Islet cell cytoplasmic antibody reactivity in midgestational human fetal pancreas

The reactivity of islet cell cytoplasmic antibodies (ICA)-positive and ICA-negative sera of recent onset type 1 diabetic patients was studied in human fetal pancreata of 12–18 weeks' gestation and compared with the reactivity of these sera in adult human control pancreata. The aims of the study were: (1) to observe the presence of ICA staining in human fetal islet cells; (2) to compare endpoint titres (in Juvenile Diabetes Foundation units) of ICA-positive patient sera in fetal pancreata and adult human control pancreata. Ten ICA-positive sera and eight ICA-negative sera from newly diagnosed diabetic patients and four sera from healthy controls were tested on three human adult and eight human fetal pancreata. As in the adult control pancreata. ICA-positive sera reacted to insulin-, glucagon-, and somatostatin-positive cells of fetal pancreata of all gestational ages. This was observed both in single cells and in cells in islet-like cell clusters. Dilution of a reference serum gave similar results in both adult and fetal pancreata. In contrast, the ICA-positive patient sera yielded a striking heterogeneity in fetal as well as in adult pancreata. However, end-point titres between adult and fetal pancreata did not differ significantly (P>0.05). In conclusion, ICA-positive sera from recent onset diabetic patients show that the expression of molecules to which ICA react is present in all islet cells and starts before week 12 of gestation.     

Islet amyloid polypeptide immunoreactivity in the human fetal pancreas

Summary Islet amyloid polypeptide is known to localize to the adult human Beta cell. We analysed the immunoreactivity for islet amyloid polypeptide in a series of 29 human fetal pancreata (9–24 weeks of gestation) with respect to age dependency and cellular localization using an antibody raised against synthetic rat islet amyloid polypeptide 12–37. Cells immunoreactive for islet amyloid polypeptide were demonstrated in low numbers from week 13 onwards while insulin positivity was already present at 9 weeks of gestation. In the age group 13–16 gestational weeks, cells positive for insulin were 20-fold more frequent than cells positive for islet amyloid polypeptide. This difference gradually disappeared with age, reaching parity in the adult gland. Double immunostaining demonstrated that all islet amyloid polypeptide immunoreactivity co-localized with insulin. Co-expression of insulin and islet amyloid polypeptide was more frequent in Beta-cell clusters (10 cells) than in single Beta cells; islet amyloid polypeptide positivity was present in 58±9% (mean±SEM; n=4) of fetal, 88±9 % (n=3) of neonatal and 100% (n=3) of adult clustered Beta cells, and only 8–18 % of the single Beta cells. The results suggest that the developing fetal Beta cells, dependent on age and localization, differ in their capacity to express detectable amounts of immunoreactive islet amyloid polypeptide. Beta-cell maturation might therefore be associated with islet amyloid polypeptide expression.     

Long-term survival of human fetal pancreatic tissue transplanted into an insulin-dependent diabetic patient

The explants of two human fetal pancreases of 15 weeks gestational age were cultured for 6-7 days before being implanted in a 29-year-old insulin-dependent diabetic woman who had received a renal graft two months previously. One pancreas was placed in the flexor muscles of the forearm whilst the other was implanted in an omental pouch. To reduce the chances of rejection the tissue was cultured in vitro, the donor of the tissue placed in the forearm was DR antigen matched with the recipient and the patient remained on cyclosporin and prednisone therapy. At 3 months a mass developed in the forearm muscle at the site of transplantation, and continued to grow. Biopsy at 13 months showed a small area of original pancreas surrounded by a large collection of mature lymphocytes and fibrous tissue. A and D cells could be seen around pancreatic ducts but B cells and acinar tissue were absent. At no stage during follow-up was plasma C-peptide detected in the recipient.     

