Pancreatic Lineage Research Articles

Activin A Decreases glucagon and arx Gene Expression in α-Cell Lines

Activin A is a potent growth and differentiation factor involved in development, differentiation, and physiological functions of the endocrine pancreas; it increases insulin and pax4 gene expression in beta-cells and can induce transdifferentiation of the exocrine acinar cell line AR42J into insulin-producing cells. We show here that Activin A decreases glucagon gene expression in the alpha-cell lines InR1G9 and alphaTC1 in a dose- and time-dependent manner and that the effect is blocked by Follistatin. This effect is also observed in adult human islets. Glucagon gene expression is inhibited at the transcriptional level by the Smad signaling pathway through the G3 DNA control element. Furthermore, Activin A decreases cell proliferation of InR1G9 and alphaTC1 cells as well as cyclin D2 and arx gene expression, whose protein product Arx has been shown to be critical for alpha-cell differentiation. Overexpression of Arx in Activin A-treated InR1G9 cells does not prevent the decrease in glucagon gene expression but corrects the inhibition of cell proliferation, indicating that Arx mediates the Activin A effects on the cell cycle. We conclude that Activin A has opposite effects on alpha-cells compared with beta-cells, a finding that may have relevance during pancreatic endocrine lineage specification and physiological function of the adult islets.

Stem-Cell Therapy for Diabetes Cure: How Close are We?

Transplantation of insulin-producing cells offers a promising therapy to treat diabetes. However, due to the limited number of donor islet cells available, researchers are looking for different sources of pancreatic islet progenitor or stem cells. A stem cell with extensive proliferative ability may provide a valuable source of islet progenitor cells. Several studies have demonstrated that a progenitor/stem-cell population can be expanded in vitro to generate large numbers of islet progenitor cells. However, efficient and directed differentiation of these cells to an endocrine pancreatic lineage has been difficult to achieve. We discuss here various pancreatic islet stem cells that we and others have obtained from embryonic, fetal or adult human tissues. We review the progress that has been achieved with pancreatic islet progenitor cell differentiation in the last 2 decades and discuss how close we are to translate this research to the clinics.

Differentiation of mouse embryonic stem cells to insulin-producing cells

Here, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem (ES) cells into insulin-producing cells. The three-step protocol comprises (i) the formation of embryoid bodies, (ii) the spontaneous differentiation of embryoid bodies into progenitor cells of ecto-, meso- and endodermal lineages, and (iii) the induction of differentiation of early progenitors into the pancreatic lineage. Differentiated cells can be obtained within approximately 33 d. Differentiation induction by growth and extracellular-matrix factors, including laminin, nicotinamide and insulin, leads to the formation of ES-derived progeny that resembles cells committed to the pancreatic lineage. During differentiation, transcript levels of genes expressed in early pancreatic cells are upregulated. Continued differentiation results in the development of C-peptide/insulin-positive islet-like clusters that release insulin upon glucose stimulation. Differentiated ES cells that overexpress the pancreatic developmental control gene Pax4 develop insulin-secretory granules and reveal functional properties with respect to the pancreas-specific ATP-modulated K+ channel and the normalization of glycemia of streptozotocin-treated diabetic mice.

Endoderm and Pancreatic Islet Lineage Differentiation from Human Embryonic Stem Cells

Human embryonic stem cells (HESCs) are a potential source of insulin-producing tissue for transplantation. Recent studies have begun to define factors that promote definitive endoderm formation from HESCs, but conditions permitting complete islet specification in vitro have not been described. Here, we study spontaneous differentiation of HESCs to definitive endoderm and pancreatic progenitor cells, and begin to determine which aspects of the protocol are required for this cell fate commitment. HESCs were differentiated in culture for up to 10 weeks, including an embryoid body (EB) formation step. Modifications to the protocol included elimination of the EB phase, varying initial cell cluster size when forming EBs, and addition of mesoderm-derived cells to EBs. Differentiated cells were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. HESCs are capable of spontaneous differentiation to cells expressing the definitive endoderm and pancreatic progenitor markers Foxa2, Sox17, and Pdx1, and ultimately, some cells express islet endocrine hormones. This differentiation occurs to a much greater extent when an EB formation step is included. Increased expression of endoderm markers during and after EB formation also correlated strongly with the size of cell clusters used to start EBs, as well as the addition of mesoderm- derived embryonic cells. This study demonstrates that a subset of differentiated HESC progeny adopt an endoderm fate and exhibit the capacity for further pancreatic lineage specification in vitro. Basal conditions were established for examining factors that can commit HESC-derived endoderm cells to specific pancreatic lineages.

