Role of Neurogenin3 phosphorylation in the reprogramming of pancreatic organoids into endocrine cells
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ABSTRACT:
PROVIDER: PRJNA472788 | ENA |
REPOSITORIES: ENA
Dataset's files
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| SRR7208400.fastq.gz | Fastqsanger.gz | |||
| SRR7208410.fastq.gz | Fastqsanger.gz | |||
| SRR7208420.fastq.gz | Fastqsanger.gz | |||
| SRR7208430.fastq.gz | Fastqsanger.gz | |||
| SRR7208440.fastq.gz | Fastqsanger.gz |
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Project description:During pancreatic development, Neurogenin3 (Ngn3) promotes the differentiation of endocrine cells, including insulin-producing β-cells. Our previous work has shown that Ngn3 phosphorylation on multiple serine-proline (SP) sites increases protein stability and endocrine differentiation (Azzarelli et al., DevCell 2017). Here, we aim to exploit our recent data to investigate whether manipulation of Ngn3 phosphostatus might improve in vitro generation of β-cells for replacement therapies in diabetes. A potential source of β-cells comes from fate conversion of exocrine pancreatic cells into the endocrine lineage, by overexpression of 3 transcription factors, Ngn3, Pdx1 and MafA. Using this approach we show that pancreatic ductal cells grown in vitro as 3D organoids can be reprogrammed to express insulin. The efficiency can be strongly enhanced by substituting wild type Ngn3 with a the un(der)phosphorylated and more active Ngn3 form (6S-A Ngn3). Here we perform transcriptomic analysis of a low number of pancreatic organoid cells expressing wild type or phosphomutant Ngn3, alone or in various combination Pdx1 and/or MafA. The analysis revealed endocrine differentiation and in particular β-like cell gene expression in the conditions where Ngn3 was in combination with both Pdx1 and MafA.
2018-12-26 | GSE114838 | GEO
Project description:The proneural transcription factor Neurogenin3 (Ngn3) plays a critical role in pancreatic endocrine cell differentiation. While previous studies have focused on Ngn3 gene function, little is known about the regulation of Ngn3 protein. Here we demonstrate that Ngn3 protein undergoes cyclin-dependent kinase (cdk)-mediated phosphorylation on multiple serine-proline sites. Replacing wild-type protein with a phosphomutant form of Ngn3 increases α-cell generation, the earliest endocrine cell type to be formed in developing pancreas. Mechanistically, preventing multi-site phosphorylation enhances Ngn3 stability and DNA binding, and promotes the expression of target genes that drive differentiation. In addition to perturbing embryonic endocrine differentiation, un(der)phosphorylated Ngn3 contributes to maintaining adult β-cell differentiation in the presence of pro-proliferation cue. Here we perform transcriptomic analysis of a low number of pancreatic organoid cells expressing wild type and phosphomutant Ngn3. Analysis of the endocrine differentiation programme activated by Ngn3 in this context revealed the cell fate plasticity of the system and demonstrated that preventing Ngn3 phosphorylation enhances endocrine gene expression.
2017-06-09 | GSE96707 | GEO
Project description:β-cell replacement has been proposed as an effective treatment for some forms of diabetes, and in vitro methods for β-cell generation are being extensively explored. A potential source of β-cells comes from fate conversion of exocrine pancreatic cells into the endocrine lineage, by overexpression of three regulators of pancreatic endocrine formation and β-cell identity, Ngn3, Pdx1 and MafA. Pancreatic ductal organoid cultures have recently been developed that can be expanded indefinitely, while maintaining the potential to differentiate into the endocrine lineage. Here, using mouse pancreatic ductal organoids, we see that co-expression of Ngn3, Pdx1 and MafA are required and sufficient to generate cells that express insulin and resemble β-cells transcriptome-wide. Efficiency of β-like cell generation can be significantly enhanced by preventing phosphorylation of Ngn3 protein and further augmented by conditions promoting differentiation. Taken together, our new findings underline the potential of ductal organoid cultures as a source material for generation of β-like cells and demonstrate that post-translational regulation of reprogramming factors can be exploited to enhance β-cell generation.
| S-EPMC6193982 | biostudies-literature
Project description:Primary human pancreatic ductal organoids (HPDO) have emerged as a model to study pancreas biology and model disease like pancreatitis and pancreatic cancer. Yet, donor material availability, genetic variability and a lack of extensive benchmarking to healthy and disease pancreas limits the range of applications. To address this gap, we established porcine pancreatic ductal organoids (PPDO) as a system from a reliable, genetically defined and easily obtainable source to model pancreatic ductal/progenitor biology. We benchmarked PPDO to HPDO and primary porcine pancreas using single-cell RNA sequencing (scRNA-Seq). We observed no overt phenotypic differences in PPDO derived from distinct developmental stages using extensive proteomics profiling, with a WNT/basal cell signaling enriched population characterizing PPDO. PPDO exhibited differentiation potential towards mature ductal cells and limited potential towards endocrine lineages. We used PPDO as a chemical screening platform to assess the safety of FDA-approved drugs and showed conserved toxicity of statins and α-adrenergic receptor inhibitors between PPDO and HPDO cultures. Overall, our results highlight the PPDO as a model for mammalian duct/progenitor applications.
