A stable analogue of glucose-dependent insulinotropic polypeptide, GIP(LysPAL16), enhances functional differentiation of mouse embryonic stem cells into cells expressing islet-specific genes and hormones

Embryonic stem (ES) cells can be differentiated into insulin-producing cells by conditioning the culture media. However, the number of insulin-expressing cells and amount of insulin released is very low. Glucose-dependent insulinotropic polypeptide (GIP) enhances the growth and differentiation of pancreatic beta-cells. This study examined the potential of the stable analogue GIP(LysPAL16) to enhance the differentiation of mouse ES cells into insulin-producing cells using a five-stage culturing strategy. Semi-quantitative PCR indicated mRNA expression of islet development markers (nestin, Pdx1, Nkx6.1, Oct4), mature pancreatic beta-cell markers (insulin, glucagon, Glut2, Sur1, Kir6.1) and the GIP receptor gene GIP-R in undifferentiated (stage 1) cells, with increasing levels in differentiated stages 4 and 5. IAPP and somatostatin genes were only expressed in differentiated stages. Immunohistochemical studies confirmed the presence of insulin, glucagon, somatostatin and IAPP in differentiated ES cells. After supplementation with GIP(LysPAL16), ES cells at stage 4 released insulin in response to secretagogues and glucose in a concentration-dependent manner, with 35-100% increases in insulin release. Cellular C-peptide content also increased by 45% at stages 4 and 5. We conclude that the stable GIP analogue enhanced differentiation of mouse ES cells towards a phenotype expressing specific beta-cell genes and releasing insulin.     

Pdx1 and Ngn3 overexpression enhances pancreatic differentiation of mouse ES cell-derived endoderm population

In order to define the molecular mechanisms regulating the specification and differentiation of pancreatic β-islet cells, we investigated the effect of upregulating Pdx1 and Ngn3 during the differentiation of the β-islet-like cells from murine embryonic stem (ES) cell-derived activin induced-endoderm. Induced overexpression of Pdx1 resulted in a significant upregulation of insulin (Ins1 and Ins2), and other pancreas-related genes. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we induced the overexpression express of Ngn3 together with Pdx1. This combination dramatically increased the level and timing of maximal Ins1 mRNA expression to approximately 100% of that found in the βTC6 insulinoma cell line. Insulin protein and C-peptide expression was confirmed by immunohistochemistry staining. These inductive effects were restricted to c-kit(+) endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions after the specification of pancreatic endoderm. Although insulin secretion was stimulated by various insulin secretagogues, these cells had only limited glucose response. Microarray analysis was used to evaluate the expression of a broad spectrum of pancreatic endocrine cell-related genes as well as genes associated with glucose responses. Taken together, these findings demonstrate the utility of manipulating Pdx1 and Ngn3 expression in a stage-specific manner as an important new strategy for the efficient generation of functionally immature insulin-producing β-islet cells from ES cells.     

Analysis of gene expressions of embryonic stem‐derived Pdx1‐expressing cells: Implications of genes involved in pancreas differentiation

We have recently reported the method by which embryonic stem (ES) cells were induced into Pdx1‐expressing cells. To gain insights into the ES cell‐derived Pdx1‐expressing cells, we examined gene expression profiles of the cells by microarray experiments. Microarray analyses followed by a comparison with the data of the cells in developing pancreatic and adult islet suggested that the ES cell‐derived Pdx1‐positive cells were immature pancreatic progenitor cells with endodermal characteristics. The analyses of the genes upregulated in the ES cell‐derived Pdx1‐positive cells would give us knowledge on early pancreatic development. Here, we first listed the genes and found that these contained not only those known to be expressed in the endoderm or pancreatic progenitor cells, but also those known to be involved in left–right axis formation. Second, we examined the gene expression patterns and found that several genes were expressed in the ventral foregut lip at the anterior intestinal portal in E8.5 embryo. Given that the Pdx1/GFP‐expressing cells are first observed in the same region at the anterior intestinal portal, these results suggest that the pancreatic progenitor cells first give rise at the ventral endoderm prior to the formation of dorsal and ventral pancreatic buds.     

An expression profile analysis of ES cell-derived definitive endodermal cells and Pdx1-expressing cells

Background  

We developed an efficient in vitro method to differentiate mouse ES cells into the definitive endoderm (DE) and then Pdx1-expressing pancreatic lineages using mesodermal-derived supporting cells, M15. Using this method, resulting ES cell-derived DE and Pdx1-expressing cells were isolated by cell sorting, and their gene expression profiles were investigated with DNA microarray. Genes that were specifically expressed in DE and/or in Pdx1-expressing cells were extracted and their expression patterns in normal embryonic development were studied.     

Sodium butyrate activates genes of early pancreatic development in embryonic stem cells

Embryonic stem (ES) cells can differentiate into any tissue, including pancreatic islet cell types. Protocols for the efficient generation of these cells in vitro could have therapeutic applications for type I diabetes. Here we describe a simple method for the differentiation of mouse ES cells into epithelial cells with a gene expression profile consistent with that expected of early pancreatic progenitors (PP). It is based on the addition of sodium butyrate, an agent known to induce chromatin rearrangements. Variations on the length of exposure to butyrate result in the generation of hepatocytes or PP-like cells. qRT-PCR indicates that butyrate induces mesendoderm/definitive endoderm, but not neuroectoderm differentiation. PPlike cells show a strong upregulation of Ipf1/Pdx1, p48, Isl-1 and Nkx6.1, but not Ngn3, NeuroD/ Beta2 or Pax4. PP-like cells also express the epithelial marker E-cadherin. Taken together, our observations suggest that butyrate stimulates early events of pancreatic specification, prior to the onset of endocrine differentiation. These findings are discussed in the context of the development of protocols for the in vitro differentiation of islets.     

Generation of insulin-expressing cells from mouse embryonic stem cells

The therapeutic potential of transplantation of insulin-secreting pancreatic beta-cells has stimulated interest in using pluripotent embryonic stem (ES) cells as a starting material from which to generate insulin secreting cells in vitro. Mature beta-cells are endodermal in origin so most reported differentiation protocols rely on the identification of endoderm-specific markers. However, endoderm development is an early event in embryogenesis that produces cells destined for the gut and associated organs in the embryo, and for the development of extra-embryonic structures such as the yolk sac. We have demonstrated that mouse ES cells readily differentiate into extra-embryonic endoderm in vitro, and that these cell populations express the insulin gene and other functional elements associated with beta-cells. We suggest that the insulin-expressing cells generated in this and other studies are not authentic pancreatic beta-cells, but may be of extra-embryonic endodermal origin.