Transcriptome profiling of microRNA-induced neurons, microRNA-induced motor neurons, and starting fibroblasts [array]

Neuronal microRNAs miR-9/9* and miR-124 (miR-9/9*-124) direct cell-fate conversion of adult human fibroblasts to post-mitotic neurons and work in concert with additional transcription factors to enable the generation of discrete neuronal subtypes. Previously, the molecular events underlying the neurogenic switch mediated by microRNAs during neuronal reprogramming were unknown. Here, we systematically dissected the neurogenic state induced by miR-9/9*-124 alone. We found that miR-9/9*-124 surprisingly stimulate reconfiguration of chromatin accessibility, DNA methylation and mRNA levels, leading to the generation of functionally excitable neurons that are not yet biased towards a particular subtype-lineage. Further subtype identity can be programmed through additional transcription factors. As such, we show ISL1 and LHX3 selectively commit conversion to a highly homogenous population of human spinal cord motor neurons. Taken together, our study reveals a modular synergism between microRNAs and transcription factors that allows lineage-specific neuronal reprogramming, providing a platform for generating distinct subtypes of human neurons. Gene expression was examined in starting human fibroblasts, microRNA-induced neurons (miNs), and microRNA-induced motor neurons (moto-miNs) after 35 days of miR-9/9*-124 and transcription factor expression, in duplicate.

神经元微RNA（microRNA）miR-9/9*与miR-124（合称miR-9/9*-124）可介导成体人类成纤维细胞向终末有丝分裂后神经元的细胞命运转换，并可与其他转录因子协同发挥作用，促成多种特异性神经元亚型的生成。此前，神经元重编程过程中由微RNA介导的神经源性转换背后的分子机制尚不明晰。本研究系统解析了仅由miR-9/9*-124诱导的神经源性细胞状态。研究发现，miR-9/9*-124可意外触发染色质开放性、DNA甲基化及mRNA表达水平的重编程，最终生成具备功能兴奋性但尚未偏向特定亚型谱系的神经元。后续可通过引入其他转录因子，对这类神经元进行进一步的亚型身份编程。据此，本研究证实ISL1与LHX3可选择性地将重编程过程导向高度均质化的人类脊髓运动神经元群体。综上，本研究揭示了微RNA与转录因子间的模块化协同作用机制，该机制可实现谱系特异性的神经元重编程，为获取不同亚型的人类神经元提供了研究平台。本研究对三组样本开展了基因表达检测：初始人类成纤维细胞、微RNA诱导神经元（miNs），以及经miR-9/9*-124与转录因子诱导培养35天后的微RNA诱导运动神经元（moto-miNs），每组设置两份生物学重复。