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. 2018 Mar 26;8:5168. doi: 10.1038/s41598-018-23512-1

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© The Author(s) 2018

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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Morphogenesis of NSC-MN and MNP-MN spheroids. (a) To test the elongation of neurites, MN spheroids were reseeded on laminin-coated dishes and migration and elongation of MNs was observed and quantified. Representative images of morphological change of NSC-MN spheroids. (b) MN neurites from NSC-MN spheroids were visualized by immunostaining of HB9, GFAP, and DAPI at three different regions (i, ii and iii). Note that the regions marked in the zoomed-out view are representative. Both soma and neurites can be seen in (i), a few soma and neurites in (ii), and only neurites in (iii). (c) after 14 days of seeding, characterization stained by HB9, GFAP, and SMI-32 (d). (e) To calculate neurite properties, image processing is conducted. (f) Quantitative comparison between NSC-MN spheroids and MNP-MN spheroids in terms of migration area, neurite length and spine density. NSC-MN spheroids have a higher potential of migration, neurite elongation, and spine density. n = 3; *P < 0.01, one-way ANOVA between NSC-MN and MNP-MN. Error bars ± SD.