Ghrelin-producing epsilon cells in the developing and adult human pancreas

Aims/hypothesis  While the mechanisms of specification and the reciprocal relationships of the four types of endocrine cell (alpha, beta, delta and pancreatic polypeptide cells) within the human endocrine pancreas are well described in adults and during fetal development, ghrelin-immunoreactive cells (epsilon cells) remain poorly understood. Methods  We studied epsilon cells in 24 human fetal pancreases between 11 and 39 weeks of development and in 32 pancreases from adult organ donors. Results  We observed single epsilon cells scattered in primitive exocrine tissue from gestational week 13 in developing pancreas. Later in the developmental process, epsilon cells started to aggregate into clusters. From gestational week 21, epsilon cells were observed located around developing islets, forming an almost continuous layer at the peripheral rim of the islets. They remain localised on the mantle of the islets, although at different amounts, in the adult pancreas. Co-production of ghrelin with insulin, glucagon or somatostatin was not detected during fetal development. Co-production with pancreatic polypeptide was evident sporadically. Epsilon cells co-produced NK2 homeobox 2 and ISL LIM homeobox 1, but not NK6 homeobox 1 and paired box 6. A quantitative analysis was performed in the adult pancreas: there was an average of 1.17 + 1.17 epsilon cells per islet, the relative epsilon cell volume was 0.14 + 0.16% and the epsilon cell mass was 0.13 + 0.15 g. Neither sex nor age affected the epsilon cell mass, although there was a significant inverse correlation with BMI. Conclusions/interpretation  During fetal development epsilon cells show an ontogenetic and morphogenetic pattern that is distinct from that of alpha and beta cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

胚胎干细胞体外定向分化为Ngn3+胰岛祖细胞的基因表达谱分析

目的 探讨胚胎干细胞体外定向分化为胰岛祖细胞的基因表达谱.方法 采用逐步诱导分化方案体外传代培养胚胎干细胞,应用RT-PCR和免疫细胞化学方法鉴定各分化阶段相关特异基因的表达,应用Illumina Mouse Ref-8 vl.1小鼠基因表达谱芯片测定胚胎干细胞来源的不同阶段的分化细胞(第4、8、15、20、22和25天)与未分化胚胎干细胞的基因组表达谱.结果 本研究得到了86个阶段特异性表达基因,6组具有相同表达趋势的基因簇,6组阶段差异表达基因组.从数量上看,差异表达基因最多的阶段是后前肠阶段(201个基因),接下来是定形内胚层细胞阶段(17个基因).结论 应用基因芯片对胰腺发育过程中具有相同表达趋势的基因和阶段差异表达基因的分析为早期胚胎发育和胰腺发育研究提供了试验依据.     

Maturation of the response of human fetal pancreatic expiants to glucose

Summary The release of insulin from the human pancreas in response to glucose is known to be either poor or absent in the fetus, whereas in the infant and adult, the response is much greater. The maturation of this response was examined systematically in this study using pancreases that were initially obtained at 14–20 weeks gestation and maintained either in culture alone or by passaging them in diabetic nude mice. Stimulation with glucose was carried out in vitro using tissue of ages ranging from 14 to 58 weeks. A response to glucose was initially seen at 25 weeks and this dramatically increased in the fetal tissue that had reached an age of 55 weeks or more. One of the nude mice used for passage developed normoglycaemia and when the pancreatic implant of age 52 weeks was removed, diabetes recurred. Our findings support the idea of the use of human fetal pancreatic tissue in the treatment of diabetes mellitus.     

Ultrastructural and immunohistochemical analysis of the 8-20 week human fetal pancreas

