Calcium mediated functional interplay between myocardial cells upon laser-induced single-cell injury: an in vitro study of cardiac cell death signaling mechanisms.

BACKGROUND: The electromechanical function of myocardial tissue depends on the intercellular communication between cardiomyocytes (CMs) as well as their crosstalk with other cell types. Cell injury, and subsequent death trigger inflammation as in myocardial infarction (MI) resulting in myocardial remodeling. Although mechanisms underlying myocardial cell death have been studied so far, the signaling events following single cell death and spontaneous response of connected cells in the myocardial tissue is still barely understood. METHODS: Here, we investigated the effect of laser-induced single cell death on Calcium (Ca2+) concentrations and transport in myocardial cell clusters in vitro. Spatial and temporal changes in intracellular Ca2+ concentrations [Ca2+]i were studied using a fluorescent calcium indicator, Fluo-4AM. Spontaneous signaling events following cell death were studied in rat embryonic cardiomyocytes and non-myocytes using separate cell culture systems. RESULTS: Cell death triggered spontaneous increase in intracellular Ca2+ levels ([Ca2+]i) of surrounding cells. The spread of the observed propagating Ca2+ signal was slow and sustained in myocytes while it was rapid and transient in fibroblasts (Fbs). Further, sustained high Ca2+ levels temporarily impaired the contractility in CMs. The cell-type specific effect of ablation was confirmed using separate cultures of CMs and Fbs. Comparing Ca2+ propagation speed in myocytes and fibroblasts, we argue for a diffusion-driven Ca2+ propagation in myocytes, but not in fibroblasts. Radial and sequential Ca2+ diffusion across the CMs through cell-cell contacts and presence of Cx43-based intercellular junctions indicated a gap junction flow of Ca2+. CONCLUSIONS: These findings illustrate the spontaneous Ca2+-mediated functional interplay in myocardial cell clusters upon mechanical injury and, further, the difference in Ca2+ signaling in cardiomyocytes and fibroblasts. Video Abstract.

Complex Size and Surface Charge Determine Nucleic Acid Transfer by Fusogenic Liposomes.

Highly efficient, biocompatible, and fast nucleic acid delivery methods are essential for biomedical applications and research. At present, two main strategies are used to this end. In non-viral transfection liposome- or polymer-based formulations are used to transfer cargo into cells via endocytosis, whereas viral carriers enable direct nucleic acid delivery into the cell cytoplasm. Here, we introduce a new generation of liposomes for nucleic acid delivery, which immediately fuse with the cellular plasma membrane upon contact to transfer the functional nucleic acid directly into the cell cytoplasm. For maximum fusion efficiency combined with high cargo transfer, nucleic acids had to be complexed and partially neutralized before incorporation into fusogenic liposomes. Among the various neutralization agents tested, small, linear, and positively charged polymers yielded the best complex properties. Systematic variation of liposomal composition and nucleic acid complexation identified surface charge as well as particle size as essential parameters for cargo-liposome interaction and subsequent fusion induction. Optimized protocols were tested for the efficient transfer of different kinds of nucleic acids like plasmid DNA, messenger RNA, and short-interfering RNA into various mammalian cells in culture and into primary tissues.

Intramembranous processing by γ-secretase regulates reverse signaling of ephrin-B2 in migration of microglia.

The Eph-ephrin system plays pivotal roles in cell adhesion and migration. The receptor-like functions of the ephrin ligands allow the regulation of intracellular processes via reverse signaling. γ-Secretase mediated processing of ephrin-B has previously been linked to activation of Src, a kinase crucial for focal adhesion and podosome phosphorylation. Here, we analyzed the role of γ-secretase in the stimulation of reverse ephrin-B2 signaling in the migration of mouse embryonic stem cell derived microglia. The proteolytic generation of the ephrin-B2 intracellular domain (ICD) by γ-secretase stimulates Src and focal adhesion kinase (FAK). Inhibition of γ-secretase decreased the phosphorylation of Src and FAK, and reduced cell motility. These effects were associated with enlargement of the podosomal surface. Interestingly, expression of ephrin-B2 ICD could rescue these effects, indicating that this proteolytic fragment mediates the activation of Src and FAK, and thereby regulates podosomal dynamics in microglial cells. Together, these results identify γ-secretase as well as ephrin-B2 as regulators of microglial migration.

