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HSC 5-FU time course

ABSTRACT: HSC (Sca+ SP) were isolated from 8-12 week C57B6 mice at various time points after treatment with 5-Fluorouracil. RNA was isolated from 50,000-100,000 FACS sorted cells and subjected to two rounds of T7 based linear amplification using Ambion's Message Amp kit. Two replicates from each time point were analyzed. http://www.plosbiology.org/plosonline/?request=get-document&doi=10.1371%2Fjournal.pbio.0020301 Keywords = HSC Keywords = proliferation Keywords = quiescence Keywords = 5-FU Keywords: time-course

ORGANISM(S): Mus musculus

PROVIDER: GSE1559 | GEO | 2004/09/28

SECONDARY ACCESSION(S): PRJNA90869

REPOSITORIES: GEO

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Project description:Schwann cells, expanded in number by exposure to growth factors in vitro, could be useful in nervous system repair. Our previous results suggest that long term exposure to heregulin and forskolin changes the functional properties of the human Schwann cells, including the ability to myelinate axons after transplantation. Here, we propose to determine the molecular changes in the Schwann cells that occur as a result of extended growth with mitogenic factors. We believe that the information obtained in these studies will provide clues about mechanisms underlying the already observed changes in function. This information will aid in the prediction of the safety and efficacy of neural repair approaches that use cultured, expanded Schwann cells. Finally this data may provide clues into the mechanisms underlying normal human Schwann cell function. To use gene array analysis to compare gene expression profiles in early and late passage human Schwann cells exposed to the growth factors heregulin and forskolin. Observed changes in the function of human Schwann cells, including their capacity for growth and differentiation, after prolonged exposure to heregulin and forskolin, are caused by changes in the gene expression profiles in these cells. Nerves from four different donors were obtained within 12-15 h postmortem, with full consent for research from the families of the donors (faxed). IRB approval not required because subjects were officially dead at nerve harvest (IRB confirmation faxed). The nerve tissue obtained consisted of the lower 15 cm of nerve extending from mid calf to ankle. Endoneurial fascicles were dissected from short (1-2 cm) nerve fragments and cultured with heregulin and forskolin for 1 week. The nerve fascicles were then treated overnight with collagenase and dispase and dissociated by gentle trituration. Cells were plated at low density on laminin coated dishes and maintained in culture with mitogens for 3 passages. Schwann cell purities during this period were > 95%. RNA was extracted from the cells at passage 1 ( i.e. after 2 population doublings) and passage 3 (i.e. after 8 population doublings) from the following donors: SN HSC 330, age 51; SN HSC 317, age 29; SN HSC 329, age 20 and SN HSC 351, age 42. Thus eight samples (2 groups, 4 independent samples per group) are to be analyzed. Because both groups in this comparison have been exposed to mitogens, differences in gene expression profiles will be interpreted as indicative of changes caused by prolonged versus short term exposure to mitogens. More RNA was prepared from each group of cells than was needed for gene array analysis to allow confirmation of differentially-expressed gene transcripts by real-time RT-PCR. Since the main purpose of this project is to detect any major changes in the molecular properties of the Schwann cells, we propose that these samples be analyzed using Affymetrix U133 + 2 arrays containing the complete human genome and request Option 1: Start to Finish Profiling of all samples. We have contacted Dr Stanley Nelson at UCLA and discussed the project with him. We request that the samples be analyzed in the UCLA facility. Keywords: time-course

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HSC 5-FU time course

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