Islet Cell Development Research Articles

Current Progress in Stem Cell Research and its Potential for Islet Cell Transplantation

Diabetes is characterized by insulin deficiency concomitant with hyperglycemia due to reduced islet cell mass and/or dysfunction. Currently, insulin replacement is the first-line treatment option for patients with type 1 and a severe form of type 2diabetes. Treatment by insulin injection is generally effective but nonphysiological, and has the potential of producing chronic complications. On the other hand, islet transplantation can maintain normoglycemia without hypoglycemic side effects, potentially freeing diabetic patients of insulin dependence. In practice, islet transplantation remains hindered by the lack of organ donors and transplant rejection concerns. Recent advances in stem cell research and regenerative medicine, however, offer promise for the clinical application of islet cell transplantation. This review article offers a critical appraisal of current molecular induction approaches, such as directed differentiation, microenvironment induction, and genetic modification, which mimic islet cell development by inducing a variety of stem cells; they include embryonic stem cells, induced pluripotent stem cells, and various tissue-derived stem cells to become functional and transplantable insulin-producing islet cells. Despite good progress, several obstacles remain to be overcome before islet transplantation can be translated into a therapy for human patients, including, but are not limited to, immunogenicity and risk of tumorogenesis. Keywords: Diabetes mellitus, immunogenicity, in vivo regeneration, islet engineering, stem cell therapy, transplantation

Current Progress in Stem Cell Research and its Potential for Islet Cell Transplantation

Diabetes is characterized by insulin deficiency concomitant with hyperglycemia due to reduced islet cell mass and/or dysfunction. Currently, insulin replacement is the first-line treatment option for patients with type 1 and a severe form of type 2 diabetes. Treatment by insulin injection is generally effective but nonphysiological, and has the potential of producing chronic complications. On the other hand, islet transplantation can maintain normoglycemia without hypoglycemic side effects, potentially freeing diabetic patients of insulin dependence. In practice, islet transplantation remains hindered by the lack of organ donors and transplant rejection concerns. Recent advances in stem cell research and regenerative medicine, however, offer promise for the clinical application of islet cell transplantation. This review article offers a critical appraisal of current molecular induction approaches, such as directed differentiation, microenvironment induction, and genetic modification, which mimic islet cell development by inducing a variety of stem cells; they include embryonic stem cells, induced pluripotent stem cells, and various tissue-derived stem cells to become functional and transplantable insulin-producing islet cells. Despite good progress, several obstacles remain to be overcome before islet transplantation can be translated into a therapy for human patients, including, but are not limited to, immunogenicity and risk of tumorogenesis.

Pituitary function and glucose tolerance in a family with a PAX6 mutation

BackgroundPAX6 is a transcription factor involved in the regulation of eye and islet cell development in humans and has also been shown to be an early marker of the pituitary gland in mice. While some subjects with PAX6 mutations were found to have impaired glucose tolerance or diabetes in two previous studies, there has been no report of systematic pituitary function assessment in these patients. AimThe objective of this report was to assess pituitary function and glucose tolerance in five related patients with a heterozygous PAX6 mutation and an unusual ocular and neurological phenotype. Subjects and methodsPituitary function (static and dynamic exploration of the five ante-pituitary axes) and glucose tolerance (oral glucose tolerance test) were explored in all patients. ResultsGlucose tolerance was normal in all patients. We found no obvious pituitary deficiency in four of the five patients. However, borderline cortisol levels were observed in three out of these patients, with subnormal values, at baseline and/or after stimulation test. Basal and stimulated cortisol levels were both more clearly diminished in one subject. ConclusionsWe report here the first complete pituitary function assessment, together with glucose tolerance evaluations, in five related patients with a PAX6 mutation. We cannot rule out subtle corticotrope deficiency induced by PAX6 mutation. The conflicting results with the literature about glucose tolerance could be explained by genotype/phenotype correlations.

