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Abbood MS, Al-Adsani AM, Al-Bustan SA. Ginger extract promotes pancreatic islets regeneration in streptozotocin-induced diabetic rats. Biosci Rep 2025; 45:BSR20241510. [PMID: 40014427 DOI: 10.1042/bsr20241510] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2024] [Revised: 01/29/2025] [Accepted: 02/26/2025] [Indexed: 03/01/2025] Open
Abstract
Ginger (Zingiber officinale) exerts an antidiabetic effect by restoring pancreatic β-cells. The present study aimed to investigate the mechanism by which ginger extract induces the regeneration of functional β-cells in diabetic rats. Sprague-Dawley rats (n=27) were divided into three groups: normal rats given double distilled water (ddH2O) (NC, n=11), diabetic rats (injected with 60 mg/kg streptozotocin) given ddH2O (DC, n=8), and diabetic rats treated with aqueous ginger extract (DG, n=8). The effect of ginger extract intake on the differential expression of neurogenin-3 (Neurog3), V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (Mafb), insulin 2 (Ins2), and glucagon (Gcg) was assessed using quantitative real-time PCR after one and eight weeks of treatment. The pancreatic insulin source was determined using immunohistochemical analysis. After one week, ginger treatment significantly up-regulated the expression of both Neurog3 and Mafb in the DG rats compared with the DC rats. However, after eight weeks, the mRNA levels of these genes dropped significantly in parallel with the up-regulation of Ins2 and Gcg expression, resulting in increased serum insulin levels, weight, and lowered fasting blood glucose levels. Immunohistochemical analysis revealed a restored β-cell mass and islet architecture in the DG group. Ginger extract exerts an antidiabetic effect by acting on pancreatic progenitors and α-cells to restore β-cell mass in streptozotocininduced diabetic rats. These findings suggest that ginger extract could be a potential stimulator of β-cell neogenesis, which provides an alternative to meet the increasing demand for exogenous insulin in patients with diabetes.
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Affiliation(s)
- Manal S Abbood
- Department of Biological Sciences, Faculty of Science, Kuwait University, Shadadiyah, Kuwait P.O. Box 5969, Safat 13060, Kuwait
| | - Amani M Al-Adsani
- Department of Biological Sciences, Faculty of Science, Kuwait University, Shadadiyah, Kuwait P.O. Box 5969, Safat 13060, Kuwait
| | - Suzanne A Al-Bustan
- Department of Biological Sciences, Faculty of Science, Kuwait University, Shadadiyah, Kuwait P.O. Box 5969, Safat 13060, Kuwait
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2
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Weng G, Martin P, Kim H, Won KJ. Integrating Prior Knowledge Using Transformer for Gene Regulatory Network Inference. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2025; 12:e2409990. [PMID: 39605181 PMCID: PMC11744656 DOI: 10.1002/advs.202409990] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/31/2024] [Revised: 10/23/2024] [Indexed: 11/29/2024]
Abstract
Gene regulatory network (GRN) inference, a process of reconstructing gene regulatory rules from experimental data, has the potential to discover new regulatory rules. However, existing methods often struggle to generalize across diverse cell types and account for unseen regulators. Here, this work presents GRNPT, a novel Transformer-based framework that integrates large language model (LLM) embeddings from publicly accessible biological data and a temporal convolutional network (TCN) autoencoder to capture regulatory patterns from single-cell RNA sequencing (scRNA-seq) trajectories. GRNPT significantly outperforms both supervised and unsupervised methods in inferring GRNs, particularly when training data is limited. Notably, GRNPT exhibits exceptional generalizability, accurately predicting regulatory relationships in previously unseen cell types and even regulators. By combining LLMs ability to distillate biological knowledge from text and deep learning methodologies capturing complex patterns in gene expression data, GRNPT overcomes the limitations of traditional GRN inference methods and enables more accurate and comprehensive understanding of gene regulatory dynamics.
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Affiliation(s)
- Guangzheng Weng
- Biotech Research and Innovation Centre (BRIC)University of CopenhagenOle Maaløes Vej 5Copenhagen2200Denmark
| | - Patrick Martin
- Department of Computational BiomedicineCedars‐Sinai Medical CenterLos AngelesCA90069USA
| | - Hyobin Kim
- Department of Computational BiomedicineCedars‐Sinai Medical CenterLos AngelesCA90069USA
| | - Kyoung Jae Won
- Department of Computational BiomedicineCedars‐Sinai Medical CenterLos AngelesCA90069USA
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3
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Zhang W, Wu L, Qu R, Liu T, Wang J, Tong Y, Bei W, Guo J, Hu X. Hesperidin activates the GLP-1R/cAMP-CREB/IRS2/PDX1 pathway to promote transdifferentiation of islet α cells into β cells Across the spectrum. Heliyon 2024; 10:e35424. [PMID: 39220963 PMCID: PMC11365324 DOI: 10.1016/j.heliyon.2024.e35424] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2023] [Revised: 07/12/2024] [Accepted: 07/29/2024] [Indexed: 09/04/2024] Open
Abstract
Background and aims In all age, FoShou as a Chinese medicinal herb has been active in various kinds of Traditional Chinese medicine formula to treating diabetes. Hesperidin (HES), the main monomeric component of FoShou, has been extensively investigated for interventions with pathogenic mechanism of diabetes as well as subsequent treatment of associated complications. Islet β-cells have an essential effect on dynamically regulating blood sugar. Functional abnormalities in these cells and their death are strongly associated with the onset of diabetes. Therefore, induction of islet endocrine cell lineage re-editing for damaged βcell replenishment would be a promising therapeutic tool. Previously, it has been found that HES can protect islet β-cells in vivo, But, the regenerative function of HES in islet β cells and its role in promoting differential non-β cells transdifferentiation into β cells and cell fate rewriting associated mechanisms remain unclear.This work focused on investigating whether HES can induce islet α cells transdifferentiation into β cells for achieving damaged β cell regeneration and the causes and possible mechanisms involved in the process. Materials and methods In brief, 60 mg/kg/d streptozotocin (STZ) was administered intraperitoneally in each male C57bL/6J mouse raised by the high-sugar and high-fat diet (HFD) to create a diabetic mouse model with severe β-cell damage. After 28 consecutive days of HES treatment (160 mg/kg; 320 mg/kg; once daily, as appropriate). Tracing the dynamics of α as well as β cell transformation, together with β cells growth and apoptosis levels during treatment by cell lineage tracing. The self-enforcing transcriptional network on which the cell lineage is based is used as a clue to explore the underlying mechanisms. Guangdong Pharmaceutical University's Animal Experiment Ethics Committee (GDPulac2019180) approved all animal experiments. Results Localization by cell lineage we find that transdifferentiated newborn β-cells derived from α cells appeared in the islet endocrine cell mass of DM mice under HES'action. Compared to the model group, expressed by Tunel staining and CXCL10 levels the overall apoptosis rate of β-cells of the pancreas were reduced,the inflammatory infiltration feedback from HE staining were lower.Ki-67 positive cells showed enhanced β-cell proliferation. Decreased HbA1c and blood glucose contents, elevated C-Peptide and insulin contents which respond to ability of nascent beta cells. Also upregulated the mRNA levels of MafA, Ngn3, PDX-1, Pax4 and Arx. Moreover, increased the expression of TGR5/cAMP-CREB/GLP-1 in mouse intestinal tissues and GLP-1/GLP-1R and cAMP-CREB/IRS2/PDX-1 in pancreatic tissues. Conclusions HES directly affects β-cells, apart from being anti-apoptotic and reducing inflammatory infiltration. HES promotes GLP-1 release by intestinal L cells by activating the TGR5 receptor in DM mouse and regulating its response element CREB signaling. GLP-1 then uses the GLP-1/GLP-1R system to act on IRS2, IRS2 as a port to influence α precursor cells to express PDX-1, with the mobilization of Pax4 strong expression than Arx so that α cell lineage is finally reversed for achieving β cell endogenous proliferation.
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Affiliation(s)
- Wang Zhang
- Guangdong Pharmaceutical University, Guangzhou, 510006, China
| | - Lele Wu
- Guangdong Pharmaceutical University, Guangzhou, 510006, China
| | - Ru Qu
- Guangdong Pharmaceutical University, Guangzhou, 510006, China
| | - Tianfeng Liu
- Guangdong Pharmaceutical University, Guangzhou, 510006, China
| | - Jiliang Wang
- Guangdong Pharmaceutical University, Guangzhou, 510006, China
| | - Ying Tong
- Guangdong Pharmaceutical University, Guangzhou, 510006, China
| | - Weijian Bei
- Guangdong Pharmaceutical University, Guangzhou, 510006, China
| | - Jiao Guo
- Guangdong Pharmaceutical University, Guangzhou, 510006, China
| | - Xuguang Hu
- Guangdong Pharmaceutical University, Guangzhou, 510006, China
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Song Y, Lu S, Gao F, Wei T, Ma W. The application of organoid models in research into metabolic diseases. Diabetes Obes Metab 2024; 26:809-819. [PMID: 38100156 DOI: 10.1111/dom.15390] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/19/2023] [Revised: 11/16/2023] [Accepted: 11/16/2023] [Indexed: 02/06/2024]
Abstract
Metabolic diseases have become a major threat to human health worldwide as a result of changing lifestyles. The exploration of the underlying molecular mechanisms of metabolic diseases and the development of improved therapeutic methods have been hindered by the lack of appropriate human experimental models. Organoids are three-dimensional in vitro models of self-renewing cells that spontaneously self-organize into structures similar to the corresponding in vivo tissues, recapitulating the original tissue function. Off-body organoid technology has been successfully applied to disease modelling, developmental biology, regenerative medicine, and tumour precision medicine. This new generation of biological models has received widespread attention. This article focuses on the construction process and research progress with regard to organoids related to metabolic diseases in recent years, and looks forward to their prospective applications.
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Affiliation(s)
- Yufan Song
- Department of Laboratory Medicine, The First Affiliated Hospital of Shandong First Medical University, Jinan, China
| | - Sumei Lu
- Department of Laboratory Medicine, The First Affiliated Hospital of Shandong First Medical University, Jinan, China
| | - Fei Gao
- Department of Laboratory Medicine, The First Affiliated Hospital of Shandong First Medical University, Jinan, China
| | - Tianshu Wei
- Department of Laboratory Medicine, The First Affiliated Hospital of Shandong First Medical University, Jinan, China
| | - Wanshan Ma
- Department of Laboratory Medicine, The First Affiliated Hospital of Shandong First Medical University, Jinan, China
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Aglan HA, Kotob SE, Mahmoud NS, Kishta MS, Ahmed HH. Bone marrow stem cell-derived β-cells: New issue for diabetes cell therapy. Tissue Cell 2024; 86:102280. [PMID: 38029457 DOI: 10.1016/j.tice.2023.102280] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2023] [Revised: 11/19/2023] [Accepted: 11/20/2023] [Indexed: 12/01/2023]
Abstract
This investigation aimed to establish the promising role of insulin-producing cells (IPCs) growing from bone marrow-mesenchymal stem cells (BM-MSCs) in relieving hyperglycemia induced in rats. BM-MSCs were differentiated into IPCs using three different protocols. The efficiency of BM-MSCs differentiation into IPCs in vitro was confirmed by detecting IPCs specific gene expression (Foxa-2, PDX-1 and Ngn-3) and insulin release assay. The in vivo study design included 3 groups of male Wistar rats; negative control group, diabetic group and IPCs-transfused group (5 ×106 cells of the most functional IPCs/rat). One month after IPCs infusion, serum glucose, insulin, c-peptide and visfatin levels as well as pancreatic glucagon level were quantified. Gene expression analysis of pancreatic Foxa-2 and Sox-17, IGF-1 and FGF-10 was done. Additionally, histological investigation of pancreatic tissue sections was performed. Our data clarified that, the most functional IPCs are those generated from BM-MSCs using differentiation protocol 3 as indicated by the significant up-regulation of Foxa-2, PDX-1 and Ngn-3 gene expression levels. These findings were further emphasized by releasing of a significant amount of insulin in response to glucose load. The transplantation of the IPCs in diabetic rats elicited significant decline in serum glucose, visfatin and pancreatic glucagon levels along with significant rise in serum insulin and c-peptide levels. Moreover, it triggered significant up-regulation in the expression levels of pancreatic Foxa-2, Sox-17, IGF-1 and FGF-10 genes versus the untreated diabetic counterpart. The histopathological examination of pancreatic tissue almost assisted the biochemical and molecular genetic analyses. These results disclose that the cell therapy holds potential to develop a new cure for DM based on the capability of BM-MSCs to generate β-cell phenotype using specific protocol.
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Affiliation(s)
- Hadeer A Aglan
- Hormones Department, Medical Research and Clinical Studies Institute, National Research Centre, Dokki, Giza, Egypt; Stem Cell Lab., Center of Excellence for Advanced Sciences, National Research Centre, Dokki, Giza, Egypt.
| | - Soheir E Kotob
- Hormones Department, Medical Research and Clinical Studies Institute, National Research Centre, Dokki, Giza, Egypt
| | - Nadia S Mahmoud
- Hormones Department, Medical Research and Clinical Studies Institute, National Research Centre, Dokki, Giza, Egypt; Stem Cell Lab., Center of Excellence for Advanced Sciences, National Research Centre, Dokki, Giza, Egypt
| | - Mohamed S Kishta
- Hormones Department, Medical Research and Clinical Studies Institute, National Research Centre, Dokki, Giza, Egypt; Stem Cell Lab., Center of Excellence for Advanced Sciences, National Research Centre, Dokki, Giza, Egypt
| | - Hanaa H Ahmed
- Hormones Department, Medical Research and Clinical Studies Institute, National Research Centre, Dokki, Giza, Egypt; Stem Cell Lab., Center of Excellence for Advanced Sciences, National Research Centre, Dokki, Giza, Egypt
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Tanday N, Tarasov AI, Moffett RC, Flatt PR, Irwin N. Pancreatic islet cell plasticity: Pathogenic or therapeutically exploitable? Diabetes Obes Metab 2024; 26:16-31. [PMID: 37845573 DOI: 10.1111/dom.15300] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/31/2023] [Revised: 09/07/2023] [Accepted: 09/18/2023] [Indexed: 10/18/2023]
Abstract
The development of pancreatic islet endocrine cells is a tightly regulated process leading to the generation of distinct cell types harbouring different hormones in response to small changes in environmental stimuli. Cell differentiation is driven by transcription factors that are also critical for the maintenance of the mature islet cell phenotype. Alteration of the insulin-secreting β-cell transcription factor set by prolonged metabolic stress, associated with the pathogenesis of diabetes, obesity or pregnancy, results in the loss of β-cell identity through de- or transdifferentiation. Importantly, the glucose-lowering effects of approved and experimental antidiabetic agents, including glucagon-like peptide-1 mimetics, novel peptides and small molecules, have been associated with preventing or reversing β-cell dedifferentiation or promoting the transdifferentiation of non-β-cells towards an insulin-positive β-cell-like phenotype. Therefore, we review the manifestations of islet cell plasticity in various experimental settings and discuss the physiological and therapeutic sides of this phenomenon, focusing on strategies for preventing β-cell loss or generating new β-cells in diabetes. A better understanding of the molecular mechanisms underpinning islet cell plasticity is a prerequisite for more targeted therapies to help prevent β-cell decline in diabetes.