Fusogenic Liposomes as Nanocarriers for the Delivery of Intracellular Proteins.

Direct delivery of proteins and peptides into living mammalian cells has been accomplished using phospholipid liposomes as carrier particles. Such liposomes are usually taken up via endocytosis where the main part of their cargo is degraded in lysosomes before reaching its destination. Here, fusogenic liposomes, a newly developed molecular carrier system, were used for protein delivery. When such liposomes were loaded with water-soluble proteins and brought into contact with mammalian cells, the liposomal membrane efficiently fused with the cellular plasma membrane delivering the liposomal content to the cytoplasm without degradation. To explore the key factors of proteofection processes, the complex formation of fusogenic liposomes and proteins of interest and the size and zeta potential of the formed fusogenic proteoliposoms were monitored. Intracellular protein delivery was analyzed using fluorescence microscopy and flow cytometry. Proteins such as EGFP, Dendra2, and R-phycoerythrin or peptides such as LifeAct-FITC and NTF2-AlexaFluor488 were successfully incorporated into mammalian cells with high efficiency. Moreover, correct functionality and faithful transport to binding sites were also proven for the imported proteins.

Keratins as the main component for the mechanical integrity of keratinocytes.

Keratins are major components of the epithelial cytoskeleton and are believed to play a vital role for mechanical integrity at the cellular and tissue level. Keratinocytes as the main cell type of the epidermis express a differentiation-specific set of type I and type II keratins forming a stable network and are major contributors of keratinocyte mechanical properties. However, owing to compensatory keratin expression, the overall contribution of keratins to cell mechanics was difficult to examine in vivo on deletion of single keratin genes. To overcome this problem, we used keratinocytes lacking all keratins. The mechanical properties of these cells were analyzed by atomic force microscopy (AFM) and magnetic tweezers experiments. We found a strong and highly significant softening of keratin-deficient keratinocytes when analyzed by AFM on the cell body and above the nucleus. Magnetic tweezers experiments fully confirmed these results showing, in addition, high viscous contributions to magnetic bead displacement in keratin-lacking cells. Keratin loss neither affected actin or microtubule networks nor their overall protein concentration. Furthermore, depolymerization of actin preserves cell softening in the absence of keratin. On reexpression of the sole basal epidermal keratin pair K5/14, the keratin filament network was reestablished, and mechanical properties were restored almost to WT levels in both experimental setups. The data presented here demonstrate the importance of keratin filaments for mechanical resilience of keratinocytes and indicate that expression of a single keratin pair is sufficient for almost complete reconstitution of their mechanical properties.

Defined 2-D microtissues on soft elastomeric silicone rubber using lift-off epoxy-membranes for biomechanical analyses.

Surface patterning with complex molecules has become a valuable tool in cell biology and biotechnology, as it enables one to control cell shape and function in culture. However, this technique for micro-contact printing is normally performed on rigid substrates, e.g. Petri dishes or glass. Despite the fact that these substrates can easily be patterned they are artificially stiff environments for cells affecting their morphology and function. Those artifacts can be avoided on tissue elasticity resembling substrates, leading to a nature like cell morphology and behavior. However, reproducible patterning of very soft elastomeric substrates is challenging. Here, we describe a simple and highly accurate method through cavities of lift-off membranes for protein patterning of silicone rubber substrates in an elasticity range down to 1.5 kPa without altering their mechanical properties. Membranes are made of epoxy resin with feature sizes that can be chosen almost arbitrarily including widths down to 5 µm and aspect ratios of 100 and more. Different feature shapes were used to actively manipulate cell adhesion, cell morphology and the actin cytoskeleton on soft substrates. Manipulation of cytoskeletal organization furthermore allowed the comparison of myofibril alignment and cellular forces of cardiac myocytes. These data could show that cell forces are largely unaffected upon active disordering of overall myofibril alignment on a single cell level while aligned multicellular systems generate cell forces in an additive manner.

Smuggling on the Nanoscale-Fusogenic Liposomes Enable Efficient RNA-Transfer with Negligible Immune Response In Vitro and In Vivo.