Smad signaling plays a key role in regulating islet cell proliferation and development

Smad signaling plays a key role in regulating islet cell proliferation and development

Label-free colorimetric immunoassay for the simple and sensitive detection of neurogenin3 using gold nanoparticles

Neurogenin3 (ngn3), as a marker for pancreatic endocrine precursor cells and an essential ingredient in the development of islet cells, was quantitatively detected for the first time. Based on a non-cross-linking specific interaction mechanism, a label-free colorimetric immunoassay for the synthetic peptide fragment of ngn3 (SKQRRSRRKKAND) using glutathione (-Glu-Cys-Gly, GSH) functionalized gold nanoparticles (GNPs) is reported. The anti-ngn3 antibody conjugated GNPs (GNP-Ab) was formed through electrostatic interaction upon the addition of anti-ngn3 antibody to the GSH-modified GNPs solution. Monobinding of the positively charged ngn3 to the negatively charged GNP-Ab will minimize the electrostatic repulsion between nanoparticles by neutralizing the surface charge, and then agglomeration is induced by an increasing salt concentration. Under the optimal conditions, the assay showed a linear response range of 50–300 ng/mL for the peptide with a detection limit being 20 ng/mL. The preliminary study on ngn3 opens up an innovative insight to detect short synthetic peptide fragment of antigen, and may own an opportunity for practical applications in clinical diagnosis and therapeutics.

Incretin physiology beyond glucagon‐like peptide 1 and glucose‐dependent insulinotropic polypeptide: cholecystokinin and gastrin peptides

Gastrin and cholecystokinin (CCK) are homologous hormone systems known to regulate gastric acid secretion, gallbladder emptying, and cell growth in the pancreas and stomach. They are, however, also involved in the development and secretory functions of pancreatic islet cells. For instance, foetal and neonatal islets express significant amounts of gastrin, and human as well as porcine islet cells express the gastrin/CCK-B receptor abundantly. Therefore, exogenous gastrin and CCK peptides stimulate insulin and glucagon secretion in man. Accordingly, endogenous hypergastrinaemia is accompanied by islet cell hyperplasia and increased insulin secretion. Conventionally, the effect of gastrointestinal hormones on insulin secretion (the incretin effect) has been defined and quantified in relation to oral versus intravenous glucose loadings. Under these unphysiological conditions, the release of gastrin and CCK and, hence, their effect on insulin secretion are modest in comparison with the effects of glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 (GLP-1). Consequently, the interest of CCK and gastrin in incretin research has for decades been limited. A few years ago, however, it was suggested that gastrin together with epidermal growth factor or later GLP-1 might stimulate beta cell growth and secretion. Recent studies have shown that the combination of gastrin and GLP-1 actually restores normoglycaemia in diabetic mice. Therefore, a short review of the incretin system in a broader functional context that includes gastrin and CCK peptides may be timely.

Circadian rhythms and diabetes.

Circadian rhythms and diabetes.

Ff1b, the SF1 ortholog, is important for pancreatic islet cell development in zebrafish

The adrenal cortex and pancreatic islets have endocrine functions, producing steroid-based hormones and insulin, respectively. Cells of the adrenal cortex originate in the mesoderm while the cells of pancreatic islets originate in the endoderm. The zebrafish is a powerful model for understanding organ development due to its ease of genetic and molecular manipulation, transparent embryos, and large number of progeny for statistically powerful experiments. Like humans, the zebrafish pancreas has both exocrine and endocrine functions; unlike humans, there is only one endocrine islet cell group, instead of multiple islets. Using an eGFP-transgenic line of zebrafish, we have observed that the steroidogenic factor 1 ( SF1) ortholog, ff1b, which is critical for adrenal cortex development and function in the zebrafish, is also implicated in zebrafish pancreatic islet development. We show that interruption of ff1b expression using an ff1b-morpholino (MO) disrupts development of insulin expressing cells. We conclude that ff1b-MO alters pancreatic islet development in zebrafish, demonstrating the utility of the zebrafish as a model for studying pancreatic development. This work is consistent with previous studies in mouse and human that have suggested SF1 participates in the vascular and ductal development of the pancreas, and disruption of SF1 function leads to abnormal development of the pancreatic islets due to poor vascularization.

