Cell Spreading Area Research Articles

Endothelial Cell Selectivity to Nanoparticles Depends on Mechanical Phenotype

AbstractEndothelial cells (ECs) elongate in the direction of blood flow, are stiffer, and are considered atheroprotective in areas of the vasculature where flow‐induced shear stress is high and unidirectional and are softer, atherogenic, and polygonal in areas experiencing oscillatory multidirectional flow. To understand the precise roles of EC mechanics and morphology in the uptake of therapeutic nanoparticles (NPs) by atherogenic endothelium, human aortic ECs are induced to adopt prescribed shapes and areas imposed by microcontact patterned adhesive islands. NP uptake per cell increases with increasing spreading area and decreases with increasing cell aspect ratio at constant cell spreading area. Biomechanical analysis shows that elongated cells exhibit higher cellular stress and stiffer membranes than cells with low aspect ratios, indicating a strong correlation between morphology, mechanical phenotype, and NP uptake. Further, ECs elongated by high laminar shear endocytosed NPs to a far lesser extent than those that are nonelongated in the chaotic, lower shear areas when cocultured in the same chamber. Results indicate that conditions leading to atherogenesis, such as low, chaotic shear‐induction of EC polygonal morphology may be used to increase the uptake of therapeutic NPs as a preventative measure against atherosclerosis.

Epithelial cells sense local stiffness via Piezo1 mediated cytoskeletal reorganization.

Local substrate stiffness is one of the major mechanical inputs for tissue organization during its development and remodeling. It is widely recognized that adherent cells use transmembrane proteins (integrins) at focal adhesions to translate ECM mechanical cues into intracellular bioprocess. Here we show that epithelial cells respond to substrate stiffening primarily via actin cytoskeleton organization, that requires activation of mechanosensitive Piezo1 channels. Piezo1 Knockdown cells eliminated the actin stress fibers that formed on stiff substrates, while it had minimal effect on cell morphology and spreading area. Inhibition of Piezo1 channels with GsMTx4 also significantly reduced stiffness-induced F-actin reorganization, suggesting Piezo1 mediated cation current plays a role. Activation of Piezo1 channels with specific agonist (Yoda1) resulted in thickening of F-actin fibers and enlargement of FAs on stiffer substrates, whereas it did not affect the formation of nascent FAs that facilitate spreading on the soft substrates. These results demonstrate that Piezo1 functions as a force sensor that couples with actin cytoskeleton to distinguish the substrate stiffness and facilitate epithelial adaptive remodeling.

How Hydrogel Stiffness Affects Adipogenic Differentiation of Mesenchymal Stem Cells under Controlled Morphology.

Matrix stiffness has been disclosed as an essential regulator of cell fate. However, it is barely studied how the matrix stiffness affects stem cell functions when cell morphology changes. Thus, in this study, the effect of hydrogel stiffness on adipogenic differentiation of human bone-marrow-derived mesenchymal stem cells (hMSCs) with controlled morphology was investigated. Micropatterns of different size and elongation were prepared by a photolithographical micropatterning technique. The hMSCs were cultured on the micropatterns and showed a different spreading area and elongation following the geometry of the underlying micropatterns. The cells with controlled morphology were embedded in agarose hydrogels of different stiffnesses. The cells showed a different level of adipogenic differentiation that was dependent on both hydrogel stiffness and cell morphology. Adipogenic differentiation became strong when the cell spreading area decreased and hydrogel stiffness increased. Adipogenic differentiation did not change with cell elongation. Therefore, cell spreading area and hydrogel stiffness could synergistically affect adipogenic differentiation of hMSCs, while cell elongation did not affect adipogenic differentiation. A change of cell morphology and hydrogel stiffness was accompanied by actin filament alignment that was strongly related to adipogenic differentiation. The results indicated that cell morphology could affect cellular sensitivity to hydrogel stiffness. The results will provide useful information for the elucidation of the interaction of stem cells and their microenvironmental biomechanical cues.

Stiff Extracellular Matrix Promotes Invasive Behaviors of Trophoblast Cells.

