Symmetry Breaking of Human Pluripotent Stem Cells (hPSCs) in Micropattern Generates a Polarized Spinal Cord-Like Organoid (pSCO) with Dorsoventral Organization.

Axis formation and related spatial patterning are initiated by symmetry breaking during development. A geometrically confined culture of human pluripotent stem cells (hPSCs) mimics symmetry breaking and cell patterning. Using this, polarized spinal cord organoids (pSCOs) with a self-organized dorsoventral (DV) organization are generated. The application of caudalization signals promoted regionalized cell differentiation along the radial axis and protrusion morphogenesis in confined hPSC colonies. These detached colonies grew into extended spinal cord-like organoids, which established self-ordered DV patterning along the long axis through the spontaneous expression of polarized DV patterning morphogens. The proportions of dorsal/ventral domains in the pSCOs can be controlled by the changes in the initial size of micropatterns, which altered the ratio of center-edge cells in 2D. In mature pSCOs, highly synchronized neural activity is separately detected in the dorsal and ventral side, indicating functional as well as structural patterning established in the organoids. This study provides a simple and precisely controllable method to generate spatially ordered organoids for the understanding of the biological principles of cell patterning and axis formation during neural development.

Cell position within human pluripotent stem cell colonies determines apical specialization via an actin cytoskeleton-based mechanism.

Human pluripotent stem cells (hPSCs) grow as colonies with epithelial-like features including cell polarity and position-dependent features that contribute to symmetry breaking during development. Our study provides evidence that hPSC colonies exhibit position-dependent differences in apical structures and functions. With this apical difference, edge cells were preferentially labeled with amphipathic dyes, which enabled separation of edge and center cells by fluorescence-activated cell sorting. Transcriptome comparison between center and edge cells showed differential expression of genes related to apicobasal polarization, cell migration, and endocytosis. Accordingly, different kinematics and mechanical dynamics were found between center and edge cells, and perturbed actin dynamics disrupted the position-dependent apical polarity. In addition, our dye-labeling approach could be utilized to sort out a certain cell population in differentiated micropatterned colonies. In summary, hPSC colonies have position-dependent differences in apical structures and properties, and actin dynamics appear to play an important role in the establishment of this position-dependent cell polarity.

Yes-Associated Protein Is Required for ZO-1-Mediated Tight-Junction Integrity and Cell Migration in E-Cadherin-Restored AGS Gastric Cancer Cells.

Yes-associated protein (YAP) regulates numerous cellular homeostasis processes and malignant transformation. We found that YAP influences ZO-1-mediated cell migration using E-cadherin-restored EC96 cells derived from gastric malignant AGS cells. Ectopic expression of E-cadherin enhanced straightforward migration of cells, in comparison to the meandering movement of parental AGS cells. In EC96 cells, YAP and ZO-1 expression increased but nuclear YAP levels and activity were reduced. Nuclear factor-κB (NF-κB) mediated the increase in ZO-1 expression, possibly stabilizing cytoplasmic YAP post-translationally. Downregulation of YAP expression using siYAP RNA or stable knock-down inhibited straightforward cell migration by fragmenting ZO-1 containing tight junctions (TJs) but not adherens junctions, implying involvement of YAP in ZO-1-mediated cell migration. The association of YAP with ZO-1 was mediated by angiomotin (AMOT) because downregulation of AMOT dissociated YAP from ZO-1 and reduced cell migration. E-cadherin restoration in malignant cancer cells induced NF-κB signaling to enhance ZO-1 expression and subsequently stabilize YAP. At high expression levels, YAP associates with ZO-1 via AMOT at TJs, influencing ZO-1-mediated cell migration and maintaining TJ integrity.

Expression of E-Cadherin in Epithelial Cancer Cells Increases Cell Motility and Directionality through the Localization of ZO-1 during Collective Cell Migration.

