A Global Transcriptome Analysis Reveals Molecular Hallmarks of Neural Stem Cell Death, Survival, and Differentiation in Response to Partial FGF-2 and EGF Deprivation

Neurosphere cell culture is a commonly used model to study the properties and potential applications of neural stem cells (NSCs). However, standard protocols to culture NSCs have yet to be established, and the mechanisms underlying NSC survival and maintenance of their undifferentiated state, in response to the growth factors FGF-2 and EGF are not fully understood. Using cultures of embryonic and adult olfactory bulb stem cells (eOBSCs and aOBSCs), we analyzed the consequences of FGF-2 and EGF addition at different intervals on proliferation, cell cycle progression, cell death and differentiation, as well as on global gene expression. As opposed to cultures supplemented daily, addition of FGF-2 and EGF every 4 days significantly reduced the neurosphere volume and the total number of cells in the spheres, mainly due to increased cell death. Moreover, partial FGF-2 and EGF deprivation produced an increase in OBSC differentiation during the proliferative phase. These changes were more evident in aOBSC than eOBSC cultures. Remarkably, these effects were accompanied by a significant upregulation in the expression of endogenous Fgf-2 and genes involved in cell death and survival (Cryab), lipid catabolic processes (Pla2g7), cell adhesion (Dscaml1), cell differentiation (Dscaml1, Gpr17, S100b, Ndrg2) and signal transduction (Gpr17, Ndrg2). These findings support that a daily supply of FGF-2 and EGF is critical to maintain the viability and the undifferentiated state of NSCs in culture, and they reveal novel molecular hallmarks of NSC death, survival and the initiation of differentiation. © 2013 Nieto-Estévez et al.Peer Reviewe

E-proteins orchestrate the progression of neural stem cell differentiation in the postnatal forebrain

Background Neural stem cell (NSC) differentiation is a complex multistep process that persists in specific regions of the postnatal forebrain and requires tight regulation throughout life. The transcriptional control of NSC proliferation and specification involves Class II (proneural) and Class V (Id1-4) basic helix-loop-helix (bHLH) proteins. In this study, we analyzed the pattern of expression of their dimerization partners, Class I bHLH proteins (E-proteins), and explored their putative role in orchestrating postnatal subventricular zone (SVZ) neurogenesis. Results Overexpression of a dominant-negative form of the E-protein E47 (dnE47) confirmed a crucial role for bHLH transcriptional networks in postnatal neurogenesis by dramatically blocking SVZ NSC differentiation. In situ hybridization was used in combination with RT-qPCR to measure and compare the level of expression of E-protein transcripts (E2-2, E2A, and HEB) in the neonatal and adult SVZ as well as in magnetic affinity cell sorted progenitor cells and neuroblasts. Our results evidence that E-protein transcripts, in particular E2-2 and E2A, are enriched in the postnatal SVZ with expression levels increasing as cells engage towards neuronal differentiation. To investigate the role of E-proteins in orchestrating lineage progression, both in vitro and in vivo gain-of-function and loss-of-function experiments were performed for individual E-proteins. Overexpression of E2-2 and E2A promoted SVZ neurogenesis by enhancing not only radial glial cell differentiation but also cell cycle exit of their progeny. Conversely, knock-down by shRNA electroporation resulted in opposite effects. Manipulation of E-proteins and/or Ascl1 in SVZ NSC cultures indicated that those effects were Ascl1 dependent, although they could not solely be attributed to an Ascl1-induced switch from promoting cell proliferation to triggering cell cycle arrest and differentiation. Conclusions In contrast to former concepts, suggesting ubiquitous expression and subsidiary function for E-proteins to foster postnatal neurogenesis, this work unveils E-proteins as being active players in the orchestration of postnatal SVZ neurogenesis.ISSN:1749-810

Neural stem cells in the adult olfactory bulb core generate mature neurons in vivo

