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Development of the Islets of Langerhans in the Human Fetal Pancreas
Abstract and Figures
The islets of Langerhans play a key role in the pathogenesis of diabetes mellitus. Investigations of the β-cells differentiation and islets formation during normal human pancreas development are necessary for the prospect of successful replacement therapies for treatment of diabetes. Studies of human pancreas development are restricted in number and include predominantly the first trimester by ethical constraints and technical difficulties. In this chapter we describe the development of human pancreatic islets and its innervation. We have investigated pancreatic specimens from 45 fetuses (8-40 weeks of gestational age), 1 infant, 2 children (3 month and 3 years old) and 10 adults (24-80 years old) from our collection. Histological and immunohistochemical methods (marking with antibodies to insulin, glucagon and five neural markers such as SNAP-25, NCAM, Neuron specific β-III tubulin, NSE and peripherin) were applied in our research. Firstly, we find spatial appearance of different types of endocrine cells clusters cytoarchitecture in the developing pancreas: single endocrine cells or their small clusters of early fetuses (8-11 weeks), murine islets with a core of β-cells surrounded by α-cells (after 12 week), bipolar islets with self aggregated β- and α-cells juxtaposed to each other (after 15-16 weeks), mosaic islets typical for adults with mixed β- and α-cells (after 25-27 weeks). We have assumed that this sequence of endocrine cells clustering may reflect the stages of human pancreatic islets morphogenesis. Secondly, we present our data on the morphological organization of pancreatic innervation during its ontogenesis. Immunopositive staining for all neural markers used in this study was found in nerve fibers and neurons in the fetal and adult pancreas. Comparative analysis of fetal and adult pancreas innervation has revealed some distinct features. Innervation of the fetal pancreas is more abundant than in adults. Neuro-endocrine interactions detected in fetal pancreas were rare in adults. Gradual branching of neural network was seen during human pancreas development. Thirdly, we describe some antigenic similarities between human pancreatic endo-crine cells and neurons. Positive immunostaining for SNAP-25, NCAM, Neuron specific β-III tubulin, NSE was detected in the cytoplasm of endocrine cells as well as in nervous elements. NSE-positive endocrine cells were first found in 12-week fetuses, NCAM- and neuron-specific β-III tubulin-positive endocrine cells - from 14-15 weeks of develop-ment, SNAP-25-positive endocrine cells - from 16 weeks of development. Besides that, the appearance of immunopositive reaction on these antibodies in endocrine cells and nervous elements was seen at different developmental stages. In agreement with previous observations, we reveal close integration and similarity between endocrine cells and nervous elements in the developing human pancreas. It has been suggested that the presence of neurons and nerve fibers is necessary for normal islet morphogenesis. © 2012 by Nova Science Publishers, Inc. All rights reserved.
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The association between islet cells and neural elements, the so-called "neuro-insular complex", has been known for centuries.
We examined the expression of beta-III tubulin, in normal pancreases from organ donors, surgical specimens of chronic pancreatitis, surgical specimens of ductal type carcinoma, isolated and purified islets of a 57-year-old male and the pancreases of adult Syrian golden hamsters by immunohistochemistry using a monoclonal antibody to beta-tubulin.
In the normal pancreas of humans and hamsters, beta-III tubulin was expressed in alpha- and beta-cells, but not in PP cells, neural fibers and gangliae. Occasionally, intra-and peri-insular neural elements were also found. In chronic pancreatitis and pancreatic cancer samples, the number of beta-cells and the immunoreactivity of the beta-III tubulin antibody in islet cells were decreased in most cases. In cultured human islets, devoid of neural elements, no correlation was found between the expression of beta-III tubulin and islet cell hormones.
Beta-III tubulin is only expressed in the islets derived from the dorsal pancreas and in neural elements. In chronic pancreatitis and pancreatic cancer swelling of intra- and peri-insular nerves occurs, possibly in response to the loss of beta-cells. The secretion of insulin and the expression of beta-tubulin seem to be regulated by nerves.
The development of efficient, reproducible protocols for directed in vitro differentiation of human embryonic stem (hES) cells into insulin-producing beta cells will benefit greatly from increased knowledge regarding the spatiotemporal expression profile of key instructive factors involved in human endocrine cell generation. Human fetal pancreases 7 to 21 weeks of gestational age, were collected following consent immediately after pregnancy termination and processed for immunostaining, in situ hybridization, and real-time RT-PCR expression analyses. Islet-like structures appear from approximately week 12 and, unlike the mixed architecture observed in adult islets, fetal islets are initially formed predominantly by aggregated insulin- or glucagon-expressing cells. The period studied (7-22 weeks) coincides with a decrease in the proliferation and an increase in the differentiation of the progenitor cells, the initiation of NGN3 expression, and the appearance of differentiated endocrine cells. The present study provides a detailed characterization of islet formation and expression profiles of key intrinsic and extrinsic factors during human pancreas development. This information is beneficial for the development of efficient protocols that will allow guided in vitro differentiation of hES cells into insulin-producing cells.
Organogenesis, the process by which organs develop from individual precursor stem cells, requires that the precursor cells proliferate, differentiate, and aggregate to form a functioning structure. This process progresses through changes in 4 dimensions: time and 3 dimensions of space-4D. Experimental analysis of organogenesis, by its nature, cuts the 4D developmental process into static, 2D histological images or into molecular or cellular markers and interactions with little or no spatial dimensionality and minimal dynamics. Understanding organogenesis requires integration of the piecemeal experimental data into a running, realistic and interactive 4D simulation that allows experimentation and hypothesis testing in silico. Here, we describe a fully executable, interactive, visual model for 4D simulation of organogenic development using the mouse pancreas as a representative case. Execution of the model provided a dynamic description of pancreas development, culminating in a structure that remarkably recapitulated morphologic features seen in the embryonic pancreas. In silico mutations in key signaling molecules resulted in altered patterning of the developing pancreas that were in general agreement with in vivo data. The modeling approach described here thus typifies a useful platform for studying organogenesis as a phenomenon in 4 dimensions.
