Specific Glial Markers Research Articles

EXTH-10. TARGETING EPHA3 AND EPHRIN A5: ADVANCED PRECISION THERAPY FOR GBM

Abstract Glioblastoma (GBM) is an aggressive brain cancer and is associated with very poor prognosis. Standard treatment involves surgical resection, post-operative radiation and TMZ chemotherapy. Further research into new therapeutic approaches which target chemo-resistant tumor propagating cells, are urgently required. The EphA3 receptor is frequently elevated in GBM, particularly in the mesenchymal subtype and expressed on tumor-initiating cells (Day et al. Cancer Cell 2013). Our data in GBM tissue shows that tumor cells expressing the high-affinity EphA3 ligand, ephrin A5; are distinct from EphA3 expressing cells. The ephrin A5-positive cells express the glial differentiation marker GFAP, are less proliferative and less stem cell-like. EphA3 is expressed on the vimentin-positive, highly proliferative cell population. Single cell RNA-sequencing revealed that EphA3 and ephrin A5 are highly expressed in recurrent GBM, and are markers of MES-like and AC-like cell states respectively. Ephrin A5 over-expression in-vivo led to extension of survival in orthotopic xenograft animal models. GBM aggressiveness and downregulation of stem cell markers. Spatial transcriptomics of xenograft tumors revealed a reduction in proliferation markers including Ki67, MCM7 and PCNA. We detected an increase in expression of AC-like cell-state with a concomitant reduction of the MES-like and NPC-like cell-states. Consistently, over-expression of ephrin-A5 in primary GBM lines led to a reduction in neurosphere formation, Ki67 staining and growth rates. Thus we propose that dual targeting of EphA3 and ephrin-A5 might better capture GBM tumour heterogeneity and lead to an extension GBM patient survival. Whilst better understanding the biology of ephrinA5 expression, we also aim to validate antibody-based approaches to perform dual targeting of EphA3 and ephrin A5, leading to an extension GBM patient survival.

A “toxic window” study on the hippocampal development of mice offspring exposed to azithromycin at different doses, courses, and time during pregnancy

BackgroundAzithromycin, one of the new-generation macrolides, is an effective medicine for the treatment of mycoplasma infection during pregnancy. Epidemiological studies have reported adverse pregnancy outcomes with prenatal azithromycin exposure (PAzE). However, the effect of PAzE on fetal hippocampal development is unclear. This study aimed to explore the effects and potential mechanism of PAzE-induced fetal hippocampal development at different doses, courses, and time. MethodPregnant mice were administered azithromycin by gavage at different doses (50, 100 or 200 mg/kg.d), different courses (gestational day (GD)15–17 for three consecutive days, or GD17 once a day) and different time (GD10-12, GD15-17). ResultsCompared with the control group, morphological development damage of the fetal hippocampus was observed in the PAzE group, with a dysbalance in neuronal proliferation and apoptosis, decreased expression of the neuronal-specific marker Snap25, NeuN, PSD95 and Map2, increased expression of the glial-specific marker Iba1, GFAP, and S-100β, and decreased expression of P2ry12. The PAzE-induced hippocampal developmental deficiency varied based on different doses, courses, and time, and the developmental toxicity was most significant in the late pregnancy, high dose, multi-course group (AZHT). The significant reduction of SOX2 and Wnt, which were related to regulation of neural progenitor cells (NPCs) proliferation in PAzE fetus compared with the control group indicated that the SOX2/Wnt signaling may be involved in PAzE-induced hippocampal developmental toxicity. ConclusionIn this study, PAzE was associated with hippocampal developmental toxicity in a variety of nerve cells. Hippocampal developmental toxicity due to azithromycin was most significant in the late pregnancy, high-dose (equivalent to maximum clinical dose) and multi-course group (AZHT). The findings provide an experimental and theoretical foundation for guiding the sensible use of medications during pregnancy and effectively assessing the risk of fetal hippocampal developmental toxicity.

