ISX-9 manipulates endocrine progenitor fate revealing conserved intestinal lineages in mouse and human organoids

Tsakmaki, Anastasia; Pedro, Patricia Fonseca; Pavlidis, Polychronis; Hayee, Bu; Bewick, Gavin

doi:10.1016/j.molmet.2020.01.012

Cited by 17 publications

(29 citation statements)

References 53 publications

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“…PDX1-expressing epithelial progenitors are essential to the development of the pancreas and intestines, giving rise to pancreatic endocrine, exocrine and ductal cells [53,54]. NGN3 is expressed in all endocrine progenitors, starting with a series of TFs whose expression regulates endocrine cell differentiation, including Nkx2.2, NeuroD1, NKX6-1, PAX4, PAX6 and ISL [55][56][57][58].…”

Section: Discussionmentioning

confidence: 99%

LncRNA Malat1 regulates iPSC-derived β-cell differentiation by targeting the miR-15b-5p/Ihh axis

Wang

Lin

et al. 2021

Preprint

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Background: Differentiation of induced pluripotent stem cell (iPSC)-derived β-like cells is a novel strategy for treatment of type 1 diabetes. Elucidation of the regulatory mechanisms of long noncoding RNAs (lncRNAs) in β-like cells derived from iPSCs is important for understanding the development of the pancreas and pancreatic β-cells and may improve the quality of β-like cells for stem cell therapy.Methods: β-like cells were derived from iPSCs in a three-step protocol. RNA sequencing and bioinformatics analysis were carried out to screen the differentially expressed lncRNAs and identify the putative target genes separately. LncRNA Malat1 was chosen for further research. Series of loss and gain of functions experiments were performed to study the biological function of this lncRNA. Quantitative real-time PCR (qRT-PCR), Western blot analysis and immunofluorescence (IF) staining were carried out to separately detect the functions of pancreatic β-cells at the mRNA and protein levels. Cytoplasmic and nuclear RNA fractionation and fluorescence in situ hybridization (FISH) were used to determine the subcellar location of lncRNA Malat1 in β-like cells. Flow cytometry and enzyme-linked immunosorbent assays (ELISAs) were performed to examine the differentiation and insulin secretion of β-like cells after stimulation with different glucose concentrations. Structural interactions between lncRNA Malat1 and miR-15b-5p and between miR-15b-5p and Ihh were detected by dual luciferase reporter assays (LRAs).Results: We found that the expression of lncRNA Malat1 declined during differentiation, and overexpression of this lncRNA notably impaired the differentiation and maturation of β-like cells derived from iPSCs in vitro and in vivo. Localized to the cytoplasm, lncRNA Malat1 could function as a competing endogenous RNA (ceRNA) of miR-15b-5p to regulate the expression of Ihh according to bioinformatics prediction, mechanistic analysis and downstream experiments.Conclusion: This study established an unreported regulatory network of lncRNA Malat1 and the miR-15b-5p/Ihh axis during the differentiation of iPSCs into β-like cells. In addition to acting as an oncogene promoting tumorigenesis, lncRNA Malat1 may be an effective and novel target for treatment of diabetes in the future.

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Section: Discussionmentioning

confidence: 99%

LncRNA Malat1 regulates iPSC-derived β-cell differentiation by targeting the miR-15b-5p/Ihh axis

Wang

Lin

et al. 2021

Preprint

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“…PDX1-expressing epithelial progenitors are essential to the development of the pancreas and intestines, giving rise to pancreatic endocrine, exocrine and ductal cells [51,52]. NGN3 is expressed in all endocrine progenitors, starting with the series of TFs whose expression regulates endocrine-cell differentiation and that includes Nkx2.2, NeuroD1, NKX6-1, PAX4, PAX6 and ISL [53][54][55][56].…”

Section: Discussionmentioning

confidence: 99%

LncRNA MALAT1 Regulates iPSC-derived Β-cell Differentiation by Targeting the miR-15b-5p/Ihh Axis

Wang

Lin

Jin

et al. 2020

Preprint

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Background: iPSCs-derived β-like cell differentiation provides a novel strategy for type 1 diabetes treatment. Clarifying the regulatory mechanisms of lncRNAs in β-like cells derived from induced pluripotent stem cells (iPSCs) is not only significant for understanding the development of pancreas or pancreatic β cells, but also helpful for improving the quality of β-like cells for stem cell therapy.Methods: β-like cells derived from iPSCs followed a three-step protocol. RNA-sequencing was carried out to screen the differentially expressed lncRNAs which was probably involved in the differentiation of pancreatic β cells. Bioinformatics was performed to analyze the putative target genes of significantly differentially expressed lncRNAs. LncRNA Malat1 was chosen for further research. Lentivirus victor, siRNA victor, antagomir victor and mimic victor were constructed for overexpression of lncRNA Malat1, knockdown of lncRNA Malat1, knockdown of miR-15b-5p and overexpression of miR-15b-5p respectively. Quantitative Real-Time PCR (qRT-PCR), Western Blot and Immunofluorescence (IF) staining were carried out to detect the functions of pancreatic β cells at mRNA and protein level separately. Cytoplasmic and nuclear RNA fractionation and Fluorescence in situ hybridization (FISH) were to ventilate the subcellar location of lncRNA Malat1 in β-like cells. Flow cytometry and ELISA were performed to examine differentiation efficiency and function of insulin secretion from β-like cells after being stimulated with different concentrations of glucose. Structural interactions between lncRNA Malat1 and miR-15b-5p and between miR-15b-5p and Ihh were detected by Dual luciferase reporter assay (LRA).Results: We found that expression of lncRNA Malat1 was on the decline during the differentiation and overexpression of this lncRNA obviously impaired the differentiation and maturation of β-like cells derived from iPSCs in vitro and in vivo. Localized to the cytoplasm, lncRNA Malat1 could function as a competing endogenous RNA (ceRNA) of miR-15b-5p to regulate the expression of Ihh according the bioinformatic prediction, mechanistic analysis and downstream experiments.Conclusion: This study built an unreported regulatory network of lncRNA Malat1 and miR-15b-5p/Ihh axis during differentiation of iPSCs into β-like cells. Except for acting as a proverbial oncogene promoting tumorigenesis, lncRNA Malat1 may provide effective and novel molecule for diabetes cell therapy in the future.

