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

signalling. These dynamic signalling cascades possibly determine the boundaries of pancreaticendoderm, i.e. expression of Pdx1, and the closely related liver endoderm.As the pancreatic buds grow and branch, the surrounding mesenchyme secretes FGF10, whichstimulates pancreatic epithelial proliferation. This mesenchymal stimulation is absolutelynecessary for maintenance of Pdx1 expression and pancreatic development, as FGF10 –/– show noPdx1 expression at E10.5 (Bhushan et al. 2001). The epithelial expression of Pdx1 and NKhomeobox transcription factor 6.1 (Nkx6.1) starts to deviate into NKX6.1 + cells found only in thecentral part of the epithelium, whereas PDX1 + /NKX6.1 – cells are found at the periphery and atE13.5 mark the acini (Jorgensen et al. 2007). These acini become the exocrine part of the pancreasthat produces and secretes digestive enzymes into the connecting ducts, releasing them to theintestine (Slack 1995). Neurogenin 3 (NGN3) + endocrine precursors delaminate from theepithelium and develop into Paired box gene 6 (Pax6)-expressing endocrine cells which form theislets of Langerhans. The endocrine cells in these islets produce hormones, which they secrete tothe bloodstream. Islets consist of five cell types: α cells producing glucagon; β cells producinginsulin; δ cells producing somatostatin; ε cells producing ghrelin and PP cells producingpancreatic polypeptide. These islets have a distinct morphology, with the β cells in the centre andthe other cell types at the periphery.Mouse ES cells in directed differentiationThe initial cell population used to generate β-like cells for cell replacement therapy may comefrom either somatic (or adult) stem cells or from ES cells. Somatic stem cells are mono- ormultipotent cells residing in most tissues and organs of the post-natal human. They are used in thetreatment of leukaemia and other haematological malignancies through bone-marrowtransplantation. Additional areas under investigation include treatment of strokes, myocardialinfarctions, corneal regeneration and epidermal gene therapy (Pellegrini et al. 2009; McCall et al.2010). There is no definitive evidence of a somatic stem cell in the pancreas and thus the focus ofcell replacement-therapy for diabetes is currently on ES cells (Madsen 2005).ES cells are pluripotent cells, i.e. they can give rise to all tissues of the embryo proper (Ohtsukaand Dalton 2008). They are isolated from the inner cell mass of a blastocyst stage embryo andunder the right culture conditions they can multiply indefinitely, while keeping their pluripotentphenotype (Evans and Kaufman 1981; Martin 1981; Ying et al. 2003a). They hold great potentialdue to their pluripotent nature, but as yet, no protocol applicable to human treatment has beendeveloped.Modelling differentiation using mouse ES cellsMouse ES (mES) cells are used for the study of directed differentiation towards definitiveendoderm, pancreatic foregut and ultimately β-like cells because they have certain advantages.Working with mES cells holds fewer ethical concerns than working with human ES (hES) cellsand there are many available tools, such as transgenic mES cell lines which can be used to testhypotheses that cannot be otherwise experimentally tested. Also, mouse embryonic developmentclosely mimics human embryonic development, suggesting that conclusions may be extrapolatedand applied to the human system. But in using mES cells for scientific purposes, it is important tobear in mind that the end product will always be a cell therapy based on hES cells, and findingswill therefore always have to be confirmed in this system. The sections below will focus on mEScells with examples from hES cell work where relevant.Derivation of mESCsThe first mES cells were derived almost 30 years ago and the first hES cell line was derived 12years ago (Evans and Kaufman 1981; Martin 1981; Thomson et al. 1998). mES cells (ES cellshereafter) are used either for generation of transgenic mice or for culturing and differentiation of(transgenic) cell lines. Mouse ES cells are derived from the ICM of the E3.5 blastocyst. They aregrown on feeder cells in the presence of serum and leukemia inhibitory factor (LIF), whichmaintains them pluripotent.11