Differentiation of mouse embryonic stem cells into insulin-producing cells: optimization to enhance the overall differentiation efficiency

Embryonic stem cells have the potential to differentiate into multiple cell types including insulin-producing cells (IPCs), which is becoming one of the promising cell sources for treating type 1 diabetes mellitus. However, in order to achieve functional stem cell-derived cells, it is important to generate more mature IPCs and to keep long-term viability post differentiation process. In this study, we varied several factors including different embryonic body culture conditions, digested cells seeding density and various coatings required for differentiation to optimize a previously established protocol to enhance the overall differentiation efficiency. Moreover, a three-dimensional in vitro collagen tissue culture system was prepared to provide a more physiological culture environment for stem cell-derived IPCs. Survivability of IPCs was examined under both static and flow conditions and low flow rate of 0.02 ml/min resulted in better survival of IPCs in in vitro three-dimensional tissues

Cerebro-vascular disruption mediated initiation and propagation of traumatic brain injury in a fluid percussion injury model

Traumatic brain injury (TBI) is a major health problem for over 3.17 million people in the US. There is no FDA-approved drug for the treatment because the injury mechanisms have not been clearly identified. The knowledge gap is addressed here by the lateral fluid percussion injury (FPI) rat model, through the understanding of layer-structured mechanisms from physical vascular rupture to acute necrosis, as well as biochemical changes in perivascular space as secondary events. Firstly, the cerebrovascular hemorrhage and related infarct volume are investigated as the primary events in moderate FPI, which is found to be increased with injury severity in FPI. The extent of coagulation is validated by the bio-distribution of fluorescent tracer in the cerebrospinal fluid (CSF) pathway after the injury. Bio-distribution of the tracer is specifically diminished at the site of coagulation, which blocks the CSF movement in subarachnoid and interstitial spaces. The pattern of coagulation is associated with the CSF blockage and correlates to necrotic cell death in and around the impact site. Different biomarkers for immune cells, neuronal death and tight junction proteins show that physical disruption of vasculature plays an important role for the acute induction of neuroinflammation and neurodegeneration in blunt TBI. Additionally, free radicals generation is found to be significantly increased in the injured hemisphere immediately post FPI and decreases over time. Upregulation of radical-generating enzymes, NADPH oxidase 1 as well as inducible nitric oxide synthase, initiates biochemical damage of the injured brain. As a result, the signatures of oxidative/nitrosative damage markers 4-HNE and 3-NT are observed in the blood brain barrier (BBB) post-TBI, with temporal changes in the injury site. Oxidative/nitrosative damage and immune cells infiltration correlate with gliosis at 4 hours and 7 days post moderate FPI. Examination of apoptosis, tau phosphorylation, and neuronal survivability at day 7 post FPI further validate neurodegeneration. Thus, it is confirmed that the acute and long-term neuroinflammation and neurodegeneration are correlated with cerebral vascular disruption. Finally, novel regenerative medicines are explored for in-situ repair with angiogenesis and neuroprotection mechanisms in the injured brain post-TBI. An injectable self-assembling peptide-based hydrogel (SAPH) appended with vascular endothelial growth factor (VEGF) mimic is used to create a regenerative microenvironment for neovascularization at the injury site. VEGF is an angiogenic and neuroprotective growth factor that is involved in the process of brain repair. Supramolecular assembly allows for thixotropy; the injectable drug delivery system provides sustained in vivo efficacy. Application of the angiogenic SAPH directly in the injury site promotes disrupted vasculature repair in and around the hydrogel implant at day 7 post-TBI. Upregulation of VEGF-receptor 2 is observed, demonstrating an angiogenic response in the presence of angiogenic SAPH. Moreover, vascular markers von-Willebrand factor (vWF) and a-smooth muscle actin (a-SMA) show a concomitant increase with blood vessels in response to the angiogenic SAPH. The neuronal rescue examination by NeuN and myelin basic protein shows that the SAPH has the potential to provide neuroprotective benefits in the long-term recovery

Moderate Exercise Enhances Endothelial Progenitor Cell Exosomes Release and Function

