Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors <em>In Vitro</em>
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The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion.These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells.Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels.Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions.Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics.While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming.Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo.Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ.The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture.Here, we describe a protocol to culture threedimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids.The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment.These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells.This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening. Video LinkThe video component of this article can be found at https://www.jove.com/video/57599/6 and intestine 7 have expanded our understanding of organogenesis, providing a tool to study developmental complexities with fewer restrictions than in vivo and in vitro models.Due to these advances in the murine organoid formation and the advent of human pluripotent stem cells, human intestinal 8 , retinal 9 , renal 10,11 , and cerebral 12 organoids have been produced, and this repertoire is only limited by the existing knowledge regarding mechanisms of development.Keywords:
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Enteroendocrine cell
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The authors of this study examine the question of whether the so-called enterochromaffin or argentaffin cells of the gastrointestinal tract should be considered as a single cell type. The systematic application of purely morphologic methods has led to the conclusion that the epithelium of the gastrointestinal mucosa comprises endocrine cells of several types. This conclusion is primarily based on the uneven and characteristic distribution of the various cell types along the intestinal tract, an observation precluding the interpretation that the different types correspond to diverse functional stages of the same cell. A specific endocrine function may be attributed to each of the given cell types recognized so far on account of their appearance and their localization in characteristic areas of the gastrointestinal tract. It is acknowledged, however, that a purely morphological study leaves room for doubt. The first cell type is probably responsible for the formation of 5-hydroxytryptamine. Cells of type II are morphologically comparable to the pancreatic A cells and may, therefore, be called intestinal A cells. Cell type III comprises intestinal D cells since their appearance corresponds to that of pancreatic D cells. Cell type IV might well be responsible for catecholamine production, whereas gastrin is in all probability produced in endocrine cell type V. As yet, the thorough morphological study of the gastrointestinal epithelium does not provide information as to additional distinct cellular sites of production of the several other hormones isolated from different parts of the gut.
Enteroendocrine cell
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Intestinal epithelium
Intestinal mucosa
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This review discusses the current concepts for the classification of gastric endocrine cells subdivided according to the type of mucosa in which they are located. In the oxyntic mucosa, the most important cell type is the ECL cell, involved in the synthesis and secretion of histamine. Proteins involved in many aspects of the biology of ECL cells including the response to the gastrin stimulus, membrane transport and docking, prevention of apoptosis, calcium homeostasis, autocrine activity, and maintenance of the differentiated cell phenotype have been localized to this cell type. Other cells of the oxyntic mucosa include: the D and EC cells producing somatostatin and serotonin, respectively, delivered through long cell processes; the X (or A-like) cells, possibly producing endothelin; and the D(1) and P cells of unknown function and possibly representing morphological variants of other cell types. In the antral mucosa, the three important cell types are represented by: the gastrin-producing G cells; the somatostatin-producing D cells, which are anatomically and functionally associated with G cells; and the serotonin-producing EC cells, which are located at the bottom of antral glands.
Enterochromaffin-like cell
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Enterochromaffin cell
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PDX1
Homeostasis
Pancreatic Islets
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This review discusses the current concepts for the classification of gastric endocrine cells subdivided according to the type of mucosa in which they are located. In the oxyntic mucosa, the most important cell type is the ECL cell, involved in the synthesis and secretion of histamine. Proteins involved in many aspects of the biology of ECL cells including the response to the gastrin stimulus, membrane transport and docking, prevention of apoptosis, calcium homeostasis, autocrine activity, and maintenance of the differentiated cell phenotype have been localized to this cell type. Other cells of the oxyntic mucosa include: the D and EC cells producing somatostatin and serotonin, respectively, delivered through long cell processes; the X (or A-like) cells, possibly producing endothelin; and the D1 and P cells of unknown function and possibly representing morphological variants of other cell types. In the antral mucosa, the three important cell types are represented by: the gastrin-producing G cells; the somatostatin-producing D cells, which are anatomically and functionally associated with G cells; and the serotonin-producing EC cells, which are located at the bottom of antral glands. Microsc. Res. Tech. 48:258–271, 2000 © 2000 Wiley-Liss, Inc.
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Maintenance of pancreatic β-cell mass and function is fundamental to glucose homeostasis and to prevent diabetes. The PI3 K-Akt-mTORC1 pathway is critical for β-cells mass and function, while PDX1 has been implicated in β-cell development, maturation, and function. Here we tested whether Akt signaling requires PDX1 expression to regulate β-cell mass, proliferation, and glucose homeostasis. In order to address that, we crossed a mouse model overexpressing constitutively active Akt mutant in β-cells (β-caAkt) with mice lacking one allele of PDX1gene (β-caAkt/pdx1+/-). While the β-caAkt mice exhibit higher plasma insulin levels, greater β-cell mass and improved glucose tolerance compared to control mice, the β-caAkt/pdx1+/- mice are hyperglycemic and intolerant to glucose. The changes in glucose homeostasis in β-caAkt/pdx1+/- were associated with a 60% reduction in β-cell mass compared to β-caAkt mice. The impaired β-cell mass in the β-caAkt/pdx1+/- mice can be explained by a lesser β-cell proliferation measured by the number of Ki67 positive β-cells. We did not observe any differences in apoptosis between β-caAkt/pdx1+/- and β-caAkt mice. In conclusion, PDX1 contributes to β-cell mass expansion and glucose metabolism induced by activation of Akt signaling.
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Endocrine cells in the gastrointestinal tract of the dog have been studied by electron microscopy.Seven types of endocrine cells were found in the gastric mucosa: the gastrinproducing G cells, serotonin-producing enterochromaffin cells, enterochromaffinlike cells, A-like cells, D cells, D, cells, and X cells.The enterochromaffin cells may be subdivided into two cell types according to the shape and structure of the secretory granules. Contrary to previous reports, the enterochromaffin-like cells were found to reach the gland lumen. Implications resulting from this observation are discussed. The X cells and D cells have so far been described as one single cell type. According to the present findings, the two cells differ distinctly in several respects and should be regarded as two separate cell types.The intestinal mucosa was found to contain the enterochromaffin cells, S cells, I cells and L cells. In addition, we observed an heretofore undescribed cell type, the secretory granules of which show a granular structure and a highly osmiophilic core. Since we were unable to find any transitory forms between these cells and established cell types, this cell type may represent a new element in the population of endocrine cells of the gastrointestinal mucosa.
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The importance of mesenchymal-epithelial interactions for normal development of the pancreas was recognized in the early 1960s, and mesenchymal signals have been shown to control the proliferation of early pancreatic progenitor cells. The mechanisms by which the mesenchyme coordinates cell proliferation and differentiation to produce the normal number of differentiated pancreatic cells are not fully understood. Here, we demonstrate that the mesenchyme positively controls the final number of β-cells that develop from early pancreatic progenitor cells. In vitro, the number of β-cells that developed from rat embryonic pancreatic epithelia was larger in cultures with mesenchyme than without mesenchyme. The effect of mesenchyme was not due to an increase in β-cell proliferation but was due to increased proliferation of early pancreatic duodenal homeobox-1 (PDX1)–positive progenitor cells, as confirmed by bromodeoxyuridine incorporation. Consequently, the window during which early PDX1+ pancreatic progenitor cells differentiated into endocrine progenitor cells expressing Ngn3 was extended. Fibroblast growth factor 10 mimicked mesenchyme effects on proliferation of early PDX1+ progenitor cells and induction of Ngn3 expression. Taken together, our results indicate that expansion of early PDX1+ pancreatic progenitor cells represents a way to increase the final number of β-cells developing from early embryonic pancreas.
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