Dachshund homologues play a conserved role in islet cell development

All metazoans use insulin to control energy metabolism, but they secrete it from different cells: neurons in the central nervous system in invertebrates and endocrine cells in the gut or pancreas in vertebrates. Despite their origins in different germ layers, all of these insulin-producing cells share common functional features and gene expression patterns. In this study, we tested the role in insulin-producing cells of the vertebrate homologues of Dachshund, a transcriptional regulator that marks the earliest committed progenitors of the neural insulin-producing cells in Drosophila. Both zebrafish and mice expressed a single dominant Dachshund homologue in the pancreatic endocrine lineage, and in both species loss of this homologue reduced the numbers of all islet cell types including the insulin-producing β-cells. In mice, Dach1 gene deletion left the pancreatic progenitor cells unaltered, but blocked the perinatal burst of proliferation of differentiated β-cells that normally generates most of the β-cell mass. In β-cells, Dach1 bound to the promoter of the cell cycle inhibitor p27Kip1, which constrains β-cell proliferation. Taken together, these data demonstrate a conserved role for Dachshund homologues in the production of insulin-producing cells.

Functional Signature of Human Islet-Derived Precursor Cells Compared to Bone Marrow-Derived Mesenchymal Stem Cells

Pancreatic islet beta-cell replenishment can be driven by epithelial cells from exocrine pancreas via epithelial-mesenchymal transition (EMT) and the reverse process MET, while specified pancreatic mesenchymal cells control islet cell development and maintenance. The role of human islet-derived precursor cells (hIPCs) in regeneration and support of endocrine islets is under investigation. Here, we analyzed hIPCs as to their immunophenotype, multilineage differentiation capacity, and gene profiling, in comparison to human bone marrow-derived mesenchymal stem cells (hBM-MSCs). hIPCs and hBM-MSCs display a common mesenchymal character and express lineage-specific marker genes upon induction toward pancreatic endocrine and mesenchymal pathways of differentiation. hIPCs can go further along endocrine pathways while lacking some core mesenchymal differentiation attributes. Significance analysis of microarray (SAM) from 5 hBM-MSC and 3 hIPC donors mirrored such differences. Candidate gene cluster analysis disclosed differential expression of key lineage regulators, indicated a HoxA gene-associated positional memory in hIPCs and hBM-MSCs, and showed as well a clear transition state from mesenchyme to epithelium or vice versa in hIPCs. Our findings raise new research platforms to further clarify the potential of hIPCs to undergo complete MET thus contributing to islet cell replenishment, maintenance, and function.

Vitamin D and Vitamin A Receptor Expression and the Proliferative Effects of Ligand Activation of These Receptors on the Development of Pancreatic Progenitor Cells Derived from Human Fetal Pancreas

The growth and development of pancreatic islet cells are regulated by various morphogens. Vitamin A modulates in vitro differentiation of islet cells and vitamin D affects beta-cell insulin secretion, while both vitamin ligands act through heterodimerization with the retinoid X receptor (RXR). However, their effects in modulating pancreatic development have not been determined. In this study, cultured human pancreatic progenitor cells (PPCs) isolated from human fetal pancreas were stimulated to differentiate into islet-like cell clusters (ICCs). RT-PCR, Western blotting and immunocytochemistry were used to examine the expression and localization of vitamin D receptor (VDR), retinoic acid receptor (RAR), and RXR in PPCs. The effects of added all-trans retinoic acid (atRA, a form of vitamin A), calcitriol (activated vitamin D) and of these ligands together on PPC cell viability, proliferation and apoptosis were assessed by MTT, BrdU and ELISA assays, respectively. Post-treatment neurogenin-3 (NGN3) expression, necessary for islet-cell lineage development, was examined by real-time RT-PCR. Results showed that RAR, RXR and VDR were expressed in PPCs. RAR and RXR were localized in nuclei, and the VDR in nuclei, cytoplasm and plasma membrane. atRA and calcitriol each increased PPC viability and proliferation; atRA additionally decreased PPC apoptosis. Co-addition of atRA and calcitriol had no additive effects on cell viability but did increase ngn3 responses. In conclusion, RAR, RXR and VDR are expressed in human fetal PPCs and PPC proliferation can be promoted by calcitriol, atRA or both together, data valuable for elucidating mechanisms underlying islet development and for developing clinical islet transplantation.