Development of the human pancreas is well-known to involve tightly controlled differentiation of pancreatic precursors to mature cells that express endocrine- or exocrine-specific protein products. However, details of human pancreatic development at the ultrastructural level are limited. The present study analyzed 8–20 week fetal age human pancreata using scanning and transmission electron microscopy (TEM), TEM immunogold and double or triple immunofluorescence staining. Primary organization of islets and acini occurred during the developmental period examined. Differentiating endocrine and exocrine cells developed from the ductal tubules and subsequently formed isolated small clusters. Extracellular matrix fibers and proteins accumulated around newly differentiated cells during their migration and cluster formation. Glycogen expression was robust in ductal cells of the pancreas from 8–15 weeks of fetal age; however, this became markedly reduced at 20 weeks, with a concomitant increase in acinar cell glycogen content. Insulin secretory granules transformed from being dense and round at 8 weeks to distinct geometric (multilobular, crystalline) structures by 14–20 weeks. Initially many of the differentiating endocrine cells were multihormonal and contained polyhormonal granules; by 20 weeks, monohormonal cells were in the majority. Interestingly, certain secretory granules in the early human fetal pancreatic cells showed positivity for both exocrine (amylase) and endocrine proteins. This combined ultrastructural and immunohistochemical study showed that, during early developmental stages, the human pancreas contains differentiating epithelial cells that associate closely with the extracellular matrix, have dynamic glycogen expression patterns and contain polyhormonal as well as mixed endocrine/exocrine granules.     

Ultrastructural and immunohistochemical analysis of the 8-20 week human fetal pancreas

Development of the human pancreas is well-known to involve tightly controlled differentiation of pancreatic precursors to mature cells that express endocrine- or exocrine-specific protein products. However, details of human pancreatic development at the ultrastructural level are limited. The present study analyzed 8–20 week fetal age human pancreata using scanning and transmission electron microscopy (TEM), TEM immunogold and double or triple immunofluorescence staining. Primary organization of islets and acini occurred during the developmental period examined. Differentiating endocrine and exocrine cells developed from the ductal tubules and subsequently formed isolated small clusters. Extracellular matrix fibers and proteins accumulated around newly differentiated cells during their migration and cluster formation. Glycogen expression was robust in ductal cells of the pancreas from 8–15 weeks of fetal age; however, this became markedly reduced at 20 weeks, with a concomitant increase in acinar cell glycogen content. Insulin secretory granules transformed from being dense and round at 8 weeks to distinct geometric (multilobular, crystalline) structures by 14–20 weeks. Initially many of the differentiating endocrine cells were multihormonal and contained polyhormonal granules; by 20 weeks, monohormonal cells were in the majority. Interestingly, certain secretory granules in the early human fetal pancreatic cells showed positivity for both exocrine (amylase) and endocrine proteins. This combined ultrastructural and immunohistochemical study showed that, during early developmental stages, the human pancreas contains differentiating epithelial cells that associate closely with the extracellular matrix, have dynamic glycogen expression patterns and contain polyhormonal as well as mixed endocrine/exocrine granules.     

Genes expressed in the developing endocrine pancreas and their importance for stem cell and diabetes research

The genes that regulate endocrine pancreas development, maintain adult endocrine cells, and stimulate progenitor/stem cells during regeneration remain largely unstudied. There is ample evidence that many of the genes involved in endocrine pancreas development also function in the homeostasis of the adult islet. In light of the potential benefits to diabetic research, it is surprising that there is little information about the genes expressed throughout the ontogeny of the endocrine pancreas. In the past few years, efforts have been made to establish the Endocrine Pancreas Consortium database (EPConDB), in which many of the genes expressed in the developing endocrine pancreas are in a database with a corresponding publicly available clone bank. In addition, advances in microarray technology now allow for a quantitative expression analysis of thousands of genes simultaneously, which makes it possible to generate a quantitative catalog of the genes expressed at each step of endocrine differentiation, from embryonic endoderm to mature beta cells. In this review, I will discuss how genes discovered by virtue of their role in endocrine pancreas development may function in the maintenance of pancreatic stem cells and the regeneration of islets. I will further summarize the recent advances in genomics-based studies of the developing endocrine pancreas and will discuss how they might impact on the discovery of diagnostics and research into stem cell-based approaches for the treatment of diabetes.     

