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Stem Cells 2003;21:111-117 www.StemCells.com
© 2003 AlphaMed Press

RAPID COMMUNICATION

High-Level Sustained Transgene Expression in Human Embryonic Stem Cells Using Lentiviral Vectors

Yue Maa, Ali Ramezanib, Rachel Lewisa, Robert G. Hawleyb,c,d, James A. Thomsona

a National Primate Research Center, and the Department of Anatomy, School of Medicine, University of Wisconsin, Madison, Wisconsin, USA; Departments of
b Hematopoiesis and
c Blood and Cell Therapy, Holland Laboratory, American Red Cross, Rockville, Maryland, USA;
d Department of Anatomy and Cell Biology, and Programs in Genetics and Molecular and Cellular Oncology, The George Washington University, Washington, DC, USA

Key Words. Human embryonic stem cells • Lentiviral vectors • Scaffold attachment region • Chromatin insulator

James A. Thomson, V.M.D, Ph.D., Wisconsin Regional Primate Research Center, University of Wisconsin, 1220 Capitol Court, Madison, Wisconsin 53715, USA. Telephone: 608-263-3585; Fax: 608-265-8984; e-mail: thomson{at}primate.wisc.edu

Here we describe the sustained expression of transgenes introduced into human embryonic stem (ES) cells using self-inactivating lentiviral vectors. At low multiplicity of infection, vesicular stomatitis virus-pseudotyped vectors containing a green fluorescent protein (GFP) transgene under the control of a human elongation factor 1{alpha} promoter transduced human ES cells at high efficiency. The majority of the transduced ES cells, which harbored low numbers of integrated vectors, continued to express GFP after 60 days of culture. Incorporation of a scaffold attachment region (SAR) from the human interferon-ß gene into the lentiviral vector backbone increased the average level of GFP expression, and inclusion of the SAR together with a chromatin insulator from the 5' end of the chicken ß-globin locus reduced the variability in GFP expression. When the transduced ES cells were induced to differentiate into CD34+ hematopoietic precursors in vitro, GFP expression was maintained with minimal silencing. The ability to efficiently introduce active transgenes into human ES cells will facilitate gain-of-function studies of early developmental processes in the human system. These results also have important implications for the possible future use of gene-modified human ES cells in transplantation and tissue regeneration applications.




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