Pancreatic development and stem cell-based regenerative medicine

After the gut endoderm is formed, subsequent permissive induction signals from the notochord allows the pancreas to emerge and growout from a specific site of the embryonic gut epithelium. The first pancreas-specific gene, pdx1, is expressed around this stage. Interaction between pancreatic epithelium and the surrounding mesenchyme allows the pancreas bud to further grow and differentiate to form ductal, exocrine or endocrine lineages. Several lines of evidence from gene knockout mice and cell lineage studies suggest that a common pancreas stem cell first gives rise to exocrine and endocrine progenitor cells. The endocrine progenitor then give rise to four different endocrine cells: alpha, beta, delta, and PP cells, although it is not known how and when these cells arise. We have focused our studies on the understanding of endodermal induction and organogenesis of the pancreas. We specifically aimed at the isolation of pancreatic stem cells and the development of functional pancreatic endocrine beta cells in culture. For this aim, we used ES cells as a model system. Here, I review the literature on the development and regeneration of the pancreas. Some recent results on growing pancreatic cells from ES (embryonic stem) cells.     

Rat pancreatic islet is formed by unification of multiple endocrine cell clusters.

The organogenesis of islets in rat pancreas was studied by three-dimensional reconstructions from serial section micrographs. On embryonic day (E) 12, an endocrine cluster consisting mainly of glucagon-expressing cells maintained connection with the pancreatic endoderm at several regions. On E15-E17, the cluster enlarged by fusion of newly formed buds. Although the proportion of insulin-expressing cells increased, they were located in the periphery of the cluster. On the day of birth, insulin-expressing cell clusters enlarged and fused to form several cores within the islet. The glucagon-expressing cell mass expanded to form a thin mantle covering the cores. During islet organogenesis, proliferation activity was high in the exocrine duct system. Moreover, the endocrine cell clusters maintained contact with the duct epithelium throughout. We conclude that the pancreatic islet is generated by the unification of multiple endocrine clusters originated from separate regions of the duct system. The mechanism of mantle-core formation is discussed.     

Xenopus cDNA microarray identification of genes with endodermal organ expression.

The endoderm is classically defined as the innermost layer of three Metazoan germ layers. During organogenesis, the endoderm gives rise to the digestive and respiratory tracts as well as associated organs such as the liver, pancreas, and lung. At present, however, how the endoderm forms the variety of cell types of digestive and respiratory tracts as well as the budding organs is not well understood. In order to investigate the molecular basis and mechanism of organogenesis and to identify the endodermal organ-related marker genes, we carried out microarray analysis using Xenopus cDNA chips. To achieve this goal, we isolated the Xenopus gut endoderm from three different stages of Xenopus organogenesis, and separated each stage of gut endoderm into anterior and posterior regions. Competitive hybridization of cDNA between the anterior and posterior endoderm regions, to screen genes that specifically expressed in the major organs, revealed 915 candidates. We then selected 104 clones for in situ hybridization analysis. Here, we report the identification and expression patterns of the 104 Xenopus endodermal genes, which would serve as useful markers for studying endodermal organ development.     

Fgf10 maintains notch activation, stimulates proliferation, and blocks differentiation of pancreatic epithelial cells.

The pancreas is an endodermally derived organ that initially appears as a dorsal and ventral protrusion of the primitive gut epithelium. The pancreatic progenitor cells present in these early pancreatic anlagen proliferate and eventually give rise to all pancreatic cell types. The fibroblast growth factor receptor (FGFR) 2b high-affinity ligand FGF10 has been linked to pancreatic epithelial cell proliferation, and we have shown previously that Notch signalling controls pancreatic cell differentiation by means of lateral inhibition. In the developing pancreas, activated intracellular Notch appears to be required for maintaining cells in the progenitor state, in part by blocking the expression of the pro-endocrine gene neurogenin 3 (ngn3), and hence endocrine cell differentiation. Here, we show that persistent expression of Fgf10 in the embryonic pancreas of transgenic mice also inhibits pancreatic cell differentiation, while stimulating pancreatic epithelial cell proliferation. We provide evidence that one of the effects of the persistent expression of Fgf10 in the developing pancreas is maintained Notch activation, which results in impaired expression of ngn3 within the pancreatic epithelium. Together, our data suggest a role for FGF10/FGFR2b signalling in regulation of pancreatic cell proliferation and differentiation and that FGF10/FGFR2b signalling affects the Notch-mediated lateral inhibition pathway.     

