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Transduction of human embryonic stem cells by ecotropic retroviral vectors.

Koch P, Siemen H, Biegler A, Itskovitz-Eldor J, Brüstle O - Nucleic Acids Res. (2006)

Bottom Line: Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo.HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression.Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure.

View Article: PubMed Central - PubMed

Affiliation: Institute of Reconstructive Neurobiology, Life and Brain Center University of Bonn and Hertie Foundation, Bonn, Germany.

ABSTRACT
The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.

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Related in: MedlinePlus

Retrovirally-transduced hES cells maintain transgene expression throughout neural differentiation. (A) Following discontinuation of G418 selection, neurospheres derived from transduced hES continue to express EGFP across several passages. Shown are EGFP-expressing neurospheres cultured for 4 weeks (five passages) without G418. (B) Following growth factor withdrawal, plated spheres show extensive neurite extension. (C–F) Plated neurospheres and single cells derived thereof express the neuronal marker antigens beta-III-tubulin (C) and MAP2ab (D and E); astrocytes can be readily detected with an antibody to GFAP (F). Both neurons and glial cells exhibit prominent EGFP expression. Scale bars: A–D: 100 μm; E and F: 20 μm.
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fig3: Retrovirally-transduced hES cells maintain transgene expression throughout neural differentiation. (A) Following discontinuation of G418 selection, neurospheres derived from transduced hES continue to express EGFP across several passages. Shown are EGFP-expressing neurospheres cultured for 4 weeks (five passages) without G418. (B) Following growth factor withdrawal, plated spheres show extensive neurite extension. (C–F) Plated neurospheres and single cells derived thereof express the neuronal marker antigens beta-III-tubulin (C) and MAP2ab (D and E); astrocytes can be readily detected with an antibody to GFAP (F). Both neurons and glial cells exhibit prominent EGFP expression. Scale bars: A–D: 100 μm; E and F: 20 μm.

Mentions: To explore whether maintenance of transgene selection pressure is required after lineage commitment, EBs were plated onto polyornithine-coated cell culture dishes and propagated in neural selection medium containing DMEM/F12, insulin (25 μg/ml), transferrin (100 μg/ml) sodium selenite (5 ng/ml), fibronectin (2.5 μg/ml), FGF2 (20 ng/ml) and G418 for a period of 10 days. After 7 days, G418 was discontinued. On day 10, neural islands containing dense neural tube-like structures were mechanically isolated and further propagated on poly-HEMA-coated tissue culture plates in DMEM/F12 supplemented with N2 (N2 media) and 10 ng/ml FGF2 as free floating neurospheres as described (27,32). Using a 100 μl pipette tip, neurospheres were dissociated in weekly intervals for a total time period of up to 3 months. Throughout this time the neurospheres displayed strong EGFP expression without evidence of transgene downregulation (Figure 3A).


Transduction of human embryonic stem cells by ecotropic retroviral vectors.

Koch P, Siemen H, Biegler A, Itskovitz-Eldor J, Brüstle O - Nucleic Acids Res. (2006)

Retrovirally-transduced hES cells maintain transgene expression throughout neural differentiation. (A) Following discontinuation of G418 selection, neurospheres derived from transduced hES continue to express EGFP across several passages. Shown are EGFP-expressing neurospheres cultured for 4 weeks (five passages) without G418. (B) Following growth factor withdrawal, plated spheres show extensive neurite extension. (C–F) Plated neurospheres and single cells derived thereof express the neuronal marker antigens beta-III-tubulin (C) and MAP2ab (D and E); astrocytes can be readily detected with an antibody to GFAP (F). Both neurons and glial cells exhibit prominent EGFP expression. Scale bars: A–D: 100 μm; E and F: 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636442&req=5

fig3: Retrovirally-transduced hES cells maintain transgene expression throughout neural differentiation. (A) Following discontinuation of G418 selection, neurospheres derived from transduced hES continue to express EGFP across several passages. Shown are EGFP-expressing neurospheres cultured for 4 weeks (five passages) without G418. (B) Following growth factor withdrawal, plated spheres show extensive neurite extension. (C–F) Plated neurospheres and single cells derived thereof express the neuronal marker antigens beta-III-tubulin (C) and MAP2ab (D and E); astrocytes can be readily detected with an antibody to GFAP (F). Both neurons and glial cells exhibit prominent EGFP expression. Scale bars: A–D: 100 μm; E and F: 20 μm.
Mentions: To explore whether maintenance of transgene selection pressure is required after lineage commitment, EBs were plated onto polyornithine-coated cell culture dishes and propagated in neural selection medium containing DMEM/F12, insulin (25 μg/ml), transferrin (100 μg/ml) sodium selenite (5 ng/ml), fibronectin (2.5 μg/ml), FGF2 (20 ng/ml) and G418 for a period of 10 days. After 7 days, G418 was discontinued. On day 10, neural islands containing dense neural tube-like structures were mechanically isolated and further propagated on poly-HEMA-coated tissue culture plates in DMEM/F12 supplemented with N2 (N2 media) and 10 ng/ml FGF2 as free floating neurospheres as described (27,32). Using a 100 μl pipette tip, neurospheres were dissociated in weekly intervals for a total time period of up to 3 months. Throughout this time the neurospheres displayed strong EGFP expression without evidence of transgene downregulation (Figure 3A).

Bottom Line: Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo.HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression.Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure.

View Article: PubMed Central - PubMed

Affiliation: Institute of Reconstructive Neurobiology, Life and Brain Center University of Bonn and Hertie Foundation, Bonn, Germany.

ABSTRACT
The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.

Show MeSH
Related in: MedlinePlus