Nanoengineering for Mechanobiology

During embryonic development, cardiac growth occurs through massive proliferation of cardiomyocytes. At birth, over 30% of these cells still actively proliferate. However, only one week after birth this percentage drops to virtually null, and subsequent cardiac growth in the postnatal life occurs through cardiomyocyte hypertrophy, sustained by increase in cytoplasm size and build up of contractile sarcomeric structures. Physiological adaptation to stress as well as maladaptive adaptation to cardiomyocyte loss and dysfunction occurs through the modulation of levels, post-translational modification and regulation of activity of sarcomeric proteins and the associated microtubuli and microfilament networks. All these functions are in direct link to sensing extracellular tension generated by powerful mechanical forces exerted onto the myocardium. Multiple evidence now indicates that both cardiomyocyte proliferation and cytoskeletal/sarcomere function are under the control of the cellular microRNA network. I will present information on how our laboratory is unraveling the function of individual miRNAs and miRNA families involved in the regulation of cardiac cell proliferation and function by a functional approach using high throughput screenings of miRNA and siRNA whole genome libraries. The identification of the molecular pathways involved in cardiomyocyte replication, mechanosensing and hypertrophic growth has obvious implications in terms of generating new small drugs or nucleic acids able to foster cardiac regeneration after myocardial infarction and to promote cardiac contractility in patients with heart failure.

Luca Braga1, Matteo Dal Ferro1,3, Miguel Mano1, Ana Eulalio1, Antonio Cannatà3, Hashim Ali1, Consuelo Torrini1, Silvia Moimas1, Andrea Raso1, Lorena Zentilin1, Serena Zacchigna2 and Mauro Giacca1
1 Molecular Medicine and 2 Cardiovascular Biology Laboratories, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy 3 Department of Cardiology, Azienda Sanitaria Universitaria Integrata Trieste, Trieste, Italy

The adult heart is capable of remodelling in response to different pathological stimuli; in most cases, a phase of compensated hypertrophy evolves into frank dysfunction and heart failure. To identify microRNAs able to prevent cardiac hypertrophy, we performed a high content functional screening for human miRNAs able to reduce neonatal cardiomyocyte (CMs) cell size using a whole-genome miRNA library. The most effective anti-hypertrophic miRNAs in the screening was hsa-miR-665. In a model of transverse abdominal aortic constriction in 8 weeks old CD1 mice, AAV9-mediated delivery of miR-665 protected against pathological cardiac hypertrophy, preserved ejection fraction, avoided LV-dilation and prevented development of heart failure. This effect was observed both when the vectors were delivered before (LVEF at 60 day after TAC: 51.3±5.8% in treated vs. 34.82±0.77% in controls; P<0.01) or after hypertrophy onset (LVEF at 60 days after TAC: 57.5±5.60% in treated vs. 28.4±15% in controls; P<0.01). Global mRNAs changes in hearts treated with miR-665 were evaluated by mRNA deep sequencing. All the genes that were found to be expressed ≤2 fold over control were tested for being direct miR-665 targets. This approach identified three sarcomeric proteins namely Enah, Fhl1 and Xirp2, which are known to be involved in mechanotransduction and myofibrillar remodelling. In addition, hsa-miR-665 ameliorated LV passive stiffness in an AngII-mediated mouse model of HFpEF, supporting the conclusion that miR-665 modulates also CMs compliance to stretch.

The generation of traction forces and their transmission to the extracellular environment supports the disseminative migration of cells from a primary tumor. In cancer cells, the periodic variation of nuclear stiffness during the cell cycle provides a functional link between efficient translocation and proliferation. However, the mechanical framework completing this picture remains unexplored. Here, the Fucci2 reporter was transfected in various human epithelial cancer cells to resolve their cell cycle phase transition. The corresponding tractions were captured by means of a recently developed, reference-free, confocal traction force microscopy (cTFM) platform. The combined approach was conducive to the analysis of phase-dependent force variation at the level of individual integrin contacts. Detected forces were invariably higher in the G1 and early S phases as compared to the ensuing late S/G2, and locally co-localized with high levels of paxillin phosphorylation. The biochemical inhibition of focal adhesion kinase (FAK) reduced paxillin phosphorylation and significantly diminished the force transmitted to the substrate. These data demonstrate a reproducible modulation of force transmission during the cell cycle progression of cancer cells. In addition, they delineate a model in which paxillin phosphorylation supports the mechanical maturation of adhesions relaying forces to the substrate.