The findings of this study show that Class III beta-tubulin is a component of the mitotic spindle in multiple cell types. Class III beta-tubulin has been widely used as a neuron-specific marker, but it has been detected also in association with breast and pancreatic cancers. In this study, we describe a novel finding of Class III beta-tubulin in a subpopulation of cells in malignant peripheral nerve sheath tumor. The findings of this study also show that Class III beta-tubulin is expressed by normal mesenchymal and epithelial cells (fibroblasts and keratinocytes), two transitional cell carcinoma cell lines, and neurofibroma Schwann cells, as shown by immunolabeling and Western transfer analysis using two different Tuj-1 antibodies that are specific for Class III beta-tubulin. The corresponding mRNA was detected using RT-PCR and whole human genome microarrays. Both antibodies localized Class III beta-tubulin to the mitotic spindle and showed a colocalization with alpha-tubulin. The immunoreaction became visible in early prophase, and the most intense immunoreaction was detected during metaphase and anaphase when microtubules were connected to the kinetochores on chromosomes. Class III beta-tubulin-specific immunoreaction lasted to the point when the midbody of cytokinesis became detectable.
The presence of autonomic nerve fibres within the islets of Langerhans of most species is well documented (cf. Kern and Grube, 1973) with both cholinergic and adrenergic plexuses running in association with the islet capillaries (Coupland, 1958; Lever, 1971) and sometimes giving rise to neuroinsular complexes (Simard, 1937; Fujita, 1959). Nerve stimulation and pharmacological studies have defined in part the role of this innervation in the control of islet hormone secretion; with glucagon secretion rising following sympathetic nerve and catecholamine stimulation, and insulin secretion increasing after parasympathetic nerve and cholinergic stimulation, and being facilitated by a β–adrenergie receptor but inhibited by an α–adrenergic receptor (cf. Woods and Porte, 1974).
The autonomic nervous system regulates hormone secretion from the endocrine pancreas, the islets of Langerhans, thus impacting glucose metabolism. The parasympathetic and sympathetic nerves innervate the pancreatic islet, but the precise innervation patterns are unknown, particularly in human. Here we demonstrate that the innervation of human islets is different from that of mouse islets and does not conform to existing models of autonomic control of islet function. By visualizing axons in three dimensions and quantifying axonal densities and contacts within pancreatic islets, we found that, unlike mouse endocrine cells, human endocrine cells are sparsely contacted by autonomic axons. Few parasympathetic cholinergic axons penetrate the human islet, and the invading sympathetic fibers preferentially innervate smooth muscle cells of blood vessels located within the islet. Thus, rather than modulating endocrine cell function directly, sympathetic nerves may regulate hormone secretion in human islets by controlling local blood flow or by acting on islet regions located downstream.
The role of neuron-specific enolase (glycolytic enzyme; marker of nerve fibers and Langerhans islet in human pancreas) in the development of type 1 diabetes mellitus was studied in autopsy specimens from 6 adult patients. Autopsied specimens of the pancreas from 7 subjects without carbohydrate metabolism disorders served as the control. Autopsied specimens of the pancreas from a child with the clinical diagnosis of type 1 diabetes mellitus, a child without carbohydrate metabolism disorders, and from 7 human fetuses of 15-40 weeks gestation were also studied. In control specimens, the neuron-specific enolase was detected in the pancreatic nerve fibers and Langerhans islets. Studies of pancreatic tissue specimens from adult patients with type 1 diabetes mellitus showed no immunopositive reaction to neuron-specific enolase in insulin-negative specimens. A possible mechanism of type 1 diabetes mellitus development is suggested.
Fluorescence histochemical studies in newborn and young golden hamster have revealed that some cells in the islets of Langerhans contain monoamine. These cells can be seen in newborns and up to ten day old animals, at least in three to five days old animals in the amine behaves like dopamine and is mostly stored in the A2-cells.
In the fetal development of the mouse pancreas, endocrine cells have been found that express more than one hormone simultaneously. Our objective was to evaluate the existence of such cells in the human fetal pancreas. We found cells coexpressing two of the major pancreatic hormones (insulin, glucagon, and somatostatin) in sections of eight midgestational (12-18 weeks) pancreata and in 0-7% of cells in single-cell suspensions from midgestational pancreata. By electron microscopy, using granule morphology and immunoelectron microscopic techniques, we could confirm these findings and even detect cells containing three hormones. Morphologically different granules contained different immunoreactivities, suggesting parallel regulation of hormone production and packaging. In six newborn pancreata (born after 22-40 weeks of gestation), we could not find any multiple-hormone-containing cells. Subsequently, we evaluated whether multiple-hormone-containing cells proliferate by using pancreatic fragments and single-cell preparations at the light and electron microscopic level (six pancreata). No endocrine hormone-containing cells incorporated bromodeoxyuridine during a 1-hr culture period, indicating that these cells have lost the ability to proliferate under the conditions chosen. We conclude that, as in mice, the human fetal pancreas of 12-18 weeks of gestation contains endocrine cells that express multiple hormones simultaneously. These (multiple) hormone-containing cells do not seem to proliferate under basal conditions.