Hypergravity enhances RBM4 expression in human bone marrow-derived mesenchymal stem cells and accelerates their differentiation into neurons.

Introduction: The regulation of stem cell differentiation is important in determining the quality of transplanted cells in regenerative medicine. Physical stimuli are involved in regulating stem cell differentiation, and in particular, research on the regulation of differentiation using gravity is an attractive choice. We have shown that microgravity is useful for maintaining undifferentiated mesenchymal stem cells (MSCs). However, the effects of hypergravity on the differentiation of MSCs, especially on neural differentiation related to neural regeneration, have not been elucidated. Methods: We induced neural differentiation of human bone marrow-derived MSCs (hbMSCs) for 10 days under normal gravity (1G) or hypergravity (3G) conditions using a gravity controller, Gravite®. HbMSCs were collected, and cell number and viability were measured 3 and 10 days after induction. RNA was also extracted from the collected hbMSCs, and the expression of neuron-associated genes and regulator markers of neural differentiation was analyzed using real-time polymerase chain reaction (PCR). Additionally, we evaluated the NF-M-positive cell rate 10 days after induction using immunofluorescent staining. Results: Neural gene expression and the NF-M-positive cell rate were increased in hbMSCs under the 3G condition 10 days after induction. mRNA expression of RNA binding motif protein 4 (RBM4) and pyruvate kinase M 1 (PKM1) in the 3G condition was also higher than that in the 1G group. Conclusions: Hypergravity can enhance RBM4 and PKM1, promoting the neural differentiation of hbMSCs.

Osteoblastic adherence regulates hematopoietic stem cell self-renewal and differentiation: a conceptional in vitro and in vivo study.

BACKGROUND: Intrinsic factors related to self-renewal regulatory factors in hematopoietic stem cells are well known; however, limited information is available on extrinsic factors, such as the cell environment. Therefore, in this study, we analyzed the regulatory mechanism of hematopoietic stem cell self-renewal, focusing on the osteoblastic niche, and examined how adherence to osteoblasts affects stem cell differentiation. METHODS: For this experimental study, we developed a co-culture system for hematopoietic stem cells and osteoblasts, such that cells adhered to osteoblasts can be separated from those that do not. Murine Sca1-positive cells were separated into groups according to whether they were attached to osteoblasts or detached from osteoblasts, and each group was then subjected to colony assays and bone marrow transplantation experiments. RESULTS: Adhered Sca1-positive cells developed more secondary colonies than non-adhered Sca1-positive cells. Furthermore, in bone marrow transplantation experiments, adhered Sca1-positive cells showed successful engraftment. We explored the role of Polycomb genes in the regulation of cell fate and found that self-renewing cells attached to osteoblasts had high Bmi-1 expression and low Mel-18 expression, while this expression was reversed in differentiating cells. CONCLUSIONS: Our results suggest that hematopoietic stem cells self-renew when they remain in osteoblastic niches after cell division. Further, when stem cells leave the niches, they undergo differentiation.

Comparisons of Neurotrophic Effects of Mesenchymal Stem Cells Derived from Different Tissues on Chronic Spinal Cord Injury Rats.

