Generation of H1 PAX6(WT/EGFP) reporter cells to purify PAX6 positive neural stem/progenitor cells
Generation of H1 PAX6(WT/EGFP) reporter cells to purify PAX6 positive neural stem/progenitor cells
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生成 H1 PAX6(WT/EGFP) 报告细胞以纯化 PAX6 阳性神经干/祖细胞
DOI：
10.1016/j.bbrc.2018.05.163
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发表时间：
2018
影响因子：
3.1
通讯作者：
Wang Linli
中科院分区：
文献类型：
--
作者：
Wu Wei;Liu Juli;Su Zhenghui;Li Zhonghao;Ma Ning;Huang Ke;Zhou Tiancheng;Wang Linli
人多能细胞（human pluripotent cells，hPSC）转化为神经细胞是未来治疗神经系统疾病的一种潜在方法。然而，这仍然受到用于从hPSC诱导神经的现有方案的效率和稳定性的限制。为了克服这一障碍，我们开发了一个报告系统，使用转录激活因子样效应核酸酶（TALEN）筛选PAX 6+神经祖细胞/干细胞。我们发现，利用TALEN同源重组技术将2A-EGFP基因敲入人胚胎干细胞H1的PAX 6外显子，可以快速、有效地建立PAX 6 WT/EGFPH 1报告细胞系。该报告细胞系经神经诱导后可分化为PAX 6和EGFP双阳性的神经祖细胞/干细胞（NPCs/NSCs）。这些PAX 6 WT/EGFPNPCs可在体外高效纯化、扩增并特异性表达于有丝分裂后神经元。利用该报告细胞系，我们还筛选出1个NPC特异性microRNA，hsa-miR-99 a-5 p，和3个ESC富集的miRNAs，hsa-miR-302 c-5 p，hsa-miR-512- 3 p和hsa-miR-518 B。基于TALEN的神经干细胞筛选系统是安全、有效的，可以帮助研究者获得足够的、纯度高的神经前体细胞用于进一步的应用。
Neural conversion from human pluripotent cells (hPSCs) is a potential therapy to neurological disease in the future. However, this is still limited by efficiency and stability of existed protocols used for neural induction from hPSCs. To overcome this obstacle, we developed a reporter system to screen PAX6+neural progenitor/stem cells using transcription activator like effector nuclease (TALEN). We found that knock-in 2 A-EGFP cassette into PAX6 exon of human embryonic stem cells H1 with TALEN-based homology recombination could establish PAX6WT/EGFPH1 reporter cell line fast and efficiently. This reporter cell line could differentiate into PAX6 and EGFP double positive neural progenitor/stem cells (NPCs/NSCs) after neural induction. Those PAX6WT/EGFPNPCs could be purified, expanded and specified to post-mitotic neuronsin vitroefficiently. With this reporter cell line, we also screened out 1 NPC-specific microRNA, hsa-miR-99a-5p, and 3 ESCs-enriched miRNAs, hsa-miR-302c-5p, hsa-miR-512–3p and hsa-miR-518 b. In conclusion, the TALEN-based neural stem cell screening system is safe and efficient and could help researcher to acquire adequate and pure neural progenitor cells for further application.