Cellular Mechanoresponse Research Articles

Multiplex, high-throughput method to study cancer and immune cell mechanotransduction

Studying cellular mechanoresponses during cancer metastasis is limited by sample variation or complex protocols that current techniques require. Metastasis is governed by mechanotransduction, whereby cells translate external stimuli, such as circulatory fluid shear stress (FSS), into biochemical cues. We present high-throughput, semi-automated methods to expose cells to FSS using the VIAFLO96 multichannel pipetting device custom-fitted with 22 G needles, increasing the maximum FSS 94-fold from the unmodified tips. Specifically, we develop protocols to semi-automatically stain live samples and to fix, permeabilize, and intracellularly process cells for flow cytometry analysis. Our first model system confirmed that the pro-apoptotic effects of TRAIL therapeutics in prostate cancer cells can be enhanced via FSS-induced Piezo1 activation. Our second system implements this multiplex methodology to show that FSS exposure (290 dyn cm−2) increases activation of murine bone marrow-derived dendritic cells. These methodologies greatly improve the mechanobiology workflow, offering a high-throughput, multiplex approach.

3D scaffolds-specific cellular mechanoresponse as a pivotal regulating factor in tissue engineering

3D scaffolds-specific cellular mechanoresponse as a pivotal regulating factor in tissue engineering

Synergistic Influence of Fibrous Pattern Orientation and Modulus on Cellular Mechanoresponse.

The fibrous extracellular matrix (ECM) is vital for tissue regeneration and impacts implanted device treatments. Previous research on fibrous biomaterials shows varying cellular reactions to surface orientation, often due to unclear interactions between surface topography and substrate elasticity. Our study addresses this gap by achieving the rapid creation of hydrogels with diverse fibrous topographies and varying substrate moduli through a surface printing strategy. Cells exhibit heightened traction force on nanopatterned soft hydrogels, particularly with randomly distributed patterns compared with regular soft hydrogels. Meanwhile, on stiff hydrogels featuring an aligned topography, optimal cellular mechanosensing is observed compared to random topography. Mechanistic investigations highlight that cellular force-sensing and adhesion are influenced by the interplay of pattern deformability and focal adhesion orientation, subsequently mediating stem cell differentiation. Our findings highlight the importance of combining substrate modulus and topography to guide cellular behavior in designing advanced tissue engineering biomaterials.

A computational bridge between traction force microscopy and tissue contraction.

Arterial wall active mechanics are driven by resident smooth muscle cells, which respond to biological, chemical, and mechanical stimuli and activate their cytoskeletal machinery to generate contractile stresses. The cellular mechanoresponse is sensitive to environmental perturbations, often leading to maladaptation and disease progression. When investigated at the single cell scale, however, these perturbations do not consistently result in phenotypes observed at the tissue scale. Here, a multiscale model is introduced that translates microscale contractility signaling into a macroscale, tissue-level response. The microscale framework incorporates a biochemical signaling network along with characterization of fiber networks that govern the anisotropic mechanics of vascular tissue. By incorporating both biochemical and mechanical components, the model is more flexible and more broadly applicable to physiological and pathological conditions. The model can be applied to both cell and tissue scale systems, allowing for the analysis of in vitro, traction force microscopy and ex vivo, isometric contraction experiments in parallel. When applied to aortic explant rings and isolated smooth muscle cells, the model predicts that active contractility is not a function of stretch at intermediate strain. The model also successfully predicts cell-scale and tissue-scale contractility and matches experimentally observed behaviors, including the hypercontractile phenotype caused by chronic hyperglycemia. The connection of the microscale framework to the macroscale through the multiscale model presents a framework that can translate the wealth of information already collected at the cell scale to tissue scale phenotypes, potentially easing the development of smooth muscle cell-targeting therapeutics.

Compressive forces stabilize microtubules in living cells.

