Horikiri Tomoko, Ohi Hiromi, Shibata Mitsuaki, Ikeya Makoto, Ueno Morio, Sotozono Chie, Kinoshita Shigeru, Sato Takahiko
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Department of Biomedical Engineering, Faculty of Life Sciences, Doshisha University, Kyotanabe, Japan.
PLoS One. 2017 Jan 20;12(1):e0170342. doi: 10.1371/journal.pone.0170342. eCollection 2017.
The neural crest is a source to produce multipotent neural crest stem cells that have a potential to differentiate into diverse cell types. The transcription factor SOX10 is expressed through early neural crest progenitors and stem cells in vertebrates. Here we report the generation of SOX10-Nano-lantern (NL) reporter human induced pluripotent stem cells (hiPS) by using CRISPR/Cas9 systems, that are beneficial to investigate the generation and maintenance of neural crest progenitor cells. SOX10-NL positive cells are produced transiently from hiPS cells by treatment with TGFβ inhibitor SB431542 and GSK3 inhibitor CHIR99021. We found that all SOX10-NL-positive cells expressed an early neural crest marker NGFR, however SOX10-NL-positive cells purified from differentiated hiPS cells progressively attenuate their NL-expression under proliferation. We therefore attempted to maintain SOX10-NL-positive cells with additional signaling on the plane and sphere culture conditions. These SOX10-NL cells provide us to investigate mass culture with neural crest cells for stem cell research.
神经嵴是产生多能神经嵴干细胞的来源,这些干细胞有分化为多种细胞类型的潜力。转录因子SOX10在脊椎动物的早期神经嵴祖细胞和干细胞中表达。在此,我们报告了通过使用CRISPR/Cas9系统生成SOX10-纳米灯笼(NL)报告基因的人诱导多能干细胞(hiPS),这有助于研究神经嵴祖细胞的产生和维持。通过用TGFβ抑制剂SB431542和GSK3抑制剂CHIR99021处理,可从hiPS细胞中短暂产生SOX10-NL阳性细胞。我们发现所有SOX10-NL阳性细胞均表达早期神经嵴标志物NGFR,然而,从分化的hiPS细胞中纯化出的SOX10-NL阳性细胞在增殖过程中其NL表达会逐渐减弱。因此,我们尝试在平面和球体培养条件下通过额外的信号传导来维持SOX10-NL阳性细胞。这些SOX10-NL细胞为我们在干细胞研究中利用神经嵴细胞进行大规模培养研究提供了条件。