Janny Landegent Peterslund:
FGF-signalling in the differentiation of mouse ES cells towards definitive endoderm

Date: 15-03-2010    Supervisor: Berthe M. Willumsen

Diabetes is a metabolic disease affecting more than 200 million people worldwide. Therapeutic transplantation with insulin-producing β cells is envisioned as a cure for diabetes, but as donors are scarce, other sources of cell material are needed. Embryonic stem (ES) cells are pluripotent cells with the potential to differentiate into all cell and tissue types of the adult body. As such, they could serve as an unlimited source of in vitro differentiated β-like cells. The present work describes how mouse ES cells, which are used as a model for human ES cells, are induced to differentiate in the early steps towards β-like cells. The work focuses on fibroblast growth factor (FGF)-signalling in relation to the formation of mesendoderm, definitive endoderm (DE) and posterior foregut endoderm. The mesendoderm is a bipotent cell population, which can differentiate into either DE or mesoderm by activinA or bone morphogenetic protein (BMP). The DE and posterior foregut endoderm are more mature cell types which will later form the pancreas and β cells.

This work is based on a serum and feeder-free cell culture system, and shows that induction of mesendoderm cell types by BMP4 or a range of activinA-concentrations depends on FGFsignalling. Blocking FGF-signalling by soluble FGF receptors (FGFRs) or small molecule inhibitors impedes formation of the mesendoderm, whereas addition of a range of FGFs increases its formation. More specifically, activation through FGFR(III)c (FGFRc)-isoforms elicits the effect, whereas activation of FGFRb-isoforms shows no effect. This correlates with the expression of especially FGFRc-isoforms in early differentiation of ES cells. On the contrary, high concentrations of FGFs reduce the number of more the mature DE cells, although active FGFR-signalling in general is necessary for DE-formation.

Surprisingly, an FGF4 knockout cell line which has previously been reported to be unable to differentiate into ectoderm and mesoderm cell lineages can readily differentiate into DE without addition of ectopic FGF4. The heterozygote cell line FGF4+/– even shows higher expression of DE marker genes than both wild type and knockout cell lines, indicating that an intermediate level of FGF-signalling is beneficial to DE-formation. Together, these results suggest that activation of FGFRc-isoforms during early mesendoderm specification followed by FGFRc activation below endogenous levels is the most attractive recipe to obtain DE formation in mouse ES cells.

Lastly, further differentiation of the DE cell population is dependent on retinoic acid and FGFsignalling, preferably by FGF7 and FGF10 which activate FGFRb-isoforms. An intermediate concentration of FGF results in a high number of cells expressing an anterior foregut marker, few cells expressing a posterior foregut marker and no cells expressing a hindgut marker. High concentrations of FGF posteriorize the DE to form hindgut.

These results provide new insight into the differential effect of FGF-signalling on mesendoderm, DE and posterior foregut endoderm formation over time. Applying this knowledge to current protocols for differentiation may prove beneficial to the end-point material, namely β-like cells.