Search

Interactions between living cells and nanoparticles have been extensively studied to enhance the delivery of therapeutics. Nanoparticles size, shape, stiffness and surface charge have been regarded as the main features able to control the fate of cell-nanoparticle interactions. However, the clinical translation of nanotherapies has so far been limited, and there is a need to better understand the biology of cell-nanoparticle interactions. This study investigated the role of cellular mechanosensitive components in cell-nanoparticle interactions. We demonstrate that the genetic and pharmacologic inhibition of yes-associated protein (YAP), a key component of cancer cell mechanosensing apparatus and Hippo pathway effector, improves nanoparticle internalization in triple-negative breast cancer cells regardless of nanoparticle properties or substrate characteristics. This process occurs through YAP-dependent regulation of endocytic pathways, cell mechanics, and membrane organization. Hence, we propose targeting YAP may sensitize triple negative breast cancer cells to chemotherapy and increase the selectivity of nanotherapy.

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level. To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young's (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice. A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

Acid-growth theory has been postulated in the 70s to explain the rapid elongation of cells in response to plant hormone auxin. More recently, it has been demonstrated that activation of the proton ATPs pump (H+-ATPs) promoting acidification of the apoplast is the principal mechanism through which hormones like auxin as well as brassinosteroids (BR) induce cell elongation. However, the impact of this acidification on the mechanical properties of the cell wall remained largely unexplored. Here, we use Atomic Force Microscopy (AFM) to demonstrate that acidification of apoplast is necessary and sufficient to induce cell elongation through cell wall relaxation. Moreover, we demonstrate that Pectin Methyl Esterase (PME) can induce both cell wall softening or stiffening in extracellular calcium dependent-manner and that tight control of PME activity is required for hypocotyl elongation.

Dairy cow feed contains, among other ingredients, soybeans, legumes, and clover, plants that are rich in phytoestrogens. Several publications have reported a positive influence of phytoestrogens on human health; however, several unfavorable effects have also been reported. In this work, a simple, selective, and eco-friendly method of phytoestrogen isolation based on the technique of noncovalent molecular imprinting was developed. Genistein was used as a template, and dopamine was chosen as a functional monomer. A layer of molecularly imprinted polymers was created in a microtitration well plate. The binding capability and selective properties of obtained molecularly imprinted polymers were investigated. The imprinted polymers exhibited higher binding affinity toward chosen phytoestrogen than did the nonimprinted polymers. A selectivity factor of 6.94 was calculated, confirming satisfactory selectivity of the polymeric layer. The applicability of the proposed sensing method was tested by isolation of genistein from a real sample of bovine milk and combined with micellar electrokinetic capillary chromatography with UV-visible detection.

In the present work, we introduce a new cell-based biosensor for detecting arrhythmias based on a novel utilization of the combination of the Atomic Force Microscope (AFM) lateral force measurement as a nanosensor with a dual 3D cardiomyocyte syncytium. Two spontaneously coupled clusters of cardiomyocytes form this. The syncytium's functional contraction behavior was assessed using video sequences analyzed with Musclemotion ImageJ/Fiji software, and immunocytochemistry evaluated phenotype composition. The application of caffeine solution induced arrhythmia as a model drug, and its spontaneous resolution was monitored by AFM lateral force recording and interpretation and calcium fluorescence imaging as a reference method describing non-synchronized contractions of cardiomyocytes. The phenotypic analysis revealed the syncytium as a functional contractile and conduction cardiac behavior model. Calcium fluorescence imaging was used to validate that AFM fully enabled to discriminate cardiac arrhythmias in this in vitro cellular model. The described novel 3D hESCs-based cellular biosensor is suitable to detect arrhythmic events on the level of cardiac contractile and conduction tissue cellular model. The resulting biosensor allows for screening of arrhythmogenic properties of tailored drugs enabling its use in precision medicine.