Quantification of Insulin Gene Expression During Development of Pancreatic Islet Cells

Despite great progress in understanding the transcriptional regulation of the development of insulin-secreting beta cells, the quantitative temporal expression of insulin gene(s) remains largely unknown. We here aimed to quantify insulin gene transcripts during development. We described bioinformatics algorithms to quantify (insulin) gene transcript abundance in sequential microarray data sets at the global level. Several molecular techniques were used to confirm our analyses. We demonstrated that the expression of insulin genes was up-regulated at approximately 14-fold, 700- to 2000-fold, and 5000- to 6000-fold in Pdx1- and Ngn3-expressing cells and adult islets compared with definitive endodermal or embryonic stem cells, respectively. The expression of multiple genes encoding molecules involved in posttranslational modifications of insulin and glucose sensing was also elevated in the same period. All islet and associated genes determined with microarray data were confirmed not only to be up-regulated by real-time quantitative reverse transcriptase polymerase chain reaction but also that the magnitude of their increase quantified with these 2 methods was statistically highly correlated. Consistent with the above, green fluorescence protein expression under the control of the mouse insulin 1 promoter could be visualized in the pancreas from embryonic day (E) 11.5, increasing progressively through E13.5 to E15.5. Our study provides a novel insight into islet developmental biology.

Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development

The transcription factor neurogenin 3 (Neurog3 or Ngn3) controls islet cell fate specification in multipotent pancreatic progenitor cells in the mouse embryo. However, our knowledge of the genetic programs implemented by Ngn3, which control generic and islet subtype-specific properties, is still fragmentary. Gene expression profiling in isolated Ngn3-positive progenitor cells resulted in the identification of the uncharacterized winged helix transcription factor Rfx6. Rfx6 is initially expressed broadly in the gut endoderm, notably in Pdx1-positive cells in the developing pancreatic buds, and then becomes progressively restricted to the endocrine lineage, suggesting a dual function in both endoderm development and islet cell differentiation. Rfx6 is found in postmitotic islet progenitor cells in the embryo and is maintained in all developing and adult islet cell types. Rfx6 is dependent on Ngn3 and acts upstream of or in parallel with NeuroD, Pax4 and Arx transcription factors during islet cell differentiation. In zebrafish, the Rfx6 ortholog is similarly found in progenitors and hormone expressing cells of the islet lineage. Loss-of-function studies in zebrafish revealed that rfx6 is required for the differentiation of glucagon-, ghrelin- and somatostatin-expressing cells, which, in the absence of rfx6, are blocked at the progenitor stage. By contrast, beta cells, whose number is only slightly reduced, were no longer clustered in a compact islet. These data unveil Rfx6 as a novel regulator of islet cell development.

The LIM-Homeodomain Protein ISL1 Activates Insulin Gene Promoter Directly through Synergy with BETA2

The LIM-homeodomain transcription factor ISL1 (islet factor 1) is essential for pancreatic islet cell and dorsal mesenchyme development. Mutations in ISL1 are associated with maturity-onset diabetes of the young and type 2 diabetes. Whether ISL1 plays a role in the insulin gene expression has not been fully elucidated. In the present study, we show that ISL1 can synergistically activate insulin gene transcription with BETA2 in pancreatic β cells. The protein–protein interactions of ISL1 and BETA2 are directly mediated by the LIM domains of ISL1 and the basic helix–loop–helix domain of BETA2. Deletion of the two LIM domains of ISL1 enhances the transcriptional activation of the insulin gene, indicating a key role for the homeodomain in activating the insulin promoter. Furthermore, ISL1 can bind with the A3/4 box in the rat insulin gene І promoter through its homeodomain. ISL1 expression is up-regulated at the mRNA level in type 2 diabetes (db/db mouse model) but down-regulated by dexamethasone in rat insulinoma cells. These results suggest that ISL1 is a transcriptional activator for insulin gene expression, and the interactions of ISL1 with BETA2 are required for the transcriptional activity of the insulin gene. Reduction in Isl1 gene expression appears to be involved in the impairment of insulin expression mediated by dexamethasone.

