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Heaton ES, Jin S. Importance of multiple endocrine cell types in islet organoids for type 1 diabetes treatment. Transl Res 2022; 250:68-83. [PMID: 35772687 PMCID: PMC11554285 DOI: 10.1016/j.trsl.2022.06.014] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/17/2022] [Revised: 06/08/2022] [Accepted: 06/21/2022] [Indexed: 11/21/2022]
Abstract
Almost 50 years ago, scientists developed the bi-hormonal abnormality hypothesis, stating that diabetes is not caused merely by the impaired insulin signaling. Instead, the presence of inappropriate level of glucagon is a prerequisite for the development of type 1 diabetes (T1D). It is widely understood that the hormones insulin and glucagon, secreted by healthy β and α cells respectively, operate in a negative feedback loop to maintain the body's blood sugar levels. Despite this fact, traditional T1D treatments rely solely on exogenous insulin injections. Furthermore, research on cell-based therapies and stem-cell derived tissues tends to focus on the replacement of β cells alone. In vivo, the pancreas is made up of 4 major endocrine cell types, that is, insulin-producing β cells, glucagon-producing α cells, somatostatin-producing δ cells, and pancreatic polypeptide-producing γ cells. These distinct cell types are involved synergistically in regulating islet functions. Therefore, it is necessary to produce a pancreatic islet organoid in vitro consisting of all these cell types that adequately replaces the function of the native islets. In this review, we describe the unique function of each pancreatic endocrine cell type and their interactions contributing to the maintenance of normoglycemia. Furthermore, we detail current sources of whole islets and techniques for their long-term expansion and culture. In addition, we highlight a vast potential of the pancreatic islet organoids for transplantation and diabetes research along with updated new approaches for successful transplantation using stem cell-derived islet organoids.
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Affiliation(s)
- Emma S Heaton
- Department of Biomedical Engineering, Thomas J. Watson School of Engineering and Applied Sciences, State University of New York at Binghamton, Binghamton, New York
| | - Sha Jin
- Department of Biomedical Engineering, Thomas J. Watson School of Engineering and Applied Sciences, State University of New York at Binghamton, Binghamton, New York; Center of Biomanufacturing for Regenerative Medicine, State University of New York at Binghamton, Binghamton, New York.
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2
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Ding L, Roeck K, Zhang C, Zidek B, Rodman E, Hernandez-Barco Y, Zhang JS, Bamlet W, Oberg A, Zhang L, Bardeesy N, Li H, Billadeau D. Nuclear GSK-3β and Oncogenic KRas Lead to the Retention of Pancreatic Ductal Progenitor Cells Phenotypically Similar to Those Seen in IPMN. Front Cell Dev Biol 2022; 10:853003. [PMID: 35646902 PMCID: PMC9136019 DOI: 10.3389/fcell.2022.853003] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Accepted: 04/11/2022] [Indexed: 11/30/2022] Open
Abstract
Glycogen synthase kinase-3β (GSK-3β) is a downstream target of oncogenic KRas and can accumulate in the nucleus in pancreatic ductal adenocarcinoma (PDA). To determine the interplay between oncogenic KRas and nuclear GSK-3β in PDA development, we generated Lox-STOP-Lox (LSL) nuclear-targeted GSK-3β animals and crossed them with LSL-KRasG12D mice under the control of the Pdx1-cre transgene—referred to as KNGC. Interestingly, 4-week-old KNGC animals show a profound loss of acinar cells, the expansion of ductal cells, and the rapid development of cystic-like lesions reminiscent of intraductal papillary mucinous neoplasm (IPMN). RNA-sequencing identified the expression of several ductal cell lineage genes including AQP5. Significantly, the Aqp5+ ductal cell pool was proliferative, phenotypically distinct from quiescent pancreatic ductal cells, and deletion of AQP5 limited expansion of the ductal pool. Aqp5 is also highly expressed in human IPMN along with GSK-3β highlighting the putative role of Aqp5+ ductal cells in human preneoplastic lesion development. Altogether, these data identify nGSK-3β and KRasG12D as an important signaling node promoting the retention of pancreatic ductal progenitor cells, which could be used to further characterize pancreatic ductal development as well as lineage biomarkers related to IPMN and PDA.
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Affiliation(s)
- Li Ding
- Division of Oncology Research, College of Medicine, Mayo Clinic, Rochester, MN, United States
- *Correspondence: Li Ding, ; Daniel Billadeau,
| | - Kaely Roeck
- Division of Oncology Research, College of Medicine, Mayo Clinic, Rochester, MN, United States
| | - Cheng Zhang
- Department of Molecular and Experimental Therapeutics, College of Medicine, Mayo Clinic, Rochester, MN, United States
| | - Brooke Zidek
- Division of Oncology Research, College of Medicine, Mayo Clinic, Rochester, MN, United States
| | - Esther Rodman
- Division of Oncology Research, College of Medicine, Mayo Clinic, Rochester, MN, United States
| | | | - Jin-San Zhang
- Division of Oncology Research, College of Medicine, Mayo Clinic, Rochester, MN, United States
- Center for Precision Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
| | - William Bamlet
- Department of Health Sciences Research, College of Medicine, Mayo Clinic, Rochester, MN, United States
| | - Ann Oberg
- Department of Health Sciences Research, College of Medicine, Mayo Clinic, Rochester, MN, United States
| | - Lizhi Zhang
- Department of Laboratory Medicine and Pathology, College of Medicine, Mayo Clinic, Rochester, MN, United States
| | - Nabeel Bardeesy
- Center for Cancer Research, Harvard Medical School, Boston, MA, United States
| | - Hu Li
- Department of Molecular and Experimental Therapeutics, College of Medicine, Mayo Clinic, Rochester, MN, United States
| | - Daniel Billadeau
- Division of Oncology Research, College of Medicine, Mayo Clinic, Rochester, MN, United States
- *Correspondence: Li Ding, ; Daniel Billadeau,
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3
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Espes D, Lau J, Carlsson PO. MECHANISMS IN ENDOCRINOLOGY: Towards the clinical translation of stem cell therapy for type 1 diabetes. Eur J Endocrinol 2017; 177:R159-R168. [PMID: 28487297 DOI: 10.1530/eje-17-0080] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2017] [Revised: 04/24/2017] [Accepted: 05/08/2017] [Indexed: 01/21/2023]
Abstract
Insulin-producing cells derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) have for long been a promising, but elusive treatment far from clinical translation into type 1 diabetes therapy. However, the field is now on the verge of moving such insulin-producing cells into clinical trials. Although stem cell therapies provide great opportunities, there are also potential risks such as teratoma formation associated with the treatment. Many considerations are needed on how to proceed with clinical translation, including whether to use hESCs or iPSCs, and whether encapsulation of tissue will be needed. This review aims to give an overview of the current knowledge of stem cell therapy outcomes in animal models of type 1 diabetes and a proposed road map towards the clinical setting with special focus on the potential risks and hurdles which needs to be considered. From a clinical point of view, transplantation of insulin-producing cells derived from stem cells must be performed without immune suppression in order to be an attractive treatment option. Although costly and highly labour intensive, patient-derived iPSCs would be the only solution, if not clinically successful encapsulation or tolerance induction protocols are introduced.
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Affiliation(s)
- Daniel Espes
- Departments of Medical Cell Biology
- Medical Sciences, Uppsala University, Uppsala, Sweden
| | - Joey Lau
- Departments of Medical Cell Biology
| | - Per-Ola Carlsson
- Departments of Medical Cell Biology
- Medical Sciences, Uppsala University, Uppsala, Sweden
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4
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Tsuchiya M, Tsuchiya K, Ohgawara H. Molecular Cloning of the Porcine Insulin cDNA Using a Monolayer Culture of Pancreatic Endocrine Cells. Cell Transplant 2017. [DOI: 10.3727/000000001783986611] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Porcine pancreatic endocrine cells are an attractive candidate for islet cell transplantation in view of the immunological properties and structural similarities of porcine insulin to human insulin. We recently established a method of isolation and a primary monolayer culture of porcine pancreatic endocrine cells. In this study, cloning of the porcine insulin cDNA was performed to clarify the genetic background of the purified isolated cells. A homology-based PCR cloning method was employed to determine the sequence using mRNA extracted from the monolayer-forming cells, and the candidate products were then determined by a homology search on the human insulin cDNA. According to the newly identified sequence, rapid amplification of cDNA ends was applied to the 5′ and 3′ ends, and the entire cDNA sequence was determined. Gene and protein expression was confirmed by Northern blotting, immunohistochemistry, and enzyme assay. To examine the transcriptional level, the cultured cells were incubated in a 20 mM D-glucose medium in the presence or absence of 5 μM forskolin. The porcine insulin cDNA exhibited a high homology to the human cDNA and showed 85% matching with the human amino acid sequence. D-Glucose at 20 mM stimulated the insulin secretion and mRNA expression, and further addition of 5 μM forskolin with the glucose was applied as the strongest stimulus in this culture system.