Persistent expression of PDX-1 in the pancreas causes acinar-to-ductal metaplasia through Stat3 activation

The transcription factor pancreatic and duodenal homeobox factor 1 (PDX-1) is expressed in pancreatic progenitor cells. In exocrine pancreas, PDX-1 is down-regulated during late development, while re-up-regulation of PDX-1 has been reported in pancreatic cancer and pancreatitis. To determine whether sustained expression of PDX-1 could affect pancreas development, PDX-1 was constitutively expressed in all pancreatic lineages by transgenic approaches. The transgenic pancreas was markedly small with the replacement of acinar cells by duct-like structures, accompanied by activated Stat3. Genetic ablation of Stat3 in the transgenic pancreas profoundly suppressed the metaplastic phenotype. These results provide a mechanism of pancreatic metaplasia by which persistent PDX-1 expression cell-autonomously induces acinar-to-ductal transition through Stat3 activation.

It‘s Endoderm…Definitively!

Evaluation of: D’Amour, KA, Agulnick AD, Eliazer S et al.: Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotech. 23, 1534–1541 (2005) [1] . Human embryonic stem cells (hESCs) offer great promise in the area of regenerative medicine because of their potential to generate vast numbers of transplantable cell types that can be used to cure degenerative diseases. The development of cell therapies for a wide variety of disease states has been severely hampered however, by the inability to control differentiation of hESCs. One area this problem applies to is the generation of pancreatic β-cells. Restoration of β-cell function in diabetic patients, by transplantation of β-cells derived from hESCs, is thought to have potential as a therapeutic strategy. A major problem however, in generating β-cells from hESCs, has been the inability to efficiently generate definitive endoderm – the precursor cell type that gives rise to pancreatic lineages in addition to those of the liver, lung, stomach, intestine and pharynx. In the absence of effective approaches that generate definitive endoderm (DE) in a controlled manner, the likelihood of producing sufficient numbers of β-cells for transplantation purposes is low. This problem appears to have been at least partially circumvented by D’Amour and co-workers who describe a simple, reliable method for the generation of DE from hESCs [1] . This report is likely to open the ‘doors of opportunity’ for the development of specified pancreatic cell types that have therapeutic potential.

Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting

To isolate putative pancreatic stem cells (PSCs) from human adult tissues of pancreas duct using serum-free, conditioned medium. The characterization of surface phenotype of these PSCs was analyzed by flow cytometry. The potential for pancreatic lineage and the capability of beta-cell differentiation in these PSCs were evaluated as well. By using serum-free medium supplemented with essential growth factors, we attempted to isolate the putative PSCs which has been reported to express nestin and pdx-1. The Matrigel(TM) was employed to evaluate the differential capacity of isolated cells. Dithizone staining, insulin content/secretion measurement, and immunohistochemistry staining were used to monitor the differentiation. Fluorescence activated cell sorting (FACS) was used to detect the phenotypic markers of putative PSCs. A monolayer of spindle-like cells was cultivated. The putative PSCs expressed pdx-1 and nestin. They were also able to differentiate into insulin-, glucagon-, and somatostatin-positive cells. The spectrum of phenotypic markers in PSCs was investigated; a similarity was revealed when using human bone marrow-derived stem cells as the comparative experiment, such as CD29, CD44, CD49, CD50, CD51, CD62E, PDGFR-alpha, CD73 (SH2), CD81, CD105(SH3). In this study, we successfully isolated PSCs from adult human pancreatic duct by using serum-free medium. These PSCs not only expressed nestin and pdx-1 but also exhibited markers attributable to mesenchymal stem cells. Although work is needed to elucidate the role of these cells, the application of these PSCs might be therapeutic strategies for diabetes mellitus.

Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development

Studies on nonmammalian vertebrate embryos have indicated that retinoic acid (RA) is required for pancreas development. We have analyzed mouse embryos carrying a null mutation of the gene encoding retinaldehyde dehydrogenase 2 (Raldh2), which controls RA synthesis. Raldh2-/- embryos specifically lack expression of Pdx1 (a homeobox gene required for pancreas development) and Prox1 in dorsal endodermal but not ventral endodermal pancreatic precursor tissues. Ventral endodermal expression of Hex is not affected in Raldh2-/- embryos, indicating that liver specification is not dependent upon RA. Also, expression of Foxa2 across the dorsoventral axis of the endoderm is not affected in Raldh2-/- embryos, indicating that a lack of RA does not cause a general defect in foregut endoderm development. Comparison of wild-type and Raldh2-/- embryos carrying an RA-reporter transgene demonstrates that RA activity is normally present throughout the endoderm except in the ventral-most region but is totally missing in endoderm of Raldh2-/- embryos. Thus, Raldh2 expressed in adjacent splanchnic lateral plate mesoderm provides an RA signal to dorsal endoderm. Dorsal Pdx1 expression is rescued in Raldh2-/- embryos by low-dose maternal administration of RA, which preferentially restores RA-reporter expression in the dorsal endoderm. Our findings demonstrate a specific role for RA in mouse embryos as a mesodermally synthesized signal needed for dorsal endodermal expression of Pdx1 during development of the dorsal pancreatic lineage.

In vitro evidence for pancreatic lineage: Ngn3 positive cells are endocrine progenitors derived from cultured islets

Further studies have been conducted to evaluate the roles of Ngn3 in adult islet maintenance and renewal. Islets were isolated from 6 - 8 week old male C57BL/6 mice. After common bile duct cannulation, the pancreas was resected and digested in collagenase V (2.5 mg/ml). Islets were then handpicked and 10 - 12 islets were plated in 60 mm culture dish and cultivated with RPMI-1640, which contained 12.5 mmol/L HEPES, 5.2 mmol/L glucose and 2% fetal bovine serum (FBS). Islet cells were analyzed by immunocytochemistry methods for A6, insulin, glucagon, nestin, Ngn3 and 5-bromo-2'-deoxy-uridine (BrdU). The results of these studies indicated that less than 15 percent of proliferated islet cells were Ngn3 expressing cells, in which about one third of the Ngn3 positive cells co-expressed A6. The existence of Ngn3 in cultured islet cells is consistent with the results from other's findings both in embryogenesis and adult islet studies. A significant finding of our study is that the existence of A6 and Ngn3 co-expressing cells in the cultured islet. A6 is a marker for identifying bile duct epithelial cell oriented hepatic progenitor cells. Islet-derived A6 cells are possibly born in the adult pancreatic duct and migrate into islets. A6 cells co-express Ngn3 when these cells commit to endocrine lineage within the islets. More interestingly, islet-derived A6 positive cells have the potential to transdifferentiate into hepatic cells. The presence of Ngn3(+) and A6(+) cells in the cultured islets suggests that the four established islet cell types arise from a common endocrine lineage residing within the adult islets. A6 and Ngn3 are useful markers for understanding intra-islet adult stem cell lineages in our future studies. This approach may allow for significant advances in understanding the IPC proliferation and differentiation, and open the possibility of using intra-islet adult stem cells for diabetes treatment.

Dissecting the role of glucocorticoids on pancreas development.

To determine whether glucocorticoids are involved in pancreas development, glucocorticoid treatment of rat pancreatic buds in vitro was combined with the analysis of transgenic mice lacking the glucocorticoid receptor (GR) in specific pancreatic cells. In vitro treatment of embryonic pancreata with dexamethasone, a glucocorticoid agonist, induced a decrease of insulin-expressing cell numbers and a doubling of acinar cell area, indicating that glucocorticoids favored acinar differentiation; in line with this, expression of Pdx-1, Pax-6, and Nkx6.1 was downregulated, whereas the mRNA levels of Ptf1-p48 and Hes-1 were increased. The selective inactivation of the GR gene in insulin-expressing beta-cells in mice (using a RIP-Cre transgene) had no measurable consequences on beta- or alpha-cell mass, whereas the absence of GR in the expression domain of Pdx-1 (Pdx-Cre transgene) led to a twofold increased beta-cell mass, with increased islet numbers and size but normal alpha-cell mass in adults. These results demonstrate that glucocorticoids play an important role in pancreatic beta-cell lineage, acting before hormone gene expression onset and possibly also modulating the balance between endocrine and exocrine cell differentiation.

Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.

