Cross-species modeling of muscular dystrophy in Caenorhabditis elegans using patient-derived extracellular vesicles.

Reliable disease models are critical for medicine advancement. Here, we established a versatile human disease model system using patient-derived extracellular vesicles (EVs), which transfer a pathology-inducing cargo from a patient to a recipient naïve model organism. As a proof of principle, we applied EVs from the serum of patients with muscular dystrophy to Caenorhabditis elegans and demonstrated their capability to induce a spectrum of muscle pathologies, including lifespan shortening and robust impairment of muscle organization and function. This demonstrates that patient-derived EVs can deliver disease-relevant pathologies between species and can be exploited for establishing novel and personalized models of human disease. Such models can potentially be used for disease diagnosis, prognosis, analyzing treatment responses, drug screening and identification of the disease-transmitting cargo of patient-derived EVs and their cellular targets. This system complements traditional genetic disease models and enables modeling of multifactorial diseases and of those not yet associated with specific genetic mutations.

Propofol Inhibits Glioma Stem Cell Growth and Migration and Their Interaction with Microglia via BDNF-AS and Extracellular Vesicles.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. GBM contains a small subpopulation of glioma stem cells (GSCs) that are implicated in treatment resistance, tumor infiltration, and recurrence, and are thereby considered important therapeutic targets. Recent clinical studies have suggested that the choice of general anesthetic (GA), particularly propofol, during tumor resection, affects subsequent tumor response to treatments and patient prognosis. In this study, we investigated the molecular mechanisms underlying propofol's anti-tumor effects on GSCs and their interaction with microglia cells. Propofol exerted a dose-dependent inhibitory effect on the self-renewal, expression of mesenchymal markers, and migration of GSCs and sensitized them to both temozolomide (TMZ) and radiation. At higher concentrations, propofol induced a large degree of cell death, as demonstrated using microfluid chip technology. Propofol increased the expression of the lncRNA BDNF-AS, which acts as a tumor suppressor in GBM, and silencing of this lncRNA partially abrogated propofol's effects. Propofol also inhibited the pro-tumorigenic GSC-microglia crosstalk via extracellular vesicles (EVs) and delivery of BDNF-AS. In conclusion, propofol exerted anti-tumor effects on GSCs, sensitized these cells to radiation and TMZ, and inhibited their pro-tumorigenic interactions with microglia via transfer of BDNF-AS by EVs.

Effect of Cocaine on Potassium-Evoked Release of Glutamate From Fetal Rat Brain Synaptosomes.

Introduction Cocaine use during pregnancy can affect fetal brain development. A fetal brain injury could happen from the direct effect of cocaine on the developing brain or from the reduction of placental perfusion from vasoconstriction, which may lead to hypoxia-ischemia. A potential mechanism for brain injury could be due to a neurotransmitter imbalance within the brain, especially glutamate. In an immature rat brain synaptosome model, we explored the additive effect of cocaine alone on glutamate release and the effect of cocaine combined with simulated hypoxic depolarization using potassium as a surrogate. Method Rat pups' brains were dissected and placed on a chilled petri dish. They then entered the experimental protocol. The suspended synaptosomes were divided equally into four experimental groups (control, high potassium "surrogate to hypoxic stimulation," cocaine, and cocaine + high K). Reversed-phase high-performance liquid chromatography analyzed glutamate with fluorescent detection Results The glutamate level was lowest in the cocaine-only group, with a level of 1.96 × 104, compared to the control and high potassium group. However, combining cocaine with high potassium seemed to generate a synergistic effect, achieving the highest glutamate level of all groups with a value of 5.31 × 104. Post hoc Conover's test for multiple pairwise-comparison between groups was done. In comparing various solutions to control, we did not find a statistically significant difference with the cocaine-only solution with a p-value of 0.074. Also, on comparing various other solutions to each other, there was no statistically significant difference between cocaine vs. cocaine + high potassium a p-value of 0.074. Conclusion Our data support the conclusion that cocaine alone does not induce glutamate release from fetal rat brain synaptosomes. Exposure to high potassium does lead to glutamate release. However, cocaine greatly enhances glutamate release in the presence of high potassium levels. This could explain how cocaine affects brain maturation during pregnancy with a low oxygen tension environment in the placenta. This hypothesis should be tested in vivo.

