracellular matrix through their nanoscale cellular sensing machinery. A simple microfabrication method for precise control and spatial patterning of the local nanoroughness on glass surfaces by using photolithography and reactive ion etching is reported. It is demonstrated that local nanoroughness as a biophysical cue could regulate a diverse array of NIH/3T3 fibroblast behaviors, including cell morphology, adhesion, proliferation, migration, and cytoskeleton contractility. The capability to control and further predict cellular responses to nanoroughness might suggest novel methods for developing biomaterials mimicking nanotopographic structures in vivo for functional tissue engineering.

UV-Modulated Substrate Rigidity for Multiscale Study of Mechanoresponsive Cellular Behaviors

Mechanical properties of the extracellular matrix (ECM) have profound effects on cellular functions. Here, we applied novel photosensitive polydimethylsiloxane (photoPDMS) chemistry to create photosensitive, biocompatible photoPDMS as a rigidity-tunable material for study of mechanoresponsive cellular behaviors. By modulating the PDMS cross-linker to monomer ratio, UV light exposure time, and postexposure baking time, we achieved a broad range of bulk Youngs modulus for photoPDMS from 0.027 to 2.48 MPa. Biocompatibility of photoPDMS was assayed, and no significant cytotoxic effect was detected as compared to conventional PDMS. We demonstrated that the bulk Youngs modulus of photoPDMS could impact cell morphology, adhesion formation, cytoskeletal structure, and cell proliferation. We further fabricated photoPDMS micropost arrays for multiscale study of mechanoresponsive cellular behaviors. Our results suggested that adherent cells could sense and respond to changes of substrate rigidity at a subfocal adhesion resolution. Together, we demonstrated the potential of photoPDMS as a photosensitive and rigidity-tunable material for mechanobiology studies.