FGF-signalling in the differentiation of mouse ES cells towards ...

AbstractProgress in embryonic stem (ES) cell research over the last decade has outlined a possible cellbased intervention strategy in diabetes based on the hierarchical differentiation of embryonicstem cells into insulin producing beta cells. The definitive endoderm (DE) cell type constitutesan essential milestone in this differentiation pathway and fibroblast growth factor (FGF)-signaling drives the formation of DE in the mouse ES cell model system. More specifically, ithas been shown that FGF4 is crucial for cells to leave the pluripotent state and differentiate toectoderm and mesoderm cell lineages. The FGF system counts several receptor isoforms with apossible functional redundant role in conducting the differentiation signal. Here, we investigatethe spatio-temporal dynamics of FGF receptor (FGFR) distribution in the forming DE and findthat FGFR(III)c-isoforms are highly represented in the whole culture, whereas FGFR2(III)band FGFR4 are found in the DE fraction, specifically. The FGFR(III)c isoform-activating FGFsinduce mesendoderm markers T and Gsc, but reduce the DE marker Sox17 whereas FGFsactivating FGF(III)b-isoforms have no effect on either cell type. Notably, FGFR(III)c isoformactivatingFGFs exhibit strong mitogenic effects on ES cells early in the differentiation periodwhere FGFs activating FGFR(III)b-isoforms have only a moderate mitogenic potentialconfined to the late differentiation period. Interestingly, when applying our DE inductionprotocol onto an FGF4 –/– cell line, we find that cells readily differentiate into endoderm cellswithout ectopic administration of FGF4. By antibody staining and qPCR analyses for definitiveand visceral endoderm markers, we show that this endoderm is definitive rather than visceral.We conclude that FGFR(III)c-isoform activation selectively drives the differentiation of mEScells towards mesendoderm and that FGF4 is dispensable for the final differentiation step intoDE.59