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The Arp 2/3 complex, a nucleator of branched actin, is shown to provide multiple distinct mechanical advantages to migrating glioblastoma cells. These include both decreased substrate stress and increased parallel focal adhesion interactions, preventing failure of cell–-substrate interactions.

Introduction: Hypertension is a major risk factor for cardiovascular diseases including cardiac hypertrophy, stroke, and heart failure. Hypertension accelerates arterial stiffening noted with natural aging. Aortic stiffness has been shown to be both a cause and a consequence of isolated systolic hypertension. Thus, it is of high clinical interest to target arterial stiffening in the context of hypertension. We have previously identified lysyl oxidase-like 2 (LOXL2) as a potential therapeutic target for treating vascular stiffening. LOXL2 is a key enzyme in the extracellular matrix that catalyzes matrix deposition and remodeling. We have previously shown that LOXL2 depletion decelerates arterial stiffening during natural aging by modulating matrix remodeling and smooth muscle cell stiffness and contractility. Our hypothesis in this study is that LOXL2 depletion is protective against hypertension induced arterial stiffening, and this was determined via the established angiotensin II (Ang II) infusion model of experimental hypertension in LOXL2+/- mouse model. Methods and results: Ang II pumps were implanted in LOXL2+/- and WT mice for a 3-week treatment. Blood pressure and pulse wave velocity were measured noninvasively to assess hypertension and aortic stiffness. Results corroborated that Ang II infusion induced hypertension in WT and LOXL2+/- mice, and that arterial stiffening was ameliorated in LOXL2+/- mice even when Ang II-induced hypertension was present. Uniaxial tensile testing was used to test the elastic properties of the aortic rings, and wire myography was used to test their vasoreactivity. These experiments showed that the increase in arterial stiffness due to Ang II-induced hypertension was driven by both matrix remodeling and VSMC response. Histological analysis supported these findings, showing increased aortic wall thickness, interlamellar distance and collagen deposition with Ang II infusion. Elevated heart weight in mice with Ang II infusion and qPCR results revealed induced cardiac hypertrophy, which was not protected by LOXL2 knockdown. Moreover, human aortic SMC and endothelial cells were cyclically stretched, to show that the overexpression of LOXL2 in the aorta under Ang II-induced hypertension is upregulated by cyclic strain. Conclusion: Arterial stiffening is increased with Ang II infusion; however, it is ameliorated in LOXL2+/- mice compared to WT despite the development of Ang II-induced hypertension. This rise in arterial stiffness is driven by both matrix remodeling and VSMC response. Cardiac hypertrophy occurred with Ang II infusion and LOXL2 knockdown was not protective against it. Future studies will continue to 1) elucidate the mechanisms involved in the regulation of LOXL2 expression in response to Ang II-induced hypertension, 2) investigate the sex differences in in vivo stiffness and vasoreactivity, and 3) study LOXL2 as a potential therapeutic target against cardiac hypertrophy. Private foundation grant: NHLBI grant R01HL148112 01 (L.S.) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The neuromuscular junction (NMJ), which is a synapse for signal transmission from motor neurons to muscle cells, has emerged as an important region because of its association with several peripheral neuropathies. In particular, mutations in

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have been reported to exhibit immature embryonic or fetal cardiomyocyte-like phenotypes. To enhance the maturation of hESC-CMs, we identified a natural steroidal alkaloid, tomatidine, as a new substance that stimulates the maturation of hESC-CMs. Treatment of human embryonic stem cells with tomatidine during cardiomyocyte differentiation stimulated the expression of several cardiomyocyte-specific markers and increased the density of T-tubules. Furthermore, tomatidine treatment augmented the number and size of mitochondria and enhanced the formation of mitochondrial lamellar cristae. Tomatidine treatment stimulated mitochondrial functions, including mitochondrial membrane potential, oxidative phosphorylation, and ATP production, in hESC-CMs. Tomatidine-treated hESC-CMs were more sensitive to doxorubicin-induced cardiotoxicity than the control cells. In conclusion, the present study suggests that tomatidine promotes the differentiation of stem cells to adult cardiomyocytes by accelerating mitochondrial biogenesis and maturation and that tomatidine-treated mature hESC-CMs can be used for cardiotoxicity screening and cardiac disease modeling.

Heart disease is one of the largest burdens to human health worldwide and has very limited therapeutic options. Engineered three-dimensional (3D) vascularized cardiac tissues have shown promise in rescuing cardiac function in diseased hearts and may serve as a whole organ replacement in the future. One of the major obstacles in reconstructing these thick myocardial tissues to a clinically applicable scale is the integration of functional vascular networks capable of providing oxygen and nutrients throughout whole engineered constructs. Without perfusion of oxygen and nutrient flow throughout the entire engineered tissue not only is tissue viability compromised, but also overall tissue functionality is lost. There are many supporting technologies and approaches that have been developed to create vascular networks such as 3D bioprinting, co-culturing hydrogels, and incorporation of soluble angiogenic factors. In this state-of-the-art review, we discuss some of the most current engineered vascular cardiac tissues reported in the literature and future directions in the field. IMPACT STATEMENT: The field of cardiac tissue engineering is rapidly evolving and is now closer than ever to having engineered tissue models capable of predicting preclinical responses to therapeutics, modeling diseases, and being used as a means of rescuing cardiac function following injuries to the native myocardium. However, a major obstacle of engineering thick cardiac tissue remains to be the integration of functional vasculature. In this review, we highlight seminal and recently published works that have influenced and pushed the field of cardiac tissue engineering toward achieving vascularized functional tissues.

