Single Endocrine Cells Research Articles

Pancreatic endocrine cell arrangement during human ontogeny

In the human pancreas, various forms of endocrine cell arrangement are found: single endocrine cells, endocrine cell clusters, and mantel, bipolar and mosaic cell (mixed) islets. Our aim was to analyse the distribution and dynamics of insulin-, glucagon- and somatostatin-containing cells within the various forms of endocrine pancreas arrangement during human prenatal development and in adults and to suggest a mechanism of change in the endocrine cell ratio in adult islets. Pancreatic autopsies derived from human foetuses from the 10th to the 40th weeks of development and from adults were examined using histological, immunohistochemical and morphometric methods. During development, the human endocrine pancreas undergoes not only de novo differentiation of endocrine cells and islet formation, but morphogenetic restructuring, which is revealed as a change of the α-, β- and δ-cell ratio in the islets. In particular, increased proportion of glucagon- and somatostatin-containing cells and decreased proportion of β-cells were shown in the largest mosaic islets in adults. Our results indicate that the distribution and proportion of α-, β- and δ-cells depend on the islets size and vascularisation. Studying of the mechanism of such restructuring may contribute to the development of new approaches in the treatment of diabetes mellitus.

Pancreatic neurogenin 3-expressing cells are unipotent islet precursors

Pancreatic islet endocrine cells arise during development from precursors expressing neurogenin 3 (Ngn3). As a population, Ngn3(+) cells produce all islet cell types, but the potential of individual Ngn3(+) cells, an issue central to organogenesis in general and to in vitro differentiation towards cell-based therapies, has not been addressed. We performed in vivo clonal analyses in mice to study the proliferation and differentiation of very large numbers of single Ngn3(+) cells using MADM, a genetic system in which a Cre-dependent chromosomal translocation labels, at extremely low mosaic efficiency, a small number of Ngn3(+) cells. We scored large numbers of progeny arising from single Ngn3(+) cells. In newborns, labeled islets frequently contained just a single tagged endocrine cell, indicating for the first time that each Ngn3(+) cell is the precursor of a single endocrine cell. In adults, small clusters of two to three Ngn3(+) progeny were detected, but all expressed the same hormone, indicating a low rate of replication from birth to adult stages. We propose a model whereby Ngn3(+) cells are monotypic (i.e. unipotent) precursors, and use this paradigm to refocus ideas on how cell number and type must be regulated in building complete islets of Langerhans.

Topographical associations between islet endocrine cells and duct epithelial cells in the adult human pancreas

The pancreatic ducts, endocrine islets and exocrine acini are three functionally related components. From birth to adulthood, the islets and ducts are regarded as independent entities. The objective of this study is to investigate the topographical associations between the islet endocrine cells and duct epithelial cells in the adult human pancreas. Panels of immunomarkers for the exocrine acinar cells (amylase), duct cells [cytokeratin 19 (CK19)], endocrine cells (chromogranin A, neurone specific enolase, synaptophysin) and islet hormones (glucagon, insulin, somatostatin, pancreatic polypeptide) were applied to sequential pancreatic tissue sections obtained from autopsy specimens of 10-nondiabetic human adults. Double immunofluorescent staining with CK19 and islet hormones was performed to confirm the islet to duct interrelationship. Sequential sectioning and immunostaining showed that 45% of the 172 islets examined appeared as single endocrine cell units or small clusters of < 10 endocrine cells on at least one plane of section. A topographical association was found between the islet endocrine cells and duct epithelial cells. Topographical associations with CK 19-stained duct cells occurred in 10.9% of the islet insulin-containing beta-cells and in 8.9% of the islet glucagon-producing alpha-cells. The frequency of topographical associations increased toward the more distally located duct systems. The CK19-stained duct cells and amylase-labelled acinar cells were less frequently in association with other islet hormone-producing cells. Topographical associations between islet endocrine cells and pancreatic duct cells are frequent in adult human pancreas. The islet-duct association suggests possible functional interactions between the two interrelated pancreatic compartments.