Tumour suppressor menin is essential for development of the pancreatic endocrine cells

Mutations of the multiple endocrine neoplasia type 1 (MEN1) gene predispose patients to MEN1 that affects mainly endocrine tissues, suggesting important physiological functions of the gene in adult endocrine cells. Homozygous disruption of Men1 in mice causes embryonic lethality, whereas the eventual involvement of the gene in embryonic development of the endocrine cells remains unknown. Here, we show that homozygous Men1 knockout mice demonstrate a reduced number of glucagon-positive cells in the E12.5 pancreatic bud associated with apoptosis, whereas the exocrine pancreas development in these mice is not affected. Our data suggest that menin is involved in the survival of the early pancreatic endocrine cells during the first developmental transition. Furthermore, chimerism assay revealed that menin has an autonomous and specific effect on the development of islet cells. In addition, using pancreatic bud culture mimicking the differentiation of alpha- and beta-cells during the second transition, we show that loss of menin leads to the failure of endocrine cell development, altered pancreatic structure and a markedly decreased number of cells expressing neurogenin 3, indicating that menin is also required at this stage of the endocrine pancreas development. Taken together, our results suggest that menin plays an indispensable role in the development of the pancreatic endocrine cells.

GeneSpeed Beta Cell: An Online Genomics Data Repository and Analysis Resource Tailored for the Islet Cell Biologist

Objective. We here describe the development of a freely available online database resource, GeneSpeed Beta Cell, which has been created for the pancreatic islet and pancreatic developmental biology investigator community. Research Design and Methods. We have developed GeneSpeed Beta Cell as a separate component of the GeneSpeed database, providing a genomics-type data repository of pancreas and islet-relevant datasets interlinked with the domain-oriented GeneSpeed database. Results. GeneSpeed Beta Cell allows the query of multiple published and unpublished select genomics datasets in a simultaneous fashion (multiexperiment viewing) and is capable of defining intersection results from precomputed analysis of such datasets (multidimensional querying). Combined with the protein-domain categorization/assembly toolbox provided by the GeneSpeed database, the user is able to define spatial expression constraints of select gene lists in a relatively rigid fashion within the pancreatic expression space. We provide several demonstration case studies of relevance to islet cell biology and development of the pancreas that provide novel insight into islet biology. Conclusions. The combination of an exhaustive domain-based compilation of the transcriptome with gene array data of interest to the islet biologist affords novel methods for multidimensional querying between individual datasets in a rapid fashion, presently not available elsewhere.

Clusterin Expression in Porcine Endocrine Cells during Islet Development

Clusterin is a well-known glycoprotein expressed by many cell types involved in multiple physiological functions. In rat pancreatic tissue it is expressed along with islet cell development and found to be involved in regeneration of pancreatic endocrine cells after various types of tissue injuries. These results led us to propose that clusterin might play a crucial role in organization and assembling processes of islet cells during pre- and postnatal development. Therefore, the aim of this study was to find out whether and in which cell type clusterin is expressed during islet cell organization in the porcine species which could play a future role in the field of xenotransplantation. For this purpose we examined the expression pattern of clusterin at different developing stages in the porcine pancreas by double-immunostaining with antibodies against chromogranin A and clusterin, and clarified whether distinct islet hormones were coexpressed with clusterin. Further, we checked by RT-PCR whether clusterin was locally expressed or possibly locally bound to the corresponding receptor. In newborn and up to 3-month-old animals clusterin was found to be expressed in a special cell type which is closely associated and intermingled with other endocrine cells. In fully developed adult islets clusterin-cells then reorganize and were found to be mainly localized in the mantle area of Langerhans islets. Double-immunostaining with antibodies against clusterin and different islet hormones such as insulin, glucagon, and somatostatin clearly demonstrate that clusterin expression was found in an own special cell type and it is also present in a subset of glucagon producing A-cells. Taken together, our results show that clusterin expression in porcine species is found in an own, as yet unidentified cell type during postnatal developmental stages, and probably labels immature precursor cells in adult animals, which finally have the potential to differentiate into glucagon-expressing cells.