The efficient and biocompatible transfer of nucleic acids into mammalian cells for research applications or medical purposes is a long-standing, challenging task. Viral transduction is the most efficient transfer system, but often entails high safety levels for research and potential health impairments for patients in medical applications. Lipo- or polyplexes are commonly used transfer systems but result in comparably low transfer efficiencies. Moreover, inflammatory responses caused by cytotoxic side effects were reported for these transfer methods. Often accountable for these effects are various recognition mechanisms for transferred nucleic acids. Using commercially available fusogenic liposomes (Fuse-It-mRNA), we established highly efficient and fully biocompatible transfer of RNA molecules for in vitro as well as in vivo applications. We demonstrated bypassing of endosomal uptake routes and, therefore, of pattern recognition receptors that recognize nucleic acids with high efficiency. This may underlie the observed almost complete abolishment of inflammatory cytokine responses. RNA transfer experiments into zebrafish embryos and adult animals fully confirmed the functional mechanism and the wide range of applications from single cells to organisms.

Fluorescent lipids: functional parts of fusogenic liposomes and tools for cell membrane labeling and visualization.

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.

PEGylation and folic-acid functionalization of cationic lipoplexes-Improved nucleic acid transfer into cancer cells.

Efficient and reliable transfer of nucleic acids for therapy applications is a major challenge. Stabilization of lipo- and polyplexes has already been successfully achieved by PEGylation. This modification reduces the interaction with serum proteins and thus prevents the lipoplexes from being cleared by the reticuloendothelial system. Problematically, this stabilization of lipoplexes simultaneously leads to reduced transfer efficiencies compared to non-PEGylated complexes. However, this reduction in transfer efficiency can be used to advantage since additional modification of PEGylated lipoplexes with functional groups enables improved selective transfer into target cells. Cancer cells overexpress folate receptors because of a significantly increased need of folate due to high cell proliferation rates. Thus, additional folate functionalization of PEGylated lipoplexes improves uptake into cancer cells. We demonstrate herein that NHS coupling chemistries can be used to modify two commercially available transfection reagents (Fuse-It-DNA and Lipofectamine® 3000) with NHS-PEG-folate for increased uptake of nucleic acids into cancer cells. Lipoplex characterization and functional analysis in cultures of cancer- and healthy cells clearly demonstrate that functionalization of PEGylated lipoplexes offers a promising method to generate efficient, stable and selective nucleic acid transfer systems.

Novel fusogenic liposomes for fluorescent cell labeling and membrane modification.

Efficient delivery of biomolecules into membranes of living cells as well as cell surface modifications are major biotechnological challenges. Here, novel liposome systems based on neutral and cationic lipids in combination with lipids modified by aromatic groups are introduced for such applications. The fusion efficiency of these liposome systems was tested on single cells in culture like HEK293, myofibroblasts, cortical neurons, human macrophages, smooth muscle cells, and even on tissue. Fusogenic liposomes enabled highly efficient incorporation of molecules into mammalian cell membranes within 1 to 30 min at fully unchanged cell growth conditions and did not affect cell behavior. We hypothesize that membrane fusions were induced in all cases by the interaction of the positively charged lipids and the delocalized electron system of the aromatic group generating local dipoles and membrane instabilities. Selected applications ranging from basic research to biotechnology are envisaged here.

Substrate Elasticity Exerts Functional Effects on Primary Microglia.