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Affiliation(s)
- Mariko Tsuchiya
- Institute of Geriatrics, Aoyama Hospital, Tokyo Women's Medical University, Tokyo, Japan
| | - Ken Tsuchiya
- Department of Medicine IV, Tokyo Women's Medical University, Tokyo, Japan
| | - Hisako Ohgawara
- Medical Research Institute, Tokyo Women's Medical University, Tokyo, Japan
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Suzuki A, Oyama K, Fukao K, Nakauchi H, Taniguchi H. Establishment of Clonal Colony-Forming Assay System for Pancreatic Stem/Progenitor Cells. Cell Transplant 2017. [DOI: 10.3727/000000002783985765] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Pluripotent stem cells found in a number of organs are usually in small cell populations. However, under adaptive stimulation, they enter the stage of growth and differentiation to compensate for the loss of differentiated cells. To analyze stem cell potential precisely, the exclusion of other differentiated cells and a clonal assay system are strongly required. In this study, we established a colony-forming assay system for pancreatic stem/progenitor cells in vitro. In this culture condition, they received signals for growth and differentiation, and formed clonal colonies including pancreatic endocrine-lineage cells, such as α and β cells. By combining this culture system with flow cytometric cell sorting, pancreatic stem/progenitor cells will be enriched, and their potential can be analyzed precisely in single cell-based experiments.
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Affiliation(s)
- Atsushi Suzuki
- Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
| | - Kazunobu Oyama
- Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
| | - Katashi Fukao
- Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
| | - Hiromitsu Nakauchi
- Department of Immunology, Institute of Basic Medical Sciences, University of Tsukuba, and CREST (Japan Science and Technology Corporation), 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
| | - Hideki Taniguchi
- Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
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6
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Abstract
Diabetes mellitus is caused by absolute (type 1) or relative (type 2) deficiency of insulin-secreting islet β cells. An ideal treatment of diabetes would, therefore, be to replace the lost or deficient β cells, by transplantation of donated islets or differentiated endocrine cells or by regeneration of endogenous islet cells. Due to their ability of unlimited proliferation and differentiation into all functional lineages in our body, including β cells, embryonic stem cells and induced pluripotent stem cells are ideally placed as cell sources for a diabetic transplantation therapy. Unfortunately, the inability to generate functional differentiated islet cells from pluripotent stem cells and the poor availability of donor islets have severely restricted the broad clinical use of the replacement therapy. Therefore, endogenous sources that can be directed to becoming insulin-secreting cells are actively sought after. In particular, any cell types in the developing or adult pancreas that may act as pancreatic stem cells (PSC) would provide an alternative renewable source for endogenous regeneration. In this review, we will summarize the latest progress and knowledge of such PSC, and discuss ways that facilitate the future development of this often controversial, but crucial research.
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Affiliation(s)
- Fang-Xu Jiang
- 1 Islet Cell Development Program, Harry Perkins Institute of Medical Research, and Centre for Medical Research, The University of Western Australia , Perth, Australia
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Minami K, Seino S. Current status of regeneration of pancreatic β-cells. J Diabetes Investig 2014; 4:131-41. [PMID: 24843642 PMCID: PMC4019265 DOI: 10.1111/jdi.12062] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/09/2013] [Accepted: 01/21/2013] [Indexed: 12/13/2022] Open
Abstract
Newly generated insulin‐secreting cells for use in cell therapy for insulin‐deficient diabetes mellitus require properties similar to those of native pancreatic β‐cells. Pancreatic β‐cells are highly specialized cells that produce a large amount of insulin, and secrete insulin in a regulated manner in response to glucose and other stimuli. It is not yet explained how the β‐cells acquire this complex function during normal differentiation. So far, in vitro generation of insulin‐secreting cells from embryonic stem cells, induced‐pluripotent stem cells and adult stem/progenitor‐like cells has been reported. However, most of these cells are functionally immature and show poor glucose‐responsive insulin secretion compared to that of native pancreatic β‐cells (or islets). Strategies to generate functional β‐cells or a whole organ in vivo have also recently been proposed. Establishing a protocol to generate fully functional insulin‐secreting cells that closely resemble native β‐cells is a critical matter in regenerative medicine for diabetes. Understanding the physiological processes of differentiation, proliferation and regeneration of pancreatic β‐cells might open the path to cell therapy to cure patients with absolute insulin deficiency.
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Affiliation(s)
- Kohtaro Minami
- Division of Cellular and Molecular Medicine Department of Physiology and Cell Biology Kobe University Graduate School of Medicine Kobe Japan
| | - Susumu Seino
- Division of Cellular and Molecular Medicine Department of Physiology and Cell Biology Kobe University Graduate School of Medicine Kobe Japan ; Division of Diabetes and Endocrinology Department of Internal Medicine Kobe University Graduate School of Medicine Kobe Japan ; Core Research for Evolutional Science and Technology (CREST) Japan Science and Technology Corp. Kawaguchi Saitama Japan
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8
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Chmielowiec J, Borowiak M. In vitro differentiation and expansion of human pluripotent stem cell-derived pancreatic progenitors. Rev Diabet Stud 2014; 11:19-34. [PMID: 25148365 DOI: 10.1900/rds.2014.11.19] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Recent progress in understanding stem cell biology has been remarkable, especially in deciphering signals that support differentiation towards tissue-specific lineages. This achievement positions us firmly at the beginning of an era of patient-specific regenerative medicine and human disease modeling. It will be necessary to equip the progress in this era with a reliable source of self-renewing progenitor cells that differentiate into functional target cells. The generation of pancreatic progenitors that mature in vivo into functional beta-cells has raised the hope for new therapeutic options in diabetes, but key challenges still remain including the production of sufficient numbers of cells for research and transplantation. Recent approaches to this problem have shown that the presence of organ- and stage-specific mesenchyme improves the generation of progenitors, from endoderm to endocrine cells. Alternatively, utilization of three-dimensional culture may improve the efficiency and yield of directed differentiation. Here, we review the current knowledge of pancreatic directed differentiation and ex vivo expansion of pancreatic progenitors, including recent advances in differentiation strategies for the generation of pancreatic progenitors, and we discuss persistent challenges which will need to be overcome before personalized cell-based therapy becomes a practical strategy.
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Affiliation(s)
- Jolanta Chmielowiec
- Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
| | - Malgorzata Borowiak
- Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
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9
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Abstract
Type 1 and some forms of type 2 diabetes mellitus are caused by deficiency of insulin-secretory islet β cells. An ideal treatment for these diseases would therefore be to replace β cells, either by transplanting donated islets or via endogenous regeneration (and controlling the autoimmunity in type 1 diabetes). Unfortunately, the poor availability of donor islets has severely restricted the broad clinical use of islet transplantation. The ability to differentiate embryonic stem cells into insulin-expressing cells initially showed great promise, but the generation of functional β cells has proven extremely difficult and far slower than originally hoped. Pancreatic stem cells (PSC) or transdifferentiation of other cell types in the pancreas may hence provide an alternative renewable source of surrogate β cells. However, the existence of PSC has been hotly debated for many years. In this review, we will discuss the latest development and future perspectives of PSC research, giving readers an overview of this controversial but important area.
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Affiliation(s)
- Fang-Xu Jiang
- Centre for Diabetes Research, Western Australian Institute for Medical Research, The University of Western Australia, 50 Murray St (Rear), Perth, WA 6000, Australia.
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10
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Lim SK, Llaguno SRA, McKay RM, Parada LF. Glioblastoma multiforme: a perspective on recent findings in human cancer and mouse models. BMB Rep 2011; 44:158-64. [PMID: 21429292 DOI: 10.5483/bmbrep.2011.44.3.158] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Gliomas are the most frequently occurring primary malignancies in the central nervous system, and glioblastoma multiforme (GBM) is the most common and most aggressive of these tumors. Despite vigorous basic and clinical studies over past decades, the median survival of patients with this disease remains at about one year. Recent studies have suggested that GBMs contain a subpopulation of tumor cells that displays stem cell characteristics and could therefore be responsible for in vivo tumor growth. We will summarize the major oncogenic pathways abnormally regulated in gliomas, and review the recent findings from mouse models that our laboratory as well as others have developed for the study of GBM. The concept of cancer stem cells in GBM and their potential therapeutic importance will also be discussed.