Cell form and function are regulated, in part, by biophysical and biochemical signals from the surrounding extracellular matrix (ECM). Emerging data suggest that cell behavior is also controlled by past microenvironments. That is, cells possess a mechanical memory that stores information from the surrounding matrix and influences cell state plasticity and robustness. If confirmed, this hypothesis has broad implications on the fundamental role of the matrix in lineage speciation, disease progression, and aging, as well as from the practical view of how cells are affected by standard in vitro culture. Here, we discuss our efforts in developing photoresponsive culture substrates that enable dynamic modulation of mechanics to investigate the role of past microenvironmental mechanics on mesenchymal stem cell and valvular interstitial cell fate. The moduli of the underlying substrate can be altered in real time and in the presence of cells with cytocompatible light. We will discuss the observation of mechanical memory in these cells and aspects of the hypothesized signaling mechanisms that regulate these processes. In the end of the talk, we will discuss recent efforts in developing array based culture systems to study the effects of past microenvironmental cues (mechanics, geometry, and ligand presentation) on cell fate toward achieving single-cell resolution of studies on mechanical memory.

Different approaches to mechanically characterize soft tissues and (bio)materials at different length-scales have been proposed to date, but a unique consensus on the optimal experimental and analysis method for obtaining physiologically relevant data for cell mechanobiology studies is still lacking. Several sample, testing and analysis variables can affect the measured sample mechanical properties (Mattei et al, Acta Biom 45:60-71, 2016). With the hypothesis that local mechanical characterization at typical cell length-scales in is critical to better characterize the biomechanical environment felt by cells in their native environment or when being cultured in-vitro, we here present the PIUMA as a tool to derive micro-mechanical properties via non-destructive nano-indentation measurements in liquids.
The PIUMA features a fiber-optical fiber-top micro-machined force transducer, which offers unique advantages when performing measurements in liquids (Chavan et al, Rev Sci Instrum 83:115110, 2012). This opto-mechanical transducer is operated by a z-axis piezoelectric motor, whose closed-loop control has been recently modified to account for the actual cantilever bending during measurement, enabling load- and displacement-controlled user-defined experiments for a wide range of mechanical tests: from quasi-static experiments to derive elastic moduli, to step-response tests (e.g. creep, stress-relaxation), dynamic mechanical analysis (DMA) and constant strain rate tests (i.e. nano-epsilon dot method, Mattei G et al, JMBBM 50:150-9, 2015) to characterize sample viscoelastic behavior – an aspect, the latter, which is becoming very important in cell mechanobiology (Chauduri et al, Nat Comm 6:6364, 2015).
Applications to hydrogels, tissues and single cells will be presented. Moreover, we recently coupled MEMS fabrication techniques to probe production to extend the range of measurable mechanical properties including advanced cell compression and adhesion measurements.

Tissues and biomaterials are generally characterised by a biphasic structure: a solid (elastic) network surrounded by an aqueous (viscous) solution. Despite the intrinsic viscoelasticity of these materials, most of the mechanobiology and tissue engineering studies in the literature are mainly focused on characterising elasticity and developing cell culture substrates with a given stiffness [Hoffman, 2012]. A few studies report on the modulation of biomaterial elasticity [Bhattacharya 2016; Shi 2007; Mensitieri 1996; Mattei 2017] using crosslinking strategies, which alter both elastic and viscous behaviour concomitantly. Therefore, our aim was to modulate material viscoelasticity by acting on the damping component, i.e. tuning the liquid phase viscosity of agarose and polyacrylamide (PAAm) gels by dissolving a constant amount of solid phase (1% w/v agarose, or 15,6% w/v acrylamide) in aqueous dextran solutions prepared at different concentrations (0, 2 and 5% w/v). Mechanical tests were performed following the epsilon-dot method [Tirella 2014], obtaining the instantaneous (Einst) and equilibrium (Eeq) elastic moduli, and the characteristic relaxation time (τ).
Increasing the solvent viscosity resulted in a significant reduction Einst and of τ, while Eeq was found to be almost independent of the solvent viscosity. In conclusion, our study demonstrates that the modulation of the liquid phase is reflected in the viscoelastic properties of the gels. The results showed also that this effect is more relevant in agarose, which presents a more elastic behaviour respect to PAAm. This suggest that the initial mechanical properties, but also the chemical behaviour, of the gels are a fundamental factor to modulate the viscoelasticity of a gel. This simple-yet-effective strategy might be beneficial to the field of mechanobiology, e.g. for studying cell response to substrate viscoelastic relaxation, while keeping the elastic cue (i.e. equilibrium modulus) constant.

