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Glioblastoma is the most common primary brain cancer in adults and represents one of the worst cancer diagnosis for the patients. Suffering from a poor prognosis and limited treatment options, tumor recurrences are virtually inevitable. Additionally, treatment resistance is very common for this disease and worsens the prognosis. These and others factors are hypothesized to be largely due to the fact that glioblastoma cells are known to be able to obtain stem-like traits and thereby driving these phenotypes. Recently, we could show that the in vitro and ex vivo treatment of glioblastoma stem-like cells with the hormonally active form of Vitamin D3, Calcitriol (1α,25(OH)2-vitamin D3) can block stemness in a subset of cell lines and reduce tumor growth. Here, we expanded our cell panel to over 40 different cultures and can show that, while half of the tested cell lines are sensitive, a quarter can be classified as high-responders. Using genetic and proteomic analysis, we further determined that treatment success can be partially explained by specific polymorphism of the Vitamin D3 receptor and that high-responders display a proteome suggestive of blockade of stemness, as well as migratory potential.

In a unique model of fluorescent based mapping of EV recipient cells, RBC-EVs were found to signal to cardiac cells and regulate gene expression in a model of ischemic heart failure., Extracellular vesicles (EVs) mediate intercellular signaling by transferring their cargo to recipient cells, but the functional consequences of signaling are not fully appreciated. RBC-derived EVs are abundant in circulation and have been implicated in regulating immune responses. Here, we use a transgenic mouse model for fluorescence-based mapping of RBC-EV recipient cells to assess the role of this intercellular signaling mechanism in heart disease. Using fluorescent-based mapping, we detected an increase in RBC-EV–targeted cardiomyocytes in a murine model of ischemic heart failure. Single cell nuclear RNA sequencing of the heart revealed a complex landscape of cardiac cells targeted by RBC-EVs, with enrichment of genes implicated in cell proliferation and stress signaling pathways compared with non-targeted cells. Correspondingly, cardiomyocytes targeted by RBC-EVs more frequently express cellular markers of DNA synthesis, suggesting the functional significance of EV-mediated signaling. In conclusion, our mouse model for mapping of EV-recipient cells reveals a complex cellular network of RBC-EV–mediated intercellular communication in ischemic heart failure and suggests a functional role for this mode of intercellular signaling.

Recovery of brain function lost to disease or in old age is a challenging task in regenerative medicine. In the last two decades, therapeutic strategies have undergone significant shifts by a succession of major discoveries from adult neural stem cells and neurogenesis to the development of induced pluripotent stem cells to technologies for reprogramming cells in vitro and in vivo. Now, extracellular vesicles, small membrane-bound vesicles released by all cells and containing lipids, proteins, and nucleic acids, emerge as the next major technological opportunity. While substantial progress has been made on their potential use in therapy and EVs have entered many clinical trials, major aspects of their physiological role, in particular regarding their influence on brain function, remain unknown. However, a better understanding of their actual in vivo function, scope of communication, and possibilities to alter cellular processes in target cells will be needed. This review places EVs in the developing landscape of strategies for cellular repair of the brain and highlights their potential by looking at some recent progress in our understanding of their function in vivo.

Summary Brain injuries, such as stroke or trauma, induce neural stem cells in the subventricular zone (SVZ) to a neurogenic response. Very little is known about the molecular cues that signal tissue damage, even over large distances, to the SVZ. Based on our analysis of gene expression patterns in the SVZ, 48 hr after an ischemic lesion caused by middle cerebral artery occlusion, we hypothesized that the presence of an injury might be transmitted by an astrocytic traveling calcium wave rather than by diffusible factors or hypoxia. Using a newly established in vitro system we show that calcium waves induced in an astrocytic monolayer spread to neural stem and progenitor cells and increase their self-renewal as well as migratory behavior. These changes are due to an upregulation of the Notch signaling pathway. This introduces the concept of propagating astrocytic calcium waves transmitting brain injury signals over long distances., Graphical Abstract, Highlights • gene profiling after MCAO suggests a role of calcium-binding proteins • Novel in vitro system to study the effects of astrocytic calcium waves on NSPCs • Astrocytic calcium waves enhance self-renewal and migration capacity of NSPCs • The Notch signaling pathway mediates effects of elevated calcium levels on NSPCs, Based on gene profiling data in a stroke model, Momma and colleagues hypothesize a role of calcium signaling in the stem cell injury response. Using a newly established in vitro model they show that astrocytic calcium waves spread to neural stem and progenitor cells, thereby increasing their self-renewal and migration capacity. These effects are mediated by the Notch/Hes1 signaling pathway.

