Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.
Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.
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DOI：
10.1038/nature18323
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发表时间：
2016-06-16
期刊：
影响因子：
64.8
通讯作者：
Quake SR
中科院分区：
文献类型：
--
作者：
Treutlein B;Lee QY;Camp JG;Mall M;Koh W;Shariati SA;Sim S;Neff NF;Skotheim JM;Wernig M;Quake SR
直接谱系重编程代表了细胞和转录组状态的显著转换。然而，单个细胞进展的中间产物在很大程度上是不确定的。在这里，我们在多个时间点使用单细胞RNA-seq来解剖从小鼠胚胎成纤维细胞（mef）到诱导神经元（iN）细胞的直接重编程。通过解构每个时间点的异质性并根据转录组相似性对细胞进行排序，我们发现分子重编程路径是显著连续的。前神经先驱因子Ascl1的过表达导致明确的初始化，导致细胞退出细胞周期，并通过不同的神经转录因子重新聚焦基因表达。最初的转录反应在成纤维细胞中是相对均匀的，这表明早期的步骤并不限制多产的重编程。相反，随着时间的推移，后来出现的竞争性肌生成程序和可变的转基因动力学似乎是直接重编程的主要效率限制。此外，不同于供体和靶细胞程序的转录状态在细胞进行生产性重编程时是短暂诱导的。我们的数据为理解谱系分化过程中的转录组状态提供了一种高分辨率的方法。
Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediates through which individual cells progress are largely undefined. Here we used single-cell RNA-seq at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts (MEFs) to induced neuronal (iN) cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts suggesting the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.