Zuzana Skubalova,1,2 Simona Rex,1,2 Martina Sukupova,1 Martin Zahalka,1 Petr Skladal,3 Jan Pribyl,3 Hana Michalkova,1,2 Akila Weerasekera,4 Vojtech Adam,1,2 Zbynek Heger1,2 1Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic; 2Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic; 3Central European Institute of Technology, Masaryk University, Brno, Czech Republic; 4Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USACorrespondence: Zbynek HegerDepartment of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, Brno CZ-613 00, Czech RepublicTel +420-5-4513-3350Fax +420-5-4521-2044Email heger@mendelu.czIntroduction: The present study reports on examination of the effects of encapsulating the tyrosine kinase inhibitors (TKIs) vandetanib and lenvatinib into a biomacromolecular ferritin-based delivery system.Methods: The encapsulation of TKIs was performed via two strategies: i) using an active reversible pH-dependent reassembly of ferritin´s quaternary structure and ii) passive loading of hydrophobic TKIs through the hydrophobic channels at the junctions of ferritin subunits. After encapsulation, ferritins were surface-functionalized with folic acid promoting active-targeting capabilities.Results: The physico-chemical and nanomechanical analyses revealed that despite the comparable encapsulation efficiencies of both protocols, the active loading affects stability and rigidity of ferritins, plausibly due to their imperfect reassembly. Biological experiments with hormone-responsive breast cancer cells (T47-D and MCF-7) confirmed the cytotoxicity of encapsulated and folate-targeted TKIs to folate-receptor positive cancer cells, but only limited cytotoxic effects to healthy breast epithelium. Importantly, the long-term cytotoxic experiments revealed that compared to the pH-dependent encapsulation, the passively-loaded TKIs exert markedly higher anticancer activity, most likely due to undesired influence of harsh acidic environment used for the pH-dependent encapsulation on the TKIs’ structural and functional properties.Conclusion: Since the passive loading does not require a reassembly step for which acids are needed, the presented investigation serves as a solid basis for future studies focused on encapsulation of small hydrophobic molecules.Keywords: drug delivery, nanomedicine, lenvatinib, vandetanib

Introduction Human induced pluripotent stem cells (hiPSC) have the ability to differentiate theoritically into any cell type. The development of organoid systems exhibiting the essential features of human organ such as liver and heart is of high interest. Optimizing the culture conditions to obtain the highest cardiac organoids efficacy is crucial. In fact, cardiac differentiation protocols have been established by essentially focusing on specific growth factors on hiPSC differentiation efficiency. However, the optimal environmental factors such as the optimal oxygen exposure to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Yet, the effect of low oxygen exposure on the molecular and functional maturity of the hiPSC-derived cardiomyocytes remains unexplored. Aims We aimed here at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hiPSCs in 2D monolayer and 3D organoids protocols. Methods hiPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed using qRT-PCR and immunofluorescence. The mitochondrial localization and metabolic properties were evaluated by high-resolution respirometry and mitochondrial staining. The intracellular Ca2+ handling and contractile properties were also monitored using confocal fluorescent microscopy and atomic force microscopy. Results Our results indicated that the 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac organoids containing hiPSC-CMs in LOE exhibited higher cardiac markers expression such as troponin T (TnTc), RyR2, Serca2a, alpha and beta heavy myosin chains. Moreover, we found enhanced contractile force, hypertrophy and steadier SR Ca2+ release reflected by a more regular spontaneous Ca2+ transients associated with a higher maximal amplitude and lower spontaneous Ca2+ events revealing a better SR Ca2+ handling in LOE. Similar beat rate, preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Conclusions Our results brought evidences that LOE is moderately beneficial for the 3D cardiac organoids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE. Introduction Human induced pluripotent stem cells (hiPSC) have the ability to differentiate theoritically into any cell type. The development of organoid systems exhibiting the essential features of human organ such as liver and heart is of high interest. Optimizing the culture conditions to obtain the highest cardiac organoids efficacy is crucial. In fact, cardiac differentiation protocols have been established by essentially focusing on specific growth factors on hiPSC differentiation efficiency. However, the optimal environmental factors such as the optimal oxygen exposure to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Yet, the effect of low oxygen exposure on the molecular and functional maturity of the hiPSC-derived cardiomyocytes remains unexplored. Aims We aimed here at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hiPSCs in 2D monolayer and 3D organoids protocols. Methods hiPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed using qRT-PCR and immunofluorescence. The mitochondrial localization and metabolic properties were evaluated by high-resolution respirometry and mitochondrial staining. The intracellular Ca2+ handling and contractile properties were also monitored using confocal fluorescent microscopy and atomic force microscopy. Results Our results indicated that the 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac organoids containing hiPSC-CMs in LOE exhibited higher cardiac markers expression such as troponin T (TnTc), RyR2, Serca2a, alpha and beta heavy myosin chains. Moreover, we found enhanced contractile force, hypertrophy and steadier SR Ca2+ release reflected by a more regular spontaneous Ca2+ transients associated with a higher maximal amplitude and lower spontaneous Ca2+ events revealing a better SR Ca2+ handling in LOE. Similar beat rate, preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Conclusions Our results brought evidences that LOE is moderately beneficial for the 3D cardiac organoids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.