Proliferation and differentiation in the human fetal endocrine pancreas

Summary The morphogenesis and growth of the endocrine pancreas has not been well investigated in man although it represents an important issue in diabetology. We examined human fetal pancreas from 12 to 41 weeks of gestation immunocytochemically to evaluate proliferative activity with the Ki-67 marker, and cytodifferentiation with cytokeratin 19 (ductal cells), synaptophysin (all endocrine cells), and insulin, glucagon, somatostatin and pancreatic polypeptide (islet cell types). Ki-67 labelling was found in all these cell types but was much higher in ductal cells than in islet cells. An intermediate population expressed synaptophysin but lacked islet hormones. With increasing gestational age the Ki-67 labelling index decreased from 17 to 4 % in ductal cells, from 9 to 1 % in synaptophysin-positive cells, and from 3 to 0.1 % in insulin- or glucagon-positive cells. From 12 to 16 weeks, all epithelial cells including the endocrine islet cells expressed cytokeratin 19. Thereafter cytokeratin 19 expression decreased and eventually disappeared from most islet cells, whereas strong expression remained in the ductal cells. We show that differentiated human islet cells have only very limited proliferative capacity, and we demonstrate the existence of transitional differentiation stages between ductal and islet cells. [Diabetologia (1997) 40: 398–404] Received: 8 November 1996 and in revised form: 7 January 1997     

Insulin secretion by fetal human pancreas in organ culture

Summary Whole fetal human pancreases of 12–22 weeks gestation, showed histological growth and differentiation in vitro over 3 weeks. At glucose concentrations of 1–4 g/l, there was no difference in insulin secretion into culture medium over 1 h. There was no stimulation of insulin release by D-glyceraldehyde, thus defective glucose-stimulated insulin release was probably not due to impairment of an early step in glycolysis. In the presence of 0.5 mmol/l dibutyryl cyclic AMP, insulin secretion was enhanced (0.188±0.030 versus 0.100±0.012 mU·mg tissue^-1·h^-1, p<0.001) independently of glucose concentrations. It thus appears that impairment of glucose-stimulated insulin release was unlikely to be due to insufficient intracellular cyclic AMP. Insulin release increased in response to tolbutamide and theophylline. Insulin secretion was stimulated in the presence of a fivefold increase in amino acid concentration (0.118±0.018 versus 0.031±0.008 mU·mg tissue ^-1·h^-1, p<0.001). There was a fourfold increase in basal insulin secretion from islets previously grown in high concentration of amino acids compared with standard culture medium, (0.284±0.052 versus 0.067±0.011 mU·mg tissue^-1·h^-1, p<0.001), emphasizing the important role of amino acids as substrates for B cell metabolism and development.     

Function and expression of somatostatin receptors of the endocrine pancreas

Somatostatin (SST) regulates multiple biological processes via five genetically distinct, G-protein coupled receptors. Clinical interest in therapy for neuroendocrine and metabolic disorders has resulted in the development of new tools for exploring the function of somatostatin receptors (SSTRs). The development of highly SSTR-selective agonists and antagonists, animal models with the deletion of individual SSTRs, as well as SSTR-specific antibodies have all been utilized in delineating SSTR functions. In the pancreas, SST is a potent regulator of insulin and glucagon secretion. Indeed, the inappropriate regulation of pancreatic A- and B-cell function in metabolic diseases provides an impetus to evaluate the SSTRs as therapeutic targets. By combining the results obtained from molecular biology, pharmacology and immunochemical studies the current review provides a summary of important recent developments which have extended our knowledge of SST actions in the endocrine pancreas.     

