Comparative analysis of gene expression and morphology of mouse embryonic stem cells differentiated towards insulin-producing cells using four different cell culture protocols.

NAUJOK O; FRANCINI F; JÖRNS A; LENZEN S

Atenas, Grecia

Congreso; 41th EASD Annual Meeting; 2005

EASD

Background and Aims: The generation of insulin-producing cells from mouse embryonic stem cells (mESC) has been the subject of several studies. However, controversial results were obtained. Thus the aim of this study was to compare the effect of four distinct differentiation protocols on the gene expression and the morphology of mESC. Material and Methods: Mouse embryonic stem cells from the D3 cell line were differentiated according to four different protocols using a combination of serum-free or fetal calf serum (FCS) supplemented media. In addition, in one protocol, the cells were cultured in spinner flasks under constant stirring. To assess the effect of these culture techniques the expression of marker genes was examined using real-time RT-PCR. In parallel the morphology of the cells was monitored by electron microscopy (EM). Results: The results obtained when the cells were differentiated according to a serum-free protocol show that the cells express marker genes for endocrine differentiation. Morphological analyses revealed that roughly 20 % of all cells were differentiated. In addition 30-40 % of the cells were prone to cell death by apoptosis (two thirds) and necrosis (one third), respectively. When the cells were instead exposed to media supplemented with 5 % FCS in the final stage of the protocol insulin gene expression decreased by around 50 %. The same decrease could also be shown for glucagon and somatostatin. But the expression of Glut-2, Sur-1, Kir6.2, Nestin and Nkx6.1 could benefit from FCS supplementation. Moreover, a significant fraction of cells with endocrine characteristics and a higher degree of differentiation were identified in EM. Nonetheless the proportion of cells which undergo apoptosis was kept unaffected. However, when the step of selection of nestin positive cells was removed from the protocol and the cells were maintained for 12-14 days in serum-free medium and subsequently in medium containing 5 % FCS the insulin expression was normalized again. The expression of other beta cell marker genes was also significantly improved. Again EM displayed a higher differentiation status of the cells. When mESC were cultured in suspension in a spinner culture protocol the expression of all genes besides Oct-4 was significantly decreased. The cells showed a well preserved embryonic phenotype in EM with no signs of differentiation. The high level of Oct-4 expression was confirmed also by the expression of a second embryonic marker gene, ERas. Conclusions: Here we report that utilisation of different culture conditions affects embryonic stem cells with respect to their differentiation towards insulin-producing cells. We were able to establish a new differentiation protocol, which, at the same time, was able to induce insulin gene expression as well as of beta cell specific genes in cells with morphological beta cell characteristics and at the same time to reduce the rate of apoptosis. The usage of spinner conditions, on the other and, maintained stem cells in an undifferentiated state. We therefore conclude that proper conditions are required for differentiation and maturation of mESC into insulin-producing cells.