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转录因子对人类胚胎干细胞的编程生成了功能性神经元,这些神经元表达上皮层和深层皮层标志物。

Transcription factor programming of human ES cells generates functional neurons expressing both upper and deep layer cortical markers.

机构信息

Laboratory of Stem Cells and Restorative Neurology, University Hospital, Lund, Sweden.

Lund Stem Cell Center, Lund University, Lund, Sweden.

出版信息

PLoS One. 2018 Oct 11;13(10):e0204688. doi: 10.1371/journal.pone.0204688. eCollection 2018.

Abstract

Human neurodegenerative disorders affect specific types of cortical neurons. Efficient protocols for the generation of such neurons for cell replacement, disease modeling and drug screening are highly warranted. Current methods for the production of cortical neurons from human embryonic stem (ES) cells are often time-consuming and inefficient, and the functional properties of the generated cells have been incompletely characterized. Here we have used transcription factor (TF) programming with the aim to induce rapid differentiation of human ES cells to layer-specific cortical neurons (hES-iNs). Three different combinations of TFs, NEUROGENIN 2 (NGN2) only, NGN2 plus Forebrain Embryonic Zinc Finger-Like Protein 2 (FEZF2), and NGN2 plus Special AT-Rich Sequence-Binding Protein 2 (SATB2), were delivered to human ES cells by lentiviral vectors. We observed only subtle differences between the TF combinations, which all gave rise to the formation of pyramidal-shaped cells, morphologically resembling adult human cortical neurons expressing cortical projection neuron (PN) markers and with mature electrophysiological properties. Using ex vivo transplantation to human organotypic cultures, we found that the hES-iNs could integrate into adult human cortical networks. We obtained no evidence that the hES-iNs had acquired a distinct cortical layer phenotype. Instead, our single-cell data showed that the hES-iNs, similar to fetal human cortical neurons, expressed both upper and deep layer cortical neuronal markers. Taken together, our findings provide evidence that TF programming can direct human ES cells towards cortical neurons but that the generated cells are transcriptionally profiled to generate both upper and deep layer cortical neurons. Therefore, most likely additional cues will be needed if these cells should adopt a specific cortical layer and area identity.

摘要

人类神经退行性疾病影响特定类型的皮质神经元。非常需要有效的方案来生成这些神经元,用于细胞替代、疾病建模和药物筛选。目前从人类胚胎干细胞 (ES 细胞) 生成皮质神经元的方法往往耗时且效率低下,并且生成细胞的功能特性尚未完全表征。在这里,我们使用转录因子 (TF) 编程,旨在快速诱导人类 ES 细胞分化为具有层特异性的皮质神经元 (hES-iNs)。我们使用了三种不同的 TF 组合,仅使用神经生成素 2 (NGN2)、NGN2 加前脑胚胎锌指样蛋白 2 (FEZF2) 以及 NGN2 加特殊富含 AT 的序列结合蛋白 2 (SATB2),通过慢病毒载体递送至人类 ES 细胞。我们仅观察到 TF 组合之间的细微差异,所有这些组合都导致了金字塔形细胞的形成,这些细胞在形态上类似于表达皮质投射神经元 (PN) 标志物并具有成熟电生理特性的成年人类皮质神经元。通过体外移植到人类器官型培养物中,我们发现 hES-iNs 可以整合到成人人类皮质网络中。我们没有发现证据表明 hES-iNs 获得了明显的皮质层表型。相反,我们的单细胞数据表明,hES-iNs 与胎儿人类皮质神经元相似,表达了上皮质和深皮质神经元标志物。总之,我们的研究结果提供了证据,表明 TF 编程可以指导人类 ES 细胞向皮质神经元分化,但生成的细胞在转录水平上呈现出产生上皮质和深皮质神经元的特征。因此,如果这些细胞要采用特定的皮质层和区域身份,很可能需要其他线索。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/58a8/6181302/dc14f4ac81a9/pone.0204688.g001.jpg

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