Author(s): Das, Avash; Valkov, Nedyalka; Salvador, Ane; Kur, Ivan; Ziegler, Olivia; Yeri, Ashish; Garcia, Fernando Camacho; Lu, Shulin; Khamesra, Aushee; Xiao, Chunyang; Rodosthenous, Rodosthenis; Li, Guoping; Srinivasan, Srimeenakshi; Toxavidis, Vasilis; Tigges, John; Laurent, Louise; Momma, Stefan; Ghiran, Ionita; Das, Saumya | Abstract: Summary Extracellular vesicles (EV) mediate intercellular signaling by transferring their cargo to recipient cells. Red blood cell (RBC)-derived EVs constitute a significant proportion of circulating EVs and have been implicated in regulating immune responses. Here, we describe a transgenic mouse model for fluorescent-based mapping of RBC-EV target cells based on the functional transfer of EV-contained Cre-recombinase to target cells. In a murine model of ischemic heart failure, we detect an increase in RBC-EV-targeted cardiomyocytes in the hearts and microglial cells in the brains. Cells targeted by RBC-EVs present an enrichment of genes implicated in cell proliferation and metabolism pathways compared to non-recombined (non-targeted) cells. Cardiomyocytes targeted by RBC-EVs are more likely to demonstrate cellular markers of DNA synthesis and proliferation, suggesting functional significance of EV-mediated signaling. In conclusion, we leverage our mouse model for mapping of RBC-EV targets in murine ischemic heart failure to demonstrate quantitative and qualitative changes in RBC-EV recipients.

Extracellular RNA (exRNA) has emerged as an important transducer of intercellular communication. Advancing exRNA research promises to revolutionize biology and transform clinical practice. Recent efforts have led to cutting-edge research and expanded knowledge of this new paradigm in cell-to-cell crosstalk; however, gaps in our understanding of EV heterogeneity and exRNA diversity pose significant challenges for continued development of exRNA diagnostics and therapeutics. To unravel this complexity, the NIH convened expert teams to discuss the current state of the science, define the significant bottlenecks, and brainstorm potential solutions across the entire exRNA research field. The NIH Strategic Workshop on Extracellular RNA Transport helped identify mechanistic and clinical research opportunities for exRNA biology and provided recommendations on high priority areas of research that will advance the exRNA field.

Glioblastoma is the most common and aggressive brain tumor, with a subpopulation of stem‐like cells thought to mediate its recurring behavior and therapeutic resistance. The epithelial–mesenchymal transition (EMT) inducing factor Zeb1 was linked to tumor initiation, invasion, and resistance to therapy in glioblastoma, but how Zeb1 functions at molecular level and what genes it regulates remain poorly understood. Contrary to the common view that EMT factors act as transcriptional repressors, here we show that genome‐wide binding of Zeb1 associates with both activation and repression of gene expression in glioblastoma stem‐like cells. Transcriptional repression requires direct DNA binding of Zeb1, while indirect recruitment to regulatory regions by the Wnt pathway effector Lef1 results in gene activation, independently of Wnt signaling. Amongst glioblastoma genes activated by Zeb1 are predicted mediators of tumor cell migration and invasion, including the guanine nucleotide exchange factor Prex1, whose elevated expression is predictive of shorter glioblastoma patient survival. Prex1 promotes invasiveness of glioblastoma cells in vivo highlighting the importance of Zeb1/Lef1 gene regulatory mechanisms in gliomagenesis.

International audience; Recently, a broader role of inhibitor of apoptosis (IAP) proteins besides their antiapoptotic functions has been described. Therefore, we investigated the effect of non-toxic concentrations of the small-molecule Smac mimetic BV6, which antagonizes IAP proteins, on differentiation of cancer stem-like cells (CSLCs) derived from primary glioblastoma (GBM) specimens. Here, we identify a novel function of BV6 in regulating differentiation of GBM CSLCs by activating NF-κB. BV6 at non-lethal doses stimulates morphological changes associated with the differentiation of GBM CSLCs. BV6 increases transcriptional activity, mRNA and protein levels of the astrocytic marker GFAP without altering expression of the neuronal marker β-III-tubulin, indicating that BV6 induces astrocytic differentiation of GBM CSLCs. Molecular studies reveal that BV6 triggers processing of the NF-κB subunit p100 to p52, nuclear translocation of p52 and p50 and increased NF-κB DNA-binding. Intriguingly, inhibition of NF-κB by overexpression of dominant-negative IκBα super-repressor (IκBα-SR) blocks the BV6-stimulated increase in GFAP and differentiation. Interestingly, this BV6-stimulated differentiation is associated with reduced expression of stemness markers such as CD133, Nanog and Sox2 in GBM CSLCs. In contrast, BV6 does not alter cell morphology, differentiation and expression of stemness markers in non-malignant neural stem cells. Importantly, BV6 treatment reduces clonogenicity of GBM CSLCs in vitro and in vivo, suppresses their tumorigenicity in orthotopic and subcutaneous mouse models and significantly increases the survival of mice. By identifying a novel role of BV6 in promoting differentiation of GBM CSLCs, these findings provide new insights into Smac mimetic-regulated non-apoptotic functions with important implications for targeting GBM CSLCs.

