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Characterization of a human iPSC-derived islet differentiation model

ABSTRACT: To explore the contribution of islet development to T2D pathogenesis, we characterised the transcriptomes of 3 human iPSC lines (from independent donors) differentiated along the islet development lineage. Whole transcriptome RNA-seq was performed at 7 developmental stages: definitive endoderm, primitive gut tube, posterior foregut (PF), pancreatic endoderm, endocrine progenitors, endocrine-like cells, and beta-like cells (BLC). Differentially-expressed (ΔExp) genes were assigned to the stage in which they were most upregulated (versus baseline iPSC profile), and used to assess the enrichment of T2D GWAS genes, as well as to predict the upstream transcription factors at each developmental stage.We found 9409 ΔExp genes across all stages, including known markers of islet development (NEUROG3, INS). Genes ΔExp in the most-differentiated (BLC) stage were significantly enriched for genes within the 99% credible sets of T2D GWAS loci. Despite this enrichment in BLC only, over 70% of genes mapping within the credible sets were ΔExp before this stage, highlighting the relevance of key T2D GWAS genes in islet development: e.g. expression of TCF7L2 peaked during the PF stage (log2FC=1.2; q=8.5×10-10). REST and LMNA are also highlighted by the analyses as having a potential function in islet development, considering their pattern of expression and those of the genes they regulate. Transcriptomic signatures derived from this iPSC differentiation model highlight T2D-associated GWAS loci, monogenic diabetes genes and potential master regulators of gene networks with plausible function in islet development. All these provide clues for developmental mechanisms contributing to T2D diabetes pathology.

PROVIDER: EGAS00001002721 | EGA |

REPOSITORIES: EGA

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Characterization of a human iPSC-derived endocrine pancreas model

Project description:Directed differentiation of stem cells offers a scalable solution to the need for human cell models recapitulating islet biology and T2D pathogenesis. We profiled mRNA expression at six stages of an induced pluripotent stem cell (iPSC) model of endocrine pancreas development from two donors, and characterized the distinct transcriptomic profiles associated with each stage. Established regulators of endodermal lineage commitment, such as SOX17 (log2 fold change [FC] compared to iPSCs=14.2, p-value=4.9x10-5) and the pancreatic agenesis gene GATA6 (log2 FC=12.1, p-value=8.6x10-5), showed transcriptional variation consistent with their known developmental roles. However, these analyses highlighted many other genes with stage-specific expression patterns, some of which may be novel drivers or markers of islet development. For example, the leptin receptor gene, LEPR, was most highly expressed in published data from in vivo-matured cells compared to our endocrine pancreas-like cells (log2 FC=5.5, p-value=2.0x10-12), suggesting a role for the leptin pathway in the maturation process. Endocrine pancreas-like cells showed significant stage-selective expression of adult islet genes, including INS, ABCC8, and GLP1R, and enrichment of relevant GO-terms (e.g. "insulin secretion"; odds ratio=4.2, p-value=1.9x10-3): however, principal component analysis indicated that in vitro-differentiated cells were more immature than adult islets. Integration of the stage-specific expression information with genetic data from T2D genome-wide association studies revealed that 46 of 82 T2D-associated loci harbor genes present in at least one developmental stage, facilitating refinement of potential effector transcripts. Together, these data show that expression profiling in an iPSC islet development model can further understanding of islet biology and T2D pathogenesis.

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ena-DATASET-OCDEM-31-05-2016-14:24:47:911-22 - samples

Project description:Directed differentiation of stem cells offers a scalable solution to the need for human cell models recapitulating islet biology and T2D pathogenesis. We profiled mRNA expression at six stages of an induced pluripotent stem cell (iPSC) model of endocrine pancreas development from two donors, and characterized the distinct transcriptomic profiles associated with each stage. Established regulators of endodermal lineage commitment, such as SOX17 (log2 fold change [FC] compared to iPSCs=14.2, p-value=4.9x10-5) and the pancreatic agenesis gene GATA6 (log2 FC=12.1, p-value=8.6x10-5), showed transcriptional variation consistent with their known developmental roles. However, these analyses highlighted many other genes with stage-specific expression patterns, some of which may be novel drivers or markers of islet development. For example, the leptin receptor gene, LEPR, was most highly expressed in published data from in vivo-matured cells compared to the endocrine pancreas-like cells (log2 FC=5.5, p-value=2.0x10-12), suggesting a role for the leptin pathway in the maturation process. Endocrine pancreas-like cells showed significant stage-selective expression of adult islet genes, including INS, ABCC8, and GLP1R, and enrichment of relevant GO-terms (e.g. “insulin secretion”; odds ratio=4.2, p-value=1.9x10-3): however, principal component analysis indicated that in vitro-differentiated cells were more immature than adult islets. Integration of the stage-specific expression information with genetic data from T2D genome-wide association studies revealed that 46 of 82 T2D-associated loci harbor genes present in at least one developmental stage, facilitating refinement of potential effector transcripts. Together, these data show that expression profiling in an iPSC islet development model can further understanding of islet biology and T2D pathogenesis.