Cell-based therapies with mesenchymal stem cells (MSCs) are considered as promising strategies for spinal cord injury (SCI). MSCs have unique characteristics due to differences in the derived tissues. However, relatively few studies have focused on differences in the therapeutic effects of MSCs derived from different tissues. In this study, the therapeutic effects of adipose tissue-derived MSCs, bone marrow-derived MSCs, and cranial bone-derived MSCs (cMSCs) on chronic SCI model rats were compared. MSCs were established from the collected adipose tissue, bone marrow, and cranial bone. Neurotrophic factor expression of each MSC type was analyzed by real-time PCR. SCI rats were established using the weight-drop method and transplanted intravenously with MSCs at 4 weeks after SCI. Hindlimb motor function was evaluated from before injury to 4 weeks after transplantation. Endogenous neurotrophic factor and neural repair factor expression in spinal cord (SC) tissue were examined by real-time PCR and western blot analyses. Although there were no differences in the expression levels of cell surface markers and multipotency, expression of Bdnf, Ngf, and Sort1 (Nt-3) was relatively higher in cMSCs. Transplantation of cMSCs improved motor function of chronic SCI model rats. Although there was no difference in the degree of engraftment of transplanted cells in the injured SC tissue, transplantation of cMSCs enhanced Bdnf, TrkB, and Gap-43 messenger RNA expression and synaptophysin protein expression in injured SC tissue. As compared with MSCs derived other tissues, cMSCs highly express many neurotrophic factors, which improved motor function in chronic SCI model rats by promoting endogenous neurotrophic and neural plasticity factors. These results demonstrate the efficacy of cMSCs in cell-based therapy for chronic SCI.

Nutritional intervention after an early assessment by a flexible endoscopic evaluation of swallowing is associated with a shorter hospital stay for patients with acute cerebral infarction: A retrospective study.

BACKGROUND AND OBJECTIVES: It is important to evaluate the swallowing function of patients with acute cerebral infarction. The effects of nutritional intervention after an early assessment by a flexible endoscopic evaluation of swallowing (FEES) were evaluated. METHODS AND STUDY DESIGN: This retrospective study included 274 patients who were hospitalized for acute cerebral infarction and underwent a FEES between 2016 and 2018. The effects of early nutritional intervention after an assessment by a FEES within 48 h from admission were evaluated. The patients were divided into a shorter hospital stay group (<30 days) and a longer group (≥30 days). A multivariate analysis was performed to identify the predictive factors for a shorter hospital stay. RESULTS: The overall patient characteristics were as follows: 166 men; median age, 81 years old; and median body mass index (BMI), 21.1 kg/m2. No significant differences in the age, sex, or BMI were found between the shorter and longer hospital stay groups. A FEES within 48 h of admission (odds ratio [OR], 2.040; 95% confidence interval [CI], 1.120-3.700; p=0.019), FILS level ≥6 at admission (OR, 2.300; 95% CI, 1.190-4.440; p=0.013), and an administered energy dose of ≥18.5 kcal/kg on hospital day 3 (OR, 2.360; 95% CI, 1.180-4.690; p=0.015) were independently associated with a hospital stay <30 days. CONCLUSIONS: Patients with acute cerebral infarction are more likely to have a shorter hospital stay (<30 days) if they undergo a FEES early after admission and receive optimal nutritional intervention.

Efficient preservation of sprouting vegetables under simulated microgravity conditions.

The effectiveness of a simulated microgravity environment as a novel method for preserving the freshness of vegetables was investigated. Three types of vegetables were selected: vegetable soybean, mung bean sprouts, and white radish sprouts. These selected vegetables were fixed on a three-dimensional rotary gravity controller, rotated slowly. The selected vegetables were stored at 25°C and 66% of relative humidity for 9, 6, or 5 d while undergoing this process. The simulated microgravity was controlled utilizing a gravity controller around 0 m s-2. The mung bean sprouts stored for 6 d under simulated microgravity conditions maintained higher thickness levels than the vegetable samples stored under normal gravity conditions (9.8 m s-2) for the same duration. The mass of all three items decreased with time without regard to the gravity environment, though the samples stored within the simulated microgravity environment displayed significant mass retention on and after 3 d for mung bean sprout samples and 1 d for white radish sprout samples. In contrast, the mass retention effect was not observed in the vegetable soybean samples. Hence, it was confirmed that the mass retention effect of microgravity was limited to sprout vegetables. As a result of analysis harnessing a mathematical model, assuming that the majority of the mass loss is due to moisture loss, a significant difference in mass reduction coefficient occurs among mung bean sprouts and white radish sprouts due to the microgravity environment, and the mass retention effect of simulated microgravity is quantitatively evaluated utilizing mathematical models. Simulated microgravity, which varies significantly from conventional refrigeration, ethylene control, and modified atmosphere, was demonstrated effective as a novel method for preserving and maintaining the freshness of sprout vegetables. This founding will support long-term space flight missions by prolonging shelf life of sprout vegetables.