The clonal isolation of putative adult pancreatic precursors has been an elusive goal of researchers seeking to develop cell replacement strategies for diabetes. We report the clonal identification of multipotent precursor cells from the adult mouse pancreas. The application of a serum-free, colony-forming assay to pancreatic cells enabled the identification of precursors from pancreatic islet and ductal populations. These cells proliferate in vitro to form clonal colonies that coexpress neural and pancreatic precursor markers. Upon differentiation, individual clonal colonies produce distinct populations of neurons and glial cells, pancreatic endocrine beta-, alpha- and delta-cells, and pancreatic exocrine and stellate cells. Moreover, the newly generated beta-like cells demonstrate glucose-dependent Ca(2+) responsiveness and insulin release. Pancreas colonies do not express markers of embryonic stem cells, nor genes suggestive of mesodermal or neural crest origins. These cells represent a previously unidentified adult intrinsic pancreatic precursor population and are a promising candidate for cell-based therapeutic strategies.

Prospective isolation of multipotent pancreatic progenitors using flow-cytometric cell sorting.

During pancreatic development, neogenesis, and regeneration, stem cells might act as a central player to generate endocrine, acinar, and duct cells. Although these cells are well known as pancreatic stem cells (PSCs), indisputable proof of their existence has not been reported. Identification of phenotypic markers for PSCs leads to their prospective isolation and precise characterization to clear whether stem cells exist in the pancreas. By combining flow cytometry and clonal analysis, we show here that a possible pancreatic stem or progenitor cell candidate that resides in the developing and adult mouse pancreas expresses the receptor for the hepatocyte growth factor (HGF) c-Met, but does not express hematopoietic and vascular endothelial antigens such as CD45, TER119, c-Kit, and Flk-1. These cells formed clonal colonies in vitro and differentiated into multiple pancreatic lineage cells from single cells. Some of them could largely expand with self-renewing cell divisions in culture, and, following cell transplantation, they differentiated into pancreatic endocrine and acinar cells in vivo. Furthermore, they produced cells expressing multiple markers of nonpancreatic organs including liver, stomach, and intestine in vitro. Our data strongly suggest that c-Met/HGF signaling plays an important role in stem/progenitor cell function in both developing and adult pancreas. By using this antigen, PSCs could be isolated prospectively, enabling a detailed investigation of stem cell markers and application toward regenerative therapies for diabetes.

Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type I diabetes

Recent findings suggest that bone marrow (BM) cells have the capacity to differentiate into a variety of cell types including endocrine cells of the pancreas. We report that BM derived cells, when cultured under defined conditions, were induced to trans-differentiate into insulin-producing cells. Furthermore, these insulin-producing cells formed aggregates that, upon transplantation into mice, acquired architecture similar to islets of Langerhans. These aggregates showed endocrine gene expression for insulin (I and II), glucagon, somatostatin and pancreatic polypeptide. Immunohistochemistry also confirmed that these aggregates were positive for insulin, somatostatin, pancreatic polypeptide and C-peptide. Also, Western and ELISA analysis demonstrated expression of proinsulin and/or secretion of active insulin upon glucose challenge. Subcapsular renal transplantation of these aggregates into hyperglycemic mice lowered circulating blood glucose levels and maintained comparatively normal glucose levels for up to 90 days post-transplantation. Graft removal resulted in rapid relapse and death in experimental animals. In addition, electron microscopy revealed these aggregates had acquired ultrastructure typically associated with mature beta (β) cells. These results demonstrate that adult BM cells are capable of trans-differentiating into a pancreatic lineage in vitro and may represent a pool of cells for the treatment of diabetes mellitus.

Flow cytometric isolation of endodermal progenitors from mouse salivary gland differentiate into hepatic and pancreatic lineages.

Experimental injury is useful to induce tissue stem cells, which may exist in small numbers under normal conditions. The salivary glands originate from the endoderm and consist of acinar and ductal epithelial cells, which have exocrine function. After salivary gland duct ligation, acinar cells disappear as a result of apoptosis, and duct epithelium subsequently proliferates. In this study, we analyzed the tissue stem cells induced by salivary gland duct ligation in mice using immunohistochemistry and flow cytometry. We sorted the Sca-1(+)/c-Kit(+) fraction from adult mice salivary glands by way of fluorescence-activated cell sorting. The sorted cells were apparently homogeneous and were designated mouse salivary gland-derived progenitors (mSGPs). mSGP cells differentiated into a hepatic lineage when cultured in matrigel. In spherical culture in the presence of glucagon-like peptide-1 (GLP-1), these cells differentiated into a pancreatic endocrine lineage. When spheroidal bodies of mSGP, 20 to 30 microm in diameter, were transplanted into liver via the portal vein, the cells integrated into hepatic cords and expressed albumin and alpha1-antitrypsin, suggesting that they had differentiated into hepatic-type cells. Moreover, ductlike structures formed by mSGP cells also appeared, epithelial cells of which were positive for cytokeratin 19. In conclusion, fluorescence-activated cell sorting (FACS) based on histologic evidence is efficient in isolating adult tissue stem cells of the salivary gland. Tissue stem cells of endodermal origin (e.g., hepatic oval cells, pancreatic epithelial progenitor cells, and salivary gland progenitor cells) have similarities in their molecular markers and tissue location. Our findings suggest the existence of common tissue stem cells in endoderm-derived organs.

