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Genome-wide gene amplification during differentiation of neural progenitor cells in vitro.

Fischer U, Keller A, Voss M, Backes C, Welter C, Meese E - PLoS ONE (2012)

Bottom Line: We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells.Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells.Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Saarland University, Homburg/Saar, Germany. ulrike.fischer@uks.eu

ABSTRACT
DNA sequence amplification is a phenomenon that occurs predictably at defined stages during normal development in some organisms. Developmental gene amplification was first described in amphibians during gametogenesis and has not yet been described in humans. To date gene amplification in humans is a hallmark of many tumors. We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells. Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells. We identified several amplified genes in neural progenitor cells that are known to be amplified in malignant tumors. There is also a striking overlap of amplified chromosomal regions between differentiating neural progenitor cells and malignant tumor cells derived from astrocytes. Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

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Related in: MedlinePlus

Whole chromosome plots.A genome-wide view of the 10× window-averaged data at 25 kb resolution is displayed for NHNP cells at day 0, day 1, day 2 and day 5 during differentiation.
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pone-0037422-g001: Whole chromosome plots.A genome-wide view of the 10× window-averaged data at 25 kb resolution is displayed for NHNP cells at day 0, day 1, day 2 and day 5 during differentiation.

Mentions: Differentiation of NHNP cells was induced by withdrawal of EGF and bFGF and supplementation of brain derived neurotrophic factor (BDNF). The expression of Tubulin beta-3 chain and GFAP was analyzed by immunofluorescence after 24 h following differentiation induction. For amplification analysis total DNA was isolated from undifferentiated NHNP sphere cells and from NHNP cells differentiated for 24 h, 2 d and 5 d respectively and analyzed on NimbleGen 720K human whole genome tiling arrays. Signal intensity data were extracted from scanned images of each array using Roche NimbleGen NimbleScan v2.6 software. After spatial correction, the Cy3 and Cy5 signal intensities were normalized using qspline normalization. Following normalization a 10× window–averaging step is applied. Window-averaging reduces the size of the data and reduces the noise in the data. For amplification detection we used the dynamic segMNT algorithm that identifies segments by minimizing the squared error relative to the segment means. To detect representative alterations and to minimize the identification of random alterations, we extracted segments with segment means greater 0.1 threshold and a size greater than 250 kb. Chromosomal regions that revealed copy number gains and match CNVs (copy number variations) present in the Database of Genomic Variants available at UCSC Genome Browser were excluded from further analysis. While we did not detect amplified regions in NHNP cells at zero time and 24 h after differentiation, we found numerous chromosomal regions with copy number gains in NHNP cells after 2 d and 5 d of differentiation. In total we found 66 amplified chromosome regions after 2 d of differentiation and 93 amplified chromosome regions after 5 d of differentiation (Table 1). We also detected 9 deleted chromosome regions after 2 d of differentiation and 30 deleted chromosome regions after 5 d of differentiation. Whole genome profiles were presented in Figure 1.


Genome-wide gene amplification during differentiation of neural progenitor cells in vitro.

Fischer U, Keller A, Voss M, Backes C, Welter C, Meese E - PLoS ONE (2012)

Whole chromosome plots.A genome-wide view of the 10× window-averaged data at 25 kb resolution is displayed for NHNP cells at day 0, day 1, day 2 and day 5 during differentiation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351388&req=5

pone-0037422-g001: Whole chromosome plots.A genome-wide view of the 10× window-averaged data at 25 kb resolution is displayed for NHNP cells at day 0, day 1, day 2 and day 5 during differentiation.
Mentions: Differentiation of NHNP cells was induced by withdrawal of EGF and bFGF and supplementation of brain derived neurotrophic factor (BDNF). The expression of Tubulin beta-3 chain and GFAP was analyzed by immunofluorescence after 24 h following differentiation induction. For amplification analysis total DNA was isolated from undifferentiated NHNP sphere cells and from NHNP cells differentiated for 24 h, 2 d and 5 d respectively and analyzed on NimbleGen 720K human whole genome tiling arrays. Signal intensity data were extracted from scanned images of each array using Roche NimbleGen NimbleScan v2.6 software. After spatial correction, the Cy3 and Cy5 signal intensities were normalized using qspline normalization. Following normalization a 10× window–averaging step is applied. Window-averaging reduces the size of the data and reduces the noise in the data. For amplification detection we used the dynamic segMNT algorithm that identifies segments by minimizing the squared error relative to the segment means. To detect representative alterations and to minimize the identification of random alterations, we extracted segments with segment means greater 0.1 threshold and a size greater than 250 kb. Chromosomal regions that revealed copy number gains and match CNVs (copy number variations) present in the Database of Genomic Variants available at UCSC Genome Browser were excluded from further analysis. While we did not detect amplified regions in NHNP cells at zero time and 24 h after differentiation, we found numerous chromosomal regions with copy number gains in NHNP cells after 2 d and 5 d of differentiation. In total we found 66 amplified chromosome regions after 2 d of differentiation and 93 amplified chromosome regions after 5 d of differentiation (Table 1). We also detected 9 deleted chromosome regions after 2 d of differentiation and 30 deleted chromosome regions after 5 d of differentiation. Whole genome profiles were presented in Figure 1.

Bottom Line: We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells.Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells.Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Saarland University, Homburg/Saar, Germany. ulrike.fischer@uks.eu

ABSTRACT
DNA sequence amplification is a phenomenon that occurs predictably at defined stages during normal development in some organisms. Developmental gene amplification was first described in amphibians during gametogenesis and has not yet been described in humans. To date gene amplification in humans is a hallmark of many tumors. We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells. Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells. We identified several amplified genes in neural progenitor cells that are known to be amplified in malignant tumors. There is also a striking overlap of amplified chromosomal regions between differentiating neural progenitor cells and malignant tumor cells derived from astrocytes. Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

Show MeSH
Related in: MedlinePlus