Early Transplantation of Human Cranial Bone-derived Mesenchymal Stem Cells Enhances Functional Recovery in Ischemic Stroke Model Rats.

We analyzed the cell characteristics, neuroprotective, and transplantation effects of human cranial bone-derived mesenchymal stem cells (hcMSCs) in ischemic stroke model rats compared with human iliac bone-derived mesenchymal stem cells (hiMSCs). The expressions of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF ) as neurotrophic factors were analyzed in both MSCs. hiMSCs or hcMSCs were intravenously administered into ischemic stroke model rats at 3 or 24 h after middle cerebral artery occlusion (MCAO) and neurological function was evaluated. The survival rate of neuroblastoma × glioma hybrid cells (NG108-15) after 3 or 24 h oxidative or inflammatory stress and the neuroprotective effects of hiMSCs or hcMSCs-conditioned medium (CM) on 3 or 24 h oxidative or inflammatory stress-exposed NG108-15 cells were analyzed. The expressions of BDNF and VEGF were higher in hcMSCs than in hiMSCs. hcMSCs transplantation at 3 h after MCAO resulted in significant functional recovery compared with that in the hiMSCs or control group. The survival rate of stress-exposed NG108-15 was lower after 24 h stress than after 3 h stress. The survival rates of NG108-15 cells cultured with hcMSCs-CM after 3 h oxidative or inflammatory stress were significantly higher than in the control group. Our results suggest that hcMSCs transplantation in the early stage of ischemic stroke suppresses the damage of residual nerve cells and leads to functional recovery through the strong expressions of neurotrophic factors. This is the first report demonstrating a functional recovery effect after ischemic stroke following hcMSCs transplantation.

Kaposi's sarcoma-associated herpesvirus is cell-intrinsically controlled in latency in microgravity.

In the next several decades, humans will explore deep space, including Mars. During long-term space flight, astronauts will be exposed to various physical stressors. Among these stressors, microgravity may compromise the immune system. Consistently, the reactivation of several latent herpesviruses has been reported in astronauts. Although herpesvirus infection status is determined by both cell-intrinsic and -extrinsic factors, it remains unclear which factors play major roles in the virus reactivation in microgravity. Here, using Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, we found that KSHV is cell-intrinsically controlled in latency in microgravity. Innate immunity appeared to be unaffected in microgravity, while the expression of some restriction factors against KSHV, such as CTCF and AMPK, was upregulated. Collectively, the infected cells in microgravity can control KSHV in latency, possibly by unimpaired innate immunity and upregulated KSHV restriction factors. This is the first pilot study of the conflicts between cell-intrinsic defense systems and viruses in microgravity and provides fundamental information regarding host-virus interactions in microgravity.

Maintenance of Neurogenic Differentiation Potential in Passaged Bone Marrow-Derived Human Mesenchymal Stem Cells Under Simulated Microgravity Conditions.