Data from transgenic mouse models show that neuronal progenitor cells (NPCs) migrate toward experimental brain tumors and modulate the course of pathology. However, the pathways whereby NPCs are attracted to CNS neoplasms are not fully understood and it is unexplored if NPCs migrate toward brain tumors (high-grade astrocytomas) in humans. We analyzed the tumor-parenchyma interface of neurosurgical resections for the presence of (NPCs) and distinguished these physiological cells from the tumor mass. We observed that polysialic acid neural cell adhesion molecule-positive NPCs accumulate at the border of high-grade astrocytomas and display a marker profile consistent with immature migratory NPCs. Importantly, these high-grade astrocytoma-associated NPCs did not carry genetic aberrations that are indicative of the tumor. Additionally, we observed NPCs accumulating in CNS metastases. These metastatic tumors are distinguished from neural cells by defined sets of markers. Transplanting murine glioma cells embedded in a cell-impermeable hollow fiber capsule into the brains of nestin-gfp reporter mice showed that diffusible factors are sufficient to induce a neurogenic reaction. In vitro, vascular endothelial growth factor (VEGF) secreted from glioma cells increases the migratory and proliferative behavior of adult human brain-derived neural stem and progenitor cells via stimulation of VEGF receptor-2 (VEGFR-2). In vivo, inhibiting VEGFR-2 signaling with a function-blocking antibody led to a reduction in NPC migration toward tumors. Overall, our data reveal a mechanism by which NPCs are attracted to CNS tumors and suggest that NPCs accumulate in human high-grade astrocytomas. Stem Cells 2014;32:244–257

In various types of cancers including glioblastoma, accumulating evidence show the existence of cancer stem-like cells (CSCs), characterized by stem cell marker expression, capability of differentiation and self-renewal, and high potential for tumor propagation in vivo. LGR5, whose expression is positively regulated by the Wnt signaling pathway, is a stem cell marker in intestinal mucosa and hair follicle in the skin. As Wnt signaling is also involved in brain development, the function of LGR5 in the maintenance of brain CSCs is to be assessed. Our study showed that the LGR5 transcript level was increased in CSCs. Co-immunofluorescence staining demonstrated the co-localization of CD133- and LGR5-positive cells in glioblastoma tissue sections. Functionally, silencing of LGR5 by lentiviral shRNA-mediated knockdown induced apoptosis in brain CSCs. Moreover, LGR5 depletion led to a downregulation of L1 cell adhesion molecule expression. In line with an important function in glioma tumorigenesis, LGR5 expression increased with glioma progression and correlated with an adverse outcome. Our findings suggest that LGR5 plays a role in maintenance and/or survival of brain CSCs.

The concept of cancer stem-like cells (CSCs) has gained considerable attention in various solid tumors including glioblastoma, the most common primary brain tumor. This sub-population of tumor cells has been intensively investigated and their role in therapy resistance as well as tumor recurrence has been demonstrated. In that respect, development of therapeutic strategies that target CSCs (and possibly also the tumor bulk) appears a promising approach in patients suffering from primary brain tumors. In the present study, we utilized RNA interference (RNAi) to screen the complete human kinome and phosphatome (682 and 180 targets, respectively) in order to identify genes and pathways relevant for the survival of brain CSCs and thereby potential therapeutical targets for glioblastoma. We report of 46 putative candidates including known survival-related kinases and phosphatases. Interestingly, a number of genes identified are involved in metabolism, especially glycolysis, such as PDK1 and PKM2 and, most prominently PFKFB4. In vitro studies confirmed an essential role of PFKFB4 in the maintenance of brain CSCs. Furthermore, high PFKFB4 expression was associated with shorter survival of primary glioblastoma patients. Our findings support the importance of the glycolytic pathway in the maintenance of malignant glioma cells and brain CSCs and imply tumor metabolism as a promising therapeutic target in glioblastoma.

