Analysis of Insulin-Producing Cells During In Vitro Differentiation From Feeder-Free Embryonic Stem Cells

Abstract

Embryonic stem (ES) cells can differentiate into many cell types and are expected to be useful for tissue engineering. Recent reports have shown that ES cells can differentiate into insulin-producing cells in response to the transient expression of the pdx-1 gene, after the removal of feeder cells. To investigate the lineage of insulin-producing cells and their in vitro differentiation, we introduced the ␤geo gene, encoding a ␤-galactosidase-neomycin phosphotransferase fusion protein under the control of the mouse insulin 2 promoter, into ES cells that had been adapted to feederfree culture, and analyzed insulin gene expression during their in vitro differentiation. We also examined the expression of transcription factors that are related to the differentiation of the pancreas. X-gal staining analysis revealed ␤-galactosidase-positive cells on the surface and in the center of the embryoid body that proliferated during differentiation. Glucose-responsive insulin-producing cells, derived from our feeder-free ES cells, expressed insulin 2, pdx-1, Pax4, and Isl1 and also the glucagon, somatostatin, and PP genes. Moreover, the genes encoding p48, amylase, and carboxypeptidase A were also expressed. These results suggest that ES cells can differentiate not only into endocrine cells but also into exocrine cells of the pancreas, without the initiation of pdx-1 expression. Diabetes 52:1163-1168, 2003 M ouse embryonic stem (ES) cells are continuous cell lines derived directly from the fetal founder tissue of the preimplantation embryo. They can be expanded in culture while retaining the functional attributes of pluripotent early embryo cells. Their capacity for multilineage differentiation is reproduced in culture, as ES cells can differentiate into a wide range of well-defined cell types. Cell transplantation to restore tissue function after disease or injury is in theory applicable to a huge variety of human diseases. Thus, the use of lineage-restricted differentiation techniques developed for ES cells will promote future cell therapy. Moreover, an in vitro ES cell differentiation system will be valuable for research into early embryonic cell lineage. We and other groups have shown that ES cells can differentiate into the endoderm cell lineage (1-5). Recently, several groups have reported that ES cells can be induced to differentiate into insulin-producing cells These reports indicate that ES cells can differentiate into insulin-producing cells, but the cell lineage of these insulin-producing cells is still unknown. Indeed, insulin gene expression has been observed not only in the pancreas but also in the yolk sac and embryonic brain in vivo (6,7). To assess the cell lineage of insulin-producing cells during their differentiation from ES cells, we have developed ES cell lines carrying a lacZ-neomycin phosphotransferase fusion gene (␤geo) under the mouse insulin 2 gene promoter and analyzed the lacZ expression pattern of clusters of differentiating ES cells. Moreover, we have examined the expression of a series of transc...

Directed differentiation of human embryonic stem cells towards a pancreatic cell fate

“…It has been shown that pancreatic cell types can be generated from mouse and non-human primate ESCs [10][11][12][13][14][15][16][17][18]. Based on these findings, recent studies have also suggested the potential of hESCs to differentiate into insulin-producing cells through spontaneous in vitro differentiation [5] or the use of a multi-stage protocol [6].…”

Detecting de novo insulin synthesis in embryonic stem cell-derived populations

“…ES-derived cell types generated in recent protocols [4][5][6][7][8][9] have remained largely uncharacterized, apart from the identification of a small proportion of insulin/C-peptide positive cells within isolated sub-populations. Some of these protocols have reported the derivation of neuronal phenotypes [4,5,8,[17][18][19] within differentiated populations whereas a more recent publication has suggested that ES-derived insulin-positive cells are actually extra-embryonic in origin [20].…”

“…These unique characteristics make ES cells a potential source of insulin-producing cells for transplantation therapy [1][2][3]. Since 2000, several important and exciting reports have been published describing the derivation of insulin-positive cells from both mouse [4][5][6][7][8][9] and, to a lesser extent, human ES cell lines [10,11]. Several of these studies have been based on the derivation of insulinproducing cells using modified protocols that were originally designed to generate neurons, on the premise that the pancreas and central nervous system share developmental pathways [12][13][14].…”

Development of novel cell lines of diabetic dysfunction model fit for cell-based screening tests of medicinal materials

“…In vitro differentiation ES cells were induced to differentiate into insulin producing cells as previously described (Moritoh et al 2003). Briefly, the procedure has 4 stages: stage 1, 2-3 days culture in ESM for multiplication; stage 2, suspension culture in ESM for 4-5 days to obtain embryoid bodies; stage 3, selection of nestin-positive cells; stage 4, induction of insulin producing cells.…”

“…Oligo(dT)-primed cDNA was prepared from 1 lg of total RNA using SuperScript reverse transcriptase (Clontech-Takara Bio, Otsu, Japan). The primers for RT-PCR were designed using the information described in Moritoh et al (2003) or mouse sequences reported elsewhere. The primer sequences used here are listed in Table 1.…”