Correction: The novel long non-coding RNA TALNEC2, regulates tumor cell growth and the stemness and radiation response of glioma stem cells.

[This corrects the article DOI: 10.18632/oncotarget.15991.].

Neuron-Glia Crosstalk Plays a Major Role in the Neurotoxic Effects of Ketamine via Extracellular Vesicles.

Background: There is a compelling evidence from animal models that early exposure to clinically relevant general anesthetics (GAs) interferes with brain development, resulting in long-lasting cognitive impairments. Human studies have been inconclusive and are challenging due to numerous confounding factors. Here, we employed primary human neural cells to analyze ketamine neurotoxic effects focusing on the role of glial cells and their activation state. We also explored the roles of astrocyte-derived extracellular vesicles (EVs) and different components of the brain-derived neurotrophic factor (BDNF) pathway. Methods: Ketamine effects on cell death were analyzed using live/dead assay, caspase 3 activity and PARP-1 cleavage. Astrocytic and microglial cell differentiation was determined using RT-PCR, ELISA and phagocytosis assay. The impact of the neuron-glial cell interactions in the neurotoxic effects of ketamine was analyzed using transwell cultures. In addition, the role of isolated and secreted EVs in this cross-talk were studied. The expression and function of different components of the BDNF pathway were analyzed using ELISA, RT-PCR and gene silencing. Results: Ketamine induced neuronal and oligodendrocytic cell apoptosis and promoted pro-inflammatory astrocyte (A1) and microglia (M1) phenotypes. Astrocytes and microglia enhanced the neurotoxic effects of ketamine on neuronal cells, whereas neurons increased oligodendrocyte cell death. Ketamine modulated different components in the BDNF pathway: decreasing BDNF secretion in neurons and astrocytes while increasing the expression of p75 in neurons and that of BDNF-AS and pro-BDNF secretion in both neurons and astrocytes. We demonstrated an important role of EVs secreted by ketamine-treated astrocytes in neuronal cell death and a role for EV-associated BDNF-AS in this effect. Conclusions: Ketamine exerted a neurotoxic effect on neural cells by impacting both neuronal and non-neuronal cells. The BDNF pathway and astrocyte-derived EVs represent important mediators of ketamine effects. These results contribute to a better understanding of ketamine neurotoxic effects in humans and to the development of potential approaches to decrease its neurodevelopmental impact.

Expanding the MECP2 network using comparative genomics reveals potential therapeutic targets for Rett syndrome.

Inactivating mutations in the Methyl-CpG Binding Protein 2 (MECP2) gene are the main cause of Rett syndrome (RTT). Despite extensive research into MECP2 function, no treatments for RTT are currently available. Here, we used an evolutionary genomics approach to construct an unbiased MECP2 gene network, using 1028 eukaryotic genomes to prioritize proteins with strong co-evolutionary signatures with MECP2. Focusing on proteins targeted by FDA-approved drugs led to three promising targets, two of which were previously linked to MECP2 function (IRAK, KEAP1) and one that was not (EPOR). The drugs targeting these three proteins (Pacritinib, DMF, and EPO) were able to rescue different phenotypes of MECP2 inactivation in cultured human neural cell types, and appeared to converge on Nuclear Factor Kappa B (NF-κB) signaling in inflammation. This study highlights the potential of comparative genomics to accelerate drug discovery, and yields potential new avenues for the treatment of RTT.

Specific Compositions of Cannabis sativa Compounds Have Cytotoxic Activity and Inhibit Motility and Colony Formation of Human Glioblastoma Cells In Vitro.

Glioblastoma multiforme (GBM) is the most lethal subtype of glioma. Cannabis sativa is used for the treatment of various medical conditions. Around 150 phytocannabinoids have been identified in C. sativa, among them Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) that trigger GBM cell death. However, the optimal combinations of cannabis molecules for anti-GBM activity are unknown. Chemical composition was determined using high-performance liquid chromatography (HPLC) and gas chromatography mass spectrometry (GC/MS). Cytotoxic activity was determined by XTT and lactate dehydrogenase (LDH) assays and apoptosis and cell cycle by fluorescence-activated cell sorting (FACS). F-actin structures were observed by confocal microscopy, gene expression by quantitative PCR, and cell migration and invasion by scratch and transwell assays, respectively. Fractions of a high-THC cannabis strain extract had significant cytotoxic activity against GBM cell lines and glioma stem cells derived from tumor specimens. A standard mix (SM) of the active fractions F4 and F5 induced apoptosis and expression of endoplasmic reticulum (ER)-stress associated-genes. F4 and F5 inhibited cell migration and invasion, altered cell cytoskeletons, and inhibited colony formation in 2 and 3-dimensional models. Combinations of cannabis compounds exert cytotoxic, anti-proliferative, and anti-migratory effects and should be examined for efficacy on GBM in pre-clinical studies and clinical trials.

