RNA profiling of PDX1+ pancreatic progenitors before or after co-culture with different types of cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Der-I Kao
PROVIDER: E-GEOD-63840 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Analysis of gene expression of Pdx-EGFP1+ pancreatic progenitors before or after co-culture at mRNA level. The hypothesis tested in the study was that the overall gene expression in Pdx1-EGFP+ does not alter after co-culture with endothelial cells. The result supported our hypothesis.
2014-12-04 | GSE63840 | GEO
Project description:Sox9/Pdx1 co-regulated target genes were identified by comparing gene expression in Sox9/Pdx1-double heterozygates versus Sox9- or Pdx1- heterozygates pancreata using microarray analysis. Pdx1LacZko (herein designated Pdx1-/+) (Offield et al. 1996) and one Sox9 allele was conditionally deleted in the developing pancreas via recombination of a Sox9-flox allele (Kist et al., 2002) using the Foxa3-Cre transgene (Lee et al., 2005). Total RNA was isolated and pooled from dorsal pancreatic epithelia of e12.5 Sox9fl/+; Foxa3-Cre (Sox9 het), Pdx1-/+ (Pdx1 het) versus Sox9fl/+; Foxa3-Cre; Pdx1-/+ (Sox9/Pdx1 double hets) littermates for three biological replicates
2015-08-07 | E-GEOD-62023 | biostudies-arrayexpress
Project description:The aim of the study was to define the specific gene expression signature of pancreatic endoderm cells (PECs), by designing a strategy for generating putative PECs (PDX1+/NKX6-1+, and posterior foregut endoderm (PFG) cells (PDX1+/NKX6-1-). Cell line: PDX1-eGFP reporter cell line (PDXeG clone 170-3) generated using the HUES-4 embryonic stem cell line. Human embryonic stem cells (hESCs) were maintained on irradiated mouse embryonic fibroblasts (MEFs). Generation of a hESC-derived PDX1-eGFP reporter cell line: A PDX1-targeting vector was constructed by inserting an eGFP-pSV40-NeoR reporter cassette into the 5â² untranslated region of the PDX1 gene upstream of the PDX1 start codon (ATG), resulting in an GFP-tagged PDX1 allele. The GFP cassette was flanked by a 12.5kb homologous arm on the 5â direction and by a 3,5kb homologous arm on the 3 direction. The final targeting construct was verified by PCR, restriction analysis and sequencing. After approximately 2 weeks emerging clones were picked, expanded and analyzed by PCR. The Neomycin cassette was deleted by using a CRE expression vector (NLS-CRE-IRES-Puro) that was transiently co-transfected with a DsRED or GFP plasmid into selected targeted cloned. 24 hours post electroporation, GFP/DsRED positive single cells were FACS sorted and plated into 96-well plates. Clones were expanded and characterized for Neo excision by PCR. Differentiation protocol: hESCs cultured on MEFs were grown until 90% confluency and differentiated into definitive endoderm (DE) by using a modified version of the DâAmour protocol (D'Amour et al., 2005). The first day of differentiation, hESCs were cultured in the presence of 100ng/ml human Activin A (Peprotech) and 25ng/ml Wnt3a (R&D systems) in RPMI medium (Life Technologies). The four following days 100ng/ml human Activin A was added together with 1x B27-insulin in RPMI medium (Life Technologies). To generate posterior foregut cells, DE cells were treated with 64 ng/ml bFGF for additional 12 days. To generate pancreatic endoderm cells, DE cells were first treated with 2uM retinoic acid (RA, Sigma Aldrich) in DMEM/F12 medium containing 1x B27 +insulin (Life Technologies) for three days and then finally treated with 64 ng/ml FGF2 in combination with 100 ng/ml Noggin for the remaining 9 days.
2017-04-05 | E-MTAB-5088 | biostudies-arrayexpress
Project description:Missense mutations in coding region of PDX1 predispose to type-2 diabetes mellitus as well as cause MODY through largely unexplored mechanisms. Here, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying heterozygous missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). Using an in vitro β-cell differentiation protocol, we demonstrated that both PDX1P33T/+, PDX1C18R/+ and PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NEURONATIN, both involved in insulin synthesis and secretion. Our results reveal mechanistic details of how diabetes-associated PDX1 point mutations impair human pancreatic endocrine lineage formation and β-cell function and contribute to pre-disposition for diabetes. Methods: In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions.