Purpose: Exercise has cardiovascular benefits which might be related to endothelial progenitor cells (EPC). Meanwhile, there is evidence suggesting that EPC-derived exosomes (EPC-EX) promote vascular repair and angiogenesis through their carried microRNA (miR)-126. In this study, we investigated whether exercise could increase the levels of circulating EPC-EX and their miR-126 cargo, and by which promote the protective function of EPC-EX on endothelial cells (EC). Methods: Plasma EPC-EX from sedentary, low, or moderate exercise mice, respectively, denoted as EPC-EXS, EPC-EXL, and EPC-EXM, were isolated using microbead-based sorting techniques and characterized by nanoparticle tracking analysis, Western blot, and quantitative real-time polymerase chain reaction assessments of biomarkers and miR-126. High glucose (25 mM) with hypoxia (1% O2) was used for inducing an EC injury model. The injured EC were treated by coculturing with vehicle, EPC-EXS, EPC-EXL, EPC-EXM, or EPC-EXM + anti–miR-126. After that, EC were used for flow cytometry analysis of apoptosis, assessments of tube formation and migration, and measurements of miR-126 level and its downstream sprouty-related protein-1 (SPRED1) and vascular endothelial growth factor (VEGF). Results: 1) Isolated EPC-EX positively expressed exosomal markers (CD63 and Tsg101) and EPC markers (CD34 and VEGFR2). 2) Exercise intensity dependently elevated plasma level of EPC, EPC-EX/EPC ratio, and miR-126 expression in EPC and EPC-EX. 3) Injured EC displayed apoptosis increment, angiogenic dysfunction and miR-126 reduction. 4) EPC-EXM had better effects than EPC-EXS and EPC-EXL on alleviating those changes of injured EC, accompanied with SPRED1 downregulation and VEGF upregulation. 5) The effects of EPC-EXM were abolished by miR-126 knockdown. Conclusions: Our data demonstrate that exercise can increase EPC-EX release and miR-126 level and enhance the effects of EPC-EX on protecting EC against injury through the SPRED1/VEGF pathway

Microvascular Endothelial Cells-Derived Microvesicles Imply in Ischemic Stroke by Modulating Astrocyte and Blood Brain Barrier Function and Cerebral Blood Flow

Background Endothelial cell (EC) released microvesicles (EMVs) can affect various target cells by transferring carried genetic information. Astrocytes are the main components of the blood brain barrier (BBB) structure in the brain and participate in regulating BBB integrity and blood flow. The interactions between ECs and astrocytes are essential for BBB integrity in homeostasis and pathological conditions. Here, we studied the effects of human brain microvascular ECs released EMVs on astrocyte functions. Additionally, we investigated the effects of EMVs treated astrocytes on regulating BBB function and cerebral ischemic damage. Results EMVs prepared from ECs cultured in normal condition (n-EMVs) or oxygen and glucose deprivation (OGD-EMVs) condition had diverse effects on astrocytes. The n-EMVs promoted, while the OGD-EMVs inhibited the proliferation of astrocytes via regulating PI3K/Akt pathway. Glial fibrillary acidic protein (GFAP) expression (marker of astrocyte activation) was up-regulated by n-EMVs, while down-regulated by OGD-EMVs. Meanwhile, n-EMVs inhibited but OGD-EMVs promoted the apoptosis of astrocytes accompanied by up/down-regulating the expression of Caspase-9 and Bcl-2. In the BBB model of ECs-astrocytes co-culture, the n-EMVs, conversely to OGD-EMVs, decreased the permeability of BBB accompanied with up-regulation of zonula occudens-1(ZO-1) and Claudin-5. In a transient cerebral ischemia mouse model, n-EMVs ameliorated, while OGD-EMVs aggravated, BBB disruption, local cerebral blood flow (CBF) reduction, infarct volume and neurological deficit score. Conclusions Our data suggest that EMVs diversely modulate astrocyte functions, BBB integrity and CBF, and could serve as a novel therapeutic target for ischemic stroke

miR-132-3p Priming Enhances the Effects of Mesenchymal Stromal Cell-Derived Exosomes on Ameliorating Brain Ischemic Injury

