Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.
Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.
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DOI：
10.7554/elife.31922
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发表时间：
2018-05-01
期刊：
影响因子：
7.7
通讯作者：
Yanik MF
中科院分区：
文献类型：
--
作者：
Azimi SM;Sheridan SD;Ghannad-Rezaie M;Eimon PM;Yanik MF
鉴定最佳转录因子表达模式以引导细胞沿所需途径分化沿着存在重大挑战。我们展示了使用动态可重构的磁性引导点样技术定位mRNA，对随时间变化的mRNA转录因子进行大规模组合筛选，以指导神经祖细胞的分化，从而实现毫米大小斑点的实验。此外，我们提出了一种时间交错的交付方法，大大减少了每个细胞交付的转录因子拷贝数的波动。我们筛选组合和时间交付的一个池的中脑特异性转录因子，以增加多巴胺能神经元的产生。我们表明，LMX 1A，FOXA 2和PITX 3的组合递送在从中脑祖细胞产生多巴胺能神经元中是非常有效的。我们发现，LMX 1A显着增加TH-表达水平时，无论是在增殖过程中或诱导神经分化后，神经祖细胞，而FOXA 2和PITX 3增加表达时，只有在诱导前交付，证明时间依赖性的因素添加。
Identification of optimal transcription factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription factor copy numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH-expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition.