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Li S, Fan L, Viktoria U, Oleksandr P, Li Z, Zhang W, Deng B. Effect of resuscitation of cryopreserved porcine adrenal glands at 26 °C on their recovery and functioning under xenotransplantation. Cryobiology 2024; 115:104895. [PMID: 38616031 DOI: 10.1016/j.cryobiol.2024.104895] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2024] [Revised: 03/28/2024] [Accepted: 04/10/2024] [Indexed: 04/16/2024]
Abstract
The study is devoted to the effect of lowered resuscitation temperature (26 °C) on cryopreserved porcine adrenal glands functional activity in vitro and in vivo under xenotransplantation. The adrenals were collected from newborn pigs, cryopreserved with 5 % DMSO at a rate of 1 °C/min, resuscitated at 26 or 37 °C for 48 h (5 % CO2, DMEM), embedded into small intestinal submucosa, and transplanted to bilaterally adrenalectomized rats. It has been shown that the glands resuscitated at 26 °C have suppressed free-radical processes and can produce cortisol and aldosterone in vitro, and may lead to elevated blood levels of these hormones. Moreover, the adrenal grafts maintain blood glucose levels and promote the formation of glycogen stores. Thus, the resuscitation at 26 °C can improve the quality of grafts and favor the introduction and application of the cryopreserved organs and tissues for transplantation in clinical and experimental practice.
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Affiliation(s)
- Shasha Li
- College of Basic Medical and Forensic Medicine, Henan University of Science and Technology, 263 Kaiyuan Avenue, Luoyang City, China.
| | - Lingling Fan
- College of Basic Medical and Forensic Medicine, Henan University of Science and Technology, 263 Kaiyuan Avenue, Luoyang City, China.
| | - Ustichenko Viktoria
- Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv, Ukraine.
| | - Pakhomov Oleksandr
- Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv, Ukraine.
| | - Zhongjie Li
- College of Basic Medical and Forensic Medicine, Henan University of Science and Technology, 263 Kaiyuan Avenue, Luoyang City, China.
| | - Wenlu Zhang
- College of Basic Medical and Forensic Medicine, Henan University of Science and Technology, 263 Kaiyuan Avenue, Luoyang City, China.
| | - Bo Deng
- College of Basic Medical and Forensic Medicine, Henan University of Science and Technology, 263 Kaiyuan Avenue, Luoyang City, China.
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Sionov RV, Ahdut-HaCohen R. A Supportive Role of Mesenchymal Stem Cells on Insulin-Producing Langerhans Islets with a Specific Emphasis on The Secretome. Biomedicines 2023; 11:2558. [PMID: 37761001 PMCID: PMC10527322 DOI: 10.3390/biomedicines11092558] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2023] [Revised: 09/06/2023] [Accepted: 09/14/2023] [Indexed: 09/29/2023] Open
Abstract
Type 1 Diabetes (T1D) is a chronic autoimmune disease characterized by a gradual destruction of insulin-producing β-cells in the endocrine pancreas due to innate and specific immune responses, leading to impaired glucose homeostasis. T1D patients usually require regular insulin injections after meals to maintain normal serum glucose levels. In severe cases, pancreas or Langerhans islet transplantation can assist in reaching a sufficient β-mass to normalize glucose homeostasis. The latter procedure is limited because of low donor availability, high islet loss, and immune rejection. There is still a need to develop new technologies to improve islet survival and implantation and to keep the islets functional. Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic progenitor cells with high plasticity that can support human pancreatic islet function both in vitro and in vivo and islet co-transplantation with MSCs is more effective than islet transplantation alone in attenuating diabetes progression. The beneficial effect of MSCs on islet function is due to a combined effect on angiogenesis, suppression of immune responses, and secretion of growth factors essential for islet survival and function. In this review, various aspects of MSCs related to islet function and diabetes are described.