Backgrounds/aims: Mesenchymal stromal cell-derived exosomes (MSC-EXs) could exert protective effects on recipient cells by transferring the contained microRNAs (miRs), and miR-132-3p is one of angiogenic miRs. However, whether the combination of MSC-EXs and miR-132-3p has better effects in ischemic cerebrovascular disease remains unknown. Methods: Mouse MSCs transfected with scrambler control or miR-132-3p mimics were used to generate MSC-EXs and miR-132-3p-overexpressed MSC-EXs (MSC-EXsmiR-132-3p). The effects of EXs on hypoxia/reoxygenation (H/R)-injured ECs in ROS generation, apoptosis, and barrier function were analyzed. The levels of RASA1, Ras, phosphorylations of PI3K, Akt and endothelial nitric oxide synthesis (eNOS), and tight junction proteins (Claudin-5 and ZO-1) were measured. Ras and PI3K inhibitors were used for pathway analysis. In transient middle cerebral artery occlusion (tMCAO) mouse model, the effects of MSC-EXs on the cerebral vascular ROS production and apoptosis, cerebral vascular density (cMVD), Evans blue extravasation, brain water content, neurological deficit score (NDS), and infarct volume were determined. Results: MSC-EXs could deliver their carried miR-132-3p into target ECs, which functionally downregulated the target protein RASA1, while upregulated the expression of Ras and the downstream PI3K phosphorylation. Compared to MSC-EXs, MSC-EXsmiR-132-3p were more effective in decreasing ROS production, apoptosis, and tight junction disruption in H/R-injured ECs. These effects were associated with increased levels of phosphorylated Akt and eNOS, which could be abolished by PI3K inhibitor (LY294002) or Ras inhibitor (NSC 23766). In the tMCAO mouse model, the infusion of MSC-EXsmiR-132-3p was more effective than MSC-EXs in reducing cerebral vascular ROS production, BBB dysfunction, and brain injury. Conclusion: Our results suggest that miR-132-3p promotes the beneficial effects of MSC-EXs on brain ischemic injury through protecting cerebral EC functions

NPC-EXs Alleviate Endothelial Oxidative Stress and Dysfunction through the miR-210 Downstream Nox2 and VEGFR2 Pathways

We have demonstrated that neural progenitor cells (NPCs) protect endothelial cells (ECs) from oxidative stress. Since exosomes (EXs) can convey the benefit of parent cells through their carried microRNAs (miRs) and miR-210 is ubiquitously expressed with versatile functions, we investigated the role of miR-210 in the effects of NPC-EXs on oxidative stress and dysfunction in ECs. NPCs were transfected with control and miR-210 scramble/inhibitor/mimic to generate NPC-EXscon, NPC-EXssc, NPC-EXsanti-miR-210, and NPC-EXsmiR-210. The effects of various NPC-EXs on angiotensin II- (Ang II-) induced reactive oxygen species (ROS) overproduction, apoptosis, and dysfunction, as well as dysregulation of Nox2, ephrin A3, VEGF, and p-VEGFR2/VEGFR2 in ECs were evaluated. Results showed (1) Ang II-induced ROS overproduction, increase in apoptosis, and decrease in tube formation ability, accompanied with Nox2 upregulation and reduction of p-VEGFR2/VEGFR2 in ECs. (2) Compared to NPC-EXscon or NPC-EXssc, NPC-EXsanti-miR-210 were less whereas NPC-EXsmiR-210 were more effective on attenuating these detrimental effects induced by Ang II in ECs. (3) These effects of NPC-EXsanti-miR-210 and NPC-EXsmiR-210 were associated with the changes of miR-210, ephrin A3, VEGF, and p-VEGFR2/VEGFR2 ratio in ECs. Altogether, the protective effects of NPC-EXs on Ang II-induced endothelial injury through miR-210 which controls Nox2/ROS and VEGF/VEGFR2 signals were studied

Progress on improving Agricultural Nitrogen use efficiency: UK-China viortual joint centers on Nitrogen Agronomy