The effect of extracellular matrix (ECM) stiffness on embryonic trophoblast cells invasion during mammalian embryo implantation remains largely unknown. In this study, we investigated the effects of ECM stiffness on various aspects of human trophoblast cell behaviors during cell-ECM interactions. The mechanical microenvironment of the uterus was simulated by fabricating polyacrylamide (PA) hydrogels with different levels of stiffness. The human choriocarcinoma (JAR) cell lineage was used as the trophoblast model. We found that the spreading area of JAR cells, the formation of focal adhesions, and the polymerization of the F-actin cytoskeleton were all facilitated with increased ECM stiffness. Significantly, JAR cells also exhibited durotactic behavior on ECM with a gradient stiffness. Meanwhile, stiffness of the ECM affects the invasion of multicellular JAR spheroids. These results demonstrated that human trophoblast cells are mechanically sensitive, while the mechanical properties of the uterine microenvironment could play an important role in the implantation process.

Poly(octamethylene citrate) Modified with Glutathione as a Promising Material for Vascular Tissue Engineering

One of the major goals of vascular tissue engineering is to develop much-needed materials that are suitable for use in small-diameter vascular grafts. Poly(1,8-octamethylene citrate) can be considered for manufacturing small blood vessel substitutes, as recent studies have demonstrated that this material is cytocompatible with adipose tissue-derived stem cells (ASCs) and favors their adhesion and viability. The work presented here is focused on modifying this polymer with glutathione (GSH) in order to provide it with antioxidant properties, which are believed to reduce oxidative stress in blood vessels. Cross-linked poly(1,8-octamethylene citrate) (cPOC) was therefore prepared by polycondensation of citric acid and 1,8-octanediol at a 2:3 molar ratio of the reagents, followed by in-bulk modification with 0.4, 0.8, 4 or 8 wt.% of GSH and curing at 80 °C for 10 days. The chemical structure of the obtained samples was examined by FTIR-ATR spectroscopy, which confirmed the presence of GSH in the modified cPOC. The addition of GSH increased the water drop contact angle of the material surface and lowered the surface free energy values. The cytocompatibility of the modified cPOC was evaluated in direct contact with vascular smooth-muscle cells (VSMCs) and ASCs. The cell number, the cell spreading area and the cell aspect ratio were measured. The antioxidant potential of GSH-modified cPOC was measured by a free radical scavenging assay. The results of our investigation indicate the potential of cPOC modified with 0.4 and 0.8 wt.% of GSH to produce small-diameter blood vessels, as the material was found to: (i) have antioxidant properties, (ii) support VSMC and ASC viability and growth and (iii) provide an environment suitable for the initiation of cell differentiation.

Impact of adhesive area on cellular traction force and spread area.

Cells integrate endogenous and exogenous mechanical forces to sense and respond to environmental signals. In particular, cell-generated microscale traction forces regulate cellular functions and impact macroscale tissue function and development. Many groups have developed tools for measuring cellular traction forces, including microfabricated post array detectors (mPADs). mPADs are a powerful tool that provides direct traction force measurements through imaging post deflections and utilizing Bernoulli-Euler beam theory. In this technical note, we investigated how mPADs presenting two different top surface areas but similar effective stiffness influence cellular spread area and traction forces for murine embryonic fibroblasts and human mesenchymal stromal cells. When focal adhesion size was restricted via mPAD top surface area, we observed a decrease in both cell spread area and cell traction forces as the mPAD top surface area decreased, but the traction force-cell area linear relationship was maintained, which is indicative of cell contractility. We conclude that the mPAD top surface area is an important parameter to consider when utilizing mPADs to measure cellular traction forces. Furthermore, the slope of the traction force-cell area linear relationship provides a useful metric to characterize cell contractility on mPADs.

Biomimetic calcium phosphate coating on medical grade stainless steel improves surface properties and serves as a drug carrier for orthodontic applications.

Recently, stainless steel (SSL) miniscrew implants have been used in orthodontic clinics as temporary anchorage devices. Although they have excellent physical properties, their biocompatibility is relatively poor. Previously, our group developed a two-phase biomimetic calcium phosphate (BioCaP) coating that can significantly improve the biocompatibility of medical devices. This study aimed to improve the biocompatibility of SSL by coating SSL surface with the BioCaP coating. Titanium (Ti) discs and SSL discs (diameter: 5mm, thickness: 1mm) were used in this study. To form an amorphous layer, the Ti discs were immersed in a biomimetic modified Tyrode solution (BMT) for 24h. The SSL discs were immersed in the same solution for 0h, 12h, 24h, 36h and 48h. To form a crystalline layer, the discs were then immersed in a supersaturated calcium phosphate solution (CPS) for 48h. The surface properties of the BioCaP coatings were analysed. In addition, bovine serum albumin (BSA) was incorporated into the crystalline layer during biomimetic mineralisation as a model protein. The morphology, chemical composition and drug loading capacity of the BioCaP coating on smooth SSL were confirmed. This coating improved roughness and wettability of SSL surface. In vitro, with the extension of BMT coating period, the cell seeding efficiency, cell spreading area and cell proliferation on the BioCaP coating were increased. These in vitro results show that the BioCaP coating can improve surface properties of smooth medical grade SSL and serve as a carrier system for bioactive agents.