A nanopatterned poly(3,4-ethylenedioxythiophene) (PEDOT):Nafion composite layer integrated with interdigitated electrodes was developed to improve the device dynamic range and sensitivity for cellular impedance spectroscopy. The nanopattern fidelity to provide cellular alignment was accessed at different mixing volumes of PEDOT to Nafion. The ion transfer rate and electrical conductivity of Nafion were improved as the mixing ratio of PEDOT increased and it provided a uniform electrical path, thus giving conformable characteristics at all spectral frequencies from 1 kHz to 100 kHz for cellular impedance spectroscopy. Computational modeling was provided to extrapolate the electrical current flow and density in the composite with respect to the different frequency ranges. These results highlight that an electrically modified Nafion nanopattern interface, combined with interdigitated electrodes, can be used for various types of impedance-based cellular biosensors in a more biomimetic and sensitive manner.

Summary Research in low Earth orbit (LEO) has become more accessible. The 2020 Biomanufacturing in Space Symposium reviewed space-based regenerative medicine research and discussed leveraging LEO to advance biomanufacturing for regenerative medicine applications. The symposium identified areas where financial investments could stimulate advancements overcoming technical barriers. Opportunities in disease modeling, stem-cell-derived products, and biofabrication were highlighted. The symposium will initiate a roadmap to a sustainable market for regenerative medicine biomanufacturing in space. This perspective summarizes the 2020 Biomanufacturing in Space Symposium, highlights key biomanufacturing opportunities in LEO, and lays the framework for a roadmap to regenerative medicine biomanufacturing in space., Sharma and colleagues recap the 2020 Biomanufacturing in Space Symposium, which reviewed space-based regenerative medicine research and discussed opportunities to leverage low Earth orbit (LEO) to advance biomanufacturing for regenerative medicine. This perspective will highlight key biomanufacturing opportunities in LEO, note current technical gaps, and discuss next steps in developing a roadmap to biomanufacturing in space for regenerative medicine applications.

Microrobots that can be precisely guided to target lesions have been studied for in vivo medical applications. However, existing microrobots have challenges in vivo such as biocompatibility, biodegradability, actuation module, and intra- and postoperative imaging. This study reports microrobots visualized with real-time x-ray and magnetic resonance imaging (MRI) that can be magnetically guided to tumor feeding vessels for transcatheter liver chemoembolization in vivo. The microrobots, composed of a hydrogel-enveloped porous structure and magnetic nanoparticles, enable targeted delivery of therapeutic and imaging agents via magnetic guidance from the actuation module under real-time x-ray imaging. In addition, the microrobots can be tracked using MRI as postoperative imaging and then slowly degrade over time. The in vivo validation of microrobot system–mediated chemoembolization was demonstrated in a rat liver with a tumor model. The proposed microrobot provides an advanced medical robotic platform that can overcome the limitations of existing microrobots and current liver chemoembolization.

Vascular dysfunction is a common cause of cardiovascular diseases characterized by the narrowing and stiffening of arteries, such as atherosclerosis, restenosis, and hypertension. Arterial narrowing results from the aberrant proliferation of vascular smooth muscle cells (VSMCs) and their increased synthesis and deposition of extracellular matrix (ECM) proteins. These, in turn, are modulated by arterial stiffness, but the mechanism for this is not fully understood. We found that survivin (an inhibitor of apoptosis) is an important regulator of stiffness-mediated ECM synthesis and intracellular stiffness in VSMCs. Whole-transcriptome analysis and cell culture experiments showed that survivin expression is upregulated in injured femoral arteries in mice and in human VSMCs cultured on stiff fibronectin-coated hydrogels. Suppressed expression of survivin in human VSMCs and mouse embryonic fibroblasts decreased the stiffness-mediated expression of ECM components implicated in arterial stiffness, namely, collagen-I, fibronectin, and lysyl oxidase. By contrast, expression of these proteins was upregulated by the overexpression of survivin in human VSMCs cultured on soft hydrogels. Atomic force microscopy analysis showed that suppressed or enhanced expression of survivin decreases or increases intracellular stiffness, respectively. These findings suggest a novel mechanism by which survivin modulates arterial stiffness.

The therapeutic efficacy of stem cells transplanted into an ischaemic brain depends primarily on the responses of the neurovascular unit. Here, we report the development and applicability of a functional neurovascular unit on a microfluidic chip as a microphysiological model of ischaemic stroke that recapitulates the function of the blood-brain barrier as well as interactions between therapeutic stem cells and host cells (human brain microvascular endothelial cells, pericytes, astrocytes, microglia and neurons). We used the model to track the infiltration of a number of candidate stem cells and to characterize the expression levels of genes associated with post-stroke pathologies. We observed that each type of stem cell showed unique neurorestorative effects, primarily by supporting endogenous recovery rather than through direct cell replacement, and that the recovery of synaptic activities is correlated with the recovery of the structural and functional integrity of the neurovascular unit rather than with the regeneration of neurons.

Poly(dimethylsiloxane) (PDMS) is a commonly used polymer in organ-on-a-chip devices and microphysiological systems. However, due to its hydrophobicity and permeability, it absorbs drug compounds, preventing accurate drug screening applications. Here, we developed an effective and facile method to prevent the absorption of drugs by utilizing a PDMS-PEG block copolymer additive and drug pretreatment. First, we incorporated a PDMS-PEG block copolymer into PDMS to address its inherent hydrophobicity. Next, we addressed the permeability of PDMS by eliminating the concentration gradient via pretreatment of the PDMS with the drug prior to experimentally testing drug absorption. The combined use of a PDMS-PEG block copolymer with drug pretreatment resulted in a mean reduction of drug absorption by 91.6% in the optimal condition. Finally, we demonstrated that the proposed method can be applied to prevent drug absorption in a PDMS-based cardiac microphysiological system, enabling more accurate drug studies.

