Yuxi Zou

Adenoviral-mediated interleukin-18 expression in mesenchymal stem cells effectively suppresses the growth of glioma in rats.

Glioma is the most common primary intracranial malignant tumor. Despite advances in surgical techniques and adjuvant radio‐ and chemotherapies, the prognosis for patients with glioma remains poor. We have explored the effects of using genetically modified mesenchymal stem cells (MSCs) to treat malignant glioma in rats. Mesenchymal stem cells isolated from Sprague–Dawley rats can directly suppress the growth of C6 cells in vitro. MSCs transplanted intratumorally can also significantly inhibit the growth of glioma and prolong survival in C6 glioma‐bearing models. MSCs producing Interleukin‐18 infected by adenoviral vector inhibited glioma growth and prolonged the survival of glioma‐bearing rats. Transplantation of IL‐18 secreting MSCs was associated with enhanced T cell infiltration and long‐term anti‐tumor immunity. Thus, IL‐18 may be an effective adoptive immunotherapy for malignant glioma. When used in conjunction with MSCs as targeting vehicles in vivo, IL‐18 may offer a promising new treatment option for malignant glioma.

Bone Marrow-Derived Mesenchymal Stem Cells Maintain the Resting Phenotype of Microglia and Inhibit Microglial Activation

Many studies have shown that microglia in the activated state may be neurotoxic. It has been proven that uncontrolled or over-activated microglia play an important role in many neurodegenerative disorders. Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown in many animal models to have a therapeutic effect on neural damage. Such a therapeutic effect is attributed to the fact that BMSCs have the ability to differentiate into neurons and to produce trophic factors, but there is little information available in the literature concerning whether BMSCs play a therapeutic role by affecting microglial activity. In this study, we triggered an inflammatory response situation in vitro by stimulating microglia with the bacterial endotoxin lipopolysaccharide (LPS), and then culturing these microglia with BMSC-conditioned medium (BMSC-CM). We found that BMSC-CM significantly inhibited proliferation and secretion of pro-inflammatory factors by activated microglia. Furthermore, we found that the phagocytic capacity of microglia was also inhibited by BMSC-CM. Finally, we investigated whether the induction of apoptosis and the production of nitric oxide (NO) were involved in the inhibition of microglial activation. We found that BMSC-CM significantly induced apoptosis of microglia, while no apoptosis was apparent in the LPS-stimulated microglia. Our study also provides evidence that NO participates in the inhibitory effect of BMSCs. Our experimental results provide evidence that BMSCs have the ability to maintain the resting phenotype of microglia or to control microglial activation through their production of several factors, indicating that BMSCs could be a promising therapeutic tool for treatment of diseases associated with microglial activation.

Passive immunization with LINGO-1 polyclonal antiserum afforded neuroprotection and promoted functional recovery in a rat model of spinal cord injury.

LINGO-1 (leucine-rich repeat and Ig domain-containing, Nogo receptor-interacting protein) is an important component of the NgR receptor complex involved in RhoA activation and axon regeneration. The authors report on passive immunization with LINGO-1 polyclonal antiserum, a therapeutic approach to overcome NgR-mediated growth inhibition after spinal cord injury (SCI). The intrathecally administered high-titer rabbit-derived antiserum can be detected around the injury site within a wide time window; it blocks LINGO-1 in vivo with high molecular specificity. In this animal model, passive immunization with LINGO-1 antiserum significantly decreased RhoA activation and increased neuronal survival. Adult rats immunized in this manner show recovery of certain hindlimb motor functions after dorsal hemisection of the spinal cord. Thus, passive immunotherapy with LINGO-1 polyclonal antiserum may represent a promising repair strategy following acute SCI.

Expression profile of cancer-related genes in human adult bone marrow–derived neural stemlike cells highlights the need for tumorigenicity study

Human adult bone marrow–derived neural stemlike cells (MDNSCs) may serve as ideal seed cells for cell replacement therapy for human neurological disorders and injuries. However, the long‐term safety of this cell population after transplantation must be thoroughly explored before clinical application, and tumorigenicity is a major concern. In this study, we generated MDNSCs capable of forming neurospherelike aggregates and with the potency to differentiate into neural lineage cells in vitro and investigated hundreds of cancer‐related genes in MDNSCs in order to determine whether there were any characteristics that could help in the evaluation of their tumorigenic potential. According to the results of testing by PCR and DNA sequencing, there were no mutations at the frequent mutation sites of tumor‐suppressor genes p53, p16, and Rb1. Of the 440 cancer‐related genes covered by Oligo GEArray Human Cancer Microarray OHS‐802, 63 were found to be significantly overexpressed compared with that in fresh normal human adult bone marrow depleted of red blood cells (RBCs). In particular, the overexpressed genes included those promoting cell proliferation and cell invasion and metastasis and members of several oncogenic signaling pathways. The overexpression of MYC, MMP2, Notch2, STC1, ITGA3, STAT5b, RhoC, and Wnt1 was also revealed by quantitative real‐time RT‐PCR. Because it has been shown that activation of some of these genes promote tumorigenesis, our findings highlight the need for further studies of long‐term tumorigenicity in MDNSCs.

