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Li X, He J, Xie K. Molecular signaling in pancreatic ductal metaplasia: emerging biomarkers for detection and intervention of early pancreatic cancer. Cell Oncol (Dordr) 2022; 45:201-225. [PMID: 35290607 DOI: 10.1007/s13402-022-00664-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/14/2022] [Indexed: 11/27/2022] Open
Abstract
Pancreatic ductal metaplasia (PDM) is the transformation of potentially various types of cells in the pancreas into ductal or ductal-like cells, which eventually replace the existing differentiated somatic cell type(s). PDM is usually triggered by and manifests its ability to adapt to environmental stimuli and genetic insults. The development of PDM to atypical hyperplasia or dysplasia is an important risk factor for pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDA). Recent studies using genetically engineered mouse models, cell lineage tracing, single-cell sequencing and others have unraveled novel cellular and molecular insights in PDM formation and evolution. Those novel findings help better understand the cellular origins and functional significance of PDM and its regulation at cellular and molecular levels. Given that PDM represents the earliest pathological changes in PDA initiation and development, translational studies are beginning to define PDM-associated cell and molecular biomarkers that can be used to screen and detect early PDA and to enable its effective intervention, thereby truly and significantly reducing the dreadful mortality rate of PDA. This review will describe recent advances in the understanding of PDM biology with a focus on its underlying cellular and molecular mechanisms, and in biomarker discovery with clinical implications for the management of pancreatic regeneration and tumorigenesis.
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Affiliation(s)
- Xiaojia Li
- Center for Pancreatic Cancer Research, The South China University of Technology School of Medicine, Guangzhou, 510006, China
- Department of Pathology, The South China University of Technology School of Medicine, Guangzhou, China
| | - Jie He
- Institute of Digestive Diseases Research, The South China University of Technology School of Medicine, Guangzhou, China
| | - Keping Xie
- Center for Pancreatic Cancer Research, The South China University of Technology School of Medicine, Guangzhou, 510006, China.
- Department of Pathology, The South China University of Technology School of Medicine, Guangzhou, China.
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2
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Reckmann AN, Tomczyk CUM, Davidoff MS, Michurina TV, Arnhold S, Müller D, Mietens A, Middendorff R. Nestin in the epididymis is expressed in vascular wall cells and is regulated during postnatal development and in case of testosterone deficiency. PLoS One 2018; 13:e0194585. [PMID: 29874225 PMCID: PMC5991371 DOI: 10.1371/journal.pone.0194585] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2017] [Accepted: 03/06/2018] [Indexed: 12/24/2022] Open
Abstract
Vascular smooth muscle cells (SMCs), distinguished by the expression of the neuronal stem cell marker nestin, may represent stem cell-like progenitor cells in various organs including the testis. We investigated epididymal tissues of adult nestin-GFP mice, rats after Leydig cell depletion via ethane dimethane sulfonate (EDS), rats and mice during postnatal development and human tissues. By use of Clarity, a histochemical method to illustrate a three-dimensional picture, we could demonstrate nestin-GFP positive cells within the vascular network. We localized nestin in the epididymis in proliferating vascular SMCs by colocalization with both smooth muscle actin and PCNA, and it was distinct from CD31-positive endothelial cells. The same nestin localization was found in the human epididymis. However, nestin was not found in SMCs of the epididymal duct. Nestin expression is high during postnatal development of mouse and rat and down-regulated towards adulthood when testosterone levels increase. Nestin increases dramatically in rats after Leydig cell ablation with EDS and subsequently low testosterone levels. Interestingly, during this period, the expression of androgen receptor in the epididymis is low and increases until nestin reaches normal levels of adulthood. Here we show that nestin, a common marker for neuronal stem cells, is also expressed in the vasculature of the epididymis. Our results give new insights into the yet underestimated role of proliferating nestin-expressing vascular SMCs during postnatal development and repair of the epididymis.
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Affiliation(s)
- Ansgar N Reckmann
- Institute of Anatomy and Cell Biology, Justus-Liebig-University Giessen, Giessen, Germany
| | - Claudia U M Tomczyk
- Institute of Anatomy and Cell Biology, Justus-Liebig-University Giessen, Giessen, Germany
| | - Michail S Davidoff
- Institute of Anatomy and Experimental Morphology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Tatyana V Michurina
- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, United States of America
- Center for Developmental Genetics and Department of Anesthesiology, Stony Brook University, Stony Brook, NY, United States of America
- Moscow Institute of Physics and Technology, Moscow, Russia
| | - Stefan Arnhold
- Institute of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University Giessen, Giessen, Germany
| | - Dieter Müller
- Institute of Anatomy and Cell Biology, Justus-Liebig-University Giessen, Giessen, Germany
| | - Andrea Mietens
- Institute of Anatomy and Cell Biology, Justus-Liebig-University Giessen, Giessen, Germany
| | - Ralf Middendorff
- Institute of Anatomy and Cell Biology, Justus-Liebig-University Giessen, Giessen, Germany
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3
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Hu W, Lu H, Wang S, Yin W, Liu X, Dong L, Chiu R, Shen L, Lu WJ, Lan F. Suppression of Nestin reveals a critical role for p38-EGFR pathway in neural progenitor cell proliferation. Oncotarget 2018; 7:87052-87063. [PMID: 27894083 PMCID: PMC5349970 DOI: 10.18632/oncotarget.13498] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2016] [Accepted: 10/14/2016] [Indexed: 12/19/2022] Open
Abstract
The expression of intermediate filament Nestin is necessary for the neural progenitor cells (NPCs) to maintain stemness, but the underlying cellular and molecular mechanism remains unclear. In this study, we demonstrated that Nestin is required for the self-renew of NPCs through activating MAPK and EGFR pathways. Knockdown of Nestin by shRNA inhibited cell cycle progression and proliferation in mouse NPCs. Moreover, suppression of Nestin reduced expression of the epidermal growth factor receptor (EGFR) in NPCs and inhibited the mitogenic effects of EGF on these cells. Treatment of NPCs with p38-MAPK inhibitor PD169316 reversed cell cycle arrest caused by the knockdown of Nestin. Our findings indicate that Nestin promotes NPC proliferation via p38-MAPK and EGFR pathways, and reveals the necessity of these pathways in NPCs self-renewal.
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Affiliation(s)
- Wentao Hu
- Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Hong Lu
- Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Shang Wang
- Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Wenhan Yin
- Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Xujie Liu
- Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing, China.,Beijing Lab for Cardiovascular Precision Medicine, Capital Medical University, Beijing, China.,The Key Laboratory of Remodeling-Related Cardiovascular Disease, Ministry of Education, Beijing, China.,Beijing Collaborative Innovation Center for Cardiovascular Disorders, Anzhen Hospital, Capital Medical University, Beijing, China.,Deparment of Radiological Medicine, Chongqing Medical University, Chongqing, China
| | - Lin Dong
- Department of Cell Biology Peking University Health Science Center, Beijing, China
| | - Richard Chiu
- Deparment of Radiology, Stanford University School of Medicine, Stanford, California, USA
| | - Li Shen
- Department of Cell Biology Peking University Health Science Center, Beijing, China
| | - Wen-Jing Lu
- Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing, China.,Beijing Lab for Cardiovascular Precision Medicine, Capital Medical University, Beijing, China.,The Key Laboratory of Remodeling-Related Cardiovascular Disease, Ministry of Education, Beijing, China.,Beijing Collaborative Innovation Center for Cardiovascular Disorders, Anzhen Hospital, Capital Medical University, Beijing, China
| | - Feng Lan
- Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing, China.,Beijing Lab for Cardiovascular Precision Medicine, Capital Medical University, Beijing, China.,The Key Laboratory of Remodeling-Related Cardiovascular Disease, Ministry of Education, Beijing, China.,Beijing Collaborative Innovation Center for Cardiovascular Disorders, Anzhen Hospital, Capital Medical University, Beijing, China
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Hepatic stellate cells derived from the nestin-positive cells in septum transversum during rat liver development. Med Mol Morphol 2018; 51:199-207. [PMID: 29380061 DOI: 10.1007/s00795-018-0183-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2017] [Accepted: 01/24/2018] [Indexed: 01/19/2023]
Abstract
Hepatic stellate cells (HSCs) play a principal role in Vitamin A metabolism and are considered the major matrix-producing cell type in the diseased liver. Rat HSCs are identified by immunohistochemistry with myogenic or mesenchymal (desmin, vimentin, and alpha-smooth muscle actin) or neural (e.g., GFAP or neuronal cell adhesion molecule) markers. Embryonic origin of rat HSCs was determined using these markers. Nestin, an intermediate filament protein originally identified in neuronal stem or progenitor cells, is widely used as a stem cell marker, including hepatic stem cells in adult rat livers. Additionally, nestin is reportedly expressed in activated HSCs during liver injury and hepatic regeneration. However, little is known about nestin expression in rat fetal liver HSCs. The present study aimed to clarify nestin-positive HSC expression during rat liver development. At embryonic day (ED) 10.5, nestin expression in mesenchymal cells adjacent to the liver bud was detected by immunohistochemistry. At ED 11.5, nestin-positive cells were also detected in desmin-positive cells appearing and increasing in intensity by ED 16.5. However, nestin-positive cells in the parenchyma decreased by ED 20.5 or later. These findings reveal that the nestin-positive HSCs during rat liver development originate from nestin-positive mesenchymal cells in the septum transversum.
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5
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Matsuda Y, Ishiwata T, Yoshimura H, Yamahatsu K, Minamoto T, Arai T. Nestin phosphorylation at threonines 315 and 1299 correlates with proliferation and metastasis of human pancreatic cancer. Cancer Sci 2017; 108:354-361. [PMID: 28002641 PMCID: PMC5378226 DOI: 10.1111/cas.13139] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2016] [Revised: 12/06/2016] [Accepted: 12/17/2016] [Indexed: 01/05/2023] Open
Abstract
The neuroepithelial stem cell marker nestin is a cytoskeletal protein that regulates cell proliferation, invasion, and stemness in various tumors, including pancreatic tumors. In the present study, we examined the expression and roles of phosphorylated nestin in pancreatic cancer cells. Nestin phosphorylation at threonines 315 (Thr315) and 1299 (Thr1299) was observed during mitosis in human pancreatic cancer cells. Nestin phosphorylation was positively correlated with a cell proliferation marker, MIB-1 expression in human pancreatic cancer samples. Transfection of MIA PaCa-2 cells with nestin mutated at Thr315 and/or Thr1299 (to suppress phosphorylation) resulted in lower proliferation rates than those in control groups. Transfecting MIA PaCa-2 cells with wild-type nestin or with nestin mutated at Thr315 increased migration and invasion. In contrast, transfection with nestin mutated at both phosphorylation sites (Thr315 and Thr1299) did not enhance cell migration or invasion. In an intra-splenic xenograft experiment using MIA PaCa-2 cells, tumors expressing the nestin double mutant formed fewer liver metastases than tumors expressing wild-type nestin. Nestin phosphorylation at these two sites was decreased upon treatment with inhibitors for cyclin dependent kinases, AKT, and Aurora in PANC-1 cells, which express a high baseline level of phosphorylated nestin. These findings suggest that phosphorylation of nestin at Thr315 and/or Thr1299 affects cell proliferation, and inhibition of both phosphorylation sites suppresses invasion and metastasis of human pancreatic cancer. Inhibiting nestin phosphorylation at these two sites may represent a novel therapeutic strategy for pancreatic cancer.
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Affiliation(s)
- Yoko Matsuda
- Department of Pathology, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, Tokyo, Japan
| | - Toshiyuki Ishiwata
- Department of Aging and Carcinogenesis, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
| | - Hisashi Yoshimura
- Division of Physiological Pathology, Department of Applied Science, School of Veterinary Nursing and Technology, Nippon Veterinary and Life Science University, Tokyo, Japan
| | - Kazuya Yamahatsu
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
| | - Toshinari Minamoto
- Divisions of Translational and Clinical Oncology and Surgical Oncology, Cancer Research Institute, Kanazawa University and Hospital, Kanazawa, Japan
| | - Tomio Arai
- Department of Pathology, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, Tokyo, Japan
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6
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Yamaguchi J, Yokoyama Y, Kokuryo T, Ebata T, Nagino M. Cells of origin of pancreatic neoplasms. Surg Today 2017; 48:9-17. [PMID: 28260136 DOI: 10.1007/s00595-017-1501-2] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2016] [Accepted: 02/07/2017] [Indexed: 12/21/2022]
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignant disease associated with poor prognosis, despite recent medical advances. It is of great importance to understand the initial events and cells of origin of pancreatic cancer to prevent the development and progression of PDAC. There are three distinct precursor lesions that develop into PDAC: pancreatic intraepithelial neoplasms (PanINs), intraductal papillary mucinous neoplasms (IPMNs), and mucinous cystic neoplasms (MCNs). Studies on genetically engineered mouse models have revealed that the initiation and development of these lesions largely depend on genetic alterations. These lesions originate from different populations in the pancreas. PanIN development seems to be the result of the transdifferentiation of acinar cells, whereas IPMNs most likely arise from the progenitor niche of the pancreatic ductal epithelium. Pancreatic carcinogenesis is dependent on various events, including gene alterations, environmental insults, and cell types. However, further studies are needed to fully understand the initial processes of pancreatic cancer.
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Affiliation(s)
- Junpei Yamaguchi
- Division of Surgical Oncology, Department of Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8560, Japan.
| | - Yukihiro Yokoyama
- Division of Surgical Oncology, Department of Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8560, Japan
| | - Toshio Kokuryo
- Division of Surgical Oncology, Department of Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8560, Japan
| | - Tomoki Ebata
- Division of Surgical Oncology, Department of Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8560, Japan
| | - Masato Nagino
- Division of Surgical Oncology, Department of Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8560, Japan
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7
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Stepwise reprogramming of liver cells to a pancreas progenitor state by the transcriptional regulator Tgif2. Nat Commun 2017; 8:14127. [PMID: 28193997 PMCID: PMC5316826 DOI: 10.1038/ncomms14127] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2015] [Accepted: 11/11/2016] [Indexed: 12/12/2022] Open
Abstract
The development of a successful lineage reprogramming strategy of liver to pancreas holds promises for the treatment and potential cure of diabetes. The liver is an ideal tissue source for generating pancreatic cells, because of its close developmental origin with the pancreas and its regenerative ability. Yet, the molecular bases of hepatic and pancreatic cellular plasticity are still poorly understood. Here, we report that the TALE homeoprotein TGIF2 acts as a developmental regulator of the pancreas versus liver fate decision and is sufficient to elicit liver-to-pancreas fate conversion both ex vivo and in vivo. Hepatocytes expressing Tgif2 undergo extensive transcriptional remodelling, which represses the original hepatic identity and, over time, induces a pancreatic progenitor-like phenotype. Consistently, in vivo forced expression of Tgif2 activates pancreatic progenitor genes in adult mouse hepatocytes. This study uncovers the reprogramming activity of TGIF2 and suggests a stepwise reprogramming paradigm, whereby a 'lineage-restricted' dedifferentiation step precedes the identity switch.
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Vieira A, Courtney M, Druelle N, Avolio F, Napolitano T, Hadzic B, Navarro-Sanz S, Ben-Othman N, Collombat P. β-Cell replacement as a treatment for type 1 diabetes: an overview of possible cell sources and current axes of research. Diabetes Obes Metab 2016; 18 Suppl 1:137-43. [PMID: 27615143 DOI: 10.1111/dom.12721] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/08/2016] [Accepted: 04/27/2016] [Indexed: 01/09/2023]
Abstract
To efficiently treat type 1 diabetes, exogenous insulin injections currently represent the main approach to counter chronic hyperglycaemia. Unfortunately, such a therapeutic approach does not allow for perfectly maintained glucose homeostasis and, in time, cardiovascular complications may arise. Therefore, seeking alternative/improved treatments has become a major health concern as an increasing proportion of type 2 diabetes patients also require insulin supplementation. Towards this goal, numerous laboratories have focused their research on β-cell replacement therapies. Herein, we will review the current state of this research area and describe the cell sources that could potentially be used to replenish the depleted β-cell mass in diabetic patients.
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Affiliation(s)
- A Vieira
- Université Côte d'Azur, CNRS, Inserm, iBV, France
| | - M Courtney
- Université Côte d'Azur, CNRS, Inserm, iBV, France
| | - N Druelle
- Université Côte d'Azur, CNRS, Inserm, iBV, France
| | - F Avolio
- Université Côte d'Azur, CNRS, Inserm, iBV, France
| | - T Napolitano
- Université Côte d'Azur, CNRS, Inserm, iBV, France
| | - B Hadzic
- Université Côte d'Azur, CNRS, Inserm, iBV, France
| | | | - N Ben-Othman
- Université Côte d'Azur, CNRS, Inserm, iBV, France
| | - P Collombat
- Université Côte d'Azur, CNRS, Inserm, iBV, France.
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9
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Kim HS, Lee MK. β-Cell regeneration through the transdifferentiation of pancreatic cells: Pancreatic progenitor cells in the pancreas. J Diabetes Investig 2016; 7:286-96. [PMID: 27330712 PMCID: PMC4847880 DOI: 10.1111/jdi.12475] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/01/2015] [Revised: 12/27/2015] [Accepted: 01/04/2016] [Indexed: 12/17/2022] Open
Abstract
Pancreatic progenitor cell research has been in the spotlight, as these cells have the potential to replace pancreatic β‐cells for the treatment of type 1 and 2 diabetic patients with the absence or reduction of pancreatic β‐cells. During the past few decades, the successful treatment of diabetes through transplantation of the whole pancreas or isolated islets has nearly been achieved. However, novel sources of pancreatic islets or insulin‐producing cells are required to provide sufficient amounts of donor tissues. To overcome this limitation, the use of pancreatic progenitor cells is gaining more attention. In particular, pancreatic exocrine cells, such as duct epithelial cells and acinar cells, are attractive candidates for β‐cell regeneration because of their differentiation potential and pancreatic lineage characteristics. It has been assumed that β‐cell neogenesis from pancreatic progenitor cells could occur in pancreatic ducts in the postnatal stage. Several studies have shown that insulin‐producing cells can arise in the duct tissue of the adult pancreas. Acinar cells also might have the potential to differentiate into insulin‐producing cells. The present review summarizes recent progress in research on the transdifferentiation of pancreatic exocrine cells into insulin‐producing cells, especially duct and acinar cells.
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Affiliation(s)
- Hyo-Sup Kim
- Division of Endocrinology and Metabolism Department of Medicine Sungkyunkwan University School of Medicine Samsung Biomedical Research Institute Samsung Medical Center Seoul Korea
| | - Moon-Kyu Lee
- Division of Endocrinology and Metabolism Department of Medicine Sungkyunkwan University School of Medicine Samsung Biomedical Research Institute Samsung Medical Center Seoul Korea
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Matsuda Y, Ishiwata T, Yoshimura H, Yamashita S, Ushijima T, Arai T. Systemic Administration of Small Interfering RNA Targeting Human Nestin Inhibits Pancreatic Cancer Cell Proliferation and Metastasis. Pancreas 2016; 45:93-100. [PMID: 26335012 DOI: 10.1097/mpa.0000000000000427] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
OBJECTIVES Nestin, a progenitor/stem cell marker, is expressed in human pancreatic cancer, where its expression correlates positively with invasiveness and metastasis. Here, we investigated the inhibition of nestin expression and the regulation of nestin expression. METHODS We analyzed the effects of small interfering RNA (siRNA) targeting nestin using pancreatic cancer cell lines. RESULTS Nestin siRNA inhibited the growth, migration, invasion, and sphere-forming ability of the pancreatic cancer cell lines. Pancreatic cancer cells cotreated with gemcitabine and nestin siRNA exhibited lower cell viability than cells treated with a control siRNA, gemcitabine alone, or nestin siRNA alone. Cells derived from the metastatic nodules of mice showed higher nestin expression than the parental cells, and nestin expression in pancreatic cancer cells was regulated by methylation of the nestin gene. In an orthotopic implantation model using mice, administration of nestin siRNA significantly decreased primary and metastatic tumor formation by human pancreatic cancer cells compared to tumor formation in control siRNA-treated mice. CONCLUSIONS Nestin plays a key role in pancreatic cancer cell metastasis and stemness and that administration of nestin siRNA may offer a novel therapeutic strategy for pancreatic cancer.
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Affiliation(s)
- Yoko Matsuda
- From the *Department of Pathology, Tokyo Metropolitan Geriatric Hospital, Itabashi-ku; †Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, Bunkyo-ku; and ‡Division of Epigenomics, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan
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11
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Nestin overexpression promotes the embryonic development of heart and brain through the regulation of cell proliferation. Brain Res 2015; 1610:1-11. [PMID: 25843934 DOI: 10.1016/j.brainres.2015.03.044] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2014] [Revised: 01/04/2015] [Accepted: 03/26/2015] [Indexed: 12/15/2022]
Abstract
Nestin, an intermediate filament protein, is a key regulator of various extracellular proteins that play important roles in cell growth and differentiation. In recent years, nestin has been widely accepted as a molecular marker for neural stem/progenitor cells. However, its function during embryogenesis remains largely unknown since its depletion is lethal after stage embryonic day 8.5 (E8.5). In order to understand the role of this protein in vivo, we compared the heart and brain tissues of control mice with those of mice overexpressing a human nestin cDNA transgene under the control of a ROSA26 promoter. In these tissues we examined the general histology and cell size, the presence of apoptotic cells by TUNEL assay, and the presence of progenitor cell markers like SOX2. Compared to controls, mouse embryos overexpressing the human nestin transgene have a larger size and display characteristic morphological changes including a larger heart and forebrain. In these tissues we found corresponding increases in the size of cardiomyocytes and brain cells, as well as indications of augmented cell proliferation. In contrast, apoptosis was not significantly altered. Co-staining brain sections with SOX2 and Ki67 showed that most of the proliferating cells in the forebrain were neural stem cells. Moreover, nestin overexpression was responsible for a marked activation of the PI3K/Akt signaling pathway. Taken together, the results of this study indicate that nestin plays an important role in the embryonic development of at least two mouse organs (heart and brain) through the regulation of cell proliferation.
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12
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Birbrair A, Zhang T, Files DC, Mannava S, Smith T, Wang ZM, Messi ML, Mintz A, Delbono O. Type-1 pericytes accumulate after tissue injury and produce collagen in an organ-dependent manner. Stem Cell Res Ther 2014; 5:122. [PMID: 25376879 PMCID: PMC4445991 DOI: 10.1186/scrt512] [Citation(s) in RCA: 211] [Impact Index Per Article: 19.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2014] [Accepted: 09/30/2014] [Indexed: 02/06/2023] Open
Abstract
Introduction Fibrosis, or scar formation, is a pathological condition characterized by excessive production and accumulation of collagen, loss of tissue architecture, and organ failure in response to uncontrolled wound healing. Several cellular populations have been implicated, including bone marrow-derived circulating fibrocytes, endothelial cells, resident fibroblasts, epithelial cells, and recently, perivascular cells called pericytes. We previously demonstrated pericyte functional heterogeneity in skeletal muscle. Whether pericyte subtypes are present in other tissues and whether a specific pericyte subset contributes to organ fibrosis are unknown. Methods Here, we report the presence of two pericyte subtypes, type-1 (Nestin-GFP-/NG2-DsRed+) and type-2 (Nestin-GFP+/NG2-DsRed+), surrounding blood vessels in lungs, kidneys, heart, spinal cord, and brain. Using Nestin-GFP/NG2-DsRed transgenic mice, we induced pulmonary, renal, cardiac, spinal cord, and cortical injuries to investigate the contributions of pericyte subtypes to fibrous tissue formation in vivo. Results A fraction of the lung’s collagen-producing cells corresponds to type-1 pericytes and kidney and heart pericytes do not produce collagen in pathological fibrosis. Note that type-1, but not type-2, pericytes increase and accumulate near the fibrotic tissue in all organs analyzed. Surprisingly, after CNS injury, type-1 pericytes differ from scar-forming PDGFRβ + cells. Conclusions Pericyte subpopulations respond differentially to tissue injury, and the production of collagen by type-1 pericytes is organ-dependent. Characterization of the mechanisms underlying scar formation generates cellular targets for future anti-fibrotic therapeutics. Electronic supplementary material The online version of this article (doi:10.1186/scrt512) contains supplementary material, which is available to authorized users.
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13
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Criscimanna A, Coudriet GM, Gittes GK, Piganelli JD, Esni F. Activated macrophages create lineage-specific microenvironments for pancreatic acinar- and β-cell regeneration in mice. Gastroenterology 2014; 147:1106-18.e11. [PMID: 25128759 DOI: 10.1053/j.gastro.2014.08.008] [Citation(s) in RCA: 85] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/02/2013] [Revised: 08/11/2014] [Accepted: 08/12/2014] [Indexed: 01/06/2023]
Abstract
BACKGROUND & AIMS Although the cells that contribute to pancreatic regeneration have been widely studied, little is known about the mediators of this process. During tissue regeneration, infiltrating macrophages debride the site of injury and coordinate the repair response. We investigated the role of macrophages in pancreatic regeneration in mice. METHODS We used a saporin-conjugated antibody against CD11b to reduce the number of macrophages in mice following diphtheria toxin receptor-mediated cell ablation of pancreatic cells, and evaluated the effects on pancreatic regeneration. We analyzed expression patterns of infiltrating macrophages after cell-specific injury or from the pancreas of nonobese diabetic mice. We developed an in vitro culture system to study the ability of macrophages to induce cell-specific regeneration. RESULTS Depletion of macrophages impaired pancreatic regeneration. Macrophage polarization, as assessed by expression of tumor necrosis factor-α, interleukin 6, interleukin 10, and CD206, depended on the type of injury. The signals provided by polarized macrophages promoted lineage-specific generation of acinar or endocrine cells. Macrophage from nonobese diabetic mice failed to provide signals necessary for β-cell generation. CONCLUSIONS Macrophages produce cell type-specific signals required for pancreatic regeneration in mice. Additional study of these processes and signals might lead to new approaches for treating type 1 diabetes or pancreatitis.
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Affiliation(s)
- Angela Criscimanna
- Department of Surgery, Division of Pediatric General and Thoracic Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center, Rangos Research Center, Pittsburgh, Pennsylvania
| | - Gina M Coudriet
- Department of Surgery, Division of Pediatric General and Thoracic Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center, Rangos Research Center, Pittsburgh, Pennsylvania
| | - George K Gittes
- Department of Surgery, Division of Pediatric General and Thoracic Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center, Rangos Research Center, Pittsburgh, Pennsylvania
| | - Jon D Piganelli
- Department of Surgery, Division of Pediatric General and Thoracic Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center, Rangos Research Center, Pittsburgh, Pennsylvania
| | - Farzad Esni
- Department of Surgery, Division of Pediatric General and Thoracic Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center, Rangos Research Center, Pittsburgh, Pennsylvania; Department of Developmental Biology, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania; University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.
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14
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Golbar HM, Izawa T, Wijesundera KK, Tennakoon AH, Katou-Ichikawa C, Tanaka M, Kuwamura M, Yamate J. Expression of nestin in remodelling of α-naphthylisothiocyanate-induced acute bile duct injury in rats. J Comp Pathol 2014; 151:255-63. [PMID: 25087881 DOI: 10.1016/j.jcpa.2014.05.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2014] [Revised: 05/12/2014] [Accepted: 05/29/2014] [Indexed: 10/25/2022]
Abstract
The function of the intermediate filament protein nestin is poorly understood. The significance of nestin expression was assessed in α-naphthylisothiocyanate (ANIT)-induced cholangiocyte injury lesions in F344 rats. Liver samples obtained from rats injected intraperitoneally with ANIT (75 mg/kg) on post-injection days 0 (control) and 1-12 were labelled immunohistochemically for expression of nestin and markers specific for mesenchymal cells (vimentin), hepatic stellate cells (HSCs) (desmin and glial fibrillary acidic protein [GFAP]), endothelial cells (rat endothelial cell antigen [RECA]-1), cholangiocytes (cytokeratin [CK] 19) and cellular proliferation (Ki67). Cholangiocyte injury led to infiltration of neutrophils and macrophages followed by aggregation of mesenchymal cells and regeneration of bile ducts. Nestin expression was detected in mesenchymal cells (vimentin positive) on days 1-7 with a peak on days 3-5 and in newly-formed RECA-1-positive endothelial cells on day 3. Nestin expression was also observed in regenerating CK19-positive cholangiocytes on days 2-5, with a peak on day 3. Labelling for Ki67 showed proliferation of cholangiocytes, mesenchymal cells and HSCs. Real-time reverse transcriptase polymerase chain reaction with microdissected samples showed significantly elevated nestin mRNA on day 3. The findings suggest an association between nestin expression and cellular proliferation. Based on these findings, it was considered that nestin-expressing mesenchymal cells, HSCs and endothelial cells may be possible progenitors of repopulating cholangiocytes. Nestin expression may serve as an indicator for cellular remodelling and behaviour of injured and repopulating bile ducts.
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Affiliation(s)
- H M Golbar
- Veterinary Pathology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku-Ourai-Kita, Izumisano City, Osaka 598-8531, Japan
| | - T Izawa
- Veterinary Pathology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku-Ourai-Kita, Izumisano City, Osaka 598-8531, Japan
| | - K K Wijesundera
- Veterinary Pathology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku-Ourai-Kita, Izumisano City, Osaka 598-8531, Japan
| | - A H Tennakoon
- Veterinary Pathology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku-Ourai-Kita, Izumisano City, Osaka 598-8531, Japan
| | - C Katou-Ichikawa
- Veterinary Pathology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku-Ourai-Kita, Izumisano City, Osaka 598-8531, Japan
| | - M Tanaka
- Veterinary Pathology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku-Ourai-Kita, Izumisano City, Osaka 598-8531, Japan
| | - M Kuwamura
- Veterinary Pathology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku-Ourai-Kita, Izumisano City, Osaka 598-8531, Japan
| | - J Yamate
- Veterinary Pathology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku-Ourai-Kita, Izumisano City, Osaka 598-8531, Japan.
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Narita K, Matsuda Y, Seike M, Naito Z, Gemma A, Ishiwata T. Nestin regulates proliferation, migration, invasion and stemness of lung adenocarcinoma. Int J Oncol 2014; 44:1118-30. [PMID: 24481417 DOI: 10.3892/ijo.2014.2278] [Citation(s) in RCA: 57] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2013] [Accepted: 01/07/2014] [Indexed: 11/05/2022] Open
Abstract
Lung cancer is the most common cancer and the most common cause of cancer-related death in the world. Nestin, a class VI intermediate filament, is known to be a cancer stem cell (CSC) marker as well as a neuroepithelial stem cell marker. High expression levels of nestin are reported in several types of cancers including lung, pancreatic and prostate cancers. Nestin is thought to regulate tumor cell proliferation, migration, invasion and CSC properties. Here, we confirmed nestin expression in non-small cell lung cancer (NSCLC): Immunohistochemical analysis in surgical specimens detected nestin protein expression in the cytoplasm of 20 of 48 adenocarcinoma (AD) cases (41.7%) and 25 of 47 squamous cell carcinoma cases (53.2%). Nestin immunoreactivity significantly correlated with not only tumor size and lymph node metastasis in NSCLC, but also poor survival in surgical patients with AD. High and moderate expression levels of nestin were confirmed in several lung AD cell lines including H1975 and PC-3. Nestin inhibition by shRNA decreased proliferation, migration, invasion and sphere formation in AD cells. Correspondingly, nestin upregulation by nestin gene transfection resulted in the opposite changes. Moreover, Akt inhibitor IV effectively decreased nestin expression via SRY-box containing protein 2 (Sox2) downregulation and overcame the enhanced sphere formation induced by nestin upregulation. Overall, our results show that nestin correlates with the aggressiveness and stemness of AD. Regulation of nestin via Akt/Sox2 is, thus, a promising candidate for novel therapeutic approaches to eradicate CSCs in lung AD.
