Extracellular matrix modulates non-viral gene transfer to mouse mesenchymal stem cells

Abstract

Non-viral gene delivery is severely limited by its efficiency. Previous studies aiming to improve the efficiency of non-viral gene delivery have focused on improving the vector system, while the cellular microenvironment where the cells reside is only beginning to be exploited as a means to enhance gene transfer. In this study, the effect of different densities of extracellular matrix proteins namely collagen I (C I), vitronectin (Vt), laminin (Lm), collagen IV (C IV), fibronectin (Fn) and ECM gel (ECMg), and their combinations on gene transfer to mouse mesenchymal stem cells (mMSCs) was studied. Protein coatings that resulted in well spread cells such as Fn, ECMg and C IV, resulted in 14.6, 7 and 6.1 fold increase in transgene expression respectively when compared to uncoated surfaces. The transgene expression was up to 90% inhibited on C I coated surface, which led to less spread cells. Interestingly, the same trend was not observed for polyplex internalization, where protein coats that resulted in less spread cells, such as C I and Vt, resulted in higher polyplex internalization. Subsequently, decreased transgene expression corresponded with inhibited trafficking of the internalized complexes to the nucleus. The effect of combining multiple ECM proteins on non-viral gene transfer was also investigated. Surfaces coated with combinations including C I resulted in inhibition of transgene expression. Furthermore surfaces coated with combination of Vt, C IV and Lm resulted in a statistically similar enhancement in transgene expression as compared to fibronectin. For all ECM combinations analyzed, the extent of cell spreading mediated by the ECM protein had a 70% correlation with the extent of overall gene transfer observed. We believe that the cellular microenvironment can be engineered to promote efficient gene transfer.

Nanotopography-mediated Reverse Uptake for siRNA Delivery into Neural Stem Cells to Enhance Neuronal Differentiation

“…The nanoparticles were assembled on the surface by centrifuging the glass substrates in a solution of positively-charged SiNPs32. This size-dependent study is critical because the nanotopographical features of the extracellular microenvironment have been shown to affect the adhesion and growth of stem cells, which in turn can influence the substrate-mediated delivery of genetic materials into stem cells25263334. For our initial studies, siRNA targeting GFP was selected to assess the efficiency of siRNA delivery and GFP knockdown in the NSCs.…”

“…ECM proteins such as laminin, fibronectin and collagen pre-adsorbed on surfaces have been previously implicated in enhancing gene delivery through endocytosis333738. Gene delivery in such systems depended primarily upon caveolae- than clathrin-mediated endocytosis37.…”

Transfection in the third dimension

“…0.5 μg DNA was added as bolus per 20,000 cells per well, as this was the same DNA amount used in our previous studies where we had shown that cell area, intracellular trafficking, endocytosis pathways and cytoskeletal dynamics modulate gene transfer in cells plated on ECM proteins in 2-D 4, 16 . In this study we wanted to know if the mechanisms of gene transfer we had observed for cells seeded in 2-D on ECM proteins, was same for cells seeded in 2-D on TCPS and in 3-D in HA hydrogels.…”

“…In addition to the type of polymer used in the vector system 26, 27 , the cell and its microenvironment also modulate gene transfer efficiency. In relation to the cell microenvironment, gene transfer efficiency has been shown to be dependent on the constitution of the matrix 4, 14 , stiffness of the matrix 5 and presentation of ligands in cellular microenvironment. In relation to the cell itself, cell proliferation, cell area, internalization, intracellular trafficking and integrin expression modulate gene transfer efficiency.…”

“…We decided to compare transfection for cells seeded on the traditional polystyrene surface used for transfection compared to cells seeded inside hydrogel scaffolds. Pathways for internalization of non-viral gene delivery systems depend on the type of polymer as vector system 36 , cell type 37 , size of the particle 38 , particle design/shape 39 and composition of the extra-cellular matrix 4, 16 . On studying the role of endocytic pathways on gene transfer, our results indicated that the internalization pathways are modulated by dimensionality of cell culture system.…”