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

摘要

Mesodermal populations can be generatedin vitrofrom 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 whetherBrachyury-expressing mesodermal cells generated using 3-D or 2-D culture systems are equivalent or, instead, have different properties. Using aBrachyury-GFP/E2-Crimson reporter mESC line, we isolatedBrachyury-GFP+mesoderm cells using flow-activated cell sorting and compared their gene expression profiles andex vivodifferentiation patterns. Quantitative real-time polymerase chain reaction analysis showed significant up-regulation ofCdx2,Foxf1andHoxb1in theBrachyury-GFP+cells isolated from the 3-D system compared with those isolated from the 2-D system. Furthermore, using anex vivomouse 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 theBrachyury-GFP+isolated from the 2-D conditions only did so to a limited degree. The high expression ofFoxf1in the 3-DBrachyury-GFP+cells combined with their tendency to differentiate into endothelial-like cells suggests that these mesodermal cells may represent lateral plate mesoderm.