Pediatric-type high-grade neuroepithelial tumors with CIC gene fusion share a common DNA methylation signature

Pediatric neoplasms in the central nervous system (CNS) show extensive clinical and molecular heterogeneity and are fundamentally different from those occurring in adults. Molecular genetic testing contributes to accurate diagnosis and enables an optimal clinical management of affected children. Here, we investigated a rare, molecularly distinct type of pediatric high-grade neuroepithelial tumor (n = 18), that was identified through unsupervised visualization of genome-wide DNA methylation array data, together with copy number profiling, targeted next-generation DNA sequencing, and RNA transcriptome sequencing. DNA and/or RNA sequencing revealed recurrent fusions involving the capicua transcriptional repressor (CIC) gene in 10/10 tumor samples analyzed, with the most common fusion being CIC::LEUTX (n = 9). In addition, a CIC::NUTM1 fusion was detected in one of the tumors. Apart from the detected fusion events, no additional oncogenic alteration was identified in these tumors. The histopathological review demonstrated a morphologically heterogeneous group of high-grade neuroepithelial tumors with positive immunostaining for markers of glial differentiation in combination with weak and focal expression of synaptophysin, CD56 and CD99. All tumors were located in the supratentorial compartment, occurred during childhood (median age 8.5 years) and typically showed early relapses. In summary, we expand the spectrum of pediatric-type tumors of the CNS by reporting a previously uncharacterized group of rare high-grade neuroepithelial tumors that share a common DNA methylation signature and recurrent gene fusions involving the transcriptional repressor CIC. Downstream functional consequences of the fusion protein CIC::LEUTX and potential therapeutic implications need to be further investigated.

Amplification of the PLAG-family genes—PLAGL1 and PLAGL2—is a key feature of the novel tumor type CNS embryonal tumor with PLAGL amplification

Pediatric central nervous system (CNS) tumors represent the most common cause of cancer-related death in children aged 0–14 years. They differ from their adult counterparts, showing extensive clinical and molecular heterogeneity as well as a challenging histopathological spectrum that often impairs accurate diagnosis. Here, we use DNA methylation-based CNS tumor classification in combination with copy number, RNA-seq, and ChIP-seq analysis to characterize a newly identified CNS tumor type. In addition, we report histology, patient characteristics, and survival data in this tumor type. We describe a biologically distinct pediatric CNS tumor type (n = 31 cases) that is characterized by focal high-level amplification and resultant overexpression of either PLAGL1 or PLAGL2, and an absence of recurrent genetic alterations characteristic of other pediatric CNS tumor types. Both genes act as transcription factors for a regulatory subset of imprinted genes (IGs), components of the Wnt/β-Catenin pathway, and the potential drug targets RET and CYP2W1, which are also specifically overexpressed in this tumor type. A derived PLAGL-specific gene expression signature indicates dysregulation of imprinting control and differentiation/development. These tumors occurred throughout the neuroaxis including the cerebral hemispheres, cerebellum, and brainstem, and were predominantly composed of primitive embryonal-like cells lacking robust expression of markers of glial or neuronal differentiation (e.g., GFAP, OLIG2, and synaptophysin). Tumors with PLAGL1 amplification were typically diagnosed during adolescence (median age 10.5 years), whereas those with PLAGL2 amplification were diagnosed during early childhood (median age 2 years). The 10-year overall survival was 66% for PLAGL1-amplified tumors, 25% for PLAGL2-amplified tumors, 18% for male patients, and 82% for female patients. In summary, we describe a new type of biologically distinct CNS tumor characterized by PLAGL1/2 amplification that occurs predominantly in infants and toddlers (PLAGL2) or adolescents (PLAGL1) which we consider best classified as a CNS embryonal tumor and which is associated with intermediate survival. The cell of origin and optimal treatment strategies remain to be defined.

The incubation of cocaine craving is dissociated from changes in glial cell markers within prefrontal cortex and nucleus accumbens of rats