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Affiliation(s)
- Neil Tanday
- Diabetes Research Centre, School of Biomedical Sciences, Ulster University, Coleraine, Northern Ireland
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany
| | - Andrei I Tarasov
- Diabetes Research Centre, School of Biomedical Sciences, Ulster University, Coleraine, Northern Ireland
| | - R Charlotte Moffett
- Diabetes Research Centre, School of Biomedical Sciences, Ulster University, Coleraine, Northern Ireland
| | - Peter R Flatt
- Diabetes Research Centre, School of Biomedical Sciences, Ulster University, Coleraine, Northern Ireland
| | - Nigel Irwin
- Diabetes Research Centre, School of Biomedical Sciences, Ulster University, Coleraine, Northern Ireland
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Beydag-Tasöz BS, D'Costa JV, Hersemann L, Lee BH, Luppino F, Kim YH, Zechner C, Grapin-Botton A. Integrating single-cell imaging and RNA sequencing datasets links differentiation and morphogenetic dynamics of human pancreatic endocrine progenitors. Dev Cell 2023; 58:2292-2308.e6. [PMID: 37591246 DOI: 10.1016/j.devcel.2023.07.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2021] [Revised: 05/20/2023] [Accepted: 07/27/2023] [Indexed: 08/19/2023]
Abstract
Basic helix-loop-helix genes, particularly proneural genes, are well-described triggers of cell differentiation, yet information on their dynamics is limited, notably in human development. Here, we focus on Neurogenin 3 (NEUROG3), which is crucial for pancreatic endocrine lineage initiation. By monitoring both NEUROG3 gene expression and protein in single cells using a knockin dual reporter in 2D and 3D models of human pancreas development, we show an approximately 2-fold slower expression of human NEUROG3 than that of the mouse. We observe heterogeneous peak levels of NEUROG3 expression and reveal through long-term live imaging that both low and high NEUROG3 peak levels can trigger differentiation into hormone-expressing cells. Based on fluorescence intensity, we statistically integrate single-cell transcriptome with dynamic behaviors of live cells and propose a data-mapping methodology applicable to other contexts. Using this methodology, we identify a role for KLK12 in motility at the onset of NEUROG3 expression.
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Affiliation(s)
- Belin Selcen Beydag-Tasöz
- The Novo Nordisk Foundation Center for Stem Cell Biology, Copenhagen 2200, Denmark; Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Saxony 01307, Germany
| | - Joyson Verner D'Costa
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Saxony 01307, Germany
| | - Lena Hersemann
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Saxony 01307, Germany
| | - Byung Ho Lee
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Saxony 01307, Germany
| | - Federica Luppino
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Saxony 01307, Germany; Center for Systems Biology Dresden Dresden 01307, Germany
| | - Yung Hae Kim
- The Novo Nordisk Foundation Center for Stem Cell Biology, Copenhagen 2200, Denmark; Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Saxony 01307, Germany
| | - Christoph Zechner
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Saxony 01307, Germany; Center for Systems Biology Dresden Dresden 01307, Germany; Cluster of Excellence Physics of Life, TU Dresden, Dresden 01062, Germany
| | - Anne Grapin-Botton
- The Novo Nordisk Foundation Center for Stem Cell Biology, Copenhagen 2200, Denmark; Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Saxony 01307, Germany; Center for Systems Biology Dresden Dresden 01307, Germany; Cluster of Excellence Physics of Life, TU Dresden, Dresden 01062, Germany; Paul Langerhans Institute Dresden of the Helmholtz Zentrum München at the University Clinic Carl Gustav Carus of Technische Universität Dresden, Helmholtz Zentrum München, Neuherberg, Germany.
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Hrovatin K, Bastidas-Ponce A, Bakhti M, Zappia L, Büttner M, Salinno C, Sterr M, Böttcher A, Migliorini A, Lickert H, Theis FJ. Delineating mouse β-cell identity during lifetime and in diabetes with a single cell atlas. Nat Metab 2023; 5:1615-1637. [PMID: 37697055 PMCID: PMC10513934 DOI: 10.1038/s42255-023-00876-x] [Citation(s) in RCA: 28] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/21/2022] [Accepted: 07/26/2023] [Indexed: 09/13/2023]
Abstract
Although multiple pancreatic islet single-cell RNA-sequencing (scRNA-seq) datasets have been generated, a consensus on pancreatic cell states in development, homeostasis and diabetes as well as the value of preclinical animal models is missing. Here, we present an scRNA-seq cross-condition mouse islet atlas (MIA), a curated resource for interactive exploration and computational querying. We integrate over 300,000 cells from nine scRNA-seq datasets consisting of 56 samples, varying in age, sex and diabetes models, including an autoimmune type 1 diabetes model (NOD), a glucotoxicity/lipotoxicity type 2 diabetes model (db/db) and a chemical streptozotocin β-cell ablation model. The β-cell landscape of MIA reveals new cell states during disease progression and cross-publication differences between previously suggested marker genes. We show that β-cells in the streptozotocin model transcriptionally correlate with those in human type 2 diabetes and mouse db/db models, but are less similar to human type 1 diabetes and mouse NOD β-cells. We also report pathways that are shared between β-cells in immature, aged and diabetes models. MIA enables a comprehensive analysis of β-cell responses to different stressors, providing a roadmap for the understanding of β-cell plasticity, compensation and demise.
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Affiliation(s)
- Karin Hrovatin
- Institute of Computational Biology, Helmholtz Zentrum München, Neuherberg, Germany
- TUM School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany
| | - Aimée Bastidas-Ponce
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany
- German Center for Diabetes Research (DZD), Neuherberg, Germany
- Medical Faculty, Technical University of Munich, Munich, Germany
| | - Mostafa Bakhti
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany
- German Center for Diabetes Research (DZD), Neuherberg, Germany
| | - Luke Zappia
- Institute of Computational Biology, Helmholtz Zentrum München, Neuherberg, Germany
- Department of Mathematics, Technical University of Munich, Garching, Germany
| | - Maren Büttner
- Institute of Computational Biology, Helmholtz Zentrum München, Neuherberg, Germany
- Genomics and Immunoregulation, Life & Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany
- Systems Medicine, Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Bonn, Germany
| | - Ciro Salinno
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany
- German Center for Diabetes Research (DZD), Neuherberg, Germany
- Medical Faculty, Technical University of Munich, Munich, Germany
| | - Michael Sterr
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany
- German Center for Diabetes Research (DZD), Neuherberg, Germany
| | - Anika Böttcher
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany
- German Center for Diabetes Research (DZD), Neuherberg, Germany
| | - Adriana Migliorini
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany
- German Center for Diabetes Research (DZD), Neuherberg, Germany
- McEwen Stem Cell Institute, University Health Network (UHN), Toronto, Ontario, Canada
| | - Heiko Lickert
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany.
- German Center for Diabetes Research (DZD), Neuherberg, Germany.
- Medical Faculty, Technical University of Munich, Munich, Germany.
| | - Fabian J Theis
- Institute of Computational Biology, Helmholtz Zentrum München, Neuherberg, Germany.
- TUM School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany.
- Department of Mathematics, Technical University of Munich, Garching, Germany.
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9
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Jiménez S, Schreiber V, Mercier R, Gradwohl G, Molina N. Characterization of cell-fate decision landscapes by estimating transcription factor dynamics. CELL REPORTS METHODS 2023; 3:100512. [PMID: 37533652 PMCID: PMC10391345 DOI: 10.1016/j.crmeth.2023.100512] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/06/2022] [Revised: 03/23/2023] [Accepted: 06/01/2023] [Indexed: 08/04/2023]
Abstract
Time-specific modulation of gene expression during differentiation by transcription factors promotes cell diversity. However, estimating their dynamic regulatory activity at the single-cell level and in a high-throughput manner remains challenging. We present FateCompass, an integrative approach that utilizes single-cell transcriptomics data to identify lineage-specific transcription factors throughout differentiation. By combining a probabilistic framework with RNA velocities or differentiation potential, we estimate transition probabilities, while a linear model of gene regulation is employed to compute transcription factor activities. Considering dynamic changes and correlations of expression and activities, FateCompass identifies lineage-specific regulators. Our validation using in silico data and application to pancreatic endocrine cell differentiation datasets highlight both known and potentially novel lineage-specific regulators. Notably, we uncovered undescribed transcription factors of an enterochromaffin-like population during in vitro differentiation toward ß-like cells. FateCompass provides a valuable framework for hypothesis generation, advancing our understanding of the gene regulatory networks driving cell-fate decisions.
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Affiliation(s)
- Sara Jiménez
- Université de Strasbourg, Strasbourg, France
- CNRS, UMR 7104, 67400 Illkirch, France
- INSERM, UMR-S 1258, 67400 Illkirch, France
- IGBMC, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67400 Illkirch, France
| | - Valérie Schreiber
- Université de Strasbourg, Strasbourg, France
- CNRS, UMR 7104, 67400 Illkirch, France
- INSERM, UMR-S 1258, 67400 Illkirch, France
- IGBMC, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67400 Illkirch, France
| | - Reuben Mercier
- Université de Strasbourg, Strasbourg, France
- CNRS, UMR 7104, 67400 Illkirch, France
- INSERM, UMR-S 1258, 67400 Illkirch, France
- IGBMC, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67400 Illkirch, France
| | - Gérard Gradwohl
- Université de Strasbourg, Strasbourg, France
- CNRS, UMR 7104, 67400 Illkirch, France
- INSERM, UMR-S 1258, 67400 Illkirch, France
- IGBMC, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67400 Illkirch, France
| | - Nacho Molina
- Université de Strasbourg, Strasbourg, France
- CNRS, UMR 7104, 67400 Illkirch, France
- INSERM, UMR-S 1258, 67400 Illkirch, France
- IGBMC, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67400 Illkirch, France
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Naina Marikar S, Al-Hasani K, Khurana I, Kaipananickal H, Okabe J, Maxwell S, El-Osta A. Pharmacological inhibition of human EZH2 can influence a regenerative β-like cell capacity with in vitro insulin release in pancreatic ductal cells. Clin Epigenetics 2023; 15:101. [PMID: 37309004 DOI: 10.1186/s13148-023-01491-z] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2023] [Accepted: 04/24/2023] [Indexed: 06/14/2023] Open
Abstract
BACKGROUND Therapeutic replacement of pancreatic endocrine β-cells is key to improving hyperglycaemia caused by insulin-dependent diabetes . Whilst the pool of ductal progenitors, which give rise to the endocrine cells, are active during development, neogenesis of islets is repressed in the human adult. Recent human donor studies have demonstrated the role of EZH2 inhibition in surgically isolated exocrine cells showing reactivation of insulin expression and the influence on the H3K27me3 barrier to β-cell regeneration. However, those studies fall short on defining the cell type active in transcriptional reactivation events. This study examines the role of the regenerative capacity of human pancreatic ductal cells when stimulated with pharmacological inhibitors of the EZH2 methyltransferase. RESULTS Human pancreatic ductal epithelial cells were stimulated with the EZH2 inhibitors GSK-126, EPZ6438, and triptolide using a 2- and 7-day protocol to determine their influence on the expression of core endocrine development marker NGN3, as well as β-cell markers insulin, MAFA, and PDX1. Chromatin immunoprecipitation studies show a close correspondence of pharmacological EZH2 inhibition with reduced H3K27me3 content of the core genes, NGN3, MAFA and PDX1. Consistent with the reduction of H3K27me3 by pharmacological inhibition of EZH2, we observe measurable immunofluorescence staining of insulin protein and glucose-sensitive insulin response. CONCLUSION The results of this study serve as a proof of concept for a probable source of β-cell induction from pancreatic ductal cells that are capable of influencing insulin expression. Whilst pharmacological inhibition of EZH2 can stimulate secretion of detectable insulin from ductal progenitor cells, further studies are required to address mechanism and the identity of ductal progenitor cell targets to improve likely methods designed to reduce the burden of insulin-dependent diabetes.
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Affiliation(s)
- Safiya Naina Marikar
- Epigenetics in Human Health and Disease Program, Baker Heart and Diabetes Institute, 75 Commercial Road, VIC, 3004, Melbourne, Australia
- Department of Diabetes, Central Clinical School, Monash University, VIC, 3004, Melbourne, Australia
- Epigenetics in Human Health and Disease Laboratory, Central Clinical School, Monash University, Melbourne, VIC, 3004, Australia
| | - Keith Al-Hasani
- Epigenetics in Human Health and Disease Program, Baker Heart and Diabetes Institute, 75 Commercial Road, VIC, 3004, Melbourne, Australia
- Department of Diabetes, Central Clinical School, Monash University, VIC, 3004, Melbourne, Australia
- Epigenetics in Human Health and Disease Laboratory, Central Clinical School, Monash University, Melbourne, VIC, 3004, Australia
| | - Ishant Khurana
- Epigenetics in Human Health and Disease Program, Baker Heart and Diabetes Institute, 75 Commercial Road, VIC, 3004, Melbourne, Australia
- Department of Diabetes, Central Clinical School, Monash University, VIC, 3004, Melbourne, Australia
- Epigenetics in Human Health and Disease Laboratory, Central Clinical School, Monash University, Melbourne, VIC, 3004, Australia
| | - Harikrishnan Kaipananickal
- Epigenetics in Human Health and Disease Program, Baker Heart and Diabetes Institute, 75 Commercial Road, VIC, 3004, Melbourne, Australia
- Department of Diabetes, Central Clinical School, Monash University, VIC, 3004, Melbourne, Australia
- Epigenetics in Human Health and Disease Laboratory, Central Clinical School, Monash University, Melbourne, VIC, 3004, Australia
| | - Jun Okabe
- Epigenetics in Human Health and Disease Program, Baker Heart and Diabetes Institute, 75 Commercial Road, VIC, 3004, Melbourne, Australia
- Department of Diabetes, Central Clinical School, Monash University, VIC, 3004, Melbourne, Australia
- Epigenetics in Human Health and Disease Laboratory, Central Clinical School, Monash University, Melbourne, VIC, 3004, Australia
| | - Scott Maxwell
- Epigenetics in Human Health and Disease Program, Baker Heart and Diabetes Institute, 75 Commercial Road, VIC, 3004, Melbourne, Australia
- Department of Diabetes, Central Clinical School, Monash University, VIC, 3004, Melbourne, Australia
- Epigenetics in Human Health and Disease Laboratory, Central Clinical School, Monash University, Melbourne, VIC, 3004, Australia
| | - Assam El-Osta
- Epigenetics in Human Health and Disease Program, Baker Heart and Diabetes Institute, 75 Commercial Road, VIC, 3004, Melbourne, Australia.
