Department of Stem Cells and Regenerative Medicine, Faculty of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
Anatomy and Cell Biology Department, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
J Cell Biochem. 2020 Jan;121(1):200-212. doi: 10.1002/jcb.29109. Epub 2019 Jul 16.
Human teratocarcinoma cell line Ntera2 (NT2) expresses dopamine signals and has shown its safe profile for clinical applications. Attempts to restore complete dopaminergic (DAergic) phenotype enabling these cells to secrete dopamine have not been fully successful so far. We applied a blend of gene transfer techniques and a defined medium to convert NT2 cells to fully DAergic. The cells were primarily engineered to overexpress the Pitx3 gene product and then cultured in a growth medium supplemented with knockout serum and retinoic acid to form embroid bodies (EBs). Trypsinization of EB colonies produced single cells ready for differentiation. Neuronal/DAergic induction was promoted by applying conditioned medium taken from engineered human astrocytomas over-secreting glial cell-derived neurotrophic factor (GDNF). Immunocytochemistry, reverse-transcription and real-time polymerase chain reaction analyses confirmed significantly induced expression of molecules involved in dopamine signaling and metabolism including tyrosine hydroxylase, Nurr1, dopamine transporter, and aromatic acid decarboxylase. High-performance liquid chromatography analysis indicated release of dopamine only from a class of fully differentiated cells expressing Pitx3 and exposed to GDNF. In addition, Pitx3 and GDNF additively promoted in vitro neuroprotection against Parkinsonian toxin. One month after transplantation to the striatum of 6-OHDA-leasioned rats, differentiated NT2 cells survived and induced significant increase in striatal volume. Besides, cell implantation improved motor coordination in Parkinson's disease (PD) rat models. Our findings highlight the importance of Pitx3-GDNF interplay in dopamine signaling and indicate that our strategy might be useful for the restoration of DAergic fate of NT2 cells to make them clinically applicable toward cell replacement therapy of PD.
人畸胎瘤细胞系 Ntera2（NT2）表达多巴胺信号，并已显示其安全的临床应用。目前，试图恢复使这些细胞能够分泌多巴胺的完整多巴胺能（DAergic）表型的尝试尚未完全成功。我们应用基因转移技术的混合物和定义的培养基将 NT2 细胞转化为完全 DAergic。这些细胞最初被设计为过表达 Pitx3 基因产物，然后在补充有敲除血清和维甲酸的生长培养基中培养以形成类胚体（EBs）。EB 菌落的胰蛋白酶消化产生准备分化的单个细胞。通过应用过表达胶质细胞衍生的神经营养因子（GDNF）的工程化人星形细胞瘤的条件培养基来促进神经元/DAergic 诱导。免疫细胞化学、逆转录和实时聚合酶链反应分析证实了参与多巴胺信号和代谢的分子的显著诱导表达，包括酪氨酸羟化酶、Nurr1、多巴胺转运体和芳香族氨基酸脱羧酶。高效液相色谱分析表明，只有表达 Pitx3 并暴露于 GDNF 的一类完全分化的细胞才会释放多巴胺。此外，Pitx3 和 GDNF 可协同促进帕金森病毒素的体外神经保护作用。将分化的 NT2 细胞移植到 6-OHDA 损伤大鼠的纹状体一个月后，细胞存活并诱导纹状体体积显著增加。此外，细胞植入改善了帕金森病（PD）大鼠模型的运动协调能力。我们的研究结果强调了 Pitx3-GDNF 相互作用在多巴胺信号中的重要性，并表明我们的策略可能对恢复 NT2 细胞的 DAergic 命运有用，以使它们可用于 PD 的细胞替代治疗。
