Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.
Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.
复制标题
DOI：
10.1016/j.nano.2015.11.015
复制
发表时间：
2016-02
期刊：
影响因子：
--
通讯作者：
Lee LJ
中科院分区：
文献类型：
--
作者：
Gallego-Perez D;Otero JJ;Czeisler C;Ma J;Ortiz C;Gygli P;Catacutan FP;Gokozan HN;Cowgill A;Sherwood T;Ghatak S;Malkoc V;Zhao X;Liao WC;Gnyawali S;Wang X;Adler AF;Leong K;Wulff B;Wilgus TA;Askwith C;Khanna S;Rink C;Sen CK;Lee LJ
安全性问题和/或电流转导方法的随机性阻碍了核重编程的临床转化。我们报告了一种新的非病毒纳米技术为基础的平台，允许确定性的大规模转染与单细胞分辨率。我们的技术的上级能力通过使用转录因子Brn 2、Ascl 1和Myt 11（BAM）的过表达对已建立的直接神经元重编程范例进行修改来证明。重编程效率与病毒方法（高达~9-12%）相当，没有衣壳大小的限制，并且具有控制质粒剂量的能力，此外还显示出相对于现有非病毒方法的上级性能。此外，增加的神经元复杂性可以通过改变BAM比率和通过在BAM混合物中包括额外的原神经基因来定制。此外，高通量NEP允许容易地询问重编程过程。我们发现BAM介导的重编程受AsclI剂量、S期细胞周期蛋白CCNA 2的调节，并且一些诱导的神经元通过巢蛋白阳性细胞阶段。将小鼠胚胎成纤维细胞加载到垂直/3D Nep装置上，在那里它们与纳米通道接触。通过纳米通道电穿孔将Brn 2a、Mytl 1和Ascl 1的混合物转染到细胞中。虽然这实现了确定性质粒递送，但存在诱导神经元产生的几个随机障碍。重编程需要在转染过程的早期表达CCNA 2。此外，在重编程过程中的几个不对称分裂导致重编程因子的数量减少。最后，至少一些诱导的神经元在终末分化成神经元之前经历巢蛋白阳性祖细胞阶段。
Safety concerns and/or the stochastic nature of current transduction approaches have hampered clinical translation of nuclear reprogramming. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9–12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage. Mouse embryonic fibroblasts are loaded onto the vertical/3D Nep device where they come in contact with the nanochannels. A cocktail of Brn2a, Mytl1, and Ascl1 is transfected into the cells by nanochannel electroporation. Although this achieves deterministic plasmid delivery, several stochastic barriers to induced neuron production exist. Reprogramming requires CCNA2 expression early during the transfection process. Furthermore, several assymetric divisions during the reprogramming process result in decreased quantities of reprogramming factors. Finally, at least some induced neurons proceed through a Nestin-positive progenitor cell stage prior to becoming terminal differentiation into a neuron.