- Department of Diabetes, Central Clinical School, Monash University, VIC, 3004, Melbourne, Australia.
- Epigenetics in Human Health and Disease Laboratory, Central Clinical School, Monash University, Melbourne, VIC, 3004, Australia.
- Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Sha Tin, Hong Kong SAR.
- Hong Kong Institute of Diabetes and Obesity, Prince of Wales Hospital, The Chinese University of Hong Kong, 3/F Lui Che Woo Clinical Sciences Building, 30‑32 Ngan Shing Street, Sha Tin, Hong Kong SAR.
- Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Sha Tin, Hong Kong SAR.
- Biomedical Laboratory Science, Department of Technology, Faculty of Health, University College Copenhagen, Copenhagen, Denmark.
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11
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Huang X, Gu W, Zhang J, Lan Y, Colarusso JL, Li S, Pertl C, Lu J, Kim H, Zhu J, Breault DT, Sévigny J, Zhou Q. Stomach-derived human insulin-secreting organoids restore glucose homeostasis. Nat Cell Biol 2023; 25:778-786. [PMID: 37106062 PMCID: PMC10859913 DOI: 10.1038/s41556-023-01130-y] [Citation(s) in RCA: 17] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2022] [Accepted: 03/15/2023] [Indexed: 04/29/2023]
Abstract
Gut stem cells are accessible by biopsy and propagate robustly in culture, offering an invaluable resource for autologous cell therapies. Insulin-producing cells can be induced in mouse gut, but it has not been possible to generate abundant and durable insulin-secreting cells from human gut tissues to evaluate their potential as a cell therapy for diabetes. Here we describe a protocol to differentiate cultured human gastric stem cells into pancreatic islet-like organoids containing gastric insulin-secreting (GINS) cells that resemble β-cells in molecular hallmarks and function. Sequential activation of the inducing factors NGN3 and PDX1-MAFA led human gastric stem cells onto a distinctive differentiation path, including a SOX4High endocrine and GalaninHigh GINS precursor, before adopting β-cell identity, at efficiencies close to 70%. GINS organoids acquired glucose-stimulated insulin secretion in 10 days and restored glucose homeostasis for over 100 days in diabetic mice after transplantation, providing proof of concept for a promising approach to treat diabetes.
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Affiliation(s)
- Xiaofeng Huang
- Division of Regenerative Medicine and Hartman Institute for Therapeutic Organ Regeneration, Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Wei Gu
- Division of Regenerative Medicine and Hartman Institute for Therapeutic Organ Regeneration, Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Jiaoyue Zhang
- Division of Regenerative Medicine and Hartman Institute for Therapeutic Organ Regeneration, Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Ying Lan
- Division of Regenerative Medicine and Hartman Institute for Therapeutic Organ Regeneration, Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Jonathan L Colarusso
- Division of Regenerative Medicine and Hartman Institute for Therapeutic Organ Regeneration, Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Sanlan Li
- Division of Regenerative Medicine and Hartman Institute for Therapeutic Organ Regeneration, Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Christoph Pertl
- Division of Regenerative Medicine and Hartman Institute for Therapeutic Organ Regeneration, Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Jiaqi Lu
- Division of Regenerative Medicine and Hartman Institute for Therapeutic Organ Regeneration, Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Hyunkee Kim
- Division of Regenerative Medicine and Hartman Institute for Therapeutic Organ Regeneration, Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Jian Zhu
- Division of Regenerative Medicine and Hartman Institute for Therapeutic Organ Regeneration, Department of Medicine, Weill Cornell Medicine, New York, NY, USA
- Department of Endocrinology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China
| | - David T Breault
- Division of Endocrinology, Boston Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, MA, USA
- Harvard Stem Cell Institute, Cambridge, MA, USA
| | - Jean Sévigny
- Département de Microbiologie-Infectiologie et d'Immunologie, Faculté de Médecine, Université Laval, Quebec City, Quebec, Canada
- Centre de recherche du CHU de Québec-Université Laval, Quebec City, Quebec, Canada
| | - Qiao Zhou
- Division of Regenerative Medicine and Hartman Institute for Therapeutic Organ Regeneration, Department of Medicine, Weill Cornell Medicine, New York, NY, USA.
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12
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Manea T, Nelson JK, Garrone CM, Hansson K, Evans I, Behrens A, Sancho R. USP7 controls NGN3 stability and pancreatic endocrine lineage development. Nat Commun 2023; 14:2457. [PMID: 37117185 PMCID: PMC10147604 DOI: 10.1038/s41467-023-38146-9] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2022] [Accepted: 04/18/2023] [Indexed: 04/30/2023] Open
Abstract
Understanding the factors and mechanisms involved in beta-cell development will guide therapeutic efforts to generate fully functional beta cells for diabetes. Neurogenin 3 (NGN3) is the key transcription factor that marks endocrine progenitors and drives beta-cell differentiation. Here we screen for binding partners of NGN3 and identify the deubiquitylating enzyme USP7 as a key regulator of NGN3 stability. Mechanistically, USP7 interacts with, deubiquitinates and stabilizes NGN3. In vivo, conditional knockout of Usp7 in the mouse embryonic pancreas causes a dramatic reduction in islet formation and hyperglycemia in adult mice, due to impaired NGN3-mediated endocrine specification during pancreatic development. Furthermore, pharmacological inhibition of USP7 during endocrine specification in human iPSC models of beta-cell differentiation decreases NGN3 expressing progenitor cell numbers and impairs beta cell differentiation. Thus, the USP7-NGN3 axis is an essential mechanism for driving endocrine development and beta-cell differentiation, which can be therapeutically exploited.
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Affiliation(s)
- Teodora Manea
- Centre for Gene Therapy and Regenerative Medicine, King's College London, London, UK
| | - Jessica Kristine Nelson
- Adult Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
- Cancer Stem Cell Laboratory, The Breast Cancer Now Toby Robins Research Centre, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK
| | | | - Karin Hansson
- Cancer Stem Cell Laboratory, The Breast Cancer Now Toby Robins Research Centre, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK
| | - Ian Evans
- Adult Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
- Cancer Stem Cell Laboratory, The Breast Cancer Now Toby Robins Research Centre, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK
| | - Axel Behrens
- Adult Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK
- Cancer Stem Cell Laboratory, The Breast Cancer Now Toby Robins Research Centre, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK
- Imperial College, Division of Cancer, Department of Surgery and Cancer, Imperial College, Exhibition Road, London, SW7 2AZ, UK
- Convergence Science Centre, Imperial College, Exhibition Road, London, SW7 2BU, UK
| | - Rocio Sancho
- Centre for Gene Therapy and Regenerative Medicine, King's College London, London, UK.
- Department of Internal Medicine III, University Hospital Carl Gustav Carus at the Technische Universität Dresden, Dresden, Germany.
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13
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Bohuslavova R, Fabriciova V, Lebrón-Mora L, Malfatti J, Smolik O, Valihrach L, Benesova S, Zucha D, Berkova Z, Saudek F, Evans SM, Pavlinkova G. ISL1 controls pancreatic alpha cell fate and beta cell maturation. Cell Biosci 2023; 13:53. [PMID: 36899442 PMCID: PMC9999528 DOI: 10.1186/s13578-023-01003-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2022] [Accepted: 03/01/2023] [Indexed: 03/12/2023] Open
Abstract
BACKGROUND Glucose homeostasis is dependent on functional pancreatic α and ß cells. The mechanisms underlying the generation and maturation of these endocrine cells remain unclear. RESULTS We unravel the molecular mode of action of ISL1 in controlling α cell fate and the formation of functional ß cells in the pancreas. By combining transgenic mouse models, transcriptomic and epigenomic profiling, we uncover that elimination of Isl1 results in a diabetic phenotype with a complete loss of α cells, disrupted pancreatic islet architecture, downregulation of key ß-cell regulators and maturation markers of ß cells, and an enrichment in an intermediate endocrine progenitor transcriptomic profile. CONCLUSIONS Mechanistically, apart from the altered transcriptome of pancreatic endocrine cells, Isl1 elimination results in altered silencing H3K27me3 histone modifications in the promoter regions of genes that are essential for endocrine cell differentiation. Our results thus show that ISL1 transcriptionally and epigenetically controls α cell fate competence, and ß cell maturation, suggesting that ISL1 is a critical component for generating functional α and ß cells.
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Affiliation(s)
- Romana Bohuslavova
- Laboratory of Molecular Pathogenetics, Institute of Biotechnology CAS, 25250, Vestec, Czechia.
| | - Valeria Fabriciova
- Laboratory of Molecular Pathogenetics, Institute of Biotechnology CAS, 25250, Vestec, Czechia
| | - Laura Lebrón-Mora
- Laboratory of Molecular Pathogenetics, Institute of Biotechnology CAS, 25250, Vestec, Czechia
| | - Jessica Malfatti
- Laboratory of Molecular Pathogenetics, Institute of Biotechnology CAS, 25250, Vestec, Czechia
| | - Ondrej Smolik
- Laboratory of Molecular Pathogenetics, Institute of Biotechnology CAS, 25250, Vestec, Czechia
| | - Lukas Valihrach
- Laboratory of Gene Expression, Institute of Biotechnology CAS, 25250, Vestec, Czechia
| | - Sarka Benesova
- Laboratory of Gene Expression, Institute of Biotechnology CAS, 25250, Vestec, Czechia
| | - Daniel Zucha
- Laboratory of Gene Expression, Institute of Biotechnology CAS, 25250, Vestec, Czechia
| | - Zuzana Berkova
- Laboratory of Pancreatic Islets, Institute for Clinical and Experimental Medicine, 14021, Prague, Czechia
| | - Frantisek Saudek
- Laboratory of Pancreatic Islets, Institute for Clinical and Experimental Medicine, 14021, Prague, Czechia
| | - Sylvia M Evans
- Department of Pharmacology; Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA, USA
| | - Gabriela Pavlinkova
- Laboratory of Molecular Pathogenetics, Institute of Biotechnology CAS, 25250, Vestec, Czechia.
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14
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Sasaki S, Miyatsuka T. Heterogeneity of Islet Cells during Embryogenesis and Differentiation. Diabetes Metab J 2023; 47:173-184. [PMID: 36631992 PMCID: PMC10040626 DOI: 10.4093/dmj.2022.0324] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/21/2022] [Accepted: 10/31/2022] [Indexed: 01/13/2023] Open
Abstract
Diabetes is caused by insufficient insulin secretion due to β-cell dysfunction and/or β-cell loss. Therefore, the restoration of functional β-cells by the induction of β-cell differentiation from embryonic stem (ES) and induced-pluripotent stem (iPS) cells, or from somatic non-β-cells, may be a promising curative therapy. To establish an efficient and feasible method for generating functional insulin-producing cells, comprehensive knowledge of pancreas development and β-cell differentiation, including the mechanisms driving cell fate decisions and endocrine cell maturation is crucial. Recent advances in single-cell RNA sequencing (scRNA-seq) technologies have opened a new era in pancreas development and diabetes research, leading to clarification of the detailed transcriptomes of individual insulin-producing cells. Such extensive high-resolution data enables the inference of developmental trajectories during cell transitions and gene regulatory networks. Additionally, advancements in stem cell research have not only enabled their immediate clinical application, but also has made it possible to observe the genetic dynamics of human cell development and maturation in a dish. In this review, we provide an overview of the heterogeneity of islet cells during embryogenesis and differentiation as demonstrated by scRNA-seq studies on the developing and adult pancreata, with implications for the future application of regenerative medicine for diabetes.
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Affiliation(s)
- Shugo Sasaki
- Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Suita, Japan
| | - Takeshi Miyatsuka
- Department of Endocrinology, Diabetes and Metabolism, Kitasato University School of Medicine, Sagamihara, Japan
- Corresponding author: Takeshi Miyatsuka https://orcid.org/0000-0003-2618-3450 Department of Endocrinology, Diabetes and Metabolism, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan E-mail:
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15
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Verhoeff K, Cuesta-Gomez N, Jasra I, Marfil-Garza B, Dadheech N, Shapiro AMJ. Optimizing Generation of Stem Cell-Derived Islet Cells. Stem Cell Rev Rep 2022; 18:2683-2698. [PMID: 35639237 DOI: 10.1007/s12015-022-10391-3] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/13/2022] [Indexed: 02/06/2023]
Abstract
Islet transplantation is a highly effective treatment for select patients with type 1 diabetes. Unfortunately, current use is limited to those with brittle disease due to donor limitations and immunosuppression requirements. Discovery of factors for induction of pluripotent stem cells from adult somatic cells into a malleable state has reinvigorated the possibility of autologous-based regenerative cell therapies. Similarly, recent progress in allogeneic human embryonic stem cell islet products is showing early success in clinical trials. Describing safe and standardized differentiation protocols with clear pathways to optimize yield and minimize off-target growth is needed to efficiently move the field forward. This review discusses current islet differentiation protocols with a detailed break-down of differentiation stages to guide step-wise controlled generation of functional islet products.
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Affiliation(s)
- Kevin Verhoeff
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
- Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada
| | - Nerea Cuesta-Gomez
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
- Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada
| | - Ila Jasra
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
- Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada
| | - Braulio Marfil-Garza
- National Institute of Medical Sciences and Nutrition Salvador Zubiran, Mexico City, and CHRISTUS-LatAm Hub - Excellence and Innovation Center, Monterrey, Mexico
| | - Nidheesh Dadheech
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
- Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada
| | - A M James Shapiro
- Department of Surgery, University of Alberta, Edmonton, Alberta, Canada.
- Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada.
- 1-002 Li Ka Shing Centre for Health Research Innovation, 112 St. NW & 87 Ave NW, Edmonton, Alberta, T6G 2E1, Canada.