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Affiliation(s)
- Kosuke Narita
- Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, Tokyo 113-8602, Japan
| | - Yoko Matsuda
- Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, Tokyo 113-8602, Japan
| | - Masahiro Seike
- Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, Tokyo 113-8603, Japan
| | - Zenya Naito
- Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, Tokyo 113-8602, Japan
| | - Akihiko Gemma
- Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, Tokyo 113-8603, Japan
| | - Toshiyuki Ishiwata
- Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, Tokyo 113-8602, Japan
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16
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Maruyama M, Yamashita Y, Kase M, Trifonov S, Sugimoto T. Lineage-specific purification of neural stem/progenitor cells from differentiated mouse induced pluripotent stem cells. Stem Cells Transl Med 2013; 2:420-33. [PMID: 23694811 PMCID: PMC3673754 DOI: 10.5966/sctm.2012-0139] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2012] [Accepted: 02/25/2013] [Indexed: 12/29/2022] Open
Abstract
Since induced pluripotent stem (iPS) cells have differentiation potential into all three germ layer-derived tissues, efficient purification of target cells is required in many fields of iPS research. One useful strategy is isolation of desired cells from differentiated iPS cells by lineage-specific expression of a drug-resistance gene, followed by drug selection. With this strategy, we purified neural stem/progenitor cells (NSCs), a good candidate source for regenerative therapy, from differentiated mouse iPS cells. We constructed a bicistronic expression vector simultaneously expressing blasticidin S resistance gene and DsRed under the control of tandem enhancer of a 257-base pair region of nestin second intron, an NSC-specific enhancer. This construct was efficiently inserted into the iPS genome by piggyBac transposon-mediated gene transfer, and the established subclone was differentiated into NSCs in the presence or absence of blasticidin S. Consequently, incubation with blasticidin S led to purification of NSCs from differentiated iPS cells. Our results suggest that a lineage-specific drug selection strategy is useful for purification of NSCs from differentiated iPS cells and that this strategy can be applied for the purification of other cell types.
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Affiliation(s)
- Masato Maruyama
- Department of Anatomy and Brain Science, Kansai Medical University, Hirakata, Osaka, Japan
| | - Yuji Yamashita
- Department of Anatomy and Brain Science, Kansai Medical University, Hirakata, Osaka, Japan
| | - Masahiko Kase
- Department of Anatomy and Brain Science, Kansai Medical University, Hirakata, Osaka, Japan
| | - Stefan Trifonov
- Department of Anatomy and Brain Science, Kansai Medical University, Hirakata, Osaka, Japan
| | - Tetsuo Sugimoto
- Department of Anatomy and Brain Science, Kansai Medical University, Hirakata, Osaka, Japan
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17
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Kopp JL, von Figura G, Mayes E, Liu FF, Dubois CL, Morris JP, Pan FC, Akiyama H, Wright CVE, Jensen K, Hebrok M, Sander M. Identification of Sox9-dependent acinar-to-ductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma. Cancer Cell 2012; 22. [PMID: 23201164 PMCID: PMC3568632 DOI: 10.1016/j.ccr.2012.10.025] [Citation(s) in RCA: 529] [Impact Index Per Article: 40.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Tumors are largely classified by histologic appearance, yet morphologic features do not necessarily predict cellular origin. To determine the origin of pancreatic ductal adenocarcinoma (PDA), we labeled and traced pancreatic cell populations after induction of a PDA-initiating Kras mutation. Our studies reveal that ductal and stem-like centroacinar cells are surprisingly refractory to oncogenic transformation, whereas acinar cells readily form PDA precursor lesions with ductal features. We show that formation of acinar-derived premalignant lesions depends on ectopic induction of the ductal gene Sox9. Moreover, when concomitantly expressed with oncogenic Kras, Sox9 accelerates formation of premalignant lesions. These results provide insight into the cellular origin of PDA and suggest that its precursors arise via induction of a duct-like state in acinar cells.
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Affiliation(s)
- Janel L. Kopp
- Departments of Pediatrics and Cellular & Molecular Medicine, University of California-San Diego, La Jolla, CA 92093-0695
| | - Guido von Figura
- Diabetes Center, Department of Medicine, University of California-San Francisco, San Francisco, CA 94143
| | - Erin Mayes
- Departments of Pediatrics and Cellular & Molecular Medicine, University of California-San Diego, La Jolla, CA 92093-0695
| | - Fen-Fen Liu
- Departments of Pediatrics and Cellular & Molecular Medicine, University of California-San Diego, La Jolla, CA 92093-0695
| | - Claire L. Dubois
- Departments of Pediatrics and Cellular & Molecular Medicine, University of California-San Diego, La Jolla, CA 92093-0695
| | - John P. Morris
- Diabetes Center, Department of Medicine, University of California-San Francisco, San Francisco, CA 94143
| | - Fong Cheng Pan
- Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232-8240
| | - Haruhiko Akiyama
- Department of Orthopedics, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo, Kyoto 606-8507, Japan
| | | | - Kristin Jensen
- Department of Pathology, Veterans Affairs Palo Alto Health Care System and Stanford University Hospital, Stanford, CA 94305
| | - Matthias Hebrok
- Diabetes Center, Department of Medicine, University of California-San Francisco, San Francisco, CA 94143
- Corresponding authors: , Telephone: (858) 246-0843, Fax: (858) 246-1579. , Telephone: (415) 514-0820, Fax: (415) 564-5813
| | - Maike Sander
- Departments of Pediatrics and Cellular & Molecular Medicine, University of California-San Diego, La Jolla, CA 92093-0695
- Corresponding authors: , Telephone: (858) 246-0843, Fax: (858) 246-1579. , Telephone: (415) 514-0820, Fax: (415) 564-5813
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18
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Rhodes JA, Criscimanna A, Esni F. Induction of mouse pancreatic ductal differentiation, an in vitro assay. In Vitro Cell Dev Biol Anim 2012; 48:641-9. [DOI: 10.1007/s11626-012-9555-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2012] [Accepted: 09/07/2012] [Indexed: 02/01/2023]
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19
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Skeletal muscle neural progenitor cells exhibit properties of NG2-glia. Exp Cell Res 2012; 319:45-63. [PMID: 22999866 DOI: 10.1016/j.yexcr.2012.09.008] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2012] [Revised: 09/13/2012] [Accepted: 09/15/2012] [Indexed: 12/16/2022]
Abstract
Reversing brain degeneration and trauma lesions will depend on cell therapy. Our previous work identified neural precursor cells derived from the skeletal muscle of Nestin-GFP transgenic mice, but their identity, origin, and potential survival in the brain are only vaguely understood. In this work, we show that Nestin-GFP+ progenitor cells share morphological and molecular markers with NG2-glia, including NG2, PDGFRα, O4, NGF receptor (p75), glutamate receptor-1(AMPA), and A2B5 expression. Although these cells exhibit NG2, they do not express other pericyte markers, such as α-SMA or connexin-43, and do not differentiate into the muscle lineage. Patch-clamp studies displayed outward potassium currents, probably carried through Kir6.1 channels. Given their potential therapeutic application, we compared their abundance in tissues and concluded that skeletal muscle is the richest source of predifferentiated neural precursor cells. We found that these cells migrate toward the neurogenic subventricular zone displaying their typical morphology and nestin-GFP expression two weeks after brain injection. For translational purposes, we sought to identify these neural progenitor cells in wild-type species by developing a DsRed expression vector under Nestin-Intron II control. This approach revealed them in nonhuman primates and aging rodents throughout the lifespan.
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20
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Hagio M, Matsuda Y, Suzuki T, Ishiwata T. Nestin regulates epithelial-mesenchymal transition marker expression in pancreatic ductal adenocarcinoma cell lines. Mol Clin Oncol 2012; 1:83-87. [PMID: 24649127 DOI: 10.3892/mco.2012.1] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2012] [Accepted: 06/04/2012] [Indexed: 01/15/2023] Open
Abstract
Nestin, a class VI intermediate filament, is a neuronal stem/progenitor cell marker that is also expressed by various types of cancer, including pancreatic ductal adenocarcinoma (PDAC). We previously detected nestin expression in approximately 30% of PDAC cases, and found that nestin promotes the migration, invasion and metastasis of cells. Findings of recent studies have shown that epithelial mesenchymal transition (EMT) is important in the invasion and migration of cancer. In the present study, we investigated whether an altered nestin expression affected the expression levels of EMT markers in PDAC cells. Two human PDAC cell lines, PK-45H and KLM-1, in which nestin was suppressed and overexpressed, respectively, were used. The expression levels of the EMT-related molecules E-cadherin, Snail, Slug and Twist were analyzed using quantitative RT-PCR. Results showed that E-cadherin expression was decreased in nestin-overexpressed KLM-1 cells, and increased in nestin-suppressed PK-45H cells. Snail gene expression in the PDAC cells was altered concomitantly with the changes in nestin expression, while the Slug gene expression was significantly decreased in nestin-overexpressed KLM-1 cells. The Twist gene expression was below the detection limit in the two PDAC cell lines. The present findings indicated that nestin may be involved in the control of cancer behaviors in PDAC via the modulation of EMT-related molecules.