Human mesenchymal stem cells (hMSCs) are considered to be able to adapt to environmental changes induced by gravity during cell expansion. In this study, we investigated neurogenic differentiation potential of passaged hMSCs under conventional gravity and simulated microgravity conditions. Immunostaining, quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), and western blot analysis of neurogenic differentiation markers, neurofilament heavy (NF-H), and microtubule-associated protein 2 (MAP2) revealed that differentiated cells from the cells cultured under simulated microgravity conditions expressed higher neurogenic levels than those from conventional gravity conditions. The levels of NF-H and MAP2 in the cells from simulated microgravity conditions were consistent during passage culture, whereas cells from conventional gravity conditions exhibited a reduction of the neurogenic levels against an increase of their passage number. In growth culture, cells under simulated microgravity conditions showed less apical stress fibers over their nucleus with fewer cells having a polarization of lamin A/C than those under conventional gravity conditions. The ratio of lamin A/C to lamin B expression in the cells under simulated microgravity conditions was constant; however, cells cultured under conventional gravity conditions showed an increase in the lamin ratio during passages. Furthermore, analysis of activating H3K4me3 and repressive H3K27me3 modifications at promoters of neuronal lineage genes indicated that cells passaged under simulated microgravity conditions sustained the methylation during serial cultivation. Nevertheless, the enrichment of H3K27me3 significantly increased in the passaged cells cultured under conventional gravity conditions. These results demonstrated that simulated microgravity-coordinated cytoskeleton-lamin reorganization leads to suppression of histone modification associated with neurogenic differentiation capacity of passaged hMSCs.

Alterations in Nuclear Lamina and the Cytoskeleton of Bone Marrow-Derived Human Mesenchymal Stem Cells Cultured Under Simulated Microgravity Conditions.

Cells sense and respond to environmental changes induced by gravity. Although reactions to conventional culture have been intensively studied, little is known about the cellular reaction to simulated microgravity conditions. Thus, in this study, we investigated the effects of simulated microgravity on human mesenchymal stem cells using a three-dimensional clinostat (Gravite®), a recently developed device used to generate simulated microgravity condition in vitro. Our time-lapse analysis shows that cells cultured under conventional culture conditions have a stretched morphology and undergo unidirectional migration, whereas cells cultured under simulated microgravity conditions undergo multidirectional migration with directional changes of cell movement. Furthermore, cells cultured under conventional culture conditions maintained their spindle shape through fibronectin fibril formation in their bodies and focal adhesion stabilization with enriched stress fibers. However, cells cultured under simulated microgravity conditions were partially contracted and the fibril structures were degraded in the cell bodies. Additionally, paxillin phosphorylation in the cells cultured under simulated microgravity conditions was more intense at the cell periphery in regions near the leading and trailing edges, but was less expressed in the cell bodies compared with that observed in cells cultured under conventional culture conditions. Furthermore, lamin A/C, a major component of the nuclear lamina, was mainly located on the apical side in cells cultured under conventional culture conditions, indicating basal-to-apical polarization. However, cells cultured under simulated microgravity conditions showed lamin A/C localization on both the apical and basal sides. Taken together, these results demonstrate that simulated microgravity-driven fibronectin assembly affects nuclear lamina organization through the spatial reorganization of the cytoskeleton.

"Microgravity" as a unique and useful stem cell culture environment for cell-based therapy.

Cell-based therapy using mesenchymal stem cells or pluripotent stem cells such as induced pluripotent stem cells has seen dramatic progress in recent years. Part of cell-based therapy are already covered by public medical insurance. Recently, researchers have attempted to improve therapeutic effects toward various diseases by cell transplantation. Culture environment is considered to be one of the most important factors affecting therapeutic effects, in particular factors such as physical stimuli, because cells have the potential to adapt to their surrounding environment. In this review, we provide an overview of the research on the effects of gravity alteration on cell kinetics such as proliferation or differentiation and on potential therapeutic effects, and we also summarize the remarkable possibilities of the use of microgravity culture in cell-based therapy for various diseases.

Stem Cell Culture in Microgravity and Its Application in Cell-Based Therapy.

Recent years have witnessed a rapid increase in space experiments. Initially, scientists focused on understanding the phenomenon of microgravity to discover countermeasures for preventing the adverse effects of microgravity on the astronauts' bodies. Lately, the application of microgravity environment has been gradually increasing with diverse objectives. Protein crystallization and three-dimensional cell culture are typical examples of microgravity application. Our recent studies suggested that microgravity is a useful tool for cell culture in cell-based therapy. In this review, we discussed microgravity-induced changes at cellular and molecular levels observed in experiments conducted during space flight or using simulated microgravity device. In addition, we summarized the utility of microgravity environment in cell-based therapy for central nervous system diseases.

