•High glucose causes blood–brain barrier disruption in the early reperfusion of stroke.•MMP-2/9-mediated extracellular ZO-1 degradation.•Caveolin-1-mediated intracellular translocation of ...ZO-1.•Autophagy-lysosome-mediated ZO-1 degradation.
Post-stroke hyperglycemia during early reperfusion increases blood–brain barrier (BBB) permeability and subsequently aggravates brain injury and clinical prognosis. The decreased level of tight junction proteins (TJPs) has been reported but the underlying mechanism remains largely elusive. Herein we designed to investigate the detailed molecular events in brain microvascular endothelial cells (BMECs) ex and in vivo. After oxygen–glucose deprivation (OGD) for 90 min and reperfusion with 8 or 16 mM glucose for 30 min, glucose at 16 mM caused significant decrease in the TJP expression and particularly ZO-1 redistribution from membrane to cytoplasm of BMECs. High glucose also markedly promoted the secretion of MMP-2/9 and oxidative/nitrosative stress, enhanced autophagy and increased the Caveolin-1 and LAMP-2 expression. Moreover, in vivo experiments demonstrated that rapamycin-enhanced autophagy further caused ZO-1 reduction and the increased BBB permeability. Therefore, high-glucose exposure in the early reperfusion causes the BBB disruption, with MMP-2/9-mediated extracellular degradation, caveolin-1-mediated intracellular translocation and autophagy-lysosome-mediated degradation of ZO-1 protein all together involved in the process. The role of MMP-2/-9 and autophagy in the modulation of paracellular permeability was confirmed by pharmacological inhibition. Therefore, our findings may provide new insights into targeting ZO-1 regulation for the purpose of significantly improving the clinical prognosis of ischemic stroke.
Cell senescence is a sign of aging and plays a significant role in the pathogenesis of age-related disorders. For cell therapy, senescence may compromise the quality and efficacy of cells, posing ...potential safety risks. Mesenchymal stem cells (MSCs) are currently undergoing extensive research for cell therapy, thus necessitating the development of effective methods to evaluate senescence. Senescent MSCs exhibit distinctive morphology that can be used for detection. However, morphological assessment during MSC production is often subjective and uncertain. New tools are required for the reliable evaluation of senescent single cells on a large scale in live imaging of MSCs.
We have developed a successful morphology-based Cascade region-based convolution neural network (Cascade R-CNN) system for detecting senescent MSCs, which can automatically locate single cells of different sizes and shapes in multicellular images and assess their senescence state. Additionally, we tested the applicability of the Cascade R-CNN system for MSC senescence and examined the correlation between morphological changes with other senescence indicators.
This deep learning has been applied for the first time to detect senescent MSCs, showing promising performance in both chronic and acute MSC senescence. The system can be a labor-saving and cost-effective option for screening MSC culture conditions and anti-aging drugs, as well as providing a powerful tool for non-invasive and real-time morphological image analysis integrated into cell production.
Chondrogenic progenitor cells (CPCs) have high self-renewal capacity and chondrogenic potential. Intra-articular delivery of purified mesenchymal stem cells (MSCs) from MRL/MpJ "superhealer" mice ...increased bone volume during repair and prevents post-traumatic arthritis. Recently, although extracellular vesicles released from MSCs have been used widely for treating OA, the application of extracellular vesicles secreted by CPCs from MRL/MpJ mice in OA therapy has never been reported. In this study, we evaluated the effects of extracellular vesicles secreted by CPCs from control CBA (CBA-EVs) and MRL/MpJ mice (MRL-EVs) on proliferation and migration of murine chondrocytes. We also determined here if weekly intra-articular injections of CBA-EVs and MRL-EVs would repair and regenerate surgically induced model in mice.
CPC surface markers were detected by flow cytometry. CBA-EVs and MRL-EVs were isolated using an ultrafiltration method. Nanoparticle tracking analysis, transmission electron microscopy, and western blots were used to identify extracellular vesicles. CBA-EVs and MRL-EVs were injected intra-articularly in a mouse model of surgical destabilization of the medial meniscus (DMM)-induced OA, and histological and immunohistochemistry analyses were used to assess the efficacy of exosome injections. We used miRNA-seq analysis to analyze the expression profiles of exosomal miRNAs derived from CBA-EVs as well as MRL-EVs. Cell-counting and scratch assays were used to evaluate the effects of CBA-EVs and MRL-EVs on proliferation and migration of murine chondrocytes, respectively. Meanwhile, a specific RNA inhibitor assesses the roles of the candidate miRNAs in CPC-EV-induced regulation of function of chondrocytes.