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Affiliation(s)
- Masahito Hagio
- Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, Tokyo 113-8602, Japan
| | - Yoko Matsuda
- Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, Tokyo 113-8602, Japan
| | - Taeko Suzuki
- Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, Tokyo 113-8602, Japan
| | - Toshiyuki Ishiwata
- Departments of Pathology and Integrative Oncological Pathology, Nippon Medical School, Tokyo 113-8602, Japan
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21
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Ectopic overexpression of Sonic Hedgehog (Shh) induces stromal expansion and metaplasia in the adult murine pancreas. Neoplasia 2012; 13:923-30. [PMID: 22028618 DOI: 10.1593/neo.11088] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2011] [Revised: 08/26/2011] [Accepted: 08/29/2011] [Indexed: 12/23/2022] Open
Abstract
Ligand-dependent activation of the Hedgehog (Hh) signaling pathway has been implicated in both tumor initiation and metastasis of pancreatic ductal adenocarcinoma (PDAC). Prior studies in genetically engineered mouse models (GEMMs) have assessed the role of Hh signaling by cell autonomous expression of a constitutively active Gli2 within epithelial cells. On the contrary, aberrant pathway reactivation in the human exocrine pancreas occurs principally as a consequence of Sonic Hh ligand (Shh) overexpression from epithelial cells. To recapitulate the cognate pathophysiology of Hh signaling observed in the human pancreas, we examined GEMM where Hh ligand is conditionally overexpressed within the mature exocrine pancreas using a tamoxifen-inducible Elastase-Cre promoter (Ela-CreERT2;LSL-mShh). We also facilitated potential cell autonomous epithelial responsiveness to secreted Hh ligand by generating compound transgenic mice with concomitant expression of the Hh receptor Smoothened (Ela-CreERT2;LSL-mShh;LSL-mSmo). Of interest, none of these mice developed intraductal precursor lesions or PDAC during the follow-up period of up to 12 months after tamoxifen induction. Instead, all animals demonstrated marked expansion of stromal cells, consistent with the previously described epithelial-to-stromal paracrine Hh signaling. Hh responsiveness was mirrored by the expression of primary cilia within the expanded mesenchymal compartment and the absence within mature acinar cells. In the absence of cooperating mutations, Hh ligand overexpression in the mature exocrine pancreas is insufficient to induce neoplasia, even when epithelial cells coexpress the Smo receptor. This autochthonous model serves as a platform for studying epithelial stromal interactions in pancreatic carcinogenesis.
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22
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Acute pancreatitis accelerates initiation and progression to pancreatic cancer in mice expressing oncogenic Kras in the nestin cell lineage. PLoS One 2011; 6:e27725. [PMID: 22140463 PMCID: PMC3225359 DOI: 10.1371/journal.pone.0027725] [Citation(s) in RCA: 58] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2011] [Accepted: 10/23/2011] [Indexed: 12/27/2022] Open
Abstract
Targeting of oncogenic Kras to the pancreatic Nestin-expressing embryonic progenitor cells and subsequently to the adult acinar compartment and Nestin-expressing cells is sufficient for the development of low grade pancreatic intraepithelial neoplasia (PanIN) between 2 and 4 months. The mice die around 6 month-old of unrelated causes, and it is therefore not possible to assess whether the lesions will progress to carcinoma. We now report that two brief episodes of caerulein-induced acute pancreatitis in 2 month-old mice causes rapid PanIN progression and pancreatic ductal adenocarcinoma (PDAC) development by 4 months of age. These events occur with similar frequency as observed in animals where the oncogene is targeted during embryogenesis to all pancreatic cell types. Thus, these data show that oncogenic Kras-driven PanIN originating in a non-ductal compartment can rapidly progress to PDAC when subjected to a brief inflammatory insult.
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23
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Matsuda Y, Naito Z, Kawahara K, Nakazawa N, Korc M, Ishiwata T. Nestin is a novel target for suppressing pancreatic cancer cell migration, invasion and metastasis. Cancer Biol Ther 2011; 11:512-23. [PMID: 21258211 DOI: 10.4161/cbt.11.5.14673] [Citation(s) in RCA: 78] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Nestin, is a class VI intermediate filament (IF) that is expressed in 30% of pancreatic ductal adenocarcinoma (PDAC) cases, and its expression in PDAC positively correlates with peripancreatic invasion. An expression vector carrying a short hairpin RNA (shRNA) targeting nestin was stably transfected into PANC-1 and PK-45H human pancreatic cancer cells, which express high nestin levels. Alterations in morphology and alignment of actin filaments and α-tubulin were examined by phase-contrast and immunocytochemistry. Effects on cell growth, migration in scratch and Boyden chamber assays, invasion, cell adhesion, and in vivo growth were determined. Differences in mRNA levels were examined by arrays. Nestin shRNA-transfected cells exhibited decreased nestin expression, a sheet-like appearance with tight cell-cell adhesion, increased expression of filamentous F-actin and E-cadherin, and attenuated migration and invasion, both of which were enhanced following nestin re-expression. Expression of α-tubulin, and in vitro cell growth and adhesion were not altered by nestin down-regulation, whereas hepatic metastases were decreased. Thus, nestin plays important roles in pancreatic cancer cell migration, invasion and metastasis by selectively modulating the expression of actin and cell adhesion molecules, and may therefore be a novel therapeutic target in PDAC.
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Affiliation(s)
- Yoko Matsuda
- Department of Pathology, Nippon Medical School, Bunkyo-ku, Tokyo, Japan
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24
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Ishiwata T, Matsuda Y, Naito Z. Nestin in gastrointestinal and other cancers: Effects on cells and tumor angiogenesis. World J Gastroenterol 2011; 17:409-18. [PMID: 21274370 PMCID: PMC3027007 DOI: 10.3748/wjg.v17.i4.409] [Citation(s) in RCA: 109] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/10/2010] [Revised: 12/01/2010] [Accepted: 12/08/2010] [Indexed: 02/06/2023] Open
Abstract
Nestin is a class VI intermediate filament protein that was originally described as a neuronal stem cell marker during central nervous system (CNS) development, and is currently widely used in that capacity. Nestin is also expressed in non-neuronal immature or progenitor cells in normal tissues. Under pathological conditions, nestin is expressed in repair processes in the CNS, muscle, liver, and infarcted myocardium. Furthermore, increased nestin expression has been reported in various tumor cells, including CNS tumors, gastrointestinal stromal tumors, pancreatic cancer, prostate cancer, breast cancer, malignant melanoma, dermatofibrosarcoma protuberances, and thyroid tumors. Nestin is reported to correlate with aggressive growth, metastasis, and poor prognosis in some tumors; however, the roles of nestin in cancer cells have not been well characterized. Furthermore, nestin is more specifically expressed in proliferating small-sized tumor vessels in glioblastoma and gastric, colorectal, and prostate cancers than are other tumor vessel markers. These findings indicate that nestin may be a marker for newly synthesized tumor vessels and a therapeutic target for tumor angiogenesis. It has received a lot of attention recently as a cancer stem cell marker in various cancer cells including brain tumors, malignant rhabdoid tumors, and uterine, cervical, prostate, bladder, head and neck, ovarian, testicular, and pancreatic cancers. The purpose of this review is to clarify the roles of nestin in cancer cells and in tumor angiogenesis, and to examine the association between nestin and cancer stem cells. Nestin has the potential to serve as a molecular target for cancers with nestin-positive cancer cells and nestin-positive tumor vasculature.
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25
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Mason MN, Mahoney MJ. Inhibition of gamma-secretase activity promotes differentiation of embryonic pancreatic precursor cells into functional islet-like clusters in poly(ethylene glycol) hydrogel culture. Tissue Eng Part A 2011; 16:2593-603. [PMID: 20236034 DOI: 10.1089/ten.tea.2010.0015] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
We assessed the ability of a gamma-secretase inhibitor to promote the in vitro differentiation of induced embryonic pancreatic precursor cell aggregates into functional islet-like clusters when encapsulated within a three-dimensional hydrogel. Undifferentiated pancreatic precursor cells were isolated from E.15 rat embryos, dissociated into single cells, and aggregated in suspension-rotation culture. Aggregates were photoencapsulated into poly(ethylene glycol) hydrogels with entrapped collagen type 1 and cultured for 14 days with or without a gamma-secretase inhibitor. Gene expression, proinsulin content, and C-peptide release were measured to determine differentiation and maturation of encapsulated precursor cell aggregates. In the control medium, scattered breakthrough beta cell differentiation was observed; however, cells remained largely insulin negative. Upon addition of a gamma-secretase inhibitor the majority of cells in clusters became insulin positive, and insulin per DNA and glucose-stimulated insulin release measurements for these cultures were comparable with those for adult rat islets. Cluster counts after culture day 14 were 88% of those initially encapsulated, demonstrating excellent cluster survival in hydrogel culture. These results indicate that concerted differentiation of pancreatic precursor cell aggregates into functionally mature islet-like clusters can be achieved in poly(ethylene glycol)-based hydrogel cultures by blocking cell contact-mediated Notch signaling with a gamma-secretase inhibitor.
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Affiliation(s)
- Mariah N Mason
- Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309, USA
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26
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Desgraz R, Bonal C, Herrera PL. β-cell regeneration: the pancreatic intrinsic faculty. Trends Endocrinol Metab 2011; 22:34-43. [PMID: 21067943 DOI: 10.1016/j.tem.2010.09.004] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2010] [Revised: 09/27/2010] [Accepted: 09/27/2010] [Indexed: 01/08/2023]
Abstract
Type I diabetes (T1D) patients rely on cumbersome chronic injections of insulin, making the development of alternate durable treatments a priority. The ability of the pancreas to generate new β-cells has been described in experimental diabetes models and, importantly, in infants with T1D. Here we discuss recent advances in identifying the origin of new β-cells after pancreatic injury, with and without inflammation, revealing a surprising degree of cell plasticity in the mature pancreas. In particular, the inducible selective near-total destruction of β-cells in healthy adult mice uncovers the intrinsic capacity of differentiated pancreatic cells to spontaneously reprogram to produce insulin. This opens new therapeutic possibilities because it implies that β-cells can differentiate endogenously, in depleted adults, from heterologous origins.
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Affiliation(s)
- Renaud Desgraz
- Department of Cell Physiology and Metabolism, University of Geneva Faculty of Medicine, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland
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27
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Mason MN, Arnold CA, Mahoney MJ. Entrapped collagen type 1 promotes differentiation of embryonic pancreatic precursor cells into glucose-responsive beta-cells when cultured in three-dimensional PEG hydrogels. Tissue Eng Part A 2010; 15:3799-808. [PMID: 19537960 DOI: 10.1089/ten.tea.2009.0148] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Development of an alternative source of functional, transplantable beta-cells to replace or supplement cadaveric tissue is critical to the future success of islet cell transplantation therapy. Embryonic pancreatic precursor cells are desirable as a renewable source of beta-cells as they are both proliferative and inherently capable of pancreatic cell differentiation. We have previously shown that precursor cells undergo selective beta-cell differentiation when dissociated and photoencapsulated in a polyethylene glycol (PEG) hydrogel network; however, these cells remained immature and were not glucose responsive. Collagen type 1 supports mature cell viability and function in many cell types and we hypothesized that incorporating it within our gels may support differentiating beta-cells and facilitate beta-cell maturation. For these studies, collagen-1 was entrapped with dissociated pancreatic precursor cells in a PEG hydrogel matrix (PEGCol) with the following key findings: (1) mature, glucose-responsive, islet-like structures differentiated from spontaneously forming precursor cell clusters in PEGCol, but not unmodified PEG, hydrogels; (2) a balance existed between providing sufficient collagen-1 signaling to support precursor cell development and providing an overabundance of adhesive sites allowing contaminating mesenchymal cells to thrive' and (3) mechanical stability provided by the PEG hydrogel platform is important for successful precursor cell culture, as PEGCol hydrogels encourage glucose responsiveness and high-insulin gene expression, while pure collagen gel cultures, with the same collagen concentration, have negligible insulin gene expression. These results indicate that PEGCol hydrogels are a useful culture platform to promote differentiation of a glucose-responsive beta-cell population from dissociated precursor cells.