• Extended-access cocaine self-administration elicits an incubation of cocaine-seeking behavior during protracted withdrawal. • Extended-access cocaine self-administration lowers ventromedial prefrontal cortex expression of several glial cell markers in early withdrawal that persist into protracted withdrawal. • Extended-access cocaine self-administration produces few effects upon the expression of glial cell markers in the dorsomedial prefrontal cortex, or nucleus accumbens subregions. The majority of neurobiological research on cocaine addiction has centered on neuronal pathology, ignoring the fact that glial cells are critical contributors to brain function. The present study examined the expression of various glial cell subtype-specific proteins within forebrain regions that exhibit functional anomalies in addiction. For this, immunoblotting was conducted for a variety of markers of astrocytes and oligodendrocytes within prefrontal cortex (PFC) and nucleus accumbens (NAC) subregions of cocaine-abstinent rats (3 or 30 days). Cocaine-experienced rats exhibited a time-dependent increase in cocaine craving. Within ventral PFC, cocaine-experienced rats exhibited reduced expression of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and the NG2(+) proteoglycan that persisted into protracted withdrawal. Within the NAC core, a persistent reduction in Cx43 expression was also observed. No statistically significant protein changes were observed for the dPFC or the NAC shell. Thus, although a history of extended cocaine-access induced an incubation of cocaine craving, the observed changes in our glial-specific markers were time-independent during withdrawal. These data from rat are consistent with imagining and post-mortem findings from humans, indicating perturbations in oligodendrocyte function in those with cocaine use disorder. As myelin is critical for normal neuronal functioning and survival, the present data suggest a reduction in the functional integrity of oligodendrocytes as a potential mechanism underpinning the enduring PFC hypofunction reported in cocaine use disorder.

The endosomal pH regulator NHE9 is a driver of stemness in glioblastoma.

A small population of self-renewing stem cells initiate tumors and maintain therapeutic resistance in glioblastoma (GBM). Given the limited treatment options and dismal prognosis for this disease, there is urgent need to identify drivers of stem cells that could be druggable targets. Previous work showed that the endosomal pH regulator NHE9 is upregulated in GBM and correlates with worse survival prognosis. Here, we probed for aberrant signaling pathways in patient-derived GBM cells and found that NHE9 increases cell surface expression and phosphorylation of multiple receptor tyrosine kinases (RTKs) by promoting their escape from lysosomal degradation. Downstream of NHE9-mediated receptor activation, oncogenic signaling pathways converged on the JAK2-STAT3 transduction axis to induce pluripotency genes Oct4 and Nanog and suppress markers of glial differentiation. We used both genetic and chemical approaches to query the role of endosomal pH in GBM phenotypes. Loss-of-function mutations in NHE9 that failed to alkalinize endosomal lumen did not increase self-renewal capacity of gliomaspheres in vitro. However, monensin, a chemical mimetic of Na+/H+ exchanger activity, and the H+ pump inhibitor bafilomycin bypassed NHE9 to directly alkalinize the endosomal lumen resulting in stabilization of RTKs and induction of Oct4 and Nanog. Using orthotopic models of primary GBM cells we found that NHE9 increased tumor initiation in vivo. We propose that NHE9 initiates inside-out signaling from the endosomal lumen, distinct from the established effects of cytosolic and extracellular pH on tumorigenesis. Endosomal pH may be an attractive therapeutic target that diminishes stemness in GBM, agnostic of specific receptor subtype.

Role of enteric glial cells in the toxicity of phycotoxins: Investigation with a tri-culture intestinal cell model

Lipophilic phycotoxins are secondary metabolites produced by phytoplankton. They can accumulate in edible filtering-shellfish and cause human intoxications, particularly gastrointestinal symptoms. Up to now, the in vitro intestinal effects of these toxins have been mainly investigated on simple monolayers of intestinal cells such as the enterocyte-like Caco-2 cell line. Recently, the combination of Caco-2 cells with mucus secreting HT29-MTX cell line has been also used to mimic the complexity of the human intestinal epithelium. Besides, enteric glial cells (EGC) from the enteric nervous system identified in the gut mucosa have been largely shown to be involved in gut functions. Therefore, using a novel model integrating Caco-2 and HT29-MTX cells co-cultured on inserts with EGC seeded in the basolateral compartment, we examined the toxicological effects of two phycotoxins, pectenotoxin-2 (PTX2) and okadaic acid (OA). Cell viability, morphology, barrier integrity, inflammation, barrier crossing, and the response of some specific glial markers were evaluated using a broad set of methodologies. The toxicity of PTX2 was depicted by a slight decrease of viability and integrity as well as a slight increase of inflammation of the Caco-2/HT29-MTX co-cultures. PTX2 induced some modifications of EGC morphology. OA induced IL-8 release and decreased viability and integrity of Caco-2/HT29-MTX cell monolayers. EGC viability was slightly affected by OA. The presence of EGC reinforced barrier integrity and reduced the inflammatory response of the epithelial barrier following OA exposure. The release of GDNF and BDNF gliomediators by EGC could be implicated in the protection observed.