Expansion of mesenchymal stem cells from human pancreatic ductal epithelium

Fibroblast-like cells emerging from cultured human pancreatic endocrine and exocrine tissue have been reported. Although a thorough phenotypic characterization of these cells has not yet been carried out, these cells have been hypothesized to be contaminating fibroblasts, mesenchyme and/or possibly beta-cell progenitors. In this study, we expanded fibroblast-like cells from adult human exocrine pancreas following islet isolation and characterized these cells as mesenchymal stem cells (MSCs) based on their cell surface antigen expression and ability to differentiate into mesoderm. Analysis by flow cytometry demonstrated that pancreatic MSCs express cell surface antigens used to define MSCs isolated from bone marrow such as CD13, CD29, CD44, CD49b, CD54, CD90 and CD105. In addition, utilizing protocols used to differentiate MSCs isolated from other somatic tissues, we successfully differentiated pancreatic MSCs into: (1) osteocytes that stained positive for alkaline phosphatase, collagen, mineralization (calcification) and expressed osteocalcin, (2) adipocytes that contained lipid inclusions and expressed fatty acid binding protein 4 and (3) chondrocytes that expressed aggrecan. We also demonstrated that pancreatic MSCs are multipotent and capable of deriving cells of endodermal origin. Pancreatic MSCs were differentiated into hepatocytes that stained positive for human serum albumin and expressed endoderm and liver-specific genes such as GATA 4 and tyrosine aminotransferase. In addition, preliminary protocols used to differentiate these cells into insulin-producing cells resulted in the expression of genes necessary for islet and beta-cell development such as Pax4 and neurogenin 3. Therefore, multipotent MSCs residing within the adult exocrine pancreas could represent a progenitor cell, which when further manipulated could result in the production of functional islet beta-cells.     

A novel stepwise differentiation of functional pancreatic exocrine cells from embryonic stem cells

Cultivation of functional pancreatic cells isolated from adult mammalian pancreas remains difficult. We developed a differentiation protocol that gradually induced the formation of mouse pancreatic exocrine cells from embryonic stem cells (ESCs). This process mimicked in vivo pancreatic development by directing cells through definitive endoderm (DE), gut tube endoderm, and pancreatic progenitor cells to differentiated cells that expressed pancreatic exocrine enzymes. Mouse ESCs were cultured in hanging drops to form embryoid bodies. Treatment of embryoid bodies with activin A induced the formation of DE cells that expressed marker mRNAs Goosecoid and Mixl1 and that were double-positive with Foxa2 and Sox17 proteins. Subsequent treatment of the DE cells by retinoic acid induced the formation of gut tube endoderm cells that expressed the specific marker Hnf1b. Expression of Goosecoid and Mixl1 was downregulated during this period. Fibroblast growth factor 7 (FGF7) promoted differentiation of PDX1-expressing pancreatic progenitor cells that also expressed Foxa2 mRNA, an endodermal marker, suggesting derivation from the DE cells. Exocrine cell differentiation was induced with FGF7, glucagon-like peptide-1, and nicotinamide. The differentiated cells expressed mature pancreatic exocrine cell mRNAs, such as Amylase, Elastase, and Carboxypeptidase A. Additionally, they produced pancreatic elastase, amylase, carboxypeptidase A, and chymotrypsin proteins that were identified in cytoplasmic granules by immunocytochemistry. Active amylase was released into the medium. Moreover, FGF7 was associated with differentiation of pancreatic exocrine cells. The findings reported here offer a novel and effective process to develop pancreatic exocrine cells from ESCs.     

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.     

Hnf6 and Tcf2 (MODY5) are linked in a gene network operating in a precursor cell domain of the embryonic pancreas

During pancreatic organogenesis endocrine cells arise from non self-renewing progenitors that express Ngn3. The precursors that give rise to Ngn3+ cells are presumably located within duct-like structures. However, the nature of such precursors is poorly understood. We show that, at E13-E18, the embryonic stage during which the major burst of beta-cell neogenesis takes place, pancreatic duct cells express Hnf1beta, the product of the maturity-onset diabetes of the young type 5 (MODY5) gene. Ngn3+ cells at this stage invariably cluster with mitotically competent Hnf1beta+ cells, and are often intercalated with these cells in the epithelium that lines the lumen of primitive ducts. We present several observations that collectively indicate that Hnf1beta+ cells are the immediate precursors of Ngn3+ cells. We furthermore show that Hnf1beta expression is markedly reduced in early pancreatic epithelial cells of Hnf6-deficient mice, in which formation of Ngn3+ cells is defective. These findings define a precursor cellular stage of the embryonic pancreas and place Hnf1beta in a genetic hierarchy that regulates the generation of pancreatic endocrine cells.     