An immunohistochemical study of the endocrine pancreas of the African ice rat, Otomys sloggetti robertsi

The African ice rat, Otomys sloggetti robertsi, is a member of the subfamily Otomyinae, in the superfamily of Muroidea, to which all rodents belong. Very little is known about this unique family of rodents. The study reported here examines the endocrine pancreas of this species using immunohistochemical techniques. The islets of Langerhans were scattered in the exocrine pancreas and tended to be quite small. Scattered single endocrine cells (mostly immunoreactive for insulin) were found in the exocrine pancreas and were not generally associated with ducts (as marked by pan-cytokeratin labeling). The normal islet architecture of insulin in the center and glucagon, somatostatin (SS) and pancreatic polypeptide (PP) in the rim was observed, but the islets tended to have 2-3 layers of glucagon immunoreactive cells. Examining for rarer endocrine cell types, we found that cocaine amphetamine regulated transcript (CART) immunoreactive cells were co-localized with SS; and peptide YY (PYY) immunoreactive cells could be found that were singly immunoreactive or co-localized with either PP or glucagon. Ghrelin cells were not found. MafA co-localized only with the insulin cells, while MafB, which localizes to the glucagon cells, also showed a low level of immunoreactivity in most insulin immunoreactive cells. The Nkx family of transcription factors (Nkx6.1 and 2.2) and PDX-1 were all detected in the pancreas in a similar manner to that seen in mouse and rat. In conclusion, the endocrine pancreas of the African ice rat is quite similar to that of other studied rodents, but these animals have more glucagon and SS cells than rat (Rattus) or mouse (Mus) species.

Quantification of mRNA in single cells and modelling of RT-qPCR induced noise.

BackgroundGene expression has a strong stochastic element resulting in highly variable mRNA levels between individual cells, even in a seemingly homogeneous cell population. Access to fundamental information about cellular mechanisms, such as correlated gene expression, motivates measurements of multiple genes in individual cells. Quantitative reverse transcription PCR (RT-qPCR) is the most accessible method which provides sufficiently accurate measurements of mRNA in single cells.ResultsLow concentration of guanidine thiocyanate was used to fully lyse single pancreatic β-cells followed by RT-qPCR without the need for purification. The accuracy of the measurements was determined by a quantitative noise-model of the reverse transcription and PCR. The noise is insignificant for initial copy numbers >100 while at lower copy numbers the noise intrinsic of the PCR increases sharply, eventually obscuring quantitative measurements. Importantly, the model allows us to determine the RT efficiency without using artificial RNA as a standard. The experimental setup was applied on single endocrine cells, where the technical and biological noise levels were determined.ConclusionNoise in single-cell RT-qPCR is insignificant compared to biological cell-to-cell variation in mRNA levels for medium and high abundance transcripts. To minimize the technical noise in single-cell RT-qPCR, the mRNA should be analyzed with a single RT reaction, and a single qPCR reaction per gene.

Mucosal endocrine cell micronests and single endocrine cells following neo-adjuvant therapy for adenocarcinoma of the distal oesophagus and oesophagogastric junction

Aims:To determine the frequency of endocrine cell micronests (ECM) and single endocrine cells (SEC) within the glandular mucosa of the distal oesophagus and oesophagogastric junction (OGJ) following neo-adjuvant therapy for...

Translational Mini-Review Series on Type 1 Diabetes: Immune-based therapeutic approaches for type 1 diabetes

Type 1 diabetes (T1D) is often considered the prototype organ-specific autoimmune disease in clinical immunology circles. The key disease features - precise destruction of a single endocrine cell type occurring on a distinct genetic and autoimmune background - have been unravelled in recent years to such an extent that there is a growing expectation that the disease should be curable. T1D is something of an orphan disease, currently managed by endocrinologists yet dependent upon the wit of immunologists, both basic and clinical, to find the best approaches to prevention and cure. Type 1 diabetes thus represents one of the most active arenas for translational research, as novel immune-based interventions find their way to the clinic. The first serious attempt at immune-based treatment for T1D was in 1984, the first at prevention in 1993; current and planned trials will take us into the next decade before reporting their results. This paper represents the first attempt at a comprehensive review of this quarter century of endeavour, documenting all the strategies that have emerged into clinical studies. Importantly, the intense clinical activity has established robust infrastructures for future T1D trials and frameworks for their design. The evident success of the monoclonal anti-CD3 antibody trials in established T1D demonstrate that modulation of islet autoimmunity in humans after the onset of overt disease can be achieved, and give some reason to be cautiously optimistic for the ability of these and other agents, alone and in combination, to provide an effective immunotherapy for the disease.