Recovery potential of transplanted oligoprogenitor cells derived from human dental pulp stem cells in Lysophosphatidyl choline demyelination mode.

The cells were cultured in DMEM-F12 medium containing 1M Retinoic acid and were treated with 5ng/ml platelet-derived growth factor, 10 ng/ml basic fibroblast growth factor for 8-10 days. The differentiation cells were examined via the expression of specific glial markers: Olig2 and O4. Cells were transplanted in to a demylinated rat corpus callosum. The alteration of the demyelination extension as well as remyelination intensity was examined via a specific myelin staining: Luxol Fast Blue and immunohistochemistry. Differentiated oligoprogenitor cells were confirmed via immunofluorescence staining with specific glial markers: Olig2 and O4. Also, the amount of demyelination was decreased and intensity of remyelination showed an increase after an engraftment of differentiated cells. Immunohistochemistry for evaluation of PLP expression proved the mature myelinating oligodendrocytes. hDPSCs can be induced to transdifferentiate into oligoprogenitor cells and respond to the routinely applied regents for glial differentiation of Mesenchymal stem cells. hDPSCs may be a valuable source for cell therapy in neurodegenerative diseases (Fig. 4, Ref. 30). Text in PDF www.elis.sk Keywords: dental pulp stem cells, lysophosphatidylcholine, corpus callosum, oligodendrocyte, differentiation.

Differentiation of bone marrow stromal stem cells seeded on silk scaffold to mature oligodendrocyte using cerebrospinal fluid

The differentiation of cultured Bone marrow stromal cells (BMSC) on silk scaffold into mature oligodendrocyte was done in the presence of cerebrospinal fluid (CSF). BMSC were isolated from Sprague-Dawley rats and were seeded on silk scaffold. The seeded cells were cultured in DMEM/F12 medium supplemented with CFS, basic fibroblast growth factor (bFGF), Retinoic acid (RA) and Epidermal growth factor (EGF). The glial differentiation was investigated using Real time-PCR and immunofluorescence techniques for specific glial markers: Oligo 2, NG2, PLP and MBP.Our dates showed that the differentiated cells expressed specific glial markers: Oligo 2, NG2, PLP and MBP. The specific mature oligodendrocyte genes were up regulated in cultured cells on silk scaffold in the presence of CSF.It is concluded that CSF leads to improve glial differentiation of seeded BMSC on silk scaffold using preparation of appropriate niche. This culture condition may be served as an efficient differentiation induction protocol for glial phenotype, with the perspective of therapeutic application in neuroregenerative medicine.

Brain Gene Expression Profiling of Individuals With Dual Diagnosis Who Died by Suicide

Objective: Dual diagnosis (DD) is the co-occurrence of at least one substance use disorder and one or more mental disorders in a given individual. Despite this comorbidity being highly prevalent and associated with adverse clinical outcomes, its neurobiology remains unclear. Furthermore, patients with DD are at higher risk for suicidal behavior in comparison with single disorder patients. Our objective was to evaluate brain gene expression patterns in individuals with DD who died by suicide. Methods: We compared the gene expression profile in the dorsolateral prefrontal cortex of suicides with DD (n = 10) to the transcriptome of suicides with substance use disorder alone (n = 10), suicides with mood disorders (MD) alone (n = 13), and suicides without mental comorbidities (n = 5). Gene expression profiles were assessed by microarrays. In addition, we performed a brain cell type enrichment to evaluate whether the gene expression profiles could reflect differences in cell type compositions among the groups. Results: When comparing the transcriptome of suicides with DD to suicides with substance use disorder alone and suicides with MD alone, we identified 255 and 172 differentially expressed genes (DEG), respectively. The overlap of DEG between both comparisons (112 genes) highlighted the presence of common disrupted pathways in substance use disorder and MD. When comparing suicides with DD to suicides without mental comorbidities, we identified 330 DEG, mainly enriched in neurogenesis. Cell type enrichment indicated higher levels of glial markers in suicides with DD compared to the other groups. Conclusions: Suicides with DD exhibited a gene expression profile distinct from that of suicides with a single disorder, being substance use disorder or MD, and suicides without mental disorders. Our results suggest alteration in the expression of genes involved in glial specific markers, glutamatergic and GABAergic neurotransmission in suicides with DD compared to suicides with a single disorder and suicides without mental comorbidities. Alterations in the expression of synaptic genes at different levels were found in substance use disorder and MD.