Two virtual joint centers for nitrogen agronomy were established between the UK and China to facilitate collaborative research aimed at improving nitrogen use efficiency (NUE) in agricultural production systems and reducing losses of reactive N to the environment. Major focus areas were improving fertilizer NUE, use of livestock manures, soil health, and policy development and knowledge exchange. Improvements to fertilizer NUE included attention to application rate in the context of yield potential and economic considerations and the potential of improved practices including enhanced efficiency fertilizers, plastic film mulching and cropping design. Improved utilization of livestock manures requires knowledge of the available nutrient content, appropriate manure processing technologies and integrated nutrient management practices. Soil carbon, acidification and biodiversity were considered as important aspects of soil health. Both centers identified a range of potential actions that could be taken to improve N management, and the research conducted has highlighted the importance of developing a systemslevel approach to assessing improvement in the overall efficiency of N management and avoiding unintended secondary effects from individual interventions. Within this context, the management of fertilizer emissions and livestock manure at the farm and regional scales appear to be particularly important targets for mitigation

Structure-function analysis of the <em>Fusarium oxysporum</em> Avr2 effector allows uncoupling of its immune-suppressing activity from recognition

Plant pathogens employ effector proteins to manipulate their hosts. Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of tomato wilt disease, produces effector protein Avr2. Besides being a virulence factor, Avr2 triggers immunity in I-2 carrying tomato (Solanum lycopersicum). Fol strains that evade I-2 recognition carry point mutations in Avr2 (e.g. Avr2R45H), but retain full virulence. Here we investigate the virulence function of Avr2 and determine its crystal structure. Transgenic tomato and Arabidopsis expressing either wild-type ΔspAvr2 (deleted signal-peptide) or the ΔspAvr2R45H variant become hypersusceptible to fungal, and even bacterial infections, suggesting that Avr2 targets a conserved defense mechanism. Indeed, Avr2 transgenic plants are attenuated in immunity-related readouts, including flg22-induced growth inhibition, ROS production and callose deposition. The crystal structure of Avr2 reveals that the protein shares intriguing structural similarity to ToxA from the wheat pathogen Pyrenophora tritici-repentis and to TRAF proteins. The I-2 resistance-breaking Avr2V41M, Avr2R45H and Avr2R46P variants cluster on a surface-presented loop. Structure-guided mutagenesis enabled uncoupling of virulence from I-2-mediated recognition. We conclude that I-2-mediated recognition is not based on monitoring Avr2 virulence activity, which includes suppression of immune responses via an evolutionarily conserved effector target, but by recognition of a distinct epitope

Phenotypic Switching of Nonpeptidergic Cutaneous Sensory Neurons following Peripheral Nerve Injury

In adult mammals, the phenotype of half of all pain-sensing (nociceptive) sensory neurons is tonically modulated by growth factors in the glial cell line-derived neurotrophic factor (GDNF) family that includes GDNF, artemin (ARTN) and neurturin (NRTN). Each family member binds a distinct GFRα family co-receptor, such that GDNF, NRTN and ARTN bind GFRα1, -α2, and -α3, respectively. Previous studies revealed transcriptional regulation of all three receptors in following axotomy, possibly in response to changes in growth factor availability. Here, we examined changes in the expression of GFRα1-3 in response to injury in vivo and in vitro. We found that after dissociation of adult sensory ganglia, up to 27% of neurons die within 4 days (d) in culture and this can be prevented by nerve growth factor (NGF), GDNF and ARTN, but not NRTN. Moreover, up-regulation of ATF3 (a marker of neuronal injury) in vitro could be prevented by NGF and ARTN, but not by GDNF or NRTN. The lack of NRTN efficacy was correlated with rapid and near-complete loss of GFRα2 immunoreactivity. By retrogradely-labeling cutaneous afferents in vivo prior to nerve cut, we demonstrated that GFRα2-positive neurons switch phenotype following injury and begin to express GFRα3 as well as the capsaicin receptor, transient receptor potential vanilloid 1(TRPV1), an important transducer of noxious stimuli. This switch was correlated with down-regulation of Runt-related transcription factor 1 (Runx1), a transcription factor that controls expression of GFRα2 and TRPV1 during development. These studies show that NRTN-responsive neurons are unique with respect to their plasticity and response to injury, and suggest that Runx1 plays an ongoing modulatory role in the adult