Tissue engineering with human induced pluripotent stem cell-derived cardiomyocytes enables unique opportunities for creating physiological models of the heart in vitro. However, there are few approaches available that can recapitulate the complex structure-function relationships that govern cardiac function at the macroscopic organ level. Here, we report a down-scaled, conical human 3D ventricular model with controllable cellular organization using multilayered, patterned cardiac sheets. Tissue engineered ventricles whose cardiomyocytes were pre-aligned parallel or perpendicular to the long axis outperformed those whose cardiomyocytes were angled or randomly oriented. Notably, the inner layers of perpendicular cardiac sheets realigned over 4 days into a parallel orientation, creating a helical transmural architecture, whereas minimal remodeling occurred in the parallel or angled sheets. Finite element analysis of engineered ventricles demonstrated that circumferential alignment leads to maximal perpendicular shear stress at the inner layer, whereas longitudinal orientation leads to maximal parallel stress. We hypothesize that cellular remodeling occurs to reduce perpendicular shear stresses in myocardium. This advanced platform provides evidence that physical forces such as shear stress drive self-organization of cardiac architecture.

Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of this data is obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) as a means to acquire in vivo estimates, directly from formalin fixed tissues, albeit with limited yield. Here we optimized each step of the xMD process including tissue retrieval, tissue transfer, film preparation, and RNA isolation to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, including the development of a non-crosslinked ethylene vinyl acetate (EVA) membrane, resulted in a 23-45 fold increase in miRNA yield, depending on cell type. By qPCR, miR-200a was increased 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143, relative to the matched non-dissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells.

Rationale: Myocardial infarction causes spatial variation in collagen organization and phenotypic diversity in fibroblasts, which regulate the heart’s ECM (extracellular matrix). The relationship between collagen structure and fibroblast phenotype is poorly understood but could provide insights regarding the mechanistic basis for myofibroblast heterogeneity in the injured heart. Objective: To investigate the role of collagen organization in cardiac fibroblast fate determination. Methods and Results: Biomimetic topographies were nanofabricated to recapitulate differential collagen organization in the infarcted mouse heart. Here, adult cardiac fibroblasts were freshly isolated and cultured on ECM topographical mimetics for 72 hours. Aligned mimetics caused cardiac fibroblasts to elongate while randomly organized topographies induced circular morphology similar to the disparate myofibroblast morphologies measured in vivo. Alignment cues also induced myofibroblast differentiation, as >60% of fibroblasts formed αSMA (α-smooth muscle actin) stress fibers and expressed myofibroblast-specific ECM genes like Postn (periostin). By contrast, random organization caused 38% of cardiac fibroblasts to express αSMA albeit with downregulated myofibroblast-specific ECM genes. Coupling topographical cues with the profibrotic agonist, TGFβ (transforming growth factor beta), additively upregulated myofibroblast-specific ECM genes independent of topography, but only fibroblasts on flat and randomly oriented mimetics had increased percentages of fibroblasts with αSMA stress fibers. Increased tension sensation at focal adhesions induced myofibroblast differentiation on aligned mimetics. These signals were transduced by p38-YAP (yes-associated protein)-TEAD (transcriptional enhanced associate domain) interactions, in which both p38 and YAP-TEAD (yes-associated protein transcriptional enhanced associate domain) binding were required for myofibroblast differentiation. By contrast, randomly oriented mimetics did not change focal adhesion tension sensation or enrich for p38-YAP-TEAD interactions, which explains the topography-dependent diversity in fibroblast phenotypes observed here. Conclusions: Spatial variations in collagen organization regulate cardiac fibroblast phenotype through mechanical activation of p38-YAP-TEAD signaling, which likely contribute to myofibroblast heterogeneity in the infarcted myocardium.

Recent advances in stem cell biology and tissue engineering have laid the groundwork for building complex tissues in a dish. We propose that these technologies are ready for a new challenge: recapitulating cardiac morphogenesis in vitro. In development, the heart transforms from a simple linear tube to a four-chambered organ through a complex process called looping. In this perspective, we re-examine heart tube looping through the lens of an engineer and argue that the linear heart tube is an advantageous starting point for tissue engineering. We summarize the structures, signaling pathways, and stresses in the looping heart, and evaluate approaches that could be used to build a linear heart tube and guide it through the process of looping.

Tissues- and organs-on-chips are microphysiological systems (MPSs) that model the architectural and functional complexity of human tissues and organs that is lacking in conventional cell monolayer cultures. While substantial progress has been made in a variety of tissues and organs, chips recapitulating immune responses have not advanced as rapidly. This review discusses recent progress in MPSs for the investigation of immune responses. To illustrate recent developments, we focus on two cases in point: immunocompetent tumor microenvironment-on-a-chip devices that incorporate stromal and immune cell components and pathomimetic modeling of human mucosal immunity and inflammatory crosstalk. More broadly, we discuss the development of systems immunology-on-a-chip devices that integrate microfluidic engineering approaches with high-throughput omics measurements and emerging immunological applications of MPSs.

