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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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Transduction of hES cells with murine retroviral vectors. (A) Transfection efficiencies of undifferentiated hES cells using optimized protocols for lipofection (L), electroporation (E) and nucleofection (N) techniques. (B) mCAT1-HA expression (green) in nucleofected hES cells cultured on matrigel. Twenty-four hours after plating, the cells show a flattened morphology typical for colonies propagated on matrigel. Nuclear expression of Oct-4 (red) reflects their undifferentiated state. (C) Schematic illustration of the two-step protocol used for transduction of hES cells with ecotropic retroviral vectors. First cells are nucleofected with a construct encoding the murine retrovirus receptor mCAT1. Twenty-four hours later they are transduced with a murine retroviral vector. Transduced cultures are either analyzed for transgene expression after 48 h or subjected to selection of permanently transduced clones. The integrated provirus expresses the EGFP transgene from the viral LTR linked to a neomycin resistance gene (neoR) by an internal ribosome entry site (IRES). (D) Forty-eight hours after infection, 16.4 ± 5.9% of the total cell population showed EGFP-expression. Normalized to the proportion of mCAT1-expressing cells determined in (A), this corresponds to a calculated transduction efficiency of ∼30% of the mCAT1-expressing cells (mean values from n = 5 independent experiments). (E–G) Transduced EGFP-positive cells continue to express the pluripotency-associated markers Oct-4 (E), Tra-1-60 (F) and Tra-1-81 (G) (all red; counterstain DAPI). (H) Five passages after transduction, four clones were subjected to Southern analysis. A single integration of the EGFP transgene could be detected in clones PK2, PK4 and PK7. Clone PK5 displays two bands, which could be the result of a double integration, a mixed clone population or a mutation of the vector provirus. Restriction analysis was performed with EcoRI (for genomic DNA) and ClaI (unique site within the retroviral vector). Scale bars: B,E: 100 μm; F: 30 μm; G: 50 μm.
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fig1: Transduction of hES cells with murine retroviral vectors. (A) Transfection efficiencies of undifferentiated hES cells using optimized protocols for lipofection (L), electroporation (E) and nucleofection (N) techniques. (B) mCAT1-HA expression (green) in nucleofected hES cells cultured on matrigel. Twenty-four hours after plating, the cells show a flattened morphology typical for colonies propagated on matrigel. Nuclear expression of Oct-4 (red) reflects their undifferentiated state. (C) Schematic illustration of the two-step protocol used for transduction of hES cells with ecotropic retroviral vectors. First cells are nucleofected with a construct encoding the murine retrovirus receptor mCAT1. Twenty-four hours later they are transduced with a murine retroviral vector. Transduced cultures are either analyzed for transgene expression after 48 h or subjected to selection of permanently transduced clones. The integrated provirus expresses the EGFP transgene from the viral LTR linked to a neomycin resistance gene (neoR) by an internal ribosome entry site (IRES). (D) Forty-eight hours after infection, 16.4 ± 5.9% of the total cell population showed EGFP-expression. Normalized to the proportion of mCAT1-expressing cells determined in (A), this corresponds to a calculated transduction efficiency of ∼30% of the mCAT1-expressing cells (mean values from n = 5 independent experiments). (E–G) Transduced EGFP-positive cells continue to express the pluripotency-associated markers Oct-4 (E), Tra-1-60 (F) and Tra-1-81 (G) (all red; counterstain DAPI). (H) Five passages after transduction, four clones were subjected to Southern analysis. A single integration of the EGFP transgene could be detected in clones PK2, PK4 and PK7. Clone PK5 displays two bands, which could be the result of a double integration, a mixed clone population or a mutation of the vector provirus. Restriction analysis was performed with EcoRI (for genomic DNA) and ClaI (unique site within the retroviral vector). Scale bars: B,E: 100 μm; F: 30 μm; G: 50 μm.

Mentions: An expression construct encoding a HA-tagged variant of the murine retrovirus receptor mCAT1 under control of the CMV promoter (22) or the CMV-enhanced chicken beta-actin promoter (CAG) were used to optimize the transfection conditions for hES cells. Lipofection and conventional electroporation yielded only moderate transfection rates. Twenty-four hours after transfection, HA-immunofluorescence was detected in 19.3 ± 4.6% of the lipofected and 34.2 ± 6.7% of the electroporated cells (3 independent experiments). To further improve transfection efficiency we explored nucleofection, a recently developed method for enhanced electroporation (Amaxa GmbH, Cologne, Germany). Under optimized conditions we achieved transient mCAT1-HA expression in 52.1 ± 8.3% of the undifferentiated hES cells (10 independent experiments; Figure 1A and B). Survival rates with Amaxa's manufacture program B-16 averaged around 70% and were thus comparable to those observed in conventional electroporation [for further information on the nucleofection of hES cells, see Siemen et al. (2005) (29)]. 94.4 ± 2.8% of the mCAT1-HA expressing cells showed nuclear Oct-4 immunofluorescence. Compared to Oct-4 expression of non-transfected cells (97 ± 1.3%), the nucleofection procedure appeared to have no significant effect on the pluripotent state of the cells.


