Open AccessDifferentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells
Author(s) -
Aflah Hanafiah,
Zhuangzhuang Geng,
Qiang Wang,
Zhonghua Gao
Publication year - 2020
Publication title -
journal of visualized experiments
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.596
H-Index - 91
ISSN - 1940-087X
DOI - 10.3791/61446
Subject(s) - embryoid body , embryonic stem cell , biology , p19 cell , nestin , progenitor cell , microbiology and biotechnology , neural stem cell , cellular differentiation , neurosphere , stem cell , retinoic acid , progenitor , adult stem cell , cell culture , genetics , gene
We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.