Although great efforts have been undertaken to develop a nanoparticle-based drug delivery system (DDS) for the treatment of solid tumors, the therapeutic outcomes are still limited. Immune cells, which possess an intrinsic ability to phagocytose nanoparticles and are recruited by tumors, can be exploited to deliver nanotherapeutics deep inside the tumors. Photothermal therapy using near-infrared light is a promising noninvasive approach for solid tumor ablation, especially when combined with chemotherapy. In this study, we design and evaluate a macrophage-based, multiple nanotherapeutics DDS, involving the phagocytosis by macrophages of both small-sized gold nanorods and anticancer drug-containing nanoliposomes. The aim is to treat solid tumors, utilizing the tumor-infiltrating properties of macrophages with synergistic photothermal-chemotherapy. Using a 3D cancer spheroid as an in vitro solid tumor model, we show that tumor penetration and coverage of the nanoparticles are both markedly enhanced when the macrophages are used. In addition, in vivo experiments involving both local and systemic administrations in breast tumor-bearing mice demonstrate that the proposed DDS can effectively target and kill the tumors, especially when the synergistic therapy is used. Consequently, this immune cell-based theranostic strategy may represent a potentially important advancement in the treatment of solid tumors.

Skeletal muscle atrophy is a well-known consequence of spaceflight. Because of the potential significant impact of muscle atrophy and muscle dysfunction on astronauts and their mission, a thorough understanding of the mechanisms of this atrophy and the development of effective countermeasures is critical. Spaceflight-induced muscle atrophy is similar to atrophy seen in many terrestrial conditions, and therefore our understanding of this form of atrophy may also contribute to the treatment of atrophy in humans on Earth. The unique environmental features humans encounter in space include the weightlessness of microgravity, space radiation, and the distinctive aspects of living in a spacecraft. The disuse and unloading of muscles in microgravity are likely the most significant factors that mediate spaceflight-induced muscle atrophy and have been extensively studied and reviewed. However, there are numerous other direct and indirect effects on skeletal muscle that may be contributing factors to the muscle atrophy and dysfunction seen as a result of spaceflight. This review offers a novel perspective on the issue of muscle atrophy in space by providing a comprehensive overview of the unique aspects of the spaceflight environment and the various ways in which they can lead to muscle atrophy. We systematically review the potential contributions of these different mechanisms of spaceflight-induced atrophy and include findings from both actual spaceflight and ground-based models of spaceflight in humans, animals, and in vitro studies.

The crawling behavior of leukocytes is driven by the cell morphology transition, which is a direct manifestation of molecular motor machinery. The topographical anisotropy and mechanical stiffness of the substrates are the main physical cues that affect leukocytes' shape generation and migratory responses. However, their combined effects on the cell morphology and motility have been poorly understood, particularly for neutrophils, which are the fastest reacting leukocytes against infections and wounds. Here, spatiotemporally correlated physical parameters are shown, which determine the neutrophil shape change during migratory processes, in response to surface topography and elasticity. Guided crawling and shape generation of individual neutrophils, activated by a uniform concentration of a chemoattractant, are analyzed by adopting elasticity-tunable micropatterning and live cell imaging techniques. Whole cell-level image analysis is performed based on a planar geometric quantification of cell shape and motility. The findings show that the pattern anisotropy and elastic modulus of the substrate induce synergic effects on the shape anisotropy, deformability, and polarization/alignment of crawling neutrophils. How the morphology-motility relationship is affected by different surface microstructures and stiffness is demonstrated. These results imply that the neutrophil shape-motility correlations can be utilized for controlling the immune cell functions with predefined physical microenvironments.

Summary: Background: Thromboembolism is a life-threatening manifestation of coronavirus disease 2019 (COVID-19). We investigated a dysfunctional phenotype of vascular endothelial cells in the lungs during COVID-19. Methods: We obtained the lung specimens from the patients who died of COVID-19. The phenotype of endothelial cells and immune cells was examined by flow cytometry and immunohistochemistry (IHC) analysis. We tested the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the endothelium using IHC and electron microscopy. Findings: The autopsy lungs of COVID-19 patients exhibited severe coagulation abnormalities, immune cell infiltration, and platelet activation. Pulmonary endothelial cells of COVID-19 patients showed increased expression of procoagulant von Willebrand factor (VWF) and decreased expression of anticoagulants thrombomodulin and endothelial protein C receptor (EPCR). In the autopsy lungs of COVID-19 patients, the number of macrophages, monocytes, and T cells was increased, showing an activated phenotype. Despite increased immune cells, adhesion molecules such as ICAM-1, VCAM-1, E-selectin, and P-selectin were downregulated in pulmonary endothelial cells of COVID-19 patients. Notably, decreased thrombomodulin expression in endothelial cells was associated with increased immune cell infiltration in the COVID-19 patient lungs. There were no SARS-CoV-2 particles detected in the lung endothelium of COVID-19 patients despite their dysfunctional phenotype. Meanwhile, the autopsy lungs of COVID-19 patients showed SARS-CoV-2 virions in damaged alveolar epithelium and evidence of hypoxic injury. Interpretation: Pulmonary endothelial cells become dysfunctional during COVID-19, showing a loss of thrombomodulin expression related to severe thrombosis and infiltration, and endothelial cell dysfunction might be caused by a pathologic condition in COVID-19 patient lungs rather than a direct infection with SARS-CoV-2. Funding: This work was supported by the Johns Hopkins University, the American Heart Association, and the National Institutes of Health.