Normal response to pregnancy in rats cured of streptozotocin diabetes by transplantation of one fetal pancreas

Summary We have investigated glucose homeostasis and insulin response to glucose in seven rats before, during and after pregnancy, who were previously successfully transplanted with a single fetal pancreas. Increased need for insulin during pregnancy provides an opportunity to test the reserve capacity of the transplanted organ. Plasma glucose in seven rats was normal before pregnancy (7.3±0.7 mmol/l), during pregnancy (6.6 ±1 mmol/l) and after parturition (6.7 ±0.3 mmol/l). Fasting plasma glucose was lower after parturition (5.1±1 mmol/l) than before pregnancy (6.1±0.7 mmol/l). The disappearance rate of injected glucose was the same before (2.3±0.2%/min) as after pregnancy (2.6±0.2%/min). Basal plasma insulin before pregnancy was elevated and there was no rise from glucose; after parturition the basal and pattern of response was normal. The total insulin content of the transplants (859±154 mU) was only 21% of that of normal rats; we conclude that this provides a reserve adequate for the needs of pregnancy.     

Long-term organ culture of large numbers of human fetal pancreata: analysis of their insulin secretion

Cultured human fetal pancreas has been transplanted into diabetic man in an attempt to cure the metabolic disorder. However, the capacity of large numbers of these organs to secrete insulin in organ culture has not been reported previously. This report sets out the characteristics of 321 human fetal pancreases of gestational age 12-20 weeks obtained over a period of 34 months, 295 of these being maintained in organ culture. Average insulin secretion was constant at 2.22 +/- 0.35 mU/plate/day over a 90-day period, the maximal duration of culture possible. Explants were lost because of infection or reduced insulin secretion. Practical guidelines for obtaining and maintaining viable explants in culture were established as follows: Insulin secretion had to be greater than 0.1 mU/plate/day. Secretory rates of this order were associated with a positive insulinogenic response to theophylline. Prostaglandin induction, suction curettage, and hysterotomy were equally suitable as methods of termination of pregnancy. The pancreas had to be obtained within 4 hours of termination of pregnancy. The tissue had to be diced into explants within a further 4 hours.     

Enrichment of Beta cells from the human fetal pancreas by fluorescence activated cell sorting with a new monoclonal antibody

Summary The aim of this study was to produce an antibody reactive to the surface of endocrine pancreatic cells and use this antibody for the purification of endocrine cells from the human fetal pancreas by fluorescence activated cell sorting. We describe such an antibody, called N1, reacting with the surface and cytoplasm of endocrine cells in the adult and fetal human pancreas (12 to 18 weeks gestational age). While unreactive to exocrine and mesenchymal cells, it was not specific for endocrine cells, as evidenced by its staining pattern in tissues other than pancreas. Almost 40% of the N1-positive pancreatic cells contained either insulin, glucagon or somatostatin. Conversely, more than 90% of each of the hormone-containing cells was N1 positive. An additional 40% of N1 positive cells, not containing other pancreatic hormones, was shown to contain islet amyloid polypeptide, synaptophysin, chromogranin, tyrosin hydroxylase or CA812. A two-step collagenase digestion protocol yielded 1.29 ± 0.17 x 10⁵ cells per mg pancreatic tissue. After Percoll gradient centrifugation, the suspension contained 15.6 ± 5.7 % (n = 25, mean ± SD) cells reactive with N1. By fluorescence activated cell sorting using the antibody N1, the single-cell suspension was enriched from 3.0 ± 1.4% to 16.2 ± 4.8% (n = 10,p < 0.01) Beta cells. Alpha and Delta cells were also enriched significantly by this procedure. The percentage of N1-positive cells increased from 17 ± 4 % to 83 ± 6 %. This preparation enriched for endocrine cells allows future studies on possible endocrine precursor cells.     

Effects of endocrine disruptors on the human fetal testis

The modern societies are exposing us to a huge variety of potentially harmful pollutants. Among these endocrine disruptors (EDs) have been especially scrutinized as several were proven to display reprotoxic effects in rodent models. In the context of high and growing concerns about the reprotoxicity of EDs, it is crucial to carry out studies in order to assess their impact on the human reproductive function. However, such evidence remains scarce. The fetal period is critical for the proper development of the testis and is known as a period of high sensitivity to many EDs. Our team has shown in 2009 that a phthalate, mono-(2-ethylhexyl) phthalate (MEHP), has a potential deleterious effect on the development of human male germ cells. This result was the first direct experimental proof of the toxic effect of an ED in human testis. More recently, we also reported that bisphenol A (BPA) impaired testosterone production in the human fetal testis. Here, we will summarize the known effects of EDs on the various cell types composing the human developing testis and discuss their relevancy to propose future directions.     