Atomic Force Microscopy (AFM) is a rather new method with increasing potential in analyzing various biosamples. Moreover, it can serve as a multi-functional device in the studies of biological specimens under physiological conditions. However, it is becoming increasingly popular among biochemists and biologists, it is not often used in cardiology. Heart disease causes millions of deaths every year. A common point in all heart diseases is the inferior function of cardiomyocytes, which are the contracting unit of the heart. Therefore, these cells are a frequent target of scientific studies. However, few of them use innovative techniques such as AFM and related methods or parallel combinations with complementary techniques such as cell potential measurements. The aim of this review is to illustrate the potential of AFM microscopy in the study of cardiac cells, comparing it with related methods and other techniques used to study the biomechanics and electrophysiology of this cell type. A better understanding of these methods may lead to a better description of the pathophysiology of the heart disease and an improved understanding of the effect of selected drugs.

Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.

BackgroundPDZ and LIM domain protein 2 (PDLIM2) is a cytoskeletal and nuclear effector that regulates the activity of several transcription factors (e.g., NF-κB, STAT), and its deregulation has been associated with oncogenesis. Our recent study identified PDLIM2 as a protein associated with the lymph node metastasis of low grade luminal A breast cancer tissues. Here, we aim to understand this association at the molecular and cellular levels.MethodsTo investigate the link between PDLIM2 and epithelial-to-mesenchymal transition (EMT), stably transduced MCF7-PDLIM2 cells, and MCF7 or MCF10A cells with PDLIM2 protein levels modified using siRNA orPDLIM2gene carrying plasmid, were used. Additionally, MCF7 and MCF10A cells were exposed to hypoxic conditions and TGFβ1 treatment. EMT was monitored using immunoblotting of EMT markers and atomic force microscopy (AFM). The role of PDLIM2 in cell migration and/or invasion was investigated using Transwell assay and xCELLigence system.ResultsFirst, we observe a positive effect of PDLIM2 overexpression on EMT in MCF7 cells, a model of luminal A tumors, using EMT markers and AFM. On the other hand, PDLIM2 helps to maintain the epithelial phenotype in MCF10A cells, a model of normal breast epithelial cells. Second, we find that exposure of the MCF7 cells to hypoxic conditions increases levels of PDLIM2 and carbonic anhydrase-9 (CA-9), a marker of the response to hypoxia. However, none of these effects are observed in the MCF10A cells. Third, PDLIM2 overexpression promotes migration, invasion, and proliferation and decreases adhesion of the MCF7 cells, but an opposite effect is observed in the MCF10A cells.ConclusionsOur data indicate that PDLIM2 plays a dual role: (i) as an EMT-supporting and hypoxia-responding oncoprotein in luminal breast cancer cells, and (ii) as an epithelial phenotype-maintaining tumor suppressor in normal epithelial breast cells.

Spinocerebellar ataxia type-1 (SCA1) is caused by an abnormally expanded polyglutamine (polyQ) tract in ataxin-1. These expansions are responsible for protein misfolding and self-assembly into intranuclear inclusion bodies (IIBs) that are somehow linked to neuronal death. However, owing to lack of a suitable cellular model, the downstream consequences of IIB formation are yet to be resolved. Here, we describe a nuclear protein aggregation model of pathogenic human ataxin-1 and characterize IIB effects. Using an inducible Sleeping Beauty transposon system, we overexpressed the ATXN1(Q82) gene in human mesenchymal stem cells that are resistant to the early cytotoxic effects caused by the expression of the mutant protein. We characterized the structure and the protein composition of insoluble polyQ IIBs which gradually occupy the nuclei and are responsible for the generation of reactive oxygen species. In response to their formation, our transcriptome analysis reveals a cerebellum-specific perturbed protein interaction network, primarily affecting protein synthesis. We propose that insoluble polyQ IIBs cause oxidative and nucleolar stress and affect the assembly of the ribosome by capturing or down-regulating essential components. The inducible cell system can be utilized to decipher the cellular consequences of polyQ protein aggregation. Our strategy provides a broadly applicable methodology for studying polyQ diseases.