The subependymal zone at the lateral ventricular wall represents a major neurogenic niche of the adult mammalian brain and continuously provides new neurons for the olfactory bulb. A mosaic of stem and progenitor cells in this niche has the potential to respond to multiple signals including growth factors such as EGF. Recent studies using long-term ventricular infusion of EGF demonstrate intense cell proliferation around the ventricular wall, implicating the presence of EGF-reactive cells also outside the classical neurogenic lateral niche. Here we show that intraventricular injection of EGF induces within minutes CREB and ERK phosphorylation in astrocyte-like progenitor cells (type B cells) and EGF receptor-expressing transit-amplifying progenitor cells-both in the striatal and septal ventricular walls. EGF infusion for 6 days induced continued CREB and ERK activation in nestin+ cells paralleled by intense periventricular cell proliferation. In addition, the ependyma became EGF receptor-immunoreactive, revealed intense CREB phosphorylation and underwent partial de-differentiation. Our results demonstrate that intraventricular application of EGF induces CREB and ERK phosphorylation along the entire ventricular walls and thus permits a direct identification of EGF-responsive cell types. They further support the notion that not only the striatal ventricular wall where the SEZ is located but also the septal ventricular wall carries latent potential for the formation of neurons and glial cells.

AIMS: The identification of factors that mobilize subsets of endogenous progenitor cells may provide new therapeutic tools to enhance the repair of ischaemic tissue. We previously identified circulating mesenchymal cells that co-express endothelial markers (so-called circulating mesoangioblasts, cMABs) in children undergoing heart surgery with cardiopulmonary bypass (CPB). However, the mechanisms by which these cells are mobilized and their origin is unclear. METHODS AND RESULTS: Circulating CD73(+)CD45(-)KDR(+) cMABs were analysed in adults undergoing heart surgery with (n = 21) or without CPB (n = 8). During surgery with CPB, cMABs are mobilized with a maximal response at the end of the operation. In contrast, off-pump heart surgery does not stimulate cMAB mobilization, indicating that the stress mediated by CPB induces the mobilization of cMAB. Circulating mesoangioblasts were enriched in blood obtained from the coronary sinus. Histologically, CD73(+) cells were detected around vessels in the heart, indicating that the heart is one of the niches of cMABs. Consistently, studies in gender mismatched bone marrow transplanted patients demonstrated that cMABs did not originate from the bone marrow. Cytokine profiling of serum samples revealed that hepatocyte growth factor (HGF) was profoundly increased at the time point of maximal mobilization of cMABs. Hepatocyte growth factor stimulated the migration of cMABs. Importantly, injection of recombinant HGF increased cMABs in rats. CONCLUSIONS: Hepatocyte growth factor induces mobilization of non-haematopoietic progenitor cells with a cardiac repair capacity. This newly identified function together with the known pleiotrophic effects of HGF makes HGF an attractive therapeutic option for the treatment of ischaemic heart disease.

Extracellular vesicles (EVs) have been shown to transfer various molecules, including functional RNA between cells and this process has been suggested to be particularly relevant in tumor-host interactions. However, data on EV-mediated RNA transfer has been obtained primarily by in vitro experiments or involving ex vivo manipulations likely affecting its biology, leaving their physiological relevance unclear. We engineered glioma and carcinoma tumor cells to express Cre recombinase showing their release of EVs containing Cre mRNA in various EV subfractions including exosomes. Transplantation of these genetically modified tumor cells into mice with a Cre reporter background leads to frequent recombination events at the tumor site. In both tumor models the majority of recombined cells are CD45+ leukocytes, predominantly Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs). In addition, multiple lineages of recombined cells can be observed in the glioma model. In the lung carcinoma model, recombined MDSCs display an enhanced immunosuppressive phenotype and an altered miRNA profile compared to their non-recombined counterparts. Cre-lox based tracing of tumor EV RNA transfer in vivo can therefore be used to identify individual target cells in the tumor microenvironment for further mechanistical or functional analysis.

