DERIVATION OF FUNCTIONAL SPINAL CORD NEURONS FROM INDUCED PLURIPOTENT STEM CELLS (iPSCs)

Induced pluripotent stem cells (iPSCs) are a class of pluripotent stem cells which are derived from somatic cells by reprogramming in the laboratory conditions. Although induced pluripotent stem cells (iPSCs) are promising cell sources for cell therapy, ethical and physiological problems of using iPSCs have not been solved yet. Therefore, the identification of alternative and safe progenitor cells prior to terminally differentiated cells is important to control differentiation capacity in cellular therapy. Motor neuron diseases are fatal, chronic, and progressive diseases that occur by destroying motor neurons. Here, we demonstrated the generation of functional motor neurons from iPSC derived NMPs using an ex vivo differentiation protocol. Spinal cord motor neurons were characterized by morphological analysis, ICC and qPCR. We examined the function of motor neurons by measurement of the calcium influx. Calcium is the important ion to transmit signals that are detected by Fluo-4 staining. Additionally, the main motor neuron function is the establishment of neuromuscular junctions. iPSCs derived SC neurons and myogenic cells were cocultured and neuromuscular junction formation was characterized. Protein profile of cultured neurons showed that hierarchical cluster analysis of day 35 neurons and day 15 neurons are more closely. Additionally, the expression of genes found in nervous system was increased at day 10, day 15 and day 35 spinal cord neurons. The outputs can be constituted as preclinical data for the treatment of the diseases caused by neuron damages including spinal cord injury and motor neuron diseases.

诱导多能干细胞（induced pluripotent stem cells，iPSCs）是一类多能干细胞，可在实验室条件下通过重编程技术由体细胞诱导获得。尽管诱导多能干细胞（iPSCs）作为细胞治疗领域极具应用前景的细胞来源，但其应用所涉及的伦理与生理学问题尚未得到解决。因此，在终末分化细胞之前筛选出替代性安全祖细胞，对于调控细胞治疗中的细胞分化能力具有重要意义。
运动神经元病是一类以运动神经元损毁为特征的致死性、慢性进行性疾病。本研究采用体外分化方案，成功由诱导多能干细胞来源的NMPs诱导获得了具有功能的运动神经元。通过形态学分析、免疫细胞化学（immunocytochemistry，ICC）与实时定量聚合酶链反应（quantitative polymerase chain reaction，qPCR）对脊髓运动神经元进行了鉴定。
我们通过钙流入检测评估了运动神经元的功能：钙是传递信号的关键离子，可通过Fluo-4染色进行检测。此外，运动神经元的核心功能之一是构建神经肌肉接头。将诱导多能干细胞来源的脊髓运动神经元与肌源性细胞共培养，对神经肌肉接头的形成进行了表征分析。
培养神经元的蛋白质谱分析结果显示，第35天神经元与第15天神经元的层级聚类结果更为相近。此外，在第10、15和35天的脊髓运动神经元中，神经系统相关基因的表达水平均显著上调。
本研究所得结果可作为包括脊髓损伤与运动神经元病在内的神经元损伤相关性疾病治疗的临床前数据。