Hao Lili, Xu Zhen, Sun Hui, Luo Wu, Yan Youchen, Wang Jing, Guo Jingyi, Liu Yizhi, Chen Shuyi
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China.
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China; Guangdong Province Key Laboratory of Brain Function and Disease, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China.
Stem Cell Res. 2017 Aug;23:61-72. doi: 10.1016/j.scr.2017.06.012. Epub 2017 Jun 29.
Obtaining and manipulating neuronal cells are critical for neural biology basic mechanism studies and translational applications. Recent advances in protocol development and mechanism dissections have made direct induction of neuronal cells from other somatic cells (iN) a promising strategy for such purposes. In this study, we established a protocol to expand a population of fibroblast-like cells from adult human retinal tissues, which can be reprogrammed into iNs by forced expression of neurogenic transcription factors. Interestingly, the combination of Ascl1, Brn2, Myt1l, and NeuroD1 transcription factors, which has been demonstrated to be sufficient to reprogram human embryonic and dermal fibroblasts into iNs, failed to reprogram the fibroblast-like cells from human retinas into iNs. Instead, supplementing Ascl1 with Pax6 sufficed to convert the cells into iNs, which exhibited a typical neuronal morphology, expressed neural marker genes, displayed active and passive neuronal membrane activities, and made synaptic communications with other neurons. Moreover, iNs converted from retina-derived fibroblast-like cells contained high ratios of γ-Aminobutyric acid- (GABA-) and tyrosine hydroxylase- (TH-) positive neurons. Thus, the present study proposes a protocol that makes use of discarded retinal tissues from eye banks for iN generation, and suggests that different sources of somatic cells require different iN induction recipes and may also affect the iN subtype outputs. Our study may also facilitate the future development of methods to convert resident cells in situ into retinal neurons for treating retinal degeneration disease purpose.
获取和操控神经元细胞对于神经生物学基础机制研究及转化应用至关重要。方案开发和机制剖析方面的最新进展使得从其他体细胞直接诱导生成神经元细胞（诱导性神经元，iN）成为实现此类目的的一种有前景的策略。在本研究中，我们建立了一种方案，可从成人视网膜组织中扩增出一群成纤维细胞样细胞，通过强制表达神经源性转录因子，这些细胞可被重编程为诱导性神经元。有趣的是，已被证明足以将人类胚胎和成纤维细胞重编程为诱导性神经元的Ascl1、Brn2、Myt1l和NeuroD1转录因子的组合，未能将来自人类视网膜的成纤维细胞样细胞重编程为诱导性神经元。相反，用Pax6补充Ascl1足以将这些细胞转化为诱导性神经元，这些诱导性神经元呈现出典型的神经元形态，表达神经标志物基因，表现出主动和被动的神经元膜活动，并与其他神经元进行突触通讯。此外，从视网膜衍生的成纤维细胞样细胞转化而来的诱导性神经元中，γ-氨基丁酸（GABA）和酪氨酸羟化酶（TH）阳性神经元的比例很高。因此，本研究提出了一种利用眼库废弃视网膜组织生成诱导性神经元的方案，并表明不同来源的体细胞需要不同的诱导性神经元诱导方法，且可能还会影响诱导性神经元亚型的产出。我们的研究也可能会促进未来将原位驻留细胞转化为视网膜神经元以治疗视网膜退行性疾病的方法的发展。