Microglia-the brain's primary immune cells-exert a tightly regulated cascade of pro- and anti-inflammatory effects upon brain pathology, either promoting regeneration or neurodegeneration. Therefore, harnessing microglia emerges as a potential therapeutic concept in neurological research. Recent studies suggest that-besides being affected by chemokines and cytokines-various cell entities in the brain relevantly respond to the mechanical properties of their microenvironment. For example, we lately reported considerable effects of elasticity on neural stem cells, regarding quiescence and differentiation potential. However, the effects of elasticity on microglia remain to be explored.Under the hypothesis that the elasticity of the microenvironment affects key characteristics and functions of microglia, we established an in vitro model of primary rat microglia grown in a polydimethylsiloxane (PDMS) elastomer-based cell culture system. This way, we simulated the brain's physiological elasticity range and compared it to supraphysiological stiffer PDMS controls. We assessed functional parameters of microglia under "resting" conditions, as well as when polarized towards a pro-inflammatory phenotype (M1) by lipopolysaccharide (LPS), or an anti-inflammatory phenotype (M2) by interleukin-4 (IL-4). Microglia viability was unimpaired on soft substrates, but we found various significant effects with a more than two-fold increase in microglia proliferation on soft substrate elasticities mimicking the brain (relative to PDMS controls). Furthermore, soft substrates promoted the expression of the activation marker vimentin in microglia. Moreover, the M2-marker CD206 was upregulated in parallel to an increase in the secretion of Insulin-Like Growth Factor-1 (IGF-1). The upregulation of CD206 was abolished by blockage of stretch-dependent chloride channels. Our data suggest that the cultivation of microglia on substrates of brain-like elasticity promotes a basic anti-inflammatory activation state via stretch-dependent chloride channels. The results highlight the significance of the omnipresent but mostly overlooked mechanobiological effects exerted on microglia and contribute to a better understanding of the complex spatial and temporal interactions between microglia, neural stem cells, and glia, in health and disease.

Changing the Way of Entrance: Highly Efficient Transfer of mRNA and siRNA via Fusogenic Nano-Carriers.

Transferring nucleic acids into mammalian cells heavily influences life science for decades. While first applications mainly dealt with DNA transfer for various purposes as e.g., plasmid encoded protein expression or generation of mutant strains, subsequent applications additionally transferred RNA molecules of mainly small lengths for specific knockdown (RNAi) or site-specific genome modification (gRNA). Significant improvements in full length mRNA generation and extension of mRNA lifetimes additionally allows their use for transient expression in latest times. For all of these types of nucleic acids the most common cell incorporation method is based on complexation and subsequent endosomal uptake. This so-called lipofection can be used theoretically for almost any mammalian cell type and a tremendous number of different product compositions exist in order to deal with drawbacks as transfer efficiency, cell type selectivity, endosomal degradation, slow uptake and cytotoxicity. In contrast, new methods transfer complexed RNA molecules directly into the cytoplasm using liposomal nano-carriers that fuse with cellular plasma membranes immediately upon contact to free functional nucleic acids directly into the cytoplasm. Here, we compare both methods in detail with special focus on robustness, short- and long-term cytotoxicity, efficiency and functionality for various types of transferred RNA. Our data clearly indicate that direct RNA incorporation via fusogenic nano-carriers circumvents most endosomal uptake-based challenges, making it to a most promising alternative for nucleic acid transfer.

Traction force microscopy with optimized regularization and automated Bayesian parameter selection for comparing cells.

Adherent cells exert traction forces on to their environment which allows them to migrate, to maintain tissue integrity, and to form complex multicellular structures during developmental morphogenesis. Traction force microscopy (TFM) enables the measurement of traction forces on an elastic substrate and thereby provides quantitative information on cellular mechanics in a perturbation-free fashion. In TFM, traction is usually calculated via the solution of a linear system, which is complicated by undersampled input data, acquisition noise, and large condition numbers for some methods. Therefore, standard TFM algorithms either employ data filtering or regularization. However, these approaches require a manual selection of filter- or regularization parameters and consequently exhibit a substantial degree of subjectiveness. This shortcoming is particularly serious when cells in different conditions are to be compared because optimal noise suppression needs to be adapted for every situation, which invariably results in systematic errors. Here, we systematically test the performance of new methods from computer vision and Bayesian inference for solving the inverse problem in TFM. We compare two classical schemes, L1- and L2-regularization, with three previously untested schemes, namely Elastic Net regularization, Proximal Gradient Lasso, and Proximal Gradient Elastic Net. Overall, we find that Elastic Net regularization, which combines L1 and L2 regularization, outperforms all other methods with regard to accuracy of traction reconstruction. Next, we develop two methods, Bayesian L2 regularization and Advanced Bayesian L2 regularization, for automatic, optimal L2 regularization. Using artificial data and experimental data, we show that these methods enable robust reconstruction of traction without requiring a difficult selection of regularization parameters specifically for each data set. Thus, Bayesian methods can mitigate the considerable uncertainty inherent in comparing cellular tractions in different conditions.

Substrate elasticity induces quiescence and promotes neurogenesis of primary neural stem cells-A biophysical in vitro model of the physiological cerebral milieu.