Nestin expression in pancreatic exocrine cell lineages

Expression of nestin has been suggested to be a characteristic of pancreatic islet stem cells. To determine whether nestin is indeed expressed in such putative cells during embryonic development, or in the adult pancreas after injury, we performed a cell lineage analysis using two independent lines of transgenic mice encoding Cre recombinase under the control of rat nestin cis-regulatory sequences, each crossed with loxP-bearing R26R mice. F1 animals produced the reporter molecule β-galactosidase only upon Cre-mediated recombination, thus solely in cells using (or having used) the transgenic nestin promoter. In early pancreatic primordia, β-galactosidase was observed in mesenchymal and epithelial cells. At later developmental stages or in adults, vast clusters of acinar cells and few ductal cells were labeled, in addition to fibroblasts and vascular cells, but no endocrine cells were tagged by β-galactosidase. This correlated with the transient expression, observed with an anti-nestin antibody, of endogenous nestin in about 5% of epithelial cells during development (whether in cord-forming arrangements or in nascent acini), and in vascular and mesenchymal structures. After partial pancreatectomy, there was a transient increase of the number of anti-nestin-labeled endothelial cells, but again, no endocrine cells bore β-galactosidase. Together, these findings show that nestin is expressed in the pancreatic exocrine cell lineage, and suggest that consistent nestin expression is not a major feature of islet endocrine progenitor cells.

Genes, signals, and lineages in pancreas development.

Type I diabetes results from the autoimmune-mediated destruction of pancreatic beta cells, which regulate blood sugar levels by secretion of insulin. Recent clinical data suggest that the disease could be cured if an adequate supply of new beta-cells were available, and one goal of pancreatic developmental biology is to understand how endogenous beta-cells are made, with the hope of making them exogenously. Much is now known about the transcriptional regulation of pancreatic organ specification, growth, and lineage allocation; less is known about intercellular signals that regulate this process, but candidates continue to emerge. Additional insights, often contradicting older models, have come from the application of new lineage-tracing techniques. Altogether, these studies also shed light on the still-elusive pancreatic stem cell, which may participate in normal organ maintenance as well as recovery from injury. A rigorous proof of the existence of such a cell, whether in vivo or in vitro, would offer real hope for the prospect of controlled beta-cell generation in a clinical setting.

Distinct expression patterns of splicing isoforms of mNumb in the endocrine lineage of developing pancreas

The pancreas is composed of three tissues: endocrine, exocrine, and duct. The endocrine/exocrine lineages diverge from the ductal lineage before E12.5 in mice, and then further separate into endocrine and exocrine precursors. These processes are regulated by differential activation of Notch1-mediated signaling, which is required to repress the expression of the pro-endocrine gene neurogenin3 ( ngn3) in the exocrine lineage. Mammalian Numb (mNumb) is an ortholog of Drosophila Numb (dNumb), which is likely to be an intracellular inhibitor of Notch signaling, and has four splicing isoforms: PTBS-PRRS, PTBL-PRRS, PTBS-PRRL, and PTBL-PRRL. Here we developed an anti-PRRL antibody, which recognizes only the PRRL forms of mNumb. We then performed immunohistochemical analyses using anti-PRRL together with anti-pan Numb, which recognizes all the isoforms of mNumb, antibodies that determine the spatio-temporal expression pattern of mNumb in the mouse fetal pancreas. mNumb PRRS and PRRL were first expressed in identical cells in the early stage of pancreatic development (i.e., E10.5), but gradually became biased. At the stage of endocrine and exocrine divergence, mNumb PRRS continued to be expressed in endocrine lineage cells, whereas PRRL was down-regulated during endocrine differentiation. Even after the endocrine/exocrine divergence, notch1 expression was sustained in endocrine lineage, where ngn3 was expressed. These results agree with the notion that mNumb PRRS has an inhibitory effect on Notch signaling, indicating its poten-tial roles in the differentiation of pancreatic endocrine lineage. In addition, islet cells, which are produced from ductal tissue, were immunostained by the anti-panNb antibody. Our present results will contribute to the understanding of the mechanisms of islet development from ductal tissue.