Both CBA-EVs and MRL-EVs stimulated chondrocyte proliferation and migration, but MRL-EVs exerted a stronger effect than CBA-EVs. The similar result was also observed in in vivo study, which indicated that injecting either CBA-EVs or MRL-EVs attenuated OA, but MRL-EVs showed a superior therapeutic effect in comparison with CBA-EVs. The results of bioinformatics analyses revealed that the differentially expressed exosomal miRNAs participated in multiple biological processes. We identified 80 significantly upregulated and 100 downregulated miRNAs. Moreover, we found that the top 20 differentially expressed exosomal miRNAs connected OA repair to processes such as AMPK signaling, regulation of autophagy, and insulin signaling. Notably, miRNA 221-3p were highly enriched in MRL-Exos and treatment with miR 221-3p inhibitor markedly decreased chondrocyte proliferation and migration induced by CBA-EVs or MRL-EVs in vitro.
This is the first study to demonstrate MRL-EVs had a greater therapeutic effect on the treatment of OA than CBA-EVs. This study will hopefully provide new insight into the pathogenesis, prevention, and treatment of OA.
Due to the narrow therapeutic window and high mortality of ischemic stroke, it is of great significance to investigate its diagnosis and therapy. We employed weighted gene coexpression network ...analysis (WGCNA) to ascertain gene modules related to stroke and used the maSigPro R package to seek the time-dependent genes in the progression of stroke. Three machine learning algorithms were further employed to identify the feature genes of stroke. A nomogram model was built and applied to evaluate the stroke patients. We analyzed single-cell RNA sequencing (scRNA-seq) data to discern microglia subclusters in ischemic stroke. The RNA velocity, pseudo time, and gene set enrichment analysis (GSEA) were performed to investigate the relationship of microglia subclusters. Connectivity map (CMap) analysis and molecule docking were used to screen a therapeutic agent for stroke. A nomogram model based on the feature genes showed a clinical net benefit and enabled an accurate evaluation of stroke patients. The RNA velocity and pseudo time analysis showed that microglia subcluster 0 would develop toward subcluster 2 within 24 h from stroke onset. The GSEA showed that the function of microglia subcluster 0 was opposite to that of subcluster 2. AZ_628, which screened from CMap analysis, was found to have lower binding energy with Mmp12, Lgals3, Fam20c, Capg, Pkm2, Sdc4, and Itga5 in microglia subcluster 2 and maybe a therapeutic agent for the poor development of microglia subcluster 2 after stroke. Our study presents a nomogram model for stroke diagnosis and provides a potential molecule agent for stroke therapy.
Previously, we established a simple method for deriving mesenchymal stem cells (MSCs) from human induced pluripotent stem cells (iPSC-MSCs). These iPSC-MSCs were capable of forming osteogenic ...structures in scaffolds and nanofibers. The objective of this study is to systematically characterize the mesenchymal characteristics of the iPSC-MSCs by comparing them to bone marrow-derived MSCs (BM-MSCs).
Two iPSC-MSC lines (named as mRNA-iPSC-MSC-YL001 and lenti-iPSC-MSC-A001) and one BM-MSC line were used for the study. Cell proliferation, presence of mesenchymal surface markers, tri-lineage differentiation capability (osteogenesis, chondrogenesis, adipogenesis), and expression of "stemness" genes were analyzed in these MSC lines.
The iPSC-MSCs were similar to BM-MSCs in terms of cell morphology (fibroblast-like) and surface antigen profile: CD29+, CD44+, CD73+, CD90+, CD105+, CD11b-, CD14-, CD31-, CD34-, CD45- and HLA-DR-. A faster proliferative capability was seen in both iPSC-MSCs lines compared to the BM-MSCs. The iPSC-MSCs showed adequate capacity of osteogenesis and chondrogenesis compared to the BM-MSCs, while less adipogenic potential was found in the iPSC-MSCs. The iPSC-MSCs and the tri-lineage differentiated cells (osteoblasts, chondrocytes, adipocytes) all lack expression of "stemness" genes: OCT4, SOX2, GDF3, CRIPTO, UTF1, DPPA4, DNMT3B, LIN28a, and SAL4.
The MSCs derived from human iPSCs with our method have advanced proliferation capability and adequate osteogenic and chondrogenic properties compared to BM-MSCs. However, the iPSC-MSCs were less efficient in their adipogenicity, suggesting that further modifications should be applied to our method to derive iPSC-MSCs more closely resembling the naïve BM-MSCs if necessary.
•Anovelquality evaluationmethod of iPSCcoloniesis proposedby fusingmulti-source features, which are extracted from three deep convolutional neural networks (DCNNs) and four traditional feature ...descriptors.•The evaluation criteria for the iPSC coloniesquality were established and validated by biological method.•The proposed method achieves high performance on the collected iPSC dataset with46500 images.
