Midbrain Dopaminergic Neurons Differentiated from Human-Induced Pluripotent Stem Cells
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.
Key wordsInduced pluripotent stem cells Cell reprogramming Cell differentiation Dopaminergic neurons Neural differentiation Dopaminergic differentiation Neurodegenerative disease models Parkinson’s disease In vitro modeling
LVP is grateful for grant support by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP -CEPID 13/08135-2), Conselho Nacional de Desenvolvimento Científico e Tecnológico/Departamento de Ciência e Tecnologia do Ministério da Saúde (CNPq/MS/DECIT- 24/2014), Banco Nacional de Desenvolvimento Econômico e Social (BNDES), Financiadora de Estudos e Projetos (FINEP), and 2010 Gaucher Generation grant program by Genzyme Corporation. HU is grateful for grant support by FAPESP (Project No. 2012/50880-4) and CNPq (Project No. 306429/2013-6). MFRF was awarded with research grants from FAPESP (2013/08028-1and 2015/18961-2) and CNPq (471999/2013-0 and 401670/2013-9). FTA (Project No. 2014/25487-3) is grateful for a doctorate fellowship granted by FAPESP. ATSS (Project No. 163310/2014-9), AOG (Project No. 141979/2014-3), and MCBG (Project No. 870458/1997-3) are grateful for a doctorate fellowship granted by CNPq. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001. Special thanks to Mark Tomishima and Faria Zafar for online advising during the establishment of this protocol.
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