Pancreatic Endocrine and Exocrine Cell Ontogeny From Renal Capsule–transplanted Embryonic Stem Cells in Streptozocin-injured Mice

In this study, we describe pancreatic cell ontogeny in renal capsule-transplanted embryonic stem cells (ES) after injury by streptozocin (STZ), showing pancreatogenesis in situ. Seven-week-old female BALB/c nude mice were treated with either a single 175- or 200-mg/kg STZ dose, a regimen that induces substantial beta-cell damage without overt hyperglycemia, and transplanted 24 hr later with 1 x 10(5) ES. Immunohistochemistry was performed on ES tissue at 15, 21, and 28 days after transplantation using antibodies against stage- and lineage-specific pancreatic markers. After 21 days, PDX-1+ pancreatic foci first appeared in the renal capsule and expressed both amylase and endocrine hormones (insulin, glucagon, and somatostatin). These foci increased in size by day 28 because of acinar and duct cell proliferation, whereas endocrine cells remained non-dividing, and made up 2-4% of ES tumor volume. PDX-1, Nkx6.1, Ngn3, and ISL-1 protein localization patterns in pancreatic foci were comparable with embryonic pancreatogenesis. A prevalence of multihormonal endocrine cells, a characteristic of adult beta-cell regeneration, indicated a possible divergence from embryonic islet cell development. The results indicate that beta-cell damage, without overt hyperglycemia, induces a process of fetal-like pancreatogenesis in renal capsule-transplanted ES, leading to beta-cell neogenesis.

Genome-Wide Mapping of in Vivo Protein-DNA Interactions

In vivo protein-DNA interactions connect each transcription factor with its direct targets to form a gene network scaffold. To map these protein-DNA interactions comprehensively across entire mammalian genomes, we developed a large-scale chromatin immunoprecipitation assay (ChIPSeq) based on direct ultrahigh-throughput DNA sequencing. This sequence census method was then used to map in vivo binding of the neuron-restrictive silencer factor (NRSF; also known as REST, for repressor element-1 silencing transcription factor) to 1946 locations in the human genome. The data display sharp resolution of binding position [+/-50 base pairs (bp)], which facilitated our finding motifs and allowed us to identify noncanonical NRSF-binding motifs. These ChIPSeq data also have high sensitivity and specificity [ROC (receiver operator characteristic) area >/= 0.96] and statistical confidence (P <10(-4)), properties that were important for inferring new candidate interactions. These include key transcription factors in the gene network that regulates pancreatic islet cell development.

Does IGF-I stimulate pancreatic islet cell growth?

Both IGF-I and its receptor (IGF-IR) are specifically expressed in various cell types of the endocrine pancreas. IGF-I has long been considered a growth factor for islet cells as it induces DNA synthesis in a glucose-dependent manner, prevents Fas-mediated autoimmune beta-cell destruction and delays onset of diabetes in non-obese diabetic (NOD) mice. Islet-specific IGF-I overexpression promotes islet cell regeneration in diabetic mice. However, in the last few years, results from most gene-targeted mice have challenged this view. For instance, combined inactivation of insulin receptor and IGF-IR or IGF-I and IGF-II genes in early embryos results in no defect on islet cell development; islet beta-cell-specific inactivation of IGF-IR gene causes no change in beta-cell mass; liver- and pancreatic-specific IGF-I gene deficiency (LID and PID mice) suggests that IGF-I exerts an inhibitory effect on islet cell growth albeit indirectly through controlling growth hormone release or expression of Reg family genes. These results need to be evaluated with potential gene redundancy, model limitations, indirect effects and ligand-receptor cross-activations within the insulin/IGF family. Although IGF-I causes islet beta-cell proliferation and neogenesis directly, what occur in normal physiology, pathophysiology or during development of an organism might be different. Locally produced and systemic IGF-I does not seem to play a positive role in islet cell growth. Rather, it is probably a negative regulator through controlling growth hormone and insulin release, hyperglycemia, or Reg gene expression. These results complicate the perspective of an IGF-I therapy for beta-cell loss.