Novel Insights on the Toxicity of Phycotoxins on the Gut through the Targeting of Enteric Glial Cells.

In vitro and in vivo studies have shown that phycotoxins can impact intestinal epithelial cells and can cross the intestinal barrier to some extent. Therefore, phycotoxins can reach cells underlying the epithelium, such as enteric glial cells (EGCs), which are involved in gut homeostasis, motility, and barrier integrity. This study compared the toxicological effects of pectenotoxin-2 (PTX2), yessotoxin (YTX), okadaic acid (OA), azaspiracid-1 (AZA1), 13-desmethyl-spirolide C (SPX), and palytoxin (PlTX) on the rat EGC cell line CRL2690. Cell viability, morphology, oxidative stress, inflammation, cell cycle, and specific glial markers were evaluated using RT-qPCR and high content analysis (HCA) approaches. PTX2, YTX, OA, AZA1, and PlTX induced neurite alterations, oxidative stress, cell cycle disturbance, and increase of specific EGC markers. An inflammatory response for YTX, OA, and AZA1 was suggested by the nuclear translocation of NF-κB. Caspase-3-dependent apoptosis and induction of DNA double strand breaks (γH2AX) were also observed with PTX2, YTX, OA, and AZA1. These findings suggest that PTX2, YTX, OA, AZA1, and PlTX may affect intestinal barrier integrity through alterations of the human enteric glial system. Our results provide novel insight into the toxicological effects of phycotoxins on the gut.

Effects of GDNF-Transfected Marrow Stromal Cells on Rats with Intracerebral Hemorrhage

Objective: The present study aimed to investigate the effects of Mesenchymal stem cells/glial cell line derived neurotrophic factor (MSCs/GDNF) transplantation on nerve reconstruction in rats with intracerebral hemorrhage. Methods: GDNF transduction to MSCs was using adenovirus vector pAdEasy-1-pAdTrack-CMV prepared. Intracerebral hemorrhage (ICH) was induced by injection of collagenase and heparin into the caudate putamen. At the third day after a collagenase-induced ICH, adult male SD rats were randomly divided into saline group, MSCs group and MSCs/GDNF group. Immunofluorescence and RT-PCR were performed to detect the differentiation of MSCs or MSCs with an adenovirus vector encoding GDNF gene in vivo and in vitro. Result: After 6 hours of induction, both MSCs and MSCs/GDNF expressed neuro or glial specific markers and synaptic-associated proteins (SYN, GAP-43, PSD-95); additionally, they secreted bioactive compounds (BDNF, NGF-β). MSCs/GDNF transplantation, compared to MSCs and saline solution injection, significantly improved neurological functions after ICH. The grafted MSCs or MSCs/GDNF survived in the striatum after 2 weeks of transplantation and expressed the neural cell-specific biomarkers NSE, MAP2, and GFAP. Conclusion: These findings demonstrate that MSCs/GDNF transplantation contributes to improved neurological function in experimental ICH rats. The mechanisms are possibly due to neuronal replacement and enhanced neurotrophic factor secretion.

Impact of Caffeine Consumption on Type 2 Diabetes-Induced Spatial Memory Impairment and Neurochemical Alterations in the Hippocampus

