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. 2015 Jun 15;10(6):e0129242. doi: 10.1371/journal.pone.0129242

Fig 3. Long term maintenance of NPC in aggregates.

Fig 3

(A) Morphology of representative E12.5 aggregates at day1, 7, 14 and 21 in culture. The size of aggregates increased with time (a-d), and the morphology of the aggregates changed from more flattened by day 7 to more three-dimensional after day 14, as demonstrated by the confocal XZ axis pictures (e-g). Immuno-staining for NPC marker, Six2 (green), and UB marker, DBA (red), shows the expansion of Six2+ NPC surrounding UB structures from day 7 up to day 21 in culture. (Scale bar = 500 μm). (B) The number of total cells and Six2+NPC in E12.5 aggregates increased in parallel from day 0 to day 7, 14 and 21, and plateaued after day 14. Data are presented as fold changes from respective cell number at day 0. (n = 3, ** p < 0.01 vs. day 0). (C) mRNA expression levels of NPC marker genes in E12.5 aggregates at day 0, 7, 14 and 21. qRT-PCR results showed a significant increase in Cited1 mRNA expression levels at day 14 and 21, while the mRNA expression levels of Six2 and Eya1 remained unchanged from day 0 to 21. Data were normalized by Gapdh expression levels and presented as fold changes from day 0. (n = 3, ** p < 0.01 vs. day 0)