Topological defects in self-assembled patterns of mesenchymal stromal cells in vitro are predictive attributes of condensation and chondrogenesis.

Mesenchymal stromal cells (MSCs) are promising therapeutic agents for cartilage regeneration, including the potential of cells to promote chondrogenesis in vivo. However, process development and regulatory approval of MSCs as cell therapy products benefit from facile in vitro approaches that can predict potency for a given production run. Current standard in vitro approaches include a 21 day 3D differentiation assay followed by quantification of cartilage matrix proteins. We propose a novel biophysical marker that is cell population-based and can be measured from in vitro monolayer culture of MSCs. We hypothesized that the self-assembly pattern that emerges from collective-cell behavior would predict chondrogenesis motivated by our observation that certain features in this pattern, namely, topological defects, corresponded to mesenchymal condensations. Indeed, we observed a strong predictive correlation between the degree-of-order of the pattern at day 9 of the monolayer culture and chondrogenic potential later estimated from in vitro 3D chondrogenic differentiation at day 21. These findings provide the rationale and the proof-of-concept for using self-assembly patterns to monitor chondrogenic commitment of cell populations. Such correlations across multiple MSC donors and production batches suggest that self-assembly patterns can be used as a candidate biophysical attribute to predict quality and efficacy for MSCs employed therapeutically for cartilage regeneration.

Artificial axons as a biomimetic 3D myelination platform for the discovery and validation of promyelinating compounds.

Multiple sclerosis (MS), a chronic neurodegenerative disease driven by damage to the protective myelin sheath, is currently incurable. Today, all clinically available treatments modulate the immune-mediated symptoms of the disease but they fail to stop neurodegeneration in many patients. Remyelination, the regenerative process of myelin repair by oligodendrocytes, which is considered a necessary step to protect demyelinated axons and stop neuronal death, is impaired in MS patients. One of the major obstacles to finding effective remyelinating drugs is the lack of biomimetic drug screening platforms that enable quantification of compounds' potential to stimulate 3D myelination in the physiologically relevant axon-like environment. To address this need, we built a unique myelination drug discovery platform, by expanding our previously developed technology, artificial axons (AAs), which enables 3D-printing of synthetic axon mimics with the geometry and mechanical properties closely resembling those of biological axons. This platform allows for high-throughput phenotypic myelination assay based on quantification of 3D wrapping of myelin membrane around axons in response to compounds. Here, we demonstrate quantification of 3D myelin wrapping by rat oligodendrocytes around the axon mimics in response to a small library of known pro-myelinating compounds. This assay shows pro-myelinating activity for all tested compounds consistent with the published in vitro and in vivo data, demonstrating predictive power of AA platform. We find that stimulation of myelin wrapping by these compounds is dose-dependent, providing a facile means to quantify the compounds' potency and efficacy in promoting myelin wrapping. Further, the ranking of relative efficacy among these compounds differs in this 3D axon-like environment as compared to a traditional oligodendrocyte 2D differentiation assay quantifying area of deposited myelin membrane. Together, we demonstrate that the artificial axons platform and associated phenotypic myelin wrapping assay afford direct evaluation of myelin wrapping by oligodendrocytes in response to soluble compounds in an axon-like environment, providing a predictive tool for the discovery of remyelinating therapies.

Complex Oxides under Simulated Electric Field: Determinants of Defect Polarization in ABO3 Perovskites.

Polarization of ionic and electronic defects in response to high electric fields plays an essential role in determining properties of materials in applications such as memristive devices. However, isolating the polarization response of individual defects has been challenging for both models and measurements. Here the authors quantify the nonlinear dielectric response of neutral oxygen vacancies, comprised of strongly localized electrons at an oxygen vacancy site, in perovskite oxides of the form ABO3 . Their approach implements a computationally efficient local Hubbard U correction in density functional theory simulations. These calculations indicate that the electric dipole moment of this defect is correlated positively with the lattice volume, which they varied by elastic strain and by A-site cation species. In addition, the dipole of the neutral oxygen vacancy under electric field increases with increasing reducibility of the B-site cation. The predicted relationship among point defect polarization, mechanical strain, and transition metal chemistry provides insights for the properties of memristive materials and devices under high electric fields.

Stiffness of targeted layer-by-layer nanoparticles impacts elimination half-life, tumor accumulation, and tumor penetration.

