RESUMO
The work with midbrain dopaminergic neurons (mDAN) differentiation might seem to be hard. There are about 40 different published protocols for mDAN differentiation, which are eventually modified according to the respective laboratory. In many cases, protocols are not fully described, failing to provide essential tips for researchers starting in the field. Considering that commercial kits produce low mDAN percentages (20-50%), we chose to follow a mix of four main protocols based on Kriks and colleagues' protocol, from which the resulting mDAN were engrafted with success in three different animal models of Parkinson's disease. We present a differential step-by-step methodology for generating mDAN directly from human-induced pluripotent stem cells cultured with E8 medium on Geltrex, without culture on primary mouse embryonic fibroblasts prior to mDAN differentiation, and subsequent exposure of neurons to rock inhibitor during passages for improving cell viability. The protocol described here allows obtaining mDAN with phenotypical and functional characteristics suitable for in vitro modeling, cell transplantation, and drug screening.
Assuntos
Diferenciação Celular , Neurônios Dopaminérgicos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Mesencéfalo/citologia , Animais , Biomarcadores , Cálcio/metabolismo , Sinalização do Cálcio , Técnicas de Cultura de Células , Separação Celular , Células Cultivadas , Neurônios Dopaminérgicos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mesencéfalo/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Doença de ParkinsonRESUMO
Magnetic responsive hydrogels composed of alginate (Alg) and xanthan gum (XG), crosslinked with Ca2+ ions, were modified by in situ magnetic nanoparticles (MNP) formation. In comparison to magnetic Alg hydrogels, magnetic Alg-XG hydrogels presented superior mechanical and swelling properties, due to the high charge density and molecular weight of XG. The loading efficiency of levodopa (LD), an important antiparkinson drug, in the Alg-XG/MNP hydrogels was the highest (64%), followed by Alg/MNP (56%), Alg-XG (53%) and Alg (28%). A static external magnetic field (EMF) of 0.4â¯T stimulated the release of LD from Alg-XG/MNP hydrogels achieving 64⯱â¯6% of the initial loading after 30â¯h. The viability, proliferation and expression of dopaminergic markers of human neuroblastoma SH-SY5Y cell on the LD loaded magnetic hydrogels were successful, particularly under EMF, which stimulated the release of LD. Overall, the results of this study provided the rational design of magnetic hydrogels for the delivery of drugs, which combined with external magnetic stimulus, might improve cell proliferation and specific differentiation.