The SDF-1α/CXCR4 Axis is Required for Proliferation and Maturation of Human Fetal Pancreatic Endocrine Progenitor Cells
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The chemokine receptor CXCR4 and ligand SDF-1α are expressed in fetal and adult mouse islets. Neutralization of CXCR4 has previously been shown to diminish ductal cell proliferation and increase apoptosis in the IFNγ transgenic mouse model in which the adult mouse pancreas displays islet regeneration. Here, we demonstrate that CXCR4 and SDF-1α are expressed in the human fetal pancreas and that during early gestation, CXCR4 colocalizes with neurogenin 3 (ngn3), a key transcription factor for endocrine specification in the pancreas. Treatment of islet like clusters (ICCs) derived from human fetal pancreas with SDF-1α resulted in increased proliferation of epithelial cells in ICCs without a concomitant increase in total insulin expression. Exposure of ICCs in vitro to AMD3100, a pharmacological inhibitor of CXCR4, did not alter expression of endocrine hormones insulin and glucagon, or the pancreatic endocrine transcription factors PDX1, Nkx6.1, Ngn3 and PAX4. However, a strong inhibition of β cell genesis was observed when in vitro AMD3100 treatment of ICCs was followed by two weeks of in vivo treatment with AMD3100 after ICC transplantation into mice. Analysis of the grafts for human C-peptide found that inhibition of CXCR4 activity profoundly inhibits islet development. Subsequently, a model pancreatic epithelial cell system (CFPAC-1) was employed to study the signals that regulate proliferation and apoptosis by the SDF-1α/CXCR4 axis. From a selected panel of inhibitors tested, both the PI 3-kinase and MAPK pathways were identified as critical regulators of CFPAC-1 proliferation. SDF-1α stimulated Akt phosphorylation, but failed to increase phosphorylation of Erk above the high basal levels observed. Taken together, these results indicate that SDF-1α/CXCR4 axis plays a critical regulatory role in the genesis of human islets.Keywords:
PDX1
The pancreatic and duodenal homeobox factor 1 (Pdx1) protein is the most pivotal transcription factor in the development of islet β cells. This study investigated the role of Pdx1 and its mechanism in differentiating induced pluripotent stem cells (iPSCs) into islet β cells. iPSCs derived from human skin fibroblasts were cultured in vitro and directionally induced to differentiate for 20 days. The expression of insulin-related genes was then detected by RT-PCR, and the expression of several differentiation-related transcription factors was assessed both before and after the differentiation process. Lastly, the specific promoter regions where Pdx1 binds were detected by ChIP. The insulin-related genes, MafA, insulin, Glut2, Nkx6.1, GCK, and Tcf1, showed increased expression during differentiation, and nearly peaked on the 20th day. Similarly, the expression of transcription factors, Pdx1, Ngn3, and Pax6 showed enhanced expression during differentiation as compared with that of the control group. ChIP experiments confirmed that Pdx1 activates the expression of the downstream transcription factors, Ngn3 and Pax6, by combined with the promoter regions of insulin (Insulin-P), Ngn3 (Ngn3-P), and Pax6 (Pax6-P). In conclusion, Pdx1 activates downstream transcription factors Ngn3 and Pax6, and may be one of the mechanisms that promote differentiation of iPSCs into islet β cells.
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PAX6
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Progenitor cells capable of self-renewal and differentiation in the adult human pancreas are an under-explored resource for regenerative medicine. Using micro-manipulation and three-dimensional colony assays we identify cells within the adult human exocrine pancreas that resemble progenitor cells. Exocrine tissues were dissociated into single cells and plated into a colony assay containing methylcellulose and 5% Matrigel. A subpopulation of ductal cells formed colonies containing differentiated ductal, acinar, and endocrine lineage cells, and expanded up to 300-fold with a ROCK inhibitor. When transplanted into diabetic mice, colonies pre-treated with a NOTCH inhibitor gave rise to insulin-expressing cells. Both colonies and primary human ducts contained cells that simultaneously express progenitor transcription factors SOX9, NKX6.1, and PDX1. In addition, in silico analysis identified progenitor-like cells within ductal clusters in a single-cell RNA sequencing dataset. Therefore, progenitor-like cells capable of self-renewal and tri-lineage differentiation either pre-exist in the adult human exocrine pancreas, or readily adapt in culture.