Melanotrope cells as a model to understand the (patho)physiological regulation of hormone secretion

Regulation of hormone secretion is a complex process that comprises the sequential participation of numerous subcellular mechanisms. Hormone secretion is dictated by extracellular stimuli that are transduced intracellularly into activation/deactivation of different mechanisms, such as hormone expression, processing and exocytosis, which will ultimately determine the precise availability of hormone to be secreted. Malfunction in any of these steps may result in deficient or excessive hormone release and the subsequent appearance of endocrine disorders. Given the complexity of this system, it is difficult to find appropriate cellular models wherein to investigate the multiple components of the secretory process in a physiologically relevant, experimentally manipulable setting. In this review, we present recent evidence on the use of the intermediate lobe (IL) of the pituitary as a powerful tool to understand different aspects of the regulated secretory pathway. IL is composed of a single endocrine cell type, alpha-melanocyte stimulating hormone (alpha-MSH)-producing melanotropes, a fact that greatly facilitates its study. Furthermore, melanotropes can be separated using classic cell separation techniques into two cell subtypes showing opposite morphophysiological phenotypes of hypo- and hypersecretory cells. Comparison of their gene expression fingerprints has unveiled the existence of certain genes preferentially expressed in each melanotrope subtype. Because of their direct participation in the secretory pathway, we postulate that characterization of these gene products in an endocrine cell type may represent novel and useful markers for reliably determining the general secretory status in an endocrine gland, as well as a valuable new tool to further investigate this complex process.

Murine prenatal expression of cholecystokinin in neural crest, enteric neurons, and enteroendocrine cells.

Cholecystokinin (CCK) is a regulatory peptide that is primarily expressed in two adult cell types: endocrine cells of the intestine and neurons of the central nervous system. To determine the ontogeny of CCK expression during intestinal organogenesis, we created a mouse strain in which the CCK gene was replaced by a lacZ reporter cassette using homologous recombination in embryonic stem cells. Initially, CCK expression in the developing intestine was limited to the myenteric plexus of the enteric nervous system. This expression pattern was widespread, extending from the proximal stomach into the colon, yet transient, being detected soon after gut tube closure [embryonic day 10.5 (E10.5)] through E15.5. Since enteric neurons are derived from the neural crest, we examined earlier (E8.5-9.5) embryos and concluded that lacZ was expressed in subpopulations of neural tube and neural crest cells. Endocrine cell expression in the intestinal epithelium occurred later, beginning at E15.5 as enteric neuronal expression was dwindling. This expression persisted to yield the adult pattern of scattered single endocrine cells in the upper small intestine. The data show that CCK is a very early marker of both neuronal and endocrine cell lineages in the developing gastrointestinal tract. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that CCK receptor transcripts were detected in embryos as early as E10.5, suggesting that CCK signaling is established early in mouse development. Dev Dyn 1999;216:190-200.

Differential effects of dopamine on two frog melanotrope cell subpopulations.

The frog intermediate lobe consists of a single endocrine cell type, the melanotrope cells, which are under the tonic inhibitory control of dopamine. Separation of dispersed pars intermedia cells in a Percoll density gradient has revealed the existence of two melanotrope cell subpopulations, referred to as high-density (HD) and low-density (LD) cells. The aim of the present study was to investigate the effects of dopamine on each of these melanotrope cell subsets. Increasing doses of dopamine, ranging from 10(-9)-10(-6) M, inhibited the release of alpha-melanocyte-stimulating hormone (alpha-MSH) in LD (but not in HD) melanotrope cells. In addition, dopamine provoked a significant reduction of the rate of acetylation of alpha-MSH in LD cells but not in HD cells. Similarly, dopamine significantly decreased the accumulation of POMC messenger RNA in LD cells, whereas it did not affect POMC gene expression in the HD melanotrope subset. On the other hand, microfluorimetric studies revealed that dopamine induced a significant reduction of KCl-stimulated cytosolic free calcium concentration in both LD and HD cells. The present study provides additional evidence for functional heterogeneity of melanotrope cells in the frog pars intermedia. Because dopamine plays a pivotal role in the regulation of alpha-MSH secretion, these data suggest the involvement of cell heterogeneity in the physiological process of background color adaptation in amphibians.

Hamster pulmonary endocrine cells with positive immunostaining for calbindin-D28K.