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Affiliation(s)
- Ronit Vogt Sionov
- The Institute of Biomedical and Oral Research (IBOR), Faculty of Dental Medicine, The Hebrew University of Jerusalem, Jerusalem 9112102, Israel
| | - Ronit Ahdut-HaCohen
- Department of Medical Neurobiology, Institute of Medical Research, Hadassah Medical School, The Hebrew University of Jerusalem, Jerusalem 9112102, Israel;
- Department of Science, The David Yellin Academic College of Education, Jerusalem 9103501, Israel
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Mohandas S, Gayatri V, Kumaran K, Gopinath V, Paulmurugan R, Ramkumar KM. New Frontiers in Three-Dimensional Culture Platforms to Improve Diabetes Research. Pharmaceutics 2023; 15:pharmaceutics15030725. [PMID: 36986591 PMCID: PMC10056755 DOI: 10.3390/pharmaceutics15030725] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2022] [Revised: 02/13/2023] [Accepted: 02/16/2023] [Indexed: 02/25/2023] Open
Abstract
Diabetes mellitus is associated with defects in islet β-cell functioning and consequent hyperglycemia resulting in multi-organ damage. Physiologically relevant models that mimic human diabetic progression are urgently needed to identify new drug targets. Three-dimensional (3D) cell-culture systems are gaining a considerable interest in diabetic disease modelling and are being utilized as platforms for diabetic drug discovery and pancreatic tissue engineering. Three-dimensional models offer a marked advantage in obtaining physiologically relevant information and improve drug selectivity over conventional 2D (two-dimensional) cultures and rodent models. Indeed, recent evidence persuasively supports the adoption of appropriate 3D cell technology in β-cell cultivation. This review article provides a considerably updated view of the benefits of employing 3D models in the experimental workflow compared to conventional animal and 2D models. We compile the latest innovations in this field and discuss the various strategies used to generate 3D culture models in diabetic research. We also critically review the advantages and the limitations of each 3D technology, with particular attention to the maintenance of β-cell morphology, functionality, and intercellular crosstalk. Furthermore, we emphasize the scope of improvement needed in the 3D culture systems employed in diabetes research and the promises they hold as excellent research platforms in managing diabetes.
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Affiliation(s)
- Sundhar Mohandas
- Department of Biotechnology, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur 603203, Tamil Nadu, India
| | - Vijaya Gayatri
- Department of Biotechnology, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur 603203, Tamil Nadu, India
| | - Kriya Kumaran
- Department of Biotechnology, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur 603203, Tamil Nadu, India
| | - Vipin Gopinath
- Department of Radiology, Molecular Imaging Program at Stanford, Canary Centre for Cancer Early Detection, Bio-X Program, Stanford University School of Medicine, Palo Alto, CA 94304, USA
- Molecular Oncology Division, Malabar Cancer Centre, Moozhikkara P.O, Thalassery 670103, Kerala, India
| | - Ramasamy Paulmurugan
- Department of Radiology, Molecular Imaging Program at Stanford, Canary Centre for Cancer Early Detection, Bio-X Program, Stanford University School of Medicine, Palo Alto, CA 94304, USA
- Correspondence: (R.P.); (K.M.R.)
| | - Kunka Mohanram Ramkumar
- Department of Biotechnology, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur 603203, Tamil Nadu, India
- Department of Radiology, Molecular Imaging Program at Stanford, Canary Centre for Cancer Early Detection, Bio-X Program, Stanford University School of Medicine, Palo Alto, CA 94304, USA
- Correspondence: (R.P.); (K.M.R.)
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In Vitro Disease Models of the Endocrine Pancreas. Biomedicines 2021; 9:biomedicines9101415. [PMID: 34680532 PMCID: PMC8533367 DOI: 10.3390/biomedicines9101415] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2021] [Revised: 09/30/2021] [Accepted: 10/05/2021] [Indexed: 12/12/2022] Open
Abstract
The ethical constraints and shortcomings of animal models, combined with the demand to study disease pathogenesis under controlled conditions, are giving rise to a new field at the interface of tissue engineering and pathophysiology, which focuses on the development of in vitro models of disease. In vitro models are defined as synthetic experimental systems that contain living human cells and mimic tissue- and organ-level physiology in vitro by taking advantage of recent advances in tissue engineering and microfabrication. This review provides an overview of in vitro models and focuses specifically on in vitro disease models of the endocrine pancreas and diabetes. First, we briefly review the anatomy, physiology, and pathophysiology of the human pancreas, with an emphasis on islets of Langerhans and beta cell dysfunction. We then discuss different types of in vitro models and fundamental elements that should be considered when developing an in vitro disease model. Finally, we review the current state and breakthroughs in the field of pancreatic in vitro models and conclude with some challenges that need to be addressed in the future development of in vitro models.