Erratum: Publisher Correction: Simulated microgravity attenuates myogenic differentiation via epigenetic regulations.

[This corrects the article DOI: 10.1038/s41526-018-0045-0.].

Simulated Microgravity Culture Enhances the Neuroprotective Effects of Human Cranial Bone-Derived Mesenchymal Stem Cells in Traumatic Brain Injury.

Fundamental cures of central nervous system (CNS) diseases are rarely achieved due to the low regenerative ability of the CNS. Recently, cell-based therapy using mesenchymal stem cells (MSCs) has been explored as an effective treatment for CNS diseases. Among the various tissue-derived MSCs, we have isolated human cranial bone-derived MSCs (cMSCs) in our laboratory. In addition, we have focused on simulated microgravity (MG) as a valuable culture environment of MSCs. However, detailed mechanisms underlying functional recovery from transplantation of MSCs cultured under MG conditions remain unclear. In this study, we investigated the therapeutic mechanisms of transplantation of cMSCs cultured under MG conditions in traumatic brain injury (TBI) model mice. Human cMSCs were cultured under 1G and MG conditions, and cMSCs cultured under MG conditions expressed significantly higher messenger RNA (mRNA) levels of hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-ß). In TBI model mice, the transplantation of cMSCs cultured under MG conditions (group MG) showed greater motor functional improvement compared with only phosphate-buffered saline administration (group PBS). Moreover, the protein expression levels of tumor necrosis factor alpha (TNF-α) and the Bcl-2-associated X protein (Bax)/b cell leukemia/lymphoma 2 protein (Bcl-2) ratio were significantly lower at brain injury sites in mice of group MG than those of group PBS. In addition, an in vitro study showed that the conditioned medium of cMSCs cultured under MG conditions significantly suppressed the cell death of NG108-15 cells exposed to oxidative or inflammatory stress through anti-inflammatory and antiapoptosis effects. These findings demonstrate that culturing cMSCs under simulated MG increases the neuroprotective effects, suggesting that simulated MG cultures may be a useful method for cell-based therapy strategies for CNS diseases.

Simulated microgravity attenuates myogenic differentiation via epigenetic regulations.

The molecular mechanisms involved in myogenic differentiation are relatively well-known. Myogenic differentiation is regulated by the sequential activation of the basic helix-loop-helix myogenic regulatory transcription factors (MRFs), and biomechanical signals play an important role in the regulation of myogenesis. In this study, we sought to determine whether simulated microgravity culture using Gravite® may affect myoblast differentiation and expression of MRF genes. Although rat myoblasts, L6 cells were differentiated to myotubes in an incubation period-dependent manner, myogenesis of L6 cells was significantly attenuated under simulated microgravity (10-3G) conditions. Real-time Reverse transcription polymerase chain reaction (RT-PCR) showed that expressions of Myog, Myf6, Mef2c, Des, and Ckm under 1 G conditions increase in an incubation period-dependent manner, and that Myod1 expression was specifically observed to increase transiently in the early phase. However, expressions of Myod1 and Myog were significantly inhibited under simulated microgravity conditions. To clarify the molecular mechanisms, L6 cells were treated with 5-AzaC, and further incubated with differentiation medium under 1 G or 10-3 G conditions. The results showed differences in expression levels of Myod1, Myog, and, as well as those of myotube thickness between 1 G and 10-3 G conditions, completely disappeared in this experimental condition. Modified HpaII tiny fragment enrichment by ligation-mediated PCR (HELP)-assay showed that kinetic changes of DNA methylation status were attenuated in simulated microgravity conditions. These results indicate that microgravity regulates myogenesis and Myod1 expression by controlling DNA methylation.

