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. 2019 Jun 21;9:9013. doi: 10.1038/s41598-019-45247-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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For directed OPC differentiation the Zfp488-ZsGreen1-hNSCs were plated as a monolayer on laminin-coated plates. In Zfp488 group, after 4–5 weeks of differentiation 74.0 ± 3.0% of ZsGreen1 positive cells were NG2⁺ (A-A2), 83.8 ± 3.8% cells were sox10⁺ (B-B2), 93.2 ± 1.2%, Olig2⁺ (C-C2) and 87.0 ± 4.2% PDGFR-alpha⁺ (D-D2). The bar graph shows the percentages of green cells that express different markers (J). We could not derive quantifiable cells expressing OPC markers from control hNSCs using this protocol. Next, the H9-derived Zfp488 NSCs were FAC sorted using ZsGreen1 fluorescence reporter using FITC-A channel on Aria II FACS (E). The sorted cells formed spheres after overnight culture in ultralow attachment plates (F) and could be easily expanded in neural expansion media and formed a monolayer after plating on laminin-coated plates (G). FACS sorted ZsGreen1-OPCs, were induced to further differentiation. The percentage of Zfp488 cells expressing O4 was 68.7 ± 4.2% at day 14 after differentiation with thyroid hormone and morphologically resembled mature oligodendrocytes (H-H2). Further differentiation for at least 1 more week resulted in approximately 59.5 ± 3.7% of ZsGreen1⁺/O1⁺ cells (I-I2).