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16
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Jeyagaran A, Lu CE, Zbinden A, Birkenfeld AL, Brucker SY, Layland SL. Type 1 diabetes and engineering enhanced islet transplantation. Adv Drug Deliv Rev 2022; 189:114481. [PMID: 36002043 PMCID: PMC9531713 DOI: 10.1016/j.addr.2022.114481] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2022] [Revised: 08/01/2022] [Accepted: 08/02/2022] [Indexed: 01/24/2023]
Abstract
The development of new therapeutic approaches to treat type 1 diabetes mellitus (T1D) relies on the precise understanding and deciphering of insulin-secreting β-cell biology, as well as the mechanisms responsible for their autoimmune destruction. β-cell or islet transplantation is viewed as a potential long-term therapy for the millions of patients with diabetes. To advance the field of insulin-secreting cell transplantation, two main research areas are currently investigated by the scientific community: (1) the identification of the developmental pathways that drive the differentiation of stem cells into insulin-producing cells, providing an inexhaustible source of cells; and (2) transplantation strategies and engineered transplants to provide protection and enhance the functionality of transplanted cells. In this review, we discuss the biology of pancreatic β-cells, pathology of T1D and current state of β-cell differentiation. We give a comprehensive view and discuss the different possibilities to engineer enhanced insulin-secreting cell/islet transplantation from a translational perspective.
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Affiliation(s)
- Abiramy Jeyagaran
- Institute of Biomedical Engineering, Department for Medical Technologies and Regenerative Medicine, Eberhard Karls University Tübingen, 72076 Tübingen, Germany; NMI Natural and Medical Sciences Institute at the University Tübingen, 72770 Reutlingen, Germany
| | - Chuan-En Lu
- Institute of Biomedical Engineering, Department for Medical Technologies and Regenerative Medicine, Eberhard Karls University Tübingen, 72076 Tübingen, Germany
| | - Aline Zbinden
- Department of Immunology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
| | - Andreas L Birkenfeld
- Department of Internal Medicine IV, University Hospital Tübingen, Tübingen, Germany; Institute for Diabetes Research and Metabolic Diseases (IDM) of the Helmholtz Center Munich at the University of Tübingen, German Center for Diabetes Research (DZD e.V.), Munich, Germany
| | - Sara Y Brucker
- Department of Women's Health, Eberhard Karls University, 72076 Tübingen, Germany
| | - Shannon L Layland
- Institute of Biomedical Engineering, Department for Medical Technologies and Regenerative Medicine, Eberhard Karls University Tübingen, 72076 Tübingen, Germany; Department of Women's Health, Eberhard Karls University, 72076 Tübingen, Germany.
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17
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An artificial LAMA2-GelMA hydrogel microenvironment for the development of pancreatic endocrine progenitors. Biomaterials 2022; 291:121882. [DOI: 10.1016/j.biomaterials.2022.121882] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2022] [Revised: 10/15/2022] [Accepted: 10/23/2022] [Indexed: 11/21/2022]
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18
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Yang L, Hu ZM, Jiang FX, Wang W. Stem cell therapy for insulin-dependent diabetes: Are we still on the road? World J Stem Cells 2022; 14:503-512. [PMID: 36157527 PMCID: PMC9350623 DOI: 10.4252/wjsc.v14.i7.503] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/17/2022] [Revised: 04/26/2022] [Accepted: 06/26/2022] [Indexed: 02/06/2023] Open
Abstract
In insulin-dependent diabetes, the islet β cells do not produce enough insulin and the patients must receive exogenous insulin to control blood sugar. However, there are still many deficiencies in exogenous insulin supplementation. Therefore, the replacement of destroyed functional β cells with insulin-secreting cells derived from functional stem cells is a good idea as a new therapeutic idea. This review introduces the development schedule of mouse and human embryonic islets. The differences between mouse and human pancreas embryo development were also listed. Accordingly to the different sources of stem cells, the important research achievements on the differentiation of insulin-secreting β cells of stem cells and the current research status of stem cell therapy for diabetes were reviewed. Stem cell replacement therapy is a promising treatment for diabetes, caused by defective insulin secretion, but there are still many problems to be solved, such as the biosafety and reliability of treatment, the emergence of tumors during treatment, untargeted differentiation and autoimmunity, etc. Therefore, further understanding of stem cell therapy for insulin is needed.
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Affiliation(s)
- Lu Yang
- Department of Endocrinology, Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen 361100, Fujian Province, China
| | - Zhu-Meng Hu
- Department of Endocrinology, Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen 361100, Fujian Province, China
| | - Fang-Xu Jiang
- Department of Endocrinology, Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen 361100, Fujian Province, China
- School of Biomedical Science, University of Western Australia, Nedlands 6009, Australia
- School of Health and Medical Sciences, Edith Cowan University, Perth 6000, Australia
| | - Wei Wang
- Department of Endocrinology, Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen 361100, Fujian Province, China.
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19
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Yang L, Hu ZM, Jiang FX, Wang W. Stem cell therapy for insulin-dependent diabetes: Are we still on the road? World J Stem Cells 2022. [DOI: 10.4252/wjsc.v14.i7.503 yang l] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
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20
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Perez-Frances M, Abate MV, Baronnier D, Scherer PE, Fujitani Y, Thorel F, Herrera PL. Adult pancreatic islet endocrine cells emerge as fetal hormone-expressing cells. Cell Rep 2022; 38:110377. [PMID: 35172145 PMCID: PMC8864465 DOI: 10.1016/j.celrep.2022.110377] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2021] [Revised: 12/07/2021] [Accepted: 01/21/2022] [Indexed: 12/13/2022] Open
Abstract
The precise developmental dynamics of the pancreatic islet endocrine cell types, and their interrelation, are unknown. Some authors claim the persistence of islet cell differentiation from precursor cells after birth (“neogenesis”). Here, using four conditional cell lineage tracing (“pulse-and-chase”) murine models, we describe the natural history of pancreatic islet cells, once they express a hormone gene, until late in life. Concerning the contribution of early-appearing embryonic hormone-expressing cells to the formation of islets, we report that adult islet cells emerge from embryonic hormone-expressing cells arising at different time points during development, without any evidence of postnatal neogenesis. We observe specific patterns of hormone gene activation and switching during islet morphogenesis, revealing that, within each cell type, cells have heterogeneous developmental trajectories. This likely applies to most maturating cells in the body, and explains the observed phenotypic variability within differentiated cell types. Such knowledge should help devising novel regenerative therapies.
Adult pancreatic islet endocrine cells arise as embryonic hormone-expressing cells No detectable islet cell differentiation from putative precursor cells after birth Some embryonic hormone-producing cells display a switch in hormone gene expression
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Affiliation(s)
- Marta Perez-Frances
- Department of Genetic Medicine & Development, iGE3 and Centre Facultaire du Diabète, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland
| | - Maria Valentina Abate
- Department of Genetic Medicine & Development, iGE3 and Centre Facultaire du Diabète, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland
| | - Delphine Baronnier
- Department of Genetic Medicine & Development, iGE3 and Centre Facultaire du Diabète, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland
| | - Philipp E Scherer
- Touchstone Diabetes Center, Departments of Internal Medicine and Cell Biology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8549, USA
| | - Yoshio Fujitani
- Laboratory of Developmental Biology & Metabolism, Institute for Molecular & Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512, Japan
| | - Fabrizio Thorel
- Department of Genetic Medicine & Development, iGE3 and Centre Facultaire du Diabète, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland
| | - Pedro L Herrera
- Department of Genetic Medicine & Development, iGE3 and Centre Facultaire du Diabète, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland.
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21
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Lange M, Bergen V, Klein M, Setty M, Reuter B, Bakhti M, Lickert H, Ansari M, Schniering J, Schiller HB, Pe'er D, Theis FJ. CellRank for directed single-cell fate mapping. Nat Methods 2022; 19:159-170. [PMID: 35027767 PMCID: PMC8828480 DOI: 10.1038/s41592-021-01346-6] [Citation(s) in RCA: 338] [Impact Index Per Article: 112.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2020] [Accepted: 11/07/2021] [Indexed: 12/20/2022]
Abstract
Computational trajectory inference enables the reconstruction of cell state dynamics from single-cell RNA sequencing experiments. However, trajectory inference requires that the direction of a biological process is known, largely limiting its application to differentiating systems in normal development. Here, we present CellRank (https://cellrank.org) for single-cell fate mapping in diverse scenarios, including regeneration, reprogramming and disease, for which direction is unknown. Our approach combines the robustness of trajectory inference with directional information from RNA velocity, taking into account the gradual and stochastic nature of cellular fate decisions, as well as uncertainty in velocity vectors. On pancreas development data, CellRank automatically detects initial, intermediate and terminal populations, predicts fate potentials and visualizes continuous gene expression trends along individual lineages. Applied to lineage-traced cellular reprogramming data, predicted fate probabilities correctly recover reprogramming outcomes. CellRank also predicts a new dedifferentiation trajectory during postinjury lung regeneration, including previously unknown intermediate cell states, which we confirm experimentally. CellRank infers directed cell state transitions and cell fates incorporating RNA velocity information into a graph based Markov process.
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Affiliation(s)
- Marius Lange
- Institute of Computational Biology, Helmholtz Center Munich, Munich, Germany.,Department of Mathematics, Technical University of Munich, Munich, Germany
| | - Volker Bergen
- Institute of Computational Biology, Helmholtz Center Munich, Munich, Germany.,Department of Mathematics, Technical University of Munich, Munich, Germany
| | - Michal Klein
- Institute of Computational Biology, Helmholtz Center Munich, Munich, Germany
| | - Manu Setty
- Program for Computational and Systems Biology, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.,Basic Sciences Division and Translational Data Science IRC, Fred Hutchinson Cancer Research Center, Seattle WA, USA
| | - Bernhard Reuter
- Department of Computer Science, University of Tübingen, Tübingen, Germany.,Zuse Institute Berlin (ZIB), Berlin, Germany
| | - Mostafa Bakhti
- Institute of Diabetes and Regeneration Research, Helmholtz Center Munich, Munich, Germany.,German Center for Diabetes Research (DZD), Neuherberg, Germany
| | - Heiko Lickert
- Institute of Diabetes and Regeneration Research, Helmholtz Center Munich, Munich, Germany.,German Center for Diabetes Research (DZD), Neuherberg, Germany
| | - Meshal Ansari
- Institute of Computational Biology, Helmholtz Center Munich, Munich, Germany.,Comprehensive Pneumology Center (CPC) / Institute of Lung Biology and Disease (ILBD), Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
| | - Janine Schniering
- Comprehensive Pneumology Center (CPC) / Institute of Lung Biology and Disease (ILBD), Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
| | - Herbert B Schiller
- Comprehensive Pneumology Center (CPC) / Institute of Lung Biology and Disease (ILBD), Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
| | - Dana Pe'er
- Program for Computational and Systems Biology, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
| | - Fabian J Theis
- Institute of Computational Biology, Helmholtz Center Munich, Munich, Germany. .,Department of Mathematics, Technical University of Munich, Munich, Germany. .,TUM School of Life Sciences Weihenstephan, Technical University of Munich, Munich, Germany.
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22
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van Roey R, Brabletz T, Stemmler MP, Armstark I. Deregulation of Transcription Factor Networks Driving Cell Plasticity and Metastasis in Pancreatic Cancer. Front Cell Dev Biol 2021; 9:753456. [PMID: 34888306 PMCID: PMC8650502 DOI: 10.3389/fcell.2021.753456] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2021] [Accepted: 10/27/2021] [Indexed: 12/15/2022] Open
Abstract
Pancreatic cancer is a very aggressive disease with 5-year survival rates of less than 10%. The constantly increasing incidence and stagnant patient outcomes despite changes in treatment regimens emphasize the requirement of a better understanding of the disease mechanisms. Challenges in treating pancreatic cancer include diagnosis at already progressed disease states due to the lack of early detection methods, rapid acquisition of therapy resistance, and high metastatic competence. Pancreatic ductal adenocarcinoma, the most prevalent type of pancreatic cancer, frequently shows dominant-active mutations in KRAS and TP53 as well as inactivation of genes involved in differentiation and cell-cycle regulation (e.g. SMAD4 and CDKN2A). Besides somatic mutations, deregulated transcription factor activities strongly contribute to disease progression. Specifically, transcriptional regulatory networks essential for proper lineage specification and differentiation during pancreas development are reactivated or become deregulated in the context of cancer and exacerbate progression towards an aggressive phenotype. This review summarizes the recent literature on transcription factor networks and epigenetic gene regulation that play a crucial role during tumorigenesis.
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Affiliation(s)
- Ruthger van Roey
- Department of Experimental Medicine 1, Nikolaus-Fiebiger Center for Molecular Medicine, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany
| | - Thomas Brabletz
- Department of Experimental Medicine 1, Nikolaus-Fiebiger Center for Molecular Medicine, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany
| | - Marc P Stemmler
- Department of Experimental Medicine 1, Nikolaus-Fiebiger Center for Molecular Medicine, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany
| | - Isabell Armstark
- Department of Experimental Medicine 1, Nikolaus-Fiebiger Center for Molecular Medicine, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany
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23
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Siehler J, Blöchinger AK, Meier M, Lickert H. Engineering islets from stem cells for advanced therapies of diabetes. Nat Rev Drug Discov 2021; 20:920-940. [PMID: 34376833 DOI: 10.1038/s41573-021-00262-w] [Citation(s) in RCA: 61] [Impact Index Per Article: 15.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/22/2021] [Indexed: 12/20/2022]
Abstract
Diabetes mellitus is a metabolic disorder that affects more than 460 million people worldwide. Type 1 diabetes (T1D) is caused by autoimmune destruction of β-cells, whereas type 2 diabetes (T2D) is caused by a hostile metabolic environment that leads to β-cell exhaustion and dysfunction. Currently, first-line medications treat the symptomatic insulin resistance and hyperglycaemia, but do not prevent the progressive decline of β-cell mass and function. Thus, advanced therapies need to be developed that either protect or regenerate endogenous β-cell mass early in disease progression or replace lost β-cells with stem cell-derived β-like cells or engineered islet-like clusters. In this Review, we discuss the state of the art of stem cell differentiation and islet engineering, reflect on current and future challenges in the area and highlight the potential for cell replacement therapies, disease modelling and drug development using these cells. These efforts in stem cell and regenerative medicine will lay the foundations for future biomedical breakthroughs and potentially curative treatments for diabetes.