The adult human brain retains the capacity to generate new neurons in the hippocampal formation (Eriksson et al., 1998) and neuronal progenitor cells (NPCs) in the forebrain (Bernier et al., 2000), but to what extent it is capable of reacting to injuries, such as ischemia, is not known. We analyzed postmortem tissue from normal and pathological human brain tissue (n= 54) to study the cellular response to ischemic injury in the forebrain. We observed that cells expressing the NPC marker polysialylated neural adhesion cell molecule (PSA-NCAM) are continuously generated in the adult human subventricular zone (SVZ) and migrate along the olfactory tracts. These cells were not organized in migrating chains as in the adult rodent rostral migratory stream, and their number was lower in the olfactory tracts of brains from old (56–81 years of age) compared with young (29 + 36 years of age) individuals. Moreover, we show that in brains of patients of advanced age (60–87 years of age), ischemia led to an elevated number of Ki-67-positive cells in the ipsilateral SVZ without concomitant apoptotic cell death. Additionally, ischemia led to an increased number of PSA-NCAM-positive NPCs close to the lateral ventricular walls, compared with brains of comparable age without obvious neuropathologic changes. These results suggest that the adult human brain retains a capacity to respond to ischemic injuries and that this capacity is maintained even in old age.

New neurons are generated from stem cells in a few regions of the adult mammalian brain. Here we provide evidence for the generation of dopaminergic projection neurons of the type that are lost in Parkinson's disease from stem cells in the adult rodent brain and show that the rate of neurogenesis is increased after a lesion. The number of new neurons generated under physiological conditions in substantia nigra pars compacta was found to be several orders of magnitude smaller than in the granular cell layer of the dentate gyrus of the hippocampus. However, if the rate of neuronal turnover is constant, the entire population of dopaminergic neurons in substantia nigra could be replaced during the lifespan of a mouse. These data indicate that neurogenesis in the adult brain is more widespread than previously thought and may have implications for our understanding of the pathogenesis and treatment of neurodegenerative disorders such as Parkinson's disease.

In most parts of the adult mammalian central nervous system cell division is a relatively rare event, which makes it difficult to study at the ultrastructural level. We designed a protocol for reliable ultrastructural identification of proliferating cells in a tissue volume using DNA-incorporated 5-bromo-2-deoxyuridine (BrdU) as a marker. After BrdU administration the tissue is fixed and embedded in hydrophilic resin (LR Gold) and then cut in serial 1–2 μm sections and mounted on glass slides. BrdU is detected at the light microscopic level using immunogold labeling followed by silver enhancement, according to a standard procedure. After detection of labeled nuclei the section is reembedded in resin on the same glass slide. The glass is then dissolved in hydrofluoric acid and labeled cells cut in ultrathin sections for further ultrastructural analysis. The technique was tested and refined in sections of the intestine containing numerous dividing cells and, once optimized, was then applied to identify the ultrastructure of slowly proliferating putative stem cells in the adult mouse spinal cord.

This study aims at evaluating the combination of the tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL)-receptor 2 (TRAIL-R2)-specific antibody Drozitumab and the Smac mimetic BV6 in preclinical glioblastoma models. To this end, the effect of BV6 and/or Drozitumab on apoptosis induction and signaling pathways was analyzed in glioblastoma cell lines, primary glioblastoma cultures and glioblastoma stem-like cells. Here, we report that BV6 and Drozitumab synergistically induce apoptosis and reduce colony formation in several glioblastoma cell lines (combination indexin vivo models including an orthotopic, intracranial mouse model, underlining the clinical relevance of these findings. Mechanistic studies reveal that BV6 and Drozitumab act in concert to trigger the formation of a cytosolic receptor-interacting protein (RIP) 1/Fas-associated via death domain (FADD)/caspase-8-containing complex and subsequent activation of caspase-8 and -3. BV6- and Drozitumab-induced apoptosis is blocked by the caspase inhibitor zVAD.fmk, pointing to caspase-dependent apoptosis. RNA interference-mediated silencing of RIP1 almost completely abolishes the BV6-conferred sensitization to Drozitumab-induced apoptosis, indicating that the synergism critically depends on RIP1 expression. In contrast, both necrostatin-1, a RIP1 kinase inhibitor, and Enbrel, a TNFα-blocking antibody, do not interfere with BV6/Drozitumab-induced apoptosis, demonstrating that apoptosis occurs independently of RIP1 kinase activity or an autocrine TNFα loop. In conclusion, the rational combination of BV6 and Drozitumab presents a promising approach to trigger apoptosis in glioblastoma, which warrants further investigation.

Our view of the central nervous system has changed dramatically over the past few years. It is now well established that new neurons are generated continuously in adult mammals, including humans. These neurons derive from self-renewing multipotent neural stem cells. The identity of these stem cells has recently been unveiled.

