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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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Following retroviral transduction, hES cells maintain transgene expression and pluripotent differentiation into derivatives of all three germ layers. (A–C) Selected clones express the pluripotency-associated markers alkaline phosphatase (A), Tra-1-60 (B, red) and Tra-1-81 (C, red) while sustaining high levels of EGFP-expression (green). (D) Under selective conditions, EGFP-expression is maintained in differentiating EBs for at least 6 weeks. (E–H) Differentiating EBs show expression of cytokeratin (E), epithelial membrane antigen (F), desmin (G) and alpha-fetoprotein (H), reflecting multi-germ layer differentiation. (I–K) Transgene expression in single cells derived from 4-week-old EBs, 4 days after plating. EGFP-positive cells co-express alpha-fetoprotein (I), smooth muscle actin (J) and epithelial membrane antigen (K) (all red). (L) Selected clones give rise to teratomas containing various somatic tissues 9 weeks after injection into immunodeficient SCID-beige mice. Scale bars: A-C: 200 μm; D-H: 50 μm; I-K: 20 μm; L: 200 μm.
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fig2: Following retroviral transduction, hES cells maintain transgene expression and pluripotent differentiation into derivatives of all three germ layers. (A–C) Selected clones express the pluripotency-associated markers alkaline phosphatase (A), Tra-1-60 (B, red) and Tra-1-81 (C, red) while sustaining high levels of EGFP-expression (green). (D) Under selective conditions, EGFP-expression is maintained in differentiating EBs for at least 6 weeks. (E–H) Differentiating EBs show expression of cytokeratin (E), epithelial membrane antigen (F), desmin (G) and alpha-fetoprotein (H), reflecting multi-germ layer differentiation. (I–K) Transgene expression in single cells derived from 4-week-old EBs, 4 days after plating. EGFP-positive cells co-express alpha-fetoprotein (I), smooth muscle actin (J) and epithelial membrane antigen (K) (all red). (L) Selected clones give rise to teratomas containing various somatic tissues 9 weeks after injection into immunodeficient SCID-beige mice. Scale bars: A-C: 200 μm; D-H: 50 μm; I-K: 20 μm; L: 200 μm.

Mentions: Transduced EGFP-positive clones continued to express markers of pluripotency, including alkaline phosphatase, Tra-1-60, Tra-1-81 (Figure 2A–C) and Oct-4. Upon withdrawal of G418, ∼50% of the cells lost visible EGFP fluorescence within five passages. Performing immunostainings with an anti-EGFP antibody, EGFP-expression could still be detected in 65.2 ± 12.2% of the cells after 4 weeks without selection pressure. These observations suggest that the transduced undifferentiated cells retain pluripotency and self-renewal capacity during prolonged cultivation, but are bound to loose retroviral transgene expression in the absence of selection pressure. To confirm that transgene expression resulted from stable integration of the provirus into the host DNA, we conducted Southern blot analyses of genomic DNA prepared from four different clones five passages after transduction. In all four clones we found genomic integration of the EGFP-expressing provirus. Whereas clones PK2, PK4 and PK7 showed a single integration of the viral transgene, clone PK5 displayed two bands, which could be the result of a double integration, a mixed clone population or a mutation of the vector provirus (Figure 1H). These data indicate that the loss of EGFP expression is not due to a loss of the transgene but may be related to transcriptional changes such as silencing of the viral LTR.


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)

Following retroviral transduction, hES cells maintain transgene expression and pluripotent differentiation into derivatives of all three germ layers. (A–C) Selected clones express the pluripotency-associated markers alkaline phosphatase (A), Tra-1-60 (B, red) and Tra-1-81 (C, red) while sustaining high levels of EGFP-expression (green). (D) Under selective conditions, EGFP-expression is maintained in differentiating EBs for at least 6 weeks. (E–H) Differentiating EBs show expression of cytokeratin (E), epithelial membrane antigen (F), desmin (G) and alpha-fetoprotein (H), reflecting multi-germ layer differentiation. (I–K) Transgene expression in single cells derived from 4-week-old EBs, 4 days after plating. EGFP-positive cells co-express alpha-fetoprotein (I), smooth muscle actin (J) and epithelial membrane antigen (K) (all red). (L) Selected clones give rise to teratomas containing various somatic tissues 9 weeks after injection into immunodeficient SCID-beige mice. Scale bars: A-C: 200 μm; D-H: 50 μm; I-K: 20 μm; L: 200 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636442&req=5

fig2: Following retroviral transduction, hES cells maintain transgene expression and pluripotent differentiation into derivatives of all three germ layers. (A–C) Selected clones express the pluripotency-associated markers alkaline phosphatase (A), Tra-1-60 (B, red) and Tra-1-81 (C, red) while sustaining high levels of EGFP-expression (green). (D) Under selective conditions, EGFP-expression is maintained in differentiating EBs for at least 6 weeks. (E–H) Differentiating EBs show expression of cytokeratin (E), epithelial membrane antigen (F), desmin (G) and alpha-fetoprotein (H), reflecting multi-germ layer differentiation. (I–K) Transgene expression in single cells derived from 4-week-old EBs, 4 days after plating. EGFP-positive cells co-express alpha-fetoprotein (I), smooth muscle actin (J) and epithelial membrane antigen (K) (all red). (L) Selected clones give rise to teratomas containing various somatic tissues 9 weeks after injection into immunodeficient SCID-beige mice. Scale bars: A-C: 200 μm; D-H: 50 μm; I-K: 20 μm; L: 200 μm.
Mentions: Transduced EGFP-positive clones continued to express markers of pluripotency, including alkaline phosphatase, Tra-1-60, Tra-1-81 (Figure 2A–C) and Oct-4. Upon withdrawal of G418, ∼50% of the cells lost visible EGFP fluorescence within five passages. Performing immunostainings with an anti-EGFP antibody, EGFP-expression could still be detected in 65.2 ± 12.2% of the cells after 4 weeks without selection pressure. These observations suggest that the transduced undifferentiated cells retain pluripotency and self-renewal capacity during prolonged cultivation, but are bound to loose retroviral transgene expression in the absence of selection pressure. To confirm that transgene expression resulted from stable integration of the provirus into the host DNA, we conducted Southern blot analyses of genomic DNA prepared from four different clones five passages after transduction. In all four clones we found genomic integration of the EGFP-expressing provirus. Whereas clones PK2, PK4 and PK7 showed a single integration of the viral transgene, clone PK5 displayed two bands, which could be the result of a double integration, a mixed clone population or a mutation of the vector provirus (Figure 1H). These data indicate that the loss of EGFP expression is not due to a loss of the transgene but may be related to transcriptional changes such as silencing of the viral LTR.

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