To gain entry into host cells, viruses have developed an exquisite tuning of their chemical and physical signatures. This entails the direct presentation or exposure of cell receptor binding sites on their surface, as well as a great variability in size and shape of viral particles. By combining live-cell microscopy and surface chemistry approaches with the development of biosensors for single molecule force measurements, we established a method to study the physical interaction of single viral particles with the cell surface. To determine the impact of particle size on clathrin mediated endocytosis (CME), we use the same approach to immobilize nanoparticles having a diameter of 20 to 1000 nm.
The critical diameter for inducing CME in a receptor independent manner is in the range of 100-300 nm, since only reoviruses (100 nm diameter) and nanoparticles of this size colocalize with clathrin-mediated structures. While the size of clathrin-mediated structures is determined by particle size, the further commitment that leads to CME is independent of cargo internalization. Further, the crucial steps for force generation are initiated by membrane bending depending on particle size, and do not rely on specific interactions with receptors at the cell surfaces and cytoskeletal forces. This suggests that a fine regulation of local, rather than global, physical events is crucial for virus uptake. Our studies provide the evidence that the early surface interactions of nanoscale objects with cells are essentially determined by a size-dependent change in membrane curvature that triggers internalization events.

We review our recent results related to the observation and modeling of cell deformation, division and migration [1]. We first discuss our experiments demonstrating that water transport in and out of the cell is needed for the formation of blebs, commonly observed protrusions in the plasma membrane driven by cortex contraction. Simulations of a model of fluid-mediated membrane-cortex deformations show that a permeable membrane is necessary for bleb formation which is otherwise impaired [2]. Next, we discuss two mechanical models for individual and multiple cell divisions: i) A three dimensional model of motor-driven chromosome congression and bi-orientation during mitosis revealing that successful cell division requires control of the total number of microtubules [3]. ii) A model for the division of stem cells in a crypt that relates stem cell population dynamics to the effect of mechanical forces acting on the spindle. We observe that the mechanically induced strategy for development is sub-optimal and crucially depends on the stiffness of the spindle [4]. Finally, we discuss the observation of universal activity bursts in collective cell migration [5].
[1] C. A. M. La Porta, S. Zapperi, The Physics of Cancer, Cambridge University Press (2017).
[2] A. Taloni et al, Volume Changes During Active Shape Fluctuations in Cells, Phys. Rev. Lett. 114, 208101 (2015).
[3] Z. Bertalan et al. Role of the Number of Microtubules in Chromosome Segregation during Cell Division. PLoS ONE 10, e0141305 (2015).
[4] Z. Bertalan et al. Modeling mechanical control of spindle orientation of intestinal crypt stem cells Journal of Theoretical Biology, 430 103 (2017).
[5] O. Chepizhko et al. Bursts of activity in collective cell migration PNAS 113 11408 (2016).

This seminar will focus on the role of the mechanical signal exerted by plasma membrane tension on cell shape changes. I will present how we unveiled that cells like fibroblasts rely on membrane tension to exert a feedback loop on several biological functions important for cell shape changes and migration. I will also discuss how this mechanical signal of tension is in fact universal and can also be at the heart of understanding immune responses like phagocytosis.