Diabetes affects the morphology and plasticity of the hippocampus, and leads to learning and memory deficits. Caffeine has been proposed to prevent memory impairment upon multiple chronic disorders with neurological involvement. We tested whether long-term caffeine consumption prevents type 2 diabetes (T2D)-induced spatial memory impairment and hippocampal alterations, including synaptic degeneration, astrogliosis, and metabolic modifications. Control Wistar rats and Goto-Kakizaki (GK) rats that develop T2D were treated with caffeine (1 g/L in drinking water) for 4 months. Spatial memory was evaluated in a Y-maze. Hippocampal metabolic profile and glucose homeostasis were investigated by 1H magnetic resonance spectroscopy. The density of neuronal, synaptic, and glial-specific markers was evaluated by Western blot analysis. GK rats displayed reduced Y-maze spontaneous alternation and a lower amplitude of hippocampal long-term potentiation when compared to controls, suggesting impaired hippocampal-dependent spatial memory. Diabetes did not impact the relation of hippocampal to plasma glucose concentrations, but altered the neurochemical profile of the hippocampus, such as increased in levels of the osmolites taurine (P < 0.001) and myo-inositol (P < 0.05). The diabetic hippocampus showed decreased density of the presynaptic proteins synaptophysin (P < 0.05) and SNAP25 (P < 0.05), suggesting synaptic degeneration, and increased GFAP (P < 0.001) and vimentin (P < 0.05) immunoreactivities that are indicative of astrogliosis. The effects of caffeine intake on hippocampal metabolism added to those of T2D, namely reducing myo-inositol levels (P < 0.001) and further increasing taurine levels (P < 0.05). Caffeine prevented T2D-induced alterations of GFAP, vimentin and SNAP25, and improved memory deficits. We conclude that caffeine consumption has beneficial effects counteracting alterations in the hippocampus of GK rats, leading to the improvement of T2D-associated memory impairment.

The collagen scaffold supports hiPSC-derived NSC growth and restricts hiPSC.

The human induced pluripotent stem cells (hiPSC) are one of the promising candidates as patient specific cell source for autologous transplantation or modeling of diseases. The collagen (Col) scaffolds have been shown suitable to create in vitro biomimetic microenvironment for human neural stem cells, but their ability to accommodate stem cells at different stages of neural differentiation has not been verified yet. In this paper we compare lineage related hiPSC during neural differentiation for their ability to colonize Col scaffold. We have also focused on modification of collagen physicochemical properties with improved mechanical and thermal stability, without loss of its biological activity. The hiPSC expressing markers of pluripotency (OCT4, SOX2, NANOG) after neural commitment are NESTIN, GFAP, PDGFR alpha, beta- TUBULIN III, MAP-2, DCX, GalC positive. We have shown, that Col scaffold was not preferable for hiPSC culture, while the neurally committed population after seeding on Col scaffolds revealed good adhesion, viability, proliferation, along with sustaining markers of neuronal and glial differentiation. The Col scaffold-based 3D culture of hiPSC-NSCs may serve as a research tool for further translational studies.

An efficient induction protocol for deriving mature oligodendrocytes from human dental stem cells.

This study examined the process of deriving mature oligodendrocytes from human dental pulp stem cells (hDPSCs) via addition of cerebrospinal fluid (CSF). The hDPSCs were cultured in the presence of retinoic acid and CSF. The oligodendrocytes were confirmed using immunocytochemistry for specific glial markers, namely Olig2 and MBP markers. The differentiated oligodendrocytes were immunopositive for Olig2 and MBP markers at the end of induction phase. It is concluded that this study indicated the glial differentiation of hDPSCs in the presence of CSF and appropriate inducers, which is a usable therapeutic technique in neuroregenerative medicine (Fig. 3, Ref. 24).

The enteric neural crest progressively loses capacity to form enteric nervous system

Cells of the vagal neural crest (NC) form most of the enteric nervous system (ENS) by a colonising wave in the embryonic gut, with high cell proliferation and differentiation. Enteric neuropathies have an ENS deficit and cell replacement has been suggested as therapy. This would be performed post-natally, which raises the question of whether the ENS cell population retains its initial ENS-forming potential with age. We tested this on the avian model in organ culture in vitro (3 days) using recipient aneural chick midgut/hindgut combined with ENS-donor quail midgut or hindgut of ages QE5 to QE10. ENS cells from young donor tissues (≤ QE6) avidly colonised the aneural recipient, but this capacity dropped rapidly 2–3 days after the transit of the ENS cell wavefront. This loss in capability was autonomous to the ENS population since a similar decline was observed in ENS cells isolated by HNK1 FACS. Using QE5, 6, 8 and 10 midgut donors and extending the time of assay to 8 days in chorio-allantoic membrane grafts did not produce ‘catch up’ colonisation. NC-derived cells were counted in dissociated quail embryo gut and in transverse sections of chick embryo gut using NC, neuron and glial marker antibodies. This showed that the decline in ENS-forming ability correlated with a decrease in proportion of ENS cells lacking both neuronal and glial differentiation markers, but there were still large numbers of such cells even at stages with low colonisation ability. Moreover, ENS cells in small numbers from young donors were far superior in colonisation ability to larger numbers of apparently undifferentiated cells from older donors. This suggests that the decline of ENS-forming ability has both quantitative and qualitative aspects. In this case, ENS cells for cell therapies should aim to replicate the embryonic ENS stage rather than using post-natal ENS stem/progenitor cells.

