CLONING AND STEM CELLS Volume 5, Number 1, 2003 © Mary Ann Liebert, Inc. Infection Efficiency of Human and Mouse Embryonic Stem Cells Using Adenoviral and Adeno-Associated Viral Vectors JOSEPH R. SMITH-ARICA, 1 ALISON J. THOMSON, 1 RAY ANSELL, 1 JOHN CHIORINI, 2 BEVERLY DAVIDSON, 3 and JIM MCWHIR 1 ABSTRACT Human and mouse embryonic stem (ES) cells have the capacity to differentiate into deriva- tives of all three germ layers, suggesting novel therapies for degenerative, metabolic, and trau- matic disorders. ES-based regenerative medicine will be further advanced by the develop- ment of reliable methods for transgene introduction and expression. Here, we show infection of human and mouse embryonic stem (ES) cells with two of the most popular vectors in gene transfer, adenovirus type 5 (Ad5) and adeno-associated virus (AAV; serotypes 2, 4, and 5). All vectors express the nuclear-localized marker gene b-galactosidase expressed from the Rous Sarcoma Virus long terminal repeat (RSV-LTR). Both Ad5 and AAV2 infected human and mouse ES cells and gave rise to b-galactosidase expression. AAV4 and 5 did not yield de- tectable levels of b-galactosidase expression. Quantitative PCR analysis of virally infected human and mouse ES cells revealed that only Ad5 and AAV2 are capable of transducing both cell-types. No viral DNA was detected in cells infected with either AAV4 or AAV5. Infection and subsequent differentiation of mouse and human ES cells with Ad5 showed that b-galac- tosidase–expressing cells were restricted to cells in the interior of the embryoid body mass. No b-galactosidase expression was observed in AAV-infected cells following differentiation. There was no difference in morphology or differentiation patterns between infected and non- infected differentiating mouse and human ES cells. Differentiation of hES cells prior to in- fection led to transduction of neuronally differentiated cells with good efficiency using all vectors. These data show that Ad5- and AAV2-based vectors are capable of infecting both hu- man and mouse ES cells, in both their undifferentiated and differentiated states, whereas AAV4 and AAV5 can infect human and mouse ES cells only following differentiation. 51 INTRODUCTION T HE ISOLATION IN CULTURE of pluripotential hu- man embryonic stem (hES) cells (Thomson et al., 1998; Reubinoff et al., 2000) provides new opportunities in regenerative medicine. ES cells have the potential to give rise to all adult cell types, and diseases arising from the failure of vir- tually any cell are in principle treatable by trans- plantation of differentiated derivatives of ES cells. ES cells also provide both an in vitro model for human development and a powerful tool for 1 Department of Gene Expression and Development, Roslin Institute, Roslin, Midlothian, United Kingdom. 2 Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Insti- tutes of Health, Bethesda, Maryland. 3 Department of Internal Medicine, Program in Gene Therapy, University of Iowa College of Medicine, Iowa City, Iowa.