2019-06-19 | GSE125590 | GEO
Project description:Objective: Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor (TF) PDX1 leads to pancreatic agenesis, whereas heterozygous mutations can cause Maturity-Onset Diabetes of the Young 4 (MODY4). Although the function of Pdx1 is well studied in pre-clinical models during insulin-producing β-cell development and homeostasis, it remains elusive how this TF controls human pancreas development by regulating a downstream transcriptional program. Furthermore, many studies reported the association between single nucleotide polymorphisms (SNPs) and T2DM and it has been shown that islet enhancers are enriched in T2DM-associated SNPs. Whether regions, harboring T2DM-associated SNPs are PDX1 bound and active at the pancreatic progenitor stage has not been reported so far. Methods: In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions. Results: ChIP-seq for PDX1 revealed a total of 8088 PDX1-bound regions that map to 5664 genes in iPSC-derived PPs. The PDX1 target regions included important pancreatic TFs, such as PDX1 itself, RFX6, HNF1B and MEIS1, which were activated during the differentiation process as revealed by the active mono-acetylated chromatin mark H3K27ac and mRNA expression profiling, suggesting that auto-regulatory feedback regulation maintains PDX1 expression and initiates a pancreatic TF program. Remarkably, we identified several PDX1 target genes that have not been reported in human so far, including RFX3, required for ciliogenesis and endocrine differentiation in mouse, and the ligand for the Notch receptor, DLL1, which is important for endocrine induction and tip-trunk patterning. The comparison of PDX1 profiles from PPs and adult human islets identified sets of stage-specific target genes, associated with early pancreas development and adult β-cell function. Furthermore, we found an enrichment of T2DM-associated SNPs in active chromatin regions from iPSC-derived PPs. Two of these SNPs fall into PDX1 occupied sites that are located in the intronic regions of TCF7L2 and HNF1B. Both of these genes are key transcriptional regulators of endocrine induction and mutations in cis-regulatory regions predispose to diabetes. Conclusions: Our data provides stage-specific target genes of PDX1 during in vitro differentiation of stem cells into pancreatic progenitors that could be useful to identify pathways and molecular targets that predispose for diabetes. In addition, we show that T2DM-associated SNPs are enriched in active chromatin regions at the pancreatic progenitor stage, suggesting that the susceptibility to T2DM might originate from imperfect execution of a β-cell developmental program.
2018-02-27 | GSE106813 | GEO
Project description:Purpose: we used next generation sequencing to analyze gene expression profiles of pancreatic tissues from KrasG12D;Pdx1-Cre and miR-301a-/-;KrasG12D;Pdx1-Cre mice treated with caerulein. The goals of this study are to compare the different gene expression profiles of pancreatic tissue between KrasG12D;Pdx1-Cre and miR-301a-/-;KrasG12D;Pdx1-Cre mice treated with caerulein.
2021-06-11 | GSE176571 | GEO
Project description:Human pluripotent stem cells were differentiated into PDX1+/NKX6-1+ Pancreatic Progenitors (PPd15 cells), which were subsequently captured and expanded in culture. These culture Pancreatic Progenitors (cPP cells) were capable of self-renewal and could be passaged up to 20 times. Furthermore, cPP cells were capable of differentiation into multiple pancreatic lineages, including c-peptide+ beta-like cells, both in vitro and in vivo.
2017-06-06 | E-MTAB-5731 | biostudies-arrayexpress
Project description:Missense mutations in coding region of PDX1 predispose to type-2 diabetes mellitus as well as cause MODY through largely unexplored mechanisms. Here, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying heterozygous missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). Using an in vitro β-cell differentiation protocol, we demonstrated that both PDX1P33T/+, PDX1C18R/+ and PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NEURONATIN, both involved in insulin synthesis and secretion. Our results reveal mechanistic details of how diabetes-associated PDX1 point mutations impair human pancreatic endocrine lineage formation and β-cell function and contribute to pre-disposition for diabetes.
2019-06-19 | GSE125769 | GEO
Project description:Missense mutations in coding region of PDX1 predispose to type-2 diabetes mellitus as well as cause MODY through largely unexplored mechanisms. Here, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying heterozygous missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). Using an in vitro β-cell differentiation protocol, we demonstrated that both PDX1P33T/+, PDX1C18R/+ and PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NEURONATIN, both involved in insulin synthesis and secretion. Our results reveal mechanistic details of how diabetes-associated PDX1 point mutations impair human pancreatic endocrine lineage formation and β-cell function and contribute to pre-disposition for diabetes.
2019-06-19 | GSE125768 | GEO
Project description:PDX1 binds and regulates numerous genes involved in human pancreatic development but also binds hepatic genes Examination of PDX1 regulated genes in pancreatic derived cell types
2014-06-21 | E-GEOD-58685 | biostudies-arrayexpress