MYH7 mutations induce changes in single cell mechanobiology.

Hypertrophic Cardiomyopathy (HCM) is characterized by thickening of the left ventricular wall and hypercontractility and has been linked to mutations in the sarcomere motor protein β-myosin (MYH7). Using single cell mechanobiology studies, we examined how the effects of single point mutations propagate to change the contractile dynamics and cellular morphology (sarcomere spacing, spread area, myofibril alignment) of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). We micropattern islands of adhesive protein to constraining the spreading and alignment of hiPSC-CM on hydrogel substrates containing fluorescent microbeads as fiducial markers for traction force microscopy (TFM). We deployed substrate stiffnesses ranging from physiological (10 kPa) to heavily diseased/fibrotic (100 kPa) to test the role of increased “afterload” in functional phenotypes. We use image and video analysis to assess the contractile dynamics of the hiPSC-CM in terms of force, power, and velocities of relaxation and contraction. For example, we assessed multiple MYH7 mutations edited into the WTC line along with isogenic controls. Some lines carried an endogenously labeled alpha-actinin GFP reporter of sarcomere structure to enable visualization of sarcomere structure and dynamics. We assessed the magnitude and dynamics of contractile force output from TFM video analysis and observed increased the contractile force when compared to the control hiPSC-CMs. We also measured significantly different dynamics in the relaxation or contraction velocities compared to control hiPSC-CMs. Interestingly, not all HCM mutant lines presented a significant increase in cell spread area, a proxy for hypertrophy, and this correlated with culture conditions, such as the size of the protein pattern constraining the cells or stiffness of the substrate. Taken together, these results suggest a role for MYH7 mutations driving remodeling of structure and function at a cell-intrinsic level via changes in mechanosignaling.

Vitronectin acts as a key regulator of adhesion and migration in human umbilical cord-derived MSCs under different stress conditions

To improve mesenchymal stem cell (MSC)-based therapy efficacy, it is critical to identify factors involved in regulating migration and adhesion of MSCs under microenvironmental stress conditions. We observed that human Wharton's jelly-derived MSCs (WJ-MSCs) exhibited increase in cell spread area and adhesion, with reduction in cellular migration under serum starvation stress. The changes in adhesion and migration characteristics were accompanied by formation of large number of super mature focal adhesions along with extensive stress fibres and altered ECM gene expression with notable induction in vitronectin (VTN) expression. NF-κβ was found to be a positive regulator of VTN expression while ERK pathway regulated it negatively. Inhibition of these signalling pathways or knocking down of VTN under serum starvation established the correlation between increase in VTN expression and increased cellular adhesion with corresponding reduction in cell migration. VTN knockdown also resulted in reduction of super mature focal adhesions and extensive stress fibres, formed under serum starvation stress. Additionally, VTN induction was not detected in hypoxia-treated WJ-MSCs, and the MSCs showed no significant change in the adhesion or migration properties under hypoxia. VTN is established as a key player which possibly regulates the adhesion and migration properties of WJ-MSCs via focal adhesion signalling.

Analysis of disordered abrasive scratches on titanium surfaces and their impact on nuclear translocation of yes-associated protein

The morphology of the metallic surface of an implant is important for its contact with bone tissue as it directly affects osteoblast functions, such as cell adhesion, proliferation, and differentiation. Firm contact between the implant and cells creates a barrier that prevents inflammation and bacterial infections. Therefore, optimizing surface morphology, such as surface roughness adjustments, is essential to improving the adhesion between the implant and cells for successful tissue regeneration. However, the manner in which the cells sense the surface roughness and morphology remains unclear. Previously, we analyzed cell adhesion behavior and observed that inhibited cell spreading can delay osteoblast functions. Therefore, assuming that the surface morphology can be sensed through cell spreading, we investigated the cell spreading area and yes-associated protein (YAP) localization in mouse osteoblasts (MC3T3-E1) on a titanium surface with disordered abrasive scratches. Surface roughness of 100–150 nm was obtained by polishing, which inhibited the cell spreading, indicating that YAP localization in the nucleus was lower than that on other surfaces. The obtained results indicate that the cells sense the surface environment based on their spreading area, which regulates cellular functions via the Hippo pathway.

