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

Abstract

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.

Materials for Neural Differentiation, Trans‐Differentiation, and Modeling of Neurological Disease

“…Using non‐viral vectors can greatly reduce cytotoxicity during transfection and improve cell survival rate and the trans‐differentiation efficiency. Lipid reagents and polysaccharides are widely used in non‐viral transfection. At the same time, the nanoscale matrix can promote cell proliferation, viability and trans‐differentiation efficiency .…”

“…Cationic polymers are valid non‐viral gene carriers that are substituted for viral vectors, which can rapidly bind to foreign DNA and sustain low cell toxicity. Polysaccharides with great water solubility and biocompatibility are another alternative gene replacement . In the latest research, Yu et al synthesized a spherical nanoscale particle as a non‐viral gene carrier, which was combined with cationized Porphyra yezoensis polysaccharide (Ed‐PYP) and plasmids that encoded the specific transcription factors, including Ascl1, Brn2, and FoxA1 (pABF), through electrostatic interaction at different ratio of 20:1, 40:1, 80:1.…”

“…It was found that the suitable weight ratio of Ed‐PYP/pABF (40:1) had the best transfection efficiency, and cell viability was maintained at ≈100% at the suitable ratio. However, when the weight ratio of Ed‐PYP/pABF increased to 150:1, there was a gradual decrease in cell survival …”

“…The safety and efficiency of trans‐differentiation are a great challenge. It is reported that developing non‐viral vectors greatly reduces the risk of trans‐differentiation and increases cell survival . Nanoscale matrices of cell culture substrates can promote the proliferation and differentiation of nerve cells .…”

FOXA1 transcription activates TFF1 to reduce 6‑OHDA‑induced dopaminergic neuron damage

“…It is also widely expressed in the hippocampus, the region where the formation of new neurons occurs in the brain ( 14 ). A previous study has shown that FOXA1 can control the differentiation of midbrain DA neurons by regulating the expression of genes that are pivotal for neural differentiation ( 15 ). Furthermore, it was found that FOXA1 expression was significantly reduced in the serum samples of 30 Indian patients with PD ( 16 ).…”