Dual expression of hTERT and VEGF prolongs life span and enhances angiogenic ability of aged BMSCs.

Previous studies have confirmed the therapeutic effects of bone marrow stromal cells (BMSCs) transplantation on cerebral ischemia. However, the proliferative, differentiative, and homing capacity of BMSC from the elderly are significantly reduced, especially after several passages expansion in vitro. In this study, by introducing lentivirus-mediated hTERT and VEGF genes to modify human BMSCs from aged donors, we observed extended lifespan, promoted angiogenic capacity while less enhanced tumorigenicity of the genetically engineering BMSCs. These results therefore suggest that the modification of aged BMSCs by dual expression of hTERT and VEGF may be used for autologous cell replacement for ischemic cerebrovascular disease in elderly patients.

Functional Analysis of Neuron-like Cells Differentiated from Neural Stem Cells Derived from Bone Marrow Stroma Cells in vitro

The transversal differentiation of bone marrow stroma cell (BMSCs) into neural stem cells (NSCs) has attracted much attention in recent years because of their therapeutic potential. However, the problem in therapeutic application of NSCs was how to confirm whether neuron-like cells differentiated from bone marrow stroma cell-derived neural stem cells (BMSCs-D-NSCs) possess corresponding functions of neurochemistry and electrophysiology. In the present study, we tried to affirm the function of neuron-like cells differentiated from BMSCs-D-NSCs in vitro. The BMSCs were harvested by gradient centrifugation in Ficoll-Paque and cultured in “NSCs medium”. Immunocytochemistry was used to detect positive expression of neuron-specific nuclear protein (NeuN) in neuron-like cells derived from the BMSCs-D-NSCs. High-pressure liquid chromatography (HPLC) was used to identify neuron-like cells by detecting excitable amino acids [aspartic acid (Asp), glutamic acid (Glu)], inhibited amino acids [glycine (Gly), gamma (γ) -aminobutyric acid (GABA), alanine (Ala)] or monoamines [noradrenaline (NE), 5-hydroxytryptamine (5-HT), dopamine (DA)]. Electrophysiological properties of the neuron-like cells were also examined using patch clamp analysis to verify their neuron-like functions. It was found that the neuron-like cells differentiated from the BMSCs-D-NSCs could express positive NeuN, synthesize and excrete amino acids, and show some typical electrophysiological properties including the typical Na+ and K+ ion channel membrane current under the voltage patch clamp condition, the typical static electrical membrane potential under the current patch clamp condition, and the differential membrane capacitance and resistance values in series between undifferentiated BMSCs-D-NSCs and differentiated neuron-like cells under the whole-cell patch clamp condition. The neuron-like cells differentiated from BMSCs-D-NSCs exhibit both neuron-like biochemical function and some corresponding electrophysiological properties.

Genetic Analysis of Interleukin-1A C(-889)T Polymorphism with Alzheimer Disease

Neuroinflammation has been implicated in the etiology of Alzheimer’s disease (AD). Many studies have suggested that C(-889) T promoter polymorphism in one of the proinflammatory cytokine interleukin-1 (IL-1) encoding gene IL-1A may be associated with AD pathogenesis. To determine whether the polymorphism contributes to the risk for late-onset AD (LOAD) in Chinese, we carried out our investigation in 344 sporadic LOAD patients and 224 healthy controls. No statistical significant association was obtained between IL-1A C(-889) T polymorphism and LOAD and no statistical difference was found between cases and controls after stratification for apolipoprotein E allele 4 (APOE ε4) status. The results reveal that it is not likely that the IL-1A C(-889) T polymorphism is involved in AD pathogenesis in the Chinese population. Further studies of the associations between other IL-1A genetic polymorphisms and AD should be performed in a larger population and biologic functional analysis of IL-1A gene is required to verify the underlying roles of IL-IA in LOAD.

Study on The Proliferation and Drug-resistance of Human Brain Tumor Stem-like Cells

Brain tumor stem-like cells (BTSLCs) have been implied to play an important role in genesis and development of glioma. However, their characteristics on proliferation and drug-resistance are uncertain thoroughly. In this experiment, some of the biological characteristics about BTSLCs were explored. Twenty cases of different grades of human glioma tissues were obtained from clinic. The primary glioma cells were collected and CD133+ cells from them were purified by magnetic cell sorting assay. The BTSLCs were identified by testing the expression of CD133, Nestin, NSE, and GFAP, along with the culture process. WST-8 assay kit was used to evaluate the proliferating situation of CD133+ cells in the different grade gliomas, and to compare the drug-resistance between the CD133+ and CD133− cells in the medium containing different concentrations of teniposide (VM-26). The results showed that the CD133+ cells could regenerate by self-renewal, then generate and different into NSE+ and GFAP+ cells, respectively. CD133+ cells in the high grade of gliomas showed the faster generation than the ones in the low grade. The number of survived CD133+ cells in the medium containing VM-26 was much more than the CD133− ones in it. Therefore, it was implied that the CD133+ BTSLCs existed in the glioma tissues possessed the more tolerant ability to the VM-26, and could proliferate much more easily in the high-grade glioma.