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Affiliation(s)
- Sang Kyun Lim
- Department of Developmental Biology, The University of Texas Southwestern Medical Center at Dallas, 75390, USA
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11
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Samuelson L, Wright N, Gerber DA. Endodermal progenitor cells isolated from mouse pancreas. ACTA ACUST UNITED AC 2011. [DOI: 10.4236/scd.2011.13005] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Stevenson KS, McGlynn L, Hodge M, McLinden H, George WD, Davies RW, Shiels PG. Isolation, characterization, and differentiation of thy1.1-sorted pancreatic adult progenitor cell populations. Stem Cells Dev 2010; 18:1389-98. [PMID: 19326970 DOI: 10.1089/scd.2008.0301] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
We have isolated a novel progenitor cell population from adult rat pancreatic ducts, termed pancreatic-derived progenitor cells (PDPCs). Here, we report the in vitro culture, selection, and characterization of Thy1.1-positive and Thy1.1-negative PDPC subpopulations. These cells exhibit bipotentiality for differentiation into both pancreatic and hepatic cell types. Significantly, they express Pdx-1. Using a serum-free FGF-4-containing differentiation protocol, we have observed a time course of both morphological and gene expression changes indicative of hepatic lineage differentiation for the Thy1.1-positive subpopulation. These cells express albumin and store glycogen, typical features of mature hepatocytes. The Thy1.1-positive subpopulation could also readily be induced to differentiate into a pancreatic lineage with characteristic morphological changes resulting in three-dimensional islet-like structures and the transcriptional expression of insulin and glucagon in addition to Pdx-1. No morphological evidence of islet-like clusters was observed using the Thy1.1-negative population. However, Thy1.1-negative cells grown in pancreatic differentiation medium did show insulin gene transcription. Glucagon was not expressed in the undifferentiated Thy1.1-negative cells, nor was it induced in vitro after differentiation. The detection of Pdx-1 transcriptional expression in both populations indicates their potential as a novel source of non-beta-cell-derived insulin.
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Affiliation(s)
- Karen S Stevenson
- Division of Cancer Sciences and Molecular Pathology, Department of Surgery, University of Glasgow, Glasgow G31 2ER, Scotland, United Kingdom
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13
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Boretti MI, Gooch KJ. Effect of extracellular matrix and 3D morphogenesis on islet hormone gene expression by Ngn3-infected mouse pancreatic ductal epithelial cells. Tissue Eng Part A 2009; 14:1927-37. [PMID: 18694323 DOI: 10.1089/ten.tea.2007.0338] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
We verified the proendocrine effects of Matrigel overlay in an adult mouse pancreatic ductal epithelial cells (PDEC) model and then decomposed the environment to delineate the specific factors responsible for this effect. Following overlay with Matrigel, supplementation of Matrigel to the culture medium, or suspension within Matrigel, neurogenin3-infected mouse PDEC underwent dramatic morphogenesis, transitioning from a two-dimensional monolayer to three-dimensional (3D) cysts. Along with these morphogenic changes, the cells displayed up to approximately sixfold increase in mRNA for the islet hormones somatostatin and ghrelin. Following overlay with collagen or suspension within collagen, PDEC also displayed similar morphogenic changes, but a much smaller increase in expression was observed (1.5- to 3-fold), suggesting that while 3D morphogenesis is capable of independently enhancing islet differentiation, biochemical factors present within Matrigel also have proendocrine effects. Following suspension within laminin gels, PDEC formed 3D cysts and also displayed an increase in islet hormone expression, similar to those cultured within Matrigel. However, medium supplemented with laminin failed to promote 3D morphogenesis of PDEC or enhance islet hormone expression, suggesting that while laminin is capable of enhancing islet hormone expression, 3D morphogenesis is required for this effect. Cell clustering appeared to maximize differentiation, as PDEC cultured on Matrigel formed aggregates and stimulated the highest expression of somatostatin and ghrelin (up to approximately 200-fold).
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Affiliation(s)
- Michael Ian Boretti
- Department of Bioengineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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14
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Xiao M, An L, Yang X, Ge X, Qiao H, Zhao T, Ma X, Fan J, Zhu M, Dou Z. Establishing a human pancreatic stem cell line and transplanting induced pancreatic islets to reverse experimental diabetes in rats. ACTA ACUST UNITED AC 2008; 51:779-88. [DOI: 10.1007/s11427-008-0109-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2007] [Accepted: 07/10/2008] [Indexed: 11/29/2022]
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15
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Schmied B, Ulrich A, Matsuzaki H, Batra S, Pour P, Schmied B, Ulrich A, Matsuzaki H, Ding X, Adrian T, Ricordi C, Moyer M. Maintenance of human islets in long term culture. Differentiation 2008. [DOI: 10.1111/j.1432-0436.2000.660403.x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
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Limbert C, Päth G, Jakob F, Seufert J. Beta-cell replacement and regeneration: Strategies of cell-based therapy for type 1 diabetes mellitus. Diabetes Res Clin Pract 2008; 79:389-99. [PMID: 17854943 DOI: 10.1016/j.diabres.2007.06.016] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/24/2007] [Accepted: 06/20/2007] [Indexed: 01/09/2023]
Abstract
Pancreatic islet transplantation has demonstrated that long-term insulin independence may be achieved in patients suffering from diabetes mellitus type 1. However, because of limited availability of islet tissue, new sources of insulin producing cells that are responsive to glucose are required. Development of pancreatic beta-cell lines from rodent or human origin has progressed slowly in recent years. Current experiments for ex vivo expansion of beta cells and in vitro differentiation of embryonic and adult stem cells into insulin producing beta-cell phenotypes led to promising results. Nevertheless, the cells generated to date lack important characteristics of mature beta cells and generally display reduced insulin secretion and loss of proliferative capacity. Therefore, much better understanding of the mechanisms that regulate expansion and differentiation of stem/progenitor cells is necessary. Here, we review recent advances in the identification of potential cellular sources, and the development of strategies to regenerate or fabricate insulin producing and glucose sensing cells that might enable future cell-based therapies of diabetes mellitus type 1.
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Affiliation(s)
- C Limbert
- Division of Endocrinology and Diabetology, Department of Internal Medicine II, University Hospital Freiburg, Freiburg, Germany
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17
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Abstract
The promising results obtained using the "Edmonton protocol" for human islet transplantation has resulted in increased interest and growth of various clinical and basic science programs worldwide. Despite these encouraging results two major drawbacks remain: first, the immunosuppressive regimen necessary to prevent the rejection of this allotransplant dramatically affects the lifestyle of the treated patients precluding its implementation in younger diabetic individuals. Second, there continues to be an inadequate amount of islet tissue available to fulfill the needs of an increasing population of diabetic patients possibly interested in receiving this type of treatment. Besides the limited number of cadaveric organ donors, the current procedure used to isolate islets from their pancreata activates metabolic processes that promote the loss of beta cells in the islets. Thus, it becomes necessary to use more than one donor for a single recipient. To fulfill the continuously growing need for more transplantable islets, an immediately available, unlimited source of islets may be found in animals, which are able to produce a type of insulin that is very similar to the human one, and carry islets in quantities that may satisfy the metabolic requirements of diabetic patients: the pigs.
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Affiliation(s)
- M Trucco
- Istituto Mediterraneo per I Trapianti e Terapie ad Alta Specializzazione, Palermo, Italy.
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18
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Lin HT, Chiou SH, Kao CL, Shyr YM, Hsu CJ, Tarng YW, Ho LLT, Kwok CF, Ku HH. Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting. World J Gastroenterol 2006; 12:4529-35. [PMID: 16874866 PMCID: PMC4125641 DOI: 10.3748/wjg.v12.i28.4529] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To isolate putative pancreatic stem cells (PSCs) from human adult tissues of pancreas duct using serum-free, conditioned medium. The characterization of surface phenotype of these PSCs was analyzed by flow cytometry. The potential for pancreatic lineage and the capability of β-cell differentiation in these PSCs were evaluated as well.
METHODS: By using serum-free medium supplemented with essential growth factors, we attempted to isolate the putative PSCs which has been reported to express nestin and pdx-1. The Matrigel™ was employed to evaluate the differential capacity of isolated cells. Dithizone staining, insulin content/secretion measurement, and immunohistochemistry staining were used to monitor the differentiation. Fluorescence activated cell sorting (FACS) was used to detect the phenotypic markers of putative PSCs.
RESULTS: A monolayer of spindle-like cells was cultivated. The putative PSCs expressed pdx-1 and nestin. They were also able to differentiate into insulin-, glucagon-, and somatostatin-positive cells. The spectrum of phenotypic markers in PSCs was investigated; a similarity was revealed when using human bone marrow-derived stem cells as the comparative experiment, such as CD29, CD44, CD49, CD50, CD51, CD62E, PDGFR-α, CD73 (SH2), CD81, CD105(SH3).
