[The feasibility of adenoviral co-transduction of BMP2 and BMP7 for the expression of recombinant human BMP2/7 heterodimer in rat bone marrow mesenchymal stem cells].

Abstract

Objective: To investigate the feasibility of rat bone marrow mesenchymal stem cells (BMSCs) as the target cell of adenovirus-mediated co-transduction of BMP2 and BMP7 genes and then facilitate the expression of recombinant BMP2/7 heterodimer protein.

Methods: 3 adult male Fischer 344 rats of about 10 weeks of age were used for harvest and in vitro culture of rat BMSCs. Recombinant adenovirus vector carrying BMP2 or BMP7 target genes were constructed with AdMax vector system, and production of high-titer adenoviruses were packaged with HEK293T cells and then concentrated with CSCl2 density-gradient ultra-centrifugation. Rat BMSCs from passage 3 were seeded in 6-well plates at the concentration of 10 000 cells/cm2.After overnight pre-culture, BMSCs were allowed to culture in 200 μl serum-free alpha MEM containing both Ad-BMP2 and Ad-BMP7 adenovirus (100 MOI of each virus). After 7 days in vitro culture, conditioned cell culture supernatants were collected and followed by immunoprecipitation through immune protein G columns pre-loaded with mouse anti-human BMP7 antibody. The resulted protein immune-precipitates were used to assay the expression of BMP2/7 heterodimers via Western Blot and ELISA assay. As a negative control, Rat BMSCs were also genetically transduced with Ad-GFP virus at a concentration of 200 MOI.

Results: Our data demonstrated that recombinant adenoviruses carrying BMP2 or BMP7 target gene was successfully reconstructed, packaged, and confirmed via Western Blot assay, which as respected, presented as an unique band at 55 000 size for BMP2 or 49 000 size for BMP7.Adenovirus Ad-GFP was used to verify the integrity of recombinant virus and its transfection efficiency in rat BMSCs, which showed well cell attachment to culture plate and had no cytotoxicity. Green fluorescent protein in BMSCs was also noted eminently under fluorescent microscope. Combined transduction with AdBMP2 plus Ad-BMP7 resulted in the formation of BMP2/7 heterodimers from rat BMSCs. Analysis of conditioned medium via Western Blot assay showed a single protein band of about 47 000 size, just as expected. Quantitative ELISA analysis presented a prominent expression of about (4.33 ± 0.42) ng/ml for recombinant BMP2/7 heterodimers. A paired t test showed significant difference (P ＜ 0.05),when compare to control groups of (0.08 ± 0.02) ng/ml.

Conclusions: As an ideal cell source for tissue engineering, rat BMSCs can be genetically modified with Ad-BMP2 plus Ad-BMP7 mediated co-transduction strategy, and efficiently produce recombinant human BMP2/7 heterodimers in vitro, which should facilitate further studies on the beneficial effect of BMP2/7 heterodimers to ex vivo osteogenesis of BMSCs