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Affiliation(s)
- Johanna Siehler
- Institute of Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany.,Technical University of Munich, Medical Faculty, Munich, Germany.,German Center for Diabetes Research (DZD), Neuherberg, Germany
| | - Anna Karolina Blöchinger
- Technical University of Munich, Medical Faculty, Munich, Germany.,German Center for Diabetes Research (DZD), Neuherberg, Germany.,Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany
| | - Matthias Meier
- Technical University of Munich, Medical Faculty, Munich, Germany.,Helmholtz Pioneer Campus, Helmholtz Zentrum München, Neuherberg, Germany
| | - Heiko Lickert
- Institute of Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany. .,Technical University of Munich, Medical Faculty, Munich, Germany. .,German Center for Diabetes Research (DZD), Neuherberg, Germany. .,Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany.
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24
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Hogrebe NJ, Maxwell KG, Augsornworawat P, Millman JR. Generation of insulin-producing pancreatic β cells from multiple human stem cell lines. Nat Protoc 2021; 16:4109-4143. [PMID: 34349281 DOI: 10.1038/s41596-021-00560-y] [Citation(s) in RCA: 113] [Impact Index Per Article: 28.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2020] [Accepted: 04/19/2021] [Indexed: 12/13/2022]
Abstract
We detail a six-stage planar differentiation methodology for generating human pluripotent stem cell-derived pancreatic β cells (SC-β cells) that secrete high amounts of insulin in response to glucose stimulation. This protocol first induces definitive endoderm by treatment with Activin A and CHIR99021, then generates PDX1+/NKX6-1+ pancreatic progenitors through the timed application of keratinocyte growth factor, SANT1, TPPB, LDN193189 and retinoic acid. Endocrine induction and subsequent SC-β-cell specification is achieved with a cocktail consisting of the cytoskeletal depolymerizing compound latrunculin A combined with XXI, T3, ALK5 inhibitor II, SANT1 and retinoic acid. The resulting SC-β cells and other endocrine cell types can then be aggregated into islet-like clusters for analysis and transplantation. This differentiation methodology takes ~34 d to generate functional SC-β cells, plus an additional 1-2 weeks for initial stem cell expansion and final cell assessment. This protocol builds upon a large body of previous work for generating β-like cells. In this iteration, we have eliminated the need for 3D culture during endocrine induction, allowing for the generation of highly functional SC-β cells to be done entirely on tissue culture polystyrene. This change simplifies the differentiation methodology, requiring only basic stem cell culture experience as well as familiarity with assessment techniques common in biology laboratories. In addition to expanding protocol accessibility and simplifying SC-β-cell generation, we demonstrate that this planar methodology is amenable for differentiating SC-β cells from a wide variety of cell lines from various sources, broadening its applicability.
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Affiliation(s)
- Nathaniel J Hogrebe
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO, USA
| | - Kristina G Maxwell
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO, USA.,Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA
| | - Punn Augsornworawat
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO, USA.,Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA
| | - Jeffrey R Millman
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO, USA. .,Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA.
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25
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Bohuslavova R, Smolik O, Malfatti J, Berkova Z, Novakova Z, Saudek F, Pavlinkova G. NEUROD1 Is Required for the Early α and β Endocrine Differentiation in the Pancreas. Int J Mol Sci 2021; 22:6713. [PMID: 34201511 PMCID: PMC8268837 DOI: 10.3390/ijms22136713] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2021] [Revised: 06/18/2021] [Accepted: 06/21/2021] [Indexed: 11/17/2022] Open
Abstract
Diabetes is a metabolic disease that involves the death or dysfunction of the insulin-secreting β cells in the pancreas. Consequently, most diabetes research is aimed at understanding the molecular and cellular bases of pancreatic development, islet formation, β-cell survival, and insulin secretion. Complex interactions of signaling pathways and transcription factor networks regulate the specification, growth, and differentiation of cell types in the developing pancreas. Many of the same regulators continue to modulate gene expression and cell fate of the adult pancreas. The transcription factor NEUROD1 is essential for the maturation of β cells and the expansion of the pancreatic islet cell mass. Mutations of the Neurod1 gene cause diabetes in humans and mice. However, the different aspects of the requirement of NEUROD1 for pancreas development are not fully understood. In this study, we investigated the role of NEUROD1 during the primary and secondary transitions of mouse pancreas development. We determined that the elimination of Neurod1 impairs the expression of key transcription factors for α- and β-cell differentiation, β-cell proliferation, insulin production, and islets of Langerhans formation. These findings demonstrate that the Neurod1 deletion altered the properties of α and β endocrine cells, resulting in severe neonatal diabetes, and thus, NEUROD1 is required for proper activation of the transcriptional network and differentiation of functional α and β cells.
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Affiliation(s)
- Romana Bohuslavova
- Institute of Biotechnology CAS, 25250 Vestec, Czech Republic; (R.B.); (O.S.); (J.M.); (Z.N.)
| | - Ondrej Smolik
- Institute of Biotechnology CAS, 25250 Vestec, Czech Republic; (R.B.); (O.S.); (J.M.); (Z.N.)
- Department of Cell Biology, Faculty of Science, Charles University, 12843 Prague, Czech Republic
| | - Jessica Malfatti
- Institute of Biotechnology CAS, 25250 Vestec, Czech Republic; (R.B.); (O.S.); (J.M.); (Z.N.)
- Department of Cell Biology, Faculty of Science, Charles University, 12843 Prague, Czech Republic
| | - Zuzana Berkova
- Laboratory of Pancreatic Islets, Institute for Clinical and Experimental Medicine, 14021 Prague, Czech Republic; (Z.B.); (F.S.)
| | - Zaneta Novakova
- Institute of Biotechnology CAS, 25250 Vestec, Czech Republic; (R.B.); (O.S.); (J.M.); (Z.N.)
| | - Frantisek Saudek
- Laboratory of Pancreatic Islets, Institute for Clinical and Experimental Medicine, 14021 Prague, Czech Republic; (Z.B.); (F.S.)
| | - Gabriela Pavlinkova
- Institute of Biotechnology CAS, 25250 Vestec, Czech Republic; (R.B.); (O.S.); (J.M.); (Z.N.)
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26
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Yeo GHT, Saksena SD, Gifford DK. Generative modeling of single-cell time series with PRESCIENT enables prediction of cell trajectories with interventions. Nat Commun 2021; 12:3222. [PMID: 34050150 PMCID: PMC8163769 DOI: 10.1038/s41467-021-23518-w] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Accepted: 04/22/2021] [Indexed: 12/20/2022] Open
Abstract
Existing computational methods that use single-cell RNA-sequencing (scRNA-seq) for cell fate prediction do not model how cells evolve stochastically and in physical time, nor can they predict how differentiation trajectories are altered by proposed interventions. We introduce PRESCIENT (Potential eneRgy undErlying Single Cell gradIENTs), a generative modeling framework that learns an underlying differentiation landscape from time-series scRNA-seq data. We validate PRESCIENT on an experimental lineage tracing dataset, where we show that PRESCIENT is able to predict the fate biases of progenitor cells in hematopoiesis when accounting for cell proliferation, improving upon the best-performing existing method. We demonstrate how PRESCIENT can simulate trajectories for perturbed cells, recovering the expected effects of known modulators of cell fate in hematopoiesis and pancreatic β cell differentiation. PRESCIENT is able to accommodate complex perturbations of multiple genes, at different time points and from different starting cell populations, and is available at https://github.com/gifford-lab/prescient .
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Affiliation(s)
- Grace Hui Ting Yeo
- Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA, USA
- Computational and Systems Biology Program, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Sachit D Saksena
- Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA, USA
- Computational and Systems Biology Program, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - David K Gifford
- Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA, USA.
- Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA.
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
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27
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Szlachcic WJ, Ziojla N, Kizewska DK, Kempa M, Borowiak M. Endocrine Pancreas Development and Dysfunction Through the Lens of Single-Cell RNA-Sequencing. Front Cell Dev Biol 2021; 9:629212. [PMID: 33996792 PMCID: PMC8116659 DOI: 10.3389/fcell.2021.629212] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2020] [Accepted: 04/06/2021] [Indexed: 12/16/2022] Open
Abstract
A chronic inability to maintain blood glucose homeostasis leads to diabetes, which can damage multiple organs. The pancreatic islets regulate blood glucose levels through the coordinated action of islet cell-secreted hormones, with the insulin released by β-cells playing a crucial role in this process. Diabetes is caused by insufficient insulin secretion due to β-cell loss, or a pancreatic dysfunction. The restoration of a functional β-cell mass might, therefore, offer a cure. To this end, major efforts are underway to generate human β-cells de novo, in vitro, or in vivo. The efficient generation of functional β-cells requires a comprehensive knowledge of pancreas development, including the mechanisms driving cell fate decisions or endocrine cell maturation. Rapid progress in single-cell RNA sequencing (scRNA-Seq) technologies has brought a new dimension to pancreas development research. These methods can capture the transcriptomes of thousands of individual cells, including rare cell types, subtypes, and transient states. With such massive datasets, it is possible to infer the developmental trajectories of cell transitions and gene regulatory pathways. Here, we summarize recent advances in our understanding of endocrine pancreas development and function from scRNA-Seq studies on developing and adult pancreas and human endocrine differentiation models. We also discuss recent scRNA-Seq findings for the pathological pancreas in diabetes, and their implications for better treatment.
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Affiliation(s)
- Wojciech J. Szlachcic
- Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland
| | - Natalia Ziojla
- Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland
| | - Dorota K. Kizewska
- Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland
| | - Marcelina Kempa
- Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland
| | - Malgorzata Borowiak
- Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, United States
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28
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Sequential progenitor states mark the generation of pancreatic endocrine lineages in mice and humans. Cell Res 2021; 31:886-903. [PMID: 33692492 DOI: 10.1038/s41422-021-00486-w] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2020] [Accepted: 01/27/2021] [Indexed: 12/12/2022] Open
Abstract
The pancreatic islet contains multiple hormone+ endocrine lineages (α, β, δ, PP and ε cells), but the developmental processes that underlie endocrinogenesis are poorly understood. Here, we generated novel mouse lines and combined them with various genetic tools to enrich all types of hormone+ cells for well-based deep single-cell RNA sequencing (scRNA-seq), and gene coexpression networks were extracted from the generated data for the optimization of high-throughput droplet-based scRNA-seq analyses. These analyses defined an entire endocrinogenesis pathway in which different states of endocrine progenitor (EP) cells sequentially differentiate into specific endocrine lineages in mice. Subpopulations of the EP cells at the final stage (EP4early and EP4late) show different potentials for distinct endocrine lineages. ε cells and an intermediate cell population were identified as distinct progenitors that independently generate both α and PP cells. Single-cell analyses were also performed to delineate the human pancreatic endocrinogenesis process. Although the developmental trajectory of pancreatic lineages is generally conserved between humans and mice, clear interspecies differences, including differences in the proportions of cell types and the regulatory networks associated with the differentiation of specific lineages, have been detected. Our findings support a model in which sequential transient progenitor cell states determine the differentiation of multiple cell lineages and provide a blueprint for directing the generation of pancreatic islets in vitro.
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29
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Zhang X, Ma Z, Song E, Xu T. Islet organoid as a promising model for diabetes. Protein Cell 2021; 13:239-257. [PMID: 33751396 PMCID: PMC7943334 DOI: 10.1007/s13238-021-00831-0] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2020] [Accepted: 01/22/2021] [Indexed: 02/06/2023] Open
Abstract
Studies on diabetes have long been hampered by a lack of authentic disease models that, ideally, should be unlimited and able to recapitulate the abnormalities involved in the development, structure, and function of human pancreatic islets under pathological conditions. Stem cell-based islet organoids faithfully recapitulate islet development in vitro and provide large amounts of three-dimensional functional islet biomimetic materials with a morphological structure and cellular composition similar to those of native islets. Thus, islet organoids hold great promise for modeling islet development and function, deciphering the mechanisms underlying the onset of diabetes, providing an in vitro human organ model for infection of viruses such as SARS-CoV-2, and contributing to drug screening and autologous islet transplantation. However, the currently established islet organoids are generally immature compared with native islets, and further efforts should be made to improve the heterogeneity and functionality of islet organoids, making it an authentic and informative disease model for diabetes. Here, we review the advances and challenges in the generation of islet organoids, focusing on human pluripotent stem cell-derived islet organoids, and the potential applications of islet organoids as disease models and regenerative therapies for diabetes.
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Affiliation(s)
- Xiaofei Zhang
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.,Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China
| | - Zhuo Ma
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.,College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Eli Song
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. .,College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China.
| | - Tao Xu
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. .,College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China. .,Guangzhou Regenerative Medicine and Health Guangdong Laboratory (Bioland Laboratory), Guangzhou, 510005, China.
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30
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Generation of high yield insulin-producing cells (IPCs) from various sources of stem cells. VITAMINS AND HORMONES 2021; 116:235-268. [PMID: 33752820 DOI: 10.1016/bs.vh.2021.02.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Type 1 diabetes mellitus occurs when beta cell mass is reduced to less than 20% of the normal level due to immune system destruction of beta cell resulting in an inability to secrete enough insulin. The prevalence of diabetes is expanding according to the American Diabetes Association and the World Health Organization (WHO), foretold to exceed 350 million by 2030. The current treatment does not cure many of the serious complications associated with the disease such as neuropathy, nephropathy, dyslipidemia, retinopathy and cardiovascular disease. Whole pancreas or isolated pancreatic islet transplantation as an alternative therapy can prevent or reduce some of the complications of diabetes. However, the shortage of matched organ or islets cells donor and alloimmune responses limit this therapeutic strategy. Recently, several reports have raised extremely promising results to use different sources of stem cells to differentiate insulin-producing cells and focus on the expansion of these alternative sources. Stem cells, due to their potential for multiple differentiation and self-renewal can differentiate into all cell types, including insulin-producing cells (IPCs). Generation of new beta cells can be achieved from various stem cell sources, including embryonic stem cells (ESCs), adult stem cells, such as mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs). Thus, this chapter discusses on the assistance of cellular reprogramming of various stem cells as candidates for the generation of IPCs using transcription factors/miRNA, cytokines/small molecules and tissue engineering.
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31
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Abdelalim EM. Modeling different types of diabetes using human pluripotent stem cells. Cell Mol Life Sci 2021; 78:2459-2483. [PMID: 33242105 PMCID: PMC11072720 DOI: 10.1007/s00018-020-03710-9] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2020] [Revised: 10/19/2020] [Accepted: 11/11/2020] [Indexed: 12/22/2022]
Abstract
Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycemia as a result of progressive loss of pancreatic β cells, which could lead to several debilitating complications. Different paths, triggered by several genetic and environmental factors, lead to the loss of pancreatic β cells and/or function. Understanding these many paths to β cell damage or dysfunction could help in identifying therapeutic approaches specific for each path. Most of our knowledge about diabetes pathophysiology has been obtained from studies on animal models, which do not fully recapitulate human diabetes phenotypes. Currently, human pluripotent stem cell (hPSC) technology is a powerful tool for generating in vitro human models, which could provide key information about the disease pathogenesis and provide cells for personalized therapies. The recent progress in generating functional hPSC-derived β cells in combination with the rapid development in genomic and genome-editing technologies offer multiple options to understand the cellular and molecular mechanisms underlying the development of different types of diabetes. Recently, several in vitro hPSC-based strategies have been used for studying monogenic and polygenic forms of diabetes. This review summarizes the current knowledge about different hPSC-based diabetes models and how these models improved our current understanding of the pathophysiology of distinct forms of diabetes. Also, it highlights the progress in generating functional β cells in vitro, and discusses the current challenges and future perspectives related to the use of the in vitro hPSC-based strategies.
