Supplementary Material for: FGF2 Signaling in Mouse Embryonic Fibroblasts Is Crucial for Self-Renewal of Embryonic Stem Cells

Human embryonic stem cells (hESCs) can be maintained undifferentiated (pluripotent) or differentiated to basically all functional cell types, depending on the culture conditions used. Culture of hESCs in the presence of medium conditioned by mouse embryonic fibroblasts (MEFs) can be used to keep hESCs undifferentiated. This observation suggests that MEFs produce factors required for the pluripotency of hESCs. The data presented here show that fibroblast growth factor 2 (FGF2) treatment of MEFs is crucial for the production of these factors. To identify the potential factors that are expressed in the presence of FGF2 in MEFs, a global expression profile analysis using microarrays was performed. This analysis indicated that 17 secreted factors are downregulated in the absence of FGF2. These factors include several ligands for known signaling receptors, extracellular proteases and components of the extracellular matrix, that may all be involved in signaling events. Surprisingly, we found that selective blocking of extracellular signal-regulated kinase (ERK) signaling by the MAPK/ERK kinase (MEK) inhibitor U0126 affected the expression of only some of the FGF2-regulated genes, suggesting FGF2-induced pathways that are independent of ERK signaling. It has been shown recently that activation of Activin/Nodal signaling and inhibition of bone morphogenetic protein signaling are required for the maintenance of pluripotency. Accordingly, among the 17 FGF2-regulated genes we found inhibin βB that can lead to the assembly of Activin B and gremlin 1 that codes for an antagonist of bone morphogenetic proteins. This study identifies potentially important factors involved in the maintenance of pluripotency in hESCs and may allow the development of defined culture conditions without contaminating material from animal cells.