Intramuscular injection of mesenchymal stem cells augments basal muscle protein synthesis after bouts of resistance exercise in male mice.

Skeletal muscle mass is critical for activities of daily living. Resistance training maintains or increases muscle mass, and various strategies maximize the training adaptation. Mesenchymal stem cells (MSCs) are multipotent cells with differential potency in skeletal muscle cells and the capacity to secrete growth factors. However, little is known regarding the effect of intramuscular injection of MSCs on basal muscle protein synthesis and catabolic systems after resistance training. Here, we measured changes in basal muscle protein synthesis, the ubiquitin-proteasome system, and autophagy-lysosome system-related factors after bouts of resistance exercise by intramuscular injection of MSCs. Mice performed three bouts of resistance exercise (each consisting of 50 maximal isometric contractions elicited by electrical stimulation) on the right gastrocnemius muscle every 48 h, and immediately after the first bout, mice were intramuscularly injected with either MSCs (2.0 × 106 cells) labeled with green fluorescence protein (GFP) or vehicle only placebo. Seventy-two hours after the third exercise bout, GFP was detected only in the muscle injected with MSCs with concomitant elevation of muscle protein synthesis. The injection of MSCs also increased protein ubiquitination. These results suggest that the intramuscular injection of MSCs augmented muscle protein turnover at the basal state after consecutive resistance exercise.

Simultaneous detection of the shuttling motion of liquid metal droplets in channels under alternating pressure and capacitive sensor signals.

Implementing a signal-switching mechanism for the selective use of integrated sensors and actuators plays a crucial role in streamlining the functionality of miniaturized devices. Here, a liquid metal droplet (LMD)-based signal-switching mechanism is introduced to achieve such functionality. Pressure modulation with a 100-µm spatial resolution enabled precise control of the position of the LMDs within a channel. After integrating the channel with asymmetrically structured electrodes, the effect of the shuttle-like movement of LMD on the temporal changes in the overall capacitance was investigated. Consequently, analysis of the capacitive peaks revealed the directional movement of the LMDs, enabling estimation of the position of the LMDs without direct observation. In addition, we achieved successful signal extraction from the capacitive sensor that was linked to the activated electrodes, thereby enabling selective data retrieval. The proposed signal-switching mechanism method achieved a detection accuracy of ~0.1 pF. The sensor's ability to simultaneously detect the LMD position and generate a signal underscores its significant potential for multiplexing in multisensing systems, particularly in concealed environments, such as in vivo settings.

Investigation of predictors for in-hospital death or long-term hospitalization in community-acquired pneumonia with risk factors for aspiration.

Background: It is known that the mortality of pneumonia in patients with risk factors for aspiration is worse than that in those without these risk factors. However, it is still unknown which risk factors for aspiration predict prognosis. Therefore, we aimed to determine which risk factors for aspiration are associated with death or prolonged hospitalization. Methods: We prospectively followed patients with community-acquired pneumonia at a single hospital providing acute to chronic care in Japan until they died or were discharged. Patients at any risk of aspiration were included. The associations between pneumonia severity, individual risk factors for aspiration, and in-hospital death or prolonged hospitalization were investigated. Overall survival was estimated by the Kaplan - Meier method, and the factors associated with in-hospital death or prolonged hospitalization were investigated by multivariate analysis using factors selected by a stepwise method. Results: In total, 765 patients with pneumonia and risk factors for aspiration were recruited. One hundred and ten patients deceased, and 259 patients were hospitalized over 27 days. In-hospital death increased as the number of risk factors for aspiration increased. In the multivariate analysis, male, impaired consciousness, acidemia, elevated blood urea nitrogen, and bedridden status before the onset of pneumonia were associated with in-hospital death (odds ratio [OR]: 2.5, 2.5, 3.6, 3.1, and 2.6; 95% confidence interval [CI]: 1.6-4.1, 1.4-4.2, 1.6-8.0, 1.9-5.0, and 1.6-4.2 respectively). In the Cox regression analysis, these factors were also associated with in-hospital death. None of the vital signs at admission were associated. Tachycardia, elevated blood urea nitrogen, hyponatremia, and bedridden status were associated with hospitalization for >27 days (OR: 4.1, 2.3, 4.3, and 2.9; 95% CI: 1.3-12.9, 1.5-3.4, 2.0-9.4, and 2.0-4.0, respectively). Conclusions: Blood sampling findings and bedridden status are useful for predicting in-hospital mortality and long-term hospitalization in patients with pneumonia and any risk factor for aspiration.

