The gene expression pattern and morphology of mouse embryonic stem cells during differentiation into insulin producing cells.

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

Munich

Congreso; 40th EASD Annual Meeting; 2004

Background and Aims: Embryonic stem cells present 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 using an improved differentiation protocol, which enriches nestin positive cells as putative pancreatic progenitor cells. Material and Methods: 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 different stages of this differentiation protocol the gene expression of a wide range of genes was examined by quantitative real-time PCR analyses. The differentiation stages were in addition also studied by electron microscopy (EM). Apoptosis was assessed during the differentiation process through quantification of caspase-3 expression as well as caspase-3 enzyme activity. Results: In the initial stages the cells were Oct4 and Pdx1 positive, but showed no significant expression of insulin and nestin. These cells exhibited typical signs of undifferentiated stem cells revealed by EM studies. In stages 2-3 many cells showed neuronal characteristics. In stage 5 the cells showed characteristics of insulin producing cells. The cells expressed nestin and insulin mRNA, but also mRNA for glucagon and somatostatin. In ultrastructural analyses stage 5 cells  showed rough endoplasmic reticulum and Golgi apparatus structures as an indication of protein biosynthesis and to a reproducible degree signs of endocrine differentiation. In addition the stage 5 cells expressed Kir6.2 and Sur1. Glut2 and Pdx1 gradually decreased during the differentiation but recovered when the medium in stage 5 was supplemented with FCS, providing an explanation for cell death of pancreatic progenitor cells under serum free conditions. Around 10 % of the cells were necrotic or apoptotic in the initial differentiation stages increasing to one third in the final stage. The majority of these cells were apoptotic. Caspase-3 activity increased two-fold from the first stage to the last stage, with a peak in stage 3. Conclusions: 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. It seems that besides the genetic approach through overexpression or gene trapping to enhance the differentiation of mESC towards insulin producing cells an improved cell culture protocol might also solve the problems of inadequate differentiation and loss of viability. Supported by the European Union in the 5th FP (project QLRT-2001-01777).