CONCLUSION: In this study, we successfully isolated PSCs from adult human pancreatic duct by using serum-free medium. These PSCs not only expressed nestin and pdx-1 but also exhibited markers attributable to mesenchymal stem cells. Although work is needed to elucidate the role of these cells, the application of these PSCs might be therapeutic strategies for diabetes mellitus.
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Affiliation(s)
- Han-Tso Lin
- Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, 11217, Taiwan, China
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Boretti MI, Gooch KJ. Induced Cell Clustering Enhances IsletβCell Formation from Human Cultures Enriched for Pancreatic Ductal Epithelial Cells. ACTA ACUST UNITED AC 2006; 12:939-48. [PMID: 16674305 DOI: 10.1089/ten.2006.12.939] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
A better understanding of the culture conditions that stimulate in vitro beta-cell differentiation from islet precursors would be useful for optimizing the production of tissue-engineered islets. In this study, high- and low-adherent substrates and high- and low-serum media were used to control the clustering of human pancreatic ductal epithelial cells and to determine its effect on their transdifferentiation to beta cells. While the initial epithelial cell cultures were devoid of any beta cells as assessed by dithizone staining, dithizone+ cells were generated during the next 3 weeks under all culture conditions. Although the rate of transdifferentiation was low, a approximately 4-fold greater number and percentage of dithizone+ cells were generated following 23-24 days of culture in the least adherent conditions (low-serum medium, low-adherent substrate), which stimulated cell clustering to the highest degree. Insulin immunohistochemistry data correlated well with the dithizone data (r(2) = 0.99), evidence that dithizone is a reliable measure of insulin+ cells. The preferential distribution of the dithizone+ cells to regions of cell aggregation and the increased efficiency of transdifferentiation in conditions that promote cell clustering suggest that cell-cell interactions and/or cell shape changes are important to the transdifferentiation of adult pancreatic ductal epithelial cells to beta cells in vitro.
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Affiliation(s)
- Michael I Boretti
- Department of Bioengineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, 19104-6392, USA
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20
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Kim BM, Kim SY, Lee S, Shin YJ, Min BH, Bendayan M, Park IS. Clusterin induces differentiation of pancreatic duct cells into insulin-secreting cells. Diabetologia 2006; 49:311-20. [PMID: 16411126 DOI: 10.1007/s00125-005-0106-2] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/30/2005] [Accepted: 10/20/2005] [Indexed: 11/27/2022]
Abstract
AIMS/HYPOTHESIS We recently reported that expression of the gene encoding clusterin (Clu) is upregulated in the regenerating pancreas, particularly in tissues undergoing differentiation. This led us to propose that clusterin participates in the cytodifferentiation of pancreatic tissue, particularly the endocrine islet cells. The aim of this study was to investigate whether clusterin induces the differentiation of duct-lining cells into insulin-secreting cells. METHODS We isolated ductal tissue from rat pancreas and cultured it to develop epithelial cell explants for transfection of the Clu cDNA as well as for treatment of clusterin protein. RESULTS The number of newly differentiated insulin cells increased 6.9-fold upon Clu overexpression compared with controls. Ins1 mRNA and peptide levels were also increased. Furthermore, glucose-stimulated insulin secretion was observed in the differentiated insulin cells. These cells were immunoreactive for insulin and C-peptide, but negative for other islet hormones and for cytokeratin-20, which indicates a fully differentiated state. Insulin cell differentiation was also increased in a dose-dependent manner by treating duct cells in culture with clusterin, indicating a growth-factor-like action of clusterin in insulin cell differentiation. CONCLUSIONS/INTERPRETATION These results suggest that clusterin can be considered as a potential morphogenic factor that promotes differentiation of pancreatic beta cells.
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Affiliation(s)
- B M Kim
- Department of Anatomy, College of Medicine, Inha University Incheon, Choong-Gu, Shinheung-Dong, Incheon 400-103, Korea
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Abstract
The most intensively studied autoimmune disorder, type 1 diabetes mellitus (DM1), has attracted perhaps the greatest interest for gene-based therapeutic and prophylactic interventions. The final clinical manifestation of this immunologically and genetically complex disease, the absence of insulin, is the major starting point for almost all the gene therapy modalities attempted to date. Insulin replacement by transplantation of islets of Langerhans or surrogate beta cells is the obvious choice, but the allogeneic nature of the transplants activates potent antidonor immunoreactivity necessitating gene and cell-based immunosuppressive strategies as an alternative to the toxic pharmacologic immunosuppressives indicated for classic solid organ transplants. Accumulating knowledge of the cellular mechanisms involved in onset, however, have yielded promising tolerance induction prophylactic approaches using genes and cells. Despite the early successes in a number of animal models, the true test of efficacy in humans remains to be demonstrated.
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Affiliation(s)
- Nick Giannoukakis
- Diabetes Institute, Pediatric Research Section, Children's Hospital of Pittsburgh and University of Pittsburgh, Rangos Research Center, Pittsburgh, PA 15213, USA
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22
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Kang EM, Zickler PP, Burns S, Langemeijer SM, Brenner S, Phang OA, Patterson N, Harlan D, Tisdale JF. Hematopoietic stem cell transplantation prevents diabetes in NOD mice but does not contribute to significant islet cell regeneration once disease is established. Exp Hematol 2005; 33:699-705. [PMID: 15911094 DOI: 10.1016/j.exphem.2005.03.008] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2004] [Revised: 02/07/2005] [Accepted: 03/11/2005] [Indexed: 11/15/2022]
Abstract
The treatment of type I diabetes by islet cell transplantation, while promising, remains restricted due to the incomplete efficacy and toxicity associated with current immunosuppression, and by limited organ availability. Given reports suggesting bone marrow derived stem cell plasticity, we sought to determine whether such cells could give rise to pancreatic islet cells in vivo. In the context of autoimmune diabetes, we transplanted unfractionated bone marrow from beta-gal trangenic donor mice into NOD mice prior to, at, and two weeks beyond the onset of disease. Successful bone marrow engraftment before diabetes onset prevented disease in all mice and for 1 year after transplant. However, despite obtaining full hematopoietic engraftment in over 50 transplanted mice, only one mouse became insulin independent, and no beta-Gal positive islets were detected in any of the mice. To test whether tolerance to islets was achieved, we injected islets obtained from the same allogeneic donor strain as the hematopoietic cells into 4 transplant recipients, and 2 had a reversion of their diabetes. Thus allogeneic bone marrow transplantation prevents autoimmune diabetes and tolerizes the recipient to donor islet grants, even in diabetic animals, yet the capacity of bone marrow derived cells to differentiate into functional islet cells, at least without additional manipulation, is limited in our model.
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Affiliation(s)
- Elizabeth M Kang
- LHD/NIAID, National Institutes of Health/DHHS, Bethesda, MD 20895, USA
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Peck AB, Yin L, Ramiya V. Animal models to study adult stem cell-derived, in vitro-generated islet implantation. ILAR J 2005; 45:259-67. [PMID: 15229373 DOI: 10.1093/ilar.45.3.259] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Type 1 diabetes (T1D) is an autoimmune disease characterized by hyperglycemia following the destruction of the insulin-producing beta cells of the pancreatic islets of Langerhans by the body's own immune system. Although routine insulin injections can provide diabetic patients with their daily insulin requirements, this treatment is not always effective in maintaining normal glucose levels. A true "cure" is considered possible only through replacement of the beta cell mass, by pancreas transplantation, islet implantation, or implantation of nonendocrine cells modified to secrete insulin. With the recent success of islet implantation to reverse T1D, this procedure has become a welcome therapy for T1D patients. Unfortunately, this procedure is hampered by the limited number of transplantation quality pancreata available for the harvesting of islets. This shortage has sparked great interest in finding a replacement for organ donation, primarily the possible use of stem cell-derived islets starting with stem cells, or alternatively the harvesting of nonhuman islets. This review focuses on progress with growing islets in the laboratory from stem cells and a comparison between this developing technology and the current use of islets harvested from nonhuman sources.