Dual-color live imaging unveils stepwise organization of multiple basal body arrays by cytoskeletons.

For mucociliary clearance of pathogens, tracheal multiciliated epithelial cells (MCCs) organize coordinated beating of cilia, which originate from basal bodies (BBs) with basal feet (BFs) on one side. To clarify the self-organizing mechanism of coordinated intracellular BB-arrays composed of a well-ordered BB-alignment and unidirectional BB-orientation, determined by the direction of BB to BF, we generated double transgenic mice with GFP-centrin2-labeled BBs and mRuby3-Cep128-labeled BFs for long-term, high-resolution, dual-color live-cell imaging in primary-cultured tracheal MCCs. At early timepoints of MCC differentiation, BB-orientation and BB-local alignment antecedently coordinated in an apical microtubule-dependent manner. Later during MCC differentiation, fluctuations in BB-orientation were restricted, and locally aligned BB-arrays were further coordinated to align across the entire cell (BB-global alignment), mainly in an apical intermediate-sized filament-lattice-dependent manner. Thus, the high coordination of the BB-array was established for efficient mucociliary clearance as the primary defense against pathogen infection, identifying apical cytoskeletons as potential therapeutic targets.

Cooling-promoted myogenic differentiation of murine bone marrow mesenchymal stem cells through TRPM8 activation in vitro.

TRPM8 agonist has been reported to promote osteogenic differentiation of mesenchymal stem cells (MSCs), therefore we evaluated whether cooling-induced activation of TRPM8 promotes myogenic differentiation of MSCs. We used 5-azacytidine as a myogenic differentiation inducer in murine bone marrow-derived MSCs. Addition of menthol, a TRPM8 agonist, to the differentiation induction medium significantly, increased the percentage of MyoD-positive cells, a specific marker of myogenic differentiation. We performed intracellular Ca2+ imaging experiments using fura-2 to confirm TRPM8 activation by cooling stimulation. The results confirmed that intracellular Ca2+ concentration ([Ca2+ ]i) increases due to TRPM8 activation, and TRPM8 antagonist inhibits increase in [Ca2+ ]i at medium temperatures below 19°C. We also examined the effect of cooling exposure time on myogenic differentiation of MSCs using an external cooling stimulus set at 17°C. The results showed that 60 min of cooling had an acceleratory effect on differentiation (2.18 ± 0.27 times). We observed that the TRPM8 antagonist counteracted the differentiation-promoting effect of the cooling. These results suggest that TRPM8 might modulate the multiple differentiation pathways of MSCs, and that cooling is an effective way of activating TRPM8, which regulates MSCs differentiation in vitro.

Transitional cell states sculpt tissue topology during lung regeneration.

Organ regeneration requires dynamic cell interactions to reestablish cell numbers and tissue architecture. While we know the identity of progenitor cells that replace lost tissue, the transient states they give rise to and their role in repair remain elusive. Here, using multiple injury models, we find that alveolar fibroblasts acquire distinct states marked by Sfrp1 and Runx1 that influence tissue remodeling and reorganization. Unexpectedly, ablation of alveolar epithelial type-1 (AT1) cells alone is sufficient to induce tissue remodeling and transitional states. Integrated scRNA-seq followed by genetic interrogation reveals RUNX1 is a key driver of fibroblast states. Importantly, the ectopic induction or accumulation of epithelial transitional states induce rapid formation of transient alveolar fibroblasts, leading to organ-wide fibrosis. Conversely, the elimination of epithelial or fibroblast transitional states or RUNX1 loss, leads to tissue simplification resembling emphysema. This work uncovered a key role for transitional states in orchestrating tissue topologies during regeneration.

