Alternative routes to induced pluripotent stem cells revealed by reprogramming of neural lineage

During the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells, the activation of pluripotency genes such as Nanog occurs after the mesenchymal to epithelial transition (MET). Here we report that both adult stem cells (neural stem cells- NSCs) and differentiated cells (astrocytes) of the neural lineage can activate Nanog in the absence of cadherin expression during reprogramming. Gene expression analysis revealed that only the Nanog+Ecadherin+ populations expressed stabilization markers and had upregulated several cell cycle genes; and were transgene independent. Inhibition of Dot1L activity, which has previously been shown to increase MET, enhanced both the numbers of Nanog+ and Nanog+E-cadherin+ colonies, suggesting a MET independent pathway for activation of Nanog in NSCs. Expressing Sox2 in MEFs prior to reprogramming does not alter the ratio of Nanog colonies that express E-cadherin obtained. Taken together these results provide a novel pathway for reprogramming taken by cells of the neural lineage. Overall design: Neural Stem Cells (NSCs) were induced to reprogram by the induction of Oct4, Sox2, c-Myc and Klf4. Reprogramming colonies that expressed either Nanog alone (N+E-) or Nanog and E-cadherin (N+E+) were sorted by flow cytometry and used as input for RNA-sequencing. There are 3 replicates each for the N+E- and N+E+ samples.

在将小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）重编程为诱导多能干细胞（induced pluripotent stem cells）的过程中，诸如Nanog这类多能性基因的激活发生于间质上皮转化（mesenchymal to epithelial transition, MET）之后。本研究发现，神经谱系来源的成体干细胞（神经干细胞，NSCs）以及分化细胞星形胶质细胞（astrocytes），均可在重编程过程中不表达钙粘蛋白（cadherin）的情况下激活Nanog。基因表达分析结果显示，仅Nanog阳性且E-钙粘蛋白阳性（Nanog+E-cadherin+）的细胞群体表达稳定化标志物，并上调了多种细胞周期相关基因，且该群体不依赖外源转基因。此前已有研究证实，抑制Dot1L的活性可增强间质上皮转化，本研究发现该处理同时增加了Nanog阳性以及Nanog阳性且E-钙粘蛋白阳性的细胞集落数量，这提示在神经干细胞中，Nanog的激活存在一条不依赖间质上皮转化的通路。在重编程前于小鼠胚胎成纤维细胞中过表达Sox2，并不会改变所获得的表达E-钙粘蛋白的Nanog阳性集落的比例。综上，本研究结果为神经谱系细胞的重编程提供了一条全新的通路。实验整体设计：通过诱导Oct4、Sox2、c-Myc及Klf4的表达，对神经干细胞（NSCs）进行重编程。利用流式细胞术分选仅表达Nanog（N+E-）或同时表达Nanog与E-钙粘蛋白（N+E+）的重编程集落，将其作为RNA测序（RNA-sequencing）的输入样本。其中N+E-与N+E+样本各设置3次生物学重复。