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“…S4), we treated small intestinal organoid cultures derived from Tph1-CFP mice with the small molecule isoxazole-9 (ISX-9) 69 . Exposure to ISX-9 activates a transcriptional program involving neurogenic differentiation 1 (NeuroD1) that promotes pancreatic b-cell differentiation and insulin production 70 as well as EEC differentiation with a specific bias towards 5-HT positive EC cells 69 . In our experimental system, increased CFP protein levels were detected in culture homogenates of EC-cell enriched (ISX-9 treated) organoids compared to the control (DMSO vehicle treated) sample as determined by the use of an antibody directed against GFP for immunoblotting (Fig.…”

Section: Caps1 and Rab3 Presynaptic Proteins Are Enriched In Mouse And Human Ec Cellsmentioning

confidence: 99%

“…We next assessed whether candidate proteins of the presynaptic release machinery were specifically enriched in homogenates of ISX-9 treated mouse and human 69 organoids. Using primary antibodies specific for CAPS-1 (SySy, Cat# 262 003) and all Rab3 isoforms (a, b, c, d; SySy #107 003), our approach revealed a prominent enrichment of CAPS-1 and members of the Rab3 protein family together with the EEC marker ChgA in homogenates from ISX-9 stimulated mouse and human organoids (Fig.…”

Section: Caps1 and Rab3 Presynaptic Proteins Are Enriched In Mouse And Human Ec Cellsmentioning

confidence: 99%

“…To stimulate EC cell differentiation in mouse organoids using the N-cyclopropyl-5-(thiophen-2-yl)-isoxazole-3-carboxamide (ISX-9) pulse protocol 69 , crypts from the small intestine of a Tph1-CFP mouse were isolated and plated in 20 µl domes of Matrigel per well of a 48-well plate. The Matrigel was polymerized for 15 min at 37 °C and 250 µl stem cell growth medium containing Wnt3a-conditioned medium [WENR; Advanced DMEM/F12 supplemented with 1 mM N-acetylcysteine, 50% Wnt3A conditioned medium, 10% R-Spondin1-conditioned medium, 5 µl of 100 ng/ml Noggin, 50 ng/ml EGF, 1x N2 supplement, 1x B27 supplement, and 10 µM Y-27632] was added.…”

Section: Small Intestine 3d Organoid Culturesmentioning

confidence: 99%

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Dissecting Functional, Structural, and Molecular Requirements for Serotonin Release from Mouse Enterochromaffin Cells

Shaaban

Maaß

Schwarze

et al. 2021

Preprint

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Serotonergic enterochromaffin (EC) cells of the gut epithelium are secretory sensory cells that communicate with vagal neurons. EC cells exhibit many features of neurons in the brain, raising the hypothesis that synapse-like contacts may mediate fast and directed signalling. To dissect functional, structural, and molecular properties underlying serotonin release from genetically identified EC cells, we employed a multidisciplinary in vitro approach combining intestinal epithelial cell and organoid cultures, electrochemistry, correlated light- and electron microscopy, and gene expression and biochemical analyses. Despite the presence of key molecules of the synaptic neurotransmitter release machinery, we found that the majority of serotonin is released with slow kinetics from large dense-core rather than small synaptic-like vesicles. While we cannot exclude synapse-like transmission between EC cells and neurons in vivo, our data support the notion that the predominant mode of serotonin secretion is similar to that of other endocrine cell types.

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ISX-9 manipulates endocrine progenitor fate revealing conserved intestinal lineages in mouse and human organoids

Cited by 17 publications

References 53 publications

LncRNA Malat1 regulates iPSC-derived β-cell differentiation by targeting the miR-15b-5p/Ihh axis

LncRNA Malat1 regulates iPSC-derived β-cell differentiation by targeting the miR-15b-5p/Ihh axis

LncRNA MALAT1 Regulates iPSC-derived Β-cell Differentiation by Targeting the miR-15b-5p/Ihh Axis

Dissecting Functional, Structural, and Molecular Requirements for Serotonin Release from Mouse Enterochromaffin Cells

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ISX-9 manipulates endocrine progenitor fate revealing conserved intestinal lineages in mouse and human organoids

Cited by 17 publications

References 53 publications

LncRNA Malat1 regulates iPSC-derived β-cell differentiation by targeting the miR-15b-5p/Ihh axis

LncRNA Malat1 regulates iPSC-derived β-cell differentiation by targeting the miR-15b-5p/Ihh axis

LncRNA MALAT1&nbsp;Regulates iPSC-derived Β-cell Differentiation by Targeting the miR-15b-5p/Ihh&nbsp;Axis

Dissecting Functional, Structural, and Molecular Requirements for Serotonin Release from Mouse Enterochromaffin Cells

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LncRNA MALAT1 Regulates iPSC-derived Β-cell Differentiation by Targeting the miR-15b-5p/Ihh Axis