The ability of cells to migrate in a specific direction governs morphogenesis and homeostasis of functional tissues in multicellular species. Directional motility requires primarily an asymmetry in interaction forces between the cell and its environment, while preserving force balance. In absence of any external cue, several mechanisms of symmetry breaking have been proposed, mostly based on the spontaneous polarisation of cytoskeleton components, and a broad range of motile behaviors has been reported. However, the direct link between the dynamics of intracellular components and cellular forces exerted on the environment remains to be determined; in particular no direct measurements of the spatio-temporal coordination of both cellular parameters and force patterns have yet been reported. Using a uni-dimensional single cell migration assay compatible with force imaging, we investigated the spatio-temporal dynamics of spontaneous symmetry breaking events in migrating cells. This analysis revealed a generic stick-slip motile behavior with stochastic initiation of directionality, occurring in absence of pre-established cytoskeleton polarity. While spreading, non polarized cells increase traction forces on the substrate, until the stochastic detachment of adhesive structures, inducing the subsequent creation of the cell rear and the initiation of migration. Hence cell migration is critically controlled by both the internal biochemical contractile machinery and the physical connections between cells and their microenvironment, and does not require cytoskeleton polarization. A theoretical model, consistent with quantitative experimental analysis of several cell types submitted to various physical or biochemical agents recapitulates the observed behaviors ranging from non polarized stochastic stick slip motion to polarized steady state migration.

Glioblastomas are extremely aggressive brain tumors with a very pour outcome, mostly due to the fact that these tumors are exceptionally invasive, penetrating into deep regions of the brain and destroying neurons leading to the rapid death of the patient (typically 10-14months). They migrate mostly by following linear brain blood vessel surface, using a saltatory linear migration mode. This type of linear migration can be recapitulated in vitro using micropatterning or electrospun fibers. We have previously shown using the rat C6 glioma cell model, that the mechanical confinement of glioma cells using micropattern and/ or increasing their cell density triggers a linear migration mode which is dependent on formins but not on Arp2/3. Specifically we have found that the formin FHOD3 was involved in this motility. In the present work we investigated the mechanical properties of glioma stem cell isolated from patients. We used micropatterns and electropsun fibers to analyze their motility and adhesive properties. We also evaluated the formin expression levels present in these cells and found that in addition to FHOD3, other formins have roles in mediating linear migration of these cells.

Cells in the body constantly experience mechanical stimuli in various organ movement, development and functions. These mechanical stimuli are often essential to proper cell function, such as migration, proliferation, differentiation[1-4]. Cells sense such mechanical stimuli through a process called mechanotransduction [1, 5, 6]. Abnormal mechanotransduction leads to several patho-genesis, such as cancer, asthma, heart disease[1, 7, 8]. In Mechanobiology studies, the advance of in vitro technologies for the application of physiologically mimicking mechanical force to cultured cells and tissue are vital due to the complex in vivo biological system. The growing interest in Mechanobiology and technology gap have motivated the development of cell stretching device in various research labs and companies [10]. However, most cell stretching systems are bulky and limited in functions. In particular, there is no cell culture chamber/dish capable of multifunctional cell stretching. A novel cell stretching device in the size of a cell culture petri dish has been invented, which is called cell stretching dish. It is capable of uniaxial as well as biaxial stretching. The cell stretching dish is characterized by high level of stretching, which can reach up to 100% (and more). It is portable and easy to be used for both cell culture and microscope imaging experiment. The device has been tested to be compatible with the high resolution objectives, for live cell as well as fixed cell immunofluorescence imaging, and can be scaled up and down to fit any inverted microscope. The device can be 3D printed or made from plastic, thus further reducing the cost of production, bringing down the complicated cell stretching system to the consumable level. The unique design, high level stretch, cheap cost will open variety of possibilities not only to researchers in mechanobiology but also broad field of biologist.

Cell proliferation and differentiation, as well as key processes in development, tumorigenesis, and wound healing, are strongly determined by the rigidity of the extracellular matrix (ECM). In this talk, I will explain how we combine molecular biology, biophysical measurements, and theoretical modelling to understand the mechanisms by which cells sense and respond to matrix rigidity. I will first discuss how the properties under force of integrin-ECM bonds, and of the adaptor protein talin, drive and regulate rigidity sensing. Then, I will analyze how signals triggered by rigidity at cell-ECM adhesions are transmitted to the nucleus, leading to the activation of the transcriptional regulator YAP.

