Overexpression of bone morphogenetic protein 3 (BMP-3) via raav-mediated gene transfer on the osteochondrogenic fate of human bone marrow-derived mesenchymal stem cells

Abstract

Purpose: Injured adult articular cartilage is not capable of reliably regenerating an original hyaline structure. To improve the intrinsic healing activities of damaged cartilage, administration of genetically modified bone marrow-derived mesenchymal stem cells (MSCs) in cartilage lesions is a potent approach to improve cartilage repair. Here, we explored the potential benefits of overexpressing the bone morphogenetic protein 3 (BMP-3) [1] via clinically relevant recombinant adeno-associated virus (rAAV) vectors upon the commitment of human MSCs (hMSCs) as future therapeutic platforms for cartilage repair. Methods: rAAV vectors were packaged, purified, and titrated as previously described [2]. rAAV-lacZ carries the E. coli β-galactosidase (lacZ) reporter gene and rAAV-hBMP-3 a human BMP-3 sequence (OriGene), both controlled by the CMV-IE promoter/enhancer [2]. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty, washed, and the pellets obtained were resuspended in DMEM, 10% FCS, 1% penicillin-streptomycin, and FGF-2 (10 ng/ml) for selection by adherence of progenitor cells and characterization of stem cell surface markers [2]. Cells (passage 2) were transduced with rAAV (rAAV-hBMP-3 or rAAV-lacZ: 40 μl each vector) or let untreated and kept either in monolayer cultures in DMEM, 10% FBS or as high-density aggregate cultures (2 x 105 cells) in chondrogenic medium (DMEM high glucose, ITS+ Premix, 1 mM pyruvate, 37.5 μg/ml ascorbate 2-phosphate, 10-7 M dexamethasone, 10 ng/ml TGF-β) for up to 21 days. Expression of BMP-3 was monitored by immunohistochemistry or by ELISA (R&D Systems) [2]. Histological analyses were performed on fixed monolayer cultures (ALP staining) or on paraffin-embedded sections of the aggregate cultures (5 μμ) (toluidine blue staining) [2]. Total RNA was extracted using the RNeasy Protect Mini kit (Qiagen) and reverse transcription was carried out with the 1st Strand cDNA Synthesis kit (Qiagen). cDNA amplification was performed via SYBR Green real-time RT-PCR [2]. Ct values were obtained for each target gene (SOX9, COL10A1) and GAPDH as a control for normalization, and fold inductions (relative to control samples) were measured using the 2-ΔΔCt method [2]. Each condition was performed in duplicate in three independent experiments. A t-test was employed with p < 0.05 considered statistically significant. Results: Successful BMP-3 overexpression was observed both in hMSC monolayer and aggregate cultures by application of rAAV-hBMP-3 relative to the control conditions (lack of vector delivery, reporter rAAV-lacZ gene transfer) over the whole period of evaluation (Fig. 1). Application of rAAV-hBMP-3 significantly increased the levels of cell proliferation in hMSC monolayer and aggregate cultures relative to the control conditions (up to 1.3- and 1.4 difference, respectively, p ≤ 0.001) (Table 1). Chondrogenic differentiation was evidenced by intense toluidine blue staining in hMSC aggregate cultures after 21 days, especially when the cells were treated with rAAV-hBMP-3 (Fig. 2). Similar results were noted regarding the osteogenic differentiation of the cells over time (Fig. 2). A real-time RT-PCR analysis revealed a reduced hypertrophic differentiation of the cells following rAAV-hBMP-3 transduction relative to the control treatments (up to 5-fold difference in COL10A1 expression, p ≤ 0.001), probably due to increased chondrogenic SOX9 gene expression levels (up to 8-fold difference, p ≤ 0.001) (Fig. 3). Conclusions: Gene transfer of BMP-3 via rAAV directs the osteochondrogenic commitment of hMSCs with a delay in hypertrophic differentiation. These Results demonstrate the possibility of modifying hMSCs by therapeutic BMP-3 gene transfer as a future, potential tool to enhance the repair processes in sites of osteochondral lesions. REFERENCES: [1] Wozney et al., Science 1988, 242:1528; [2] Venkatesan et al., Stem Cell Res Ther 2012, 3:22. ACKNOWLEDGMENTS: Work funded by the Deutsche Arthrose-Hilfe. The authors wish to thank Mary B. Goldring for helpful discussion.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)