Nanoparticle (NP) stiffness has been shown to significantly impact circulation time and biodistribution in anticancer drug delivery. In particular, the relationship between particle stiffness and tumor accumulation and penetration in vivo is an important phenomenon to consider in optimizing NP-mediated tumor delivery. Layer-by-layer (LbL) NPs represent a promising class of multifunctional nanoscale drug delivery carriers. However, there has been no demonstration of the versatility of LbL systems in coating systems with different stiffnesses, and little is known about the potential role of LbL NP stiffness in modulating in vivo particle trafficking, although NP modulus has been recently studied for its impact on pharmacokinetics. LbL nanotechnology enables NPs to be functionalized with uniform coatings possessing molecular tumor-targeting properties, independent of the NP core stiffness. Here, we report that the stiffness of LbL NPs is directly influenced by the mechanical properties of its underlying liposomal core, enabling the modulation and optimization of LbL NP stiffness while preserving LbL NP outer layer tumor-targeting and stealth properties. We demonstrate that the stiffness of LbL NPs has a direct impact on NP pharmacokinetics, organ and tumor accumulation, and tumor penetration-with compliant LbL NPs having longer elimination half-life, higher tumor accumulation, and higher tumor penetration. Our findings underscore the importance of NP stiffness as a design parameter in enhancing the delivery of LbL NP formulations.

Dissolvable Gelatin-Based Microcarriers Generated through Droplet Microfluidics for Expansion and Culture of Mesenchymal Stromal Cells.

Microcarriers are synthetic particles used in bioreactor-based cell manufacturing of anchorage-dependent cells to promote proliferation at efficient physical volumes, mainly by increasing the surface area-to-volume ratio. Mesenchymal stromal cells (MSCs) are adherent cells that are used for numerous clinical trials of autologous and allogeneic cell therapy, thus requiring avenues for large-scale cell production at efficiently low volumes and cost. Here, a dissolvable gelatin-based microcarrier is developed for MSC expansion. This novel microcarrier shows comparable cell attachment efficiency and proliferation rate when compared to several commercial microcarriers, but with higher harvesting yield due to the direct dissolution of microcarrier particles and thus reduced cell loss at the cell harvesting step. Furthermore, gene expression and in vitro differentiation suggest that MSCs cultured on gelatin microcarriers maintain trilineage differentiation with similar adipogenic differentiation efficiency and higher chondrogenic and osteogenic differentiation efficiency when compared to MSCs cultured on 2D planar polystyrene tissue culture flask; on the contrary, MSCs cultured on conventional microcarriers appear to be bipotent along osteochondral lineages whereby adipogenic differentiation potential is impeded. These results suggest that these gelatin microcarriers are suitable for MSC culture and expansion, and can also potentially be extended for other types of anchorage-dependent cells.

Localized characterization of brain tissue mechanical properties by needle induced cavitation rheology and volume controlled cavity expansion.

Changes in the elastic properties of brain tissue have been correlated with injury, cancers, and neurodegenerative diseases. However, discrepancies in the reported elastic moduli of brain tissue are persistent, and spatial inhomogeneities complicate the interpretation of macroscale measurements such as rheology. Here we introduce needle induced cavitation rheology (NICR) and volume-controlled cavity expansion (VCCE) as facile methods to measure the apparent Young's modulus E of minimally manipulated brain tissue, at specific tissue locations and with sub-millimeter spatial resolution. For different porcine brain regions and sections analyzed by NICR, we found E to be 3.7 ± 0.7 kPa and 4.8 ± 1.0 kPa for gray matter, and white matter, respectively. For different porcine brain regions and sections analyzed by VCCE, we found E was 0.76 ± 0.02 kPa for gray matter and 0.92 ± 0.01 kPa for white matter. Measurements from VCCE were more similar to those obtained from macroscale shear rheology (0.75 ± 0.06 kPa) and from instrumented microindentation of white matter (0.97 ± 0.40 kPa) and gray matter (0.86 ± 0.20 kPa). We attributed the higher stiffness reported from NICR to that method's assumption of a cavitation instability due to a neo-Hookean constitutive response, which does not capture the strain-stiffening behavior of brain tissue under large strains, and therefore did not provide appropriate measurements. We demonstrate via both analytical modeling of a spherical cavity and finite element modeling of a needle geometry, that this strain stiffening may prevent a cavitation instability. VCCE measurements take this stiffening behavior into account by employing an incompressible one-term Ogden model to find the nonlinear elastic properties of the tissue. Overall, VCCE afforded rapid and facile measurement of nonlinear mechanical properties of intact, healthy mammalian brain tissue, enabling quantitative comparison among brain tissue regions and also between species. Finally, accurate estimation of elastic properties for this strain stiffening tissue requires methods that include appropriate constitutive models of the brain tissue response, which here are represented by inclusion of the Ogden model in VCCE.

