Abstract
IntroductionA combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia.MethodsTransduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-α-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated.ResultsThe second generation lentiviral vector rHIV-pWPT-EF1-α-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression.ConclusionsCollectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies.
Highlights
A combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs)
Collectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification
At multiplicity of infection (MOI) 100, this vector gave transduction efficiencies of 90-98% at 7, 14, 21 and 28 days respectively (Figure 1A), and total green fluorescent protein (GFP) values of over 75,000 at all time points (Figure 1B). This vector consistently mediated high level, sustained GFP transgene expression levels with no significant reduction throughout the time frame analysed. This was in contrast to the rHIV-cPPT-CMV-GFP-W and rHIVPPT-phosphoglycerate kinase (PGK)-GFP-W lentiviral vectors, which were tested in parallel, and consistently gave percentage GFP positives of 1-5% and 35-40% (Figure 1A), and total GFP values of 4,552-186 and 28,866-15,806 (Figure 1B) respectively
Summary
A combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). Mesenchymal stem cells (MSCs) represent a significant area of interest in the field of cell therapy They are a multipotent, self-renewing cell population isolated from bone marrow, capable of differentiation into cells of different lineages including chondrocytes, osteoblasts and benefit of MSCs as a gene delivery system is their lack of immunogenicity, which would confer significant advantage to gene therapy techniques, as the MSC itself may prevent a vector-induced host immune response [5,6,7]. A more prolonged lifespan may be necessary for MSCs to exert a desired therapeutic effect in vivo In this respect, we hypothesize that increasing MSC survival capacity by genetic modification could afford the MSC a longer time in which to mediate repair and recovery both by direct and paracrine means, thereby enhancing their in vivo therapeutic value
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