Human adipose-derived stem cells (hADSCs) are increasingly presumed to be a prospective stem cell source for cell replacement therapy in various degenerative and/or traumatic diseases. The potential of trans-differentiating hADSCs into motor neuron cells indisputably provides an alternative way for spinal cord injury (SCI) treatment. In the present study, a stepwise and efficient hADSC trans-differentiation protocol with retinoic acid (RA), sonic hedgehog (SHH), and neurotrophic factors were developed. With this protocol hADSCs could be converted into electrophysiologically active motoneuron-like cells (hADSC-MNs), which expressed both a cohort of pan neuronal markers and motor neuron specific markers. Moreover, after being primed for neuronal differentiation with RA/SHH, hADSCs were transplanted into SCI mouse model and they survived, migrated, and integrated into injured site and led to partial functional recovery of SCI mice. When ablating the transplanted hADSC-MNs harboring HSV-TK-mCherry overexpression system with antivirial Ganciclovir (GCV), functional relapse was detected by motor-evoked potential (MEP) and BMS assays, implying that transplanted hADSC-MNs participated in rebuilding the neural circuits, which was further confirmed by retrograde neuronal tracing system (WGA). GFP-labeled hADSC-MNs were subjected to whole-cell patch-clamp recording in acute spinal cord slice preparation and both action potentials and synaptic activities were recorded, which further confirmed that those pre-conditioned hADSCs indeed became functionally active neurons in vivo. As well, transplanted hADSC-MNs largely prevented the formation of injury-induced cavities and exerted obvious immune-suppression effect as revealed by preventing astrocyte reactivation and favoring the secretion of a spectrum of anti-inflammatory cytokines and chemokines. Our work suggests that hADSCs can be readily transformed into MNs in vitro, and stay viable in spinal cord of the SCI mouse and exert multi-therapeutic effects by rebuilding the broken circuitry and optimizing the microenvironment through immunosuppression.
Background/Aims: Doublecortin-like kinase 1 (DCLK1) is emerging as a tumor-specific stem cell marker in pancreatic cancer (PC). MicroRNA-195 (miR-195) plays an important role in many types of tumors. However, the roles of DCLK1 in cancer and miRNAs that directly regulate DCLK1 have not been elucidated. The goal of this study is to assess the effects of miR-195 on inhibiting DCLK1 and to clarify the regulating mechanism of miR-195-DCLK1 in PC cells. Methods: The expression of DCLK1 protein and miR-195 in PC tissues and adjacent healthy pancreatic tissues was detected by Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively and the correlation between overall survival of PC patients and expression of DCLK1 was measured by Kaplan-Meier analysis. Bioinformatics tools were used to identify the target gene of miR-195. Effects of miR-195 and DCLK1 on proliferation and cell cycle of PC cells were analyzed by MTT, colony formation assays and flow cytometry. Transwell and wound-healing experiments were employed to examine the cellular migration and invasion. A xenograft mouse model was also used to test the effects of miR-195 on tumor growth and metastasis in vivo. Results: The expression level of DCLK1 and miR-195 shows an inverse correlation in PC tissues and cell lines. A higher DCLK1 level is associated with higher TNM (tumor, node, and metastasis) stage, higher rate of lymph node metastasis, and poor survival. Luciferase reporter assay shows that miR-195 directly targets DCLK1. Overexpression of miR-195 inhibits proliferation, migration and invasion of PC cells, whereas downregulation of miR-195 has an opposite role. These actions were similar to the effects of knockdown and overexpression of DCLK1, respectively. Conclusions: These data suggest that miR-195 has tumor suppressor roles in PC by targeting DCLK1. MiR-195-DCLK1 pathway may provide insight into PC progression and represent a novel, promising diagnostic and therapeutic target for PC.
Spinal cord injury (SCI) involves diverse injury responses in different cell types in a temporally and spatially specific manner. Here, using single-cell transcriptomic analyses combined with classic anatomical, behavioral, electrophysiological analyses, we report, with single-cell resolution, temporal molecular and cellular changes in crush-injured adult mouse spinal cord. Data revealed pathological changes of 12 different major cell types, three of which infiltrated into the spinal cord at distinct times post-injury. We discovered novel microglia and astrocyte subtypes in the uninjured spinal cord, and their dynamic conversions into additional stage-specific subtypes/states. Most dynamic changes occur at 3-days post-injury and by day-14 the second wave of microglial activation emerged, accompanied with changes in various cell types including neurons, indicative of the second round of attacks. By day-38, major cell types are still substantially deviated from uninjured states, demonstrating prolonged alterations. This study provides a comprehensive mapping of cellular/molecular pathological changes along the temporal axis after SCI, which may facilitate the development of novel therapeutic strategies, including those targeting microglia.