In the brain, neural stem cells (NSC) are tightly regulated by external signals and biophysical cues mediated by the local microenvironment or "niche." In particular, the influence of tissue elasticity, known to fundamentally affect the function of various cell types in the body, on NSC remains poorly understood. We, accordingly, aimed to characterize the effects of elastic substrates on critical NSC functions. Primary rat NSC were grown as monolayers on polydimethylsiloxane- (PDMS-) based gels. PDMS-coated cell culture plates, simulating the physiological microenvironment of the living brain, were generated in various degrees of elasticity, ranging from 1 to 50 kPa; additionally, results were compared with regular glass plates as usually used in cell culture work. Survival of NSC on the PDMS-based substrates was unimpaired. The proliferation rate on 1 kPa PDMS decreased by 45% compared with stiffer PMDS substrates of 50 kPa (p < 0.05) whereas expression of cyclin-dependent kinase inhibitor 1B/p27Kip1 increased more than two fold (p < 0.01), suggesting NSC quiescence. NSC differentiation was accelerated on softer substrates and favored the generation of neurons (42% neurons on 1 kPa PDMS vs. 25% on 50 kPa PDMS; p < 0.05). Neurons generated on 1 kPa PDMS showed 29% longer neurites compared with those on stiffer PDMS substrates (p < 0.05), suggesting optimized neuronal maturation and an accelerated generation of neuronal networks. Data show that primary NSC are significantly affected by the mechanical properties of their microenvironment. Culturing NSC on a substrate of brain-like elasticity keeps them in their physiological, quiescent state and increases their neurogenic potential.

Chemically defined, ultrasoft PDMS elastomers with selectable elasticity for mechanobiology.

Living animal cells are strongly influenced by the mechanical properties of their environment. To model physiological conditions ultrasoft cell culture substrates, in some instances with elasticity (Young's modulus) of only 1 kPa, are mandatory. Due to their long shelf life PDMS-based elastomers are a popular choice. However, uncertainty about additives in commercial formulations and difficulties to reach very soft materials limit their use. Here, we produced silicone elastomers from few, chemically defined and commercially available substances. Elastomers exhibited elasticities in the range from 1 kPa to 55 kPa. In detail, a high molecular weight (155 kg/mol), vinyl-terminated linear silicone was crosslinked with a multifunctional (f = 51) crosslinker (a copolymer of dimethyl siloxane and hydrosilane) by a platinum catalyst. The following different strategies towards ultrasoft materials were explored: sparse crosslinking, swelling with inert silicone polymers, and, finally, deliberate introduction of dangling ends into the network (inhibition). Rheological experiments with very low frequencies led to precise viscoelastic characterizations. All strategies enabled tuning of stiffness with the lowest stiffness of ~1 kPa reached by inhibition. This system was also most practical to use. Biocompatibility of materials was tested using primary cortical neurons from rats. Even after several days of cultivation no adverse effects were found.

Measuring cellular traction forces on non-planar substrates.

Animal cells use traction forces to sense the mechanics and geometry of their environment. Measuring these traction forces requires a workflow combining cell experiments, image processing and force reconstruction based on elasticity theory. Such procedures have already been established mainly for planar substrates, in which case one can use the Green's function formalism. Here we introduce a workflow to measure traction forces of cardiac myofibroblasts on non-planar elastic substrates. Soft elastic substrates with a wave-like topology were micromoulded from polydimethylsiloxane and fluorescent marker beads were distributed homogeneously in the substrate. Using feature vector-based tracking of these marker beads, we first constructed a hexahedral mesh for the substrate. We then solved the direct elastic boundary volume problem on this mesh using the finite-element method. Using data simulations, we show that the traction forces can be reconstructed from the substrate deformations by solving the corresponding inverse problem with an L1-norm for the residue and an L2-norm for a zeroth-order Tikhonov regularization. Applying this procedure to the experimental data, we find that cardiac myofibroblast cells tend to align both their shapes and their forces with the long axis of the deformable wavy substrate.

Biotin-conjugated fusogenic liposomes for high-quality cell purification.