Microtubules are cytoskeleton components with unique mechanical and dynamic properties. They are rigid polymers that alternate phases of growth and shrinkage. Nonetheless, the cells can display a subset of stable microtubules, but it is unclear whether microtubule dynamics and mechanical properties are related. Recent in vitro studies suggest that microtubules have mechano-responsive properties, being able to stabilize their lattice by self-repair on physical damage. Here we study how microtubules respond to cycles of compressive forces in living cells and find that microtubules become distorted, less dynamic and more stable. This mechano-stabilization depends on CLASP2, which relocates from the end to the deformed shaft of microtubules. This process seems to be instrumental for cell migration in confined spaces. Overall, these results demonstrate that microtubules in living cells have mechano-responsive properties that allow them to resist and even counteract the forces to which they are subjected, being a central mediator of cellular mechano-responses.

Mechanical activation of lung epithelial cells through the ion channel Piezo1 activates the metalloproteinases ADAM10 and ADAM17 and promotes growth factor and adhesion molecule release

In the lung, pulmonary epithelial cells undergo mechanical stretching during ventilation. The associated cellular mechanoresponse is still poorly understood at the molecular level. Here, we demonstrate that activation of the mechanosensitive cation channel Piezo1 in a human epithelial cell line (H441) and in primary human lung epithelial cells induces the proteolytic activity of the metalloproteinases ADAM10 and ADAM17 at the plasma membrane. These ADAMs are known to convert cell surface expressed proteins into soluble and thereby play major roles in proliferation, barrier regulation and inflammation. We observed that chemical activation of Piezo1 promotes cleavage of substrates that are specific for either ADAM10 or ADAM17. Activation of Piezo1 also induced the synthesis and ADAM10/17-dependent release of the growth factor amphiregulin (AREG). In addition, junctional adhesion molecule A (JAM-A) was shed in an ADAM10/17-dependent manner resulting in a reduction of cell contacts. Stretching experiments combined with Piezo1 knockdown further demonstrated that mechanical activation promotes shedding via Piezo1. Most importantly, high pressure ventilation of murine lungs increased AREG and JAM-A release into the alveolar space, which was reduced by a Piezo1 inhibitor. Our study provides a novel link between stretch-induced Piezo1 activation and the activation of ADAM10 and ADAM17 in lung epithelium. This may help to understand acute respiratory distress syndrome (ARDS) which is induced by ventilation stress and goes along with perturbed epithelial permeability and release of growth factors.

Portal Hypertension in Nonalcoholic Fatty Liver Disease: Challenges and Paradigms.

Portal hypertension in cirrhosis is defined as an increase in the portal pressure gradient (PPG) between the portal and hepatic veins and is traditionally estimated by the hepatic venous pressure gradient (HVPG), which is the difference in pressure between the free-floating and wedged positions of a balloon catheter in the hepatic vein. By convention, HVPG≥10 mmHg indicates clinically significant portal hypertension, which is associated with adverse clinical outcomes. Nonalcoholic fatty liver disease (NAFLD) is a common disorder with a heterogeneous clinical course, which includes the development of portal hypertension. There is increasing evidence that portal hypertension in NAFLD deserves special considerations. First, elevated PPG often precedes fibrosis in NAFLD, suggesting a bidirectional relationship between these pathological processes. Second, HVPG underestimates PPG in NAFLD, suggesting that portal hypertension is more prevalent in this condition than currently believed. Third, cellular mechanoresponses generated early in the pathogenesis of NAFLD provide a mechanistic explanation for the pressure-fibrosis paradigm. Finally, a better understanding of liver mechanobiology in NAFLD may aid in the development of novel pharmaceutical targets for prevention and management of this disease.

Soft, strong, tough, and durable protein-based fiber hydrogels

Load-bearing soft tissues normally show J-shaped stress-strain behaviors with high compliance at low strains yet high strength at high strains. They have high water content but are still tough and durable. By contrast, naturally derived hydrogels are weak and brittle. Although hydrogels prepared from synthetic polymers can be strong and tough, they do not have the desired bioactivity for emerging biomedical applications. Here, we present a thermomechanical approach to replicate the combinational properties of soft tissues in protein-based photocrosslinkable hydrogels. As a demonstration, we create a gelatin methacryloyl fiber hydrogel with soft tissue-like mechanical properties, such as low Young's modulus (0.1 to 0.3 MPa), high strength (1.1 ± 0.2 MPa), high toughness (9,100 ± 2,200 J/m3), and high fatigue resistance (2,300 ± 500 J/m2). This hydrogel also resembles the biochemical and architectural properties of native extracellular matrix, which enables a fast formation of 3D interconnected cell meshwork inside hydrogels. The fiber architecture also regulates cellular mechanoresponse and supports cell remodeling inside hydrogels. The integration of tissue-like mechanical properties and bioactivity is highly desirable for the next-generation biomaterials and could advance emerging fields such as tissue engineering and regenerative medicine.