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Affiliation(s)
- Ammon B Peck
- Department of Pathology, Immunology and Laboratory Medicine, University of Florida, College of Medicine, Gainesville, FL, USA
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24
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Abstract
Type 1 diabetes is one of the more costly chronic diseases of children and adolescents throughout North America and Europe, exhibiting an average estimated prevalence rate of nearly 0.2%. It occurs in genetically predisposed individuals when the immune system attacks and destroys specifically the insulin-producing beta cells of the pancreatic islets of Langerhans. While routine insulin therapy can provide diabetic patients with their daily insulin requirements, non-compliance and undetected hyperglycemic excursions often lead to subsequent long-term microvascular and macrovascular complications. The only real cure for type 1 diabetes is replacement of the beta cell mass, currently being accomplished through ecto-pancreatic transplantation and islet implantation. Both of these procedures suffer from a chronic shortage of available donor tissue in comparison to the number of potential recipients. To circumvent this need, three alternative approaches are being intensively investigated: (1) the production of surrogate cells by genetically modifying non-endocrine cells to secrete insulin in response to glucose challenge; (2) the trans-differentiation of non-endocrine stem/progenitor cells or mature cells to glucose-responsive adult tissue; and (3) the regulated differentiation of islet stem/progenitor cells to produce large numbers of mature, functional islets. In recent years, each of these approaches has made impressive advances, leading to the most important question, 'how soon will this new science be available to the patient?' In the present review, we discuss some of the recent advances, focusing primarily on the differentiation of islet stem cells to functional endocrine pancreas that may form the basis for future treatment.
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Affiliation(s)
- Ammon B Peck
- Department of Pathology, Immumology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville 32610, USA.
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25
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Semino CE, Kasahara J, Hayashi Y, Zhang S. Entrapment of migrating hippocampal neural cells in three-dimensional peptide nanofiber scaffold. ACTA ACUST UNITED AC 2005; 10:643-55. [PMID: 15165480 DOI: 10.1089/107632704323061997] [Citation(s) in RCA: 147] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Isolation and expansion of self-renewing neural cells ex vivo are required for neural tissue repair in regenerative medicine. Neurogenesis occurs in restricted areas of postnatal mammalian brain including dentate gyrus and subventricular zone. We developed a simple method to entrap migrating neural cells (potential neuroprogenitors) from postnatal hippocampal organotypic cultures in three-dimensional (3-D) peptide nanofiber scaffolds. A few hours after placing the hippocampal slices in culture, cell proliferation activity at the "interface zone" between the tissue slice and the membrane culture surface was observed. Pulse-chase experiments using 5-bromodeoxyuridine (BrdU), which measures mitotic activity, showed that a number of cells incorporated BrdU at the interface zone. The number of BrdU(+) cells increased exponentially during the first 3 days of exposure to the label. The BrdU(+) cells also stained positive for glial fibrillary acidic protein (2.2 +/- 0.5%), a marker for astroglia; and for betaIII tubulin (7.3 +/- 2.8%) and nestin (2.7 +/- 0.9%), markers for neural progenitors. When hippocampal slices were cultured on a peptide nanofiber scaffold layer (~500 microm thick), a more extended interface zone between each tissue slice and the scaffold was formed. Moreover, the migrating BrdU(+) cell population entrapped in the 3-D peptide scaffold was readily isolated by mechanically disrupting the scaffold and then used for conventional 2-D culture systems for further studies. This simple method may be useful not only in developing technology for neural progenitor cell isolation and enrichment in vitro, but also for expanding cells for cell-based therapies of regenerative medicine.
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Affiliation(s)
- Carlos E Semino
- Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
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26
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Gershengorn MC, Hardikar AA, Wei C, Geras-Raaka E, Marcus-Samuels B, Raaka BM. Epithelial-to-Mesenchymal Transition Generates Proliferative Human Islet Precursor Cells. Science 2004; 306:2261-4. [PMID: 15564314 DOI: 10.1126/science.1101968] [Citation(s) in RCA: 354] [Impact Index Per Article: 16.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Insulin-expressing beta cells, found in pancreatic islets, are capable of generating more beta cells even in the adult. We show that fibroblast-like cells derived from adult human islets donated postmortem proliferate readily in vitro. These mesenchymal-type cells, which exhibit no hormone expression, can then be induced to differentiate into hormone-expressing islet-like cell aggregates, which reestablishes the epithelial character typical of islet cells. Immunohistochemistry, in situ hybridization, and messenger RNA measurements in single cells and cell populations establish the transition of epithelial cells within islets to mesenchymal cells in culture and then to insulin-expressing epithelial cells.
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Affiliation(s)
- Marvin C Gershengorn
- Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-8029, USA.
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27
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Affiliation(s)
- Timothy S Sadiq
- Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
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28
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Abstract
Spain ranks number one in organ donors (35 per million per yr). Although the prevalence of diabetes is low (100,000 type 1 diabetic patients and 2 million type 2 diabetic patients), the expected number of patients receiving islet transplants should be estimated at 200 per year. Islet replacement represents a promising cure for diabetes and has been successfully applied in a limited number of type 1 diabetic patients, resulting in insulin independence for periods longer than 3 yr. However, it has been difficult to obtain sufficient numbers of islets from cadaveric donors. Interesting alternatives include acquiring renewable sources of cells using either embryonic or adult stem cells to overcome the islet scarcity problem. Stem cells are capable of extensive proliferation rates and are capable of differentiating into other cell types of the body. In particular, totipotent stem cells are capable of differentiating into all cell types in the body, whereas pluripotent stem cells are limited to the development of a certain number of differentiated cell types. Insulin-producing cells have been obtained from both embryonic and adult stem cells using several approaches. In animal models of diabetes, the therapeutic application of bioengineered insulin-secreting cells derived from stem cells has delivered promising results. This review will summarize the different approaches that have been used to obtain insulin-producing cells from embryonic and adult stem cells and highlights the key points that will allow in vitro differentiation and subsequent transplantation in the future.
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Affiliation(s)
- Enrique Roche
- Institute of Bioengineering, Campus de San Juan, University Miguel Hernandez, San Juan, Alicante, Spain
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29
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Linning KD, Tai MH, Madhukar BV, Chang CC, Reed DN, Ferber S, Trosko JE, Olson LK. Redox-mediated enrichment of self-renewing adult human pancreatic cells that possess endocrine differentiation potential. Pancreas 2004; 29:e64-76. [PMID: 15367896 DOI: 10.1097/00006676-200410000-00015] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
OBJECTIVES The limited availability of transplantable human islets has stimulated the development of methods needed to isolate adult pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation. The objective of this study was to determine whether modulation of intracellular redox state with N-acetyl-L-cysteine (NAC) would allow for the propagation of pancreatic stem/progenitor cells from adult human pancreatic tissue. METHODS Cells were propagated from human pancreatic tissue using a serum-free, low-calcium medium supplemented with NAC and tested for their ability to differentiate when cultured under different growth conditions. RESULTS Human pancreatic cell (HPC) cultures coexpressed alpha-amylase, albumin, vimentin, and nestin. The HPC cultures, however, did not express other genes associated with differentiated pancreatic exocrine, duct, or endocrine cells. A number of transcription factors involved in endocrine cell development including Beta 2, Islet-1, Nkx6.1, Pax4, and Pax6 were expressed at variable levels in HPC cultures. In contrast, pancreatic duodenal homeobox factor 1 (Pdx-1) expression was extremely low and at times undetectable. Overexpression of Pdx-1 in HPC cultures stimulated somatostatin, glucagon, and carbonic anhydrase expression but had no effect on insulin gene expression. HPC cultures could form 3-dimensional islet-like cell aggregates, and this was associated with expression of somatostatin and glucagon but not insulin. Cultivation of HPCs in a differentiation medium supplemented with nicotinamide, exendin-4, and/or LY294002, an inhibitor of phosphatidylinositol-3 kinase, stimulated expression of insulin mRNA and protein. CONCLUSION These data support the use of intracellular redox modulation for the enrichment of pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation.
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Affiliation(s)
- Katrina D Linning
- Department of Physiology, Michigan State University, East Lansing, Michigan 48824, USA
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30
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Abstract
Development of a multicellular organism is accomplished through a series of events that are preprogrammed in the genome. These events encompass cellular proliferation, lineage commitment, lineage progression, lineage expression, cellular inhibition, and regulated apoptosis. The sequential progression of cells through these events results in the formation of the differentiated cells, tissues, and organs that constitute an individual. Although most cells progress through this sequence during development, a few cells leave the developmental continuum to become reserve precursor cells. The reserve precursor cells are involved in the continual maintenance and repair of the tissues and organs throughout the life span of the individual. Until recently it was generally assumed that the precursor cells in postnatal individuals were limited to lineage-committed progenitor cells specific for various tissues. However, studies by Young, his colleagues, and others have demonstrated the presence of two categories of precursor cells that reside within the organs and tissues of postnatal animals. These two categories of precursor cells are lineage-committed (multipotent, tripotent, bipotent, and unipotent) progenitor cells and lineage-uncommitted pluripotent (epiblastic-like, ectodermal, mesodermal, and endodermal) stem cells. These reserve precursor cells provide for the continual maintenance and repair of the organism after birth.