Endothelial dysfunction, which includes endothelial oxidative damage and vascular inflammation, is a key initiating step in the pathogenesis of atherosclerosis (AS) and an independent risk factor for this disorder. Intracellular chloride channel 1 (CLIC1), a novel metamorphic protein, acts as a sensor of cell oxidation and is involved in inflammation. In this study, we hypothesize that CLIC1 plays an important role in AS. Apolipoprotein E-deficient mice were supplied with a normal diet or a high-fat and high-cholesterol diet for 8 weeks. Overexpressed CLIC1 was associated with the accelerated atherosclerotic plaque development, amplified oxidative stress, and in vivo release of inflammatory cytokines. We subsequently examined the underlying molecular mechanisms through in vitro experiments. Treatment of cultured human umbilical vein endothelial cells (HUVECs) with H2O2 induced endothelial oxidative damage and enhanced CLIC1 expression. Suppressing CLIC1 expression through gene knocked-out (CLIC1−/−) or using the specific inhibitor indanyloxyacetic acid-94 (IAA94) reduced ROS production, increased SOD enzyme activity, and significantly decreased MDA level. CLIC1−/− HUVECs exhibited significantly reduced expression of TNF-α and IL-1β as well as ICAM-1 and VCAM-1 at the protein levels. In addition, H2O2 promoted CLIC1 translocation to the cell membrane and insertion into lipid membranes, whereas IAA94 inhibited CLIC1 membrane translocation induced by H2O2. By contrast, the majority of CLIC1 did not aggregate on the cell membrane in normal HUVECs, and this finding is consistent with the changes in cytoplasmic chloride ion concentration. This study demonstrates for the first time that CLIC1 is overexpressed during AS development both in vitro and in vivo and can regulate the accumulation of inflammatory cytokines and production of oxidative stress. Our results also highlight that deregulation of endothelial functions may be associated with the membrane translocation of CLIC1 and active chloride-selective ion channels in endothelial cells.
Spinal cord injury (SCI) remains a severe condition with an extremely high disability rate. The challenges of SCI repair include its complex pathological mechanisms and the difficulties of neural regeneration in the central nervous system. In the past few decades, researchers have attempted to completely elucidate the pathological mechanism of SCI and identify effective strategies to promote axon regeneration and neural circuit remodeling, but the results have not been ideal. Recently, new pathological mechanisms of SCI, especially the interactions between immune and neural cell responses, have been revealed by single-cell sequencing and spatial transcriptome analysis. With the development of bioactive materials and stem cells, more attention has been focused on forming intermediate neural networks to promote neural regeneration and neural circuit reconstruction than on promoting axonal regeneration in the corticospinal tract. Furthermore, technologies to control physical parameters such as electricity, magnetism and ultrasound have been constantly innovated and applied in neural cell fate regulation. Among these advanced novel strategies and technologies, stem cell therapy, biomaterial transplantation, and electromagnetic stimulation have entered into the stage of clinical trials, and some of them have already been applied in clinical treatment. In this review, we outline the overall epidemiology and pathophysiology of SCI, expound on the latest research progress related to neural regeneration and circuit reconstruction in detail, and propose future directions for SCI repair and clinical applications.
Spinal cord injury (SCI) leads to the loss of sensory, motor, and autonomic function. We aimed to identify the therapeutic targets of-SCI by bioinformatics analysis. The gene expression profile of GSE20907 was downloaded from gene expression omnibus database. By comparing gene expression profiles with control samples, we screened out several differentially expressed genes (DEGs) in 3 days, 2 weeks and 1 month post-SCI. The pathway enrichment and protein-protein interaction (PPI) network analysis for the identified DEGs were performed. Then, transcription factors and microRNAs for DEGs were predicted. We found that up-regulated DEGs mainly participated in cell cycle, oxidative phosphorylation and immune-related pathways; while down-regulated DEGs were mainly involved in oxidative phosphorylation and central nervous system disease signaling pathways. In the constructed PPI network, Bub1, Vascular endothelial growth factor, Topoisomerase IIα (TOP2a) and Cdc20 showed better correspondence with cell cycle, repair system and nerve system. Furthermore, the up-regulated genes (Arpc1b, CD74 and Brd2) significantly mapped to the target genes of transcription factors. The down-regulated genes of 3 days post-injury and the up-regulated genes of 2 weeks post-injury were significantly enriched as the target genes of microRNAs (miR-129 and miR-124). In conclusion, our results may provide guidelines to discuss the collaboration of PPI network in carcinogenesis of SCI.
BackgroundThe purpose was to explore possible risk factors of facet joint violation induced by adjacent superior vertebral pedicle screw during the minimally invasive surgery transforaminal lumbar interbody fusion (MIS-TLIF).MethodsA total of 69 patients with lumbar degenerative disease, who underwent MIS-TLIF were retrospectively reviewed. Postoperative computed tomography images were used to assess the facet joint violation. The correlation of facet joint violations with gender, age, body mass index (BMI), the adjacent superior vertebral level, fusion segment numbers, position of screw insertion, straight leg-raising test (SLRT) results, clinical diseases and renal dysfunction were analyzed by Chi-square tests and binary logistic regression analysis.ResultsThe incidence of adjacent superior facet joint violations was 25.4 %. Chi-square test showed the patients with age <60 and high BMI (≥30 kg/m2) were more prone to have facet joint violations (P = 0.007; P = 0.006). The single segment fusion presented more facet joint violations than the double segments fusion (P = 0.048). The vertebral pedicle screw implant location at L5 showed more facet joint violations compared with that at L3 and L4 (P = 0.035). No correlation was found between gender, screw implant position, SLRT results, clinical diseases and renal dysfunction and facet joint violations. Logistic regression analysis revealed that age <60 years (OR: 2.902; 95 % CI 1.227–6.864; P = 0.015) and BMI ≥30 kg/m2 (OR: 2.825; 95 % CI 1.191–6.700; P = 0.018 < 0.05) were significantly associated with facet joint violation.ConclusionThese results found a high incidence of adjacent superior vertebral facet joint violation in the MIS-TLIF. Age <60 and BMI ≥30 kg/m2 might be risk factors of facet joint violation.Evidence level: Level 4.
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