Mechanosensitivity of Human Oligodendrocytes.

Oligodendrocytes produce and repair myelin, which is critical for the integrity and function of the central nervous system (CNS). Oligodendrocyte and oligodendrocyte progenitor cell (OPC) biology is modulated in vitro by mechanical cues within the magnitudes observed in vivo. In some cases, these cues are sufficient to accelerate or inhibit terminal differentiation of murine oligodendrocyte progenitors. However, our understanding of oligodendrocyte lineage mechanobiology has been restricted primarily to animal models to date, due to the inaccessibility and challenges of human oligodendrocyte cell culture. Here, we probe the mechanosensitivity of human oligodendrocyte lineage cells derived from human induced pluripotent stem cells. We target phenotypically distinct stages of the human oligodendrocyte lineage and quantify the effect of substratum stiffness on cell migration and differentiation, within the range documented in vivo. We find that human oligodendrocyte lineage cells exhibit mechanosensitive migration and differentiation. Further, we identify two patterns of human donor line-dependent mechanosensitive differentiation. Our findings illustrate the variation among human oligodendrocyte responses, otherwise not captured by animal models, that are important for translational research. Moreover, these findings highlight the importance of studying glia under conditions that better approximate in vivo mechanical cues. Despite significant progress in human oligodendrocyte derivation methodology, the extended duration, low yield, and low selectivity of human-induced pluripotent stem cell-derived oligodendrocyte protocols significantly limit the scale-up and implementation of these cells and protocols for in vivo and in vitro applications. We propose that mechanical modulation, in combination with traditional soluble and insoluble factors, provides a key avenue to address these challenges in cell production and in vitro analysis.

Mechanical regulation of oligodendrocyte biology.

Oligodendrocytes (OL) are a subset of glial cells in the central nervous system (CNS) comprising the brain and spinal cord. The CNS environment is defined by complex biochemical and biophysical cues during development and response to injury or disease. In the last decade, significant progress has been made in understanding some of the key biophysical factors in the CNS that modulate OL biology, including their key role in myelination of neurons. Taken together, those studies offer translational implications for remyelination therapies, pharmacological research, identification of novel drug targets, and improvements in methods to generate human oligodendrocyte progenitor cells (OPCs) and OLs from donor stem cells in vitro. This review summarizes current knowledge of how various physical and mechanical cues affect OL biology and its implications for disease, therapeutic approaches, and generation of human OPCs and OLs.

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain.

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.

Emergent heterogeneity in putative mesenchymal stem cell colonies: Single-cell time lapsed analysis.

Bone marrow stromal cells (BMSCs) include a subset of stem cells that are considered promising for developmental studies and therapeutic applications. While it is appreciated generally that BMSC populations can exhibit morphological and functional heterogeneity upon in vitro culture expansion, the potential for heterogeneity within a single colony forming unit-generated ostensibly from a single mother cell-is less explored but is critical to design of both fundamental studies and cell therapy production. Here we observed BMSC colony formation in real time via time lapsed optical imaging and analysis, to quantify whether and how heterogeneity emerged over multiple cell divisions spanning the duration of a typical colony formation unit assay. These analyses demonstrate that such colonies are neither homogeneous subpopulations of stem cells nor necessarily derived from single originating cells. While the mechanisms for and causes of this intracolony heterogeneity are not understood fully, we further demonstrate that extensive cell-cell contacts do not correlate with senescence, but that media exchange was concurrent with diversification in even the most uniform single-cell-derived colonies. These direct quantitative observations and visualizations of colony formation provide new insights that are motivated by significant implications for both basic research and stem cell-based therapies.

Probing Mechanical Properties of Brain in a Tuberous Sclerosis Model of Autism.