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Murray HE, Zafar A, Qureshi KM, Paget MB, Bailey CJ, Downing R. The potential role of multifunctional human amniotic epithelial cells in pancreatic islet transplantation. J Tissue Eng Regen Med 2021; 15:599-611. [PMID: 34216434 DOI: 10.1002/term.3214] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2020] [Accepted: 04/23/2021] [Indexed: 11/08/2022]
Abstract
Pancreatic islet cell transplantation has proven efficacy as a treatment for type 1 diabetes mellitus, chiefly in individuals who are refractory to conventional insulin replacement therapy. At present its clinical use is restricted, firstly by the limited access to suitable donor organs but also due to factors associated with the current clinical transplant procedure which inadvertently impair the long-term functionality of the islet graft. Of note, the physical, biochemical, inflammatory, and immunological stresses to which islets are subjected, either during pretransplant processing or following implantation are detrimental to their sustained viability, necessitating repeated islet infusions to attain adequate glucose control. Progressive decline in functional beta (β)-cell mass leads to graft failure and the eventual re-instatement of exogenous insulin treatment. Strategies which protect and/or preserve optimal islet function in the peri-transplant period would improve clinical outcomes. Human amniotic epithelial cells (HAEC) exhibit both pluripotency and immune-privilege and are ideally suited for use in replacement and regenerative therapies. The HAEC secretome exhibits trophic, anti-inflammatory, and immunomodulatory properties of relevance to islet graft survival. Facilitated by β-cell supportive 3D cell culture systems, HAEC may be integrated with islets bringing them into close spatial arrangement where they may exert paracrine influences that support β-cell function, reduce hypoxia-induced islet injury, and alter islet alloreactivity. The present review details the potential of multifunctional HAEC in the context of islet transplantation, with a focus on the innate capabilities that may counter adverse events associated with the current clinical transplant protocol to achieve long-term islet graft function.
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Affiliation(s)
- Hilary E Murray
- The Islet Research Laboratory, Worcester Clinical Research Unit, Worcestershire Acute Hospitals NHS Trust, Worcester, UK
| | - Ali Zafar
- The Islet Research Laboratory, Worcester Clinical Research Unit, Worcestershire Acute Hospitals NHS Trust, Worcester, UK.,Queen Elizabeth Hospital Birmingham, University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK
| | - Khalid M Qureshi
- The Islet Research Laboratory, Worcester Clinical Research Unit, Worcestershire Acute Hospitals NHS Trust, Worcester, UK.,Bradford Royal Infirmary, Bradford Teaching Hospitals NHS Foundation Trust, Bradford, UK
| | - Michelle B Paget
- The Islet Research Laboratory, Worcester Clinical Research Unit, Worcestershire Acute Hospitals NHS Trust, Worcester, UK
| | - Clifford J Bailey
- Diabetes Research, School of Life and Health Sciences, Aston University, Birmingham, UK
| | - Richard Downing
- The Islet Research Laboratory, Worcester Clinical Research Unit, Worcestershire Acute Hospitals NHS Trust, Worcester, UK
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Ruhela A, Kasinathan GN, Rath SN, Sasikala M, Sharma CS. Electrospun freestanding hydrophobic fabric as a potential polymer semi-permeable membrane for islet encapsulation. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2020; 118:111409. [PMID: 33255012 DOI: 10.1016/j.msec.2020.111409] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/29/2020] [Revised: 07/04/2020] [Accepted: 08/11/2020] [Indexed: 01/13/2023]
Abstract