Heart disease is the leading cause of death worldwide and imposes a significant burden on healthcare systems globally. A major hurdle to the development of more effective therapeutics is the reliance on animal models that fail to faithfully recapitulate human pathophysiology. The predictivity of in vitro models that lack the complexity of in vivo tissue remain poor as well. To combat these issues, researchers are developing organ-on-a-chip models of the heart that leverage the use of human induced pluripotent stem cell-derived cardiomyocytes in combination with novel platforms engineered to better recapitulate tissue- and organ-level physiology. The integration of novel biosensors into these platforms is also a critical step in the development of these models, as they allow for increased throughput, real-time and longitudinal phenotypic assessment, and improved efficiency during preclinical disease modeling and drug screening studies. These platforms hold great promise for both improving our understanding of heart disease as well as for screening potential therapeutics based on clinically relevant endpoints with better predictivity of clinical outcomes. In this review, we describe state-of-the-art heart-on-a-chip platforms, the integration of novel biosensors into these models for real-time and continual monitoring of tissue-level physiology, as well as their use for modeling heart disease and drug screening applications. We also discuss future perspectives and further advances required to enable clinical trials-on-a-chip and next-generation precision medicine platforms.

Matrix nanotopographical cues are known to regulate the structure and function of somatic cells derived from human pluripotent stem cell (hPSC) sources. High-throughput electrophysiological analysis of excitable cells derived from hPSCs is possible via multielectrode arrays (MEAs), but conventional MEA platforms use flat substrates and do not reproduce physiologically-relevant tissue-specific architecture. To address this issue, we developed a high-throughput nanotopographically-patterned multielectrode array (nanoMEA) by integrating conductive, ion-permeable, nanotopographic patterns with 48-well MEA plates, and investigated the effect of substrate-mediated cytoskeletal organization on hPSC-derived cardiomyocyte and neuronal function at scale. Using our nanoMEA platform, we found patterned hPSC-derived cardiac monolayers exhibit both enhanced structural organization and greater sensitivity to treatment with calcium blocking or conduction inhibiting compounds when subjected to high-throughput dose-response studies. Similarly, hPSC-derived neurons grown on nanoMEA substrates exhibit faster migration and neurite outgrowth speeds, greater co-localization of pre- and post-synaptic markers, and enhanced cell-cell communication, only revealed through examination of data sets derived from multiple technical replicates. The presented data highlight the nanoMEA as a new tool to facilitate high-throughput, electrophysiological analysis of ordered cardiac and neuronal monolayers, which can have important implications for preclinical analysis of excitable cell function.

Charcot-Marie-Tooth disease type 2D (CMT2D), is a hereditary peripheral neuropathy caused by mutations in the gene encoding glycyl-tRNA synthetase (GARS1). Here, human induced pluripotent stem cell (hiPSC)-based models of CMT2D bearing mutations in GARS1 and their use for the identification of predictive biomarkers amenable to therapeutic efficacy screening is described. Cultures containing spinal cord motor neurons generated from this line exhibited network activity marked by significant deficiencies in spontaneous action potential firing and burst fire behavior. This result matched clinical data collected from a patient bearing a GARS1(P724H) mutation and was coupled with significant decreases in acetylated α-tubulin levels and mitochondrial movement within axons. Treatment with HDAC6 inhibitors, tubastatin A and CKD504, improved mitochondrial movement and α-tubulin acetylation in these cells. Furthermore, CKD504 treatment enhanced population-level electrophysiological activity, highlighting its potential as an effective treatment for CMT2D.

In contrast to microfluidic devices, bulky syringe pumps are widely used to deliver a small amount of solution with high accuracy. Miniaturizing the syringe pump is difficult due to the scale effect in the microscale where the friction of the piston–cylinder is dominant and there are few high-power microactuators. To solve these problems, an on-chip microsyringe pump without mechanical sliding parts and with high power sources is proposed. The microsyringe pump utilizes the interface between water and oil (electro-conjugate fluid, ECF) instead of a piston and an electrohydrodynamic (EHD) flow driven by ECF in place of a linear actuator. ECF as a functional fluid has two capabilities: a) making the water–oil interface in microchannels and b) generating an active ECF flow at an applied voltage to withdraw and infuse aqueous solution by the interface. To control the flow direction, ECF-driven leakless on/off microvalves are also integrated. It is demonstrated that the proposed ECF microsyringe pump synchronized with the ECF on/off microvalves can control the withdrawing and infusing of aqueous solution with high resolution and precision. The experiments prove the feasibility of the microsyringe pump to be embedded as a module for the precise and linear control of flow rates in microfluidic devices.

Water droplets sitting between wires are ubiquitous in nature and industry, often showing irregular (non-spherical) droplet shapes. To understand their topological singularity and evaporation mechanism, measuring volume changes of irregular water droplets is essential but highly challenging for small-volume water droplets. Here we experimentally explore topological heterogeneity and evaporation dynamics for irregular water droplets between wires with four-dimensional X-ray microtomography that directly provides images in three spatial dimensions as a function of time, enabling us to get three-dimensional structural and geometric information changes with time. We find that the topological heterogeneity of an irregular droplet is due to the local contact lines and the evaporation dynamics of an irregular droplet is governed by the effective contact radius. This study may offer an opportunity to understand how the topological heterogeneity contributes to the evaporation dynamics of irregular water droplets.