2025-11-10 | PXD068063 | Pride
Project description:Aims/hypothesis: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it. Methods: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming. Results: Ngn3 stimulates duct cells to express a focused set of genes that are abundant in islet endocrine cells and/or neural tissues. This neuro-endocrine shift, however, covers a minor fraction (5%) of the estimated genome-wide transcriptome difference between duct and islet endocrine cells. Interestingly, transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1. Conclusions/interpretation: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes. We used Affymetrix HG133A and HG133B to get a comprehensive view on the reprogramming potential in vitro of human pancreatic duct cell cultures (n=3-4) at 3 and 14/20 days after ectopic adenoviral expression of murine neurogenin 3 as compared to GFP-expressing control vectors. The microarray analysis was performed on 3 independent samples that each contained RNA extracted from a pool of 3 independent donor pancreata. The total number of non-selected donor organs is 9. Transcripts were considered as differentially regulated by Ngn3 when 1.5 fold (LCB, unpaired P < 0.05) up- or down-regulated in AdGFP-Ngn3 versus AdGFP controls, at 3 and/or 14 dpi. Transcripts that showed differential expression between day 3 and day 14 in AdGFP-Ngn3 duct cells but not in AdGFP control cells, were also considered Ngn3-regulated
2012-07-10 | E-GEOD-30802 | biostudies-arrayexpress
Project description:Gene expression profiles from ALDH high cells sorted from expanded adult human pancreatic organoids are more similar to fetal pancreatic tissue and ALDH high cells sorted from expanded fetal human pancreatic organoids than to adult human islets or adult islet-depleted exocrine tissue.
2019-01-08 | GSE108854 | GEO
Project description:MicroRNA (miRNA) expression profiles for pancreatic endocrine tumors were examined to investigate the miRNA involvement in pancreatic carcinogenesis. miRNA microarray analysis identified statistical unique profiles, which could discriminate pancreatic cancers from noncancerous pancreas tissues.
2007-05-11 | E-TABM-48 | biostudies-arrayexpress
Project description:Aims/hypothesis: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it. Methods: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming. Results: Ngn3 stimulates duct cells to express a focused set of genes that are abundant in islet endocrine cells and/or neural tissues. This neuro-endocrine shift, however, covers a minor fraction (5%) of the estimated genome-wide transcriptome difference between duct and islet endocrine cells. Interestingly, transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1. Conclusions/interpretation: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes. We used Affymetrix HG133A and HG133B to get a comprehensive view on the reprogramming potential in vitro of human pancreatic duct cell cultures (n=3-4) at 3 and 14/20 days after ectopic adenoviral expression of murine neurogenin 3 as compared to GFP-expressing control vectors.
2012-07-11 | GSE30802 | GEO
Project description:NEUROD1 is a transcription factor that helps maintain a mature phenotype of pancreatic β cells. Disruption of Neurod1 during pancreatic development causes severe neonatal diabetes; however, the exact role of NEUROD1 in the differentiation programs of endocrine cells is unknown. Here, we report a crucial role of the NEUROD1 regulatory network in endocrine lineage commitment and differentiation. Mechanistically, transcriptome and chromatin landscape analyses demonstrate that Neurod1 inactivation triggers a downregulation of endocrine differentiation transcription factors and upregulation of non-endocrine genes within the Neurod1-deficient endocrine cell population, disturbing endocrine identity acquisition. Neurod1 deficiency altered the H3K27me3 histone modification pattern in promoter regions of differentially expressed genes, which resulted in gene regulatory network changes in the differentiation pathway of endocrine cells, compromising endocrine cell potential, differentiation, and functional properties.
2023-08-17 | GSE236559 | GEO
Project description:Organogenesis is a complex process that relies on a dynamic interplay between extrinsic factors originating from the microenvironment and tissue-specific intrinsic factors. For pancreatic endocrine cells, the local niche consists of acinar and ductal cells as well as neuronal, immune, endothelial, and stromal cells. Hematopoietic cells have been detected in human pancreas as early as 6 post-conception weeks, but whether they play a role during human endocrinogenesis remains unknown. To investigate this, we performed single nuclei RNA sequencing of the second trimester human pancreas and identified a wide range of hematopoietic cells, including two distinct subsets of tissue-resident macrophages. Leveraging this discovery, we developed a co-culture system of human embryonic stem cell-derived endocrine-macrophage organoids to model their interaction in vitro. Here, we show that macrophages support the differentiation and viability of endocrine cells in vitro and enhance tissue engraftment, highlighting their potential role in tissue engineering strategies for diabetes.
2024-10-14 | GSE230403 | GEO