One of the significant problems associated with islet encapsulation for type 1 diabetes treatment is the loss of islet functionality or cell death after transplantation because of the unfavorable environment for the cells. In this work, we propose a simple strategy to fabricate electrospun membranes that will provide a favorable environment for proper islet function and also a desirable pore size to cease cellular infiltration, protecting the encapsulated islet from immune cells. By electrospinning the wettability of three different biocompatible polymers: cellulose acetate (CA), polyethersulfone (PES), and polytetrafluoroethylene (PTFE) was greatly modified. The contact angle of electrospun CA, PES, and PTFE increased to 136°, 126°, and 155° as compared to 55°, 71°, and 128° respectively as a thin film, making the electrospun membranes hydrophobic. Commercial porous membranes of PES and PTFE show a contact angle of 30° and 118°, respectively, confirming the hydrophobicity of electrospun membranes is due to the surface morphology induced by electrospinning. In- vivo results confirm that the induced hydrophobicity and surface morphology of electrospun membranes impede cell attachment, which would help in maintaining the 3D circular morphology of islet cell. More importantly, the pore size of 0.3-0.6 μm obtained due to the densely packed structure of nanofibers, will be able to restrict immune cells but would allow free movement of molecules like insulin and glucose. Therefore, electrospun polymer fibrous membranes as fabricated in this work, with hydrophobic and porous properties, make a strong case for successful islet encapsulation.
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Affiliation(s)
- Aakanksha Ruhela
- Creative & Advanced Research Based On Nanomaterials (CARBON) Laboratory, Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Kandi 502285, Telangana, India
| | - Gokula Nathan Kasinathan
- Regenerative Medicine and Stem Cell Laboratory (RMS), Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Kandi 502285, Telangana, India
| | - Subha N Rath
- Regenerative Medicine and Stem Cell Laboratory (RMS), Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Kandi 502285, Telangana, India
| | - M Sasikala
- Asian Healthcare Foundation, Gachibowli, Hyderabad 500032, Telangana, India
| | - Chandra S Sharma
- Creative & Advanced Research Based On Nanomaterials (CARBON) Laboratory, Department of Chemical Engineering, Indian Institute of Technology Hyderabad, Kandi 502285, Telangana, India.
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Klak M, Gomółka M, Dobrzański T, Tymicki G, Cywoniuk P, Kowalska P, Kosowska K, Bryniarski T, Berman A, Dobrzyń A, Idaszek J, Święszkowski W, Wszoła M. Irradiation with 365 nm and 405 nm wavelength shows differences in DNA damage of swine pancreatic islets. PLoS One 2020; 15:e0235052. [PMID: 32584858 PMCID: PMC7316267 DOI: 10.1371/journal.pone.0235052] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2019] [Accepted: 06/07/2020] [Indexed: 01/29/2023] Open
Abstract
INTRODUCTION 3D printing is being used more extensively in modern biomedicine. One of the problems is selecting a proper crosslinking method of bioprinted material. Amongst currently used techniques we can distinguish: physical crosslinking (e.g. Ca2+ and Sr2+) and chemical crosslinking-the UV light crosslinking causing the biggest discussion. UV radiation is selectively absorbed by DNA, mainly in the UV-B region but also (to some extent) in UV-A and UV-C regions. DNA excitement results in typical photoproducts. The amount of strand breaks may vary depending on the period of exposition, it can also differ when cells undergo incubation after radiation. AIM The aim of this study was to show whether and how the time of irradiation with 405 nm and 365 nm wavelengths affect DNA damage in cell lines and micro-organs (pancreatic islets). MATERIALS AND METHODS The degree of DNA damage caused by different wavelengths of radiation (405 nm and 365 nm) was evaluated by a comet assay. The test was performed on fibroblasts, alpha cells, beta cells and porcine pancreatic islets after 24 hours incubation period. Samples without radiation treatment were selected as a control group. Results analysis consisted of determining the percent of cells with damaged DNA and the tail intensity evaluation. RESULTS The degree of DNA damage in pancreatic islets after exposure to 405 nm wavelength oscillated between 2% and 6% depending on the tested time period (10 - 300 seconds). However, treating islets using 365 nm wavelength resulted in damage up to 50%. This clearly shows significantly less damage when using 405 nm wavelength. Similar results were obtained for the tested cell lines. CONCLUSIONS Crosslinking with 405 nm is better for pancreatic islets than crosslinking with 365 nm UV light.