Gene editing technologies hold great potential to enhance our ability to model inheritable neurodegenerative diseases. Specifically, engineering multiple amyotrophic lateral sclerosis (ALS) mutations into isogenic cell populations facilitates determination of whether different causal mutations cause pathology via shared mechanisms, and provides the capacity to separate these mechanisms from genotype-specific effects. As gene-edited, cell-based models of human disease become more commonplace, there is an urgent need to verify that these models constitute consistent and accurate representations of native biology. Here, commercially sourced, induced pluripotent stem cell-derived motor neurons from Cellular Dynamics International, edited to express the ALS-relevant mutations TDP-43M337V and TDP-43Q331K were compared with in-house derived lines engineered to express the TDP-43Q331K mutation within the WTC11 background. Our results highlight electrophysiological and mitochondrial deficits in these edited cells that correlate with patient-derived cells, suggesting a consistent cellular phenotype arising from TDP-43 mutation. However, significant differences in the transcriptomic profiles and splicing behavior of the edited cells underscores the need for careful comparison of multiple lines when attempting to use these cells as a means to better understand the onset and progression of ALS in humans.

Author's summary Cardiovascular disease is a prevalent cause of mortality and morbidity, largely due to the limited ability of cardiomyocytes to proliferate. Existing therapies for cardiac regeneration include cell-based therapies and bioactive molecules. However, delivery remains one of the major challenges impeding such therapies from having significant clinical impact. Recent advancements in biomaterials-based approaches for cardiac regeneration have shown promise in improving cardiac function, promoting angiogenesis, and reducing adverse immune response in both human clinical trials and animal studies. These advances in therapeutic delivery via extracellular vesicles, cardiac patches, and hydrogels have the potential to enable clinical impact of cardiac regeneration therapies., The limited ability of cardiomyocytes to proliferate is a major cause of mortality and morbidity in cardiovascular diseases. There exist therapies for cardiac regeneration that are cell-based as well as that involve bioactive molecules. However, delivery remains one of the major challenges impeding such therapies from having clinical impact. Recent advancements in biomaterials-based approaches for cardiac regeneration have shown promise in clinical trials and animal studies in improving cardiac function, promoting angiogenesis, and reducing adverse immune response. This review will focus on current clinical studies of three contemporary biomaterials-based approaches for cardiac regeneration (extracellular vesicles, injectable hydrogels, and cardiac patches), remaining challenges and shortcomings to be overcome, and future directions for the use of biomaterials to promote cardiac regeneration.

In humankind’s endeavor to explore beyond our planet and travel further into space, we are now at the threshold of an era in which it is possible to move to and from low Earth orbit (LEO) with increasing ease and reduced cost. Through the International Space Station (ISS) U.S. National Laboratory, investigators from industry, academia, and government can easily access the unique LEO environment on the ISS to conduct research and development (R&D) activities in ways not possible on Earth. A key advantage of the LEO environment for life sciences research is the ability to conduct experiments in sustained microgravity conditions. The ability to conduct long-term research in microgravity enables opportunities for novel, fundamental studies in tissue engineering and regenerative medicine, including research on stem cell proliferation and differentiation, biofabrication, and disease modeling using microphysiological systems (MPS) that build on prior research using simulated microgravity conditions (Grimm, D., et al. 2018). Over the last decade, space-based research has demonstrated that microgravity informs our knowledge of fundamental biology and accelerates advancements in health care and medical technologies (International Space Station 2019). The benefits provided by conducting biomedical research in LEO may lead to breakthroughs not achievable on Earth. We are now at a transition point, in which nations are changing their approach to space-based R&D. The focus is shifting from government-funded fundamental science toward the expansion of privately funded R&D with terrestrial application and economic value that will drive a robust marketplace for innovation and manufacturing in LEO. Making this long-term transition requires public-private participation and near-term funding to support critical R&D to leverage the benefits of the LEO environment and de-risk space-based research. Studies conducted on the ISS over the past several years have indicated that one area with potential significant economic value and benefit to life on Earth is space-based biomanufacturing, or the use of biological and nonbiological materials to produce commercially relevant biomolecules and biomaterials for use in preclinical, clinical, and therapeutic applications. We must take advantage of the remaining lifetime of the ISS as a valuable LEO platform to demonstrate this economic value and Earth benefit. By facilitating access to the space station, the ISS National Lab is uniquely positioned to enable the R&D necessary to bridge the gap between the initial discovery phase of space-based biomedical research and the development of a sustainable, investment-worthy biomanufacturing market in LEO supported by future commercial platforms. Through a joint effort, the Center for the Advancement of Science in Space (CASIS), which manages the ISS National Lab, and the University of Pittsburgh’s McGowan Institute for Regenerative Medicine brought together thought leaders from around the U.S. for a Biomanufacturing in Space Symposium that consisted of a series of working sessions to review data from past space-based tissue engineering and regenerative medicine research, discuss relevant current space-based R&D in this area, and consider potential future markets to address the questions: What are the most promising opportunities to leverage the ISS to advance space-based biomanufacturing moving forward? What are the current gaps or barriers that, if overcome, could clear pathways toward private investment in LEO as a valued site for research, development, and production activity? And, most importantly: For which opportunities do the most compelling value propositions exist? The goal of the Biomanufacturing in Space Symposium was to help identify the specific areas in which government and industry investment would be most likely to stimulate advancements that overcome barriers. This would lead to a more investment-ready landscape for private interests to enter the market and fuel exponential growth. The symposium was meant to serve as the first step in developing a roadmap to a sustainable market for biomanufacturing in space. The symposium identified and prioritized multiple key R&D opportunities to advance space-based biomanufacturing. These opportunities fall in the areas of disease modeling, stem cells and stem-cell-derived products, and biofabrication. Additionally, symposium participants highlighted the critical need for additional data to help validate and de-risk these opportunities and concluded that approaches such as automation, artificial intelligence (AI), and machine learning will be needed to produce and capture the required data. Symposium participants also came to a consensus that public-private partnerships and funding will be needed to advance the opportunities toward a biomanufacturing marketplace in LEO. This paper will summarize the current state of the science and technology on the ISS and in the fields of tissue engineering and regenerative medicine; provide an overview of biomanufacturing R&D in space to date; review the goals of the Biomanufacturing in Space Symposium; highlight the key commercial opportunities and gaps identified during the symposium; provide information on potential market sizes; and briefly discuss the next steps in developing a roadmap to biomanufacturing in space.

