Embryonic stem cells (ES cells) represent a population of self renewing pluripotent cells, capable of differentiating into derivatives of the 3 embryonic germ layers and are therefore a promising source material for generation of replacement pancreatic beta cells for transplantation in type 1 diabetes. ES cells can be induced to undergo unregulated differentiation when cultured as floating clusters (embryoid bodies). Successful generation of pancreatic endocrine cells from ES cells has thus far relied on embryoid body formation as an initial step. However, selection and expansion of sufficient quantities of insulin -secreting cells has remained a problem. Data presented here show a novel route for ES cell differentiation avoiding embryoid body formation and resulting in a cell population that expresses markers of pancreatic endocrine cell differentiation. The CCE mouse ES cell line was cultured in standard ES cell medium. Cultures were grown to high confluence to create large differentiated colonies which spread to form a monolayer of morphologically identical cells. This process was repeated on 3 separate occasions. RT-PCR analysis of differentiated cultures revealed expression of markers of early pancreas differentiation (HNF3beta and pdx-1) in all spontaneously differentiated cell (SDC) cultures but very low expression in undifferentiated cells. Insulin and glucagon expression was detected in one of the SDC cultures and components of the beta-cell ATP sensitive potassium (KATP) channel , Kir6.2 and SUR1, were detected in all SDC cultures. Functional KATP channels were shown to be present in SDC cells by calcium microfluorimetry. The sulphonylurea, tolbutamide, induced reversible intracellular calcium elevations in 7% of SDC cells tested.
In conclusion, spontaneously differentiated ES cells express a number of islet mRNAs that may represent a pool of cells with restricted gene expression that have the propensity for islet cell differentiation given the appropriate conditions.
03 - 05 Nov 2003
Society for Endocrinology