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2021-11-03 | PXD018887 | Pride

Insulin deficiency distorts human pancreatic endocrine cell composition during development

Project description:Objective: The developmental effects of mutations in genes associated with monogenic diabetes on human pancreas development is not well understood. More specifically, if insulin gene recessive mutations influence the human endocrine lineage segregation still needs to be investigated. Methods: We generated a novel knock-in H2B-Cherry reporter human induced pluripotent stem cell (iPSCs) line expressing no insulin upon differentiation to stem cell-derived (SC-) β cells in vitro. This cell line enabled us to have a model mimicking the extremely reduced insulin levels in patients with recessive insulin mutations. We combined immunostaining, Western blotting and proteomics analysis to characterize the SC-islets from this iPSC line. Furthermore, we leveraged FACS analysis and imaging to explore the impact of insulin shortage on human endocrine cell induction, composition and proliferation. Results: We found that lack of insulin hampers insulin receptor (IR) signaling in SC-islets but increases the IR sensitivity. Furthermore, insulin deficiency showed no effects on human endocrine lineage induction. However, lack of insulin skewed the SC-islet cell composition. We found an increased in SC-β cell number at the expense of SC-α cell differentiation in the absence of insulin. Finally, insulin shortage reduced the rate of SC-β cell proliferation but had no impact of the expansion of SC-α cells. Conclusions: We provided evidence of the developmental impacts of reduced insulin levels on human β cell characteristics and endocrine lineage formation. These findings help to better understand the pathomechanisms of recessive insulin mutations during embryonic development and also shed some lights on the possible physiological function of this hormone coordinating human islet cell composition and architecture during endocrinogenesis.

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Project description:Ngn3 is a master regulator of pancreatic endocrine development. It is necessary for the creation of all endocrine cells in mice. Little is known about the genes that act downstream of the transcription factor Ngn3 in pancreas endocrine development to specify each of the endocrine lineages. As a consequence, little is known about the genes involved in early development and the specification of the beta cell. We used microarrays to identify Ngn3 downstream genes that are involved in early and ectopic beta cell development in Xenopus laevis. We overexpressed Ngn3 in the Xenopus early endoderm and analyzed the genes that are upregulated four hours after. Xenopus laevis embryos were injected with Ngn3-GR mRNA at the eight-cell stage in the two dorsal vegetal blastomeres. Embryos at stage 12 were either treated with dexamethasone (+DEX) for four hours or no DEX (-DEX). The endoderm was dissected at stage 15. We looked for genes upregulated in the +DEX samples compared to the -DEX samples.

2012-03-31 | E-GEOD-29017 | biostudies-arrayexpress

Pancreatic progenitor epigenome maps prioritize type 2 diabetes risk genes with roles in development

Project description:Genetic variants associated with type 2 diabetes (T2D) risk affect gene regulation in metabolically relevant tissues, such as pancreatic islets. Here, we investigated contributions of regulatory programs active during pancreatic development to T2D risk. Interrogation of chromatin maps from developmental precursors throughout pancreatic differentiation of human embryonic stem cells (hESCs) identifies enrichment of T2D variants in pancreatic progenitor-specific stretch enhancers that are not active in islets. Genes associated with progenitor-specific stretch enhancers are predicted to regulate developmental processes, most notably tissue morphogenesis. Through gene editing in hESCs, we demonstrate that progenitor-specific enhancers harboring T2D-associated variants regulate cell polarity genes LAMA1 and CRB2. Knockdown of lama1 or crb2 in zebrafish embryos causes a defect in pancreas morphogenesis and impairs islet cell development. Together, our findings reveal that a subset of T2D risk variants specifically affects pancreatic developmental programs, suggesting that dysregulation of developmental processes can predispose to T2D.

2021-01-19 | GSE149148 | GEO

A comparative study of endoderm differentiation in humans and chimpanzees

Project description:There is substantial interest in the genetic regulatory framework that is established in early human development, and in the evolutionary forces that shaped early developmental processes in humans. Progress in these areas has been slow because it is difficult to obtain relevant biological samples. Recent technological developments in the generation and differentiation of inducible pluripotent stem cells (iPSCs) provide the ability to develop in vitro models of early human and non-human primates developmental stages. We have previously established matched iPSC panels from humans and chimpanzees. Using these panels, we comparatively characterized gene regulatory changes through a four-day timecourse differentiation of iPSCs (day 1) into primary streak (day 2), endoderm progenitors (day 3), and definitive endoderm (day 4). As might be expected, we found that differentiation stage (in effect, cell type) is the major driver of variation in gene expression levels in our study, followed by species. We then identified thousands of differentially expressed genes between humans and chimpanzees in each differentiation stage. Yet, when we considered gene-specific dynamic regulatory trajectories throughout the timecourse, we found that 75% of genes, including nearly all known endoderm developmental markers, have conserved trajectories in the two species. Interestingly, we observed a marked reduction of both intra- and inter-species variation in gene expression levels in primitive streak samples compared to the iPSCs, with a recovery of variation in endoderm progenitors. The reduction in variation in gene expression levels at a specific developmental stage, paired with the high degree of conservation of temporal expression across species, is consistent with the dynamics of developmental canalization. Overall, we conclude that endoderm development in iPSC-based models are highly conserved and canalized between humans and our closest evolutionary relative.

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2016-09-22 | E-MTAB-5061 | biostudies-arrayexpress

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