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Abstract Pancreas duodenum homeobox-1 (PDX-1) is a pancreas-specific homeodomain transcription factor that regulates embryonic pancreas development, the expression of genes essential for glucose metabolism, and the differentiation, survival, and function of insulin-producing cells in the endocrine pancreas. Expression of PDX-1 is confined primarily to the developing embryonic pancreas and the adult pancreatic β cells, and PDX-1 function is essential for the proper development of both the endocrine and the exocrine pancreas and for normal metabolic function in the adult. Mutations in the PDX-1 gene are associated with multiple metabolic phenotypes in humans.
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Diabetes has been a worldwide healthcare problem for many years. Current methods of treating diabetes are still largely directed at symptoms, aiming to control the manifestations of the pathology. This creates an overall need to find alternative measures that can impact on the causes of the disease, reverse diabetes, or make it more manageable. Understanding the role of key players in the pathogenesis of diabetes and the related β-cell functions is of great importance in combating diabetes. PDX1 is a master regulator in pancreas organogenesis, the maturation and identity preservation of β-cells, and of their role in normal insulin function. Mutations in the PDX1 gene are correlated with many pancreatic dysfunctions, including pancreatic agenesis (homozygous mutation) and MODY4 (heterozygous mutation), while in other types of diabetes, PDX1 expression is reduced. Therefore, alternative approaches to treat diabetes largely depend on knowledge of PDX1 regulation, its interaction with other transcription factors, and its role in obtaining β-cells through differentiation and transdifferentiation protocols. In this article, we review the basic functions of PDX1 and its regulation by genetic and epigenetic factors. Lastly, we summarize different variations of the differentiation protocols used to obtain β-cells from alternative cell sources, using PDX1 alone or in combination with various transcription factors and modified culture conditions. This review shows the unique position of PDX1 as a potential target in the genetic and cellular treatment of diabetes.
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AimsEndothelial progenitor cells (EPCs) play a pivotal role in endothelial repair after artery injury. The chemokine receptor CXCR4 is a key modulator of the homing of EPCs to impaired artery and reendothelialization. In this study, we addressed the hypothesis that CXCR4 gene transfer could enhance the reendothelialization capacity of EPCs.
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Aldh1b1 regulates pancreas maturation in the mouse embryo affecting islet functionality in the adult
Aldehyde dehydrogenase activity is increasingly associated with stem and progenitor cells but its functional significance remains uncertain. We identified Aldh1b1 as a marker for pancreas progenitor cells. Aldh1b1 is exclusively expressed in the emerging pancreatic buds and later in progenitors cells of the developing pancreatic epithelium. Its expression persists in rare progenitor like cells in the adult pancreas and therefore Aldh1b1 expression is associated with pancreas stem and progenitor cells during development and in the adult.
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A recent study has shown that deletion of beta-catenin within the pancreatic epithelium results in a loss of pancreas mass. Here, we show that ectopic stabilization of beta-catenin within mouse pancreatic epithelium can have divergent effects on both organ formation and growth. Robust stabilization of beta-catenin during early organogenesis drives changes in hedgehog and Fgf10 signaling and induces a loss of Pdx1 expression in early pancreatic progenitor cells. Together, these perturbations in early pancreatic specification culminate in a severe reduction of pancreas mass and postnatal lethality. By contrast, inducing the stabilized form of beta-catenin at a later time point in pancreas development causes enhanced proliferation that results in a dramatic increase in pancreas organ size. Taken together, these data suggest a previously unappreciated temporal/spatial role for beta-catenin signaling in the regulation of pancreas organ growth.
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