Expression and regulatory roles of lncRNAs in G-CIMP-low vs G-CIMP-high Glioma: an in-silico analysis.

BACKGROUND: Clinically relevant glioma subtypes, such as the glioma-CpG island methylator phenotype (G-CIMP), have been defined by epigenetics. In this study, the role of long non-coding RNAs in association with the poor-prognosis G-CMIP-low phenotype and the good-prognosis G-CMIP-high phenotype was investigated. Functional associations of lncRNAs with mRNAs and miRNAs were examined to hypothesize influencing factors of the aggressive phenotype. METHODS: RNA-seq data on 250 samples from TCGA's Pan-Glioma study, quantified for lncRNA and mRNAs (GENCODE v28), were analyzed for differential expression between G-CIMP-low and G-CIMP-high phenotypes. Functional interpretation of the differential lncRNAs was performed by Ingenuity Pathway Analysis. Spearman rank order correlation estimates between lncRNA, miRNA, and mRNA nominated differential lncRNA with a likely miRNA sponge function. RESULTS: We identified 4371 differentially expressed features (mRNA = 3705; lncRNA = 666; FDR ≤ 5%). From these, the protein-coding gene TP53 was identified as an upstream regulator of differential lncRNAs PANDAR and PVT1 (p = 0.0237) and enrichment was detected in the "development of carcinoma" (p = 0.0176). Two lncRNAs (HCG11, PART1) were positively correlated with 342 mRNAs, and their correlation estimates diminish after adjusting for either of the target miRNAs: hsa-miR-490-3p, hsa-miR-129-5p. This suggests a likely sponge function for HCG11 and PART1. CONCLUSIONS: These findings identify differential lncRNAs with oncogenic features that are associated with G-CIMP phenotypes. Further investigation with controlled experiments is needed to confirm the molecular relationships.

Frequency and Analysis of Unplanned Extubation in Coronavirus Disease 2019 Patients.

OBJECTIVES: To determine if patients with coronavirus disease 2019 had a greater number of unplanned extubations resulting in reintubations than in patients without coronavirus disease 2019. DESIGN: Retrospective cohort study comparing the frequency of unplanned extubations resulting in reintubations in a group of coronavirus disease 2019 patients to a historical (noncoronavirus disease 2019) control group. SETTING: This study was conducted at Henry Ford Hospital, an academic medical center in Detroit, MI. The historical noncoronavirus disease 2019 patients were treated in the 68 bed medical ICU. The coronavirus disease 2019 patients were treated in the coronavirus disease ICU, which included the 68 medical ICU beds, 18 neuro-ICU beds, 32 surgical ICU beds, and 40 cardiovascular ICU beds, as the medical ICU was expanded to these units at the peak of the pandemic in Detroit, MI. PATIENTS: The coronavirus disease 2019 cohort included patients diagnosed with coronavirus disease 2019 who were intubated for respiratory failure from March 12, 2020, to April 13, 2020. The historic control (noncoronavirus disease 2019) group consisted of patients who were admitted to the medical ICU in the year spanning from November 1, 2018 to October 31, 2019, with a need for mechanical ventilation that was not related to surgery or a neurologic reason. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: To identify how many patients in each cohort had unplanned extubations, an electronic medical records query for patients with two intubations within 30 days was performed, in addition to a review of our institutional quality and safety database of reported self-extubations. Medical charts were manually reviewed by board-certified anesthesiologists to confirm each event was an unplanned extubation followed by a reintubation within 24 hours. There was a significantly greater incidence of unplanned extubations resulting in reintubation events in the coronavirus disease 2019 cohort than in the noncoronavirus disease 2019 cohort (coronavirus disease 2019 cohort: 167 total admissions with 22 events-13.2%; noncoronavirus disease 2019 cohort: 326 total admissions with 14 events-4.3%; p < 0.001). When the rate of unplanned extubations was expressed per 100 intubated days, there was not a significant difference between the groups (0.88 and 0.57, respectively; p = 0.269). CONCLUSIONS: Coronavirus disease 2019 patients have a higher incidence of unplanned extubation that requires reintubation than noncoronavirus disease 2019 patients. Further study is necessary to evaluate the variables that contribute to this higher incidence and clinical strategies that can reduce it.

miR-504 modulates the stemness and mesenchymal transition of glioma stem cells and their interaction with microglia via delivery by extracellular vesicles.

