Dataset Information

Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the Capacity for Endocrine Cell Fate.

ABSTRACT: Neurogenin 3 (NGN3) is necessary and sufficient for endocrine differentiation during pancreatic development and is expressed by a population of progenitor cells that give rise exclusively to hormone-secreting cells within islets. NGN3 protein can be detected in the adult rodent pancreas only following certain types of injury, when it is transiently expressed by exocrine cells undergoing reprogramming to an endocrine cell fate. Here, NGN3 protein can be detected in 2% of acinar and duct cells in living biopsies of histologically normal adult human pancreata and 10% in cadaveric biopsies of organ donor pancreata. The percentage and total number of NGN3+ cells increase during culture without evidence of proliferation or selective cell death. Isolation of highly purified and viable NGN3+ cell populations can be achieved based on coexpression of the cell surface glycoprotein CD133. Transcriptome and targeted expression analyses of isolated CD133+ / NGN3+ cells indicate that they are distinct from surrounding exocrine tissue with respect to expression phenotype and Notch signaling activity, but retain high level mRNA expression of genes indicative of acinar and duct cell function. NGN3+ cells have an mRNA expression profile that resembles that of mouse early endocrine progenitor cells. During in vitro differentiation, NGN3+ cells express genes in a pattern characteristic of endocrine development and result in cells that resemble beta cells on the basis of coexpression of insulin C-peptide, chromogranin A and pancreatic and duodenal homeobox 1. NGN3 expression in the adult human exocrine pancreas marks a dedifferentiating cell population with the capacity to take on an endocrine cell fate. These cells represent a potential source for the treatment of diabetes either through ex vivo manipulation, or in vivo by targeting mechanisms controlling their population size and endocrine cell fate commitment.

SUBMITTER: Gomez DL

PROVIDER: S-EPMC4545947 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the Capacity for Endocrine Cell Fate.

Gomez Danielle L DL O'Driscoll Marci M Sheets Timothy P TP Hruban Ralph H RH Oberholzer Jose J McGarrigle James J JJ Shamblott Michael J MJ

PloS one 20150819 8

Neurogenin 3 (NGN3) is necessary and sufficient for endocrine differentiation during pancreatic development and is expressed by a population of progenitor cells that give rise exclusively to hormone-secreting cells within islets. NGN3 protein can be detected in the adult rodent pancreas only following certain types of injury, when it is transiently expressed by exocrine cells undergoing reprogramming to an endocrine cell fate. Here, NGN3 protein can be detected in 2% of acinar and duct cells in ...[more]

PMID: 26288179

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Project description:Pancreatic stellate cells (PaSCs) are non-endocrine, mesenchymal-like cells that reside within the peri-pancreatic tissue of the rodent and human pancreas. PaSCs regulate extracellular matrix (ECM) turnover in maintaining the integrity of pancreatic tissue architecture. Although there is evidence indicating that PaSCs are involved in islet cell survival and function, its role in islet cell differentiation during human pancreatic development remains unclear. The present study examines the expression pattern and functional role of PaSCs in islet cell differentiation of the developing human pancreas from late 1st to 2nd trimester of pregnancy. The presence of PaSCs in human pancreata (8-22 weeks of fetal age) was characterized by ultrastructural, immunohistological, quantitative RT-PCR and western blotting approaches. Using human fetal PaSCs derived from pancreata at 14-16 weeks, freshly isolated human fetal islet-epithelial cell clusters (hIECCs) were co-cultured with active or inactive PaSCs in vitro. Ultrastructural and immunofluorescence analysis demonstrated a population of PaSCs near ducts and newly formed islets that appeared to make complex cell-cell dendritic-like contacts. A small subset of PaSCs co-localized with pancreatic progenitor-associated transcription factors (PDX1, SOX9, and NKX6-1). PaSCs were highly proliferative, with significantly higher mRNA and protein levels of PaSC markers (desmin, αSMA) during the 1st trimester of pregnancy compared to the 2nd trimester. Isolated human fetal PaSCs were identified by expression of stellate cell markers and ECM. Suppression of PaSC activation, using all-trans retinoic acid (ATRA), resulted in reduced PaSC proliferation and ECM proteins. Co-culture of hIECCs, directly on PaSCs or indirectly using Millicell® Inserts or using PaSC-conditioned medium, resulted in a reduction the number of insulin+ cells but a significant increase in the number of amylase+ cells. Suppression of PaSC activation or Notch activity during the co-culture resulted in an increase in beta-cell differentiation. This study determined that PaSCs, abundant during the 1st trimester of pancreatic development but decreased in the 2nd trimester, are located near ductal and islet structures. Direct and indirect co-cultures of hIECCs with PaSCs suggest that activation of PaSCs has opposing effects on beta-cell and exocrine cell differentiation during human fetal pancreas development, and that these effects may be dependent on Notch signaling.

