Functional comparison of distinct Brachyury⁺ states in a renal differentiation assay

Mesodermal populations can be generated in vitro from mouse embryonic stem cells (mESCs) using three-dimensional (3-D) aggregates called embryoid bodies or two-dimensional (2-D) monolayer culture systems. Here, we investigated whether Brachyury-expressing mesodermal cells generated using 3-D or 2-D culture systems are equivalent or, instead, have different properties. Using a Brachyury-GFP/E2-Crimson reporter mESC line, we isolated Brachyury-GFP ⁺ mesoderm cells using flow-activated cell sorting and compared their gene expression profiles and ex vivo differentiation patterns. Quantitative real-time polymerase chain reaction analysis showed significant up-regulation of Cdx2, Foxf1 and Hoxb1 in the Brachyury-GFP⁺ cells isolated from the 3-D system compared with those isolated from the 2-D system. Furthermore, using an ex vivo mouse kidney rudiment assay, we found that, irrespective of their source, Brachyury-GFP⁺ cells failed to integrate into developing nephrons, which are derived from the intermediate mesoderm. However, Brachyury-GFP⁺ cells isolated under 3-D conditions appeared to differentiate into endothelial-like cells within the kidney rudiments, whereas the Brachyury-GFP⁺ isolated from the 2-D conditions only did so to a limited degree. The high expression of Foxf1 in the 3-D Brachyury-GFP⁺ cells combined with their tendency to differentiate into endothelial-like cells suggests that these mesodermal cells may represent lateral plate mesoderm.