Glioblastoma (GBM) is a highly aggressive tumor with poor prognosis. A small subpopulation of glioma stem cells (GSCs) has been implicated in radiation resistance and tumor recurrence. In this study we analyzed the expression of miRNAs associated with the functions of GSCs using miRNA microarray analysis of these cells compared with human neural stem cells. These analyses identified gene clusters associated with glioma cell invasiveness, axonal guidance, and TGF-ß signaling. miR-504 was significantly downregulated in GSCs compared with NSCs, its expression was lower in GBM compared with normal brain specimens and further decreased in the mesenchymal glioma subtype. Overexpression of miR-504 in GSCs inhibited their self-renewal, migration and the expression of mesenchymal markers. The inhibitory effect of miR-504 was mediated by targeting Grb10 expression which acts as an oncogene in GSCs and GBM. Overexpression of exogenous miR-504 resulted also in its delivery to cocultured microglia by GSC-secreted extracellular vesicles (EVs) and in the abrogation of the GSC-induced polarization of microglia to M2 subtype. Finally, miR-504 overexpression prolonged the survival of mice harboring GSC-derived xenografts and decreased tumor growth. In summary, we identified miRNAs and potential target networks that play a role in the stemness and mesenchymal transition of GSCs and the miR-504/Grb10 pathway as an important regulator of this process. Overexpression of miR-504 exerted antitumor effects in GSCs as well as bystander effects on the polarization of microglia via delivery by EVs.

Synthetic PreImplantation Factor (sPIF) induces posttranslational protein modification and reverses paralysis in EAE mice.

An autoimmune response against myelin protein is considered one of the key pathogenic processes that initiates multiple sclerosis (MS). The currently available MS disease modifying therapies have demonstrated to reduce the frequency of inflammatory attacks. However, they appear limited in preventing disease progression and neurodegeneration. Hence, novel therapeutic approaches targeting both inflammation and neuroregeneration are urgently needed. A new pregnancy derived synthetic peptide, synthetic PreImplantation Factor (sPIF), crosses the blood-brain barrier and prevents neuro-inflammation. We report that sPIF reduces paralysis and de-myelination of the brain in a clinically-relevant experimental autoimmune encephalomyelitis mice model. These effects, at least in part, are due to post-translational modifications, which involve cyclic AMP dependent protein kinase (PKA), calcium-dependent protein kinase (PKC), and immune regulation. In terms of potential MS treatment, sPIF was successfully tested in neurodegenerative animal models of perinatal brain injury and experimental autoimmune encephalitis. Importantly, sPIF received a FDA Fast Track Approval for first in human trial in autommuninty (completed).

sPIF promotes myoblast differentiation and utrophin expression while inhibiting fibrosis in Duchenne muscular dystrophy via the H19/miR-675/let-7 and miR-21 pathways.

