Neural differentiation of fibroblasts induced by intracellular co-delivery of Ascl1, Brn2 and FoxA1 via a non-viral vector of cationic polysaccharide.

Direct reprogramming of other somatic cells into neurons is an alternative strategy for the recovery of an injured nervous system. In this work, we developed a new non-viral gene carrier based on Porphyra yezoensis polysaccharide (PYP). After modification with ethylenediamine, the cationized PYP (Ed-PYP) was combined with plasmids encoding Ascl1, Brn2 and FoxA1 to form spherical nanoscale particles (Ed-PYP-pABF nanoparticles). Cytotoxicity assays proved that Ed-PYP-pABF nanoparticles had a better safety profile than Lipofectamine 2000 and polyetherimide. Characterization tests illustrated that the Ed-PYP-pABF nanoparticles at an Ed-PYP:pABF weight ratio of 40:1 is a potential candidate for gene delivery, which was further supported by Western blot and plasmid encoding enhanced green fluorescence protein transfection. Based on this transfection strategy, we co-delivered pABF to 3T6 cells using Ed-PYP. ELISA indicated that the levels of brain-derived neurotrophic factor, nerve growth factors and sonic hedgehog reached a maximum at 14 days after the last transfection. Immunofluorescence and Western blot further exhibited positive expression of neurofilament 200, Nestin, glial fibrillary acidic protein, growth associated protein-43, β-3tubulin, and microtubule associated protein 2, proving the successful conversion of 3T6 cells into neurons. Taken together, these results illustrated that a natural polysaccharide-based gene co-delivery system is a promising strategy for neural reprogramming.