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)

Transduction of hES cells with murine retroviral vectors. (A) Transfection efficiencies of undifferentiated hES cells using optimized protocols for lipofection (L), electroporation (E) and nucleofection (N) techniques. (B) mCAT1-HA expression (green) in nucleofected hES cells cultured on matrigel. Twenty-four hours after plating, the cells show a flattened morphology typical for colonies propagated on matrigel. Nuclear expression of Oct-4 (red) reflects their undifferentiated state. (C) Schematic illustration of the two-step protocol used for transduction of hES cells with ecotropic retroviral vectors. First cells are nucleofected with a construct encoding the murine retrovirus receptor mCAT1. Twenty-four hours later they are transduced with a murine retroviral vector. Transduced cultures are either analyzed for transgene expression after 48 h or subjected to selection of permanently transduced clones. The integrated provirus expresses the EGFP transgene from the viral LTR linked to a neomycin resistance gene (neoR) by an internal ribosome entry site (IRES). (D) Forty-eight hours after infection, 16.4 ± 5.9% of the total cell population showed EGFP-expression. Normalized to the proportion of mCAT1-expressing cells determined in (A), this corresponds to a calculated transduction efficiency of ∼30% of the mCAT1-expressing cells (mean values from n = 5 independent experiments). (E–G) Transduced EGFP-positive cells continue to express the pluripotency-associated markers Oct-4 (E), Tra-1-60 (F) and Tra-1-81 (G) (all red; counterstain DAPI). (H) Five passages after transduction, four clones were subjected to Southern analysis. A single integration of the EGFP transgene could be detected in clones PK2, PK4 and PK7. Clone PK5 displays two bands, which could be the result of a double integration, a mixed clone population or a mutation of the vector provirus. Restriction analysis was performed with EcoRI (for genomic DNA) and ClaI (unique site within the retroviral vector). Scale bars: B,E: 100 μm; F: 30 μm; G: 50 μm.
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Related In: Results  -  Collection

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fig1: Transduction of hES cells with murine retroviral vectors. (A) Transfection efficiencies of undifferentiated hES cells using optimized protocols for lipofection (L), electroporation (E) and nucleofection (N) techniques. (B) mCAT1-HA expression (green) in nucleofected hES cells cultured on matrigel. Twenty-four hours after plating, the cells show a flattened morphology typical for colonies propagated on matrigel. Nuclear expression of Oct-4 (red) reflects their undifferentiated state. (C) Schematic illustration of the two-step protocol used for transduction of hES cells with ecotropic retroviral vectors. First cells are nucleofected with a construct encoding the murine retrovirus receptor mCAT1. Twenty-four hours later they are transduced with a murine retroviral vector. Transduced cultures are either analyzed for transgene expression after 48 h or subjected to selection of permanently transduced clones. The integrated provirus expresses the EGFP transgene from the viral LTR linked to a neomycin resistance gene (neoR) by an internal ribosome entry site (IRES). (D) Forty-eight hours after infection, 16.4 ± 5.9% of the total cell population showed EGFP-expression. Normalized to the proportion of mCAT1-expressing cells determined in (A), this corresponds to a calculated transduction efficiency of ∼30% of the mCAT1-expressing cells (mean values from n = 5 independent experiments). (E–G) Transduced EGFP-positive cells continue to express the pluripotency-associated markers Oct-4 (E), Tra-1-60 (F) and Tra-1-81 (G) (all red; counterstain DAPI). (H) Five passages after transduction, four clones were subjected to Southern analysis. A single integration of the EGFP transgene could be detected in clones PK2, PK4 and PK7. Clone PK5 displays two bands, which could be the result of a double integration, a mixed clone population or a mutation of the vector provirus. Restriction analysis was performed with EcoRI (for genomic DNA) and ClaI (unique site within the retroviral vector). Scale bars: B,E: 100 μm; F: 30 μm; G: 50 μm.
Mentions: An expression construct encoding a HA-tagged variant of the murine retrovirus receptor mCAT1 under control of the CMV promoter (22) or the CMV-enhanced chicken beta-actin promoter (CAG) were used to optimize the transfection conditions for hES cells. Lipofection and conventional electroporation yielded only moderate transfection rates. Twenty-four hours after transfection, HA-immunofluorescence was detected in 19.3 ± 4.6% of the lipofected and 34.2 ± 6.7% of the electroporated cells (3 independent experiments). To further improve transfection efficiency we explored nucleofection, a recently developed method for enhanced electroporation (Amaxa GmbH, Cologne, Germany). Under optimized conditions we achieved transient mCAT1-HA expression in 52.1 ± 8.3% of the undifferentiated hES cells (10 independent experiments; Figure 1A and B). Survival rates with Amaxa's manufacture program B-16 averaged around 70% and were thus comparable to those observed in conventional electroporation [for further information on the nucleofection of hES cells, see Siemen et al. (2005) (29)]. 94.4 ± 2.8% of the mCAT1-HA expressing cells showed nuclear Oct-4 immunofluorescence. Compared to Oct-4 expression of non-transfected cells (97 ± 1.3%), the nucleofection procedure appeared to have no significant effect on the pluripotent state of the cells.

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