Purification of defined cell populations from mixed primary cell sources is essential for many biomedical and biotechnological applications but often very difficult to accomplish due to missing specific surface markers. In this study, we developed a new approach for efficient cell population separation based on the specific membrane fusion characteristics of distinct cell types upon treatment with fusogenic liposomes. When such liposomes are conjugated with biotin, specific cell populations can be efficiently surface functionalized by biotin after liposomal treatment while other populations remain unlabeled. Due to the high affinity of biotin for avidin-like proteins, biotin functionalized cells are ideal targets for conjugation of e.g. avidin tagged magnetic beads, fluorophores or antibodies with bioanalytical relevance. Here, based on the differential biotinylation of distinct cell populations high quality separation of cardiac fibroblasts from myocytes, and cerebromicrovascular endothelial cells from fibroblasts was successfully established.

Surface topography enhances differentiation of mesenchymal stem cells towards osteogenic and adipogenic lineages.

Surface topography impacts on cell growth and differentiation, but it is not trivial to generate defined surface structures and to assess the relevance of specific topographic parameters. In this study, we have systematically compared in vitro differentiation of mesenchymal stem cells (MSCs) on a variety of groove/ridge structures. Micro- and nano-patterns were generated in polyimide using reactive ion etching or multi beam laser interference, respectively. These structures affected cell spreading and orientation of human MSCs, which was also reflected in focal adhesions morphology and size. Time-lapse demonstrated directed migration parallel to the nano-patterns. Overall, surface patterns clearly enhanced differentiation of MSCs towards specific lineages: 15 µm ridges increased adipogenic differentiation whereas 2 µm ridges enhanced osteogenic differentiation. Notably, nano-patterns with a periodicity of 650 nm increased differentiation towards both osteogenic and adipogenic lineages. However, in absence of differentiation media surface structures did neither induce differentiation, nor lineage-specific gene expression changes. Furthermore, nanostructures did not affect the YAP/TAZ complex, which is activated by substrate stiffness. Our results provide further insight into how structuring of tailored biomaterials and implant interfaces - e.g. by multi beam laser interference in sub-micrometer scale - do not induce differentiation of MSCs per se, but support their directed differentiation.

Plasma membrane functionalization using highly fusogenic immune activator liposomes.

Cell surface functionalization and target molecule incorporation into living cell membranes without functional damage represent major biotechnological challenges. One possible way to achieve these goals is to induce cell membrane fusion with an artificial membrane containing molecules equipped with reactive groups or ligands. In this work we developed a carrier system to incorporate lipopolysaccharide (LPS), an immune cell activating molecule from Gram-negative bacteria, into mammalian membranes. LPS is not present in untreated mammalian cells which hence are not detectable by the immune system. Here, we demonstrate the successful incorporation of LPS into fusogenic liposomes (FLs) and subsequent incorporation into mammalian plasma membranes using these FLs. Additionally, the presence of LPS in cell membranes was probed by the addition of non-activated macrophages. A high concentration of LPS in the plasma membrane of immortalized fibroblasts activated the immune cells, which in turn started to eliminate LPS-exhibiting cells. Our method for cellular membrane functionalization is a promising tool for biomedical applications and could provide the basis for specific cell targeting approaches.

Matrix elasticity, replicative senescence and DNA methylation patterns of mesenchymal stem cells.

Matrix elasticity guides differentiation of mesenchymal stem cells (MSCs) but it is unclear if these effects are only transient - while the cells reside on the substrate - or if they reflect persistent lineage commitment. In this study, MSCs were continuously culture-expanded in parallel either on tissue culture plastic (TCP) or on polydimethylsiloxane (PDMS) gels of different elasticity to compare impact on replicative senescence, in vitro differentiation, gene expression, and DNA methylation (DNAm) profiles. The maximal number of cumulative population doublings was not affected by matrix elasticity. Differentiation towards adipogenic and osteogenic lineage was increased on soft and rigid biomaterials, respectively - but this propensity was no more evident if cells were transferred to TCP. Global gene expression profiles and DNAm profiles revealed relatively few differences in MSCs cultured on soft or rigid matrices. Furthermore, only moderate DNAm changes were observed upon culture on very soft hydrogels of human platelet lysate. Our results support the notion that matrix elasticity influences cellular behavior while the cells reside on the substrate, but it does not have major impact on cell-intrinsic lineage determination, replicative senescence or DNAm patterns.