531 0007-4888/08/1464-0531 © 2008 Springer Science+Business Media, Inc. Viral Vectors for Stable Transduction of Human Mesenchymal Stem Cells: Systems Based on Adeno-Associated Viruses and Lentiviruses A. V. Shakhbazau, I. N. Sevyaryn*, N. V. Goncharova*, V. V. Grinev**, S. M. Kosmacheva*, and M. P. Potapnev Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 4, pp. 216-218, November, 2008 Original article submitted July 10, 2008 We compared the efficiency of transduction with lentivectors based on HIV-1 and adeno-associated viruses serotype 2 and stability of transgene expression in human mesenchymal bone marrow stem cells. Key Words: mesenchymal stem cells; lentivirus vectors; transduction Institute of Genetics and Cytology, National Academy of Sciences of Belarus; *Center of Hematology and Transfusiology; **Belorussian State University, Minsk, Belarussia. Address for correspondence: shakhbazau@gmail.com. A. V. Shakhbasov. The prospects of clinical use of mesenchymal stem cells (MSC) from human bone marrow (BM) modi- fied by therapeutically useful transgenes dictate the need in evaluation of the most effective system for transgene delivery. The advantage of vectors based on recombinant lentiviruses and adeno-associated viruses is stable transduction and long-term ex- pression of the transgene in primary cell cultures including MSC. The aim of the present study was to compare the efficiency of transduction with lentivectors ba- sed on HIV-1 and adeno-associated viruses sero- type 2 and stability of transgene expression in MSC from human BM. MATERIALS AND METHODS Vector plasmids used for virus assembly were gene- rated in Escherichia coli strain DH5a under stand- ard conditions (medium LB containing 10 g/liter tryptone, 10 g/liter NaCl, and 5 g/liter yeast extract, pH 7.2, 100 mg/liter ampicillin, in dark, at 37 o C). Preparative isolation and purification of plasmids for cell transfection were performed using Endo- Free Plasmid Maxi kits (Qiagen) according to ma- nufacturer’s instructions. For isolation of MSC, BM aspirate was sepa- rated by centrifugation in Ficoll gradient, the mono- nuclear fraction was collected, washed twice with PBS, and seeded in MEM medium (α-modification) supplemented with 10% fetal calf serum (FBS; Hy- Clone) [1]. MSC selected by adhesion to plastic were immunotyped using antibodies to CD90, CD105, CD45, and CD34. The cells were cultured in a CO 2 incubator (5% CO 2 ) at 37 o C. The growth medium was replaced after 2 days. HEK 293T cells were cultured under similar conditions in DMEM with 10% FBS (HyClone). Transfection of HEK 293T cells was carried out in 6-well plates using ExGen 500 reagent (Fer- mentas) or by the calcium phosphate method. In transduction experiments, supernatant of the pro- ducent strain purified from the cells and debris by filtration was used as the source of recombinant viruses. Supernatant of a strain co-transfected with a transfer vector and one of the two necessary hel- per plasmids served as the control. GFP fluores- cence was detected on a Carl Zeiss Axio Imager M1 microscope. The percent of transfected and trans- duced cells was determined on a FACScan cyto- meter (BD). Cell Technologies in Biology and Medicine, No. 4, November, 2008