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Embryonic Stem Cells/Induced Pluripotent Stem Cells
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Excision of Reprogramming Transgenes Improves the Differentiation Potential of iPS Cells Generated with a Single Excisable Vector†‡§
Article first published online: 10 NOV 2009
DOI: 10.1002/stem.255
Copyright © 2009 AlphaMed Press
Additional Information
How to Cite
Sommer, C. A., Sommer, A. G., Longmire, T. A., Christodoulou, C., Thomas, D. D., Gostissa, M., Alt, F. W., Murphy, G. J., Kotton, D. N. and Mostoslavsky, G. (2010), Excision of Reprogramming Transgenes Improves the Differentiation Potential of iPS Cells Generated with a Single Excisable Vector. STEM CELLS, 28: 64–74. doi: 10.1002/stem.255
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Author contributions: C.A.S.: Conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; A.G.S.: Conception and design, collection and assembly of data, data analysis and interpretation; T.L., C.C., D.D.T., M.G., and F.W.A.: Collection and assembly of data; G.J.M: Conception and design, data analysis and interpretation; D.N.K and G.M.: Conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript.
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Disclosure of potential conflicts of interest is found at the end of this article.
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First published online in STEM CELLS EXPRESS November 10, 2009.
Publication History
- Issue published online: 12 JAN 2010
- Article first published online: 10 NOV 2009
- Accepted manuscript online: 10 NOV 2009 12:00AM EST
- Manuscript Accepted: 3 NOV 2009
- Manuscript Received: 1 JUL 2009
Keywords:
- Induced pluripotent stem cells;
- Reprogramming;
- Excisable single lentiviral vector;
- Stem cell cassette;
- Endoderm
Abstract
The residual presence of integrated transgenes following the derivation of induced pluripotent stem (iPS) cells is highly undesirable. Here we demonstrate efficient derivation of iPS cells free of exogenous reprogramming transgenes using an excisable polycistronic lentiviral vector. A novel version of this vector containing a reporter fluorochrome allows direct visualization of vector excision in living iPS cells in real time. We find that removal of the reprogramming vector markedly improves the developmental potential of iPS cells and significantly augments their capacity to undergo directed differentiation in vitro. We further propose that methods to efficiently excise reprogramming transgenes with minimal culture passaging, such as those demonstrated here, are critical since we find that iPS cells may acquire chromosomal abnormalities, such as trisomy of chromosome 8, similar to embryonic stem cells after expansion in culture. Our findings illustrate an efficient method for the generation of transgene-free iPS cells and emphasize the potential beneficial effects that may result from elimination of integrated reprogramming factors. In addition, our results underscore the consequences of long-term culture that will need to be taken into account for the clinical application of iPS cells. STEM CELLS 2010;28:64–74