分化的细胞可以被重新编程为胚胎干细胞样细胞,称为诱导多能干细胞(iPSCs)。其中自然发育分化过程逆转。尚不清楚在重编程期间是否可以分离和鉴定多谱系细胞。在目前的研究中,我们检测到谱系标记的表达,孤立的神经谱系,并鉴定了iPSC形成过程中的相关microRNA。我们的结果表明,当使用转录因子(TF)对小鼠胚胎成纤维细胞进行iPSC形成时,神经外胚层在形成集落之前比中胚层和定形内胚层更早出现。在第3天,细胞表达Sox1和Nestin,并且具有与向身份神经胚层谱系过渡一致的超微结构。荧光激活细胞分选分析显示在神经祖细胞标记阳性细胞中出现峰值(40%)。当随后在神经前体细胞培养基中培养时,这些细胞增殖缓慢,变得圆形和聚集,生成神经元和神经胶质。全基因组microRNA(miRNA)分析鉴定了45种差异调节的miRNA。分子网络分析证明这些miRNA验证了6,047个实验性mRNA靶标。对mRNA靶标的GO功能注释分析表明,大多数基因与神经发生有关,比如生长锥,神经元细胞体,神经元投影,和细胞连接突触。观察到蛋白质-蛋白质相互作用的网络,这表明神经谱系重编程相关靶标的关键节点是Sall1,Foxa2,Nf2,Ctnnb1,Shh,和Bmpr1a。因此,这些数据表明,TF可以通过神经外胚层驱动体细胞向多能状态重编程。此外,神经谱系重编程系统可以解决miRNAs如何影响其靶位点。
Differentiated cells can be reprogrammed to embryonic stem cell-like cells called induced pluripotent stem cells (iPSCs), in which the natural developmental differentiation process is reversed. It is unclear whether the multi-lineage cells can be isolated and identified during reprogramming. In the current study, we detected the expression of lineage markers, isolated neural lineages, and identified the related microRNAs during iPSC formation. Our results demonstrated that a
neuroectoderm appeared earlier than mesoderm and definitive endoderm before forming colonies when mouse embryonic fibroblasts were subjected to iPSC formation using transcription factors (TFs). On day 3, the cells expressed Sox1 and Nestin and had ultrastructure consistent with the transition to identity neural germ layer lineage. Fluorescence-activated cell sorting analysis revealed a peak (40%) in neural progenitor marker-positive cells. When subsequently cultured in a neural precursor cell medium, these cells proliferated slowly, became round and aggregated, generating into neurons and glia. Genome-wide microRNA (miRNA) analysis identified 45 differentially regulated miRNAs. Molecular network analysis demonstrated that these miRNAs validated 6,047 experimental mRNA targets. The GO functional annotation analysis of mRNA targets revealed that most genes were related to neurogenesis, such as growth cone, neuronal cell body, neuron projection, and cell junction synapse. The network of protein-protein interactions was observed, which demonstrated that key nodes of neural lineage reprogramming-associated targets were Sall1, Foxa2, Nf2, Ctnnb1, Shh, and Bmpr1a. Therefore, these data suggested that TFs can drive the reprogramming of somatic cells towards a pluripotent state via
neuroectoderm. Moreover, the neural lineage reprogramming system can address how miRNAs influence their target sites.