Influence on the Osteogenic Activity of the Human Bone Marrow Mesenchymal Stem Cells Transfected by Liposome-Mediated Recombinant Plasmid pIRES-hBMP2-hVEGF165 In Vitro

Wang

Experimental and Therapeutic Medicine

et al. 2013

Bone marrow-derived mesenchymal stem cells (MSCs) are dominant seed cell sources for bone regeneration. Bone morphogenetic proteins (BMPs) initiate cartilage and bone formation in a sequential cascade. Vascular endothelial growth factor (VEGF) is an essential coordinator of extracellular matrix remodeling, angiogenesis and bone formation. In the present study, the effects of the vascular endothelial growth factor 165 (VEGF165) and bone morphogenetic protein 2 (BMP2) genes on bone regeneration were investigated by the lentivirus-mediated cotransfection of the two genes into rat bone marrow-derived MSCs. The successful co-expression of the two genes in the MSCs was confirmed using quantitative polymerase chain reaction (qPCR) and western blot analysis. The results of alizarin red and alkaline phosphatase (ALP) staining at 14 days subsequent to transfection showed that the area of staining in cells transfected with BMP2 alone was higher than that in cells transfected with BMP2 and VEGF165 or untransfected control cells, while the BMP2 + VEGF165 group showed significantly more staining than the untransfected control. This indicated that BMP2 alone exhibited a stronger effect in bone regeneration than BMP2 in combination with VEGF165. Similarly, in inducing culture medium, the ALP activity of the BMP2 + VEGF165 group was notably suppressed compared with that of the BMP2 group. The overexpression of VEGF165 inhibited BMP2-induced MSC differentiation and osteogenesis in vitro. Whether or not local VEGF gene therapy is likely to affect bone regeneration in vivo requires further investigation.

Section: Discussionmentioning

confidence: 99%

Effects of BMP2 and VEGF165 on the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells

Wang

Experimental and Therapeutic Medicine

et al. 2013

“…However, the expression decreased with a prolonged time after selection. Other studies also established a method for transfecting the human VEGF 165 gene into MSCs in which the target gene and protein could be expressed effectively (Guo-ping et al 2010;Tarnowski et al 2010).…”

Section: Discussionmentioning

confidence: 99%

VEGF165 expressing bone marrow mesenchymal stem cells differentiate into hepatocytes under HGF and EGF induction in vitro

Yan

Xiao

et al. 2012

Cytotechnology

A short half-life and low levels of growth factors in an injured microenvironment necessitates the sustainable delivery of growth factors and stem cells to augment the regeneration of injured tissues. Our aim was to investigate the ability of VEGF 165 expressing bone marrow mesenchymal stem cells (BMMSCs) to differentiate into hepatocytes when cultured with hepatocyte growth factor (HGF) and epidermal growth factor (EGF) in vitro. We isolated, cultured and identified rabbit BMMSCs, then electroporated the BMMSCs with VEGF 165 -pCMV6-AC-GFP plasmid. G418 was used to select transfected cells and the efficiency was up to 70%. The groups were then divided as follows: Group A was electroporated with pCMV6-AC-GFP plasmid ? HGF ? EGF and Group B was electroporated with VEGF 165 -pCMV6-AC-GFP plasmid ?HGF ? EGF. After 14 days, BMMSCs were induced into short spindle and polygonal cells. Alpha-fetoprotein (AFP) was positive and albumin (ALB) was negative in Group A, while both AFP and ALB were positive in group B on day 10. AFP and ALB in both groups were positive on day 20, but the quantity of AFP in group B decreased with prolonged time and was about 43.5% less than group A. The quantity of the ALB gene was increased with prolonged time in both groups. However, there was no significant difference between group A and B on day 10 and 20. Our results demonstrated that VEGF 165 -pCMV6-AC-GFP plasmid modified BMMSCs still had the ability to differentiate into hepatocytes. The VEGF 165 gene promoted BMMSCs to differentiate into hepatocyte-like cells under the induction of HGF and EGF, and reduced the differentiation time. These results have implications for cell therapies.

“…Gene combinations can be co-delivered as a mixture of different single expression cassette plasmids, as co-expression plasmids with multiple independent transcriptional entities (Kawai et al, 2009) and as plasmids encoding poly-cistronic mRNA with intra-ribosomal entry sites (IRES) (Gurtu et al, 1996;Guo-ping et al, 2010). Multi-cistronic plasmids allow the co-expression of multiple factors from the same plasmid molecule via bidirectional, or separated unidirectional, gene expression cassettes and enable the simultaneous expression of multiple therapeutic genes at the target site.…”

Section: Constitutive and Inducible Co-expression Systems For Non-virmentioning

confidence: 99%

Constitutive and inducible co-expression systems for non-viral osteoinductive gene therapy

Feichtinger

Hacobian²,

Hofmann³

et al. 2014

eCM

Tissue regenerative gene therapy requires expression strategies that deliver therapeutic effective amounts of transgenes. As physiological expression patterns are more complex than high-level expression of a singular therapeutic gene, we aimed at constitutive or inducible co-expression of 2 transgenes simultaneously.Co-expression of human bone morphogenetic protein 2 and 7 (BMP2/7) from constitutively expressing and doxycycline inducible plasmids was evaluated in vitro in C2C12 cells with osteocalcin reporter gene assays and standard assays for osteogenic differentiation. The constitutive systems were additionally tested in an in vivo pilot for ectopic bone formation after repeated naked DNA injection to murine muscle tissue.Inductor controlled differentiation was demonstrated in vitro for inducible co-expression. Both co-expression systems, inducible and constitutive, achieved significantly better osteogenic differentiation than single factor expression. The potency of the constitutive co-expression systems was dependent on relative expression cassette topology. In vivo, ectopic bone formation was demonstrated in 6/13 animals (46 % bone formation efficacy) at days 14 and 28 in hind limb muscles as proven by in vivo µCT and histological evaluation.In vitro findings demonstrated that the devised single vector BMP2/7 co-expression strategy mediates superior osteoinduction, can be applied in an inductor controlled fashion and that its efficiency is dependent on expression cassette topology. In vivo results indicate that co-expression of BMP2/7 applied by non-viral naked DNA gene transfer effectively mediates bone formation without the application of biomaterials, cells or recombinant growth factors, offering a promising alternative to current treatment strategies with potential for clinical translation in the future.