Induced pluripotent stem cells (iPSCs) have great potential as the basis of regenerative medicine. In this paper, we propose an automatic quality evaluation model based on multi-source feature ensemble learning to divide the iPSC colonies into three categories: good, medium and bad.
First, we obtained iPSCs samples using a Sendai virus reprogramming method. Second, we collected the bright field-images of iPSC colonies and processed them with adaptive gamma transform and data enhancement. The evaluation for the iPSC colony quality was further verified with living cell fluorescent staining, currently accepted as the optimal biological method. Third, multi-source features were extracted using three deep convolutional neural networks (DCNNs) and four traditional feature descriptors. Finally, we utilized a support vector machine (SVM) to perform classification. Before feeding into the SVM, the features were processed by principal component analysis algorithm to save computational cost and training time.
Experimental results on the collected iPSC dataset (46,500 images) show that the proposed method could obtain 95.55% classification accuracy.
Our study could provide a method to efficiently and quickly judge the biological quality of a single iPSC colony or populations and facilitate the large-scale iPSC manufacturing.
Chondrocyte dysfunction occurs during the development of osteoarthritis (OA), typically resulting from a deleterious increase in oxidative stress. Accordingly, strategies for arresting oxidative ...stress-induced chondrocyte dysfunction may lead to new potential therapeutic targets for OA treatment. Forkhead box O (FoxO) transcription factors have recently been shown to play a protective role in chondrocyte dysfunction through the regulation of inflammation, autophagy, aging, and oxidative stress. They also regulate growth, maturation, and matrix synthesis in chondrocytes. In this review, we discuss the recent progress made in the field of oxidative stress-induced chondrocyte dysfunction. We also discuss the protective role of FoxO transcription factors as potential molecular targets for the treatment of OA. Understanding the function of FoxO transcription factors in the OA pathology may provide new insights that will facilitate the development of next-generation therapies to prevent OA development and to slow OA progression.
Graphical Abstract
Induced pluripotent stem cell-derived mesenchymal stem cells (iMSC) and primary MSC comparison: to show the advantages and applications of iMSC.
Mesenchymal stem cells (MSC) ...isolated from different tissue sources exhibit multiple biological effects and have shown promising therapeutic effects in a broad range of diseases. In order to fulfill their clinical applications in context of precision medicine, however, more detailed molecular characterization of diverse subgroups and standardized scalable production of certain functional subgroups would be highly desired. Thus far, the generation of induced pluripotent stem cell (iPSC)-derived MSC (iMSC) seems to provide the unique opportunity to solve most obstacles that currently exist to prevent the broad application of MSC as an advanced medicinal product. The features of iMSC include their single cell clone origins, and defined and controllable cultural conditions for their derivation and proliferation. Still, comprehensive research of the molecular and functional heterogeneity of iMSC, just like MSC from any other tissue types, would be required. Furthered on previous efforts on iMSC differentiation and expansion platform and transcriptomic studies, advantages of single cell multi-omics analysis and other up-to-dated technologies would be taken in order to elucidate the molecular origin and regulation of heterogeneity and to obtain iMSC subgroups homogeneous enough for particular clinical conditions. In this perspective, the current obstacles in MSC applications, the advantages of iMSC over MSC and their implications for biological research and clinical applications will be discussed.
The purpose of this study was to determine the functional recovery of the transplanted induced pluripotent stem cells in a rat model of Huntington's disease with use of 18F-FDG microPET/CT imaging.
...In a quinolinic acid-induced rat model of striatal degeneration, induced pluripotent stem cells were transplanted into the ipsilateral lateral ventricle ten days after the quinolinic acid injection. The response to the treatment was evaluated by serial 18F-FDG PET/CT scans and Morris water maze test. Histological analyses and Western blotting were performed six weeks after stem cell transplantation.
After induced pluripotent stem cells transplantation, higher 18F-FDG accumulation in the injured striatum was observed during the 4 to 6-weeks period compared with the quinolinic acid-injected group, suggesting the metabolic recovery of injured striatum. The induced pluripotent stem cells transplantation improved learning and memory function (and striatal atrophy) of the rat in six week in the comparison with the quinolinic acid-treated controls. In addition, immunohistochemical analysis demonstrated that transplanted stem cells survived and migrated into the lesioned area in striatum, and most of the stem cells expressed protein markers of neurons and glial cells.
Our findings show that induced pluripotent stem cells can survive, differentiate to functional neurons and improve partial striatal function and metabolism after implantation in a rat Huntington's disease model.
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