Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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An analysis of global gene expression reveals molecular and signalling pathways hallmarks of neural stem cell survival and expansion in response to FGF-2 and EGF

ABSTRACT: The culture of neural stem cells (NSCs) as floating neurospheres has become widely used as an experimental model to analyse the properties of NSCs. Although the neurosphere model has existed for two decades, there is still no standard protocol to grow NSCs in this way. Thus, we have analysed the consequences of the frequency of growth factor (FGF-2 and EGF) addition to embryonic and adult olfactory bulb stem cells (eOBSCs and aOBSCs) cultures, specifically in terms of proliferation, cell cycle progression, death and differentiation, as well as on global changes in gene expression and signaling pathways. We found that addition of FGF-2 and EGF every two or four days rather than daily significantly reduces the volume of the neurospheres and the total number of cells, changes that were more evident in aOBSC than in eOBSC cultures. The reduction in neurosphere size was mainly due to an increase in cell death and occurs without major changes in the cell cycle parameters tested. Moreover, partial deprivation of FGF-2 and EGF produces a mild increase in aOBSC differentiation during the proliferative phase. Remarkably, these effects were accompanied by a significant upregulation in the expression of genes involved in cell death regulation (Cryab), lipid catabolic processes (Pla2g7), cell adhesion (Dscaml1), cell differentiation (Dscaml1, Gpr17, S100b) and signal transduction (Gpr17, Ndrg2), among others. These findings support that continuous supply of FGF-2 and EGF is critical to maintain the viability/survival of NSCs in culture and reveals novel molecular hallmarks of NSC maintenance/survival and expansion in response to these growth factors. Total RNA was extracted from aOBSC cultures using the Trizol reagent (Invitrogen) and purified with Qiagen RNeasy Mini Kit separation columns (Qiagen). The RNA was sent to the Genomic Unit of CNB (Centro Nacional de Biotecnologia, Madrid, Spain). RNA integrity was corroborated by using Bioanalyzer. Then, cDNA was synthesized and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA, http://www.affymetrix.com) which contain a total of 45101 transcripts to assess and compare the overall gene expression profiles. 9 samples were analyzed. Ctr: Control mouse adult olfatory bulb stem cells (aOBSC) cultured with daily added Fgf2 and Egf, 3 biological rep C2: Mouse adult olfatory bulb stem cells (aOBSC) cultured with Fgf2 and Egf added every two days, 3 biological rep C4: Mouse adult olfatory bulb stem cells (aOBSC) cultured with Fgf2 and Egf added every four days, 3 biological rep

ORGANISM(S): Mus musculus

SUBMITTER: Marcos AraÃºzo-Bravo

PROVIDER: E-GEOD-37516 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The culture of neural stem cells (NSCs) as floating neurospheres has become widely used as an experimental model to analyse the properties of NSCs. Although the neurosphere model has existed for two decades, there is still no standard protocol to grow NSCs in this way. Thus, we have analysed the consequences of the frequency of growth factor (FGF-2 and EGF) addition to embryonic and adult olfactory bulb stem cells (eOBSCs and aOBSCs) cultures, specifically in terms of proliferation, cell cycle progression, death and differentiation, as well as on global changes in gene expression and signaling pathways. We found that addition of FGF-2 and EGF every two or four days rather than daily significantly reduces the volume of the neurospheres and the total number of cells, changes that were more evident in aOBSC than in eOBSC cultures. The reduction in neurosphere size was mainly due to an increase in cell death and occurs without major changes in the cell cycle parameters tested. Moreover, partial deprivation of FGF-2 and EGF produces a mild increase in aOBSC differentiation during the proliferative phase. Remarkably, these effects were accompanied by a significant upregulation in the expression of genes involved in cell death regulation (Cryab), lipid catabolic processes (Pla2g7), cell adhesion (Dscaml1), cell differentiation (Dscaml1, Gpr17, S100b) and signal transduction (Gpr17, Ndrg2), among others. These findings support that continuous supply of FGF-2 and EGF is critical to maintain the viability/survival of NSCs in culture and reveals novel molecular hallmarks of NSC maintenance/survival and expansion in response to these growth factors. Total RNA was extracted from aOBSC cultures using the Trizol reagent (Invitrogen) and purified with Qiagen RNeasy Mini Kit separation columns (Qiagen). The RNA was sent to the Genomic Unit of CNB (Centro Nacional de Biotecnologia, Madrid, Spain). RNA integrity was corroborated by using Bioanalyzer. Then, cDNA was synthesized and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA, http://www.affymetrix.com) which contain a total of 45101 transcripts to assess and compare the overall gene expression profiles.

Project description:In this study, we demonstrate that insulin is produced not only in the mammalian pancreas but also in adult neuronal cells derived from hippocampus and olfactory bulb. Paracrine Wnt3 plays an essential role in promoting the active expression of insulin in both hippocampus and olfactory bulb-derived neural stem cells. Our analysis indicates that the balance between Wnt3, which triggers the expression of insulin via NeuroD1 transcription factor, and IGFBP-4, which inhibits the original Wnt3 action, is regulated depending on the diabetic status. We also show that adult neural progenitors derived from diabetic animals retain the ability to give rise to insulin-producing cells and that grafting neuronal progenitors into the pancreas of diabetic animals reduces glucose levels. This study provides an example of a simple and direct use of adult stem cells from one organ to another, without introducing additional inductive genes. In this study, we demonstrate that insulin is produced not only in the mammalian pancreas but also in adult neuronal cells derived from hippocampus and olfactory bulb. Paracrine Wnt3 plays an essential role in promoting the active expression of insulin in both hippocampus and olfactory bulb-derived neural stem cells. Our analysis indicates that the balance between Wnt3, which triggers the expression of insulin via NeuroD1 transcription factor, and IGFBP-4, which inhibits the original Wnt3 action, is regulated depending on the diabetic status. We also show that adult neural progenitors derived from diabetic animals retain the ability to give rise to insulin-producing cells and that grafting neuronal progenitors into the pancreas of diabetic animals reduces glucose levels. This study provides an example of a simple and direct use of adult stem cells from one organ to another, without introducing additional inductive genes. Total four different samples, gene expressions in hippocampal derived neural stem cells (HPC NSC), that in Olfactory bulb-derived neural stem cells (OB NSC), that in neurons derived from the HPC NSCs (HPC Neu) and that in neurons derived from the OB NSCs (OB Neu) were independently analyzed. Three independent experiments were performed to prepare each cell sample, and the extracted total RNAs from each cell source were mixed to apply following microarray analysis (Four independent RNA sample; HPC NSC, OB NSC, HPC Neu and OB Neu).

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An analysis of global gene expression reveals molecular and signalling pathways hallmarks of neural stem cell survival and expansion in response to FGF-2 and EGF

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