Tofoli Fabiano Araújo, Semeano Ana Teresa Silva, Oliveira-Giacomelli Ágatha, Gonçalves Maria Carolina Bittencourt, Ferrari Merari F R, Veiga Pereira Lygia, Ulrich Henning
Institute of Biosciences, University of São Paulo, São Paulo, Brazil.
Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil.
Methods Mol Biol. 2019;1919:97-118. doi: 10.1007/978-1-4939-9007-8_8.
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.
中脑多巴胺能神经元（mDAN）分化的研究工作似乎颇具难度。目前已发表了约40种不同的mDAN分化方案，最终会根据各个实验室的情况进行修改。在许多情况下，方案并未得到充分描述，未能为该领域的新手研究人员提供关键提示。鉴于商业试剂盒产生的mDAN比例较低（20%-50%），我们选择基于克里克斯及其同事的方案，综合四种主要方案，由此产生的mDAN已成功移植到三种不同的帕金森病动物模型中。我们提出了一种差异化的分步方法，可直接从在Geltrex上用E8培养基培养的人诱导多能干细胞中生成mDAN，在mDAN分化之前无需在原代小鼠胚胎成纤维细胞上培养，并且在传代过程中让神经元接触ROCK抑制剂以提高细胞活力。此处描述的方案能够获得具有适合体外建模、细胞移植和药物筛选的表型和功能特征的mDAN。