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Affiliation(s)
- Henry E Young
- Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia 31207, USA.
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31
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Suzuki A, Nakauchi H, Taniguchi H. Prospective isolation of multipotent pancreatic progenitors using flow-cytometric cell sorting. Diabetes 2004; 53:2143-52. [PMID: 15277399 DOI: 10.2337/diabetes.53.8.2143] [Citation(s) in RCA: 181] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
During pancreatic development, neogenesis, and regeneration, stem cells might act as a central player to generate endocrine, acinar, and duct cells. Although these cells are well known as pancreatic stem cells (PSCs), indisputable proof of their existence has not been reported. Identification of phenotypic markers for PSCs leads to their prospective isolation and precise characterization to clear whether stem cells exist in the pancreas. By combining flow cytometry and clonal analysis, we show here that a possible pancreatic stem or progenitor cell candidate that resides in the developing and adult mouse pancreas expresses the receptor for the hepatocyte growth factor (HGF) c-Met, but does not express hematopoietic and vascular endothelial antigens such as CD45, TER119, c-Kit, and Flk-1. These cells formed clonal colonies in vitro and differentiated into multiple pancreatic lineage cells from single cells. Some of them could largely expand with self-renewing cell divisions in culture, and, following cell transplantation, they differentiated into pancreatic endocrine and acinar cells in vivo. Furthermore, they produced cells expressing multiple markers of nonpancreatic organs including liver, stomach, and intestine in vitro. Our data strongly suggest that c-Met/HGF signaling plays an important role in stem/progenitor cell function in both developing and adult pancreas. By using this antigen, PSCs could be isolated prospectively, enabling a detailed investigation of stem cell markers and application toward regenerative therapies for diabetes.
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Affiliation(s)
- Atsushi Suzuki
- Gene Expression Laboratories, The Salk Institute for Biological Studies, La Jolla, California, USA
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32
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Wu F, Jagir M, Powell JS. Long-term correction of hyperglycemia in diabetic mice after implantation of cultured human cells derived from fetal pancreas. Pancreas 2004; 29:e23-9. [PMID: 15211120 DOI: 10.1097/00006676-200407000-00064] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Type I diabetes is characterized by destruction of insulin-producing beta-islet cells in the pancreas resulting in hyperglycemia and associated morbidity. The successful treatment of diabetes by transplanted islets has resulted in renewed efforts to identify methods to augment islet availability. One approach is to identify and expand islet precursor cells able to later differentiate into functional endocrine cells. A population of cytokeratin 19-negative, vimentin-positive, insulin-negative, glucagon-negative, and nestin-positive cells was cultured from human fetal pancreas and passaged for over 20 population doublings. These cells were stimulated to form cell aggregates when grown on poly-D-lysine (PDL)-coated surfaces and then evaluated for differentiation potential using in vivo function as a surrogate marker for the presence of differentiated precursor cells. Streptozotocin-induced diabetic SCID mice implanted with PDL-induced cell aggregates were able to maintain glucose concentrations below 200 mg/dL for over 70 days (n = 5). In addition, human C-peptide was detectable in implanted animals but not in control animals. These findings show that a population of human fetal pancreas-derived cells (1) can be cultured and expanded in vitro, (2) can maintain the ability to differentiate into beta-islet-like cells, and (3) can correct hyperglycemia in a mouse model of diabetes. Further improvements in isolation, culture, and differentiation of human pancreas-derived beta-cell precursors may one day help to provide a novel source of islets for use in transplantation therapy to treat type I diabetes.
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Affiliation(s)
- Fred Wu
- University of California, Davis Medical Center, UC Davis Cancer Center, Sacramento, California 95817, USA.
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33
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Stimulation of pancreatic islet neogenesis: a possible treatment for type 1 and type 2 diabetes. ACTA ACUST UNITED AC 2004. [DOI: 10.1097/01.med.0000125482.65536.0a] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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Song KH, Ko SH, Ahn YB, Yoo SJ, Chin HM, Kaneto H, Yoon KH, Cha BY, Lee KW, Son HY. In vitro transdifferentiation of adult pancreatic acinar cells into insulin-expressing cells. Biochem Biophys Res Commun 2004; 316:1094-100. [PMID: 15044097 DOI: 10.1016/j.bbrc.2004.02.153] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2004] [Indexed: 01/04/2023]
Abstract
Despite a recent breakthrough in human islet transplantation for treating diabetes mellitus, the limited availability of insulin-producing tissue is still a major obstacle. Here, we studied whether adult pancreatic acinar cells have the potential to transdifferentiate into islet or beta cells. Pancreatic acini were isolated from 7- to 8-weeks-old male Sprague-Dawley rats and cultured in suspension. Within 1 week, most of the acinar cells lost amylase expression and converted to cells with a duct cell phenotype. Insulin-positive cells were also observed, mainly at the periphery of the acini-derived spheroids. Insulin gene and protein expression was increased. Presence of a few insulin-positive cells coexpressing cytokeratins suggests that a spontaneous acinar to ductal cell transdifferentiation process was further going on towards beta cells. This study provides the first evidence that adult pancreatic acinar cells could be differentiated into insulin-expressing cells in vitro.
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Affiliation(s)
- Ki-Ho Song
- Department of Internal Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
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35
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Roche E, Soria B. Generation of new islets from stem cells. Cell Biochem Biophys 2004. [DOI: 10.1007/bf02739017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
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36
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Gu G, Wells JM, Dombkowski D, Preffer F, Aronow B, Melton DA. Global expression analysis of gene regulatory pathways during endocrine pancreatic development. Development 2003; 131:165-79. [PMID: 14660441 DOI: 10.1242/dev.00921] [Citation(s) in RCA: 200] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
To define genetic pathways that regulate development of the endocrine pancreas, we generated transcriptional profiles of enriched cells isolated from four biologically significant stages of endocrine pancreas development: endoderm before pancreas specification, early pancreatic progenitor cells, endocrine progenitor cells and adult islets of Langerhans. These analyses implicate new signaling pathways in endocrine pancreas development, and identified sets of known and novel genes that are temporally regulated, as well as genes that spatially define developing endocrine cells from their neighbors. The differential expression of several genes from each time point was verified by RT-PCR and in situ hybridization. Moreover, we present preliminary functional evidence suggesting that one transcription factor encoding gene (Myt1), which was identified in our screen, is expressed in endocrine progenitors and may regulate alpha, beta and delta cell development. In addition to identifying new genes that regulate endocrine cell fate, this global gene expression analysis has uncovered informative biological trends that occur during endocrine differentiation.
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Affiliation(s)
- Guoqiang Gu
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
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37
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Giannoukakis N, Trucco M. Current status and prospects for gene and cell therapeutics for type 1 diabetes mellitus. Rev Endocr Metab Disord 2003; 4:369-80. [PMID: 14618022 DOI: 10.1023/a:1027306213563] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Affiliation(s)
- Nick Giannoukakis
- Department of Pathology and Diabetes Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
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38
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Gao R, Ustinov J, Pulkkinen MA, Lundin K, Korsgren O, Otonkoski T. Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture. Diabetes 2003; 52:2007-15. [PMID: 12882917 DOI: 10.2337/diabetes.52.8.2007] [Citation(s) in RCA: 198] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
We have reproduced a previously described method for the in vitro generation of endocrine cells in adult human pancreatic tissue culture. The aim of this study was to characterize the nature of pancreatic progenitor cells and to identify the factors necessary for their differentiation in this model. During monolayer expansion, two types of cells proliferated sequentially; first cytokeratin 19 (CK19)-positive ductal epithelial cells and then nestin-positive fibroblastoid cells. After the bromodeoxyuridine-labeled cells were traced in differentiated islet buds, some of the proliferating ductal cells had differentiated into endocrine cells, whereas nestin-positive cells could not give rise to endocrine tissue. Serum-free culture was found to be an absolute requirement for the endocrine differentiation to occur. Also, overlay of the cells with Matrigel was essential, whereas nicotinamide had a potentiating effect. The in vitro-generated islet buds released insulin in response to glucose nearly as efficiently as native islets. When transplanted under the kidney capsule of nude mice, only one of five grafts demonstrated further growth with foci of both endocrine and exocrine differentiation. Our results support the previous notion that pancreatic progenitor cells represent a subpopulation of ductal epithelial cells. No evidence was found for the development of endocrine cells from nestin-positive stem cells.