The spatiotemporal organization of the eukaryotic genome is dynamic and contributes to the regulation of gene expression, but how does it respond to the changes of cell morphology is an unexplored area of research.
To study the interplay between cell architecture and nuclear organization we are combining tools typical of experimental biophysics, like image analysis and micro-patterning, with chromosome painting. Micro-patterning of adhesive molecules is a powerful technique that, by specifically defining the available area of spreading for a cell, allows to control its geometry. Chromosome painting enables instead to visualize chromosomes in the nucleus. Combining these two techniques, we can measure the positioning of a chromosome in the nucleus of cells with a determined geometry.
Our preliminary results suggest that imposing to cells an asymmetric shape, like a teardrop, which mimics a polarized state, generates an asymmetric distribution of chromosomes along the axis of symmetry. Some chromosomes are indeed more frequently positioned towards the front of the cell, the curved part of the teardrop, while some others are more frequently found close to the back. Similarly, when cells are plated on lines, a geometry previously used to study cell migration, an asymmetric distribution of chromosomes along the “nucleus to Golgi” axis of polarity is observed.
Forces and flows are necessary to maintain a specific geometry or to move. Is a portion of this tension/dynamics somehow transmitted to the genome affecting its spatial organization?
We would be delighted to have the opportunity to discuss this question with the community.

ATR (Ataxia Telangiectasia Related) is an essential kinase involved in sensing DNA damage and initiating appropriate repair response. ATR is also known to regulate nuclear integrity by sensing mechanical stress at the nuclear envelope. We observe that depletion of ATR leads to nuclear deformations, abnormal chromosomal architecture, altered nuclear stiffness, defective nuclear mechanotransduction; collectively indicating loss of nuclear integrity and homeostasis. Consequently, due to defective nuclear dynamics, cells lacking ATR are defective in 2D migration and in 3D migration through constrictions due. Cancer cells with reduced ATR have decreased survival rate, fail to survive the circulation through the blood stream and to efficiently invade host organs. Recent reports have associated higher levels of ATR with severe phenotypes and more invasive cancers supporting our observation. In summary, we report a novel role of ATR in maintaining of nuclear integrity against mechanical and topological insults. We propose that ATR has duel role in tumorogenisis, as a tumor suppressor by maintaining genomic stability and as a facilitator of tumor progression by supporting cell survival in extreme mechano-environmental alterations.

Collective motility, a widely recognized mode of migration during embryogenesis, wound repair and cancer, is ruled by both biochemical and physical interactions that cells establish among each other and with their environment. An emerging theoretical framework to interpret and integrate these interactions in unifying principles is the notion of cell jamming. During collective motility cells can flow like a fluid, but as density rises, the motion of each cell is constrained by the crowding due to its neighbors, forcing them to move in groups. At a critical density, motility ceases and collectives rigidify undergoing a liquid (unjammed)-to-solid (jammed) transition, herein referred to as UJT. This transition is thought to ensure proper development of barrier properties in epithelial tissues, but also to act as a tumor suppressive mechanism. The reverse, JUT might, instead, represent an alternative gateway to cell migration, enabling tissues to escape the caging imposed by the crowded cellular landscape of mature epithelia. The general validity of this physical framework and of JUT, however, remains to be verified. Even less understood is how cells control such phase transitions.
Here, inspired by our recent findings that endocytic perturbations impact upon dynamic and mechanical properties of multicellular assemblies, reawakening motility in jammed epithelia, we will discuss key open issues on the pathophysiology of jamming transition. We will focus specifically on the molecular bases of unjamming of solid-like epithelia and discuss whether the transition between “solid” and “liquid” locomotory states is a general framework, alternative or complementary to epithelial-to-mesenchymal transition (EMT), to account for the plastic remodeling of epithelial tissues in pathology.