Notochord repression of endodermal Sonic hedgehog permits pancreas development

Notochord signals to the endoderm are required for development of the chick dorsal pancreas. Sonic hedgehog (SHH) is normally absent from pancreatic endoderm, and we provide evidence that notochord, in contrast to its effects on adjacent neuroectoderm where SHH expression is induced, represses SHH expression in adjacent nascent pancreatic endoderm. We identify activin-βB and FGF2 as notochord factors that can repress endodermal SHH and thereby permit expression of pancreas genes including Pdx1 and insulin. Endoderm treatment with antibodies that block hedgehog activity also results in pancreatic gene expression. Prevention of SHH expression in prepancreatic dorsal endoderm by intercellular signals, like activin and FGF, may be critical for permitting early steps of chick pancreatic development.     

The tyrosine hydroxylase gene is expressed in endoderm and pancreas of early quail embryos

The initial expression of the gene encoding tyrosine hydroxylase (TH) was studied in the trunk of quail embryos by in situ hybridization. We detected the presence of quail TH mRNA on embryonic day 3.5 (E3.5) in the sympathetic ganglia and aortic plexus, both neural crest derived structures. In contrast, the TH gene was expressed much earlier in the endodermal layer of E2 embryos, i. e. from the 8-somite stage onwards. TH mRNA was found also in the pancreatic bud, an endoderm-derived structure. The TH protein and catecholamines were subsequently looked for in these structures. TH immunoreactivity was found in cells of E2 explanted endoderm, but no catecholamine histofluorescence was observed before or after a few days in culture. TH-positive cells were also detected in cultures of pancreatic rudiments, explanted from E3 to E6 quail embryos. We suggest that the TH-positive cells of the endoderm are the progenitors of the catecholaminergic cells of the pancreas and of the enterochromaffin cells of the gut. The hypothesis that the TH-positive cells of the endoderm are involved in the expression of the catecholaminergic phenotype by neural crest cells is discussed.     

Conversion of embryonic stem cells into pancreatic beta-cell surrogates guided by ontogeny

Cellular therapies to treat Type 1 diabetes are being devised and the use of human embryonic stem cells (hESCs) offers a solution to the issue of supply, because hESCs can be maintained in a pluripotent state indefinitely. Furthermore, hESCs have advantages in terms of their plasticity and reduced immunogenicity. Several strategies that have so far been investigated indicate that hESCs are capable of differentiating into insulin producing beta-cell surrogates. However the efficiency of the differentiation procedures used is generally quite low and the cell populations derived are often highly heterogenous. A strategy that appears to have long term potential is to design differentiation procedures based on the ontogeny of the beta-cell. The focus of this strategy is to replicate signaling processes that are known to be involved in the maturation of a beta-cell. The earliest pancreatic progenitors found in the developing vertebrate fetus are produced via a process known as gastrulation and form part of the definitive endoderm germ layer. hESCs have recently been differentiated into definitive endoderm with high efficiency using a differentiation procedure that mimics the signaling that occurs during gastrulation and the formation of the definitive endoderm. Subsequent events during pancreas development involve a section of the definitive endoderm forming into pancreatic epithelium, which then branches into the pancreatic mesenchyme to form islet clusters of endocrine cells. A proportion of the endocrine precursor cells within islets develop into insulin producing beta-cells. The challenge currently is to design hESC differentiation procedures that mimic the combined events of these stages of beta-cell development.     

Hex expression suggests a role in the development and function of organs derived from foregut endoderm.

Hex is a divergent homeobox gene expressed as early as E4.5 in the mouse and in a pattern that suggests a role in anterior-posterior patterning. Later in embryogenesis, Hex is expressed in the developing thyroid, lung, and liver. We now show Hex expression during thymus, gallbladder, and pancreas development and in the adult thyroid, lung, and liver. At E10.0, Hex is expressed in the 3rd pharyngeal pouch, from which the thymus originates, the endodermal cells of liver that are invading the septum transversum, the thyroid, the dorsal pancreatic bud, and gallbladder primoridum. At E13.5, expression is maintained at high levels in the thyroid, liver, epithelial cells lining the pancreatic and extrahepatic biliary ducts and is present in both the epithelial and mesenchymal cells of the lung. Expression in the thymus at this age is less than in the other organs. In the E16.5 embryo, expression persists in the thyroid, pancreatic, and bile duct epithelium, lung, and liver, with thymic expression dropping to barely detectable levels. By E18.5, expression in the thyroid and bile ducts remains high, whereas lung expression is markedly decreased. At this age, expression in the pancreas and thymus is no longer present. Finally, we show the cell types in the adult thyroid, lung, and liver that express Hex in the mature animal. Our results provide more detail on the potential role of Hex in the development of several organs derived from foregut endoderm and in the maintenance of function of several of these organs in the mature animal.