bioRxiv preprint doi: https://doi.org/10.1101/239913; this version posted December 26, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 of 44 + 1 Comparing the differentiation potential of Brachyury mesodermal 2 cells generated from 3-D and 2-D culture systems 3 Jing Zhou 1, Antonius Plagge 1 and Patricia Murray 1,* 4 1 Institute of Translational Medicine, University of Liverpool, Liverpool L69 3BX, UK; E-mails: 5
[email protected] (J.Z.);
[email protected] (A.P.);
[email protected] 6 (P.M.) 7 * Correspondence:
[email protected] 8 Abstract 9 Mesodermal populations can be generated in vitro from mouse embryonic stem cells (mESCs) using 10 three-dimensional (3-D) aggregates called embryoid bodies or two-dimensional (2-D) monolayer 11 culture systems. Here, we investigated whether Brachyury-expressing mesodermal cells generated 12 using 3-D or 2-D culture systems are equivalent, or instead, have different properties. Using a 13 Brachyury-GFP/E2-Crimson reporter mESC line, we isolated Brachyury-GFP+ mesoderm cells using 14 flow-activated cell sorting and compared their gene expression profiles and ex vivo differentiation 15 patterns. Quantitative RT-PCR analysis showed significant up-regulation of Cdx2, Foxf1 and Hoxb1 in 16 the Brachyury-GFP+ cells isolated from the 3-D system compared with those isolated from the 2-D 17 system.