Morphological analysis and lentiviral transduction of fetal monkey bone marrow-derived mesenchymal stem cells.
We explored the transduction kinetics of HIV-1-derived lentiviral vectors containing the CMV, EF1alpha, 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 EF1alpha 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 EF1alpha 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 EF1alpha 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 > EF1alpha > PGK) without inhibiting differentiation potential, although the cell cycle was transiently altered at high MOI
Lee
Chang I
CI
California National Primate Research Center, University of California, Davis, California 95616-8542, USA.
Kohn
Donald B
DB
Ekert
Jason E
JE
Tarantal
Alice F
AF
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002453:Cell Cycle; D002455:Cell Division; D005333:Fetus / Q000166:cytology; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics; D007120:Immunochemistry; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008253:Macaca mulatta / Q000196:embryology*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D010012:Osteogenesis; D011401:Promoter Regions, Genetic; D014161:Transduction, Genetic; D014746:Vimentin / Q000235:genetics / Q000378:metabolism
D008164:Luminescent Proteins; D014746:Vimentin; D049452:Green Fluorescent Proteins