How sticky? How tight? How hot? Imaging probes for fluid viscosity, membrane tension and temperature measurements at the cellular level.

We review the progress made in imaging probes for three important physical parameters: viscosity, membrane tension, and temperature, all of which play important roles in many cellular processes. Recent evidences showed that cell migration speed can be modulated by extracellular fluid viscosity; membrane tension contributes to the regulation of cell motility, exo-/endo-cytosis, and cell spread area; and temperature affects neural activity and adipocyte differentiation. We discuss the techniques implementing imaging-based probes to measure viscosity, membrane tension, and temperature at subcellular resolution dynamically. The merits and shortcomings of each technique are examined, and the future applications of the recently developed techniques are also explored.

Cellular nanomechanics derived from pattern-dependent focal adhesion and cytoskeleton to balance gene transfection of malignant osteosarcoma

Gene transfection was supposed to be the most promising technology to overcome the vast majority of diseases and it has been popularly reported in clinical applications of gene therapy. In spite of the rapid development of novel transfection materials and methods, the influence of morphology-dependent nanomechanics of malignant osteosarcoma on gene transfection is still unsettled. In this study, cell spreading and adhesion area was adjusted by the prepared micropatterns to regulate focal adhesion (FA) formation and cytoskeletal organization in osteosarcoma cells. The micropattern-dependent FA and cytoskeleton could induce different cellular nanomechanics to affect cell functions. Our results indicated that transfection efficiency was improved with enlarging FA area and cell nanomechanics in micropatterned osteosarcoma. The difference of gene transfection in micropatterned cells was vigorously supported by cellular internalization capacity, Ki67 proliferation ability and YAP mechanotranduction through the regulation of focal adhesion and cytoskeletal mechanics. This study is an attempt to disclose the relationship of cell nanomechanics and gene transfection for efficient gene delivery and develop multifunctional nanomedicine biomaterials for accurate gene therapy in osteosarcoma cells.

Force- and cell state-dependent recruitment of Piezo1 drives focal adhesion dynamics and calcium entry.

Mechanosensing is an integral part of many physiological processes including stem cell differentiation, fibrosis, and cancer progression. Two major mechanosensing systems-focal adhesions and mechanosensitive ion channels-can convert mechanical features of the microenvironment into biochemical signals. We report here unexpectedly that the mechanosensitive calcium-permeable channel Piezo1, previously perceived to be diffusive on plasma membranes, binds to matrix adhesions in a force-dependent manner, promoting cell spreading, adhesion dynamics, and calcium entry in normal but not in most cancer cells tested except some glioblastoma lines. A linker domain in Piezo1 is needed for binding to adhesions, and overexpression of the domain blocks Piezo1 binding to adhesions, decreasing adhesion size and cell spread area. Thus, we suggest that Piezo1 is a previously unidentified component of focal adhesions in nontransformed cells that catalyzes adhesion maturation and growth through force-dependent calcium signaling, but this function is absent in most cancer cells.

Influence of precursor deficiency sites for borate incorporation on the structural and biological properties of boronated hydroxyapatite

The biological properties of hydroxyapatite (HA) are significantly influenced by its compositional characteristics especially doping elements and/or Ca/P ratio, which can be altered by precursor chemistry. In this study, a group of boronated (B-incorporated) hydroxyapatite (BHA) was synthesized using a precipitation method by setting the Ca/P ratio to the stoichiometric value of HA (1.67), while altering the precursor chemistry by adjusting either (Ca + B)/P (Ca-deficient precursor, BC) or Ca/(P + B) (P-deficient precursor, BP). After heat-treatment, the partial decomposition of the BC was observed, forming tricalcium phosphate as the byproduct, however, the BP showed phase stability at all temperatures. The B-ionic species in the form of (BO2)− and (BO3)3− were incorporated into the HA structure at the (PO4)3− and (OH)− positions, respectively. The incorporation of the B species also facilitated the incorporation of (CO3)2− groups specifically in the BPs. This is the first finding on BHA reporting that preferential (CO3)2− incorporation depends on the precursor chemistry used. As a result, osteoblast adhesion was superior on the BPs compared to pure HA owing to the carbonated structure, increasing cell spreading area. As such, this in vitro study highlighted that the present P-deficient precursor approach for synthesizing BHA improved biocompatibility properties and should, thus, be further considered for the next-generation of improved orthopedic applications.