Although previous studies suggest that neural stem cells (NSCs) exist in the adult olfactory bulb (OB), their location, identity, and capacity to generate mature neurons in vivo has been little explored. Here, we injected enhanced green fluorescent protein (EGFP)-expressing retroviral particles into the OB core of adult mice to label dividing cells and to track the differentiation/maturation of any neurons they might generate. EGFP-labeled cells initially expressed adult NSC markers on days 1 to 3 postinjection (dpi), including Nestin, GLAST, Sox2, Prominin-1, and GFAP. EGFP+ -doublecortin (DCX) cells with a migratory morphology were also detected and their abundance increased over a 7-day period. Furthermore, EGFP-labeled cells progressively became NeuN+ neurons, they acquired neuronal morphologies, and they became immunoreactive for OB neuron subtype markers, the most abundant representing calretinin expressing interneurons. OB-NSCs also generated glial cells, suggesting they could be multipotent in vivo. Significantly, the newly generated neurons established and received synaptic contacts, and they expressed presynaptic proteins and the transcription factor pCREB. By contrast, when the retroviral particles were injected into the subventricular zone (SVZ), nearly all (98%) EGFP+ -cells were postmitotic when they reached the OB core, implying that the vast majority of proliferating cells present in the OB are not derived from the SVZ. Furthermore, we detected slowly dividing label-retaining cells in this region that could correspond to the population of resident NSCs. This is the first time NSCs located in the adult OB core have been shown to generate neurons that incorporate into OB circuits in vivo

Papel de IGF-I en la migración y neurogénesis de la zona subventricular y el bulbo olfatorio

Tesis doctoral inédita realizada en la Universidad Autónoma de Madrid, Facultad de Medicina. Departamento de Anatomía, Histología y Neurociencia y el Instituto Cajal (CSIC) . Fecha de lectura: 27 de Abril de 201

Lack of adrenomedullin affects growth and differentiation of adult neural stem/progenitor cells

Adrenomedullin (AM) is a peptide hormone involved in the modulation of cellular growth, migration, apoptosis, and angiogenesis. These characteristics suggest that AM is involved in the control of neural stem/progenitor cell (NSPC) biology. To explore this hypothesis, we have obtained NSPC from the olfactory bulb of adult wild-type animals and brain conditional knockouts for adm, the gene that produces AM. Knockout NSPC contain higher levels of hyperpolymerized tubulin and more abundant filopodia than adm-containing cells, resulting in a different morphology in culture, whereas the size of the knockout neurospheres is smaller than that of the wild-types. Proliferation studies have demonstrated that adm-null NSPC incorporate less 5?-bromodeoxyuridine (BrdU) than their wild-type counterparts. In contrast, BrdU studies in the olfactory bulb of adult animals show more labeled cells in adm-null mice that in wild-types, suggesting that a compensatory mechanism exists that guarantees the sufficient production of neural cells in this organ. In NSPC differentiation tests, lack of adm results in significantly lower proportions of neurons and astrocytes and higher proportions of oligodendrocytes. The oligodendrocytes produced from adm-null neurospheres present an immature phenotype with fewer and shorter processes than adm-containing oligo-dendrocytes. Thus, AM is an important factor in regulating the proliferation and differentiation of adult NSPC and might be used to modulate stem cell renewal and fate in protocols destined to produce neural cells for regenerative therapies. © Springer-Verlag 2010.Peer Reviewe

Expert Consensus on Predictive Parameters for the Occurrence of a Fall and a Contralateral Hip Fracture 1 and 3 Years After Hip Fracture: A Delphi Survey