We have examined the distribution of calcium-binding proteins (CaBPs) in adult and fetal lungs of Syrian golden hamsters (Mesocricetus auratus) using immunostaining with confocal laser microscopy and electron microscopy. Single and grouped (neuroepithelial body; NEB) endocrine cells were distributed from bronchi to alveolar ducts in the adult lung. Serial frozen sections immunostained for CaBPs in combination with immunostaining for endocrine markers such as calcitonin gene-related peptide, serotonin, PGP9.5, and synaptophysin revealed that positive immunostaining for calbindin-D28K (CB-D28K) was seen in single endocrine cells and NEBs. However, other so-called EF-hand family CaBPs, parvalbumin and calretinin, were not detected. Electron microscopically, positive immunoreaction for CB-D28K was mainly in the organelle-free cytoplasmic matrix of endocrine cells, and partly in nuclei and associated with secretory granules and endoplasmic reticulum. In fetal developing lungs, endocrine cells appeared first on gestational day 13, and they were positive for all the endocrine markers used. However, pulmonary endocrine cells were positively immunostained for CB-D28K from gestational days 15 and 16 onward. In summary, our observations suggest that CB-D28K is a useful marker for endocrine cells of the lung, and CB-D28K could function as a mediator of endocrine stimulation or calcium homeostasis in pulmonary endocrine cells.

Ductal Cyst Formation in Collagen-Embedded Adult Human Islet Preparations: A Means to the Reproduction of Nesidioblastosis In Vitro

Neogenesis of endocrine islets from ductal epithelium termed nesidioblastosis has been described in vivo after various experimental conditions (90% pancreatectomy or pancreas wrapping in the rodent) and in clinical pathologies. In the adult regenerating pancreas, a proliferation and organization of ductal epithelium into tubular structures precedes its differentiation into endocrine cells. Reproduction of nesidioblastosis in vitro may provide a novel approach to human islet propagation in vitro. With this aim, adult human islet preparations were cultured in diverse three-dimensional (3D) gels in the presence of serum. After 3-5 days in rat tail collagen gels, proliferating (bromodeoxyuridine-positive) cystic structures appeared associated with islets and as isolated spheres. Percentage labeling indexes of the cysts were 4.1, 18.7, 15.4, and 13.3% after 3, 5, 7, and 10 days of culture, respectively. Immunohistochemistry confirmed the ductal (carbohydrate antigen 19-9) and epithelial (keratin-1) nature of the cysts. No cysts were formed in agarose gels or Vitrogen 100, whereas the cyst number was increased by the quantity of serum (20% > 10%) and gels rich in extracellular matrix components and growth factors (Matrigel). The latter lead to tubular networks. Single endocrine islet cells were observed in the ductal cysts after 7 (2.8%) to 10 (5.6%) days in rat tail collagen. Our observations paralleled the changes characteristic of the regenerating pancreas in vivo. 3D culture may permit the identification of matrix and media constituents promoting the neogenesis of islets and may be the means to increase the mass of endocrine tissue obtained from adult cadaveric pancreases for transplantation.

Hamster pulmonary endocrine cells with neural cell adhesion molecule (NCAM) immunostaining

Pulmonary endocrine cells of Syrian golden hamster were stained for neural cell adhesion molecule (NCAM) with indirect fluorescent immunostaining and observed with a confocal laser scanning microscope equipped with an argon laser. Sections 100 microns thick of hamster lung fixed with phosphate-buffered 4% paraformaldehyde were prepared. The sections were incubated with rat monoclonal antibody against NCAM, followed by fluorescence-labeled antibody against rat immunoglobulin. Some were doubly immunostained for NCAM and one of the following endocrine markers: neuron-specific enolase, calcitonin gene-related peptide and serotonin. Expression of NCAM in the hamster airway epithelium was seen in cell nests resembling neuroepithelial bodies (NEBs). NCAM immunostaining was positive at the lateral cell borders between the cells composing the nest, but negative at the border with the adjacent, presumably non-endocrine cells. Double immunostaining confirmed that the grouped cells with NCAM immunoreactivity were of an endocrine nature, but that single endocrine cells did not show NCAM immunoreactivity. An electron microscopic study with NCAM immunostaining confirmed the light microscopic study. These suggest that NCAM expression could be important for the morphogenesis of NEBs. A confocal laser microscope was used to make three-dimensional images of NEBs after NCAM immunostaining and the spatial interaction between NEBs and the surrounding microenvironment was studied.

Endocrine tissue associated with the pancreatic ductal system: A light and electron microscopic study of the adult rat pancreas with special reference to a new endocrine arrangement

A substantial part of the endocrine pancreas has been previously described as being located either close to the excretory ducts as small clusters of endocrine cells and as Islets of Langerhans, or associated with the ducts as single endocrine cells scattered through the ductal epithelium. Four Wistar white adult rats were sacrificed and perfused via the thoracic aorta with 2.5% glutaraldehyde. After the usual treatment for the transmission electron microscopy, pieces of pancreas were sectioned consecutively for light microscopy. Consecutive ultrathin sections were performed in the most interesting cases. The observations previously reported were confirmed. In addition, a new endocrine arrangement was detected and described as buds of endocrine cells (mainly B-cells) protruding from the ductal epithelium into the surrounding tissue. The authors propose to explain the endocrine buds as components of the gastro-entero-pancreatic system or as a stage of an endocrine pancreatic "neo-histogenesis" occurring in the adult rat pancreas.