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Affiliation(s)
- Mariah N Mason
- Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309, USA
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28
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Insulin-producing cells derived from stem/progenitor cells: therapeutic implications for diabetes mellitus. Med Mol Morphol 2009; 42:195-200. [DOI: 10.1007/s00795-009-0471-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2009] [Accepted: 09/14/2009] [Indexed: 10/20/2022]
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29
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Abstract
The β-cells of the pancreas are responsible for insulin production and their destruction results in type I diabetes. β-cell maintenance, growth and regenerative repair is thought to occur predominately, if not exclusively, through the replication of existing β-cells, not via an adult stem cell. It was recently found that all β-cells contribute equally to islet growth and maintenance. The fact that all β-cells replicate homogeneously makes it possible to set up straightforward screens for factors that increase β-cell replication either In vitro or in vivo. It is possible that a circulating factor may be capable of increasing β-cell replication or that intrinsic cell cycle regulators may affect β-cell growth. An improved understanding of the in vivo maintenance and growth of β-cells will facilitate efforts to expand β-cells In vitro and may lead to new treatments for diabetes.
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Affiliation(s)
- Kristen Brennand
- Department of Stem Cell and Regenerative Biology, HHMI and Harvard University, Harvard Stem Cell Institute, Cambridge, MA, USA
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Takamori Y, Mori T, Wakabayashi T, Nagasaka Y, Matsuzaki T, Yamada H. Nestin-positive microglia in adult rat cerebral cortex. Brain Res 2009; 1270:10-8. [DOI: 10.1016/j.brainres.2009.03.014] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2008] [Revised: 02/27/2009] [Accepted: 03/05/2009] [Indexed: 11/25/2022]
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Abstract
The number of patients worldwide suffering from the chronic disease diabetes mellitus is growing at an alarming rate. Insulin-secreting beta-cells in the islet of Langerhans are damaged to different extents in diabetic patients, either through an autoimmune reaction present in type 1 diabetic patients or through inherent changes within beta-cells that affect their function in patients suffering from type 2 diabetes. Cell replacement strategies via islet transplantation offer potential therapeutic options for diabetic patients. However, the discrepancy between the limited number of donor islets and the high number of patients who could benefit from such a treatment reflects the dire need for renewable sources of high-quality beta-cells. Human embryonic stem cells (hESCs) are capable of self-renewal and can differentiate into components of all three germ layers, including all pancreatic lineages. The ability to differentiate hESCs into beta-cells highlights a promising strategy to meet the shortage of beta-cells. Here, we review the different approaches that have been used to direct differentiation of hESCs into pancreatic and beta-cells. We will focus on recent progress in the understanding of signaling pathways and transcription factors during embryonic pancreas development and how this knowledge has helped to improve the methodology for high-efficiency beta-cell differentiation in vitro.
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Affiliation(s)
- Tingxia Guo
- Department of Medicine, Diabetes Center, University of California, San Francisco, San Francisco, California 94143, USA
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32
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Fendrich V, Waldmann J, Feldmann G, Schlosser K, König A, Ramaswamy A, Bartsch DK, Karakas E. Unique expression pattern of the EMT markers Snail, Twist and E-cadherin in benign and malignant parathyroid neoplasia. Eur J Endocrinol 2009; 160:695-703. [PMID: 19176646 DOI: 10.1530/eje-08-0662] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
BACKGROUND Epithelial and mesenchymal transitions (EMT) are essential for embryonic development and progression of non-invasive tumor cells into malignant, metastatic carcinomas. During embryogenesis, the parathyroid glands develop from pharyngeal pouches and migrate to their final destinations, densely enclosed by mesenchymal neural crest cells. In this study, we examined the expression of the EMT markers Snail, Twist and E-cadherin in normal parathyroid glands and benign and malignant parathyroid diseases. METHODS Using immunohistochemistry, we compared expression of E-cadherin, Snail and Twist in 25 patients with parathyroid adenoma, 25 patients with parathyroid hyperplasia, and nine patients with parathyroid cancer with normal parathyroid glands. RESULTS Normal parathyroid glands, parathyroid adenomas, and parathyroid hyperplasias showed a typical membranous E-cadherin staining pattern. Expression of Snail was found in 22/25 parathyroid adenomas and in all parathyroid hyperplasias. Twist was expressed in 22/25 of parathyroid adenomas and in 20/25 parathyroid hyperplasias. Snail and Twist positive cells were homogeneously distributed throughout the gland. However, in all nine parathyroid carcinomas, membranous E-cadherin staining was lost. In addition, the expression pattern of Snail and Twist was changed and mostly limited to the invasive front of cancer tissue samples. CONCLUSION Expression of Snail and Twist at the invasive front and consecutive loss of E-cadherin in parathyroid carcinomas suggests a key role of EMT in the tumorigenesis of this cancer. The unique expression pattern could help to distinguish between an adenoma and a non-metastatic carcinoma. Loss of E-cadherin and change of the expression pattern of Snail and Twist together should result in an en bloc resection or a close follow-up.
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Affiliation(s)
- Volker Fendrich
- Department of Surgery, Philipps-University Marburg, Marburg, Germany.
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Bhagwandin VJ, Shay JW. Pancreatic cancer stem cells: fact or fiction? BIOCHIMICA ET BIOPHYSICA ACTA 2009; 1792:248-59. [PMID: 19233264 PMCID: PMC2670354 DOI: 10.1016/j.bbadis.2009.02.007] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/14/2008] [Revised: 02/07/2009] [Accepted: 02/10/2009] [Indexed: 12/11/2022]
Abstract
The terms cancer-initiating or cancer stem cells have been the subject of great interest in recent years. In this review we will use pancreatic cancer as an overall theme to draw parallels with historical findings to compare to recent reports of stem-like characteristics in pancreatic cancer. We will cover such topics as label-retaining cells (side-population), ABC transporter pumps, telomerase, quiescence, cell surface stem cell markers, and epithelial-mesenchymal transitions. Finally we will integrate the available findings into a pancreatic stem cell model that also includes metastatic disease.
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Affiliation(s)
- Vikash J. Bhagwandin
- University of California, San Francisco, G.W. Hooper Research Foundation, 513 Parnassus Avenue, San Francisco, CA 94143-0552
| | - Jerry W. Shay
- University of Texas Southwestern Medical Center, Department of Cell Biology, 5323 Harry Hines Boulevard, Dallas, TX 75390-9039
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Wang H, Wang S, Hu J, Kong Y, Chen S, Li L, Li L. Oct4 is expressed in Nestin-positive cells as a marker for pancreatic endocrine progenitor. Histochem Cell Biol 2009; 131:553-63. [PMID: 19224238 DOI: 10.1007/s00418-009-0560-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/22/2009] [Indexed: 01/18/2023]
Abstract
There are abundant progenitor cells in the developing pancreas, but molecular markers for these cells are lacking. Octamer-binding transcription factor-4 (Oct4) is an important transcription factor for keeping the features of self-renewal and pluripotency of embryonic stem cells. It's well known that Oct4, as a totipotent stem cells marker, just is expressed in totipotent stem cells. In the present study, we collected ten human fetal pancreases, and found that Oct4 mRNA and protein were expressed in human fetal pancreas samples by RT-PCR, western blot and immunohistochemistry assays. Using double-staining, we demonstrated that Oct4 was not co-expressed with Chromogranin A (a peptide expressed in endocrine cells), but partially co-expressed with Ngn3 (a transcription factor expressed in pancreatic endocrine precursor cells) and Nestin (a intermediate filament, Nestin-positive cells isolated from islets can be induced to express insulin) in human fetal pancreases. Indeed, we prepared Nestin-positive cells from human fetal pancreas by cell selection, and found that these cells expressed Oct4 and Ngn3. The Nestin-positive cells displayed a rapid duplication and could differentiate into osteoblasts, fat and endocrine cells in vitro. These results indicated that the Nestin-positive cells in the fetal age should be pancreatic progenitor cells. Overall, our study suggested that Oct4 was a marker for pancreatic endocrine progenitor.
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Affiliation(s)
- Hong Wang
- Stem Cell Research Center, Health Science Center, Peking University, No. 38 Xueyuan Road, Haidian District, Beijing, China.
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Raikwar SP, Zavazava N. Insulin producing cells derived from embryonic stem cells: are we there yet? J Cell Physiol 2008; 218:256-63. [PMID: 18932230 DOI: 10.1002/jcp.21615] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Derivation of insulin producing cells (IPCs) from embryonic stem (ES) cells provides a potentially innovative form of treatment for type 1 diabetes. Here, we discuss the current state of the art, unique challenges, and future directions on generating IPCs.
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Affiliation(s)
- Sudhanshu P Raikwar
- Division of Allergy and Immunology, Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
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Sordi V, Bertuzzi F, Piemonti L. Diabetes mellitus: an opportunity for therapy with stem cells? Regen Med 2008; 3:377-97. [PMID: 18462060 DOI: 10.2217/17460751.3.3.377] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
In both Type 1 and 2 diabetes, insufficient numbers of insulin-producing beta-cells are a major cause of defective control of blood glucose and its complications. Restoration of damaged beta-cells by endocrine pancreas regeneration would be an ideal therapeutic option. The possibility of generating insulin-secreting cells with adult pancreatic stem or progenitor cells has been investigated extensively. The conversion of differentiated cells such as hepatocytes into beta-cells is being attempted using molecular insights into the transcriptional make-up of beta-cells. Additionally, the enhanced proliferation of beta-cells in vivo or in vitro is being pursued as a strategy for regenerative medicine for diabetes. Advances have also been made in directing the differentiation of embryonic stem cells into beta-cells. Although progress is encouraging, major gaps in our understanding of developmental biology of the pancreas and adult beta-cell dynamics remain to be bridged before a therapeutic application is made possible.