Background: Early identification of people at risk for a contralateral hip fracture would be desirable to favorably influence patients’ prognosis. A recent systematic review failed to depict stringent patterns of risk parameters to be used for decision- making in clinical practice. Objective: To perform a consensus study using the Delphi method to reach an expert consensus on predictive parameters for the occurrence of a fall and a contralateral hip fracture 1 and 3 years after hip fracture. Methods: A list of potential members of the expert panel was identified based on the authors’ list of a recently conducted systematic review. Participating experts were asked to name parameters determining the probability for a fall and a contralateral hip fracture 1 and 3 years after an occurred hip fracture, separately. Additionally, we asked how those stated parameters should be measured. All mentioned parameters were compiled and sent back to the experts asking them to weight each single parameter by assigning a number between 1 (not important) and 10 (very important). The survey was conducted online using the REDCap software package. We defined expert agreement if the interquartile range of attributed weights for a parameter was 2. A relevant parameter had at least a median weight of 8. Results: Twelve experts from 7 countries completed the survey. Presence of fall history and mental and general health status were considered relevant irrespective of the outcome. For falling within 1 and 3 years, the number of medications and residential status were considered relevant, while for fractures within 1 and 3 years, osteoporosis management was considered important. Conclusion: Using the insights gained in this consensus study, empiric studies need to be set up assessing the prognostic value of the selected parameters

Expert Consensus on Predictive Parameters for the Occurrence of a Fall and a Contralateral Hip Fracture 1 and 3 Years After Hip Fracture: A Delphi Survey

Background: Early identification of people at risk for a contralateral hip fracture would be desirable to favorably influence patients’ prognosis. A recent systematic review failed to depict stringent patterns of risk parameters to be used for decision- making in clinical practice. Objective: To perform a consensus study using the Delphi method to reach an expert consensus on predictive parameters for the occurrence of a fall and a contralateral hip fracture 1 and 3 years after hip fracture. Methods: A list of potential members of the expert panel was identified based on the authors’ list of a recently conducted systematic review. Participating experts were asked to name parameters determining the probability for a fall and a contralateral hip fracture 1 and 3 years after an occurred hip fracture, separately. Additionally, we asked how those stated parameters should be measured. All mentioned parameters were compiled and sent back to the experts asking them to weight each single parameter by assigning a number between 1 (not important) and 10 (very important). The survey was conducted online using the REDCap software package. We defined expert agreement if the interquartile range of attributed weights for a parameter was 2. A relevant parameter had at least a median weight of 8. Results: Twelve experts from 7 countries completed the survey. Presence of fall history and mental and general health status were considered relevant irrespective of the outcome. For falling within 1 and 3 years, the number of medications and residential status were considered relevant, while for fractures within 1 and 3 years, osteoporosis management was considered important. Conclusion: Using the insights gained in this consensus study, empiric studies need to be set up assessing the prognostic value of the selected parameters

Fibroblast growth factor-2 increases the expression of neurogenic genes and promotes the migration and differentiation of neurons derived from transplanted neural stem/progenitor cells

The capacity of neural stem cells (NSC) to generate different types of neurons and glia depends on the action of intrinsic determinants and extracellular signals. Here, we isolated adult olfactory bulb stem cells (aOBSC) that express nestin, RC2 and Sox2, and that have the capacity to generate neurons possessing mature features in culture and in vivo. The differentiation of aOBSC into neurons and glia, as well as their genetic profile, was compared to that of embryonic OBSC (eOBSC) and ganglionic eminence stem cells (GESC). While these eOBSC express neurogenin (Ngn) 1 and 2, two telencephalic dorsal markers, GESC only express Ngn2. Adult OBSC express either little or no detectable Ngn1 and 2, and they produced significantly fewer neurons in culture than eOBSC. By contrast, Dlx2 transcripts (a telencephalic ventral marker) were only clearly detected in GESC. When transplanted into the early postnatal P5-P7 OB, each of the three populations gave rise to cells with a distinct pattern of neuronal migration and/or dendritic arborization. Overall, these findings suggest that cultured NSC partially maintain their regional and temporal specification. Notably, significant neuronal migration and differentiation were only observed in vivo when the NSC were briefly exposed to fibroblast growth factor-2 (FGF-2) before grafting, a treatment that enhanced the neurogenin expression. Hence, the migration and maturation of neurons derived from transplanted NSC can be promoted by upregulating neurogenic gene expression with FGF-2. © 2009 IBRO.Peer Reviewe

