Comparison Infection Efficiency Of Type 5 With Chimeric 5/F35 Adenoviral Vectors To Human Bone Marrow Mesenchymal Stem Cells

[Objective] To compare infection efficiency of type 5 with chimeric 5/F35 adenovirus vectors to the human bone marrow mesenchymal stem cells (hBMSCs) in vitro.[Methods] The experiments were conducted at the Laboratory of Department of Hematopathy of Beijing General Hospital of PLA from March 2008 to October 2008. hBMNCs (Human Bone Marrow Mononuclear Cells) from bone marrow blood of healthy volunteers were isolated by Lymphocyte Separation Media and hBMSCs were cultured withα-MEM medium by density-grade centrifugation and wall-adhesion method. Phenotypes of the cells were identified by flow cytometry. With inducing differentiation of the hBMSCs into adipocytes and osteoblasts, the adipocyte and osteoblast inductors were added into the medium, respectively, after the third passage of hBMSCs was inoculated into a 24-well plate at the density of 1×104 cells/per well and adhered at the plastic walls. Alkaline phosphatase and Oil-Red-O staining were used to verify the differentiation of hBMSCs to adipocyte and osteoblast, respectively. To detect infection efficiency of type 5 adenovirus encoding enhanced fluorescent protein (Ad5 EGFP) and chimeric 5/F35 adenovirus encoding EGFP (Ad 5/F35 EGFP) to hBMSCs and human leukemia cell lines (K562,U937 and HL60), the infection rates for hBMSCs were detected by flow cytometry and fluorescence microscopy. Two days after the cells was infected with Ad5 EGFP or Ad 5/F35 EGFP, and the rates of living cells were counted by trypan blue dye exclusion at the different titres of the adenoviral vectors.[Results] Analysis by flow cytometry showed that the positive rates of CD166, CD29, CD73, CD31, CD45, CD34, CD14 were 95.08%, 99.53%, 72.26%, 1.50%, 2.02%, 3.80%, and 4.94%, respectively, in the hBMSCs at our culture systems. Alkaline phosphatase staining and Oil-Red-O staining were positive 14 days after the addition of corresponding inductors into the hBMSCs, suggesting differentiation of the hBMSCs into adipocytes and osteoblasts, respectivly. EGFP-positive hBMSCs were (0.72±0.14) %, (4.97±0.46) %, (9.80±3.43), and (45.53±6.32) %, respectively, at the multiple of infection (MOI) of 5, 20, 100, and 400 two days after the infection of hBMSCs with Ad 5-EGFP, whereas the positive cells were (24.31±10.55) %, (55.19±13.73) %, (87.68±9.5) %, and (96.57±5.64) % with Ad 5/F35-EGFP. The survival rate of hBMSCs was more than 95% at the MOIs of 5, 20, and 100. At the MOI of 400, however, the survival rate was significantly lower, lese than 90%. The transfection efficiencies of Ad5/F35 vector to human leukemia cell lines (K562, U937 and HL60) were more than 50% at MOI=100, whereas Ad5 vector could not effectively transfect human leukemia cell lines (K562, U937 and HL60).[Conclusion] There are differences in infection efficiencies of the two adenoviral vectors, type 5 and chimeric 5/F35 adenoviral vectors encoding EGFP, to hBMSCs. Infection rates to hBMSCs by Ad5 EGFP or Ad 5/F35 EGFP are positive relation with the MOIs of viruses, a higher MOI will lead a higher infection rate to hBMSCs. At a same MOI, Ad5/F35 EGFP has obviously higher infection efficiency to hBMSCs and human leukemia cell lines (K562,U937 and HL60) than Ad5, suggesting that chimeric 5/F35 adenoviral vectors for hBMSC-based cell or gene therapy might offer better advantages than type 5 adenoviral vectors.