Salivary gland progenitor cells induced by duct ligation differentiate into hepatic and pancreatic lineages

Tissue damage can be assessed based on regenerative responses, including progenitor cell proliferation. In the salivary gland, tissue damage induced by ligation of main ducts leads to the disappearance of acinar cells and to marked proliferation of ductal cells. Reopening of the ducts leads to repopulation of acinar cells within 1 to 2 weeks, which suggests activation of tissue progenitor cells in a damaged state. Because submandibular glands derive from the endoderm and ectoderm, we investigated the possibility of the presence of endodermal progenitor cells. We cultured cells obtained from the ligated salivary gland and identified colonies of epithelium-like cells. We singled out and purified the cells by limited dilution, and one of the cells designated SGP-1 was used for further experiments. The SGP-1 expresses both α6β1 integrin and cytoplasmic laminin. The hematopoietic stem cell marker CD34 and hepatic oval cell markers such as albumin, α-fetoprotein (AFP), and cytokeratin 19 are all negative. However, when SGP-1 cells were transplanted into the liver via the portal vein, these cells were integrated into hepatic trabecula and produced albumin. When SGP-1 cells formed clusters on type I collagen-coated dishes, they differentiated into endodermal lineage and 2 major types of clusters appeared: one contained cells positive for AFP and/or albumin (hepatic cluster) and the other positive for glucagon and/or insulin (pancreatic cluster). On laminin-coated dishes, SGP-1 selectively differentiated into hepatic-type cells. In conclusion, the multipotent progenitor cells isolated from the rat salivary gland have characteristics of tissue stem cells and can differentiate into cells of endodermal lineages. (H epatology 2003;38:104-113.)

Phenotypic Determination and Characterization of Nestin-Positive Precursors Derived from Human Fetal Pancreas

Demand for donations to meet the requirements of pancreas or islet transplantation has prompted the search for alternative sources of β-cell replacement therapy. Earlier studies identified nestin-positive islet-derived progenitor cells (NIPs) residing in human pancreas. In the present study, we isolated and cultured human fetal NIPs that express stem cell marker ABCG2/BCRP1. In confluent cultures, NIPs formed three-dimensional islet-like cell clusters (ICCs). During differentiation, NIP-derived ICCs showed numerous pancreatic lineage transcripts including insulin, whereas ABCG2 and nestin expression fell concomitantly. In addition, ICCs displayed the ability to reverse hyperglycemia in diabetic NOD-SCID mice, as well as infiltrate and form well-differentiated structures in normal mice. These cells can be cloned repeatedly and maintained in long-term culture. Our studies are the first to show NIPs derived from human fetal pancreas, which may have significant implications for future applications in stem cell therapy of diabetes.

Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells.

Mouse embryonic stem (ES) cells differentiate into cells of all three primary germ layers including endodermal cells that produce insulin in vitro. We show that constitutive expression of Pax4 (Pax4(+)), and to a lesser extent Pdx1 (Pdx1(+)), affects the differentiation of ES cells and significantly promote the development of insulin-producing cells. In Pax4 overexpressing R1 ES cells, isl-1, ngn3, insulin, islet amyloid polypeptide, and glucose transporter 2 (Glut-2) mRNA levels increase significantly. The number of nestin-expressing (nestin+) cells also increases. Constitutive Pax4 expression combined with selection of nestin+ cells and histotypic culture conditions give rise to spheroids containing insulin-positive granules typical of embryonal and adult beta cells. In response to glucose, Pax4(+) and wild-type ES-derived cells release insulin. Transplantation of these cells into streptozotocin-treated diabetic mice results in a normalization of blood glucose levels. We conclude that constitutive expression of Pax4 in combination with histotypic cultivation facilitates ES cell differentiation into the pancreatic lineage, which leads to the formation of islet-like spheroid structures that produce increased levels of insulin.