Germany now has a substantial number of new biotechnology firms, with the number steadily increasing. The institutional framework has been slow to develop for this novel form of company, but many are now emerging and will certainly play an important part in the dynamics of the system. At the same time, the science base in this area has gained considerably in breadth and quality. The essential questions that arise from these recent developments are: (i) what prevented these new biotechnology firms from growing earlier and what is the current situation? (ii) What changes have occurred which have stimulated this growth? (iii) How are biotechnology companies going to develop further and what are the implications for Germany's pharmaceutical industry and wider economy? A database for biotechnology firms in Germany was set up of which a subset was used to analyse the current state of development. The following conclusions were reached: (i) Germany now has a substantial number of new biotechnology firms and the numbers are steadily increasing. (ii) Their collaborations with and proximity to academic centres of excellence suggests they are well embedded in the German research system. However, their sectoral composition sets them apart from their American counterparts, with greater bias towards instrumentation and environmental biotechnology, both areas of German industrial strength. (iii) Since the mid-1980s there has been continuous, if slow, adaptation to the institutional framework supporting biotechnology. These changes have finally resulted in an effective network of industry, academic and government links and have helped to promote both an increasingly strong scientific performance and the development of new firms. The authors suggest that, although these developments do not conform to the Anglo-Saxon entrepreneurial model in which new firms effectively forge new industries, the German evolutionary approach to innovation may still be holding its ground.

New neurons are continuously added in specific regions of the adult mammalian central nervous system. These neurons are derived from multipotent stem cells whose identity has been enigmatic. In this work, we present evidence that ependymal cells are neural stem cells. Ependymal cells give rise to a rapidly proliferating cell type that generates neurons that migrate to the olfactory bulb. In response to spinal cord injury, ependymal cell proliferation increases dramatically to generate migratory cells that differentiate to astrocytes and participate in scar formation. These data demonstrate that ependymal cells are neural stem cells and identify a novel process in the response to central nervous system injury.

In various types of cancers including glioblastoma, accumulating evidence show the existence of cancer stem‐like cells (CSCs), characterized by stem cell marker expression, capability of differentiation and self‐renewal, and high potential for tumor propagation in vivo. LGR5, whose expression is positively regulated by the Wnt signaling pathway, is a stem cell marker in intestinal mucosa and hair follicle in the skin. As Wnt signaling is also involved in brain development, the function of LGR5 in the maintenance of brain CSCs is to be assessed. Our study showed that the LGR5 transcript level was increased in CSCs. Co‐immunofluorescence staining demonstrated the co‐localization of CD133‐ and LGR5‐positive cells in glioblastoma tissue sections. Functionally, silencing of LGR5 by lentiviral shRNA‐mediated knockdown induced apoptosis in brain CSCs. Moreover, LGR5 depletion led to a downregulation of L1 cell adhesion molecule expression. In line with an important function in glioma tumorigenesis, LGR5 expression increased with glioma progression and correlated with an adverse outcome. Our findings suggest that LGR5 plays a role in maintenance and/or survival of brain CSCs.

Primary astrocytomas of grade 3 or 4 according to the classification system of the World Health Organization (high-grade astrocytomas or HGAs) are preponderant among adults and are almost invariably fatal despite the use of multimodal therapy. Here we show that the juvenile brain has an endogenous defense mechanism against HGAs. Neural precursor cells (NPCs) migrate to HGAs, reduce glioma expansion and prolong survival time by releasing endovanilloids that activate the vanilloid receptor (transient receptor potential vanilloid subfamily member-1 or TRPV1) on HGA cells. TRPV1 is highly expressed in tumor and weakly expressed in tumor-free brain. TRPV1 stimulation triggers tumor cell death through the branch of the endoplasmic reticulum stress pathway that is controlled by activating transcription factor-3 (ATF3). The antitumorigenic response of NPCs is lost with aging. NPC-mediated tumor suppression can be mimicked in the adult brain by systemic administration of the synthetic vanilloid arvanil, suggesting that TRPV1 agonists have potential as new HGA therapeutics.

Vesicular stomatitis virus (VSV)-based oncolytic virotherapy has the potential to significantly improve the prognosis of aggressive malignancies such as brain cancer. However, VSV's inherent neurotoxicity has hindered clinical development so far. Given that this neurotropism is attributed to the glycoprotein VSV-G, VSV was pseudotyped with the nonneurotropic envelope glycoprotein of the lymphocytic choriomeningitis virus (LCMV-GP→VSV-GP). Compared to VSV, VSV-GP showed enhanced infectivity for brain cancer cells in vitro while sparing primary human and rat neurons in vitro and in vivo , respectively. In conclusion, VSV-GP has a much wider therapeutic window than VSV and is thus more suitable for clinical applications, especially in the brain.

