Dissertação para obtenção do Grau de Mestre em
BiotecnologiaThe brain derived neurotrophic factor holds neuroprotective and neurogenic roles. The presence of this factor is believed to present highly beneficial effects on injured cells within several neurological disorders, where the levels of this neurotrophin are usually drastically decreased. Neural stem cells are multipotent, self-renewing cells with the ability to differentiate into the three main type of cells within the central nervous system - neurons, astrocytes and oligodendrocytes. The combination of gene therapy, through the introduction of therapeutic genes into the desired cells, with cell therapy aiming for the replacement of damaged cells within a given disorder, hold great promise in modern regenerative medicine.
The aim of this work was the overexpression of the brain derived neurotrophic factor in neural stem cells. For this, mouse and human neural stem cells were transfected with one of three non-viral techniques - microporation, lipid and cationic-polymer based strategies. Human NSC were efficiently transfected using the commercial lipid-based transfection reagent Lipofectamine 2000 with an efficiency of 35%, maintaining their differentiation potential. Cells within the differentiation process were efficiently transfected with 13% efficiency. Cell viability has always remained above 70% after the lipofection process. The transfection with the BDNF gene resulted in neurons with longer primary neurites, and more secondary neurites than control cells, which hints at the promotion of neurite outgrowth and ramification of neurons by this neurotrophin.
Finally, healthy cells were exposed to toxic concentrations of glutamate. Conditioned media containing secreted BDNF from transfected cells was able to protect these cells from glutamate-induced neurotoxicity, as well as reducing the levels of expression of the pro-apoptotic protein caspase7 to near-control levels.
Overall, this work provides the first evidences of the successful use of BDNF-overexpressing NSC, based on a non-viral gene delivery approach for decreasing neurotoxicity
