Abstract
Ex vivo gene therapy offers enormous potential for cell-based therapies, however, cumbersome in vitro cell culture conditions have limited its use in clinical practice. We have optimized an innovative strategy for the transient transfection of bone morphogenetic protein-2 (BMP-2) expressing plasmids in suspended human stem cells within 5-min that enables efficient loading of the transfected cells into a 3D hydrogel system. Such a short incubation time for lipid-based DNA nanoparticles (lipoplexes) reduces cytotoxicity and at the same time reduces the processing time for cells to be transplanted. The encapsulated human mesenchymal stromal/stem cells (hMSCs) transfected with BMP-2 plasmid demonstrated high expression of an osteogenic transcription factor, namely RUNX2, but not the chondrogenic factor (SOX9), within the first three days. This activation was also reflected in the 7-day and 21-day experiment, which clearly indicated the induction of osteogenesis but not chondrogenesis. We believe our transient transfection method demonstrated in primary MSCs can be adapted for other therapeutic genes for different cell-based therapeutic applications.
Highlights
Gene therapy holds great promise to treat a variety of human conditions and has been clinically evaluated for treating several diseases such as cancer, cardiovascular disease and immunological conditions, as well as for developing drugs for regenerative medicine [1,2]
We have recently developed injectable hydrogels that can sequester recombinant human bone morphogenetic protein-2 and promote bone formation in vivo, mimicking the natural behavior of the extracellular matrix [4]
We have developed an optimal method for ex vivo transfection of plasmid DNA in human mesenchymal stromal/stem cells (hMSCs) followed by encapsulation in Hyaluronic acid (HA) hydrogel
Summary
Gene therapy holds great promise to treat a variety of human conditions and has been clinically evaluated for treating several diseases such as cancer, cardiovascular disease and immunological conditions, as well as for developing drugs for regenerative medicine [1,2]. We have recently developed injectable hydrogels that can sequester recombinant human bone morphogenetic protein-2 (rhBMP-2) and promote bone formation in vivo, mimicking the natural behavior of the extracellular matrix [4] Though this is very promising, delivering osteogenic genes locally at the defect site, instead of the protein, will act as a reservoir for the protein that will reduce the systemic toxicity and lower the need for supra-physiological dosages of therapeutic proteins [5]. Since cationic lipid-based reagents are known to promote cytosolic delivery of the cargo molecules within 5–15 min of endocytosis, we envisioned that a short incubation time with cells and lipoplexes could deliver the cargo molecules to the cytosol [20] For this purpose, we selected LipofectamineTM 2000 as the transfection reagent.
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