Cell Stretcher Assay to Analyze Mechanoresponses to Cyclic Stretching.

In their natural environment, most cells and tissues are continuously exposed to cyclic mechanical strain. Sensing these stimuli by mechanosensory proteins and subsequent conversion into a variety of biological responses (referred to as mechanotransduction) are key processes for tissue homeostasis, survival, and differentiation. Perturbations of underlying signaling pathways lead to severe diseases in vivo (Urciuoli E, Peruzzi B, Int J Mol Sci 21(24). https://doi.org/10.3390/ijms21249426, (2020)). In addition, cellular mechanoresponses to cyclic stretching of an isolated single cell differ from those of a cell monolayer, network, or even three-dimensional tissue. Since these processes depend on various physical and biological parameters, the development of a precise, well-characterized, and highly reproducible but also easily tunable stretcher assay is indispensable. Here, we describe the fabrication of defined elastic substrates and their application in cyclic stretching of cultured cells in a custom-made cell stretcher device. We focus on the detailed description of the system and provide a possibility for mechanoresponse characterization, using the analysis of actin stress fiber orientation as exemplary mechanoresponse to cyclic stretching of adherent cells.

Mechanotransduction in the pathogenesis of non-alcoholic fatty liver disease.

Mechanobiology is a domain of interdisciplinary research that aims to explore the impact of physical force, applied externally or internally, on cell and tissue function, including development, growth, and differentiation. Mechanotransduction is a term that describes how cells sense physical forces (such as compression, stretch, and shear stress), convert them into biochemical signals, and mount adaptive responses integrated by the nucleus. There is accumulating evidence that mechanical forces extensively inform the biological behaviour of liver cells in health and disease. Recent research has elucidated many cellular and molecular mechanisms involved in this process including the pleiotropic control and diverse effects of the paralogous transcription co-activators YAP/TAZ, which play a prominent role in mechanotransduction. The liver sinusoids represent a unique microenvironment in which cells are exposed to mechanical cues originating in the cytoskeleton and at interfaces with adjacent cells, the extracellular matrix, and vascular or interstitial fluids. In non-alcoholic fatty liver disease (NAFLD), hepatocellular lipid accumulation and ballooning, activation of inflammatory responses, dysfunction of liver sinusoidal endothelial cells, and transdifferentiation of hepatic stellate cells into a pro-contractile and pro-fibrotic phenotype have been associated with aberrant cycles of mechanosensing and mechanoresponses. The downstream consequences of disrupted mechanical homeostasis likely contribute to the progression of NAFLD and promote the development of portal hypertension, cirrhosis, and hepatocellular carcinoma. Identification of molecular targets involved in pathogenic mechanotransduction will allow for the development of novel strategies to prevent the progression of liver disease in NAFLD.

A Metal-Ion-Incorporated Mussel-Inspired Poly(Vinyl Alcohol)-Based Polymer Coating Offers Improved Antibacterial Activity and Cellular Mechanoresponse Manipulation.

Cobalt (CoII) ions have been an attractive candidate for the biomedical modification of orthopedic implants for decades. However, limited research has been performed into how immobilized CoII ions affect the physical properties of implant devices and how these changes regulate cellular behavior. In this study we modified biocompatible poly(vinyl alcohol) with terpyridine and catechol groups (PVA‐TP‐CA) to create a stable surface coating in which bioactive metal ions could be anchored, endowing the coating with improved broad‐spectrum antibacterial activity against Escherichia coli and Staphylococcus aureus, as well as enhanced surface stiffness and cellular mechanoresponse manipulation. Strengthened by the addition of these metal ions, the coating elicited enhanced mechanosensing from adjacent cells, facilitating cell adhesion, spreading, proliferation, and osteogenic differentiation on the surface coating. This dual‐functional PVA‐TP‐CA/Co surface coating offers a promising approach for improving clinical implantation outcomes.