Openable artificial intestinal tract device integrated with a permeable filter for evaluating drug permeation through cells.

Organs-on-chips using cultured cells have been developed and applied for evaluating in vitro biological phenomena. We previously reported an openable artificial intestinal tract system, as an in vitro model of the small intestine, for in vitro drug screening. The intestinal tract device could be transformed using an integrated artificial muscle actuator. An initial flat state was suitable for cell culture, and the transformed tubular structure was used as a fluidic channel for perfusion tests. The previously developed intestinal tract system could be used to evaluate drug absorption by cells through perfusion testing. This study presents an improved artificial intestinal tract system for analysis of drug permeation, in addition to absorption. Permeable filters were integrated into the intestinal tract device. Integration of additional filters into the design of the existing artificial muscle actuator was accomplished by considering device performance and available filter locations. Filter permeability was evaluated by perfusion testing. MDCK-II cells were cultured on the device and visually and electrically evaluated. The openable device, equipped with new functions for further pharmacokinetic analysis, could perform and evaluate drug disposition using cultured cells. We anticipate that the improved, openable organ-on-a-chip device system will contribute to advances in in vitro drug screening technology.

Enhancement of solute diffusion in microdroplets using microrotors under rotational magnetic field.

In vertical contact control (VCC), a microdroplet array selectively contacts with an opposite microdroplet array. Generally, VCC is useful for the dispenser mechanism based on solute diffusion between microdroplet pairs. However, sedimentation due to gravity can cause an inhomogeneous distribution of solutes in microdroplets. Therefore, it is necessary to enhance solute diffusion to achieve the accurate dispensing of a large quantity of solute in the direction opposite to that of gravity. Herein, we applied a rotational magnetic field to the microrotors in microdroplets to enhance the solute diffusion in microdroplets. Driven by microrotors, the rotational flow can generate a homogeneous distribution of solutes in microdroplets. We analyzed the diffusion dynamics of solutes using a phenomenological model, and the results showed that the rotation of microrotors can increase the diffusion constant of solutes.

Efficient Adeno-associated Virus-mediated Transgenesis in Alveolar Stem Cells and Associated Niches.

Targeted delivery of transgenes to tissue-resident stem cells and related niches offers avenues for interrogating pathways and editing endogenous alleles for therapeutic interventions. Here, we survey multiple adeno-associated virus (AAV) serotypes, administered via intranasal and retroorbital routes in mice, to target lung alveolar stem cell niches. We found that AAV5, AAV4, and AAV8 efficiently and preferentially transduce alveolar type-2 stem cells (AT2s), endothelial cells, and PDGFRA+ fibroblasts, respectively. Notably, some AAVs show different cell tropisms depending on the route of administration. Proof-of-concept experiments reveal the versatility of AAV5-mediated transgenesis for AT2-lineage labeling, clonal cell tracing after cell ablation, and conditional gene inactivation in both postnatal and adult mouse lungs in vivo. AAV6, but not AAV5, efficiently transduces both mouse and human AT2s in alveolar organoid cultures. Furthermore, AAV5 and AAV6 can be used to deliver guide RNAs and transgene cassettes for homologous recombination in vivo and ex vivo, respectively. Using this system coupled with clonal derivation of AT2 organoids, we demonstrate efficient and simultaneous editing of multiple loci, including targeted insertion of a payload cassette in AT2s. Taken together, our studies highlight the powerful utility of AAVs for interrogating alveolar stem cells and other specific cell types both in vivo and ex vivo.

Threshold magnetic field as a universal criterion for the selective transport of magnetized particles in microdroplets.