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Affiliation(s)
- Essam M Abdelalim
- Diabetes Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), PO Box 34110, Doha, Qatar.
- College of Health and Life Sciences, Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), Education City, Doha, Qatar.
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32
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Verhoeff K, Henschke SJ, Marfil-Garza BA, Dadheech N, Shapiro AMJ. Inducible Pluripotent Stem Cells as a Potential Cure for Diabetes. Cells 2021; 10:cells10020278. [PMID: 33573247 PMCID: PMC7911560 DOI: 10.3390/cells10020278] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2021] [Revised: 01/22/2021] [Accepted: 01/24/2021] [Indexed: 02/07/2023] Open
Abstract
Over the last century, diabetes has been treated with subcutaneous insulin, a discovery that enabled patients to forego death from hyperglycemia. Despite novel insulin formulations, patients with diabetes continue to suffer morbidity and mortality with unsustainable costs to the health care system. Continuous glucose monitoring, wearable insulin pumps, and closed-loop artificial pancreas systems represent an advance, but still fail to recreate physiologic euglycemia and are not universally available. Islet cell transplantation has evolved into a successful modality for treating a subset of patients with ‘brittle’ diabetes but is limited by organ donor supply and immunosuppression requirements. A novel approach involves generating autologous or immune-protected islet cells for transplant from inducible pluripotent stem cells to eliminate detrimental immune responses and organ supply limitations. In this review, we briefly discuss novel mechanisms for subcutaneous insulin delivery and define their shortfalls. We describe embryological development and physiology of islets to better understand their role in glycemic control and, finally, discuss cell-based therapies for diabetes and barriers to widespread use. In response to these barriers, we present the promise of stem cell therapy, and review the current gaps requiring solutions to enable widespread use of stem cells as a potential cure for diabetes.
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Affiliation(s)
- Kevin Verhoeff
- Department of Surgery, University of Alberta, Edmonton, AB T6G 2B7, Canada;
- Correspondence: ; Tel.: +1-780-984-1836
| | - Sarah J. Henschke
- Department of Emergency Medicine, University of Saskatchewan, Saskatoon, SK S7N 0W8, Canada;
| | | | - Nidheesh Dadheech
- Alberta Diabetes Institute, University of Alberta, Edmonton, AB T6G 2B7, Canada;
| | - Andrew Mark James Shapiro
- FRCS (Eng) FRCSC MSM FCAHS, Clinical Islet Transplant Program, Alberta Diabetes Institute, Department of Surgery, Canadian National Transplant Research Program, Edmonton, AB T6G 2B7, Canada;
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33
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Balboa D, Iworima DG, Kieffer TJ. Human Pluripotent Stem Cells to Model Islet Defects in Diabetes. Front Endocrinol (Lausanne) 2021; 12:642152. [PMID: 33828531 PMCID: PMC8020750 DOI: 10.3389/fendo.2021.642152] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Accepted: 02/03/2021] [Indexed: 12/17/2022] Open
Abstract
Diabetes mellitus is characterized by elevated levels of blood glucose and is ultimately caused by insufficient insulin production from pancreatic beta cells. Different research models have been utilized to unravel the molecular mechanisms leading to the onset of diabetes. The generation of pancreatic endocrine cells from human pluripotent stem cells constitutes an approach to study genetic defects leading to impaired beta cell development and function. Here, we review the recent progress in generating and characterizing functional stem cell-derived beta cells. We summarize the diabetes disease modeling possibilities that stem cells offer and the challenges that lie ahead to further improve these models.
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Affiliation(s)
- Diego Balboa
- Regulatory Genomics and Diabetes, Centre for Genomic Regulation, The Barcelona Institute of Science and Technology, Barcelona, Spain
- *Correspondence: Diego Balboa,
| | - Diepiriye G. Iworima
- Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada
- School of Biomedical Engineering, The University of British Columbia, Vancouver, BC, Canada
| | - Timothy J. Kieffer
- Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada
- School of Biomedical Engineering, The University of British Columbia, Vancouver, BC, Canada
- Department of Surgery, University of British Columbia, Vancouver, BC, Canada
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Tracing the cellular basis of islet specification in mouse pancreas. Nat Commun 2020; 11:5037. [PMID: 33028844 PMCID: PMC7541446 DOI: 10.1038/s41467-020-18837-3] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2019] [Accepted: 09/15/2020] [Indexed: 02/07/2023] Open
Abstract
Pancreatic islets play an essential role in regulating blood glucose level. Although the molecular pathways underlying islet cell differentiation are beginning to be resolved, the cellular basis of islet morphogenesis and fate allocation remain unclear. By combining unbiased and targeted lineage tracing, we address the events leading to islet formation in the mouse. From the statistical analysis of clones induced at multiple embryonic timepoints, here we show that, during the secondary transition, islet formation involves the aggregation of multiple equipotent endocrine progenitors that transition from a phase of stochastic amplification by cell division into a phase of sublineage restriction and limited islet fission. Together, these results explain quantitatively the heterogeneous size distribution and degree of polyclonality of maturing islets, as well as dispersion of progenitors within and between islets. Further, our results show that, during the secondary transition, α- and β-cells are generated in a contemporary manner. Together, these findings provide insight into the cellular basis of islet development. The cellular basis of islet morphogenesis and fate allocation remain unclear. Here, the authors use a R26-CreER-R26R-Confetti mouse line to follow quantitatively the clonal dynamics of islet formation showing how, during the secondary transition, islet progenitors amplify through rounds of stochastic cell division before becoming restricted to α and β cell sublineages.
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35
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Sharon N, Vanderhooft J, Straubhaar J, Mueller J, Chawla R, Zhou Q, Engquist EN, Trapnell C, Gifford DK, Melton DA. Wnt Signaling Separates the Progenitor and Endocrine Compartments during Pancreas Development. Cell Rep 2020; 27:2281-2291.e5. [PMID: 31116975 DOI: 10.1016/j.celrep.2019.04.083] [Citation(s) in RCA: 87] [Impact Index Per Article: 17.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2018] [Revised: 01/23/2019] [Accepted: 04/17/2019] [Indexed: 10/26/2022] Open
Abstract
In vitro differentiation of pluripotent cells into β cells is a promising alternative to cadaveric-islet transplantation as a cure for type 1 diabetes (T1D). During the directed differentiation of human embryonic stem cells (hESCS) by exogenous factors, numerous genes that affect the differentiation process are turned on and off autonomously. Manipulating these reactions could increase the efficiency of differentiation and provide a more complete control over the final composition of cell populations. To uncover in vitro autonomous responses, we performed single-cell RNA sequencing on hESCs as they differentiate in spherical clusters. We observed that endocrine cells and their progenitors exist beside one another in separate compartments that activate distinct genetic pathways. WNT pathway inhibition in the endocrine domain of the differentiating clusters reveals a necessary role for the WNT inhibitor APC during islet formation in vivo. Accordingly, WNT inhibition in vitro causes an increase in the proportion of differentiated endocrine cells.
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Affiliation(s)
- Nadav Sharon
- Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
| | - Jordan Vanderhooft
- Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
| | | | - Jonas Mueller
- Computer Science and Artificial Intelligence Laboratory, MIT, Cambridge, MA 02412, USA
| | - Raghav Chawla
- Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA; Division of Hematology/Oncology, Seattle Children's Hospital, Seattle, WA 98105, USA; Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Quan Zhou
- Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
| | - Elise N Engquist
- Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
| | - Cole Trapnell
- Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA; Molecular & Cellular Biology Program, University of Washington, Seattle, WA 98195, USA
| | - David K Gifford
- Computer Science and Artificial Intelligence Laboratory, MIT, Cambridge, MA 02412, USA
| | - Douglas A Melton
- Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA.
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36
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Bi H, Karanth SS, Ye K, Stein R, Jin S. Decellularized Tissue Matrix Enhances Self-Assembly of Islet Organoids from Pluripotent Stem Cell Differentiation. ACS Biomater Sci Eng 2020; 6:4155-4165. [PMID: 33463310 DOI: 10.1021/acsbiomaterials.0c00088] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Regenerating human islet organoids from stem cells remains a significant challenge because of our limited knowledge on cues essential for developing the endocrine organoids in vitro. In this study, we discovered that a natural material prepared from a decellularized rat pancreatic extracellular matrix (dpECM) induces the self-assembly of human islet organoids during induced pluripotent stem cell (iPSC) pancreatic differentiation. For the first time, we demonstrated that the iPSC-derived islet organoids formed in the presence of the dpECM are capable of glucose-responsive secretion of both insulin and glucagon, two major hormones that maintain blood glucose homeostasis. The characterization of the organoids revealed that the organoids consisted of all major endocrine cell types, including α, β, δ, and pancreatic polypeptide cells, that were assembled into a tissue architecture similar to that of human islets. The exposure of iPSCs to the dpECM during differentiation resulted in considerably elevated expression of key pancreatic transcription factors such as PDX-1, MAFA, and NKX6.1 and the production of all major hormones, including insulin, glucagon, somatostatin, and pancreatic polypeptide from stem cell-derived organoids. This study highlights the importance of natural, bioactive biomaterials for building microenvironments crucial to regenerating islet organoids from stem cells.
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Affiliation(s)
- Huanjing Bi
- Department of Biomedical Engineering, Binghamton University, State University of New York (SUNY), Binghamton, New York 13902, United States
| | - Soujanya S Karanth
- Department of Biomedical Engineering, Binghamton University, State University of New York (SUNY), Binghamton, New York 13902, United States
| | - Kaiming Ye
- Department of Biomedical Engineering, Binghamton University, State University of New York (SUNY), Binghamton, New York 13902, United States.,Center of Biomanufacturing for Regenerative Medicine, Binghamton University, State University of New York (SUNY), Binghamton, New York 13902, United States
| | - Roland Stein
- Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, United States
| | - Sha Jin
- Department of Biomedical Engineering, Binghamton University, State University of New York (SUNY), Binghamton, New York 13902, United States.,Center of Biomanufacturing for Regenerative Medicine, Binghamton University, State University of New York (SUNY), Binghamton, New York 13902, United States
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37
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Mahaddalkar PU, Scheibner K, Pfluger S, Ansarullah, Sterr M, Beckenbauer J, Irmler M, Beckers J, Knöbel S, Lickert H. Generation of pancreatic β cells from CD177 + anterior definitive endoderm. Nat Biotechnol 2020; 38:1061-1072. [PMID: 32341565 DOI: 10.1038/s41587-020-0492-5] [Citation(s) in RCA: 63] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Accepted: 03/13/2020] [Indexed: 01/08/2023]
Abstract
Methods for differentiating human pluripotent stem cells to pancreatic and liver lineages in vitro have been limited by the inability to identify and isolate distinct endodermal subpopulations specific to these two organs. Here we report that pancreatic and hepatic progenitors can be isolated using the surface markers CD177/NB1 glycoprotein and inducible T-cell costimulatory ligand CD275/ICOSL, respectively, from seemingly homogeneous definitive endoderm derived from human pluripotent stem cells. Anterior definitive endoderm (ADE) subpopulations identified by CD177 and CD275 show inverse activation of canonical and noncanonical WNT signaling. CD177+ ADE expresses and synthesizes the secreted WNT, NODAL and BMP antagonist CERBERUS1 and is specified toward the pancreatic fate. CD275+ ADE receives canonical Wnt signaling and is specified toward the liver fate. Isolated CD177+ ADE differentiates more homogeneously into pancreatic progenitors and into more functionally mature and glucose-responsive β-like cells in vitro compared with cells from unsorted differentiation cultures.
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Affiliation(s)
- Pallavi U Mahaddalkar
- Institute of Diabetes and Regeneration Research, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany.,Institute of Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany
| | - Katharina Scheibner
- Institute of Diabetes and Regeneration Research, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany
| | - Sandra Pfluger
- Institute of Diabetes and Regeneration Research, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany
| | - Ansarullah
- Institute of Diabetes and Regeneration Research, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany
| | - Michael Sterr
- Institute of Diabetes and Regeneration Research, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany.,Institute of Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany
| | - Julia Beckenbauer
- Institute of Diabetes and Regeneration Research, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany
| | - Martin Irmler
- Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany
| | - Johannes Beckers
- Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany.,Chair of Experimental Genetics, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany.,German Center for Diabetes Research (DZD), Neuherberg, Germany
| | | | - Heiko Lickert
- Institute of Diabetes and Regeneration Research, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany. .,Institute of Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany. .,German Center for Diabetes Research (DZD), Neuherberg, Germany. .,β-Cell Biology, Technische Universität München, School of Medicine, Klinikum Rechts der Isar, Munich, Germany.
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38
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Yu XX, Xu CR. Understanding generation and regeneration of pancreatic β cells from a single-cell perspective. Development 2020; 147:147/7/dev179051. [PMID: 32280064 DOI: 10.1242/dev.179051] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2019] [Accepted: 02/20/2020] [Indexed: 12/12/2022]
Abstract
Understanding the mechanisms that underlie the generation and regeneration of β cells is crucial for developing treatments for diabetes. However, traditional research methods, which are based on populations of cells, have limitations for defining the precise processes of β-cell differentiation and trans-differentiation, and the associated regulatory mechanisms. The recent development of single-cell technologies has enabled re-examination of these processes at a single-cell resolution to uncover intermediate cell states, cellular heterogeneity and molecular trajectories of cell fate specification. Here, we review recent advances in understanding β-cell generation and regeneration, in vivo and in vitro, from single-cell technologies, which could provide insights for optimization of diabetes therapy strategies.