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Affiliation(s)
- Valeria Sordi
- Laboratory of Experimental Surgery, San Raffaele Scientific Institute, via Olgettina 60, 20132 Milan, Italy
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37
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Hedgehog signaling is required for effective regeneration of exocrine pancreas. Gastroenterology 2008; 135:621-31. [PMID: 18515092 PMCID: PMC2666349 DOI: 10.1053/j.gastro.2008.04.011] [Citation(s) in RCA: 153] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/04/2007] [Revised: 02/22/2008] [Accepted: 04/10/2008] [Indexed: 01/27/2023]
Abstract
Although both endocrine and the exocrine pancreas display a significant capacity for tissue regeneration and renewal, the existence of progenitor cells in the adult pancreas remains uncertain. Using a model of cerulein-mediated injury and repair, we demonstrate that mature exocrine cells, defined by expression of an Elastase1 promoter, actively contribute to regenerating pancreatic epithelium through formation of metaplastic ductal intermediates. Acinar cell regeneration is associated with activation of Hedgehog (Hh) signaling, as assessed by up-regulated expression of multiple pathway components, as well as activation of a Ptch-lacZ reporter allele. Using both pharmacologic and genetic techniques, we also show that the ability of mature exocrine cells to accomplish pancreatic regeneration is impaired by blockade of Hh signaling. Specifically, attenuated regeneration in the absence of an intact Hh pathway is characterized by persistence of metaplastic epithelium expressing markers of pancreatic progenitor cells, suggesting an inhibition of redifferentiation into mature exocrine cells. Given the known role of Hh signaling in exocrine pancreatic cancer, these findings may provide a mechanistic link between injury-induced activation of pancreatic progenitors and subsequent pancreatic neoplasia.
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Abstract
The past two decades have witnessed an explosion in our understanding of pancreatic cancer, and it is now clear that pancreatic cancer is a disease of inherited (germ-line) and somatic gene mutations. The genes mutated in pancreatic cancer include KRAS2, p16/CDKN2A, TP53, and SMAD4/DPC4, and these are accompanied by a substantial compendium of genomic and transcriptomic alterations that facilitate cell cycle deregulation, cell survival, invasion, and metastases. Pancreatic cancers do not arise de novo, and three distinct precursor lesions have been identified. Experimental models of pancreatic cancer have been developed in genetically engineered mice, which recapitulate the multistep progression of the cognate human disease. Although the putative cell of origin for pancreatic cancer remains elusive, minor populations of cells with stem-like properties have been identified that appear responsible for tumor initiation, metastases, and resistance of pancreatic cancer to conventional therapies.
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Affiliation(s)
- Anirban Maitra
- Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
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Rovira M, Jané-Valbuena J, Marchand M, Savatier P, Real FX, Skoudy A. Viral-mediated coexpression of Pdx1 and p48 regulates exocrine pancreatic differentiation in mouse ES cells. CLONING AND STEM CELLS 2008; 9:327-38. [PMID: 17907943 DOI: 10.1089/clo.2006.0064] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Embryonic stem cells (ES) can spontaneously activate a pancreatic differentiation program in vitro, although with low efficiency. The aim was to improve such process by using viral mediated gene transduction. In this study, we have examined the suitability of using viral vectors to express key transcriptional factors involved in pancreatic development. ES cell lines that constitutively express Pdx1, a homeodomain protein involved in both exocrine and endocrine pancreatic development and differentiation, were established using a lentiviral vector. These cells were additionally infected with an adenovirus expressing p48, a bHLH factor that is also crucial for pancreatic development and acinar differentiation. Quantitative RT-PCR analysis demonstrated an increase in the expression of exocrine genes, including those coding for both digestive enzymes and transcription factors. Immunocytochemical staining also revealed an increase in the number of amylase-expressing cell clusters. However, other important genes involved in acinar cell maturation (i.e., Mist1) were not modulated under these conditions, suggesting that the cells display features of immature exocrine cells or because of an uncoupled gene expression of the exocrine differentiation program. Importantly, this effect was selective for the acinar lineage as the expression of a large set of endocrine markers remained unchanged. Therefore, combined expression of key genes involved in pancreatic development may be a promising approach to generate mature pancreatic exocrine cells.
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Affiliation(s)
- Meritxell Rovira
- Cell and Molecular Biology Unit, Institut Municipal d'Investigació Mèdica (IMIM), Dr Aiguader 88, Barcelona, Spain
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40
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Masui T, Long Q, Beres TM, Magnuson MA, MacDonald RJ. Early pancreatic development requires the vertebrate Suppressor of Hairless (RBPJ) in the PTF1 bHLH complex. Genes Dev 2007; 21:2629-43. [PMID: 17938243 DOI: 10.1101/gad.1575207] [Citation(s) in RCA: 129] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
PTF1a is an unusual basic helix-loop-helix (bHLH) transcription factor that is required for the development of the pancreas. We show that early in pancreatic development, active PTF1a requires interaction with RBPJ, the vertebrate Suppressor of Hairless, within a stable trimeric DNA-binding complex (PTF1). Later, as acinar cell development begins, RBPJ is swapped for RBPJL, the constitutively active, pancreas-restricted paralog of RBPJ. Moreover, the Rbpjl gene is a direct target of the PTF1 complex: At the onset of acinar cell development when the Rbpjl gene is first induced, a PTF1 complex containing RBPJ is bound to the Rbpjl promoter. As development proceeds, RBPJL gradually replaces RBPJ in the PTF1 complex bound to Rbpjl and appears on the binding sites for the complex in the promoters of other acinar-specific genes, including those for the secretory digestive enzymes. A single amino acid change in PTF1a that eliminates its ability to bind RBPJ (but does not affect its binding to RBPJL) causes pancreatic development to truncate at an immature stage, without the formation of acini or islets. These results indicate that the interaction between PTF1a and RBPJ is required for the early stage of pancreatic growth, morphogenesis, and lineage fate decisions. The defects in pancreatic development phenocopy those of Ptf1a-null embryos; thus, the first critical function of PTF1a is in the context of the PTF1 complex containing RBPJ. Action within an organ-specific transcription factor is a previously unknown function for RBPJ and is independent of its role in Notch signaling.
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Affiliation(s)
- Toshihiko Masui
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA
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42
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Kleeberger W, Bova GS, Nielsen ME, Herawi M, Chuang AY, Epstein JI, Berman DM. Roles for the stem cell associated intermediate filament Nestin in prostate cancer migration and metastasis. Cancer Res 2007; 67:9199-206. [PMID: 17909025 PMCID: PMC3072059 DOI: 10.1158/0008-5472.can-07-0806] [Citation(s) in RCA: 147] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The intermediate filament protein Nestin identifies stem/progenitor cells in adult tissues, but the function of Nestin is poorly understood. We investigated Nestin expression and function in common lethal cancers. Nestin mRNA was detected in cell lines from small cell lung, and breast cancers, and particularly elevated in cell lines derived from prostate cancer metastases. Whereas the androgen-independent lines PC3, 22RV1, and DU145 all expressed Nestin transcripts under standard culture conditions, the androgen-dependent line LnCaP expressed Nestin only on androgen withdrawal. We confirmed associations of Nestin expression, androgen withdrawal, and metastatic potential by immunohistochemical analysis of samples from 254 prostate cancer patients. Cytoplasmic Nestin protein was readily identifiable in prostate cancer cells from 75% of patients with lethal androgen-independent disease, even in cancer sampled from the prostate itself. However, Nestin expression was undetectable in localized androgen-deprived tumors and in metastases without prior androgen deprivation. To address its function, we reduced Nestin levels with short hairpin RNAs, markedly inhibiting in vitro migration and invasion in prostate cancer cells but leaving cell growth intact. Nestin knockdown also diminished metastases 5-fold compared with controls despite uncompromised tumorigenicity at the site of inoculation. These results specify a function for Nestin in cell motility and identify a novel pathway for prostate cancer metastasis. Activity of this pathway may be selected by the extraprostatic environment or, as supported by our data, may originate within the prostate after androgen deprivation. Further dissection of this novel Nestin migration pathway may lead to strategies to prevent and neutralize metastatic spread.
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Affiliation(s)
- Wolfram Kleeberger
- Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - G. Steven Bova
- Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
- Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
- Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
- Department of Health Information Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Matthew E. Nielsen
- Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Mehsati Herawi
- Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Ai-Ying Chuang
- Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Jonathan I. Epstein
- Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
- Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
- Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - David M. Berman
- Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
- Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
- Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
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43
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Ohike N, Sato M, Hisayuki T, Imataka H, Sato S, Wada Y, Saito K, Takahashi M, Tajiri T, Kunimura T, Morohoshi T. Immunohistochemical analysis of nestin and c-kit and their significance in pancreatic tumors. Pathol Int 2007; 57:589-93. [PMID: 17685930 DOI: 10.1111/j.1440-1827.2007.02143.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The purpose of the present study was to clarify the difference of expression of two stem cell markers, nestin and c-kit, among various pancreatic epithelial tumors and evaluate their utility. Immunohistochemistry was done for 99 surgically resected pancreatic tumor specimens, including 20 ductal adenocarcinoma (DAC), two undifferentiated carcinomas (UC), 31 intraductal papillary-mucinous neoplasms (IPMN), six mucinous cystic neoplasms (MCN), five serous cystadenomas (SCA), six acinar cell carcinomas, two pancreatoblastoma (PB), eight solid-pseudopapillary neoplasms (SPN), and 19 endocrine neoplasms (EN). Nestin was widely expressed in four SPN, one PB, one SCA, sarcoma areas in two UC, one MCN, and one DAC, and an area of oncocytic component in one IPMN. Some of these SPN, SCA and sarcomatous or oncocytic components in which nestin was expressed, also coexpressed c-kit. Additionally, partial (scattered) expression of c-kit was observed in ductal elements of 16 DAC, eight IPMN, five MCN, and one UC, one SCA, and three EN. The eight c-kit-positive IPMN included four of 23 adenoma-to-border lesions and four of eight non-invasive-to-invasive carcinomas. The three EN were all carcinomas. These indicate that expression of two stem cell markers is different by tumor type, but the utility of judging direction or degree of differentiation and malignant grade on the basis of their expression status is suggested.
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Affiliation(s)
- Nobuyuki Ohike
- First Department of Pathology, Showa University School of Medicine, Shinagawa-ku, Tokyo, Japan.
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Abstract
In both type 1 and type 2 diabetes, insufficient numbers of insulin-producing beta cells are a major cause of defective control of blood glucose and its complications. Accordingly, therapies that increase functional beta-cell mass may offer a cure for diabetes. Efforts to achieve this goal explore several directions. Based on the realization that beta cells are capable of significant proliferation throughout adult life, the enhanced proliferation of beta cells in vivo or in vitro is pursued as a strategy for regenerative medicine for diabetes. Alternatively, the conversion of differentiated cells such as hepatocytes into beta cells is being attempted using molecular insights into the transcriptional makeup of beta cells. Advances were also made in directing the differentiation of embryonic stem cells into beta cells. Although progress is encouraging, major gaps in our understanding of developmental biology of the pancreas and adult beta-cell dynamics remain to be closed before a therapeutic application is made possible.