Transportation of magnetized particles (MPs) against gravity is possible by applying a magnetic field to the particles. This transport phenomenon of MPs in microdroplets can be quantitatively assessed by determining the contribution of individual forces acting on the MPs. We studied the selective transportation of MPs in microdroplets. MPs in microdroplets were transported in the opposite direction to gravity when we applied an external magnetic field larger than a threshold value. We modulated the intensity of the external magnetic field and selectively manipulated the MPs. As a result, MPs were separated into different microdroplets based on their magnetic properties. Our quantitative investigation of transport dynamics shows that the threshold magnetic field depends only on the magnetic susceptibility and the density of MPs. This is a universal criterion for the selective transport of magnetized targets such as magnetized cells in microdroplets.

Rotation and transportation of liquid crystal droplets for visualizing electric properties of microstructured electrodes.

The spatial resolution of typical sensor probes is sufficient for measuring the average electric properties of microelectrical devices, but they are unable to measure the distribution with a spatial precision. Liquid crystal droplets (LCDs) are promising candidate for visualizing the distribution. When voltage is applied, the LCDs show rotational and translational behaviors which depend on the location of LCDs within the devices. We demonstrate that by comparing the experimental and numerical results, the electric field and electrostatic energy distribution are visualized by rotating and transporting LCDs, with a spatial resolution of 10 µm and a detection accuracy of 5 µV/µm. In addition, we produced an array of LCDs by designing periodic modulation of the electrostatic energy density in the model device. These findings show that the LCDs serve as a periodic modulator of the refractive index as well as a sensor for the observation of electric properties of microelectrical devices.

Directional touch sensing for stiffness singularity search in an object using microfinger with tactile sensor.

Palpation is widely used as the initial medical diagnosis. Integration of micro tactile sensors and artificial muscles enables a soft microfinger for active touch sensing using its bending actuation. Active touch sensing by pushing-in motion of microfinger enables to evaluate stiffness distribution on an elastic object. Due to its compactness, the microfinger can enter a narrow space, such as gastrointestinal and abdominal spaces in a body. However, a microfinger can only touch and sense limited points. We aim at efficient method for searching a stiffness singular part in an elastic object by the directional touch sensing of a microfinger. This study presents a microfinger for active touch sensing using bending and push-in actuation and proposes an algorithm utilizing directivity in touch sensing by a microfinger for efficient localization of the stiffness singular part in an object. A gelatin block structure with a small rigid ball was prepared and touch sensed by the microfinger. Consequently, the position of the buried rigid ball could be efficiently identified based on the proposed algorithm. This result implies that the proposed method has potential applications in endoscopic medical diagnosis, particularly in identifying tumor positions.

Protein C activity as a potential prognostic factor for nursing home-acquired pneumonia.

INTRODUCTION: Despite the poor prognosis for nursing home acquired pneumonia (NHAP), a useful prognostic factor is lacking. We evaluated protein C (PC) activity as a predictor of in-hospital death in patients with NHAP and community-acquired pneumonia (CAP). METHODS: This prospective, observational study included all patients hospitalized with pneumonia between July 2007 and December 2012 in a single hospital. We measured PC activity at admission and investigated whether it was different between survivors and non-survivors. We also examined whether PC activity < 55% was a predictor for in-hospital death of pneumonia by logistic regression analysis with CURB-65 items (confusion, blood urea >20 mg/dL, respiratory rate >30/min, and blood pressure <90/60 mmHg, age >65). When it was a useful prognostic factor for pneumonia, we combined PC activity with the existing prognostic scores, the pneumonia severity index (PSI) and CURB-65, and analyzed its additional effect by comparing the areas under the receiver operating characteristic curves (AUCs) of the modified and original scores. RESULTS: Participants comprised 75 NHAP and 315 CAP patients. PC activity was lower among non-survivors than among survivors in NHAP and all-pneumonia (CAP+NHAP). PC activity <55% was a useful prognostic predictor for NHAP (Odds ratio 7.39 (95% CI; 1.59-34.38), and when PSI or CURB-65 was combined with PC activity, the AUC improved (from 0.712 to 0.820 for PSI, and 0.657 to 0.734 for CURB-65). CONCLUSIONS: PC activity was useful for predicting in-hospital death of pneumonia, especially in NHAP, and became more useful when combined with the PSI or CURB-65.