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Affiliation(s)
- Xin-Xin Yu
- Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China
| | - Cheng-Ran Xu
- Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China
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39
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Hogrebe NJ, Augsornworawat P, Maxwell KG, Velazco-Cruz L, Millman JR. Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells. Nat Biotechnol 2020; 38:460-470. [PMID: 32094658 PMCID: PMC7274216 DOI: 10.1038/s41587-020-0430-6] [Citation(s) in RCA: 237] [Impact Index Per Article: 47.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2019] [Accepted: 01/09/2020] [Indexed: 12/31/2022]
Abstract
Generation of pancreatic β cells from human pluripotent stem cells (hPSCs) holds promise as a cell replacement therapy for diabetes. In this study, we establish a link between the state of the actin cytoskeleton and the expression of pancreatic transcription factors that drive pancreatic lineage specification. Bulk and single-cell RNA sequencing demonstrated that different degrees of actin polymerization biased cells toward various endodermal lineages and that conditions favoring a polymerized cytoskeleton strongly inhibited neurogenin 3-induced endocrine differentiation. Using latrunculin A to depolymerize the cytoskeleton during endocrine induction, we developed a two-dimensional differentiation protocol for generating human pluripotent stem-cell-derived β (SC-β) cells with improved in vitro and in vivo function. SC-β cells differentiated from four hPSC lines exhibited first- and second-phase dynamic glucose-stimulated insulin secretion. Transplantation of islet-sized aggregates of these cells rapidly reversed severe preexisting diabetes in mice at a rate close to that of human islets and maintained normoglycemia for at least 9 months.
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Affiliation(s)
- Nathaniel J Hogrebe
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO, USA
| | - Punn Augsornworawat
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO, USA
- Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA
| | - Kristina G Maxwell
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO, USA
- Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA
| | - Leonardo Velazco-Cruz
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO, USA
| | - Jeffrey R Millman
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO, USA.
- Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA.
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40
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Alessandra G, Algerta M, Paola M, Carsten S, Cristina L, Paolo M, Elisa M, Gabriella T, Carla P. Shaping Pancreatic β-Cell Differentiation and Functioning: The Influence of Mechanotransduction. Cells 2020; 9:E413. [PMID: 32053947 PMCID: PMC7072458 DOI: 10.3390/cells9020413] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2019] [Revised: 01/29/2020] [Accepted: 02/07/2020] [Indexed: 02/08/2023] Open
Abstract
Embryonic and pluripotent stem cells hold great promise in generating β-cells for both replacing medicine and novel therapeutic discoveries in diabetes mellitus. However, their differentiation in vitro is still inefficient, and functional studies reveal that most of these β-like cells still fail to fully mirror the adult β-cell physiology. For their proper growth and functioning, β-cells require a very specific environment, the islet niche, which provides a myriad of chemical and physical signals. While the nature and effects of chemical stimuli have been widely characterized, less is known about the mechanical signals. We here review the current status of knowledge of biophysical cues provided by the niche where β-cells normally live and differentiate, and we underline the possible machinery designated for mechanotransduction in β-cells. Although the regulatory mechanisms remain poorly understood, the analysis reveals that β-cells are equipped with all mechanosensors and signaling proteins actively involved in mechanotransduction in other cell types, and they respond to mechanical cues by changing their behavior. By engineering microenvironments mirroring the biophysical niche properties it is possible to elucidate the β-cell mechanotransductive-regulatory mechanisms and to harness them for the promotion of β-cell differentiation capacity in vitro.
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Affiliation(s)
- Galli Alessandra
- Department of Excellence of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, 20134 Milan, Italy
| | - Marku Algerta
- Department of Excellence of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, 20134 Milan, Italy
| | - Marciani Paola
- Department of Excellence of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, 20134 Milan, Italy
| | - Schulte Carsten
- CIMAINA, Department of Physics, Università degli Studi di Milano, 20133 Milan, Italy
| | - Lenardi Cristina
- CIMAINA, Department of Physics, Università degli Studi di Milano, 20133 Milan, Italy
| | - Milani Paolo
- CIMAINA, Department of Physics, Università degli Studi di Milano, 20133 Milan, Italy
| | - Maffioli Elisa
- Department of Veterinary Medicine, Università degli Studi di Milano, 20133 Milan, Italy
| | - Tedeschi Gabriella
- Department of Veterinary Medicine, Università degli Studi di Milano, 20133 Milan, Italy
| | - Perego Carla
- Department of Excellence of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, 20134 Milan, Italy
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41
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Solorzano-Vargas RS, Bjerknes M, Wang J, Wu SV, Garcia-Careaga MG, Pitukcheewanont P, Cheng H, German MS, Georgia S, Martín MG. Null mutations of NEUROG3 are associated with delayed-onset diabetes mellitus. JCI Insight 2020; 5:127657. [PMID: 31805014 DOI: 10.1172/jci.insight.127657] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2019] [Accepted: 11/21/2019] [Indexed: 01/15/2023] Open
Abstract
Biallelic mutations of the gene encoding the transcription factor NEUROG3 are associated with a rare disorder that presents in neonates as generalized malabsorption - due to a complete absence of enteroendocrine cells - followed, in early childhood or beyond, by insulin-dependent diabetes mellitus (IDDM). The commonly delayed onset of IDDM suggests a differential requirement for NEUROG3 in endocrine cell generation in the human pancreas versus the intestine. However, previously identified human mutations were hypomorphic and, hence, may have had residual function in pancreas. We report 2 patients with biallelic functionally null variants of the NEUROG3 gene who nonetheless did not present with IDDM during infancy but instead developed permanent IDDM during middle childhood ages. The variants showed no evidence of function in traditional promoter-based assays of NEUROG3 function and also failed to exhibit function in a variety of potentially novel in vitro and in vivo molecular assays designed to discern residual NEUROG3 function. These findings imply that, unlike in mice, pancreatic endocrine cell generation in humans is not entirely dependent on NEUROG3 expression and, hence, suggest the presence of unidentified redundant in vivo pathways in human pancreas capable of yielding β cell mass sufficient to maintain euglycemia until early childhood.
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Affiliation(s)
- R Sergio Solorzano-Vargas
- Division of Gastroenterology and Nutrition, Department of Pediatrics, Mattel Children's Hospital and David Geffen School of Medicine at UCLA, Los Angeles, California, USA
| | - Matthew Bjerknes
- Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
| | - Jiafang Wang
- Division of Gastroenterology and Nutrition, Department of Pediatrics, Mattel Children's Hospital and David Geffen School of Medicine at UCLA, Los Angeles, California, USA
| | - S Vincent Wu
- Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, USA.,Department of Medicine, UCLA, Los Angeles, California, USA
| | | | - Pisit Pitukcheewanont
- Division of Endocrinology, Department of Pediatrics, Children's Hospital Los Angeles and University of Southern California, Los Angeles, California, USA
| | - Hazel Cheng
- Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
| | - Michael S German
- Diabetes Center and.,Department of Medicine, UCSF, San Francisco, California, USA
| | - Senta Georgia
- Division of Endocrinology, Department of Pediatrics, Children's Hospital Los Angeles and University of Southern California, Los Angeles, California, USA
| | - Martín G Martín
- Division of Gastroenterology and Nutrition, Department of Pediatrics, Mattel Children's Hospital and David Geffen School of Medicine at UCLA, Los Angeles, California, USA.,Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Los Angeles, California, USA
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42
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Xu Y, Chen J, Zhou H, Wang J, Song J, Xie J, Guo Q, Wang C, Huang Q. Effects and mechanism of stem cells from human exfoliated deciduous teeth combined with hyperbaric oxygen therapy in type 2 diabetic rats. Clinics (Sao Paulo) 2020; 75:e1656. [PMID: 32520222 PMCID: PMC7247751 DOI: 10.6061/clinics/2020/e1656] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/28/2019] [Accepted: 02/10/2020] [Indexed: 01/06/2023] Open
Abstract
OBJECTIVES Mesenchymal stem cells (MSCs) are potentially ideal for type 2 diabetes treatment, owing to their multidirectional differentiation ability and immunomodulatory properties. Here we investigated whether the stem cells from human exfoliated deciduous teeth (SHED) in combination with hyperbaric oxygen (HBO) could treat type 2 diabetic rats, and explored the underlying mechanism. METHODS SD rats were used to generate a type 2 diabetes model, which received stem cell therapy, HBO therapy, or both together. Before and after treatment, body weight, blood glucose, and serum insulin, blood lipid, pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-6), and urinary proteins were measured and compared. After 6 weeks, rats were sacrificed and their organs were subjected to hematoxylin and eosin staining and immunofluorescence staining for insulin and glucagon; apoptosis and proliferation were analyzed in islet cells. Structural changes in islets were observed under an electron microscope. Expression levels of Pdx1, Ngn3, and Pax4 mRNAs in the pancreas were assessed by real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS In comparison with diabetic mice, those treated with the combination or SHE therapy showed decreased blood glucose, insulin resistance, serum lipids, and pro-inflammatory cytokines and increased body weight and serum insulin. The morphology and structure of pancreatic islets improved, as evident from an increase in insulin-positive cells and a decrease in glucagon-positive cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining of islet cells revealed the decreased apoptosis index, while Ki67 and proliferating cell nuclear antigen staining showed increased proliferation index. Pancreatic expression of Pdx1, Ngn3, and Pax4 was upregulated. CONCLUSION SHED combined with HBO therapy was effective for treating type 2 diabetic rats. The underlying mechanism may involve SHED-mediated increase in the proliferation and trans-differentiation of islet β-cells and decrease in pro-inflammatory cytokines and apoptosis of islets.
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Affiliation(s)
- Yifeng Xu
- Department of Endocrinology, Changhai Hospital, the First Affiliated Hospital of the Naval Medical University, Shanghai 200433, China
- Department of Endocrinology, Air Force Hospital of Northern Theater Command of PLA, Shenyang 110042, China
| | - Jin Chen
- Department of Endocrinology, Changhai Hospital, the First Affiliated Hospital of the Naval Medical University, Shanghai 200433, China
| | - Hui Zhou
- Department of Out-patient, Changning retired cadre retreat of Shanghai garrison command, Shanghai 200050, China
| | - Jing Wang
- Department of Endocrinology, Changhai Hospital, the First Affiliated Hospital of the Naval Medical University, Shanghai 200433, China
- Department of Internal Medcine, Hotan Country People’s Hospital of Xinjiang, Hotan Country 848000, China
| | - Jingyun Song
- Department of Endocrinology, Changhai Hospital, the First Affiliated Hospital of the Naval Medical University, Shanghai 200433, China
| | - Junhao Xie
- Department of Endocrinology, Changhai Hospital, the First Affiliated Hospital of the Naval Medical University, Shanghai 200433, China
| | - Qingjun Guo
- Department of Endocrinology, Changhai Hospital, the First Affiliated Hospital of the Naval Medical University, Shanghai 200433, China
| | - Chaoqun Wang
- Department of Endocrinology, Changhai Hospital, the First Affiliated Hospital of the Naval Medical University, Shanghai 200433, China
| | - Qin Huang
- Department of Endocrinology, Changhai Hospital, the First Affiliated Hospital of the Naval Medical University, Shanghai 200433, China
- *Corresponding author. E-mail:
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43
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Abstract
A comprehensive understanding of mechanisms that underlie the development and function of human cells requires human cell models. For the pancreatic lineage, protocols have been developed to differentiate human pluripotent stem cells (hPSCs) into pancreatic endocrine and exocrine cells through intermediates resembling in vivo development. In recent years, this differentiation system has been employed to decipher mechanisms of pancreatic development, congenital defects of the pancreas, as well as genetic forms of diabetes and exocrine diseases. In this review, we summarize recent insights gained from studies of pancreatic hPSC models. We discuss how genome-scale analyses of the differentiation system have helped elucidate roles of chromatin state, transcription factors, and noncoding RNAs in pancreatic development and how the analysis of cells with disease-relevant mutations has provided insight into the molecular underpinnings of genetically determined diseases of the pancreas.
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Affiliation(s)
- Bjoern Gaertner
- Departments of Pediatrics and Cellular & Molecular Medicine, Pediatric Diabetes Research Center, University of California, San Diego, La Jolla, California 92093, USA
| | - Andrea C Carrano
- Departments of Pediatrics and Cellular & Molecular Medicine, Pediatric Diabetes Research Center, University of California, San Diego, La Jolla, California 92093, USA
| | - Maike Sander
- Departments of Pediatrics and Cellular & Molecular Medicine, Pediatric Diabetes Research Center, University of California, San Diego, La Jolla, California 92093, USA
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44
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Salinno C, Cota P, Bastidas-Ponce A, Tarquis-Medina M, Lickert H, Bakhti M. β-Cell Maturation and Identity in Health and Disease. Int J Mol Sci 2019; 20:E5417. [PMID: 31671683 PMCID: PMC6861993 DOI: 10.3390/ijms20215417] [Citation(s) in RCA: 54] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2019] [Revised: 10/28/2019] [Accepted: 10/28/2019] [Indexed: 12/15/2022] Open
Abstract
The exponential increase of patients with diabetes mellitus urges for novel therapeutic strategies to reduce the socioeconomic burden of this disease. The loss or dysfunction of insulin-producing β-cells, in patients with type 1 and type 2 diabetes respectively, put these cells at the center of the disease initiation and progression. Therefore, major efforts have been taken to restore the β-cell mass by cell-replacement or regeneration approaches. Implementing novel therapies requires deciphering the developmental mechanisms that generate β-cells and determine the acquisition of their physiological phenotype. In this review, we summarize the current understanding of the mechanisms that coordinate the postnatal maturation of β-cells and define their functional identity. Furthermore, we discuss different routes by which β-cells lose their features and functionality in type 1 and 2 diabetic conditions. We then focus on potential mechanisms to restore the functionality of those β-cell populations that have lost their functional phenotype. Finally, we discuss the recent progress and remaining challenges facing the generation of functional mature β-cells from stem cells for cell-replacement therapy for diabetes treatment.
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Affiliation(s)
- Ciro Salinno
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
- German Center for Diabetes Research (DZD), D-85764 Neuherberg, Germany.
- Institute of Stem Cell Research, Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
- School of Medicine, Technical University of Munich, 81675Munich, Germany.
| | - Perla Cota
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
- German Center for Diabetes Research (DZD), D-85764 Neuherberg, Germany.
- Institute of Stem Cell Research, Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
- School of Medicine, Technical University of Munich, 81675Munich, Germany.
| | - Aimée Bastidas-Ponce
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
- German Center for Diabetes Research (DZD), D-85764 Neuherberg, Germany.
- Institute of Stem Cell Research, Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
- School of Medicine, Technical University of Munich, 81675Munich, Germany.
| | - Marta Tarquis-Medina
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
- German Center for Diabetes Research (DZD), D-85764 Neuherberg, Germany.
- Institute of Stem Cell Research, Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
- School of Medicine, Technical University of Munich, 81675Munich, Germany.
| | - Heiko Lickert
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
- German Center for Diabetes Research (DZD), D-85764 Neuherberg, Germany.