Atomic force microscopy (AFM) helps to describe and explain the mechanobiological properties of living cells on the nanoscale level under physiological conditions. The stiffness of cells is an important parameter reflecting cell physiology. Here, we have provided the first study of the stiffness of cryopreserved cells during post-thawing regeneration using AFM combined with confocal fluorescence microscopy. We demonstrated that the nonfrozen cell stiffness decreased proportionally to the cryoprotectant concentration in the medium. AFM allowed us to map cell surface reconstitution in real time after a freeze/thaw cycle and to monitor the regeneration processes at different depths of the cell and even different parts of the cell surface (nucleus and edge). Fluorescence microscopy showed that the cytoskeleton in fibroblasts, though damaged by the freeze/thaw cycle, is reconstructed after long-term plating. Confocal microscopy confirmed that structural changes affect the nuclear envelopes in cryopreserved cells. AFM nanoindentation analysis could be used as a noninvasive method to identify cells that have regenerated their surface mechanical properties with the proper dynamics and to a sufficient degree. This identification can be important particularly in the field of in vitro fertilization and in future cell-based regeneration strategies.

Duchenne muscular dystrophy (DMD) is a severe genetic disorder characterized by the lack of functional dystrophin. DMD is associated with progressive dilated cardiomyopathy, eventually leading to heart failure as the main cause of death in DMD patients. Although several molecular mechanisms leading to the DMD cardiomyocyte (DMD-CM) death were described, mostly in mouse model, no suitable human CM model was until recently available together with proper clarification of the DMD-CM phenotype and delay in cardiac symptoms manifestation. We obtained several independent dystrophin-deficient human pluripotent stem cell (hPSC) lines from DMD patients and CRISPR/Cas9-generated DMD gene mutation. We differentiated DMD-hPSC into cardiac cells (CC) creating a human DMD-CC disease model. We observed that mutation-carrying cells were less prone to differentiate into CCs. DMD-CCs demonstrated an enhanced cell death rate in time. Furthermore, ion channel expression was altered in terms of potassium (Kir2.1 overexpression) and calcium handling (dihydropyridine receptor overexpression). DMD-CCs exhibited increased time of calcium transient rising compared to aged-matched control, suggesting mishandling of calcium release. We observed mechanical impairment (hypocontractility), bradycardia, increased heart rate variability, and blunted β-adrenergic response connected with remodeling of β-adrenergic receptors expression in DMD-CCs. Overall, these results indicated that our DMD-CC models are functionally affected by dystrophin-deficiency associated and recapitulate functional defects and cardiac wasting observed in the disease. It offers an accurate tool to study human cardiomyopathy progression and test therapies in vitro.

We focused on the biomechanical and morphological characteristics of prostate cancer cells and their changes resulting from the effect of docetaxel, cisplatin, and long-term zinc supplementation. Cell population surviving the treatment was characterized as follows: cell stiffness was assessed by atomic force microscopy, cell motility and invasion capacity were determined by colony forming assay, wound healing assay, coherence-controlled holographic microscopy, and real-time cell analysis. Cells of metastatic origin exhibited lower height than cells derived from the primary tumour. Cell dry mass and CAV1 gene expression followed similar trends as cell stiffness. Docetaxel- and cisplatin-surviving cells had higher stiffness, and decreased motility and invasive potential as compared to non-treated cells. This effect was not observed in zinc(II)-treated cells. We presume that cell stiffness changes may represent an important overlooked effect of cisplatin-based anti-cancer drugs. Atomic force microscopy and confocal microscopy data images used in our study are available for download in the Zenodo repository (https://zenodo.org/, Digital Object Identifiers:10.5281/zenodo.1494935).

Cardiomyocytes (CM) placed on microelectrode array (MEA) were simultaneously probed with cantilever from atomic force microscope (AFM) system. This electric / nanomechanical combination in real time recorded beating force of the CMs cluster and the triggering electric events. Such "organ-on-a-chip" represents a tool for drug development and disease modeling. The human pluripotent stem cells included the WT embryonic line CCTL14 and the induced dystrophin deficient line reprogrammed from fibroblasts of a patient affected by Duchenne Muscular Dystrophy (DMD, complete loss of dystrophin expression). Both were differentiated to CMs and employed with the AFM/MEA platform for diseased CMs’ drug response testing and DMD characterization. The dependence of cardiac parameters on extracellular Ca2+ was studied. The differential evaluation explained the observed effects despite variability of biological samples. The β-adrenergic stimulation (isoproterenol) and antagonist trials (verapamil) addressed ionotropic and chronotropic cell line-dependent features. For the first time, a distinctive beating-force relation for DMD CMs was measured on the 3D cardiac in vitro model.

