Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Human embryonic stem cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage

ABSTRACT: All data are derived from the same batch of human ES cells (line H9, WA-09). With the exception of the â??UDâ?? file all files represent neural cell types derived from the undifferentiated hESCs. Following mechanical isolation, rosettes were maintained for various passages in the presence of defined growth factors and morphogen factors as indicated below at the R-NSC stage. Cells at the NSCFGF/EGF stage were derived from mechanically isolated neural rosettes that were purified and long-term expanded as attached monolayer cultures in serum free medium in the presence of FGF2/EGF. Experiment Overall Design: All data are derived from the same batch of human ES cells (line H9, WA-09). With the exception of the â??UDâ?? file all files represent neural cell types derived from the undifferentiated hESCs. Following mechanical isolation, rosettes were maintained for various passages in the presence of defined growth factors and morphogen factors as indicated below at the R-NSC stage. Cells at the NSCFGF/EGF stage were derived from mechanically isolated neural rosettes that were purified and long-term expanded as attached monolayer cultures in serum free medium in the presence of FGF2/EGF.

ORGANISM(S): Homo sapiens

SUBMITTER: Agnes Viale

PROVIDER: E-GEOD-9921 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Adult mouse subventricular zone (SVZ) neural progenitor cells (NPCs) retain the capacity to generate multiple lineages in vitro and in vivo. Thus far, the mechanisms involved in the regulation of these cells have not been well elucidated. We have carried out RNA profiling of adult SVZ cell cultures undergoing differentiation, to identify pathways that regulate progenitor cell proliferation and to define a set of transcripts that can be used as molecular tools in the drug discovery process. We carried out a stepwise stratification of the results to identify transcripts specifically enriched in NPCs and validated some of these using comparative literature analysis, quantitative PCR and immunological techniques. The results show a set of transcription factors, secreted molecules, and plasma membrane markers which are differentially regulated during differentiation. Pathway analysis highlights alterations in IGF, Wnt and TGFbeta signalling cascades. Further characterization of these components could provide greater insight into the mechanisms involved in the regulation of neurogenesis in the adult brain. Mouse subventricular zone neural stem cells cultures were prepared from C57Bl6/J mice (Johansson et al.,1999) and cultured in suspension in DFEM/F12 1:1 supplemented with PSF, B27 supplement and EGF/ FGF-2 at 20ng/ml. RNA was isolated from either expanding neurospheres in the presence of FGF2 and EGF (Reynolds and Weiss, 1992; Gritti et al., 1999), or from cultures induced to differentiate upon growth factor withdrawal or serum exposure, either after 24 hours (in suspension, to avoid changes induced by substrate attachment) or 5 days (as adherent cultures in poly-lysine and laminin-coated plates). Experiments were conducted in triplicate from independent batches of SVZ preparations. As a control for potential effects of media changes, a set of identical cultures were included where cells were grown for a further 24 hours in fresh media containing FGF2 and EGF (20 ng/ml each, normal growth media, NGM). Fluorescently labelled aRNA from individual samples were competitively hybridised against a pool of labelled aRNA from undifferentiated reference (starting) material on custom Agilent microarrays representing ~22,500 mouse transcripts. Technical, fluor-reversed hybridisations were performed for every sample.

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Transcription profiling of Human embryonic stem cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage

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