5-Aminolevulinic Acid -Mediated Photodynamic Therapy of Human Glioma Cells In Vitro

ObjectiveTo investigate the effect of 5-aminolevulinic acid (ALA) mediated photodynamic therapy (PDT) on U251 human glioma cells in vitro.MethodsU251 human glioma cells were routinely cultured and then treated with ALA, a type of photosensitizer, at various concentrations followed by light irradiation. The PDT-induced phototoxicity of the cells was determined by a MTT assay. In addition, cells were treated with ALA at a fixed concentration and subjected to various doses of light irradiation.ResultsWith the same light dosage (25.0 J/cm2), the cell survival rates were 70.16%±5.02%, 50.19%±4.79%, 34.97%±5.34%, 27.04%±4.34%, and 24.26% ±2.76% at ALA concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 mM, respectively (F =279.88, P =0.0000). But the survival rates of the cells incubated with 2.0 mM ALA compared to those with 4.0 mM ALA (27.04%±4.34% vs 24.26%±2.76%) showed no significant difference (P=0.611 ). At a single ALA concentration, the cell survival rates were 83.48% ± 6.79%, 68.09%±6.02%, 33.75%± 6.70%, 23.34%± 5.08% and 15.14%± 3.60% for light doses of 6.25,12.5, 25.0,50.0, and 100 J/cm2, respectively (F=422.03, P=0.0000). Without exposure to light, however, the cell survival rates were 96.64% ±6.56%, 97.71% ±5.48%, 98.10% ±6.25%, 99.44% ±7.02%, and 95.86% ±7.80% for ALA concentrations at 0.25, 0.5, 1.0, 2.0, and 4.0 mM, respectively (F=0.68,P =0.6085). Without ALA in the medium, the cell survival rates were 98.74% ±6.20%, 96.49% ±7.13%, 97.60% ±5.94%, 95.70%±4.86%, 98.08%±6.26% for light doses of 6.25, 12.5, 25.0, 50.0, and 100 J/cm2, respectively (F=0.6400, P=0.6368).ConclusionThe PDT damage to the U251 cells increased with ALA concentration within a relative lower range, but then plateaued at higher concentrations. PDT damage was proportional to the doses of irradiated light. Without ALA, the light alone caused no photodynamic damage and ALA itself was nontoxic. The ALA-induced PDT appears to be a promising therapy for glioma.

Oncostatin M-induced upregulation of SDF-1 improves Bone marrow stromal cell migration in a rat middle cerebral artery occlusion stroke model

Abstract Bone marrow‐derived mesenchymal stem cells (BMSCs) exhibit potential regenerative effects on the injured brain. However, these effects are constrained by their limited ability to migrate to the injured site. Oncostatin M (OSM) has been shown to affect the proliferation and migration of mesenchymal stem cells. Therefore, in the present study, we explored whether OSM improves BMSC migration and secretion of growth factors and cytokines in a rat middle cerebral artery occlusion (MCAO) stroke model. The effect of OSM on the proliferation and apoptosis of rat BMSCs was first assessed in vitro, and the gene and secretion levels of factors related to cell nutrition and migration, such as SDF‐1 and VEGF, were detected. To further explore underlying pathways triggered by OSM, BMSCs were treated with OSM in the presence or absence of inhibitors of the STAT3 and ERK pathways. Effects of OSM on SDF‐1 expression in astrocytes and BMSC migration were also evaluated. In the rat MCAO model, OSM secretion levels were detected in the brain for up to 72 h after model establishment. Ventricle injection of OSM alone or OSM combined with caudal vein graft of BMSCs was then performed in MCAO stroke rats. After 72 h, production of SDF‐1 and grafted BMSCs was detected in the lesion areas of the brain, and the nerve function score was evaluated. We found that the production of OSM continually increased in the brains of MCAO rats from 12 h to 72 h. OSM significantly upregulated SDF‐1 in BMSCs via the STAT3 and ERK pathways and significantly promoted the expression of VEGF and MMP‐2. OSM also promoted the secretion of SDF‐1 in astrocytes through the STAT3 and ERK pathways to in turn enhance BMSC migration. Combination treatment with OSM and BMSCs in MCAO rats increased the migration efficiency of BMSCs in the brain, which significantly improved neurofunctional recovery while reducing the expression of inflammatory mediators and promoting the secretion of nutrition factors. Overall, these results show that OSM is highly expressed in the brains of MCAO stroke rats and can upregulate SDF‐1 to promote BMSC migration. Thus, combination treatment with OSM and BMSCs improves the graft efficiency of BMSCs and neurofunctional recovery. HighlightsOncostatin M (OSM) enhances bone marrow mesenchymal stem cell (BMSC) migration.OSM upregulates SDF‐1 in astrocytes and BMSCs via the STAT3 and ERK pathways.OSM is upregulated in the brain of MCAO stroke rats.OSM enhances the therapeutic efficiency of BMSCs in the ischemic brain.