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Affiliation(s)
- Ru Gao
- Program of Developmental and Reproductive Biology, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland
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39
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Bottino R, Lemarchand P, Trucco M, Giannoukakis N. Gene- and cell-based therapeutics for type I diabetes mellitus. Gene Ther 2003; 10:875-89. [PMID: 12732873 DOI: 10.1038/sj.gt.3302015] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Type 1 diabetes mellitus, an autoimmune disorder is an attractive candidate for gene and cell-based therapy. From the use of gene-engineered immune cells to induce hyporesponsiveness to autoantigens to islet and beta cell surrogate transplants expressing immunoregulatory genes to provide a local pocket of immune privilege, these strategies have demonstrated proof of concept to the point where translational studies can be initiated. Nonetheless, along with the proof of concept, a number of important issues have been raised by the choice of vector and expression system as well as the point of intervention; prophylactic or therapeutic. An assessment of the current state of the science and potential leads to the conclusion that some strategies are ready for safety trials while others require varying degrees of technical and conceptual refinement.
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Affiliation(s)
- R Bottino
- Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
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Wells JM. Genes expressed in the developing endocrine pancreas and their importance for stem cell and diabetes research. Diabetes Metab Res Rev 2003; 19:191-201. [PMID: 12789652 DOI: 10.1002/dmrr.364] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
The genes that regulate endocrine pancreas development, maintain adult endocrine cells, and stimulate progenitor/stem cells during regeneration remain largely unstudied. There is ample evidence that many of the genes involved in endocrine pancreas development also function in the homeostasis of the adult islet. In light of the potential benefits to diabetic research, it is surprising that there is little information about the genes expressed throughout the ontogeny of the endocrine pancreas. In the past few years, efforts have been made to establish the Endocrine Pancreas Consortium database (EPConDB), in which many of the genes expressed in the developing endocrine pancreas are in a database with a corresponding publicly available clone bank. In addition, advances in microarray technology now allow for a quantitative expression analysis of thousands of genes simultaneously, which makes it possible to generate a quantitative catalog of the genes expressed at each step of endocrine differentiation, from embryonic endoderm to mature beta cells. In this review, I will discuss how genes discovered by virtue of their role in endocrine pancreas development may function in the maintenance of pancreatic stem cells and the regeneration of islets. I will further summarize the recent advances in genomics-based studies of the developing endocrine pancreas and will discuss how they might impact on the discovery of diagnostics and research into stem cell-based approaches for the treatment of diabetes.
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Affiliation(s)
- James M Wells
- Division of Developmental Biology, Children's Hospital Research Foundation, Cincinnati, OH 45229-3039, USA.
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Abstract
Type 1 (insulin-dependent) diabetes mellitus results from selective immune-mediated destruction of pancreatic islet beta cells. Strategies to prevent or reverse the development of diabetes can be divided into three groups, depending on whether they focus on beta-cell protection, regeneration or replacement. Prevention of immune beta-cell destruction involves either halting the immune attack directed against beta cells or making beta cells better able to withstand immune attack, for example, by making them resistant to free radical damage. The recent identification of beta-cell growth factors and development of stem cell technologies provides an alternative route to the reversal of diabetes, namely beta-cell regeneration. Interestingly, stem cell-derived islets appear to be less sensitive to recurrent immune destruction that is normally seen in response to islet transplantation. The last alternative is beta-cell replacement or substitution. This covers a wide range of interventions including human whole pancreas transplantation, xenotransplantation, genetically modified beta cells, mechanical insulin sensing and delivery devices, and the artificial pancreas. This review describes recent advances in each of these research areas and aims to provide clinicians with an idea of where and when an effective strategy to prevent or reverse diabetes development will become available.
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Affiliation(s)
- Nikolai Petrovsky
- Autoimmunity Research Unit, Canberra Hospital and Medical Informatics Centre, University of Canberra, ACT, Australia.
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Huang H, Tang X. Phenotypic determination and characterization of nestin-positive precursors derived from human fetal pancreas. J Transl Med 2003; 83:539-47. [PMID: 12695557 DOI: 10.1097/01.lab.0000062890.40534.1c] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Demand for donations to meet the requirements of pancreas or islet transplantation has prompted the search for alternative sources of beta-cell replacement therapy. Earlier studies identified nestin-positive islet-derived progenitor cells (NIPs) residing in human pancreas. In the present study, we isolated and cultured human fetal NIPs that express stem cell marker ABCG2/BCRP1. In confluent cultures, NIPs formed three-dimensional islet-like cell clusters (ICCs). During differentiation, NIP-derived ICCs showed numerous pancreatic lineage transcripts including insulin, whereas ABCG2 and nestin expression fell concomitantly. In addition, ICCs displayed the ability to reverse hyperglycemia in diabetic NOD-SCID mice, as well as infiltrate and form well-differentiated structures in normal mice. These cells can be cloned repeatedly and maintained in long-term culture. Our studies are the first to show NIPs derived from human fetal pancreas, which may have significant implications for future applications in stem cell therapy of diabetes.
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Affiliation(s)
- Haixia Huang
- Department of Cell Biology, Shanghai Second Medical University, Shanghai, China
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Giannoukakis N, Robbins PD. Gene and cell therapies for diabetes mellitus: strategies and clinical potential. BioDrugs 2003; 16:149-73. [PMID: 12102644 DOI: 10.2165/00063030-200216030-00001] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The last 5 years have witnessed an explosion in the use of genes and cells as biomedicines. While primarily aimed at cancer, gene engineering and cell therapy strategies have additionally been used for Mendelian, neurodegenerative and metabolic disorders. The main focus of gene and cell therapy strategies in metabolism has been diabetes mellitus. This disease is a disorder of glucose homeostasis, either due to the immune-mediated eradication of pancreatic beta cells in the islets of Langerhans (type 1 diabetes) or resulting from insulin resistance and obesity syndromes where the insulin-producing capability of the beta cell is ultimately exhausted in the face of insensitivity to the effects of insulin in the peripheral glucose-utilising tissues (type 2 diabetes). A significant number of animal studies have demonstrated the potential in restoring normoglycaemia by islet transplantation in the context of immunoregulation achieved by gene transfer of immunoregulatory genes to allo- and xenogeneic islets ex vivo. Additionally, gene and cell therapy has also been used to induce tolerance to auto- and alloantigens and to generate the tolerant state in autoimmune rodent animal models of type 1 diabetes or rodent recipients of allogeneic/xenogeneic islet transplants. The achievements of gene and cell therapy in type 2 diabetes are less evident, but seminal studies promise that this modality can be relevant to treat and perhaps prevent the underlying causes of the disease. Here we present an overview of the current status of gene and cell therapy for type 1 and 2 diabetes and we propose potential therapeutic options that could be clinically useful. For type 1 diabetes, transplantation of islets engineered to evade or suppress the recipient immune response is the most readily-available technology today. A number of gene delivery vectors encoding proteins that impair a variety of immune cells have already been examined and proven versatile. More challenging but, nonetheless, just over the horizon are attempts to promote tolerance to islet allografts. Type 2 diabetes will likely require a better understanding of the processes that determine insulin sensitivity in the periphery. Targeting tissues such as muscle and fat with vectors encoding genes whose products promote insulin sensitivity and glucose uptake is an approach that does not carry with it the side-effects often associated with pharmacologic agents currently in use. In the end, progress in vector design, elucidation of antigen-specific immunity and insulin sensitivity will provide the framework for gene drug use in the treatment of type 1 and type 2 diabetes.
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Affiliation(s)
- Nick Giannoukakis
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
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Lee KM, Nguyen C, Ulrich AB, Pour PM, Ouellette MM. Immortalization with telomerase of the Nestin-positive cells of the human pancreas. Biochem Biophys Res Commun 2003; 301:1038-44. [PMID: 12589817 DOI: 10.1016/s0006-291x(03)00086-x] [Citation(s) in RCA: 124] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Cells expressing the neuronal stem cell marker Nestin are present in the human pancreas but the biological role of these cells has yet to be resolved. We report here the establishment with the catalytic subunit of human telomerase (hTERT) of a line of normal human cells representing this cell type. Primary human cells derived from the ducts of the pancreas were transduced with an hTERT cDNA. The infected cells became positive for telomerase, failed to senesce, and were still proliferating after more than 150 doublings. The immortalized cells were positive for the expression of Nestin (at both the mRNA and protein levels) and were found to be free of cancer-associated changes: diploid and expressing wild type p16(INK4a), p53, and K-Ras. An established line of normal human cells representing this cell type should be of great value to help define the biological properties of this novel cell type.
