1. ¹California National Primate Research Center, University of California, Davis, California 95616-8542, USA
2. ²Division of Research Immunology/BMT, Childrens Hospital Los Angeles, Los Angeles, California, 90027-6016,USA
3. ³Department of Pediatrics, University of California, Davis, California 95616-8542, USA

Correspondence: Alice F. Tarantal, California National Primate Research Center, Pedrick and Hutchison Roads, University of California, Davis, CA 95616-8542. Fax: (530)-754-4286. E-mail: aftarantal@primate.ucdavis.edu

Received 30 April 2003; Accepted 29 September 2003.

Abstract

We explored the transduction kinetics of HIV-1-derived lentiviral vectors containing the CMV, EF1, or PGK promoter expressing EGFP in fetal rhesus monkey bone marrow-derived mesenchymal stem cells (rhMSC). Studies included the effects of transduction (MOI 0–100) on growth, cell cycle, and differentiation toward an osteogenic lineage. Flow cytometric analysis indicated an approximate 8- to 10-fold greater quantity of EGFP-expressing rhMSC when cells were transduced with the CMV or EF1 promoter compared to PGK, although quantitative PCR revealed no differences at the DNA level. The CMV promoter initially expressed 10- to 100-fold higher levels of EGFP compared to EF1 or PGK, respectively, at increasing MOI, although a significant decline in transgene expression was observed posttransduction and with advancing passage (P < 0.01), whereas a significant increase in the level of expression was observed over time with the EF1 promoter. At an MOI of 100, a transient arrest at the S phase of the cell cycle was observed for both vector constructs. Transduced rhMSC differentiated toward an osteogenic lineage comparable to untransduced rhMSC and showed equivalent levels of alkaline phosphatase activity. These findings suggest that the SIN HIV-1-derived lentiviral vectors used in these studies can efficiently transduce rhMSC in vitro (CMV > EF1 > PGK) without inhibiting differentiation potential, although the cell cycle was transiently altered at high MOI