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

involved in WNT-signalling and maintenance of the pluripotent state (Chang et al. 2010). Thus,here it seems that a high cell density may inhibit differentiation, which has also been suggestedby Smith and co-workers (Smith et al. 1992). Similarly, we propose inhibition by high celldensities in the FGF4 –/– ES cell culture, and suggest low seeding densities for optimaldifferentiation.DE patterningIt seems rather symptomatic that we successfully reach 40-60% SOX17 + DE cells, but havelimited success in further patterning this DE to PDX1-expressing posterior foregut. We saw aninduction of PDX1-expressing cells when adding RA and FGF (and Cyclopamine), but nevermore than 2-4% on average. Competing groups have successfully obtained 32% PDX1 + cells inhES cell cultures by using some of the same posterior foregut-inducing factors, basing theirscientific approach on the same hypotheses as we do (Johannesson et al. 2009; Ameri et al.2010). Therefore the reason for our inefficient Pdx1-GFP induction shall possibly be soughtelsewhere.The Pdx1-GFP cell line we use has shown a nice expression pattern within the posterior foregutregion when injected into mouse blastocysts to form chimaeras (Tino Klein, unpublished data).The cell line works in vivo and is pluripotent, and we therefore expect it to also work in vitro.One point could be that we have optimized the DE-induction step in the Sox17-GFP and not thePdx1-GFP cell line. Data from hES cells indicate that the outcome of a differentiation protocolvaries much between cell lines (D'Amour et al. 2006; Mfopou et al. 2010). If this is also thecase for mES cells, we will have to redo optimization of DE-induction in our Pdx1-GFP cellline to have the best starting material. However, our protocol induces 2-4% Pdx1 + cells in E14,Pdx1-LacZ and Pdx1-GFP cell lines, showing robustness of the protocol.A developmental-based explanation is that the DE we have is simply not the ‘correct’ one.Using our protocol, we get many SOX2 + cells, suggesting that the DE we have after 5 days inhigh concentrations of activin may be somehow pre-patterned to respond to patterning factorspredominantly by induction of anterior foregut, marked by SOX2.Finally, there could be one or more components in our basic medium or medium supplementsthat inhibit differentiation. This problem could be overcome by changing the basic medium,medium supplements and the culture dish coating individually to decipher which may beinhibitory.Cell replacement therapy as a future cure for TIDMES cells are not the only source of β cells or β-like cells envisioned as material for futuretransplantation in the treatment or even cure for diabetes. Some perspectives of the variousalternatives are discussed below.Xeno-transplantationXeno-transplantation of islets of Langerhans from pig to primate is being investigated as atreatment for type I diabetes. The use of pigs is promising, as their vascular physiology issimilar to that of humans and they are relatively cheap to breed. Furthermore, the so-calledmini-pigs weighing app. 120 kg are similar to humans in organ and body sizes and can beinbred to homozygosity at e.g. the porcine major histocompatibility complex (MCH). The latterholds a great potential for manipulation to create customized donor organs/ cells and overcomesome of the problems of immune-responses normally seen in transplantation. This could bedone by introducing porcine MHC genes into the bone marrow of the recipient human inducingmixed chimaerism thereby introducing immunological tolerance to the xenograft (Hoerbelt andMadsen 2004). Alternatively, pigs could be genetically manipulated not to express geneproducts to which the recipient immune system reacts. Of major concern in xeno-86