- Institute of Stem Cell Research, Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
- School of Medicine, Technical University of Munich, 81675Munich, Germany.
| | - Mostafa Bakhti
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
- German Center for Diabetes Research (DZD), D-85764 Neuherberg, Germany.
- Institute of Stem Cell Research, Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
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45
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Lee K, Kim H, Lee J, Oh CM, Song H, Kim H, Koo SH, Lee J, Lim A, Kim H. Essential Role of Protein Arginine Methyltransferase 1 in Pancreas Development by Regulating Protein Stability of Neurogenin 3. Diabetes Metab J 2019; 43:649-658. [PMID: 30968621 PMCID: PMC6834834 DOI: 10.4093/dmj.2018.0232] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/12/2018] [Accepted: 11/24/2018] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND Protein arginine methyltransferase 1 (PRMT1) is a major enzyme responsible for the formation of methylarginine in mammalian cells. Recent studies have revealed that PRMT1 plays important roles in the development of various tissues. However, its role in pancreas development has not yet been elucidated. METHODS Pancreatic progenitor cell-specific Prmt1 knock-out (Prmt1 PKO) mice were generated and characterized for their metabolic and histological phenotypes and their levels of Neurog3 gene expression and neurogenin 3 (NGN3) protein expression. Protein degradation assays were performed in mPAC cells. RESULTS Prmt1 PKO mice showed growth retardation and a severely diabetic phenotype. The pancreatic size and β-cell mass were significantly reduced in Prmt1 PKO mice. Proliferation of progenitor cells during the secondary transition was decreased and endocrine cell differentiation was impaired. These defects in pancreas development could be attributed to the sustained expression of NGN3 in progenitor cells. Protein degradation assays in mPAC cells revealed that PRMT1 was required for the rapid degradation of NGN3. CONCLUSION PRMT1 critically contributes to pancreas development by destabilizing the NGN3 protein.
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Affiliation(s)
- Kanghoon Lee
- Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea
| | - Hyunki Kim
- Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea
| | - Joonyub Lee
- Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea
| | - Chang Myung Oh
- Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea
- Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Korea
| | - Heein Song
- Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea
| | - Hyeongseok Kim
- Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea
| | - Seung Hoi Koo
- Division of Life Sciences, Korea University, Seoul, Korea
| | - Junguee Lee
- Department of Pathology, Daejeon St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Daejeon, Korea
| | - Ajin Lim
- Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea
| | - Hail Kim
- Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea
- KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Korea.
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46
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Cejas P, Drier Y, Dreijerink KMA, Brosens LAA, Deshpande V, Epstein CB, Conemans EB, Morsink FHM, Graham MK, Valk GD, Vriens MR, Castillo CFD, Ferrone CR, Adar T, Bowden M, Whitton HJ, Da Silva A, Font-Tello A, Long HW, Gaskell E, Shoresh N, Heaphy CM, Sicinska E, Kulke MH, Chung DC, Bernstein BE, Shivdasani RA. Enhancer signatures stratify and predict outcomes of non-functional pancreatic neuroendocrine tumors. Nat Med 2019; 25:1260-1265. [PMID: 31263286 PMCID: PMC6919319 DOI: 10.1038/s41591-019-0493-4] [Citation(s) in RCA: 113] [Impact Index Per Article: 18.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2018] [Accepted: 05/21/2019] [Indexed: 12/29/2022]
Abstract
Most pancreatic neuroendocrine tumors (PNETs) do not produce excess hormones and are therefore considered 'non-functional'1-3. As clinical behaviors vary widely and distant metastases are eventually lethal2,4, biological classifications might guide treatment. Using enhancer maps to infer gene regulatory programs, we find that non-functional PNETs fall into two major subtypes, with epigenomes and transcriptomes that partially resemble islet α- and β-cells. Transcription factors ARX and PDX1 specify these normal cells, respectively5,6, and 84% of 142 non-functional PNETs expressed one or the other factor, occasionally both. Among 103 cases, distant relapses occurred almost exclusively in patients with ARX+PDX1- tumors and, within this subtype, in cases with alternative lengthening of telomeres. These markedly different outcomes belied similar clinical presentations and histology and, in one cohort, occurred irrespective of MEN1 mutation. This robust molecular stratification provides insight into cell lineage correlates of non-functional PNETs, accurately predicts disease course and can inform postoperative clinical decisions.
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Affiliation(s)
- Paloma Cejas
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.,Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, MA, USA.,Translational Oncology Laboratory, Hospital La Paz Institute for Health Research, Madrid, Spain
| | - Yotam Drier
- Broad Institute of Harvard and MIT, Cambridge, MA, USA. .,Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. .,Lautenberg Center for Immunology and Cancer Research, Hebrew University, Faculty of Medicine, Jerusalem, Israel.
| | - Koen M A Dreijerink
- Department of Endocrine Oncology, UMC Utrecht Cancer Center, Utrecht, the Netherlands.,Department of Internal Medicine, Amsterdam UMC, Amsterdam, the Netherlands
| | | | - Vikram Deshpande
- Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
| | | | - Elfi B Conemans
- Department of Endocrine Oncology, UMC Utrecht Cancer Center, Utrecht, the Netherlands.,Department of Internal Medicine, Amsterdam UMC, Amsterdam, the Netherlands
| | - Folkert H M Morsink
- Department of Pathology, UMC Utrecht Cancer Center, Utrecht, the Netherlands
| | - Mindy K Graham
- Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Gerlof D Valk
- Department of Endocrine Oncology, UMC Utrecht Cancer Center, Utrecht, the Netherlands
| | - Menno R Vriens
- Department of Surgical Oncology, UMC Utrecht Cancer Center, Utrecht, the Netherlands
| | | | - Cristina R Ferrone
- Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
| | - Tomer Adar
- Department of Gastroenterology Division, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
| | - Michaela Bowden
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
| | | | | | - Alba Font-Tello
- Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, MA, USA
| | - Henry W Long
- Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, MA, USA
| | | | - Noam Shoresh
- Broad Institute of Harvard and MIT, Cambridge, MA, USA
| | - Christopher M Heaphy
- Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Ewa Sicinska
- Department of Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA, USA
| | - Matthew H Kulke
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.,Departments of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA
| | - Daniel C Chung
- Department of Gastroenterology Division, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
| | - Bradley E Bernstein
- Broad Institute of Harvard and MIT, Cambridge, MA, USA. .,Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
| | - Ramesh A Shivdasani
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. .,Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, MA, USA. .,Departments of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA.
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47
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Abstract
PURPOSE OF REVIEW Pancreatic β-cells play a critical role in whole-body glucose homeostasis by regulating the release of insulin in response to minute by minute alterations in metabolic demand. As such, β-cells are staunchly resilient but there are circumstances where they can become functionally compromised or physically lost due to pathophysiological changes which culminate in overt hyperglycemia and diabetes. RECENT FINDINGS In humans, β-cell mass appears to be largely defined in the postnatal period and this early replicative and generative phase is followed by a refractory state which persists throughout life. Despite this, efforts to identify physiological and pharmacological factors which might re-initiate β-cell replication (or cause the replenishment of β-cells by neogenesis or transdifferentiation) are beginning to bear fruit. Controlled manipulation of β-cell mass in humans still represents a holy grail for therapeutic intervention in diabetes, but progress is being made which may lead to ultimate success.
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Affiliation(s)
- Giorgio Basile
- Islet Cell and Regenerative Biology, Joslin Diabetes Center, Department of Medicine, Harvard Medical School, Harvard Stem Cell Institute, Boston, MA 02215, USA
| | - Rohit N. Kulkarni
- Islet Cell and Regenerative Biology, Joslin Diabetes Center, Department of Medicine, Harvard Medical School, Harvard Stem Cell Institute, Boston, MA 02215, USA
| | - Noel G. Morgan
- Institute of Biomedical & Clinical Science, University of Exeter Medical School, Exeter EX2 5DW, UK
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48
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Tritschler S, Büttner M, Fischer DS, Lange M, Bergen V, Lickert H, Theis FJ. Concepts and limitations for learning developmental trajectories from single cell genomics. Development 2019; 146. [DOI: 10.1242/dev.170506] [Citation(s) in RCA: 132] [Impact Index Per Article: 22.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/30/2023]
Abstract
ABSTRACT
Single cell genomics has become a popular approach to uncover the cellular heterogeneity of progenitor and terminally differentiated cell types with great precision. This approach can also delineate lineage hierarchies and identify molecular programmes of cell-fate acquisition and segregation. Nowadays, tens of thousands of cells are routinely sequenced in single cell-based methods and even more are expected to be analysed in the future. However, interpretation of the resulting data is challenging and requires computational models at multiple levels of abstraction. In contrast to other applications of single cell sequencing, where clustering approaches dominate, developmental systems are generally modelled using continuous structures, trajectories and trees. These trajectory models carry the promise of elucidating mechanisms of development, disease and stimulation response at very high molecular resolution. However, their reliable analysis and biological interpretation requires an understanding of their underlying assumptions and limitations. Here, we review the basic concepts of such computational approaches and discuss the characteristics of developmental processes that can be learnt from trajectory models.
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Affiliation(s)
- Sophie Tritschler
- Institute of Computational Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, 85764 Neuherberg, Germany
- TUM School of Life Sciences Weihenstephan, Technical University of Munich, 85353 Freising, Germany
| | - Maren Büttner
- Institute of Computational Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
- Department of Mathematics, Technische Universität München, 85748 Garching, Germany
| | - David S. Fischer
- Institute of Computational Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
- TUM School of Life Sciences Weihenstephan, Technical University of Munich, 85353 Freising, Germany
| | - Marius Lange
- Institute of Computational Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
- Department of Mathematics, Technische Universität München, 85748 Garching, Germany
| | - Volker Bergen
- Institute of Computational Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
- Department of Mathematics, Technische Universität München, 85748 Garching, Germany
| | - Heiko Lickert
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, 85764 Neuherberg, Germany
- German Center for Diabetes Research, 85764 Neuherberg, Germany
- Institute of Stem Cell Research, Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Fabian J. Theis
- Institute of Computational Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany
- Department of Mathematics, Technische Universität München, 85748 Garching, Germany
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49
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Huijbregts L, Petersen MBK, Berthault C, Hansson M, Aiello V, Rachdi L, Grapin-Botton A, Honore C, Scharfmann R. Bromodomain and Extra Terminal Protein Inhibitors Promote Pancreatic Endocrine Cell Fate. Diabetes 2019; 68:761-773. [PMID: 30655386 DOI: 10.2337/db18-0224] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/23/2018] [Accepted: 01/07/2019] [Indexed: 11/13/2022]
Abstract
Bromodomain and extraterminal (BET) proteins are epigenetic readers that interact with acetylated lysines of histone tails. Recent studies have demonstrated their role in cancer progression because they recruit key components of the transcriptional machinery to modulate gene expression. However, their role during embryonic development of the pancreas has never been studied. Using mouse embryonic pancreatic explants and human induced pluripotent stem cells (hiPSCs), we show that BET protein inhibition with I-BET151 or JQ1 enhances the number of neurogenin3 (NEUROG3) endocrine progenitors. In mouse explants, BET protein inhibition further led to increased expression of β-cell markers but in the meantime, strongly downregulated Ins1 expression. Similarly, although acinar markers, such as Cpa1 and CelA, were upregulated, Amy expression was repressed. In hiPSCs, BET inhibitors strongly repressed C-peptide and glucagon during endocrine differentiation. Explants and hiPSCs were then pulsed with BET inhibitors to increase NEUROG3 expression and further chased without inhibitors. Endocrine development was enhanced in explants with higher expression of insulin and maturation markers, such as UCN3 and MAFA. In hiPSCs, the outcome was different because C-peptide expression remained lower than in controls, but ghrelin expression was increased. Altogether, by using two independent models of pancreatic development, we show that BET proteins regulate multiple aspects of pancreatic development.
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Affiliation(s)
- Lukas Huijbregts
- INSERM U1016, Institut Cochin, Université Paris Descartes, Paris, France
| | - Maja Borup Kjær Petersen
- Novo Nordisk Foundation Center for Stem Cell Biology (DanStem), University of Copenhagen, Copenhagen, Denmark
| | - Claire Berthault
- INSERM U1016, Institut Cochin, Université Paris Descartes, Paris, France
| | | | - Virginie Aiello
- INSERM U1016, Institut Cochin, Université Paris Descartes, Paris, France
| | - Latif Rachdi
- INSERM U1016, Institut Cochin, Université Paris Descartes, Paris, France
| | - Anne Grapin-Botton
- Novo Nordisk Foundation Center for Stem Cell Biology (DanStem), University of Copenhagen, Copenhagen, Denmark
| | - Christian Honore
- Department of Stem Cell Biology, Novo Nordisk A/S, Måløv, Denmark
| | - Raphael Scharfmann
- INSERM U1016, Institut Cochin, Université Paris Descartes, Paris, France
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50
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Abstract
Diabetes mellitus is a multifactorial disease affecting increasing numbers of patients worldwide. Progression to insulin-dependent diabetes mellitus is characterized by the loss or dysfunction of pancreatic β-cells, but the pathomechanisms underlying β-cell failure in type 1 diabetes mellitus and type 2 diabetes mellitus are still poorly defined. Regeneration of β-cell mass from residual islet cells or replacement by β-like cells derived from stem cells holds great promise to stop or reverse disease progression. However, the development of new treatment options is hampered by our limited understanding of human pancreas organogenesis due to the restricted access to primary tissues. Therefore, the challenge is to translate results obtained from preclinical model systems to humans, which requires comparative modelling of β-cell biology in health and disease. Here, we discuss diverse modelling systems across different species that provide spatial and temporal resolution of cellular and molecular mechanisms to understand the evolutionary conserved genotype-phenotype relationship and translate them to humans. In addition, we summarize the latest knowledge on organoids, stem cell differentiation platforms, primary micro-islets and pseudo-islets, bioengineering and microfluidic systems for studying human pancreas development and homeostasis ex vivo. These new modelling systems and platforms have opened novel avenues for exploring the developmental trajectory, physiology, biology and pathology of the human pancreas.
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Affiliation(s)
- Mostafa Bakhti
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany.
- Institute of Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany.
- German Center for Diabetes Research (DZD), Neuherberg, Germany.
| | - Anika Böttcher
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany.
- Institute of Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany.
- German Center for Diabetes Research (DZD), Neuherberg, Germany.
| | - Heiko Lickert
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany.
- Institute of Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany.
- German Center for Diabetes Research (DZD), Neuherberg, Germany.
- Technical University of Munich, Medical Faculty, Munich, Germany.
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