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Affiliation(s)
- K M Lee
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198, USA
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Tai MH, Olson LK, Madhukar BV, Linning KD, Van Camp L, Tsao MS, Trosko JE. Characterization of gap junctional intercellular communication in immortalized human pancreatic ductal epithelial cells with stem cell characteristics. Pancreas 2003; 26:e18-26. [PMID: 12499933 DOI: 10.1097/00006676-200301000-00025] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/10/2022]
Abstract
INTRODUCTION Gap junctional intercellular communication has been implicated in the homeostatic regulation of cell growth, differentiation, and apoptosis. Cancer cells, which have been viewed as "partially blocked stem cells," and which lack the ability for growth control, terminal differentiation, and apoptosis, also lack functional gap junctional communication. AIMS AND METHODOLOGY A clone of a human pancreatic ductal epithelial cell line, H6c7, derived after immortalization with human papilloma virus, was used to examine gap junctional intercellular communication and the ability to differentiate under different growth conditions. RESULTS The cells showed characteristic epithelial morphology on standard tissue culture dishes. When placed on Matrigel they showed phenotypical changes with extensive ductal organization and budding structures. In growth medium containing hormones and growth factors, these cells were gap junctional intercellular communication (GJIC)-incompetent. In the presence of c-AMP elevating agents, isobutylmethylxanthine, and forskolin, in basal medium that did not contain the hormones and growth factors, the cells became GJIC-competent and expressed connexin43 gap junction protein within 48 hours after treatment. RT-PCR analyses of the cells under different growth conditions showed that the cells expressed, and genes when cultured in the basal medium with c-AMP elevating agents. They also expressed the gene that did not change with c-AMP treatment. H6c7 cells also have the capacity to turn on an ectopic insulin promoter reporter gene. CONCLUSION Our data suggest that the immortalized H6c7 cells retain stem-like characteristics and have the potential to differentiate into duct-like structures and perhaps insulin-producing cells.
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Affiliation(s)
- Mei-Hui Tai
- Department of Pediatrics and Human Development, Michigan State University, East Lansing, Michigan 48824, USA
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Tsuchiya M, Tsuchiya K, Iwami Y, Ohgawara H. Analysis of gene expression and insulin secretion by monolayer-forming adult porcine pancreatic endocrine cells. Pancreas 2003; 26:71-5. [PMID: 12499920 DOI: 10.1097/00006676-200301000-00012] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/10/2022]
Abstract
INTRODUCTION We recently established a method of isolation and primary monolayer culture of porcine pancreatic endocrine cells that involves the use of nicotinamide. AIM To obtain genetic information on cultured porcine endocrine cells and to examine cell function in relation to insulin secretion during long-term culture. METHODOLOGY Gene expression of insulin and several transcription factors, including PDX-1, Beta2/NeuroD, Pax6, and Nkx6.1, was assessed by reverse transcription-polymerase chain reaction analysis, and the insulin protein level was estimated by immunohistochemistry and enzyme assay during a 12-week period. RESULTS During the culture period, insulin accumulation in the medium at 5 weeks had decreased by almost half the level of accumulation in the first week. In contrast to the alteration of secretory function, insulin gene expression was maintained for at least 12 weeks, and regulatory transcription factors were expressed at the same levels until 9 weeks. These observations suggest that gene expression is not involved in the cause of decreased baseline insulin secretion. Moreover, although the insulin response to high glucose and potassium loading was maintained, the magnitude of the responses to both stimuli was attenuated in the late period of culture. Insulin secretion tended to decrease in our culture system, and the secretory response to pharmacological stimulation was attenuated despite maintenance of messenger RNA expression of insulin and other islet-specific genes for at least 9 weeks in vitro. CONCLUSION These findings indicate that cell integrity is maintained and that the alteration in insulin secretion must be explained by another mechanism.
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Affiliation(s)
- Mariko Tsuchiya
- Institute of Geriatrics, Aoyama Hospital, Medical Research Institute, Tokyo Women's Medical University, Tokyo, Japan
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Gu G, Brown JR, Melton DA. Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis. Mech Dev 2003; 120:35-43. [PMID: 12490294 DOI: 10.1016/s0925-4773(02)00330-1] [Citation(s) in RCA: 180] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Lineage tracing follows the progeny of labeled cells through development. This technique identifies precursors of mature cell types in vivo and describes the cell fate restriction steps they undergo in temporal order. In the mouse pancreas, direct cell lineage tracing reveals that Pdx1- expressing progenitors in the early embryo give rise to all pancreatic cells. The progenitors for the mature pancreatic ducts separate from the endocrine/exocrine tissues before E12.5. Expression of Ngn3 and pancreatic polypeptide marks endocrine cell lineages during early embryogenesis, and these cells behave as transient progenitors rather than stem cells. In adults, Ngn3 is expressed within the endocrine islets, and the NGN3+ cells seem to contribute to pancreatic islet renewal. These results indicate the stage at which each progenitor population is restricted to a particular fate and provide markers for isolating progenitors to study their growth, differentiation, and the genes necessary for their development.
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Affiliation(s)
- Guoqiang Gu
- Department of Molecular and Cell Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA
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Bottino R, Balamurugan AN, Giannoukakis N, Trucco M. Islet/pancreas transplantation: challenges for pediatrics. Pediatr Diabetes 2002; 3:210-23. [PMID: 15016150 DOI: 10.1034/j.1399-5448.2002.30408.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Beta cell replacement is a valid alternative to exogenous insulin injections to treat type 1 diabetic patients. The rate of success obtained after whole-pancreas transplantation, performed alone or in combination with kidney, and, as shown recently, by islet transplantation, justifies optimism and sets the stage for a larger clinical application of these approaches. Lifetime immunosuppression, however, required to protect the graft against recurrent autoimmune destruction and allorejection, raises serious doubts about the safety of its employment in children. While it is evident that children may be helped even more than adults by the possibility to correct diabetic metabolic disorders without exogenous insulin, and to lower in a more effective way the chance to develop secondary complications, the drawbacks of the currently used immunosuppressive drugs largely overcome the potential benefits. A great step forward for immediate applicability of transplantation to children involves the optimization of tolerogenic protocols and a better understanding of the concept of immune ignorance. Functional tolerance should be sufficient to entail the absence of immune reactivity against self- and graft antigens, while maintaining immune reactivity against other non-self, non-donor antigens. In addition, novel strategies aimed at utilizing surrogate beta cells obtained from non-islet cells, or by genetic manipulation of beta-cell precursors merit consideration as the use of xenogeneic donors. However, much work is still needed for their safe clinical implementation.
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Affiliation(s)
- Rita Bottino
- Diabetes Institute, Rangos Research Center, Children's Hospital of Pittsburgh, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213, USA
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Peck AB, Cornelius JG, Chaudhari M, Shatz D, Ramiya VK. Use of in vitro-generated, stem cell-derived islets to cure type 1 diabetes: how close are we? Ann N Y Acad Sci 2002; 958:59-68. [PMID: 12021084 DOI: 10.1111/j.1749-6632.2002.tb02947.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Recent successes in treating type 1 diabetic patients with islet transplantation portends a future need for an increase in available islets. Ductal structures of the adult pancreas contain multipotent stem cells that, under the proper in vitro conditions, can both self-renew and differentiate into functional islets of Langerhans. In vitro-generated islets exhibit temporal changes in mRNA transcripts for islet-associated markers as well as regulated insulin responses following glucose challenge. When implanted into diabetic mice, in vitro-generated islets induce neovascularization and reverse insulin-dependent diabetes. The possibility of growing functional endocrine pancreas from stem cells provides new opportunities to produce large numbers of islets, even autologous islets, for use as implants.
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Affiliation(s)
- A B Peck
- Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
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50
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Abstract
Pancreatic cancer has an extremely poor prognosis and lacks early diagnostic and therapeutic possibilities, mainly because of its silent course and explosive fatal outcome. The histogenesis of the disease and early biochemical and genetic alterations surrounding carcinogenesis are still controversial. In vitro studies offer a useful tool to study physiologic, pathophysiologic, differentiation, and transformation processes of cells and to understand some of these shortcomings. The extreme difficulties in isolating individual pancreatic cells and their purification by maintaining their native characteristics have limited research in this area. This review is intended to present and discuss the current availability of rodent and pancreatic cell lines, their differences as well as the difficulties, limitations, and characteristics of these cultured cells. Discussed are in vitro models; ductal, islet, and acinar cell culture; cell differentiation; cell transformation, including genetic and chromosomal alterations; as well as tumor cell markers. Also addressed are the advantages and problems associated with the cell culture in humans and rodents. Advancements in tissue culture technique and molecular biology offer steady progress in this important line of research. The improved methods not only promise the establishment of beta-cell cultures for the treatment of diabetes, but also for studying sequential genetic alterations during pancreatic carcinogenesis and in understanding the tumor cell origin.
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Affiliation(s)
- Alexis B Ulrich
- Eppley Cancer Center and the Department of Pathology and Microbiology, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, U.S.A
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