The human mechanosensitive ion channel PIEZO1 gates in response to membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently, little is known about the plasma membrane localization and organization of PIEZO1, but previous work suggests that membrane cholesterol content is a key determinant of PIEZO1 function. Using a previously characterized PIEZO1-GFP fusion protein (hP1-1591-GFP,), we investigated the effect of the cholesterol depleting agent methyl-β-Cyclodextrin (mβCD) on the membrane organization and the response of PIEZO1 to mechanical force in HEK-293 cells. STORM super-resolution imaging revealed at the nanoscale that PIEZO1 channels associate in the membrane as clusters, as previously inferred by electrophysiological data. Both cluster size and diffusion rates, as determined using TIRF microscopy, were modulated by treatment with mβCD (5 mM). In addition, electrophysiological recordings in the cell-attached configuration revealed that mβCD caused a rightward shift in the PIEZO1 pressure-response curve and increased channel latency in response to mechanical stimuli.

Here we use DNA-based nanosensors to investigate mechanisms of B cell antigen extraction from the immune synapse. B cells play a critical role in immune responses by producing antibodies against the foreign pathogens that they encounter. Their responses are initiated when the B cell receptor binds antigen displayed on the surface of an antigen-presenting cell in a cell-cell contact called the immune synapse. This binding event triggers the B cell to internalize, process, and present the antigen to helper T cells, which are required for full B cell activation and clonal selection. High-affinity B cells receive more T cell help than low-affinity B cells do, suggesting that affinity discrimination during antigen internalization is essential for high-affinity antibody responses. The mechanisms of antigen recognition, discrimination, and uptake remain a topic of debate, with physical extraction through mechanical forces and enzymatic liberation through lysosome secretion proposed as efficient ways for B cells to acquire antigen. To investigate B cell extraction mechanisms, we developed DNA-based nanosensors to interrogate antigen extraction from both artificial substrates and live antigen-presenting cells. We show that B cells acquire antigen primarily through mechanical force, and resort to enzymatic liberation only if force-dependent extraction fails. The use of mechanical force renders B cells sensitive to physical properties of the antigen-presenting cell. We find that stiff antigen-presenting cells promote strong B cell pulling forces and stringent affinity discrimination, while flexible antigen-presenting cells allow B cells to extract low-affinity antigen using weak forces. Thus, mechanical properties of the immune synapse regulate B cell antigen extraction, suggesting that distinct physical properties of antigen-presenting cells support different stages of B cell responses.

Collective cell migration is fundamental throughout development, wound healing and in many diseases. Although much effort has focused on cell-cell junctions, a role for physical confinement in collective cell migration has been recognized but not yet investigated. Here we show that the area of adhesion to the substrate is a key biophysical parameter that regulates lamellipodial protrusive forces, cell traction stresses, and consequently migrating cell velocity. Using adhesive microstripes to mimic the 2D confinement experienced by follower cells during epithelial sheet migration, we show that confined keratocytes displayed an elongated morphology with a similar cytoskeletal organization to that observed in follower cells, suggesting that adhesive area confinement alone is sufficient to lead to a reorganization of actin and microtubule networks without the need for intercellular adhesive cues. Normalization of migration velocity in cells moving on adhesive lines to their total adhesive area demonstrates that cell velocity is directly correlated to adhesive area. Closer examination revealed that adhesive area confinement reduces protrusive forces in the lamellipodium, decreases the number of focal complexes at the leading edge and prevents the formation of mature focal adhesions at the trailing edge required for retraction of the cell body. The combination of small protrusive forces in thick leading edges with low traction forces exerted at the trailing edge generate less effective forward propelling forces that lead to reduced migration velocities in confined cells. Additionally, adhesive microstripes offered an ideal environment for studying the dynamics underlying the escaping process of leader cells and to reveal the distinct roles of front and rear in this process. Together, these results identify substrate area confinement as a key determinant of the cell speed in collective migration.