A Metal‐Ion‐Incorporated Mussel‐Inspired Poly(Vinyl Alcohol)‐Based Polymer Coating Offers Improved Antibacterial Activity and Cellular Mechanoresponse Manipulation

AbstractCobalt (CoII) ions have been an attractive candidate for the biomedical modification of orthopedic implants for decades. However, limited research has been performed into how immobilized CoII ions affect the physical properties of implant devices and how these changes regulate cellular behavior. In this study we modified biocompatible poly(vinyl alcohol) with terpyridine and catechol groups (PVA‐TP‐CA) to create a stable surface coating in which bioactive metal ions could be anchored, endowing the coating with improved broad‐spectrum antibacterial activity against Escherichia coli and Staphylococcus aureus, as well as enhanced surface stiffness and cellular mechanoresponse manipulation. Strengthened by the addition of these metal ions, the coating elicited enhanced mechanosensing from adjacent cells, facilitating cell adhesion, spreading, proliferation, and osteogenic differentiation on the surface coating. This dual‐functional PVA‐TP‐CA/Co surface coating offers a promising approach for improving clinical implantation outcomes.

"Raspberry" Hierarchical Topographic Features Regulate Human Mesenchymal Stem Cell Adhesion and Differentiation via Enhanced Mechanosensing.

An understanding of cellular mechanoresponses to well-defined synthetic topographic features is crucial for the fundamental research and biomedical applications of stem cells. Structured biointerfaces, in particular the ones with nanometer and/or micrometer surficial features, have drawn more attention in the past few decades. However, it is still difficult to integrate nanostructures and microstructures onto the synthesized biointerfaces to mimic the hierarchical architecture of the native extracellular matrix (ECM). Herein, a series of "raspberry"-like hierarchical surfaces with well-defined nanofeatures and tunable nano/microfeatures have been achieved via a catecholic polymer coating technique. Cellular responses to these hierarchical interfaces were systemically studied, indicating that the nanofeatures on the raspberry surfaces significantly enhanced the mechanosensing of human mesenchymal stem cells (hMSCs) to interfacial physical cues. Cell mechanotransduction was further investigated by analyzing focal adhesion assembling, cytoskeleton organization, cell nuclear mechanics, and transcriptional activity. The results suggest that nanosize surficial features could increase cellular mechanosensing to environment physical cues. The mechanotransduction and cell fate specification were greatly enhanced by the ECM mimicking nano/microhierarchical biointerfaces but the features should be in an optimized size.

Keloid fibroblasts have elevated and dysfunctional mechanotransduction signaling that is independent of TGF-β

BackgroundFibroblasts found in keloid tissues are known to present an altered sensitivity to microenvironmental stimuli. However, the impact of changes in extracellular matrix stiffness on phenotypes of normal fibroblasts (NFs) and keloid fibroblasts (KFs) is poorly understood. ObjectivesInvestigation the impact of matrix stiffness on NFs and KFs mainly via detecting yes-associated protein (YAP) expression. MethodsWe used fibronectin-coated polyacrylamide hydrogel substrates with a range from physiological to pathological stiffness values with or without TGF-β (fibrogenic inducer). Atomic force microscopy was used to measure the stiffness of fibroblasts. Cellular mechanoresponses were screened by immunocytochemistry, Western blot and Luminex assay. ResultsKFs are stiffer than NFs with greater expression of α-SMA. In NFs, YAP nuclear translocation was induced by increasing matrix stiffness as well as by stimulation with TGF-β. In contrast, KFs showed higher baseline levels of nuclear YAP that was not responsive to matrix stiffness or TGF-β. TGF-β1 induced p-SMAD3 in both KFs and NFs, demonstrating the pathway was functional and not hyperactivated in KFs. Moreover, blebbistatin suppressed α-SMA expression and cellular stiffness in KFs, linking the elevated YAP signaling to keloid phenotype. ConclusionsThese data suggest that whilst normal skin fibroblasts respond to matrix stiffness in vitro, keloid fibroblasts have elevated activation of mechanotransduction signaling insensitive to the microenvironment. This elevated signaling appears linked to the expression of α-SMA, suggesting a direct link to disease pathogenesis. These findings suggest changes to keloid fibroblast phenotype related to mechanotransduction contribute to disease and may be a useful therapeutic target.