Production of Regulatory Factors in the Respiratory System of Vertebrates

Among the different cell types present in the respiratory tract of the vertebrates, some (epithelial, endothelial, neural) specialise in the production of regulatory factors. Endocrine cells occur either single, spread throughout the epithelial lining, or in innervated groups, called 'neuroepithelial bodies' (NEBs). In mammals, these endocrine cells may be involved in lung maturation during perinatal life and in chemoreception. A neuroendocrine diffuse system is present in the respiratory organs of all classes of vertebrates. In amphibians and reptiles, single endocrine cells as well as NEBs are located in the apices of the lung septa. The respiratory tract shows nerve fibres immunoreactive to several neuropeptides. Since some neurons and fibres contain NO synthase a broad evolutionary presence of NO-releasing neurons, probably involved in the control of relaxation, is suggested.

Analysis of the secretion from single anterior pituitary cells by cell immunoblot assay.

The CIBA is very useful in quantifying hormone secretion from single endocrine cells. Variations of this assay can extend its power for evaluating the proportion of hormone-secreting cells in a cell population, determining the responsiveness of individual cells to a particular secretagogue and identifying multihormones that are simultaneously secreted by the same cells. By using the CIBA and its variations, mammotrophs were shown to be heterogeneous with respect to the responsiveness to DA and TRH and cosecretion of PRL and GH. Furthermore, the fact that SP secretion from pituitary cells was increased by thyroidectomy via an increase in the number of SP-secreting cells indicates the possibility that the CIBA can clarify mechanisms underlying changes in hormone secretion and intercellular signaling at the cellular basis.

Long‐Term Monitoring of Depolarization‐Induced Exocytosis from Adrenal Medullary Chromaffin Cells and Pancreatic Islet B Cells Using “Perforated Patch Recording”a

(1) Membrane capacitance measurements using perforated patch recording offer the possibility of studying the process of depolarization-secretion coupling (DSC) in single endocrine cells with unprecedented time resolution and stability. (2) Early results with catecholamine-secreting adrenal chromaffin cells and insulin-secreting pancreatic B cells support longstanding ideas that the Ca(2+)-dependent processes underlying DSC are fundamentally similar to those of nerve terminals. (3) Future experiments using these approaches should prove useful in sorting out those effects of humoral substances that have a predominant effect on excitability and Ca2+ entry from those that affect the secretory process itself.

Cytosolic Ca 2+, exocytosis, and endocytosis in single melanotrophs of the rat pituitary

We have monitored cytosolic [Ca 2+] with fura-2 and exocytosis by measuring the membrane capacitance, and we have studied the influence of cytosolic [Cal 2+] on secretion in single endocrine cells. As in neurons, cytosolic Ca 2+ is sufficient to trigger exocytosis. The rate of secretion grows with the fourth or fifth power of cytosolic [Ca 2+], and paired stimuli reveal facilitation. Cal 2+ influx through voltage-sensitive Cal 2+ channels can stimulate secretion 1000-fold over the basal levels measured biochemically. Unlike neurons, however, melanotrophs continue to secrete for seconds after a depolarizing pulse, while they extrude or sequester the Cat 2+ that has entered through Cat+ channels. Following episodes of secretion, pituitary cells can retrieve membrane with half-times around 30 s at 32°C, even in the absence of cytosolic K +.

Endocrine cells in human Bartholin’s glands

Endocrine cells were investigated in human Bartholin's glands by use of histochemical, immunohistochemical and ultrastructural methods. Endocrine cells represent normal constituents of these glands, being mainly distributed throughout the transitional epithelium of the major excretory duct; however, single elements are dispersed among the acinar lobules. Serotonin-, calcitonin-, katacalcin-, bombesin- and alpha-hCG-immunoreactive cells were recognized, with serotonin-immunoreactive cells predominating. Co-expression of calcitonin, katacalcin or alpha-hCG with serotonin was observed in single endocrine cells. At the ultrastructural level, these cells are richly granulated and show typical neuroendocrine features. Bartholin's glands display an endocrine profile quite similar to that of other cloacal-derived tissues.

Allotransplantation of dispersed single pancreatic endocrine cells in diabetic rats

Allotransplantation of dispersed single pancreatic endocrine cells in diabetic rats