Optimization of methacrylated gelatin /layered double hydroxides nanocomposite cell-laden hydrogel bioinks with high printability for 3D extrusion bioprinting.

Layered double hydroxides (LDHs) offer unique source of inspiration for design of bone mimetic biomaterials due to their superior mechanical properties, drug delivery capability and regulation cellular behaviors, particularly by divalent metal cations in their structure. Three-dimensional (3D) bioprinting of LDHs holds great promise as a novel strategy thanks to highly tunable physiochemical properties and shear-thinning ability of LDHs, which allow shape fidelity after deposition. Herein, we introduce a straightforward strategy for extrusion bioprinting of cell laden nanocomposite hydrogel bioink of gelatin methacryloyl (GelMA) biopolymer and LDHs nanoparticles. First, we synthesized LDHs by co-precipitation process and systematically examined the effect of LDHs addition on printing parameters such as printing pressure, extrusion rate, printing speed, and finally bioink printability in creating grid-like constructs. The developed hydrogel bioinks provided precise control over extrudability, extrusion uniformity, and structural integrity after deposition. Based on the printability and rheological analysis, the printability could be altered by controlling the concentration of LDHs, and printability was found to be ideal with the addition of 3 wt % LDHs. The addition of LDHs resulted in remarkably enhanced compressive strength from 652 kPa (G-LDH0) to 1168 kPa (G-LDH3). It was shown that the printed nanocomposite hydrogel scaffolds were able to support encapsulated osteoblast survival, spreading, and proliferation in the absence of any osteoinductive factors taking advantage of LDHs. In addition, cells encapsulated in G-LDH3 had a larger cell spreading area and higher cell aspect ratio than those encapsulated in G-LDH0. Altogether, the results demonstrated that the developed GelMA/LDHs nanocomposite hydrogel bioink revealed a high potential for extrusion bioprinting with high structural fidelity to fabricate implantable 3D hydrogel constructs for repair of bone defects.

Syndecan-4 and stromal cell-derived factor-1 alpha functionalized endovascular scaffold facilitates adhesion, spreading and differentiation of endothelial colony forming cells and functions under flow and shear stress conditions.

Acellular vascular scaffolds with capture molecules have shown great promise in recruiting circulating endothelial colony forming cells (ECFCs) to promote in vivo endothelialization. A microenvironment conducive to cell spreading and differentiation following initial cell capture are key to the eventual formation of a functional endothelium. In this study, syndecan-4 and stromal cell-derived factor-1 alpha were used to functionalize an elastomeric biomaterial composed of poly(glycerol sebacate), Silk Fibroin and Type I Collagen, termed PFC, to enhance ECFC-material interaction. Functionalized PFC (fPFC) showed significantly greater ECFCs capture capability under physiological flow. Individual cell spreading area on fPFC (1474 ± 63 μm2 ) was significantly greater than on PFC (1187 ± 54 μm2 ) as early as 2 h, indicating enhanced cell-material interaction. Moreover, fPFC significantly upregulated the expression of endothelial cell specific markers such as platelet endothelial cell adhesion molecule (24-fold) and Von Willebrand Factor (11-fold) compared with tissue culture plastic after 7 days, demonstrating differentiation of ECFCs into endothelial cells. fPFC fabricated as small diameter conduits and tested using a pulsatile blood flow bioreactor were stable and maintained function. The findings suggest that the new surface functionalization strategy proposed here results in an endovascular material with enhanced endothelialization.

The effect of pore size on cell behavior in mesoporous bioglass scaffolds for bone regeneration

The material properties of bone tissue repair scaffolds dictate the interaction between the material and tissues or cells in vivo. The interface between them at the nanoscale can regulate cell adhesion, migration, and differentiation behaviors, thereby reshaping osteogenic properties of the material in vivo. In this study, a series of 3 nm, 8 nm and 20 nm mesoporous bioactive glass scaffolds (MSX-3, MSX-8 and MSX-20) were prepared by employing sol-gel technique and active self-assembly, with mesoporous bioglass serving as matrix. We then further examined effects of different mesopore pore sizes on the material biology of scaffolds and effects of osteogenic properties both in vitro and in vivo. The prepared multi-level micro-nano-structured bone repair scaffolds all had ∼300 μm macropores and possessed good biocompatibility and in vitro mineralizing activity. In vitro mineralization experiments proved that a large amount of calcium and phosphate salts were deposited on the surfaces of MSX-3 and MSX-8 scaffolds, while MSX-20 had conspicuous adsorption/deposition effects on organic compounds such as proteins in solution. MSX-20 multi-level micro-nano-structured bone repair scaffold could significantly promote the cell adhesion of BMSCs, and the cell spreading area was significantly larger than that of MSX-3 and MSX-8 scaffolds. MSX-20 multi-level micro-nano structured bone repair scaffold had a good sustained release effect on BMP-2, which could promote the osteogenic differentiation effect of BMP-2 on BMSCs. In vivo animal experiments also showed that MSX-20 loaded with BMP-2 could significantly promote in vivo osteogenesis. These findings suggest that when the mesopore pore size is 20 nm, the scaffold can be more conductive to bone regeneration due to the enhanced adhesion of stem cells and more sustained release of BMP-2.

