CONICET | Buscador de Institutos y Recursos Humanos

Background and Aims: Embryonic stem (ES) cells are a potential source for insulin-producing cells, which have the potential to differentiate into tissues from all three germ layers. The scarcity of 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. It was therefore the aim of this study to develop a differentiation protocol, which drives differentiation of ES cells towards insulin-producing cells and to combine this with a technique for separation of these insulin-positive cells. Materials and Methods: A 2.9 kb promoter fragment of the Krt19 gene (cytokeratin 19) was cloned to drive the expression of eYFP in stably transfected ES-D3 cells. eYFP+ and eYFP- cells were FACS sorted on d12, d19 and d26 of differentiation and gene expression was examined by qPCR analyses. The differentiation stages were additionally studied by electron microscopy (EM) and immunohistochemistry.: A 2.9 kb promoter fragment of the Krt19 gene (cytokeratin 19) was cloned to drive the expression of eYFP in stably transfected ES-D3 cells. eYFP+ and eYFP- cells were FACS sorted on d12, d19 and d26 of differentiation and gene expression was examined by qPCR analyses. The differentiation stages were additionally studied by electron microscopy (EM) and immunohistochemistry. Results: The pCK19-eYFP construct was successfully integrated into ES cells. Analysis of the gene expression of the endogenous cytokeratin 19 revealed a 4 to 7 fold higher cytokeratin 19 expression in eYFP+ cells when compared to eYFP- cells. Carbonic anhydrase 2, as a second marker for ductal cell progeny, showed a 2 fold increase in eYFP+ cells at day 12 and a 9 fold increase in expression in eYFP+ cells at day 26 when compared to eYFP- cells. Quantitative analysis of gene expression revealed a low expression of insulin in eYFP+ and eYFP- cells up to day 19. A significant 80 fold increase in insulin gene expression was observed in eYFP+ cells on day 26 as compared to day 12 and day 19. Compared with eYFP- cells insulin gene expression on day 26 was higher by a factor of 38, whereas the level of insulin gene expression in eYFP- cells did not change significantly. Glucagon gene expression was not detectable at day 12 and day 19. While glucagon gene expression remained barely detectable at day 26 in eYFP+ cells, it increased to appreciable levels in eYFP- cells 12 times higher than in eYFP+ cells. Ultrastructure of differentiated eYFP+ cells, in contrast to eYFP- cells, exhibited clear signs of differentiation comprising subcellular organelles for synthesis, processing, storage and release of insulin. Moreover, eYFP+ cells showed positive staining for C-peptide and insulin in the cytoplasm providing evidence for ongoing active pre-proinsulin biosynthesis and for insulin processing, while CK19- cells were negative for C-peptide.: The pCK19-eYFP construct was successfully integrated into ES cells. Analysis of the gene expression of the endogenous cytokeratin 19 revealed a 4 to 7 fold higher cytokeratin 19 expression in eYFP+ cells when compared to eYFP- cells. Carbonic anhydrase 2, as a second marker for ductal cell progeny, showed a 2 fold increase in eYFP+ cells at day 12 and a 9 fold increase in expression in eYFP+ cells at day 26 when compared to eYFP- cells. Quantitative analysis of gene expression revealed a low expression of insulin in eYFP+ and eYFP- cells up to day 19. A significant 80 fold increase in insulin gene expression was observed in eYFP+ cells on day 26 as compared to day 12 and day 19. Compared with eYFP- cells insulin gene expression on day 26 was higher by a factor of 38, whereas the level of insulin gene expression in eYFP- cells did not change significantly. Glucagon gene expression was not detectable at day 12 and day 19. While glucagon gene expression remained barely detectable at day 26 in eYFP+ cells, it increased to appreciable levels in eYFP- cells 12 times higher than in eYFP+ cells. Ultrastructure of differentiated eYFP+ cells, in contrast to eYFP- cells, exhibited clear signs of differentiation comprising subcellular organelles for synthesis, processing, storage and release of insulin. Moreover, eYFP+ cells showed positive staining for C-peptide and insulin in the cytoplasm providing evidence for ongoing active pre-proinsulin biosynthesis and for insulin processing, while CK19- cells were negative for C-peptide. Conclusion: Cytokeratin 19 is a marker for pancreatic duct cells. Duct cells have been considered to be a pool for newly derived beta cells during organogenesis of the pancreas. Following this assumption, we have set up a separation strategy using FACS sorting of fluorescent eYFP+ and non-fluorescent eYFP- cells. Gene expression analysis of the endogenous CK19 revealed, that this gene is predominantly expressed in the FACS-sorted eYFP+ population. Thus, our selection procedure purifies ductal progenitor cells and separates them successfully from an eYFP-, non-ductal cell population. A detailed molecular, biochemical and morphological analysis revealed that eYFP+ cells represent a population of differentiated ES cells, which express the insulin prohormone gene, and process, store and release insulin.: Cytokeratin 19 is a marker for pancreatic duct cells. Duct cells have been considered to be a pool for newly derived beta cells during organogenesis of the pancreas. Following this assumption, we have set up a separation strategy using FACS sorting of fluorescent eYFP+ and non-fluorescent eYFP- cells. Gene expression analysis of the endogenous CK19 revealed, that this gene is predominantly expressed in the FACS-sorted eYFP+ population. Thus, our selection procedure purifies ductal progenitor cells and separates them successfully from an eYFP-, non-ductal cell population. A detailed molecular, biochemical and morphological analysis revealed that eYFP+ cells represent a population of differentiated ES cells, which express the insulin prohormone gene, and process, store and release insulin.