Abstract A068: Targeting glioblastoma stem cells through a MET inhibitor

Abstract Background: Glioblastoma (GBM) is the most common and aggressive human brain cancer with high relapse rate, lacking any targeted therapy. The current therapy consisting of surgery in combination with radiotherapy and the DNA-alkylating agent temozolomide has led to median survival increase. Nevertheless, many patients, after early treatment response, experience drug resistance and tumor progression. Recent advances identified cancer stem cell subpopulation as the cause of treatment failure, suggesting that the direct targeting of this cell subset may represent an innovative approach to improve the therapy efficacy. Based on studies indicating the role of the tyrosine kinase receptor MET in GBM stem cells, we examined the effects of the rational combination of radiotherapy with a MET inhibitor against stem-like cells from a human GBM cell line. Methods: Stem-like cells from U87MG cell line were sorted using PKH-67-labeling method for isolating slow-dividing cells (PKH67+), and analyzed for cell proliferation index by WST assay, clonogenic potential, and expression of stemness-related transcription factors by RT-PCR. Effects by the MET inhibitor alone and in combination with radiotherapy (IR) were evaluated on cell proliferation by Trypan Blue exclusion test, on apoptosis, cell cycle progression, GFAP protein expression by flow cytometry and on neurosphere-forming efficiency. The mechanisms underlying the effects induced by the combined treatment were dissected by biochemical and RT-PCR assays. Results: The PKH-67+ cells recapitulated the functional properties of stem-like cells, as slower cell proliferation index, higher clonogenic capacity, and expression of stemness-transcription factors than the negative counterpart. They also displayed a marked radioresistance dependent on MET signalling activation and characterized by the enrichment in stem-like cells as indicated by the upregulation of stemness-related transcription factor expression Nanog and Sox2, and enhanced susceptibility to MET inhibitor. The single-agent therapy with the inhibitor induced cell proliferation inhibition with an early cell cycle arrest in G2/M phase and a late cytotoxic effect. The drug alone negatively affected the neurosphere-forming efficiency and the clonogenic capacity. Following IR, the daily treatment with the inhibitor of MET significantly caused time-dependent cell proliferation inhibition, associated with an increased percentage of apoptotic cells and cells positive for GFAP, a glial differentiation marker, as compared to single-agent treatments. The biochemical analysis confirmed that MET inhibition remarkably impaired the radioresistance, and parallelly reduced the content of stem-like cells, as indicated either by the negative regulation of Nanog and Sox2 genes or the positive regulation of GFAP. Conclusions: Our results provide evidence that targeting MET with a specific inhibitor might interfere with MET-mediated radiotherapy resistance, thus sensitizing the GBM-stem-like cells to radiation, depleting the pool, and parallelly inducing their reprogramming towards more differentiated subtype. Citation Format: Anna Rossini, Evelyn Oliva Savoia, Eleonora Cicoria, Lorenzo Castagnoli, Serenella Pupa, Filippo de Braud, Massimo Di Nicola. Targeting glioblastoma stem cells through a MET inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A068.

Neurogenesis from Sox2 expressing cells in the adult cerebellar cortex

We identified a rare undifferentiated cell population that is intermingled with the Bergmann glia of the adult murine cerebellar cortex, expresses the stem cell markers Sox2 and Nestin, and lacks markers of glial or neuronal differentiation. Interestingly, such Sox2+ S100− cells of the adult cerebellum expanded after adequate physiological stimuli in mice (exercise), and Sox2+ precursors acquired positivity for the neuronal marker NeuN over time and integrated into cellular networks. In human patients, SOX2+ S100− cells similarly increased in number after relevant pathological insults (infarcts), suggesting a similar expansion of cells that lack terminal glial differentiation.