Causes of autism spectrum disorders (ASD) are understood poorly, making diagnosis and treatment challenging. While many studies have investigated the biochemical and genetic aspects of ASD, whether and how mechanical characteristics of the autistic brain can modulate neuronal connectivity and cognition in ASD are unknown. Previously, it has been shown that ASD brains are characterized by abnormal white matter and disorganized neuronal connectivity; we hypothesized that these significant cellular-level structural changes may translate to changes in the mechanical properties of the autistic brain or regions therein. Here, we focused on tuberous sclerosis complex (TSC), a genetic disorder with a high penetrance of ASD. We investigated mechanical differences between murine brains obtained from control and TSC cohorts at various deformation length- and time-scales. At the microscale, we conducted creep-compliance and stress relaxation experiments using atomic force microscope(AFM)-enabled indentation. At the mesoscale, we conducted impact indentation using a pendulum-based instrumented indenter to extract mechanical energy dissipation metrics. At the macroscale, we used oscillatory shear rheology to quantify the frequency-dependent shear moduli. Despite significant changes in the cellular organization of TSC brain tissue, we found no corresponding changes in the quantified mechanical properties at every length- and time-scale explored. This investigation of the mechanical characteristics of the brain has broadened our understanding of causes and markers of TSC/ASD, while raising questions about whether any mechanical differences can be detected in other animal models of ASD or other disease models that also feature abnormal brain structure.

Improving hematopoietic recovery through modeling and modulation of the mesenchymal stromal cell secretome.

BACKGROUND: Efficient and sustained hematopoietic recovery after hematopoietic stem cell or bone marrow transplantation is supported by paracrine signaling from specific subpopulations of mesenchymal stromal cells (MSCs). Here, we considered whether in vitro mechanopriming of human MSCs could be administered to predictively and significantly improve in vivo hematopoietic recovery after irradiation injury. METHODS: First, we implemented regression modeling to identify eight MSC-secreted proteins that correlated strongly with improved rescue from radiation damage, including hematopoietic recovery, in a murine model of hematopoietic failure. Using these partial least squares regression (PLSR) model parameters, we then predicted recovery potential of MSC populations that were culture expanded on substrata of varying mechanical stiffness. Lastly, we experimentally validated these predictions using an in vitro co-culture model of hematopoiesis and using new in vivo experiments for the same irradiation injury model used to generate survival predictions. RESULTS: MSCs grown on the least stiff (elastic moduli ~ 1 kPa) of these polydimethylsiloxane (PDMS) substrata secreted high concentrations of key proteins identified in regression modeling, at concentrations comparable to those secreted by minor subpopulations of MSCs shown previously to be effective in supporting such radiation rescue. We confirmed that these MSCs expanded on PDMS could promote hematopoiesis in an in vitro co-culture model with hematopoietic stem and progenitor cells (HSPCs). Further, MSCs cultured on PDMS of highest stiffness (elastic moduli ~ 100 kPa) promoted expression of CD123+ HSPCs, indicative of myeloid differentiation. Systemic administration of mechanoprimed MSCs resulted in improved mouse survival and weight recovery after bone marrow ablation, as compared with both standard MSC expansion on stiffer materials and with biophysically sorted MSC subpopulations. Additionally, we observed recovery of white blood cells, platelets, and red blood cells, indicative of complete recovery of all hematopoietic lineages. CONCLUSIONS: These results demonstrate that computational techniques to identify MSC biomarkers can be leveraged to predict and engineer therapeutically effective MSC phenotypes defined by mechanoprimed secreted factors, for translational applications including hematopoietic recovery.

High-velocity micro-particle impact on gelatin and synthetic hydrogel.

The high-velocity impact response of gelatin and synthetic hydrogel samples is investigated using a laser-based microballistic platform for launching and imaging supersonic micro-particles. The micro-particles are monitored during impact and penetration into the gels using a high-speed multi-frame camera that can record up to 16 images with nanosecond time resolution. The trajectories are compared with a Poncelet model for particle penetration, demonstrating good agreement between experiments and the model for impact in gelatin. The model is further validated on a synthetic hydrogel and the applicability of the results is discussed. We find the strength resistance parameter in the Poncelet model to be two orders of magnitude higher than in macroscopic experiments at comparable impact velocities. The results open prospects for testing high-rate behavior of soft materials on the microscale and for guiding the design of drug delivery methods using accelerated microparticles.

Mechanical Strain Alters Cellular and Nuclear Dynamics at Early Stages of Oligodendrocyte Differentiation.

Mechanical and physical stimuli including material stiffness and topography or applied mechanical strain have been demonstrated to modulate differentiation of glial progenitor and neural stem cells. Recent studies probing such mechanotransduction in oligodendrocytes have focused chiefly on the biomolecular components. However, the cell-level biophysical changes associated with such responses remain largely unknown. Here, we explored mechanotransduction in oligodendrocyte progenitor cells (OPCs) during the first 48 h of differentiation induction by quantifying the biophysical state in terms of nuclear dynamics, cytoskeleton organization, and cell migration. We compared these mechanophenotypic changes in OPCs exposed to both chemical cues (differentiation factors) and mechanical cues (static tensile strain of 10%) with those exposed to only those chemical cues. We observed that mechanical strain significantly hastened the dampening of nuclear fluctuations and decreased OPC migration, consistent with the progression of differentiation. Those biophysical changes were accompanied by increased production of the intracellular microtubule network. These observations provide insights into mechanisms by which mechanical strain of physiological magnitude could promote differentiation of progenitor cells to oligodendrocytes via inducing intracellular biophysical responses over hours to days post induction.