Cardiomyocyte contraction and relaxation are important parameters of cardiac function altered in many heart pathologies. Biosensing of these parameters represents an important tool in drug development and disease modeling. Human embryonic stem cells and especially patient specific induced pluripotent stem cell-derived cardiomyocytes are well established as cardiac disease model.. Here, a live stem cell derived embryoid body (EB) based cardiac cell syncytium served as a biorecognition element coupled to the microcantilever probe from atomic force microscope thus providing reliable micromechanical cellular biosensor suitable for whole-day testing. The biosensor was optimized regarding the type of cantilever, temperature and exchange of media; in combination with standardized protocol, it allowed testing of compounds and conditions affecting the biomechanical properties of EB. The studied effectors included calcium , drugs modulating the catecholaminergic fight-or-flight stress response such as the beta-adrenergic blocker metoprolol and the beta-adrenergic agonist isoproterenol. Arrhythmogenic effects were studied using caffeine. Furthermore, with EBs originating from patient's stem cells, this biosensor can help to characterize heart diseases such as dystrophies.

Atomic force microscopy (AFM) is not only a high-resolution imaging technique but also a sensitive tool able to study biomechanical properties of bio-samples (biomolecules, cells) in native conditions-i.e., in buffered solutions (culturing media) and stable temperature (mostly 37 °C). Micromechanical transducers (cantilevers) are often used to map surface stiffness distribution, adhesion forces, and viscoelastic parameters of living cells; however, they can also be used to monitor time course of cardiomyocytes contraction dynamics (e.g. beating rate, relaxation time), together with other biomechanical properties. Here we describe the construction of an AFM-based biosensor setup designed to study the biomechanical properties of cardiomyocyte clusters, through the use of standard uncoated silicon nitride cantilevers. Force-time curves (mechanocardiograms, MCG) are recorded continuously in real time and in the presence of cardiomyocyte-contraction affecting drugs (e.g., isoproterenol, metoprolol) in the medium, under physiological conditions. The average value of contraction force and the beat rate, as basic biomechanical parameters, represent pharmacological indicators of different phenotype features. Robustness, low computational requirements, and optimal spatial sensitivity (detection limit 200 pN, respectively 20 nm displacement) are the main advantages of the presented method.

Atomic force microscopy (AFM) is not only a high-resolution imaging technique but also a sensitive tool able to study biomechanical properties of bio-samples (biomolecules, cells) in native conditions—i.e., in buffered solutions (culturing media) and stable temperature (mostly 37 °C). Micromechanical transducers (cantilevers) are often used to map surface stiffness distribution, adhesion forces, and viscoelastic parameters of living cells; however, they can also be used to monitor time course of cardiomyocytes contraction dynamics (e.g. beating rate, relaxation time), together with other biomechanical properties. Here we describe the construction of an AFM-based biosensor setup designed to study the biomechanical properties of cardiomyocyte clusters, through the use of standard uncoated silicon nitride cantilevers. Force-time curves (mechanocardiograms, MCG) are recorded continuously in real time and in the presence of cardiomyocyte-contraction affecting drugs (e.g., isoproterenol, metoprolol) in the medium, under physiological conditions. The average value of contraction force and the beat rate, as basic biomechanical parameters, represent pharmacological indicators of different phenotype features. Robustness, low computational requirements, and optimal spatial sensitivity (detection limit 200 pN, respectively 20 nm displacement) are the main advantages of the presented method.

Hippo effectors YAP/TAZ act as on–off mechanosensing switches by sensing modifications in extracellular matrix (ECM) composition and mechanics. The regulation of their activity has been described by a hierarchical model in which elements of Hippo pathway are under the control of focal adhesions (FAs). Here we unveil the molecular mechanism by which cell spreading and RhoA GTPase activity control FA formation through YAP to stabilize the anchorage of the actin cytoskeleton to the cell membrane. This mechanism requires YAP co-transcriptional function and involves the activation of genes encoding for integrins and FA docking proteins. Tuning YAP transcriptional activity leads to the modification of cell mechanics, force development and adhesion strength, and determines cell shape, migration and differentiation. These results provide new insights into the mechanism of YAP mechanosensing activity and qualify this Hippo effector as the key determinant of cell mechanics in response to ECM cues., The transcriptional co-activator YAP is known to operate downstream of mechanical signals arising from the cell niche. Here the authors demonstrate that YAP controls cell mechanics, force development and adhesion strength by promoting the transcription of genes related to focal adhesions.

