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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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FISH and immunofluorescence analysis.FISH with GINS2 specific BAC (pink) and simultaneous immunofluorescence staining with GFAP (green) revealed GINS2 amplification in cells with beginning GFAP expression after 5 d of differentiation (A). FISH with CDK4 specific BAC (pink) and immunofluorescence staining with GFAP revealed CDK4 amplification in cells with beginning GFAP expression after 2 d of differentiation (B). FISH with CDK4 specific BAC (pink) and immunofluorescence staining with Tubulin-ß-III-chain (green) revealed CDK4 amplification in cells with beginning Tubulin-ß-III-chain expression after 11 d of differentiation (C and D). Nuclei were counterstained with DAPI. Size calibration bar = 5 µm.
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pone-0037422-g004: FISH and immunofluorescence analysis.FISH with GINS2 specific BAC (pink) and simultaneous immunofluorescence staining with GFAP (green) revealed GINS2 amplification in cells with beginning GFAP expression after 5 d of differentiation (A). FISH with CDK4 specific BAC (pink) and immunofluorescence staining with GFAP revealed CDK4 amplification in cells with beginning GFAP expression after 2 d of differentiation (B). FISH with CDK4 specific BAC (pink) and immunofluorescence staining with Tubulin-ß-III-chain (green) revealed CDK4 amplification in cells with beginning Tubulin-ß-III-chain expression after 11 d of differentiation (C and D). Nuclei were counterstained with DAPI. Size calibration bar = 5 µm.

Mentions: While cells with strong GFAP expression showed a normal copy number for all genes tested, e.g. CDK4 and GINS2, cells with weak GFAP expression showed amplifications of CDK4 and GINS2 after 2 d and 5 d (Figures 4 A, B). While cells with a weak GFAP expression were localized in or near the sphere, cells that had migrated out of the sphere revealed a stronger expression of the differentiation marker GFAP and a differentiated morphology of the cell body with typical appendages. Similar results were obtained with immunofluorescence staining using the differentiation marker Tubulin beta-3 chain. Notably, after 11 days of differentiation we still found CDK4 amplification in NHNP cells with weak Tubulin beta-3 chain expression (Figure 4 C, D).


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)

FISH and immunofluorescence analysis.FISH with GINS2 specific BAC (pink) and simultaneous immunofluorescence staining with GFAP (green) revealed GINS2 amplification in cells with beginning GFAP expression after 5 d of differentiation (A). FISH with CDK4 specific BAC (pink) and immunofluorescence staining with GFAP revealed CDK4 amplification in cells with beginning GFAP expression after 2 d of differentiation (B). FISH with CDK4 specific BAC (pink) and immunofluorescence staining with Tubulin-ß-III-chain (green) revealed CDK4 amplification in cells with beginning Tubulin-ß-III-chain expression after 11 d of differentiation (C and D). Nuclei were counterstained with DAPI. Size calibration bar = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037422-g004: FISH and immunofluorescence analysis.FISH with GINS2 specific BAC (pink) and simultaneous immunofluorescence staining with GFAP (green) revealed GINS2 amplification in cells with beginning GFAP expression after 5 d of differentiation (A). FISH with CDK4 specific BAC (pink) and immunofluorescence staining with GFAP revealed CDK4 amplification in cells with beginning GFAP expression after 2 d of differentiation (B). FISH with CDK4 specific BAC (pink) and immunofluorescence staining with Tubulin-ß-III-chain (green) revealed CDK4 amplification in cells with beginning Tubulin-ß-III-chain expression after 11 d of differentiation (C and D). Nuclei were counterstained with DAPI. Size calibration bar = 5 µm.
Mentions: While cells with strong GFAP expression showed a normal copy number for all genes tested, e.g. CDK4 and GINS2, cells with weak GFAP expression showed amplifications of CDK4 and GINS2 after 2 d and 5 d (Figures 4 A, B). While cells with a weak GFAP expression were localized in or near the sphere, cells that had migrated out of the sphere revealed a stronger expression of the differentiation marker GFAP and a differentiated morphology of the cell body with typical appendages. Similar results were obtained with immunofluorescence staining using the differentiation marker Tubulin beta-3 chain. Notably, after 11 days of differentiation we still found CDK4 amplification in NHNP cells with weak Tubulin beta-3 chain expression (Figure 4 C, D).

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