ICA69 is expressed equally in the human endocrine and exocrine pancreas

Summary Islet cell autoantigen 69 kDa (ICA69) has been reported as a polypeptide antigen expressed in pancreatic beta cells, and autoimmunity against this antigen has been associated with insulin-dependent diabetes mellitus. We have studied the cell type specificity and ontogeny of ICA69 gene expression in man. The ICA69 gene was expressed in all adult human tissues. The level of expression was three-to five-times higher in the pancreas than in the brain, liver, intestine, kidney, spleen, lung or adrenal glands. Pancreatic ICA69 expression increased with age, adult levels being five times higher than the levels present at 13 weeks of gestation. Total RNA from four separate preparations of isolated human islets revealed levels of ICA69 mRNA similar to those found in the pancreas as a whole, although another islet antigen, glutamic acid decarboxylase 65, was highly enriched in the islets. In situ hybridization and immunohistochemical staining of sections of the fetal and adult pancreas revealed expression of the ICA69 gene and protein throughout the acinar, ductal, and islet tissue, but not in the mesenchyme. Analysis of ICA69 mRNA levels in human cell lines indicated expression in neural, endothelial and epithelial cells, but not in fibroblasts. In conclusion, ICA69, although highest in the pancreas, is widely distributed in other human tissues, excluding connective tissue. Within the human pancreas, ICA69 is not enriched in the islets or in the beta cells.Abbreviations GAD Glutamic acid decarboxylase - BSA bovine serum albumin - GLUT2 glucose transporter 2 - bp base pair - PBS phosphate buffered saline - IDDM insulin-dependent diabetes mellitus - HAEC human aortic endothelial cells - ICA islet cell autoantibodies     

Fresh human islet transplantation to replace pancreatic endocrine function in type 1 diabetic patients

The aim of this study was to evaluate the feasibility of islet allografts in patients with type 1 diabetes melititus. Six patients received human islets from either one or two donors via the portal vein, after (n=4) or simultaneously with (n=2) a kidney graft. The patients with functioning kidney grafts (nos. 1–4) were already on triple immunosuppressive therapy (cyclosporine A, azathioprine, prednisone). Prednisone was increased to 60 mg/day for 15 days after the islet transplant in patient 1. Patient 2–4 and the patients who underwent a simultaneous kidney-islets graft (nos. 5, 6) also received antilymphocyte globulin. Intravenous insulin was given for the first 15 days to maintain blood glucose concentrations within the normal range. Patient 1 rejected the islets within 15 days of islet transplantation. In patient 2, a 25% reduction in insulin requirement was observed and 12 months after transplantation post-prandial serum C-peptide was 1.5 ng/ml. In patient 3, the insulin requirement decreased from 40 to 8 units/day with a post-prandial serum C-peptide of 4.1 ng/ml 12 months after islet transplantation. In patient 4 the post-prandial secretion of C-peptide increased to 6.4 ng/ml. Six months after the islet infusion, insulin therapy was discontinued and HbA_1c, 24-h metabolic profile and oral glucose tolerance test remained within the normal range. He had remained off insulin for 5 months until recently, when foot gangrene paralleled a worsening of post-prandial glycaemic control. Twelve months after transplantation he is receiving 8 units insulin/day. Patients 5 and 6 received a simultaneous kidney and islet graft and 6 months after transplantation their post-prandial C-peptide secretion peaks were 2.5 and 1.9 ng/ml respectively. Their daily insulin requirement was not significantly modified. In conclusion, these results show that an adequate number of human islets injected intraportally in type 1 diabetic patients can replace the pancreatic endocrine function and can lead to insulin independence.