Insulin expressing cells from differentiated embryonic stem cells are not beta cells

Aim/hypothesis. Embryonic stem (ES) cells have been proposed as a potential source of tissue for transplantation for the treatment of Type 1 diabetes. However, studies showing differentiation of beta cells from ES cells are controversial. The aim of this study was to characterise the insulin-expressing cells differentiated in vitro from ES cells and to assess their suitability for the treatment of diabetes. Methods. ES cell-derived insulin-expressing cells were characterised by means of immunocytochemistry, RT-PCR and functional analyses. Activation of the Insulin I promoter during ES-cell differentiation was assessed in ES-cell lines transfected with a reporter gene. ES cell-derived cultures were transplanted into STZ-treated SCID-beige mice and blood glucose concentrations of diabetic mice were monitored for 3 weeks. Results. Insulin-stained cells differentiated from ES cells were devoid of typical beta-cell granules, rarely showed immunoreactivity for C-peptide and were mostly apoptotic. The main producers of proinsulin/insulin in these cultures were neurons and neuronal precursors and a reporter gene under the control of the insulin I promoter was activated in cells with a neuronal phenotype. Insulin was released into the incubation medium but the secretion was not glucose-dependent. When the cultures were transplanted in diabetic mice they formed teratomas and did not reverse the hyperglycaemic state. Conclusions/Interpretation. Our studies show that insulin-positive cells in vitro-differentiated from ES cells are not beta cells and suggest that alternative protocols, based on enrichment of ES cell-derived cultures with cells of the endodermal lineage, should be developed to generate true beta cells for the treatment of diabetes.