Isolated cardiomyocytes have been used as valid and useful model in experimental cardiology research for decades. The cell contraction function is usually measured via expensive and complex instruments which can either damage the cell or take much time for setting up. In contrary, recent development of optical microscopy and digital cameras suggests utilization of touch-less cardiomyocyte video acquisition in connection with advanced image processing techniques for evaluation of the cell contraction process. The proposed paper presents an automatic membrane detection method via computer processing of acquired video-sequences by utilization of an active contour model. Evaluation of detected cell area is used for estimation of cardiomyocyte contraction function. The method is evaluated utilizing the comparison with contraction measurement performed via atomic force microscopy technique.

In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are able to differentiate into functional cardiomyocytes (CMs). Traditional suspension method for 3D embryoid bodies (EBs) formation results in heterogeneous EB population. Here, we hypothesized that forming homogenous EB in size and shape allows studying the mechano-biological properties of spontaneously beating clusters containing CMs. Using a defined number of single cells in AggreWell plate, we formed homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors. We checked the expression of cardiac markers by qRT-PCR, immunocytochemistry and western blotting in both beating EBs and enzymatically-dissociated CMs and found increased expression of MYH6, MYH7 and RYR2 genes as well as sarcomeric pattern for cardiac troponin T and alpha-actinin. Using atomic force microscopy-based technique, we measured the beating properties of hESC-EBs and hiPSC-EBs and found a slower beat rate in hESC-CMs when compared to hiPSC-CMs (51 ± 5 vs 74 ± 7 bpm) and similar contraction force (31 ± 7 vs 39 ± 9 nN). Both cell types respond upon stimulation or inhibition of beta-adrenergic pathway and caffeine. Using Ca2+ imaging we also demonstrated the functionality of RyR2 to release Ca2+ from the sarcoplasmic reticulum and evaluated the contribution of IP3R. Thus, upon xestospongin C and histamine (IP3R modulators), spontaneous Ca2+ transients are maintained confirming the role of RyR2. Using the patch-clamp technique, we evaluated the cardiac population constituting the EBs and found that more than 75% of excitable cells exhibit atrial-like action potentials. Our results indicated that the mechano-biological properties of homogenous beating EBs can be investigated. They also indicated that spontaneous beating EBs contain mostly functional and organized atrial CMs.

In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes (CMs). Protocols for cardiac differentiation of hESCs and hiPSCs include formation of the three-dimensional cell aggregates called embryoid bodies (EBs). The traditional suspension method for EB formation from clumps of cells results in an EB population heterogeneous in size and shape. In this study we show that forced aggregation of a defined number of single cells on AggreWell plates gives a high number of homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors in the media. For cardiac differentiation, we used three hESC lines and one hiPSC line. Our contracting EBs and the resulting CMs express cardiac markers, namely myosin heavy chain α and β, cardiac ryanodine receptor/calcium release channel, and cardiac troponin T, shown by real-time polymerase chain reaction and immunocytochemistry. Using Ca(2+) imaging and atomic force microscopy, we demonstrate the functionality of RyR2 to release Ca(2+) from the sarcoplasmic reticulum as well as reliability in contractile and beating properties of hESC-EBs and hiPSC-EBs upon the stimulation or inhibition of the β-adrenergic pathway.

Stem cell-derived cardiomyocytes (CMs) hold great hopes for myocardium regeneration because of their ability to produce functional cardiac cells in large quantities. They also hold promise in dissecting the molecular principles involved in heart diseases and also in drug development, owing to their ability to model the diseases using patient-specific human pluripotent stem cell (hPSC)-derived CMs. The CM properties essential for the desired applications are frequently evaluated through morphologic and genotypic screenings. Even though these characterizations are necessary, they cannot in principle guarantee the CM functionality and their drug response. The CM functional characteristics can be quantified by phenotype assays, including electrophysiological, optical, and/or mechanical approaches implemented in the past decades, especially when used to investigate responses of the CMs to known stimuli (eg, adrenergic stimulation). Such methods can be used to indirectly determine the electrochemomechanics of the cardiac excitation-contraction coupling, which determines important functional properties of the hPSC-derived CMs, such as their differentiation efficacy, their maturation level, and their functionality. In this work, we aim to systematically review the techniques and methodologies implemented in the phenotype characterization of hPSC-derived CMs. Further, we introduce a novel approach combining atomic force microscopy, fluorescent microscopy, and external electrophysiology through microelectrode arrays. We demonstrate that this novel method can be used to gain unique information on the complex excitation-contraction coupling dynamics of the hPSC-derived CMs.