Bio-sourced and biodegradable polymers for additive manufacturing could enable the rapid fabrication of parts for a broad spectrum of applications ranging from healthcare to aerospace. However, a limited number of these materials are suitable for vat photopolymerization processes. Herein, we report a two-step additive manufacturing process to fabricate robust protein-based constructs using a commercially available laser-based SLA printer. Methacrylated bovine serum albumin (MA-BSA) was synthesized and formulated into aqueous resins that were used to print complex 3D objects with a resolution comparable to a commercially available resin. The MA-BSA resins were characterized by rheometry to determine the viscosity and the cure rate, as both of these parameters can ultimately be used to predict the printability of the resin. In the first step of patterning these materials, the MA-BSA resin was 3D printed, and in the second step, the printed construct was thermally cured to denature the globular protein and increase the intermolecular noncovalent interactions. Thus, the final 3D printed part was comprised of both chemical and physical cross-links. Compression studies of hydrated and dehydrated constructs demonstrated a broad range of compressive strengths and Young’s moduli that could be further modulated by adjusting the type and amount of co-monomer. The printed hydrogel constructs demonstrated good cell viability (> 95%) after a 21-day culture period. These MA-BSA resins are expected to be compatible with other vat photopolymerization techniques including digital light projection (DLP) and continuous liquid interface production (CLIP).

Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX. Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.

Mozzetta and Tedesco preview work from the Murry laboratory yielding insight into cardiac laminopathy pathogenesis mechanisms by analyzing chromatin compartment dynamics in a haploinsufficient model of the disease., Lamins A and C are intermediate filaments that provide structural support to the nuclear envelope and regulate gene expression. In this issue, Bertero et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201902117) report that although lamin A/C haploinsufficient cardiomyocytes show disease-associated phenotypes, those changes cannot be explained by alterations in chromatin compartmentalization.

Collective cell migration occurs in many patho-physiological states, including wound healing and invasive cancer growth. The integrity of the expanding epithelial sheets depends on extracellular cues, including cell-cell and cell-matrix interactions. We show that the nano-scale topography of the extracellular matrix underlying epithelial cell layers can strongly affect the speed and morphology of the fronts of the expanding sheet, triggering partial and complete epithelial-mesenchymal transitions (EMTs). We further demonstrate that this behavior depends on the mechano-sensitivity of the transcription regulator YAP and two new YAP-mediated cross-regulating feedback mechanisms: Wilms Tumor-1-YAP-mediated downregulation of E-cadherin, loosening cell-cell contacts, and YAP-TRIO-Merlin mediated regulation of Rho GTPase family proteins, enhancing cell migration. These YAP-dependent feedback loops result in a switch-like change in the signaling and the expression of EMT-related markers, leading to a robust enhancement in invasive cell spread, which may lead to a worsened clinical outcome in renal and other cancers., Reorganisation of the extracellular matrix (ECM) controls processes involving epithelial-mesenchymal transition (EMT). Here, the authors show that EMT occurring in epithelial cells on a fabricated nano-engineered cell adhesion surface is triggered by mechanical cues from the ECM.

Aims Heart failure invariably affects patients with various forms of muscular dystrophy (MD), but the onset and molecular sequelae of altered structure and function resulting from full-length dystrophin (Dp427) deficiency in MD heart tissue are poorly understood. To better understand the role of dystrophin in cardiomyocyte development and the earliest phase of Duchenne muscular dystrophy (DMD) cardiomyopathy, we studied human cardiomyocytes differentiated from induced pluripotent stem cells (hiPSC-CMs) obtained from the urine of a DMD patient. Methods and results The contractile properties of patient-specific hiPSC-CMs, with no detectable dystrophin (DMD-CMs with a deletion of exon 50), were compared to CMs containing a CRISPR-Cas9 mediated deletion of a single G base at position 263 of the dystrophin gene (c.263delG-CMs) isogenic to the parental line of hiPSC-CMs from a healthy individual. We hypothesized that the absence of a dystrophin-actin linkage would adversely affect myofibril and cardiomyocyte structure and function. Cardiomyocyte maturation was driven by culturing long-term (80–100 days) on a nanopatterned surface, which resulted in hiPSC-CMs with adult-like dimensions and aligned myofibrils. Conclusions Our data demonstrate that lack of Dp427 results in reduced myofibril contractile tension, slower relaxation kinetics, and to Ca2+ handling abnormalities, similar to DMD cells, suggesting either retarded or altered maturation of cardiomyocyte structures associated with these functions. This study offers new insights into the functional consequences of Dp427 deficiency at an early stage of cardiomyocyte development in both patient-derived and CRISPR-generated models of dystrophin deficiency.

