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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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Schematic images of formation of neuronal and vascular networks. (a) The multi-channel microfluidic device consists of 14 independent gel channels separated by and adjacent to 15 medium channels. (b,c) Vascular and neuronal networks were formed in collagen gel on a petridish and in a microfluidic device by co-culturing motor neuron spheroids (MN spheroids) and endothelial cells (HUVEC, iPS-EC). (d) Characterization of differentiation into MN progenitor cells. Immunostaining of Tuj1 and Oligo2 showed that after 7 days of differentiation from neural stem cells, cells start to express Tuj1 which is an early neuronal marker, but not Oligo2 which is late MN progenitor marker. Mature MNs can be obtained by day 28. Phase contrast image shows the morphology of motor neurons on day 35. (e) Characterization of iPS-EC stained by VE-cadherin. (f) Time course of MN differentiation from human neural stem cells (ES-derived: H9). For formation of NSC derived MN spheroids (NSC-MN spheroids), NSCs were seeded into spheroid formation plate supplemented with NSC growth medium. Then, RA, SHH, bFGF, and Activin A were added on day 1 to induce differentiation. On day 18, BDNF and GDNF were added to promote maturation of MNs. For formation of progenitor-derived MN spheroids (MNP-MN spheroid), after NSCs were cultured in a Petri dish for 18 days, spheroids were formed. After ~30 days, MN spheroids and ECs were encapsulated in collagen gel within a microfluidic device and on Petri dish or the formation of MN and EC networks.