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Affiliation(s)
- M. Klak
- Foundation of Research and Science Development, Warsaw, Poland
| | - M. Gomółka
- Foundation of Research and Science Development, Warsaw, Poland
| | - T. Dobrzański
- Foundation of Research and Science Development, Warsaw, Poland
| | - G. Tymicki
- Foundation of Research and Science Development, Warsaw, Poland
| | - P. Cywoniuk
- Foundation of Research and Science Development, Warsaw, Poland
| | - P. Kowalska
- Foundation of Research and Science Development, Warsaw, Poland
| | - K. Kosowska
- Foundation of Research and Science Development, Warsaw, Poland
| | - T. Bryniarski
- Foundation of Research and Science Development, Warsaw, Poland
| | - A. Berman
- Foundation of Research and Science Development, Warsaw, Poland
| | - A. Dobrzyń
- Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland
| | - J. Idaszek
- Faculty of Materials Science and Engineering, Warsaw University of Technology, Warsaw, Poland
| | - W. Święszkowski
- Faculty of Materials Science and Engineering, Warsaw University of Technology, Warsaw, Poland
| | - M. Wszoła
- Foundation of Research and Science Development, Warsaw, Poland
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Gaetani R, Aude S, DeMaddalena LL, Strassle H, Dzieciatkowska M, Wortham M, Bender RHF, Nguyen-Ngoc KV, Schmid-Schöenbein GW, George SC, Hughes CCW, Sander M, Hansen KC, Christman KL. Evaluation of Different Decellularization Protocols on the Generation of Pancreas-Derived Hydrogels. Tissue Eng Part C Methods 2020; 24:697-708. [PMID: 30398401 DOI: 10.1089/ten.tec.2018.0180] [Citation(s) in RCA: 50] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Different approaches have investigated the effects of different extracellular matrices (ECMs) and three-dimensional (3D) culture on islet function, showing encouraging results. Ideally, the proper scaffold should mimic the biochemical composition of the native tissue as it drives numerous signaling pathways involved in tissue homeostasis and functionality. Tissue-derived decellularized biomaterials can preserve the ECM composition of the native tissue making it an ideal scaffold for 3D tissue engineering applications. However, the decellularization process may affect the retention of specific components, and the choice of a proper detergent is fundamental in preserving the native ECM composition. In this study, we evaluated the effect of different decellularization protocols on the mechanical properties and biochemical composition of pancreatic ECM (pECM) hydrogels. Fresh porcine pancreas tissue was harvested, cut into small pieces, rinsed in water, and treated with two different detergents (sodium dodecyl sulfate [SDS] or Triton X-100) for 1 day followed by 3 days in water. Effective decellularization was confirmed by PicoGreen assay, Hoescht, and H&E staining, showing no differences among groups. Use of a protease inhibitor (PI) was also evaluated. Effective decellularization was confirmed by PicoGreen assay and hematoxylin and eosin (H&E) staining, showing no differences among groups. Triton-treated samples were able to form a firm hydrogel under appropriate conditions, while the use of SDS had detrimental effects on the gelation properties of the hydrogels. ECM biochemical composition was characterized both in the fresh porcine pancreas and all decellularized pECM hydrogels by quantitative mass spectrometry analysis. Fibrillar collagen was the major ECM component in all groups, with all generated hydrogels having a higher amount compared with fresh pancreas. This effect was more pronounced in the SDS-treated hydrogels when compared with the Triton groups, showing very little retention of other ECM molecules. Conversely, basement membrane and matricellular proteins were better retained when the tissue was pretreated with a PI and decellularized in Triton X-100, making the hydrogel more similar to the native tissue. In conclusion, we showed that all the protocols evaluated in the study showed effective tissue decellularization, but only when the tissue was pretreated with a PI and decellularized in Triton detergent, the biochemical composition of the hydrogel was closer to the native tissue ECM. Impact Statement The article compares different methodologies for the generation of a pancreas-derived hydrogel for tissue engineering applications. The biochemical characterization of the newly generated hydrogel shows that the material retains all the extracellular molecules of the native tissue and is capable of sustaining functionality of the encapsulated beta-cells.