Collective cell migration of epithelial tumor cells is one of the important factors for elucidating cancer metastasis and developing novel drugs for cancer treatment. Especially, new roles of E-cadherin in cancer migration and metastasis, beyond the epithelial-mesenchymal transition, have recently been unveiled. Here, we quantitatively examined cell motility using micropatterned free edge migration model with E-cadherin re-expressing EC96 cells derived from adenocarcinoma gastric (AGS) cell line. EC96 cells showed increased migration features such as the expansion of cell islands and straightforward movement compared to AGS cells. The function of tight junction proteins known to E-cadherin expression were evaluated for cell migration by knockdown using sh-RNA. Cell migration and straight movement of EC96 cells were reduced by knockdown of ZO-1 and claudin-7, to a lesser degree. Analysis of the migratory activity of boundary cells and inner cells shows that EC96 cell migration was primarily conducted by boundary cells, similar to leader cells in collective migration. Immunofluorescence analysis showed that tight junctions (TJs) of EC96 cells might play important roles in intracellular communication among boundary cells. ZO-1 is localized to the base of protruding lamellipodia and cell contact sites at the rear of cells, indicating that ZO-1 might be important for the interaction between traction and tensile forces. Overall, dynamic regulation of E-cadherin expression and localization by interaction with ZO-1 protein is one of the targets for elucidating the mechanism of collective migration of cancer metastasis.

Study on the Expansion Dynamics of MDCK Epithelium by Interstitial Flow Using a Traction Force-Measurable Microfluidic Chip.

The movement of collective cells is affected through changes in physical interactions of cells in response to external mechanical stimuli, including fluid flow. Most tissues are affected by fluid flow at the interstitial level, but few studies have investigated the physical effects in collective cells affected by a low flow rate. In this study, collective cell migration of Madin-Darby canine kidney (MDCK) epithelial cells was investigated under static or interstitial flow (0, 0.1, and 1 µL/min) using a traction microfluidic device. The optimization of calculation of cellular traction forces was first achieved by changing interrogation window size from the fluorescent bead images. Migration analysis of cell collectives patterned with a 700 µm circular shape reveals that cells under the slow flow (0.1 and 1 µL/min) showed the inhibitory migration by decreasing cell island size and cellular speed compared to that of static condition. Analysis of cellular forces shows that level of traction forces was lower in the slow flow condition (~20 Pa) compared to that of static condition (~50 Pa). Interestingly, the standard deviation of traction force of cells was dramatically decreased as the flow rate increased from 0 to 1 µL/min, which indicates that flow affects the distribution of cellular traction forces among cell collectives. Cellular tension was increased by 50% in the cells under the fluid flow rate of 1 µL/min. Treatment of calcium blocker increased the migratory speed of cells under the flow condition, whereas there is little change of cellular forces. In conclusion, it has been shown that the interstitial flow inhibited the collective movement of epithelial cells by decreasing and re-distributing cellular forces. These findings provide insights into the study of the effect of interstitial flow on cellular behavior, such as development, regeneration, and morphogenesis.

Development of Magnetic Torque Stimulation (MTS) Utilizing Rotating Uniform Magnetic Field for Mechanical Activation of Cardiac Cells.

Regulation of cell signaling through physical stimulation is an emerging topic in biomedicine. BACKGROUND: While recent advances in biophysical technologies show capabilities for spatiotemporal stimulation, interfacing those tools with biological systems for intact signal transfer and noncontact stimulation remains challenging. Here, we describe the use of a magnetic torque stimulation (MTS) system combined with engineered magnetic particles to apply forces on the surface of individual cells. MTS utilizes an externally rotating magnetic field to induce a spin on magnetic particles and generate torsional force to stimulate mechanotransduction pathways in two types of human heart cells-cardiomyocytes and cardiac fibroblasts. METHODS: The MTS system operates in a noncontact mode with two magnets separated (60 mm) from each other and generates a torque of up to 15 pN µm across the entire area of a 35-mm cell culture dish. The MTS system can mechanically stimulate both types of human heart cells, inducing maturation and hypertrophy. RESULTS: Our findings show that application of the MTS system under hypoxic conditions induces not only nuclear localization of mechanoresponsive YAP proteins in human heart cells but also overexpression of hypertrophy markers, including ß-myosin heavy chain (ßMHC), cardiotrophin-1 (CT-1), microRNA-21 (miR-21), and transforming growth factor beta-1 (TGFß-1). CONCLUSIONS: These results have important implications for the applicability of the MTS system to diverse in vitro studies that require remote and noninvasive mechanical regulation.