Active tactile sensing of small insect force by a soft microfinger toward microfinger-insect interactions.

Human-robot interaction technology has contributed to improving sociality for humanoid robots. At scales far from human scales, a microrobot can interact with an environment in a small world. Microsensors have been applied to measurement of forces by flying or walking insects. Meanwhile, most previous works focused on the measurement of the behavior of insects. Here, we propose microrobot-insect interactions by soft microfingers integrated with artificial muscle actuators and tactile sensors, which has been developed for a haptic teleoperation robot system. A soft pneumatic balloon actuator acts as the artificial muscle, and a flexible strain sensor using a liquid metal provides tactile sensing. Force interaction between a pill bug and the microfinger could be accomplished. The microfinger (12 mm × 3 mm × 490 µm) can move and touch an insect, and it can detect reaction force from an insect. The measured reaction force from the legs of a pill bug as a representative insect was less than 10 mN. This paper presents a microfinger as an end effector for the active sensing of reaction force from a small insect. We anticipate that our results will lead to further evaluation of small living things as well as technology development for human-environment interaction.

Quality evaluation of cell spheroids for transplantation by monitoring oxygen consumption using an on-chip electrochemical device.

Three-dimensional cell spheroids are superior cell-administration form for cell-based therapy which generally exhibit superior functionality and long-term survival after transplantation. Here, we nondestructively measured the oxygen consumption rate of cell spheroids using an on-chip electrochemical device (OECD) and examined whether this rate can be used as a marker to estimate the quality of cell spheroids. Cell spheroids containing NanoLuc luciferase-expressing mouse mesenchymal stem cell line C3H10T1/2 (C3H10T1/2/Nluc) were prepared. Spheroids of high or low quality were prepared by altering the medium change frequency. After transplantation into mice, the high-quality C3H10T1/2/Nluc spheroids exhibited a higher survival rate than the low-quality ones. The oxygen consumption rate of the high-quality C3H10T1/2/Nluc spheroids was maintained at high levels, whereas that of the low-quality spheroids decreased with time. These results indicate that OECD-based measurement of the oxygen consumption rate can be used to estimate the quality of cell spheroids without destructive analysis of the spheroids.

Multi-apical polarity of alveolar stem cells and their dynamics during lung development and regeneration.

Epithelial cells of diverse tissues are characterized by the presence of a single apical domain. In the lung, electron microscopy studies have suggested that alveolar type-2 epithelial cells (AT2s) en face multiple alveolar sacs. However, apical and basolateral organization of the AT2s and their establishment during development and remodeling after injury repair remain unknown. Thick tissue imaging and electron microscopy revealed that a single AT2 can have multiple apical domains that enface multiple alveoli. AT2s gradually establish multi-apical domains post-natally, and they are maintained throughout life. Lineage tracing, live imaging, and selective cell ablation revealed that AT2s dynamically reorganize multi-apical domains during injury repair. Single-cell transcriptome signatures of residual AT2s revealed changes in cytoskeleton and cell migration. Significantly, cigarette smoke and oncogene activation lead to dysregulation of multi-apical domains. We propose that the multi-apical domains of AT2s enable them to be poised to support the regeneration of a large array of alveolar sacs.

Temperature gradient sensing mechanism using liquid crystal droplets with 0.1-mK-level detection accuracy and high spatial resolution.

We proposed the detection mechanism of the micro-levels of temperature gradient in a micro-electromechanical system using the unidirectional rotation of cholesteric-liquid crystal (Ch-LC) droplets. Ch-LC droplets in the presence of an isotropic phase subjected to a heat flux rotate with a speed proportional to the magnitude of the temperature gradient. We further quantified the temperature gradient-to-torque conversion efficiency to apply the thermomechanical cross-correlation to the detection of temperature gradient. Then, we observed the rotational behavior of Ch-LC droplets after introducing them onto model devices containing patterned Au thin-film electrodes. Direct electric current applied to these Au electrodes results in unidirectional rotation of the Ch-LC droplets in response to heat flux generated from the Au electrodes. By evaluating the possible temperature gradient detection resolution using Ch-LC droplet rotation, we show that Ch-LC droplets can achieve both high spatial resolution (~ 10 µm) and high detection accuracy (~ 0.1 mK/µm).