Rat Cranial Bone-Derived Mesenchymal Stem Cell Transplantation Promotes Functional Recovery in Ischemic Stroke Model Rats.

The functional disorders caused by central nervous system (CNS) diseases, such as ischemic stroke, are clinically incurable and current treatments have limited effects. Previous studies suggested that cell-based therapy using mesenchymal stem cells (MSCs) exerts therapeutic effects for ischemic stroke. In addition, the characteristics of MSCs may depend on their sources. Among the derived tissues of MSCs, we have focused on cranial bones originating from the neural crest. We previously demonstrated that the neurogenic potential of human cranial bone-derived MSCs (cMSCs) was higher than that of human iliac bone-derived MSCs. Therefore, we presumed that cMSCs have a higher therapeutic potential for CNS diseases. However, the therapeutic effects of cMSCs have not yet been elucidated in detail. In the present study, we aimed to demonstrate the therapeutic effects of transplantation with rat cranial bone-derived MSCs (rcMSCs) in ischemic stroke model rats. The mRNA expression of brain-derived neurotrophic factor and nerve growth factor was significantly stronger in rcMSCs than in rat bone marrow-derived MSCs (rbMSCs). Ischemic stroke model rats in the rcMSC transplantation group showed better functional recovery than those in the no transplantation and rbMSC transplantation groups. Furthermore, in the in vitro study, the conditioned medium of rcMSCs significantly suppressed the death of neuroblastoma × glioma hybrid cells (NG108-15) exposed to oxidative and inflammatory stresses. These results suggest that cMSCs have potential as a candidate cell-based therapy for CNS diseases.

Mesenchymal Stem Cell-Based Therapy Improves Lower Limb Movement After Spinal Cord Ischemia in Rats.

BACKGROUND: Spinal cord ischemia is a devastating complication after thoracic and thoracoabdominal aortic operations. In this study, we aimed to investigate the effects of mesenchymal stem cells (MSCs), which have regenerative capability and exert paracrine actions on damaged tissues, injected into rat models of spinal cord ischemia-reperfusion injury. METHODS: Forty-five Sprague-Dawley rats were divided into sham, phosphate-buffered saline (PBS), and MSC groups. Spinal cord ischemia was induced in the latter two groups by balloon occlusion of the thoracic aorta. MSCs and PBS were then immediately injected into the left carotid artery of the MSC and PBS groups, respectively. Hindlimb motor function was evaluated at 6 and 24 hours. The spinal cord was removed at 24 hours after ischemia-reperfusion injury, and histologic and immunohistochemical analyses and real-time polymerase chain reaction assessments were performed. RESULTS: Rats in the MSC and PBS groups showed flaccid paraparesis/paraplegia postoperatively. Hindlimb function was significantly better at 6 and 24 hours after ischemia-reperfusion injury in the MSC group than in the PBS group (p < 0.05). The number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive neuron cells in the spinal cord and the ratio of Bax to Bcl2 were significantly larger (p < 0.05) in the PBS group than in the MSC group. The injected MSCs were observed in the spinal cord 24 hours after ischemia-reperfusion injury. CONCLUSIONS: The MSC therapy by transarterial injection immediately after spinal cord ischemia-reperfusion injury may improve lower limb function by preventing apoptosis of neuron cells in the spinal cord.

Neuromagnetic evaluation of a communication support system for hearing-impaired patients.

Hearing-impaired patients often encounter obstacles in communication. Not all of them wear hearing aids, citing issues with usage difficulty and discomfort in wearing. To overcome these difficulties, a new endeavor was started to improve sound intelligibility from the speaker's side. The present study objectively evaluated an intelligible-hearing (IH) loudspeaker by means of magnetoencephalography. Magnetic counterparts of mismatch negativity (MMNm) to pronunciation ('mi' and 'ni') were recorded and compared when they were transmitted from the IH loudspeaker and from a normal-hearing loudspeaker. On using the IH loudspeaker, the peak latency was found to be significantly shortened. In the case of hearing-impaired participants, marked MMNm responses were observed only when the IH loudspeaker was used. These findings suggest that improving sound intelligibility may be a supportive and rehabilitative approach for hearing-impaired patients.