Development of a 3-D Physical Dynamics Monitoring System Using OCM with DVC for Quantification of Sprouting Endothelial Cells Interacting with a Collagen Matrix.

The extracellular matrix (ECM) plays a key role during cell migration, proliferation, and differentiation by providing adhesion sites and serving as a physical scaffold. Elucidating the interaction between the cell and ECM can reveal the underlying mechanisms of cellular behavior that are currently unclear. Analysis of the deformation of the ECM due to cell-matrix interactions requires microscopic, three-dimensional (3-D) imaging methods, such as confocal microscopy and second-harmonic generation microscopy, which are currently limited by phototoxicity and bleaching as a result of the point-scanning approach. In this study, we suggest the use of optical coherence microscopy (OCM) as a live-cell, volumetric, fast imaging tool for analyzing the deformation of fibrous ECM. We optimized such OCM parameters as the sampling rate to obtain images of the best quality that meet the requirements for robust digital volume correlation (DVC) analysis. Visualization and analysis of the mechanical interaction between collagen ECM and human umbilical vein endothelial cells (HUVECs) show that cellular adhesion during protrusion can be analyzed and quantified. The advantages of OCM, such as fine isotropic spatial resolution, fast time resolution, and low phototoxicity, make it the ideal optic tool for 3-D traction force microscopy.

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Traction microscopy with integrated microfluidics: responses of the multi-cellular island to gradients of HGF.

Collective cellular migration plays a central role in development, regeneration, and metastasis. In these processes, mechanical interactions between cells are fundamental but measurement of these interactions is often hampered by technical limitations. To overcome some of these limitations, here we describe a system that integrates microfluidics with traction microscopy (TM). Using this system we can measure simultaneously, and in real time, migration speeds, tractions, and intercellular tension throughout an island of confluent Madin-Darby canine kidney (MDCK) cells. The cell island is exposed to hepatocyte growth factor (HGF) at a controlled gradient of concentrations; HGF is known to elicit epithelial-to-mesenchymal transition (EMT) and cell scattering. As expected, the rate of expansion of the cell island was dependent on the concentration of HGF. Higher concentrations of HGF reduced intercellular tensions, as expected during EMT. A novel finding, however, is that the effects of HGF concentration and its gradient were seen within an island. This integrated experimental system thus provides an integrated tool to better understand cellular forces during collective cellular migration under chemical gradients.

Quantification of focal adhesion dynamics of cell movement based on cell-induced collagen matrix deformation using second-harmonic generation microscopy.

Mechanical interactions of living cells with the surrounding environment via focal adhesion (FA) in three dimensions (3-D) play a key role in dynamic biological events, such as tissue regeneration, wound healing, and cancer invasion. Recently, several methods for observing 3-D cell-extracellular matrix (ECM) interactions have been reported, lacking solid and quantitative analysis on the dynamics of the physical interaction between the cell and the ECM. We measured the submicron displacements of ECM deformation in 3-D due to protrusion-retraction dynamics during cell migration, using second-harmonic generation without labeling the matrix structures. We then quantitatively analyzed the mechanical deformation between the ECM and the cells based on spatiotemporal volumetric correlations. The greatest deformations within the collagen matrix were found to occur at sites of colocalization of the FA site-related proteins vinculin and actin, which confirms that FA sites play a critical role in living cells within the ECM as a point for adhesion, traction, and migration. We believe that this modality can be used in studies of cell-ECM interaction during angiogenesis, wound healing, and metastasis.