IGF-I promotes neuronal migration and positioning in the olfactory bulb and the exit from the subventricular zone

52 p.-8 fig.-4 supl.fig.While insulin-like growth factor-I (IGF-I) supports neuronal and glial differentiation in the CNS, it is largely unknown whether IGF-I also influences neuronal migration and positioning. We show here that the pattern of olfactory bulb (OB) layering is altered in Igf-I −/− mice. In these animals, Tbr1+-glutamatergic neurons are misplaced in the mitral cell layer (ML) and the external plexiform layer (EPL). In addition, there are fewer interneurons in the glomerular layer and the EPL of the Igf-I −/− mice, and fewer newborn neurons are incorporated into the OB from the forebrain subventricular zone (SVZ). Indeed, neuroblasts accumulate in the postnatal/adult SVZ of Igf-I −/− mice. Significantly, the positioning of Tbr1+-cells in a primitive ML is stimulated by IGF-I in cultured embryonic OB slices, an effect that is partially repressed by the phosphoinositide 3-kinase (PI3K) inhibitor. In OB cell cultures, IGF-I increases the phosphorylation of disabled1 (P-Dab1), an adaptor protein that is a target of Src family kinases (SFK) in the reelin signalling pathway, whereas reduced P-Dab1 levels were found in Igf-I −/− mice. Neuroblast migration from the rostral migratory stream (RMS) explants of postnatal Igf-I −/− was similar to that from Igf-I +/+ explants. However, cell migration was significantly enhanced by IGF-I added to the explants, an effect that was repressed by PI3K and SFK inhibitors. These findings suggest that IGF-I promotes neuronal positioning in the OB and support a role for IGF-I in stimulating neuroblast exit from the SVZ into the RMS, thereby promoting the incorporation of newly formed neurons into the OBThis work was funded by grants from the Spanish Ministry of Education and Science (MEC; SAF2004-05798 and BFU2007-61230), the ‘Comunidad de Madrid’ (CM; GR/SAL/0835/2004) and the ‘Instituto de Salud Carlos III’ (ISCIII; CIBERNED CB06/05/0065) to C.V.-A., and from the MEC (BMC 2004-0152) to F. de P., A.H.-C., M.J.Y.-B. and E.V.-V were supported by the MEC, the CM and CIBERNEDPeer reviewe

Pax6 Is essential for the maintenance and multi-lineage differentiation of neural stem cells, and for neuronal incorporation into the adult olfactory bulb

© 2014 Mary Ann Liebert, Inc. The paired type homeobox 6 (Pax6) transcription factor (TF) regulates multiple aspects of neural stem cell (NSC) and neuron development in the embryonic central nervous system. However, less is known about the role of Pax6 in the maintenance and differentiation of adult NSCs and in adult neurogenesis. Using the +/SeyDey mouse, we have analyzed how Pax6 heterozygosis influences the self-renewal and proliferation of adult olfactory bulb stem cells (aOBSCs). In addition, we assessed its influence on neural differentiation, neuronal incorporation, and cell death in the adult OB, both in vivo and in vitro. Our results indicate that the Pax6 mutation alters Nestin+-cell proliferation in vivo, as well as self-renewal, proliferation, and survival of aOBSCs in vitro although a subpopulation of +/SeyDey progenitors is able to expand partially similar to wild-type progenitors. This mutation also impairs aOBSC differentiation into neurons and oligodendrocytes, whereas it increases cell death while preserving astrocyte survival and differentiation. Furthermore, Pax6 heterozygosis causes a reduction in the variety of neurochemical interneuron subtypes generated from aOBSCs in vitro and in the incorporation of newly generated neurons into the OB in vivo. Our findings support an important role of Pax6 in the maintenance of aOBSCs by regulating cell death, self-renewal, and cell fate, as well as in neuronal incorporation into the adult OB. They also suggest that deregulation of the cell cycle machinery and TF expression in aOBSCs which are deficient in Pax6 may be at the origin of the phenotypes observed in this adult NSC population.Peer Reviewe