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Affiliation(s)
- Shay Porat
- The Department of Cellular Biochemistry and Human Genetics, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
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45
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Kedees MH, Guz Y, Vuguin PM, Vargas C, Cui L, Steiner DF, Charron MJ, Teitelman G. Nestin expression in pancreatic endocrine and exocrine cells of mice lacking glucagon signaling. Dev Dyn 2007; 236:1126-33. [PMID: 17366624 PMCID: PMC4287279 DOI: 10.1002/dvdy.21112] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Nestin, a marker of neural stem cells, is also expressed by cells located in the epithelium of the pancreatic primordium and by a subpopulation of exocrine cells but not by endocrine cells. These findings raised the possibility that the pancreatic epithelium is heterogeneous and comprised of subpopulations of exocrine/nestin-positive and endocrine/nestin-negative precursor cells. We examined this issue in two mutant mouse models characterized by protracted expression of several embryonal properties in islet cells. One mutant line comprises mice lacking mature glucagon due to abrogation of proprotein convertase-2 (PC2(-/-)), responsible for the conversion of proglucagon into glucagon, while the second line consists of mice with a global deletion of the glucagon receptor (Gcgr(-/-)). We demonstrate that nestin is transiently expressed by acinar cells and by insulin and glucagon cells of islets of both lines of mice. In addition, the lack of glucagon signaling increased nestin mRNA levels in pancreas of mutant embryos and adult mice. We conclude that nestin+ cells located in the pancreatic primordium generate the cells of the endocrine and exocrine lineages. Furthermore, our results suggest that nestin expression is regulated by glucagon signaling.
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Affiliation(s)
- Mamdouh H. Kedees
- Department of Anatomy and Cell Biology, SUNY-Downstate Medical Center, Brooklyn, New York
| | - Yelena Guz
- Department of Anatomy and Cell Biology, SUNY-Downstate Medical Center, Brooklyn, New York
| | - Patricia M. Vuguin
- Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York
- Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York
| | - Carlos Vargas
- Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York
| | - Lingguang Cui
- Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York
| | - Donald F. Steiner
- Department of Biochemistry, University of Chicago, Chicago, Illinois
| | - Maureen J. Charron
- Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York
| | - Gladys Teitelman
- Department of Anatomy and Cell Biology, SUNY-Downstate Medical Center, Brooklyn, New York
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Carrière C, Seeley ES, Goetze T, Longnecker DS, Korc M. The Nestin progenitor lineage is the compartment of origin for pancreatic intraepithelial neoplasia. Proc Natl Acad Sci U S A 2007; 104:4437-42. [PMID: 17360542 PMCID: PMC1810329 DOI: 10.1073/pnas.0701117104] [Citation(s) in RCA: 106] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
To determine the cell compartment in which initial oncogenic mutations occur in pancreatic ductal adenocarcinoma (PDAC), we generated a mouse model in which endogenous expression of mutated Kras (Kras(G12D)) was initially directed to a population of pancreatic exocrine progenitors characterized by the expression of Nestin. Targeting of oncogenic Kras to such a restricted cell compartment was sufficient for the formation of pancreatic intraepithelial neoplasias (PanINs), putative precursors to PDAC. PanINs appeared with the same grade and frequency as observed when Kras(G12D) was targeted to the whole pancreas by a Pdx1-driven Cre recombinase strategy. Thus, the Nestin cell lineage is highly responsive to Kras oncogenic activation and may represent the elusive progenitor population in which PDAC arises.
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Affiliation(s)
- Catherine Carrière
- *Departments of Medicine, and Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755
- Norris Cotton Comprehensive Cancer Center at Dartmouth Hitchcock Medical Center, Lebanon, NH 03756; and
- To whom correspondence may be addressed at:
Department of Medicine, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756. E-mail:
or
| | - Elliott S. Seeley
- *Departments of Medicine, and Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755
- Norris Cotton Comprehensive Cancer Center at Dartmouth Hitchcock Medical Center, Lebanon, NH 03756; and
| | - Tobias Goetze
- *Departments of Medicine, and Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755
- Norris Cotton Comprehensive Cancer Center at Dartmouth Hitchcock Medical Center, Lebanon, NH 03756; and
| | | | - Murray Korc
- *Departments of Medicine, and Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755
- Norris Cotton Comprehensive Cancer Center at Dartmouth Hitchcock Medical Center, Lebanon, NH 03756; and
- To whom correspondence may be addressed at:
Department of Medicine, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756. E-mail:
or
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47
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Bernardo AS, Barrow J, Hay CW, McCreath K, Kind AJ, Schnieke AE, Colman A, Hart AW, Docherty K. Presence of endocrine and exocrine markers in EGFP-positive cells from the developing pancreas of a nestin/EGFP mouse. Mol Cell Endocrinol 2006; 253:14-21. [PMID: 16698177 DOI: 10.1016/j.mce.2006.03.003] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/20/2006] [Revised: 03/06/2006] [Accepted: 03/10/2006] [Indexed: 01/08/2023]
Abstract
In order to purify and characterize nestin-positive cells in the developing pancreas a transgenic mouse was generated, in which the enhanced green fluorescent protein (EGFP) was driven by the nestin second intronic enhancer and upstream promoter. In keeping with previous studies on the distribution of nestin, EGFP was expressed in the developing embryo in neurones in the brain, eye, spinal cord, tail bud and glial cells in the small intestine. In the pancreas there was no detectable EGFP at embryonic day 11.5 (E11.5). EGFP expression appeared at E12.5 and increased in intensity through E14.5, E18.5 and post-natal day 1. Flow cytometry was used to quantify and purify the EGFP positive population in the E15.5 pancreas. The purified (96%) EGFP-expressing cells, which represent 20% of the total cell population, were shown by RT/PCR to express exocrine cell markers (amylase and P48) and endocrine cell markers (insulin 1, insulin 2, and Ngn3). They also expressed, at a lower level, PDX-1, Isl-1, and the islet hormones pancreatic polypeptide, glucagon and somatostatin as well as GLUT2, the stem cell marker ABCG2 and PECAM, a marker of endothelial cells. It was further shown by immunocytochemistry of the E15.5 pancreas that EGFP colocalised in separate subpopulations of cells that expressed nestin, insulin and amylase. These results support the conclusion that nestin expressing cells can give rise to both endocrine and exocrine cells. The ability to purify these putative progenitor cells may provide further insights into their properties and function.
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Affiliation(s)
- Andreia S Bernardo
- School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK
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48
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Abstract
PURPOSE OF REVIEW Neoplasms of the endocrine pancreas, commonly referenced as pancreatic islet cell tumors, are rare, often well differentiated endocrine neoplasms, whose biology remains poorly characterized. This article reviews the current clinical management of pancreatic islet cell tumors and describes the molecular events that have been studied to guide future therapies of these peculiar neoplasms. RECENT FINDINGS While some islet cell tumors arise in association with the MEN-1 syndrome, the majority of these neoplasms are sporadic lesions whose underlying genetic and molecular events remain largely unknown. Recent work has identified changes in gene expression occurring in metastatic and non-metastatic islet cell tumors, which appear to correlate with the occurrence of lymph node and liver metastases. Epigenetic alterations of select tumor suppressor genes may influence patient survival, and the presence of gene promoter methylation may be used as a prognostic marker system. In addition, multiple molecular alterations, including changes in expression of cellular proteins with migratory, cell cycle or angiogenic functions, have been demonstrated to influence islet cell tumor growth, invasion and metastatic spread. SUMMARY Understanding the molecular events underlying the biology of pancreatic islet cell tumors will aid the development of accurate prognostic markers and will guide improved therapeutic modalities in the future.
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Affiliation(s)
- Michael G House
- Department of Surgery, The Johns Hopkins Hospital, Baltimore, Maryland 21231, USA
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Duval D, Trouillas M, Thibault C, Dembelé D, Diemunsch F, Reinhardt B, Mertz AL, Dierich A, Boeuf H. Apoptosis and differentiation commitment: novel insights revealed by gene profiling studies in mouse embryonic stem cells. Cell Death Differ 2006; 13:564-75. [PMID: 16311515 DOI: 10.1038/sj.cdd.4401789] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF starvation leads to apoptosis of some of the ES-derived differentiated cells, together with p38alpha mitogen-activated protein kinase (MAPK) activation. Apoptosis, but not morphological cell differentiation, is blocked by a p38 inhibitor, PD169316. To further understand the mechanism of action of this compound, we have identified its specific targets by microarray studies. We report on the global expression profiles of genes expressed at 3 days upon LIF withdrawal (d3) compared to pluripotent cells and of genes whose expression is modulated at d3 under anti-apoptotic conditions. We showed that at d3 without LIF cells express, earlier than anticipated, specialized cell markers and that when the apoptotic process was impaired, expression of differentiation markers was altered. In addition, functional tests revealed properties of anti-apoptotic proteins not to alter cell pluripotency and a novel role for metallothionein 1 gene, which prevents apoptosis of early differentiated cells.
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Affiliation(s)
- D Duval
- UMR5096-CNRS/UP/IRD, Perpignan, France
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Baharvand H, Jafary H, Massumi M, Ashtiani SK. Generation of insulin-secreting cells from human embryonic stem cells. Dev Growth Differ 2006; 48:323-32. [PMID: 16759282 DOI: 10.1111/j.1440-169x.2006.00867.x] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
A growth factor-mediated selection method was used to obtained insulin-secreting cells from human embryonic stem cells (hESC; Royan H1). Our resultant cells were positive for dithizone, a zinc-chelating agent known to selectively stain pancreatic beta cells and immunoreactive for antibodies against insulin, glucagon, and C-peptide. Semi-quantitative reverse transcription-polymerase chain reaction detected expression of proinsulin, insulin and other pancreatic beta-cell-related genes, such as Nkx6.1, Is11, Glut2, Pax4, and prohormone convertase2 (PC2). Moreover, glucagon, somatostatin, K(ATP)-channel genes KIR6.2 and SUR1, islet amyloid polypeptide (IAPP), PC1/3, and glucokinase (GCK) were expressed in the differentiating hESC in a developmental stage-dependent manner. Also, the addition of glucose to the culture medium triggered insulin release from differentiated cells, but transmission electron microscopy of the differentiated cells did not show typical beta-cell granules, even though secretary granules were detected. The results showed that hESC have the ability to transcribe and process insulin, but further improvements of the current method are required to generate a sufficient source of true beta cells for the treatment of diabetes mellitus.
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Affiliation(s)
- Hossein Baharvand
- Department of Stem Cells, Royan Institute, PO Box 19395-4644, Tehran, Iran.
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