Directional migration of mesenchymal stem cells under an SDF-1α gradient on a microfluidic device.

Homing of peripheral stem cells is regulated by one of the most representative homing factors, stromal cell-derived factor 1 alpha (SDF-1α), which specifically binds to the plasma membrane receptor CXCR4 of mesenchymal stem cells (MSCs) in order to initiate the signaling pathways that lead to directional migration and homing of stem cells. This complex homing process and directional migration of stem cells have been mimicked on a microfluidic device that is capable of generating a chemokine gradient within the collagen matrix and embedding endothelial cell (EC) monolayers to mimic blood vessels. On the microfluidic device, stem cells showed directional migration toward the higher concentration of SDF-1α, whereas treatment with the CXCR4 antagonist AMD3100 caused loss of directionality of stem cells. Furthermore, inhibition of stem cell's main migratory signaling pathways, Rho-ROCK and Rac pathways, caused blockage of actomyosin and lamellipodia formation, decreasing the migration distance but maintaining directionality. Stem cell homing regulated by SDF-1α caused directional migration of stem cells, while the migratory ability was affected by the activation of migration-related signaling pathways.

Homogenizing cellular tension by hepatocyte growth factor in expanding epithelial monolayer.

Hepatocyte growth factor (HGF) induces cell migration and scattering by mechanisms that are thought to tip a local balance of competing physical forces; cell-to-cell and cell-to-substrate forces. In this local process, HGF is known to attenuate local cadherin-dependent adhesion forces for cell-cell junction development and enhance local integrin-dependent contractile forces for pulling neighboring cells apart. Here we use an expanding island of confluent Madin-Darby canine kidney (MDCK) cells as a model system to quantify the collective cell migration. In the absence of HGF, cell trajectories are highly tortuous whereas in the presence of HGF, they become far less so, resembling free expansion of a gas. At the level of cell-to-cell junctions, HGF attenuates the linkage of stress fibers to cell-to-cell junctions with concomitant decrease in intercellular stress. At the level of cell-to-substrate junctions, HGF augments the linkage of stress fibers to cell-to-substrate junctions with no apparent effect on traction. Together, HGF induces both structural changes in the actin-bound junctional protein complex and physical forces spanning multicellular clusters, which further promotes the expansion of confluent cellular layer.

Cellular Contraction and Polarization Drive Collective Cellular Motion.

Coordinated motions of close-packed multicellular systems typically generate cooperative packs, swirls, and clusters. These cooperative motions are driven by active cellular forces, but the physical nature of these forces and how they generate collective cellular motion remain poorly understood. Here, we study forces and motions in a confined epithelial monolayer and make two experimental observations: 1) the direction of local cellular motion deviates systematically from the direction of the local traction exerted by each cell upon its substrate; and 2) oscillating waves of cellular motion arise spontaneously. Based on these observations, we propose a theory that connects forces and motions using two internal state variables, one of which generates an effective cellular polarization, and the other, through contractile forces, an effective cellular inertia. In agreement with theoretical predictions, drugs that inhibit contractility reduce both the cellular effective elastic modulus and the frequency of oscillations. Together, theory and experiment provide evidence suggesting that collective cellular motion is driven by at least two internal variables that serve to sustain waves and to polarize local cellular traction in a direction that deviates systematically from local cellular velocity.

Combination of three angiogenic growth factors has synergistic effects on sprouting of endothelial cell/mesenchymal stem cell-based spheroids in a 3D matrix.

