Adenovirus‐mediated BMP2 expression in human bone marrow stromal cells

Abstract

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.

Osteoinduction by ex vivo adenovirus-mediated BMP2 delivery is independent of cell type

“…BM-MSCs were derived from discarded human bone marrow from healthy donors as previously described. 11,12 Cells were isolated by gradient density centrifugation on Ficoll-Paquet PLUS (Amersham Pharmacia Biotech, Piscataway, NJ, USA), plated at 5 Â 10 6 cells/cm 2 in the medium above, and enzymatically collected before becoming confluent. 13 …”

“…14 A replication-defective human Ad5 with an adenovirus type 35 fiber, which also contains a cDNA for BMP2 in the E1 region (Ad5F35-BMP2), was constructed, propagated, and purified as previously described. 11,12,14 Control vectors included: Ad5-lacZ, an Ad5 vector containing a CMV promoterdriven b-galactosidase expression cassette in region E1; and Ad5F35-HM4, lacking a transgene cassette. Particle to plaque-forming unit (PFU) ratios were 12.5, 56, 20, and 562 for Ad5F35-BMP2, Ad5-BMP2, Ad5-lacZ, and Ad5F35-HM4, respectively.…”

“…11 Briefly, cells were infected at the specified multiplicity in DMEM supplemented with 2% FBS and the antibiotic-antimycotic. Virus was allowed to adsorb overnight and then the media was adjusted to 10% FBS.…”

“…Recently, we have described, a novel recombinant chimeric adenoviral BMP2 vector, 11,12 which consists of Ad5 containing a fiber gene of adenovirus type 35 (Ad5F35-BMP2). Comparison of Ad5F35-BMP2 with Ad5-BMP2 demonstrated the ability of the former to elicit significantly higher expression of BMP2 in vitro in human bone marrow MSCs (BM-MSCs).…”

“…Using nonobese diabetic severe immunodeficiency (NOD/SCID) mice, BM-MSCs transduced with Ad5F35-BMP2 demonstrated significantly greater potential to induce heterotopic bone formation than the same cells transduced with Ad5-BMP2. 11,12 We have previously demonstrated 12 by FACS analysis with a monoclonal antibody against the Coxsackie-adenovirus receptor (CAR) that human BM-MSCs are deficient in CAR that is a prerequisite for transduction with Ad5-BMP2, whereas Ad5F35-BMP2 transduces cells in a CAR-independent fashion.…”