A novel method to differentiate rat embryonic stem cells into dopaminergic neurons in vitro

作者：Gu, Xin; Shi, Zhifeng; Hu, Jie^*

来源：International Journal of Clinical and Experimental Medicine, 2017, 10(4): 6081-6090.

• Rat embryonic stem cells (rESCs)
• embryoid bodies (EBs)
• dopaminergic (DA) neurons
• sonic hedgehog (Shh)
• fibroblast growth factors (FGF8)

Objective: To develop an efficient protocol to differentiate the rat embryonic stem cells (rESCs) into dopaminergic (DA) neurons, thus providing a new insight for the study and development of embryonic stem cells (ESCs) and cell replacement therapy. Methods: We made a "4-step" protocol which was a modified EB (embryoid body)based method, to differentiate rESCs into DA neurons via adding sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8) sequentially. The 4 steps include: 1) enrichment of rESCs; 2) EBs formation; 3) neural induction; 4) DA neurons derivation. Immunofluorescence staining and qRT-PCR were applied to identify DA neurons. Results: By our "4-step" protocol, ventral midbrain DA neurons could be successfully differentiated from rESCs within 18 days. Combined treatment of Shh and FGF8 was essential for successful induction of DA neurons. Especially, the concentration of Shh was a critical point for DA neurons generation, and 200 ng/ml was highly recommended. Conclusion: Our study demonstrates that DA neurons can be induced from rESCs with an efficient and less time-consuming method based on EBs formation with sequential addition of Shh and FGF8, which named "4-step" protocol. And this novel method is of great potential for the treatment of DA system disorders and the study of related drugs discovery as a new renewable source of functional DA neurons.

• 出版日期2017
• 单位复旦大学