Combinations of angiogenic growth factors have been shown to have synergistic effects on angiogenesis and natural wound healing in various animal models. Each growth factor has unique roles during angiogenesis; vascular endothelial growth factor (VEGF) plays a key role during the initial step of angiogenesis, whereas PDGF functions in the maturation of blood vessels. We used a combination of three angiogenic growth factors to increase angiogenesis in vitro and in vivo. We chose VEGF as a basic factor and added platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) to induce angiogenesis in three in vitro and in vivo models: 3D angiogenesis assay, 3D co-culture, and matrigel plug implantation assay. Cell proliferation was significantly higher in co-cultured cells treated with PDGF + VEGF + FGF than in the control, single, or dual combination groups. mRNA expression of α-smooth muscle actin (α-SMA), von Willebrand factor (vWF), and CD105 was higher in the triple group (PDGF + VEGF + FGF) than in control, single, or dual combination groups. In the PDGF + VEGF + FGF group, the length and number of branches of spheroids was also significantly higher than in the control, single, or dual combination groups. Furthermore, in a nude mouse model, α-SMA expression was significantly higher in the PDGF + VEGF + FGF group than in other groups. In conclusion, the addition of PDGF and FGF to VEGF showed synergistic effects on angiogenesis in vitro and in vivo. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1535-1543, 2016.

Regeneration of chronic myocardial infarction by injectable hydrogels containing stem cell homing factor SDF-1 and angiogenic peptide Ac-SDKP.

Regeneration of chronic myocardial infarction (CMI) is one of the challenging issues due to its limited regeneration activity compared to acute or sub-acute stage. In this study, we examined whether combination of stem cell homing factor (SDF-1) and angiogenic peptides (Ac-SDKP) injected with biomimetic hydrogels promote regeneration of cardiac function in a CMI model. We evaluated the regeneration of chronically infarcted myocardium using injectable biomimetic hydrogels containing two therapeutic factors; stromal-derived factor-1 (SDF-1) and Ac-SDKP for stem cell homing and angiogenesis, respectively. Injection of the two therapeutic factors into the infarct region of the left ventricle showed that the biomimetic hydrogels containing two therapeutic factor exhibited significantly improved left ventricle function, increased angiogenesis, decreased infarct size and greatest wall thickness within the infarct region at 4 weeks post-treatment. From these results, it is clear that hydrogels containing two therapeutic factors showed synergistic effects on regeneration in the chronic heart failure model. In conclusion, these results suggest that combination of stem cell homing factor with angiogenic peptides recruit stem cells to the microenvironments, increase the expression of angiogenic genes, enhance the matured vessel formation and improve the cardiac function in chronic MI.

Cellular behavior in micropatterned hydrogels by bioprinting system depended on the cell types and cellular interaction.

The fabrication of patterned microstructures within three-dimensional (3D) matrices is a challenging subject in tissue engineering and regenerative medicine. A 3D, free-moving bioprinting system was developed and hydrogels were patterned by varying the process parameters of z-axis moving velocity and ejection velocity. The patterning of hydrogel based microfibers in a 3D matrigel was achieved with dimensions of 4.5 mm length and widths from 79 to 200 µm. Hyaluronan-based hydrogels mixed with fibroblasts (L929), mouse endothelial cells (MS1), or human mesenchymal stem cells (hMSCs) were patterned using a 3D moving axis bioprinter and cell behavior was monitored in culture for up to 16 days. L929 and MS1 cells and hMSCs in patterned hydrogel revealed cell-cell interactions and a morphological dependency on cell types. HMSCs formed spheres through cell aggregation, while L929 cells increased in cellular mass without cell aggregation and MS1 dispersed into the matrix instead of aggregating. The aggregation of hMSCs was attenuated by treatment with Rho kinase (ROCK) inhibitor and cadherin antibody. This reflected the close relationship between cell aggregation and migration with RhoA and cell-cell adhesion molecules. Angiogenic-specific gene expression profiles showed that expression of CD105 decreased to 22% in the ROCK inhibitor group compared to control group. These results showed that cell-based patterns in a 3D matrix are highly dependent on both cell aggregation and migration over time.