Rationale : Complementation of pluripotency genes may improve adult stem cell functions. Objectives : Here we show that clonally expandable, telomerase expressing progenitor cells can be isolated from peripheral blood of children. The surface marker profile of the clonally expanded cells is distinct from hematopoietic or mesenchymal stromal cells, and resembles that of embryonic multipotent mesoangioblasts. Cell numbers and proliferative capacity correlated with donor age. Isolated circulating mesoangioblasts (cMABs) express the pluripotency markers Klf4, c-Myc, as well as low levels of Oct3/4, but lack Sox2. Therefore, we tested whether overexpression of Sox2 enhances pluripotency and facilitates differentiation of cMABs in cardiovascular lineages. Methods and Results : Lentiviral transduction of Sox2 (Sox-MABs) enhanced the capacity of cMABs to differentiate into endothelial cells and cardiomyocytes in vitro. Furthermore, the number of smooth muscle actin positive cells was higher in Sox-MABs. In addition, pluripotency of Sox-MABs was shown by demonstrating the generation of endodermal and ectodermal progenies. To test whether Sox-MABs may exhibit improved therapeutic potential, we injected Sox-MABs into nude mice after acute myocardial infarction. Four weeks after cell therapy with Sox-MABs, cardiac function was significantly improved compared to mice treated with control cMABs. Furthermore, cell therapy with Sox-MABs resulted in increased number of differentiated cardiomyocytes, endothelial cells, and smooth muscle cells in vivo. Conclusions : The complementation of Sox2 in Oct3/4-, Klf4-, and c-Myc-expressing cMABs enhanced the differentiation into all 3 cardiovascular lineages and improved the functional recovery after acute myocardial infarction.

Transplanted hematopoietic cells have previously been shown to contribute to cells of other tissues by cell fusion. We wanted to elucidate whether this phenomenon of cell fusion also occurs under physiological conditions. Using a transgenic mouse reporter system to irreversibly label cells of the hematopoietic lineage, we were able to test their contribution to other tissues in the absence of any additional and potentially confounding factors such as irradiation or chemoablation. We found genetically marked, fused Purkinje neurons as well as hepatocytes in numbers comparable to previous bone marrow transplantation studies. The number of fused Purkinje neurons increased after intrathecal administration of bacterial lipopolysaccharide, suggesting that cell fusion can be induced by inflammation. In contrast to previous studies, however, genetically labeled Purkinje neurons never contained more than one nucleus, and we found only a single cell containing two Y-chromosomes in a male mouse. Consistent with results from the mouse model and unlike human bone marrow transplant recipients, postmortem adult human cerebelli of nontransplanted individuals were devoid of binucleated or polyploid Purkinje neurons. Therefore, our data suggests that fusion of hematopoietic cells with Purkinje neurons is only transient and does not lead to stable heterokaryon formation under noninvasive conditions.

When stimulated by inflammation, peripheral blood cells signal directly to neurons in the brain via the transfer of functional RNA enclosed in extracellular vesicles., Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation., Author Summary Peripheral infections leading to an inflammatory response can initiate signaling from the hematopoietic system to various organs including the brain. The traditional view of this communication between blood and brain is that individual factors are released by immune cells that in turn bind to neuronal or nonneuronal target cells in the brain where they exert their effects. By using a genetic tracing system, we now show that extracellular vesicles, small membrane structures that can contain a multitude of different molecules, can transfer functional RNA directly from blood cells to neurons. Although this type of signaling is highly restricted in the healthy animal, inflammatory injuries increase both the frequency of transfer and the range of the neuronal target populations in the brain. By showing altered miRNA profiles in neurons receiving extracellular vesicle cargo, we predict a complex regulation of gene expression in neural cells in response to peripheral inflammation.

This review gives an overview of current issues concerning the application of the concept of the stem cell niche to the adult mammalian brain. It describes how the niche manifests itself at different structural levels as well as the main applications that are influenced by this concept. Finally, special regard is given to what is known for the adult human brain and how far the findings from lower animals can be applied in harnessing the regenerative potential of stem cells for therapy.