It is a general concern that mechanical forces, such as gravity, tension, compression, hydrostatic pressure, govern the cell growth and shape within tissues and organs under physiological and pathological conditions. Additionally, traction forces generated by cells may markedly influence many biological processes such as the stem cells self-renewal and differentiation. The hypothesis is that stem cells sense the mechanical loading through a diverse group of membrane-anchored mechanosensors (e.g. stretch-activated ion channels, integrins). These mechanical stimuli are then converted to biochemical signals by triggering a step-by-step activation of downstream signaling involving the cytoskeleton and the nucleoskeleton components. The convergence of these pathways results in the expression of a gene profile that direct the stem cells fate.
Hence, elucidating mechanotransduction pathways may shed light to complex biological phenomena such as the stem cells' specification and determination processes and developmental behaviors.
With this aim, we have explored the mechanotransduction axes in ex vivo tissue engineering modeling generated by combining different types of stem cells and biomaterials under growth culture condition. The latter, allows to provide informative microenvironments mimicking a physiological niche, allowing stem cells to interpret the biomaterial instructions and modify their fate accordingly.
We demonstrated that the interaction of stem cells and PLLA-derivative films steers cells toward a selected phenotype depending on the characteristics of the films. Thus, human umbilical cord matrix stem cells cultured on Poly (L-lactide) acid (PLLA) and PLLA/Multi Walled Carbon Nanotubes (MWCNTs) acquired a spheroids organization and based on the content of MWCNTs within the PLLA polymer, were steered toward a Primitive Endoderm-like phenotype (PLLA/1MWCNTs) instead of an Epiblast-like phenotype (PLLA polymer).

During the last decades, tools for rapid characterization of large cell quantities have become indispensable not only for basic research but also clinical diagnostics. The gold standard for cell characterization is flow cytometry. Its success is closely tied to the availability of fluorescent labels. But what if there is no molecular marker known for the cells of interest? Or if the label changes cell function? Or cells shall be used for transplantation? As an attractive alternative, deformability cytometry exploits cell mechanics as a sensitive, inherent, label-free functional marker, but lacks the specificity provided by a fluorescent signal. Here we present real-time fluorescence and deformability cytometry (RT-FDC), the ideal, combined system. It facilitates fluorescence detection as in conventional flow cytometry, extended by 1D analysis of spatial information encoded in the fluorescence pulse shape, and adds bright field imaging for mechanical phenotyping of single cells - all in real-time at rates of 100 cells/s. We show utility of RT-FDC for the most common fluorescent labels: Fluorescent surface markers (CD34) are used to separate human hematopoietic stem and progenitor cells (HSPCs) from an unpurified apheresis sample as harvested for bone marrow transplantations. Membrane permeant dyes identify reticulocytes by their ribonucleic acid (RNA) content in a blood sample. And endogenously expressed fluorescent proteins (FUCCI) reveal cell cycle phases in an unsynchronized sample of retinal pigment epithelial cells (RPE1). In addition, we can now also directly correlate mechanical characteristics with fluorescence intensity and localization for each single cell to improve correct classification. In future, this combined approach could establish mechanical phenotyping as equivalent to fluorescent labeling, or even identify subpopulations invisible to molecular labels.

Regulation of F-actin dynamics is key for multiple aspects of cell biology. Recent work indicates that this is particularly significant for cells to correctly sense the mechanical properties of the microenvironment, and to respond by regulating the YAP/TAZ transcriptional coactivators, which ultimately control cell behavior in response to mechanical cues. Yet, whether the connection between F-actin dynamics and YAP/TAZ signaling is relevant in mammalian tissues remains unaddressed. We will present data on a novel mouse knockout system enabling cell-autonomous regulation of F-actin dynamics and tissue mechanics, where we observe a potent upregulation of YAP/TAZ activity, leading to tissue hyperproliferation and organ overgrowth. This unveils an unprecedented function for the F-actin cytoskeleton in the regulation of mammalian tissue homeostasis.

The thrombogenicity of artificial materials comprising ventricular assist devices (VADs) limits their long-term integration in the human body. A living endothelium covering the luminal surface can provide a safe interface working compatibly with blood and circumventing this problem. However, the survival of endothelial cells is endangered by non-physiological hemodynamic conditions generated by VAD function, including high wall shear stress and deformation. Here, we introduce a surface topography comprising hexagonal honeycomb shelters in which cells remodel to generate coherently organized patterns of subcellular compartments. The resulting metasurface shows resistance to supraphysiological loads maintaining endothelium integrity and avoiding local discontinuities.

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