Glucocorticoid counteracts cellular mechanoresponses by LINC01569-dependent glucocorticoid receptor-mediated mRNA decay.

Mechanical stimuli on cells and mechanotransduction are essential in many biological and pathological processes. Glucocorticoid is an important hormone, roles, and mechanisms of which in cellular mechanotransduction remain unknown. Here, we report that glucocorticoid counteracted cellular mechanoresponses dependently on a novel long noncoding RNA (lncRNA), LINC01569 Further, LINC01569 mediated glucocorticoid effects on mechanotransduction by destabilizing messenger RNA (mRNA) of mechanosensors including early growth response protein 1 (EGR1), Cbp/P300-interacting transactivator 2 (CITED2), and bone morphogenic protein 7 (BMP7) in glucocorticoid receptor-mediated mRNA decay (GMD) manner. Mechanistically, LINC01569 directly bound to the GMD factor Y-box-binding protein 1 (YBX1). Then, the LINC01569-YBX1 complex was guided to the mRNAs of EGR1, CITED2, and BMP7 through specific LINC01569-mRNA interaction, thereby contributing to the successful assembly of GMD complex and triggering GMD. Our results uncovered roles of glucocorticoid in cellular mechanotransduction and novel lncRNA-dependent GMD machinery and provided potential strategy for early intervention in mechanical disorder-associated diseases.

Mechanical and Physical Regulation of Fibroblast-Myofibroblast Transition: From Cellular Mechanoresponse to Tissue Pathology.

Fibroblasts are cells present throughout the human body that are primarily responsible for the production and maintenance of the extracellular matrix (ECM) within the tissues. They have the capability to modify the mechanical properties of the ECM within the tissue and transition into myofibroblasts, a cell type that is associated with the development of fibrotic tissue through an acute increase of cell density and protein deposition. This transition from fibroblast to myofibroblast—a well-known cellular hallmark of the pathological state of tissues—and the environmental stimuli that can induce this transition have received a lot of attention, for example in the contexts of asthma and cardiac fibrosis. Recent efforts in understanding how cells sense their physical environment at the micro- and nano-scales have ushered in a new appreciation that the substrates on which the cells adhere provide not only passive influence, but also active stimulus that can affect fibroblast activation. These studies suggest that mechanical interactions at the cell–substrate interface play a key role in regulating this phenotype transition by changing the mechanical and morphological properties of the cells. Here, we briefly summarize the reported chemical and physical cues regulating fibroblast phenotype. We then argue that a better understanding of how cells mechanically interact with the substrate (mechanosensing) and how this influences cell behaviors (mechanotransduction) using well-defined platforms that decouple the physical stimuli from the chemical ones can provide a powerful tool to control the balance between physiological tissue regeneration and pathological fibrotic response.

Energy expenditure during cell spreading influences the cellular response to matrix stiffness

Cells respond to the mechanical properties of the extracellular matrix (ECM) through formation of focal adhesions (FAs), re-organization of the actin cytoskeleton and adjustment of cell contractility. These are energy-demanding processes, but a potential causality between mechanical cues (matrix stiffness) and cellular (energy) metabolism remains largely unexplored. Here, we cultured human mesenchymal stem cells (hMSCs) on stiff (20 kPa) or soft (1 kPa) substrate and demonstrate that cytoskeletal reorganization and FA formation spreading on stiff substrates lead to a drop in intracellular ATP levels, correlating with activation of AMP-activated protein kinase (AMPK). The resulting increase in ATP levels further facilitates cell spreading and reinforces cell tension of the steady state, and coincides with nuclear localization of YAP/TAZ and Runx2. While on soft substrates (1 kPa), lowered ATP levels limit these cellular mechanoresponses. Furthermore, genetic ablation of AMPK lowered cellular ATP levels on stiff substrate and strongly reduced responses to substrate stiffness. Together, these findings reveal a hitherto unidentified relationship between energy expenditure and the cellular mechanoresponse, and point to AMPK as a key mediator of stem cell fate in response to ECM mechanics.