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Affiliation(s)
- Roberto Gaetani
- Department of Bioengineering, University of California San Diego, La Jolla, California.,Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, California
| | - Soraya Aude
- Department of Bioengineering, University of California San Diego, La Jolla, California.,Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, California
| | - Lea Lara DeMaddalena
- Department of Bioengineering, University of California San Diego, La Jolla, California.,Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, California
| | - Heinz Strassle
- Department of Bioengineering, University of California San Diego, La Jolla, California.,Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, California
| | - Monika Dzieciatkowska
- Department of Biochemistry and Molecular Genetics, University of Colorado, Aurora, Colorado
| | - Matthew Wortham
- Departments of Pediatrics and Cellular and Molecular Medicine, Pediatric Diabetes Research Center, University of California San Diego, La Jolla, California
| | - R Hugh F Bender
- Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, California
| | - Kim-Vy Nguyen-Ngoc
- Departments of Pediatrics and Cellular and Molecular Medicine, Pediatric Diabetes Research Center, University of California San Diego, La Jolla, California
| | | | - Steven C George
- Department of Biomedical Engineering, University of California, Davis, Davis, California
| | - Christopher C W Hughes
- Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, California.,Department of Biomedical Engineering, University of California, Irvine, Irvine, California.,Chao Comprehensive Cancer Center, University of California, Irvine, Irvine, California.,Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California, Irvine, Irvine, California.,Center for Complex Biological Systems, University of California, Irvine, Irvine, California.,Department of Molecular Biology & Biochemistry, University of California, Irvine, Irvine, California
| | - Maike Sander
- Departments of Pediatrics and Cellular and Molecular Medicine, Pediatric Diabetes Research Center, University of California San Diego, La Jolla, California
| | - Kirk C Hansen
- Department of Biochemistry and Molecular Genetics, University of Colorado, Aurora, Colorado
| | - Karen L Christman
- Department of Bioengineering, University of California San Diego, La Jolla, California.,Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, California
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Urbanczyk M, Zbinden A, Layland SL, Duffy G, Schenke-Layland K. Controlled Heterotypic Pseudo-Islet Assembly of Human β-Cells and Human Umbilical Vein Endothelial Cells Using Magnetic Levitation. Tissue Eng Part A 2019; 26:387-399. [PMID: 31680653 PMCID: PMC7187983 DOI: 10.1089/ten.tea.2019.0158] [Citation(s) in RCA: 39] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
β-Cell functionality and survival are highly dependent on the cells' microenvironment and cell–cell interactions. Since the pancreas is a highly vascularized organ, the crosstalk between β-cells and endothelial cells (ECs) is vital to ensure proper function. To understand the interaction of pancreatic β-cells with vascular ECs, we sought to investigate the impact of the spatial distribution on the interaction of human cell line-based β-cells (EndoC-βH3) and human umbilical vein endothelial cells (HUVECs). We focused on the evaluation of three major spatial distributions, which can be found within human islets in vivo, in tissue-engineered heterotypic cell spheroids, so-called pseudo-islets, by controlling the aggregation process using magnetic levitation. We report that heterotypic spheroids formed by spontaneous aggregation cannot be maintained in culture due to HUVEC disassembly over time. In contrast, magnetic levitation allows the formation of stable heterotypic spheroids with defined spatial distribution and significantly facilitated HUVEC integration. To the best of our knowledge, this is the first study that introduces a human-only cell line-based in vitro test system composed of a coculture of β-cells and ECs with a successful stimulation of β-cell secretory function monitored by a glucose-stimulated insulin secretion assays. In addition, we systematically investigate the impact of the spatial distribution on cocultures of human β-cells and ECs, showing that the architecture of pseudo-islets significantly affects β-cell functionality.