Characterizing viscoelastic mechanical properties of highly compliant polymers and biological tissues using impact indentation.

Precise and accurate measurement of viscoelastic mechanical properties becomes increasingly challenging as sample stiffness decreases to elastic moduli <1 kPa, largely due to difficulties detecting initial contact with the compliant sample surface. This limitation is particularly relevant to characterization of biological soft tissues and compliant gels. Here, we employ impact indentation which, in contrast to shear rheology and conventional indentation, does not require contact detection a priori, and present a novel method to extract viscoelastic moduli and relaxation time constants directly from the impact response. We first validate our approach by using both impact indentation and shear rheology to characterize polydimethylsiloxane (PDMS) elastomers of stiffness ranging from 100 s of Pa to nearly 10 kPa. Assuming a linear viscoelastic constitutive model for the material, we find that the moduli and relaxation times obtained from fitting the impact response agree well with those obtained from fitting the rheological response. Next, we demonstrate our validated method on hydrated, biological soft tissues obtained from porcine brain, murine liver, and murine heart, and report the equilibrium shear moduli, instantaneous shear moduli, and relaxation time constants for each tissue. Together, our findings provide a new and straightforward approach capable of probing local mechanical properties of highly compliant viscoelastic materials with millimeter scale spatial resolution, mitigating complications involving contact detection or sample geometric constraints. STATEMENT OF SIGNIFICANCE: Characterization and optimization of mechanical properties can be essential for the proper function of biomaterials in diverse applications. However, precise and accurate measurement of viscoelastic mechanical properties becomes increasingly difficult with increased compliance (particularly for elastic moduli <1 kPa), largely due to challenges detecting initial contact with the compliant sample surface and measuring response at short timescale or high frequency. By contrast, impact indentation has highly accurate contact detection and can be used to measure short timescale (glassy) response. Here, we demonstrate an experimental and analytical method that confers significant advantages over existing approaches to extract spatially resolved viscoelastic moduli and characteristic time constants of biological tissues (e.g., brain and heart) and engineered biomaterials.

Engineered 3D-printed artificial axons.

Myelination is critical for transduction of neuronal signals, neuron survival and normal function of the nervous system. Myelin disorders account for many debilitating neurological diseases such as multiple sclerosis and leukodystrophies. The lack of experimental models and tools to observe and manipulate this process in vitro has constrained progress in understanding and promoting myelination, and ultimately developing effective remyelination therapies. To address this problem, we developed synthetic mimics of neuronal axons, representing key geometric, mechanical, and surface chemistry components of biological axons. These artificial axons exhibit low mechanical stiffness approaching that of a human axon, over unsupported spans that facilitate engagement and wrapping by glial cells, to enable study of myelination in environments reflecting mechanical cues that neurons present in vivo. Our 3D printing approach provides the capacity to vary independently the complex features of the artificial axons that can reflect specific states of development, disease, or injury. Here, we demonstrate that oligodendrocytes' production and wrapping of myelin depend on artificial axon stiffness, diameter, and ligand coating. This biofidelic platform provides direct visualization and quantification of myelin formation and myelinating cells' response to both physical cues and pharmacological agents.

Atomic Resolution Imaging of Nanoscale Chemical Expansion in PrxCe1-xO2-δ during In Situ Heating.