Elliptical supra-cellular topographies regulate stem cells migratory pattern and osteogenic differentiation

In living systems, the extracellular environment is structured in a hierarchal order assembling into tissues in a myriad of shapes and complex geometries. Residing within the extracellular matrix, cells are presented and influenced by geometrical cues at several scales. While there is an emerging body of evidence that substrate with symmetric supra-cellular scale geometries (e.g. cylinders and spheres) influence the cell behavior, the effect of physiologically relevant, non-symmetric geometries with varying mean curvatures remain unexplored. In this study, we systematically explore the migratory and differentiation behavior of adipose derived stem cells (ADSCs) on arrays of elliptical cylinders (up to 80 × cell size) with varying mean curvature made from hydroxyapatite. Here, we report a new substrate-driven cell response, which we term “ridge-effect” that leads to osteogenic differentiation and nuclear deformation of cells adhered on regions of highest mean curvature at the ridge of the elliptical cylinders. This phenomenon is observed in both expansion and osteogenic medium. Live imaging combined with functional analysis shows that cells travel along-side the zero mean curvature direction on elliptical cylinders and significantly promote expression of collagen I and osteocalcin compared to a flat surface, in the absence of osteogenic supplements. Altogether, this work identifies supra-cellular scale topographies, and suggest the “ridge-effect” as a physical cue for guiding cellular mechanoresponse and promoting osteogenic differentiation. This knowledge could be utilized as an important biomaterial design parameter for the development of biomedical interfaces and bone scaffolds in tissue engineering and regenerative medicine.

Correction to Surface Roughness and Substrate Stiffness Synergize To Drive Cellular Mechanoresponse.

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionNEXTORIGINAL ARTICLEThis notice is a correctionCorrection to Surface Roughness and Substrate Stiffness Synergize To Drive Cellular MechanoresponseYong HouYong HouMore by Yong Hou, Leixiao Yu*Leixiao YuMore by Leixiao Yuhttp://orcid.org/0000-0001-8804-948X, Wenyan XieWenyan XieMore by Wenyan Xie, Luis Cuellar CamachoLuis Cuellar CamachoMore by Luis Cuellar Camachohttp://orcid.org/0000-0002-9218-2678, Man ZhangMan ZhangMore by Man Zhang, Zhiqin ChuZhiqin ChuMore by Zhiqin Chu, Qiang Wei*Qiang WeiMore by Qiang Weihttp://orcid.org/0000-0001-5194-8262, and Rainer HaagRainer HaagMore by Rainer Haaghttp://orcid.org/0000-0003-3840-162XCite this: Nano Lett. 2020, 20, 5, 4059Publication Date (Web):April 9, 2020Publication History Published online9 April 2020Published inissue 13 May 2020https://doi.org/10.1021/acs.nanolett.0c01294Copyright © 2020 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views955Altmetric-Citations3LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Surface Roughness and Substrate Stiffness Synergize To Drive Cellular Mechanoresponse.

Material surface topographic features have been shown to be crucial for tissue regeneration and surface treatment of implanted devices. Many biomaterials were investigated with respect to the response of cells on surface roughness. However, some conclusions even conflicted with each other due to the unclear interplay of surface topographic features and substrate elastic features as well as the lack of mechanistic studies. Herein, wide-scale surface roughness gradient hydrogels, integrating the surface roughness from nanoscale to microscale with controllable stiffness, were developed via soft lithography with precise surface morphology. Based on this promising platform, we systematically studied the mechanosensitive response of human mesenchymal stem cells (MSCs) to a broad range of roughnesses (200 nm to 1.2 μm for Rq) and different substrate stiffnesses. We observed that MSCs responded to surface roughness in a stiffness-dependent manner by reorganizing the surface hierarchical structure. Surprisingly, the cellular mechanoresponse and osteogenesis were obviously enhanced on very soft hydrogels (3.8 kPa) with high surface roughness, which was comparable to or even better than that on smooth stiff substrates. These findings extend our understanding of the interactions between cells and biomaterials, highlighting an effective noninvasive approach to regulate stem cell fate via synergetic physical cues.