Effect of different serum concentration on gene expression and differentiation pattern of mouse embryonic stem cells to insulin-producing cells.

FRANCINI F; NAUJOK O; TIEDGE M; JÖRNS A; LENZEN S

Prien

Congreso; Post -EASD Symposium 2004 EASD Islet Study Group; 2004

Embryonic stem cells represent an unlimited source of cells, which have the potential to differentiate into tissues from all three germ layers. The shortage of transplantable donor islets of Langerhans for therapy of type 1 diabetes mellitus has focused research on the development of surrogate cells with characteristics similar to those of pancreatic beta cells. The aim of this study was to examine the gene expression and the morphology of mouse embryonic stem cells under different fetal calf serum (FCS) concentrations in the last differentiation step. Mouse embryonic stem cells (mESC) from the D3 cell line were differentiated with a modified so-called Lumelsky protocol (Science 18, 1389-1394, 2001). In  the different differentiation stages the gene expression of endocrine and stem cell markers was examined by quantitative real-time PCR analyses. Signs of differentiation or cell death, both necrosis and apoptosis, were ultrastructurally analyzed. Apoptosis was assessed during the differentiation process through quantification of pro-caspase-3 expression as well as caspase-3 enzyme activity. In the first stage the cells expressed Oct4 and Pdx1 as stem cell markers with a very low expression level of insulin and the lowest values for caspase-3 activity. This last fact was  morphologically confirmed. Ultrastructurally these cells were undifferentiated stem cells with poorly developed cell organelles. During the differentiation steps, in stages 2-3, the cells acquired neuronal characteristics with an increase in nestin expression and the highest values in caspase-3 activity (20-30 folds increase over stage 1 value). In stage 5 the cells presented characteristics of insulin-producing cells with the highest level of insulin mRNA, but also mRNA for glucagon and somatostatin. Even when we observed a negative effect of the increase in FCS concentration in the medium on the gene expression for endocrine markers, especially insulin, the ultrastructural studies demonstrated a higher differentiation of the rough endoplasmic reticulum and Golgi apparatus structures as an indication of protein biosynthesis. Caspase-3 activity decreased to a two-fold level compared with stage 1. At 5-7,5% FCS concentration in the medium, the pro-caspase-3 mRNA expression reached the lowest values in parallel with the well differentiated structure observed by EM.  It could be shown that this protocol is able to drive differentiation of mESC towards cells with characteristics of insulin-producing cells but that special attention is required with respect to the optimization of differentiation conditions. Supported by the European Union in the 5th FP.