Thin film nonstoichiometric oxides enable many high-temperature applications including solid oxide fuel cells, actuators, and catalysis. Large concentrations of point defects (particularly, oxygen vacancies) enable fast ionic conductivity or gas exchange kinetics in these materials but also manifest as coupling between lattice volume and chemical composition. This chemical expansion may be either detrimental or useful, especially in thin film devices that may exhibit enhanced performance through strain engineering or decreased operating temperatures. However, thin film nonstoichiometric oxides can differ from bulk counterparts in terms of operando defect concentrations, transport properties, and mechanical properties. Here, we present an in situ investigation of atomic-scale chemical expansion in PrxCe1-xO2-δ (PCO), a mixed ionic-electronic conducting oxide relevant to electrochemical energy conversion and high-temperature actuation. Through a combination of electron energy loss spectroscopy and transmission electron microscopy with in situ heating, we characterized chemical strains and changes in oxidation state in cross sections of PCO films grown on yttria-stabilized zirconia (YSZ) at temperatures reaching 650 °C. We quantified, both statically and dynamically, the nanoscale chemical expansion induced by changes in PCO redox state as a function of position and direction relative to the film-substrate interface. Additionally, we observed dislocations at the film-substrate interface, as well as reduced cation localization to threading defects within PCO films. These results illustrate several key aspects of atomic-scale structure and mechanical deformation in nonstoichiometric oxide films that clarify distinctions between films and bulk counterparts and that hold several implications for operando chemical expansion or "breathing" of such oxide films.

Cell-Extracellular Matrix Mechanobiology: Forceful Tools and Emerging Needs for Basic and Translational Research.

Extracellular biophysical cues have a profound influence on a wide range of cell behaviors, including growth, motility, differentiation, apoptosis, gene expression, adhesion, and signal transduction. Cells not only respond to definitively mechanical cues from the extracellular matrix (ECM) but can also sometimes alter the mechanical properties of the matrix and hence influence subsequent matrix-based cues in both physiological and pathological processes. Interactions between cells and materials in vitro can modify cell phenotype and ECM structure, whether intentionally or inadvertently. Interactions between cell and matrix mechanics in vivo are of particular importance in a wide variety of disorders, including cancer, central nervous system injury, fibrotic diseases, and myocardial infarction. Both the in vitro and in vivo effects of this coupling between mechanics and biology hold important implications for clinical applications.

Temporal Variation in Single-Cell Power-Law Rheology Spans the Ensemble Variation of Cell Population.

Changes in the cytoskeletal organization within cells can be characterized by large spatial and temporal variations in rheological properties of the cell (e.g., the complex shear modulus G∗). Although the ensemble variation in G∗ of single cells has been elucidated, the detailed temporal variation of G∗ remains unknown. In this study, we investigated how the rheological properties of individual fibroblast cells change under a spatially confined environment in which the cell translational motion is highly restricted and the whole cell shape remains unchanged. The temporal evolution of single-cell rheology was probed at the same measurement location within the cell, using atomic force microscopy-based oscillatory deformation. The measurements reveal that the temporal variation in the power-law rheology of cells is quantitatively consistent with the ensemble variation, indicating that the cell system satisfies an ergodic hypothesis in which the temporal statistics are identical to the ensemble statistics. The autocorrelation of G∗ implies that the cell mechanical state evolves in the ensemble of possible states with a characteristic timescale.

Mechanical Strain Promotes Oligodendrocyte Differentiation by Global Changes of Gene Expression.

Differentiation of oligodendrocyte progenitor cells (OPC) to oligodendrocytes and subsequent axon myelination are critical steps in vertebrate central nervous system (CNS) development and regeneration. Growing evidence supports the significance of mechanical factors in oligodendrocyte biology. Here, we explore the effect of mechanical strains within physiological range on OPC proliferation and differentiation, and strain-associated changes in chromatin structure, epigenetics, and gene expression. Sustained tensile strain of 10-15% inhibited OPC proliferation and promoted differentiation into oligodendrocytes. This response to strain required specific interactions of OPCs with extracellular matrix ligands. Applied strain induced changes in nuclear shape, chromatin organization, and resulted in enhanced histone deacetylation, consistent with increased oligodendrocyte differentiation. This response was concurrent with increased mRNA levels of the epigenetic modifier histone deacetylase Hdac11. Inhibition of HDAC proteins eliminated the strain-mediated increase of OPC differentiation, demonstrating a role of HDACs in mechanotransduction of strain to chromatin. RNA sequencing revealed global changes in gene expression associated with strain. Specifically, expression of multiple genes associated with oligodendrocyte differentiation and axon-oligodendrocyte interactions was increased, including cell surface ligands (Ncam, ephrins), cyto- and nucleo-skeleton genes (Fyn, actinins, myosin, nesprin, Sun1), transcription factors (Sox10, Zfp191, Nkx2.2), and myelin genes (Cnp, Plp, Mag). These findings show how mechanical strain can be transmitted to the nucleus to promote oligodendrocyte differentiation, and identify the global landscape of signaling pathways involved in mechanotransduction. These data provide a source of potential new therapeutic avenues to enhance OPC differentiation in vivo.