Overexpression of GDF5 through an Adenovirus Vector Stimulates Osteogenesis of Human Mesenchymal Stem Cells in vitro and in vivo

Abstract

The use of stem cells combined with gene therapy could be an important way to facilitate bone regeneration. In this study, the aim was to investigate the potential of growth and differentiation factor-5 (GDF5) to genetically manipulate human mesenchymal stem cells (hMSCs) for bone regeneration. Recombinant adenovirus Ad-GDF5 and Ad-GFP were constructed and identified, and the titer of both were determined. Third-passage hMSCs were infected with adenovirus, and the expression of GDF5 was confirmed by detection of GFP-positive cells, GDF5 mRNA levels, Western blotting, and enzyme-linked immunosorbent assay (ELISA). hMSCs at passage 3 were divided into four groups: (1) an experimental group infected with Ad-GDF5, (2) a positive control group cultured with osteogenic differentiation medium, (3) a control group infected with Ad-GFP cultured with standard medium, and (4) a blank control group cultured with standard medium. Evaluation of cell morphology and proliferation, analysis of the expression of genes related to osteogenic differentiation, von Kossa staining, and immunofluorescent staining of collagen I were used to investigate the osteogenesis of cells among the groups. After culturing the cells for 2 days under each corresponding condition, the cells were detached and subcutaneously injected into the backs of nude mice to evaluate bone formation. Samples were collected for histological staining, protein Western blotting, and micro-computer tomography. When infected with Ad-GDF5, hMSCs could overexpress GDF5 for a prolonged period in vitro and reach a concentration of 160 ng/ml. Cells infected with Ad-GDF5 or cultured in osteogenic medium displayed osteogenic differentiation based on their histological and cellular properties and on their gene and protein expression patterns. Furthermore, Ad-GDF5 showed a better ability to upregulate the expression of collagen I, alkaline phosphatase, and osteocalcin mRNA than the osteogenic medium. Furthermore, Ad-GDF5 expression was associated with enhanced bone formation in vivo. Our findings suggest that hMSCs infected with Ad-GDF5 can differentiate in an osteogenic direction and may be a promising cell source for bone regeneration.

Fabrication of a Cu/Zn co-incorporated calcium phosphate scaffold-derived GDF-5 sustained release system with enhanced angiogenesis and osteogenesis properties

“…GDF-5 was proved to stimulate osteogenic differentiation by enhancing both ALP activity and mineral formation in vitro. 18,51 Also, relative study showed that the gene expressions of Runx2 and OCN were enhanced by GDF-5 addition in a dosedependent manner. 52 Our results are in accordance with these studies, indicating that GDF-5 may have a positive role in osteogenesis.…”

CpG methylation regulates allelic expression of GDF5 by modulating binding of SP1 and SP3 repressor proteins to the osteoarthritis susceptibility SNP rs143383

“…Mice with targeted overexpression of GDF5 in cartilage have a reduced chondrocyte proliferative zone and expanded pre-hypertrophic and hypertrophic zones of the growth plate with increased expression of differentiation markers Ihh and Col10a1 (Tsumaki et al 1999). Overexpression of human GDF5 in human mesenchymal stem cells enhances chondrocyte differentiation and hypertrophy (Coleman et al 2013), induces tenogenic differentiation (Tan et al 2012) and stimulates osteogenesis and bone formation (Cheng et al 2012). GDF5 plays an important role in postnatal joint homeostasis and repair (Luyten 1997), and injection of recombinant human GDF5 into the nucleus pulposus of rabbits after anular puncture of the intervertebral discs is reported to have a reparative effect Chujo et al 2006).…”

Gene-modified mesenchymal stromal cells: A VIP experience

“…Cell Source Transplantation Therapeutic Gene Animal Model Ryu CH et al 64) Bone marrow-derived MSCs (human) Xenogenic IFN-β EAE Cobo M et al 60) Adipose-derived MSCs (mice) Allogenic VIP EAE Payne NL et al 65) Adipose-derived MSCs (human) Xenogenic IL-10 EAE Payne NL et al 66) Adipose-derived MSCs (human) Xenogenic IL-4 EAE Mohajeri M et al 67) Bone marrow-derived MSCs (human) Xenogenic FOXP3 EAE Lu Z et al 68) Unknown (human) Xenogenic CNTF EAE Table 1 Gene-Cell Therapy approaches for the treatment of experimental autoimmune encephalomyelitis (EAE) using GM-MSCs tion myocardial repair 41,42) , cartilage repair [43][44][45] and osteogenesis 46) .…”