Non-Viral Delivery of the Gene for Glial Cell Line-Derived Neurotrophic Factor to Mesenchymal Stem Cells In Vitro Via a Collagen Scaffold

Abstract

Recent advances in tissue engineering that combine an extracellular matrix-like scaffold with therapeutic molecules, cells, DNA encoding therapeutic proteins, or a combination of the three hold promise for treating defects in the brain resulting from a penetrating injury or tumor resection. The purpose of this study was to investigate a porous sponge-like collagen scaffold for non-viral delivery of a plasmid encoding for glial cell line-derived neurotrophic factor (pGDNF) to rat marrow stromal stem cells (also referred to as mesenchymal stem cells, MSCs). The effects of the following parameters on GDNF synthesis in the three-dimensional (3D) constructs were evaluated and compared with results in monolayer culture: initial plasmid load (2-50 microg pGDNF), ratio of a lipid transfection reagent to plasmid (5:10), culture environment during the transfection (static and dynamic), and cell density. The level of gene expression in the collagen scaffolds achieved therapeutic levels that had previously been found to support survival of dopaminergic and trigeminal neurons in vitro. For the highest loading of plasmid (50 microg), the level of GDNF protein remained six to seven times above the control level after 2 weeks, a significant difference. Cell density in the scaffold was of importance for an early increase in GDNF production, with accumulated GDNF being approximately 60% greater after 9 days of culture when scaffolds were initially seeded with 2 million rat MSCs compared to 500,000 cells. Application of orbital shaking during the 4 h of transfection had a positive effect on the production of GDNF on 3D constructs but not of the same magnitude as reported in monolayer studies. Overall, these results demonstrate that the combination of tissue engineering and non-viral transfection of MSCs for the over-expression of GDNF is a promising approach for the long-term production of GDNF and probably for neurotrophic factors in general.

Gene delivery to human bone marrow mesenchymal stem cells by microporation

“…During the last 10 years several authors have reported promising results regarding the use of cationic lipids (Bolliet et al, 2008), polymers , dendrimers (Santos et al, 2009) and different electroporation techniques (Aluigi et al, 2006;Ferreira et al, 2008) to deliver plasmid DNA (pDNA) into MSC derived from BM, cord blood or adipose tissue. When compared to other non-viral methods, conventional electroporation has led to higher transfection efficiencies to MSC although encompassed with high cellular mortality, which might be due to the high electrode surface area (Kim et al, 2008).…”

Novel Magnetic Hydroxyapatite Nanoparticles as Non‐Viral Vectors for the Glial Cell Line‐Derived Neurotrophic Factor Gene

“…Cell Culture and Transfection: Rat MSCs were harvested from both the femurs and tibiae of 6 young Lewis rats (<6 weeks old) as previously described [62]. The cells from the 6 rats were cultured separately throughout the experiment.…”

Neural ECM mimetics

“…Collagen's inherent integrin binding sites are advantageous as they promote the migration and differentiation of neuronal cells (Bradshaw et al, 1995). Modification of collagen scaffolds via the addition of neurotrophic factors (Han et al, 2009;Houweling et al, 1998), other pharmacologically active substances (Bolliet et al, 2008), or cells ( Joosten et al, 2004) has been described to increase axonal regeneration and functional recovery after spinal cord trauma. Despite the fact that collagen per se is a suitable substrate for neuronal growth, it is also important to consider the role of collagen as the basement membrane constituent of the lesion scar that develops after a traumatic CNS injury (Fawcett et al, 2012;Klapka and Muller, 2006).…”