Visual Abstract, Highlights • Many cardiomyopathy families have genetic variants whose significance is unknown. We studied a novel (E848G) mutation in MYH7, a sarcomeric protein. • Patient-specific induced pluripotent stem cell–derived cardiomyocytes and engineered heart tissues recapitulated the contractile dysfunction. • Overexpression of the E848G allele in MYH7-null induced pluripotent stem cell–derived cardiomyocytes confirms the causality of the E848G variant. • The E848G allele disrupts the protein–protein interaction between MYH7 and cardiac myosin binding protein C, presenting a potential mechanism of action. • Assessing the pathogenicity of new MYH7 variants by overexpressing them in a null background should accelerate their screening for disease causality., Summary A novel myosin heavy chain 7 mutation (E848G) identified in a familial cardiomyopathy was studied in patient-specific induced pluripotent stem cell–derived cardiomyocytes. The cardiomyopathic human induced pluripotent stem cell–derived cardiomyocytes exhibited reduced contractile function as single cells and engineered heart tissues, and genome-edited isogenic cells confirmed the pathogenic nature of the E848G mutation. Reduced contractility may result from impaired interaction between myosin heavy chain 7 and cardiac myosin binding protein C.

SUMMARYThe gut microbiota benefits the host by limiting enteric pathogen expansion (colonization resistance) partially via the production of inhibitory metabolites. Propionate, a short-chain fatty acid produced by microbiota members, is proposed to mediate colonization resistance against Salmonella enterica serovar Typhimurium (S. Tm). Here, we show that S. Tm overcomes the inhibitory effects of propionate by using it as a carbon source for anaerobic respiration. We determined that propionate metabolism provides an inflammation-dependent colonization advantage to S. Tm during infection. Such benefit was abolished in the intestinal lumen of Salmonella-infected germ-free mice. Interestingly, S. Tm propionate-mediated intestinal expansion was restored when germ-free mice were monocolonized with Bacteroides thetaiotaomicron (B. theta), a prominent propionate producer in the gut, but not when mice were monocolonized with a propionate production-deficient B. theta strain. Taken together, our results reveal a novel strategy used by S. Tm to mitigate colonization resistance by metabolizing microbiota-derived propionate.

In this paper, we report a simple and facile method to fabricate nanomolded Nafion thin films with tunable mechanical, and electrical properties. To achieve this, we combine a novel thermal evaporation-induced capillary force lithography method with swelling process to obtain enhanced pattern fidelity in nanomolded Nafion films. We demonstrate that structural fidelity and mechanical properties of patterned Nafion thin films can be modulated by changing fabrication parameters such as swelling time, Nafion polymer concentration, and curing temperature. Interestingly, we also find that impedance properties of nanomolded Nafion thin films are associated with the Nafion polymer concentration and curing temperature. In particular, 20% Nafion thin films exhibit greater impedance stability and lower impedance values than 5% Nafion thin films at lower frequencies. Moreover, curing temperature-specific impedance changes are observed. These results suggest that capillary lithography can be used to fabricate Nafion nanostructures with high pattern fidelity capable of modifying mechanical and electrical properties of Nafion thin films.

Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post-translational cancer hallmark and the consequences thereof remain elusive. Here we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In both cancer cell cultures and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes revealed that fucosylation of the antioxidant PON1 is a critical component of the therapy-induced secretome. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Non-specific and PON1-specific secretome de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.

Stem cell therapy is emerging as a promising treatment option to restore a neurological function after ischemic stroke. Despite the growing number of candidate stem cell types, each with unique characteristics, there is a lack of experimental platform to systematically evaluate their neurorestorative potential. When stem cells are transplanted into ischemic brain, the therapeutic efficacy primarily depends on the response of the neurovascular unit (NVU) to these extraneous cells. In this work, we developed an ischemic stroke microphysiological system (MPS) with a functional NVU on a microfluidic chip. Our new chip design facilitated the incorporated cells to form a functional blood-brain barrier (BBB) and restore their in vivo-like behaviors in both healthy and ischemic conditions. We utilized this MPS to track the transplanted stem cells and characterize their neurorestorative behaviors reflected in gene expression levels. Each type of stem cells showed unique neurorestorative effects, primarily through supporting the endogenous recovery, rather than through direct cell replacement. And the recovery of synaptic activities, critical for neurological function, was more tightly correlated with the recovery of the structural and functional integrity in NVU, rather than with the regeneration of neurons itself.

Depth estimation using RGB images plays an important role in many computer vision applications, such as pose tracking for navigation, computational photography, and 3D reconstruction. Depth estimation using a single camera has relatively low accuracy compared to that using a conventional stereo camera system and time-of-flight (ToF) sensor. Nowadays, cameras in smartphone have the optical image stabilization system and can rotate to improve image quality such as image stabilization and image deblur. In this paper, we propose a novel depth estimation technique using a single camera equipped with tilting mechanism. Instead of motion compensation for optical image stabilization, a typical usage of tilting mechanism, we exploit it to capture multi-view images from a single viewpoint, by rotating the camera module in varying angles. The captured images have inside-out views, which is more challenging in depth estimation than out-side in views. We train a network based on structure-from-motion algorithm for depth estimation of a scene to achieve high accuracy depth maps. A synthetic dataset is created from 3D indoor models to train and validate the network. Evaluation results demonstrate that we achieve state-of-the-art performance in depth estimation using a single camera.

Point-of-View videos recorded by Augmented Reality Glasses contain jitters because they are acquired under users’ actions in varying environments. Applying video stabilization on such videos is difficult due to weakness of conventional keypoint-based motion estimation to environmental conditions. They are prone to fail to track in low-textured or dark environments. To overcome this limitation, we propose a neural network-based motion estimation method for video stabilization. Our network predicts frame-to-frame motion in high accuracy by focusing on global camera motion, while ignoring local motion caused by moving objects. Motion prediction takes only up to 10 ms so that we achieve real-time stabilization on modern smartphones hardware. We demonstrate our method outperforms keypoint-based motion estimation and the quality of estimated motion is good enough for video stabilization. Our network is trainable without ground truth and easily scalable to large datasets.