ATRT-05. PRECLINICAL EFFICACY OF RADIATION-FREE TREATMENT OF ATYPICAL TERATOID/RHABDOID TUMORS BY COMBINED MEK AND MELK INHIBITION

AbstractAtypical teratoid/rhabdoid tumors (AT/RT) are highly malignant tumors that occur both in- and outside the central nervous system, predominantly in young children. Although therapeutic interventions, combining surgery with intensive chemotherapy and conformal radiotherapy, have shown curative potential, the long-term consequences of these treatment modalities in young children are often severe. Therefore, there is a dire need for novel, targeted therapeutic strategies that potentially reduce or eliminate chemo- and radiotherapy, especially for children under three years of age. Genetically, AT/RT is characterized by a biallelic inactivation of SMARCB1 or SMARCA4, causing aberrant chromatin remodeling and thereby expression of a variety of oncogenes. Recently, the importance of the mitogen-activated protein kinase (MAPK) pathway in the pathogenesis of AT/RT has been demonstrated, with preclinical efficacy of MEK inhibition in AT/RT cells. Preclinical patient-derived AT/RT models are largely lacking. We developed a patient-derived primary cell line and xenograft model, VUMC-AT/RT-01, from a surgical specimen of an AT/RT patient that had not received prior therapy. Immunohistochemistry was performed on xenograft tumors, revealing typical loss of SMARCB1 expression as well as heterogeneous expression of glial, epithelial, mesenchymal and neuronal differentiation markers. In silico analysis of gene expression revealed strong upregulation of MELK in AT/RT tumors compared to normal brain tissues. These high levels of MELK were subsequently confirmed in our xenograft model. Inhibition of MELK with the small molecule OTSSP167 effectively reduced proliferation and induced cell death in primary AT/RT neurospheres. Combined treatment of primary AT/RT cells with OTSSP167 and the MEK inhibitor Trametinib showed strongly synergistic cytotoxicity at low nanomolar concentrations. Treatment of mice carrying VUMC-AT/RT-01-Fluc xenografts with OTSSP167 and Trametinib confirmed this synergy in vivo. We conclude that combined MEK and MELK inhibition represents a promising targeted therapy strategy for AT/RT that might ultimately reduce or eliminate the need for radiation and intensive chemotherapy.

Transdifferentiation of Human Dental Pulp Stem Cells Into Oligoprogenitor Cells.

Introduction:The nerve fibers in central nervous system are surrounded by myelin sheet which is formed by oligodendrocytes. Cell therapy based on oligodendrocytes and their precursors transplantation can hold a promising alternative treatment for myelin sheet repair in demyelinating diseases.Methods:Human Dental Pulp Stem Cells (hDPSCs) are noninvasive, autologous and easy available source with multipotency characteristics, so they are in focus of interest in regenerative medicine. In the present study, hDPSCs were differentiated into oligoprogenitor using glial induction media, containing Retinoic Acid (RA), basic Fibroblast Growth Factor (bFGF), Platelet-Derived Growth Factor (PDGF), N2 and B27. The differentiated Oligoprogenitor Cells (OPCs) were evaluated for nestin, Olig2, NG2 and O4 using immunocytochemistry. Also, the expression of nestin, Olig2 and PDGFR-α gens (neuroprogenitor and oligoprogenitor markers) were investigated via RT-PCR technique.Results:The results indicate that glial differentiation medium induces the generation of oligoprogenitor cells as revealed via exhibition of specific glial markers, including Olig2, NG2 and O4. The expersion of nestin gene (neuroprogenitor marker) and Olig2 and PDGFR-α genes (oligoprogentor markers) were detected in treated hDPSCs at the end of the induction stage.Conclusion:hDPSCs can be induced to transdifferentiate into oligoprogenitor cells and respond to the routinely applied regents for glial differentiation of mesanchymal stem cells. These data suggest the hDPSCs as a valuable source for cell therapy in neurodegenerative diseases.