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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.

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.

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.

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.