The characteristics of human cranial bone marrow mesenchymal stem cells.

Recently, cell-based therapy has attracted attention for treatment of central nervous system (CNS) disorders. Bone marrow-derived mesenchymal stem cells (BMSCs) are considered to have good engraftment potential. Therefore, more efficient and less invasive methods to obtain donor cells are required. Here, we established human BMSCs from cranial bone waste (cBMSCs) obtained following routine neurosurgical procedures. cBMSCs and cells obtained from the iliac crest (iBMSCs, standard BMSCs) showed expression of cell surface markers associated with mesenchymal stem cells and multipotency traits such as differentiation into osteogenic and adipogenic lineages. cBMSCs showed higher expression of the neural crest-associated mRNAs p75, Slug, and Snail than iBMSCs. Neurogenic induced cells from cBMSCs expressed the neural markers nestin, Pax6, neurofilament (NF)-L, and NF-M as seen with RT-PCR, and NF-M protein as seen with western blotting at higher levels than cells from iBMSCs. Immunostaining showed a significantly greater proportion of NF-M-positive cells in the population of induced cBMSCs compared with the population of iBMSCs. Thus, cBMSCs showed a greater tendency to differentiate into neuron-like cells than iBMSCs.

Interactive effects of cell therapy and rehabilitation realize the full potential of neurogenesis in brain injury model.

The therapeutic effect of rehabilitation after cell therapy for brain injury remains unclear. Here, we report the neural stem/progenitor cells transplantation into a brain injury mouse model followed by treadmill exercise training. Among all experimental groups, mice that underwent transplantation and treadmill exercise demonstrated significant functional motor and electrophysiological improvement. Transplanted cells at the brain injury site were observed and differentiated into neurons and astrocytes. Transplanted cells significantly differentiated into neurons in the mice that underwent transplantation and treadmill exercise compared with those treated with only transplantation. Furthermore, the expression of brain-derived neurotrophic factor and growth-associated protein 43 mRNAs were significantly up-regulated in the mice that underwent transplantation and treadmill exercise than in those in other experimental groups during the early recovery stage. These results suggest that rehabilitation after neural stem/progenitor cell transplantation enhances neurogenesis and promotes the recovery of motor function in brain injury model mice.

Electrical stimulation accelerates neuromuscular junction formation through ADAM19/neuregulin/ErbB signaling in vitro.

The mechanism by which electrical stimulation affects formation of neuromuscular junctions (NMJs) remains unknown. NG108-15, a neural cell line, is commonly used in in vitro co-culture models of myotubes to observe synapse formation; therefore, we employed this model to observe the effects of electrical stimulation on NMJ formation. Initially, L6 cells were differentiated and NG108-15 cells were then added to the same culture dish. After 2 and 3 days of co-culture, the cells were electrically stimulated at 50 V and 0.5 Hz for 0, 5, 30, and 60 min (C, ES5, ES30, and ES60 groups, respectively) and were analyzed after co-culture for 4 days. Immunofluorescence experiments showed significantly increased aggregation of acetylcholine receptors and inhibition of neural outgrowth in the ES30 and ES60 groups. Furthermore, ADAM19 and phospho-ErbB3 were found to be specifically localized in co-cultured NG108-15 cells. Immunoblotting demonstrated that synapsin 1, ADAM19 precursor and its activated form, phospho-ErbB3, and ERK1 protein levels had increased in an electrical stimulation period-dependent manner. Thus, we found that electrical stimulation accelerated NMJ formation, possibly through activation of ADAM19/neuregulin/ErbB signaling in NG108-15 cells.