Human induced pluripotent stem cells (iPSC) have opened new avenues for regenerative medicine. Consequently, iPSC-derived motor neurons have emerged as potentially viable therapies for spinal cord injuries and neurodegenerative disorders including Amyotrophic Lateral Sclerosis. However, direct clinical application of iPSC bears in itself the risk of tumorigenesis and other unforeseeable genetic or epigenetic abnormalities. Employing RNA-seq technology, we identified and characterized gene regulatory networks triggered by in vitro chemical reprogramming of iPSC into cells with the molecular features of motor neurons (MNs) whose function in vivo is to innervate effector organs. We present meta-transcriptome signatures of 5 cell types: iPSCs, neural stem cells, motor neuron progenitors, early motor neurons, and mature motor neurons. In strict response to the chemical stimuli, along the MN differentiation axis we observed temporal downregulation of tumor growth factor-β signaling pathway and consistent activation of sonic hedgehog, Wnt/β-catenin, and Notch signaling. Together with gene networks defining neuronal differentiation (neurogenin 2, microtubule-associated protein 2, Pax6, and neuropilin-1), we observed steady accumulation of motor neuron-specific regulatory genes, including Islet-1 and homeobox protein HB9. Interestingly, transcriptome profiling of the differentiation process showed that Ca2+ signaling through cAMP and LPC was downregulated during the conversion of the iPSC to neural stem cells and key regulatory gene activity of the pathway remained inhibited until later stages of motor neuron formation. Pathways shaping the neuronal development and function were well-represented in the early motor neuron cells including, neuroactive ligand-receptor interactions, axon guidance, and the cholinergic synapse formation. A notable hallmark of our in vitro motor neuron maturation in monoculture was the activation of genes encoding G-coupled muscarinic acetylcholine receptors and downregulation of the ionotropic nicotinic acetylcholine receptors expression. We observed the formation of functional neuronal networks as spontaneous oscillations in the extracellular action potentials recorded on multi-electrode array chip after 20 days of differentiation. Detailed transcriptome profile of each developmental step from iPSC to motor neuron driven by chemical induction provides the guidelines to novel therapeutic approaches in the re-construction efforts of muscle innervation.

中文翻译：

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体外 iPSC 到运动神经元分化发育原理的全局转录组谱

人类诱导多能干细胞 (iPSC) 为再生医学开辟了新途径。因此，iPSC 衍生的运动神经元已成为脊髓损伤和神经退行性疾病（包括肌萎缩侧索硬化症）的潜在可行疗法。然而，iPSC 的直接临床应用本身就存在肿瘤发生和其他不可预见的遗传或表观遗传异常的风险。利用 RNA-seq 技术，我们鉴定并表征了由 iPSC 体外化学重编程为具有运动神经元 (MNs) 分子特征的细胞触发的基因调控网络，其体内功能是支配效应器官。我们展示了 5 种细胞类型的元转录组特征：iPSC、神经干细胞、运动神经元祖细胞、早期运动神经元和成熟运动神经元。严格响应化学刺激，沿着 MN 分化轴，我们观察到肿瘤生长因子-β 信号通路的时间下调和声波刺猬蛋白、Wnt/β-连环蛋白和 Notch 信号通路的一致激活。连同定义神经元分化的基因网络（神经元2、微管相关蛋白2、Pax6 和neuropilin-1），我们观察到运动神经元特异性调节基因的稳定积累，包括胰岛1 和同源框蛋白HB9。有趣的是，分化过程的转录组分析表明，在 iPSC 转化为神经干细胞期间，通过 cAMP 和 LPC 的 Ca2+ 信号传导被下调，并且该途径的关键调控基因活性一直受到抑制，直到运动神经元形成的后期阶段。塑造神经元发育和功能的途径在早期运动神经元细胞中得到了很好的体现，包括神经活性配体-受体相互作用、轴突引导和胆碱能突触形成。我们在单一培养中体外运动神经元成熟的一个显着标志是编码 G 偶联毒蕈碱乙酰胆碱受体的基因的激活和离子型烟碱乙酰胆碱受体表达的下调。我们观察到功能性神经元网络的形成，作为在分化 20 天后记录在多电极阵列芯片上的细胞外动作电位的自发振荡。