Primary astrocytomas of grade 3 or 4 according to the classification system of the World Health Organization (high-grade astrocytomas or HGAs) are preponderant among adults and are almost invariably fatal despite the use of multimodal therapy. Here we show that the juvenile brain has an endogenous defense mechanism against HGAs. Neural precursor cells (NPCs) migrate to HGAs, reduce glioma expansion and prolong survival time by releasing endovanilloids that activate the vanilloid receptor (transient receptor potential vanilloid subfamily member-1 or TRPV1) on HGA cells. TRPV1 is highly expressed in tumor and weakly expressed in tumor-free brain. TRPV1 stimulation triggers tumor cell death through the branch of the endoplasmic reticulum stress pathway that is controlled by activating transcription factor-3 (ATF3). The antitumorigenic response of NPCs is lost with aging. NPC-mediated tumor suppression can be mimicked in the adult brain by systemic administration of the synthetic vanilloid arvanil, suggesting that TRPV1 agonists have potential as new HGA therapeutics. Citation Format: Michael Synowitz, Kristin Stock, Jitender Kumar, Stefania Petrosino, Roberta Imperatore, Ewan St J Smith, Peter Wend, Bettina Purfürst, Ulrike A Nuber, Ulf Gurok, Vitali Matyash, Joo-Hee Wälzlein, Sridhar R Chirasani, Gunnar Dittmar, Benjamin F Cravatt, Stefan Momma, Gary R Lewin, Alessia Ligresti, Luciano De Petrocellis, Luigia Cristino, Joerg Christian Tonn, Vincenzo Di Marzo, Helmut Kettenmann, Rainer Glass. Neural precursor cells induce cell death of high-grade astrocytomas through stimulation of TRPV1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 215. doi:10.1158/1538-7445.AM2013-215

Until the end of 1987, generation was not a goal of the acord project. As a result, the specifications for the acord system were written with an analysis-based system in mind rather than for a system which would allow for both analysis and generation. Furthermore, there were a lot of shortcuts within the specifications, like mapping similar words to the same semantic, or mapping complex information to atoms, which were aimed at facilitating analysis but which makes it in some cases impossible to design a generator which would generate adequate sentences from some input semantic representation. In 1987 when it was decided to augment the acord prototype with generators for French, English and German, the situation was the following.

We propose an architecture for the organisation of linguistic knowledge which allows to (1) separately formulate generalizations for different types of linguistic information, and (2) state interrelations between partial information belonging to different levels of description. We use typed feature structures for encoding linguistic knowledge. We show the application of this representational device for the architecture of linguistic knowledge sources for multilingual generation. As an example, we describe the use of interacting collocational and syntactic constraints in the generation of French and German sentences.

Das Papier beschreibt einen Algorithmus zur strukturgesteuerten Generierung aus f-Strukturen der LFG. Anhand von Beispielen werden Adaquatheitsbedingungen fur den Generierungsprozes formuliert, die zeigen, das die Eingabe-f-Struktur als komplexes Constraint fur die Ableitung einer Zeichenkette betrachtet werden mus. Es werden Methoden fur eine effiziente Behandlung dieses Constraints entwickelt, die eine effiziente Methode zur Behandlung der bekannten constraining equations der LFG mit einschliesen. Aus der Problemstellung der Generierung resultiert damit eine neue Motivation fur die Verwendung von Constraints in einem unifikationsbasierten Formalismus.

OBJECTIVE: Data from transgenic mouse models show that neuronal progenitor cells (NPCs) can migrate towards experimental brain tumors and modulate the course of pathology. However, the pathways attracting NPCs to CNS neoplasms were unknown and it was unexplored if NPCs migrate towards metastatic brain tumors in humans. METHODS: We analyzed the tumor-parenchyma interface of neurosurgical resections for the presence of (NPCs) and distinguished these physiological cells from the tumor mass ( by fluorescence in situ hybridization, FISH). Also, we performed experiments with transgenic mouse models, cell-culture (primary NPC- and glioma cultures of human and murine origin), cell migration assays and immunohistochemistry. RESULTS: We observed that PSA-NCAM-positive NPCs accumulate at CNS metastases. These metastatic tumors are distinguished from neural cells by defined sets of markers. Transplanting murine glioma cells embedded in a cell impermeable hollow fiber capsule into the brains of nestin-gfp reporter mice shows that diffusible factors are sufficient to induce a neurogenic reaction. In vitro, vascular endothelial growth factor (VEGF) secreted from glioma cells increases the migratory and proliferative behavior of adult human brain derived neural stem and progenitor cells via stimulation of VEGF receptor-2 (VEGFR-2). In vivo, inhibiting VEGFR-2 signaling with a function-blocking antibody leads to a reduction in NPC migration in response to tumors. CONCLUSION: Overall, our data reveal a mechanism for NPC attraction to CNS tumors and suggest that NPCs accumulate in human high-grade astrocytomas.