Mesenchymal Stem Cells Promote IL-6 Secretion and Suppress the Gene Expression of Proinflammatory Cytokines in Contractile C2C12 Myotubes.

Sarcopenia is not only a major cause of disability but also a risk factor for obesity and diabetes in elderly persons. Exercise is an effective method for improving the sarcopenic condition by inducing the secretion of interleukin (IL)-6, which has the capacities to both promote muscle hypertrophy and regulate lipid metabolism and glucose homeostasis, by skeletal muscle. We previously showed that mesenchymal stem cells (MSCs) promote IL-6 secretion by lipopolysaccharide-stimulated C2C12 mouse skeletal muscle myotubes via paracrine mechanisms. Therefore, in this study, we investigated the effect of paracrine actions of MSCs on IL-6 and proinflammatory cytokine expression in contractile C2C12 myotubes by applying electrical stimulation. IL-6 secretion by C2C12 myotubes was increased by electrical stimulation, and a more significant increase in IL-6 secretion was observed in electrically stimulated C2C12 myotubes cultured in conditioned medium from MSCs. The activation of nuclear factor-κB in C2C12 myotubes was also promoted by the combination of conditioned medium from MSCs and electrical stimulation. Moreover, the increases in tumor necrosis factor-α and IL-1ß mRNA expression in C2C12 myotubes induced by electrical stimulation were suppressed by culture in conditioned medium from MSCs. The present findings suggest that MSCs transplantation or injection of their extracellular vesicles improve the therapeutic effect of exercise against sarcopenia without exacerbating inflammation.

Defined conditions for long-term expansion of murine and human alveolar epithelial stem cells in three-dimensional cultures.

Alveolar type 2 cells (AT2s) serve as stem cells of the alveoli and restore cell numbers after injury. Here, we describe a detailed protocol for the isolation, purification, and culture of murine and human AT2s. We have developed chemically defined and stroma-free culture conditions that enable expansion and maintenance of AT2s. The culture conditions are scalable and compatible with high-throughput chemical and genetic screenings and can potentially be used to generate large AT2 numbers for cell-based therapies. For complete details on the use and execution of this protocol, please refer to Katsura et al. (2020).

Investigation of Brain Function-Related Myokine Secretion by Using Contractile 3D-Engineered Muscle.

Brain function-related myokines, such as lactate, irisin, and cathepsin B (CTSB), are upstream factors that control brain-derived neurotrophic factor (BDNF) expression and are secreted from skeletal muscle by exercise. However, whether irisin and CTSB are secreted by muscle contraction remains controversial. Three-dimensional (3D)-engineered muscle (3D-EM) may help determine whether skeletal muscle contraction leads to the secretion of irisin and CTSB, which has never been identified with the addition of drugs in conventional 2D muscle cell cultures. We aimed to investigate the effects of electrical pulse stimulation (EPS)-evoked muscle contraction on irisin and CTSB secretion in 3D-EM. The 3D-EM, which consisted of C2C12 myoblasts and type-1 collagen gel, was allowed to differentiate for 2 weeks and divided into the control and EPS groups. EPS was applied at 13 V, 66 Hz, and 2 msec for 3 h (on: 5 s/off: 5 s). Irisin and CTSB secretion into the culture medium was measured by Western blotting. Irisin secretion was significantly increased following EPS (p < 0.05). However, there was no significant difference in CTSB secretion between the two groups. The present study suggests that irisin may be a contractile muscle-derived myokine, but CTSB is not secreted by EPS-evoked muscle contractile stimulation in 3D-EM.