Project description:Context: Previously, we identified chromograninA positive hormone-negative (CPHN) cells in high frequency in human fetal and neonatal pancreas, likely representing nascent endocrine precursor cells. Here, we characterize the putative endocrine fate and replicative status of these newly formed cells. Objective: To establish the replicative frequency and transcriptional identity of CPHN cells, extending our observation on CPHN cell frequency to a larger cohort of fetal and infant pancreas. Design, Setting, and Participants: 8 fetal, 19 infant autopsy pancreata were evaluated for CPHN cell frequency; 12 fetal, 24 infant/child pancreata were evaluated for CPHN replication and identity. Results: CPHN cell frequency decreased 84% (islets) and 42% (clusters) from fetal to infant life. Unlike the beta-cells at this stage, CPHN cells were rarely observed to replicate (0.2 ± 0.1 vs. 4.7 ± 1.0%, CPHN vs. islet hormone positive cell replication, p < 0.001), indicated by the lack of Ki67 expression in CPHN cells whether located in the islets or in small clusters, and with no detectable difference between fetal and infant groups. While the majority of CPHN cells express (in overall compartments of pancreas) the pan-endocrine transcription factor NKX2.2 and beta-cell specific NKX6.1 in comparable frequency in fetal and infant/child cases (81.9 ± 6.3 vs. 82.8 ± 3.8% NKX6.1+-CPHN cells of total CPHN cells, fetal vs. infant/child, p = 0.9; 88.0 ± 4.7 vs. 82.1 ± 5.3% NKX2.2+-CPHN cells of total CPHN cells, fetal vs. infant/child, p = 0.4), the frequency of clustered CPHN cells expressing NKX6.1 or NKX2.2 is lower in infant/child vs. fetal cases (1.2 ± 0.3 vs. 16.7 ± 4.7 clustered NKX6.1+-CPHN cells/mm2, infant/child vs. fetal, p < 0.01; 2.7 ± 1.0 vs. 16.0 ± 4.0 clustered NKX2.2+-CPHN cells/mm2, infant/child vs. fetal, p < 0.01). Conclusions: The frequency of CPHN cells declines steeply from fetal to infant life, presumably as they differentiate to hormone-expressing cells. CPHN cells represent a non-replicative pool of endocrine precursor cells, a proportion of which are likely fated to become beta-cells. Precis : CPHN cell frequency declines steeply from fetal to infant life, as they mature to hormone expression. CPHN cells represent a non-replicative pool of endocrine precursor cells, a proportion of which are likely fated to become beta-cells.

Project description:BackgroundReports of exocrine-to-endocrine reprogramming through expression or stabilization of the transcription factor neurogenin 3 (NGN3) have generated renewed interest in harnessing pancreatic plasticity for therapeutic applications. NGN3 is expressed by a population of endocrine progenitor cells that give rise exclusively to hormone-secreting cells within pancreatic islets and is necessary and sufficient for endocrine differentiation during development. In the adult human pancreas, NGN3 is expressed by dedifferentiating exocrine cells with a phenotype resembling endocrine progenitor cells and the capacity for endocrine differentiation in vitro. Neurotrophic tyrosine kinase receptor type 2 (TRKB), which regulates neuronal cell survival, differentiation and plasticity, was identified as highly overexpressed in the NGN3 positive cell transcriptome compared to NGN3 negative exocrine cells. This study was designed to determine if NGN3 is regulated by TRKB signaling in the adult human exocrine pancreas.MethodsTranscriptome analysis, quantitative reverse transcriptase polymerase chain reaction (RTPCR) and immunochemistry were used to identify TRKB isoform expression in primary cultures of human islet-depleted exocrine tissue and human cadaveric pancreas biopsies. The effects of pharmacological modulation of TRKB signaling on the expression of NGN3 were assessed by Student's t-test and ANOVA.ResultsApproximately 30 % of cultured exocrine cells and 95 % of NGN3+ cells express TRKB on their cell surface. Transcriptome-based exon splicing analyses, isoform-specific quantitative RTPCR and immunochemical staining demonstrate that TRKB-T1, which lacks a tyrosine kinase domain, is the predominant isoform expressed in cultured exocrine tissue and is expressed in histologically normal cadaveric pancreas biopsies. Pharmacological inhibition of TRKB significantly decreased the percentage of NGN3+ cells, while a TRKB agonist significantly increased this percentage. Inhibition of protein kinase B (AKT) blocked the effect of the TRKB agonist, while inhibition of tyrosine kinase had no effect. Modulation of TRKB and AKT signaling did not significantly affect the level of NGN3 mRNA.ConclusionsIn the adult human exocrine pancreas, TRKB-T1 positively regulates NGN3 independent of effects on NGN3 transcription. Targeting mechanisms controlling the NGN3+ cell population size and endocrine cell fate commitment represent a potential new approach to understand pancreas pathobiology and means whereby cell populations could be expanded for therapeutic purposes.

Dataset Information

Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the Capacity for Endocrine Cell Fate.

Publications

Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the Capacity for Endocrine Cell Fate.

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