Substrate Stiffness Controls the Cell Cycle of Human Mesenchymal Stem Cells Via Cellular Traction

The microenvironment of human mesenchymal stem cells (hMSCs) regulates their self-renewal and differentiation properties. Previously, it was shown that hMSCs remain quiescent on soft (0.25 kPa) polyacrylamide (PA) gels, but re-enter the cell cycle on a stiff (7.5 kPa) gel. However, how cells behave on intermediate stiffness and what intracellular factors transmit mechanical changes to cell interior, thereby regulating cell cycle, remained unknown. In this work, we have demonstrated that PA gels of stiffness between 1 kPa and 5 kPa act as a mechanical switch from quiescence to proliferation in the cell cycle of hMSCs. Further studies with ROCK inhibitor Y-27632 have revealed that contractile proteins, but not cell spread area, account for the sensitivity of hMSCs towards substrate stiffness, and hence correlates with their changes in cell cycle. These observations therefore suggest that substrate stiffness regulates hMSC proliferation through contractile forces, as generated by cellular contractile proteins in a unique pattern that is distinct from the two other cell types studied here, C2C12 and 3T3.

Morphological Dependence of Breast Cancer Cell Responses to Doxorubicin on Micropatterned Surfaces.

Cell morphology has been widely investigated for its influence on the functions of normal cells. However, the influence of cell morphology on cancer cell resistance to anti-cancer drugs remains unclear. In this study, micropatterned surfaces were prepared and used to control the spreading area and elongation of human breast cancer cell line. The influences of cell adhesion area and elongation on resistance to doxorubicin were investigated. The percentage of apoptotic breast cancer cells decreased with cell spreading area, while did not change with cell elongation. Large breast cancer cells had higher resistance to doxorubicin, better assembled actin filaments, higher DNA synthesis activity and higher expression of P-glycoprotein than small breast cancer cells. The results suggested that the morphology of breast cancer cells could affect their resistance to doxorubicin. The influence was correlated with cytoskeletal organization, DNA synthesis activity and P-glycoprotein expression.

Covariation of Pluripotency Markers and Biomechanical Properties in Mouse Embryonic Stem Cells.

Pluripotent cells are subject to much interest as a source of differentiated cellular material for research models, regenerative medical therapies and novel applications such as lab-cultured meat. Greater understanding of the pluripotent state and control over its differentiation is therefore desirable. The role of biomechanical properties in directing cell fate and cell behavior has been increasingly well described in recent years. However, many of the mechanisms which control cell morphology and mechanical properties in somatic cells are absent from pluripotent cells. We leveraged naturally occurring variation in biomechanical properties and expression of pluripotency genes in murine ESCs to investigate the relationship between these parameters. We observed considerable variation in a Rex1-GFP expression reporter line and found that this variation showed no apparent correlation to cell spreading morphology as determined by circularity, Feret ratio, phase contrast brightness or cell spread area, either on a parameter-by-parameter basis, or when evaluated using a combined metric derived by principal component analysis from the four individual criteria. We further confirmed that cell volume does not co-vary with Rex1-GFP expression. Interestingly, we did find that a subpopulation of cells that were readily detached by gentle agitation collectively exhibited higher expression of Nanog, and reduced LmnA expression, suggesting that elevated pluripotency gene expression may correlate with reduced adhesion to the substrate. Furthermore, atomic force microscopy and quantitative fluorescent imaging revealed a connection between cell stiffness and Rex1-GFP reporter expression. Cells expressing high levels of Rex1-GFP are consistently of a relatively low stiffness, while cells with low levels of Rex1-GFP tend toward higher stiffness values. These observations indicate some interaction between pluripotency gene expression and biomechanical properties, but also support a strong role for other interactions between the cell culture regime and cellular biomechanical properties, occurring independently of the core transcriptional network that supports pluripotency.