Duchenne muscular dystrophy (DMD) is a progressive, lethal, X-linked disease of skeletal and cardiac muscles caused by mutations in the dystrophin gene. Loss of dystrophin leads to muscle fiber damage and impairment of satellite cell asymmetric division, which are essential for muscle regeneration. These processes ultimately result in muscle wasting and the replacement of the degenerating muscles by fibrogenic cells, a process that leads to the generation of fibrotic tissues. Preimplantation factor (PIF) is an evolutionary conserved 15-amino acid peptide secreted by viable mammalian embryos. Synthetic PIF (sPIF) reproduces the protective/regenerative effects of the endogenous peptide in immune disorders and transplantation models. In this study, we demonstrated that sPIF treatment promoted mouse and human myoblast differentiation and inhibited the expression of collagen 1A1, collagen 1A2, and TGF-ß in DMD patient-derived myoblasts. Additionally, sPIF increased the expression of utrophin, a homolog of dystrophin protein. sPIF effects were mediated via the upregulation of lncRNA H19 and miR-675 and downregulation of let-7. sPIF also inhibited the expression of miR-21, a major fibrosis regulator. The administration of sPIF in mdx mice significantly decreased serum creatine kinase and collagen I and collagen IV expression in the diaphragm, whereas it increased utrophin expression in the diaphragm, heart and quadriceps muscles. In conclusion, sPIF promoted the differentiation of DMD myoblasts, increased utrophin expression via the H19/miRNA-675/let-7 pathway, and reduced muscle fibrosis possibly via the upregulation of miR-675 and inhibition of miR-21 expression. These findings strongly support pursuing sPIF as a potential therapeutic agent for DMD. Moreover, the completion of an sPIF phase I safety trial will further promote the use of sPIF for the treatment of muscular dystrophies.

Placenta-derived mesenchymal stromal cells and their exosomes exert therapeutic effects in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a degenerative lethal, X-linked disease of skeletal and cardiac muscles caused by mutations in the dystrophin gene. Cell therapy using different cell types, including mesenchymal stromal cells (MSCs), has been considered as a potential approach for the treatment of DMD. MSCs can be obtained from autologous sources such as bone marrow and adipose tissues or from allogeneic placenta and umbilical cord. The safety and therapeutic impact of these cells has been demonstrated in pre-clinical and clinical studies and their functions are attributed to paracrine effects that are mediated by secreted cytokines and extracellular vesicles. Here, we studied the therapeutic effects of placenta-derived MSCs (PL-MSCs) and their secreted exosomes using mouse and human myoblasts from healthy controls, Duchenne patients and mdx mice. Treatment of myoblasts with conditioned medium or exosomes secreted by PL-MSCs increased the differentiation of these cells and decreased the expression of fibrogenic genes in DMD patient myoblasts. In addition, these treatments also increased the expression of utrophin in these cells. Using a quantitative miR-29c reporter, we demonstrated that the PL-MSC effects were partly mediated by the transfer of exosomal miR-29c. Intramuscular transplantation of PL-MSCs in mdx mice resulted in decreased creatine kinase levels. PL-MSCs significantly decreased the expression of TGF-ß and the level of fibrosis in the diaphragm and cardiac muscles, inhibited inflammation and increased utrophin expression. In vivo imaging analyses using MSCs labeled with gold nanoparticles or fluorescent dyes demonstrated localization of the cells in the muscle tissues up to 3 weeks post treatment. Altogether, these results demonstrate that PL-MSCs and their secreted exosomes have important clinical applications in cell therapy of DMD partly via the targeted delivery of exosomal miR-29c.

Therapeutic Effect of Astroglia-like Mesenchymal Stem Cells Expressing Glutamate Transporter in a Genetic Rat Model of Depression.

Recent studies have proposed that abnormal glutamatergic neurotransmission and glial pathology play an important role in the etiology and manifestation of depression. It was postulated that restoration of normal glutamatergic transmission, by enhancing glutamate uptake, may have a beneficial effect on depression. We examined this hypothesis using unique human glial-like mesenchymal stem cells (MSCs), which in addition to inherent properties of migration to regions of injury and secretion of neurotrophic factors, were differentiated to express high levels of functional glutamate transporters (excitatory amino acid transporters; EAAT). Additionally, gold nanoparticles (GNPs), which serve as contrast agents for CT imaging, were loaded into the cells for non-invasive, real-time imaging and tracking of MSC migration and final location within the brain. MSC-EAAT (2×105; 10 µl) were administered (i.c.v.) to Flinder Sensitive Line rats (FSLs), a genetic model for depression, and longitudinal behavioral and molecular changes were monitored. FSL rats treated with MSC-EAAT showed attenuated depressive-like behaviors (measured by the forced swim test, novelty exploration test and sucrose self-administration paradigm), as compared to controls. CT imaging, Flame Atomic Absorption Spectroscopy analysis and immunohistochemistry showed that the majority of MSCs homed specifically to the dentate gyrus of the hippocampus, a region showing structural brain changes in depression, including loss of glial cells. mRNA and protein levels of EAAT1 and BDNF were significantly elevated in the hippocampus of MSC-EAAT-treated FSLs. Our findings indicate that MSC-EAATs effectively improve depressive-like manifestations, possibly in part by increasing both glutamate uptake and neurotropic factor secretion in the hippocampus.

Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles.

Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell-related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore convened a Workshop on this topic to discuss the opportunities and challenges associated with development of EV-based therapeutics at the preclinical and clinical levels. This review outlines topic-specific action items that, if addressed, will enhance the development of best-practice models for EV therapies. Stem Cells Translational Medicine 2017;6:1730-1739.

PIF* promotes brain re-myelination locally while regulating systemic inflammation- clinically relevant multiple sclerosis M.smegmatis model.

Neurologic disease diagnosis and treatment is challenging. Multiple Sclerosis (MS) is a demyelinating autoimmune disease with few clinical forms and uncertain etiology. Current studies suggest that it is likely caused by infection(s) triggering a systemic immune response resulting in antigen/non-antigen-related autoimmune response in central nervous system (CNS). New therapeutic approaches are needed. Secreted by viable embryos, PreImplantation Factor (PIF) possesses a local and systemic immunity regulatory role. Synthetic PIF (PIF) duplicates endogenous peptide's protective effect in pre-clinical autoimmune and transplantation models. PIF protects against brain hypoxia-ischemia by directly targeting microglia and neurons. In chronic experimental autoimmune encephalitis (EAE) model PIF reverses paralysis while promoting neural repair. Herein we report that PIF directly promotes brain re-myelination and reverses paralysis in relapsing remitting EAE MS model. PIF crosses the blood-brain barrier targeting microglia. Systemically, PIF decreases pro-inflammatory IL23/IL17 cytokines, while preserving CNS-specific T-cell repertoire. Global brain gene analysis revealed that PIF regulates critical Na+/K+/Ca++ ions, amino acid and glucose transport genes expression. Further, PIF modulates oxidative stress, DNA methylation, cell cycle regulation, and protein ubiquitination while regulating multiple genes. In cultured astrocytes, PIF promotes BDNF-myelin synthesis promoter and SLC2A1 (glucose transport) while reducing deleterious E2F5, and HSP90ab1 (oxidative stress) genes expression. In cultured microglia, PIF increases anti-inflammatory IL10 while reducing pro-inflammatory IFNγ expression. Collectively, PIF promotes brain re-myelination and neuroprotection in relapsing remitting EAE MS model. Coupled with ongoing, Fast-Track FDA approved clinical trial, NCT#02239562 (immune disorder), current data supports PIF's translation for neurodegenerative disorders therapy.

The novel long non-coding RNA TALNEC2, regulates tumor cell growth and the stemness and radiation response of glioma stem cells.

Despite advances in novel therapeutic approaches for the treatment of glioblastoma (GBM), the median survival of 12-14 months has not changed significantly. Therefore, there is an imperative need to identify molecular mechanisms that play a role in patient survival. Here, we analyzed the expression and functions of a novel lncRNA, TALNEC2 that was identified using RNA seq of E2F1-regulated lncRNAs. TALNEC2 was localized to the cytosol and its expression was E2F1-regulated and cell-cycle dependent. TALNEC2 was highly expressed in GBM with poor prognosis, in GBM specimens derived from short-term survivors and in glioma cells and glioma stem cells (GSCs). Silencing of TALNEC2 inhibited cell proliferation and arrested the cells in the G1\S phase of the cell cycle in various cancer cell lines. In addition, silencing of TALNEC2 decreased the self-renewal and mesenchymal transformation of GSCs, increased sensitivity of these cells to radiation and prolonged survival of mice bearing GSC-derived xenografts. Using miRNA array analysis, we identified specific miRNAs that were altered in the silenced cells that were associated with cell-cycle progression, proliferation and mesenchymal transformation. Two of the downregulated miRNAs, miR-21 and miR-191, mediated some of TALNEC2 effects on the stemness and mesenchymal transformation of GSCs. In conclusion, we identified a novel E2F1-regulated lncRNA that is highly expressed in GBM and in tumors from patients of short-term survival. The expression of TALNEC2 is associated with the increased tumorigenic potential of GSCs and their resistance to radiation. We conclude that TALNEC2 is an attractive therapeutic target for the treatment of GBM.

Design principles for noninvasive, longitudinal and quantitative cell tracking with nanoparticle-based CT imaging.

