Enrichment of human embryonic stem cell-derived V3 interneurons using an Nkx2-2 gene-specific reporter.
Enrichment of human embryonic stem cell-derived V3 interneurons using an Nkx2-2 gene-specific reporter.
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DOI：
10.1038/s41598-023-29165-z
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发表时间：
2023-02-03
影响因子：
4.6
通讯作者：
中科院分区：
文献类型：
--
作者：
V3脊髓中间神经元是控制运动功能的脊髓回路的关键元件。然而，迄今为止，还没有从人多能干细胞衍生纯V3群体的有效方法。在这里，我们报告了一种分化和分离脊髓V3中间神经元的方法，结合外源性因子介导的分化和磁激活细胞分选。我们发现V3祖细胞的分化可以用更高浓度的Sonic Hedgehog激动剂以及以3D格式培养细胞来增强。为了能够从混合分化培养物中纯化V3祖细胞，我们开发了转基因报告基因，其中V3特异性基因Nkx 2 -2的调节区的一部分驱动膜标记物CD 14的表达。我们发现，在人类细胞中，NKX 2 -2最初表现出与运动神经元祖细胞标记物的共标记，但随着分化培养的进行，V3特异性出现。在这些较晚的分化时间点，我们能够富集用CD 14标记的V3祖细胞至~ 95%纯度，并使它们成熟为有丝分裂后V3中间神经元。V3中间神经元的这种纯化工具将用于体外疾病建模、正常人神经发育的研究和脊髓疾病的潜在细胞疗法。
V3 spinal interneurons are a key element of the spinal circuits, which control motor function. However, to date, there are no effective ways of deriving a pure V3 population from human pluripotent stem cells. Here, we report a method for differentiation and isolation of spinal V3 interneurons, combining extrinsic factor-mediated differentiation and magnetic activated cell sorting. We found that differentiation of V3 progenitors can be enhanced with a higher concentration of Sonic Hedgehog agonist, as well as culturing cells in 3D format. To enable V3 progenitor purification from mixed differentiation cultures, we developed a transgene reporter, with a part of the regulatory region of V3-specific gene Nkx2-2 driving the expression of a membrane marker CD14. We found that in human cells, NKX2-2 initially exhibited co-labelling with motor neuron progenitor marker, but V3 specificity emerged as the differentiation culture progressed. At these later differentiation timepoints, we were able to enrich V3 progenitors labelled with CD14 to ~ 95% purity, and mature them to postmitotic V3 interneurons. This purification tool for V3 interneurons will be useful for in vitro disease modeling, studies of normal human neural development and potential cell therapies for disorders of the spinal cord.
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影响因子：
46.9
作者：
Qi Y;Zhang XJ;Renier N;Wu Z;Atkin T;Sun Z;Ozair MZ;Tchieu J;Zimmer B;Fattahi F;Ganat Y;Azevedo R;Zeltner N;Brivanlou AH;Karayiorgou M;Gogos J;Tomishima M;Tessier-Lavigne M;Shi SH;Studer L
通讯作者：
Studer L
影响因子：
16.6
作者：
Du, Zhong-Wei;Chen, Hong;Liu, Huisheng;Lu, Jianfeng;Qian, Kun;Huang, CindyTzu-Ling;Zhong, Xiaofen;Fan, Frank;Zhang, Su-Chun
通讯作者：
Zhang, Su-Chun
影响因子：
46.9
作者：
Hockemeyer, Dirk;Soldner, Frank;Beard, Caroline;Gao, Qing;Mitalipova, Maisam;DeKelver, Russell C.;Katibah, George E.;Amora, Ranier;Boydston, Elizabeth A.;Zeitler, Bryan;Meng, Xiangdong;Miller, Jeffrey C.;Zhang, Lei;Rebar, Edward J.;Gregory, Philip D.;Urnov, Fyodor D.;Jaenisch, Rudolf
通讯作者：
Jaenisch, Rudolf
DOI：
10.1242/dev.097188
发表时间：
2014-02
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Machado CB;Kanning KC;Kreis P;Stevenson D;Crossley M;Nowak M;Iacovino M;Kyba M;Chambers D;Blanc E;Lieberam I
通讯作者：
Lieberam I
影响因子：
11.8
作者：
Lei, Qiubo;Jeong, Yongsu;Matise, Michael P.
通讯作者：
Matise, Michael P.