Contradictory results in clinical trials are preventing the advancement and implementation of cell-based therapy. To explain such results, there is a need to uncover the mystery regarding the fate of the transplanted cells. To answer this need, we developed a technique for noninvasive in vivo cell tracking, which uses gold nanoparticles as contrast agents for CT imaging. Herein, we investigate the design principles of this technique for intramuscular transplantation of therapeutic cells. Longitudinal studies were performed, displaying the ability to track cells over long periods of time. As few as 500 cells could be detected and a way to quantify the number of cells visualized by CT was demonstrated. Moreover, monitoring of cell functionality was demonstrated on a mouse model of Duchenne muscular dystrophy. This cell-tracking technology has the potential to become an essential tool in pre-clinical as well as clinical trials and to advance the future of cell therapy.

Mesenchymal stem cells enhance the oncolytic effect of Newcastle disease virus in glioma cells and glioma stem cells via the secretion of TRAIL.

BACKGROUND: Newcastle disease virus (NDV) is an avian paramyxovirus, which selectively exerts oncolytic effects in cancer cells. Mesenchymal stem cells (MSCs) have been reported to affect tumor growth and deliver anti-tumor agents to experimental glioblastoma (GBM). Here, we explored the effects of NDV-infected MSCs derived from different sources, on glioma cells and glioma stem cells (GSCs) and the mechanisms involved in their effects. METHODS: The glioma cell lines (A172 and U87) and primary GSCs that were generated from GBM tumors were used in this study. MSCs derived from bone marrow, adipose tissue or umbilical cord were infected with NDV (MTH-68/H). The ability of these cells to deliver the virus to glioma cell lines and GSCs and the effects of NDV-infected MSCs on cell death and on the stemness and self-renewal of GSCs were examined. The mechanisms involved in the cytotoxic effects of the NDV-infected MSCs and their influence on the radiation sensitivity of GSCs were examined as well. RESULTS: NDV induced a dose-dependent cell death in glioma cells and a low level of apoptosis and inhibition of self-renewal in GSCs. MSCs derived from bone marrow, adipose and umbilical cord that were infected with NDV delivered the virus to co-cultured glioma cells and GSCs. Conditioned medium of NDV-infected MSCs induced higher level of apoptosis in the tumor cells compared with the apoptosis induced by their direct infection with similar virus titers. These results suggest that factor(s) secreted by the infected MSCs sensitized the glioma cells to the cytotoxic effects of NDV. We identified TRAIL as a mediator of the cytotoxic effects of the infected MSCs and demonstrated that TRAIL synergized with NDV in the induction of cell death in glioma cells and GSCs. Moreover, conditioned medium of infected MSCs enhanced the sensitivity of GSCs to γ-radiation. CONCLUSIONS: NDV-infected umbilical cord-derived MSCs may provide a novel effective therapeutic approach for targeting GSCs and GBM and for sensitizing these tumors to γ-radiation.

Repurposing phenformin for the targeting of glioma stem cells and the treatment of glioblastoma.

Glioblastoma (GBM) is the most aggressive primary brain tumor with poor prognosis. Here, we studied the effects of phenformin, a mitochondrial complex I inhibitor and more potent chemical analog of the diabetes drug metformin on the inhibition of cell growth and induction of apoptosis of glioma stem cells (GSCs) using both in vitro and in vivo models. Phenformin inhibited the self-renewal of GSCs, decreased the expression of stemness and mesenchymal markers and increased the expression of miR-124, 137 and let-7. Silencing of let-7 abrogated phenformin effects on the self-renewal of GSCs via a pathway associated with inhibition of H19 and HMGA2 expression. Moreover, we demonstrate that phenformin inhibited tumor growth and prolonged the overall survival of mice orthotopically transplanted with GSCs. Combined treatments of phenformin and temozolomide exerted an increased antitumor effect on GSCs in vitro and in vivo. In addition, dichloroacetate, an inhibitor of the glycolysis enzyme pyruvate dehydrogenase kinase, that decreases lactic acidosis induced by biguanides, enhanced phenformin effects on the induction of cell death in GSCs and prolonged the survival of xenograft-bearing mice. Our results demonstrate for the first time that phenformin targets GSCs and can be efficiently combined with current therapies for GBM treatment and GSC eradication.