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Affiliation(s)
- Max Urbanczyk
- Department of Women's Health, Research Institute for Women's Health, Eberhard Karls University Tübingen, Tübingen, Germany
| | - Aline Zbinden
- Department of Women's Health, Research Institute for Women's Health, Eberhard Karls University Tübingen, Tübingen, Germany
| | - Shannon L Layland
- Department of Women's Health, Research Institute for Women's Health, Eberhard Karls University Tübingen, Tübingen, Germany
| | - Garry Duffy
- Department of Anatomy, School of Medicine, College of Medicine, Nursing and Health Sciences, National University of Ireland Galway, Galway, Ireland
| | - Katja Schenke-Layland
- Department of Women's Health, Research Institute for Women's Health, Eberhard Karls University Tübingen, Tübingen, Germany.,The Natural and Medical Sciences Institute (NMI) at the University of Tübingen, Reutlingen, Germany.,Cluster of Excellence iFIT (EXC 2180) "Image-Guided and Functionally Instructed Tumor Therapies," Eberhard Karls University Tübingen, Tübingen, Germany.,Department of Medicine/Cardiology, Cardiovascular Research Laboratories, University of California, Los Angeles, California
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10
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Pancreatic ductal cells may have a negative effect on human islet transplantation. PLoS One 2019; 14:e0220064. [PMID: 31323061 PMCID: PMC6641198 DOI: 10.1371/journal.pone.0220064] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2019] [Accepted: 07/07/2019] [Indexed: 02/07/2023] Open
Abstract
AIM To evaluate the effect of pancreatic ductal cells on experimental human islet transplantation. MATERIALS AND METHODS Isolated islets were additionally purified by handpicking. Ductal cells were purified by magnetic cell sorting and then clustered into ductal pancreatospheres (DPS). Islets, DPS, and islets + DPS (100 islets + 75 DPS, or 100 islets + 200 DPS) were cultured and glucose-stimulated insulin secretion, β-cell apoptosis, and gene expression was determined. Islets and islets + DPS preparations (800 islets + 600 DPS) were transplanted to streptozotocin-treated immunodeficient mice and glycemia, graft morphometry, and gene expression were determined. RESULTS Insulin stimulation index was higher in islets than in islets co-cultured with DPS (5.59 ± 0.93 vs 4.02 ± 0.46; p<0.05). IL1B and CXCL11 expression was higher in 100 islets + 200 DPS than in islets (p<0.01), and IL-1β was detected in supernatants collected from DPS and islets + DPS preparations, but not in islets. Hyperglycemia developed in 33% and 67% of mice transplanted with islets or with islets + DPS respectively. β-cell mass was 26% lower in islets + DPS than in islets grafts (p>0.05), and the ratio β-/endocrine non-β-cell mass was lower in islets + DPS grafts (islets: 2.05 ± 0.18, islets + DPS: 1.35 ± 0.15; p<0.01). IL1B and IL1RN expression was significantly higher in islets + DPS grafts. CONCLUSIONS Islet preparations enriched with ductal cells have a lower insulin stimulation index in vitro and achieved a worse metabolic outcome after transplantation. Inflammation may mediate the deleterious effects of ductal cells on islet cells.
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11
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Kumar M, Gupta P, Bhattacharjee S, Nandi SK, Mandal BB. Immunomodulatory injectable silk hydrogels maintaining functional islets and promoting anti-inflammatory M2 macrophage polarization. Biomaterials 2018; 187:1-17. [DOI: 10.1016/j.biomaterials.2018.09.037] [Citation(s) in RCA: 58] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2018] [Revised: 08/28/2018] [Accepted: 09/23/2018] [Indexed: 02/08/2023]
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12
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Huang PJ, Qu J, Saha P, Muliana A, Kameoka J. Microencapsulation of beta cells in collagen micro-disks via circular pneumatically actuated soft micro-mold (cPASMO) device. Biomed Phys Eng Express 2018. [DOI: 10.1088/2057-1976/aae55e] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
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Lemos NE, Brondani LDA, Dieter C, Rheinheimer J, Bouças AP, Leitão CB, Crispim D, Bauer AC. Use of additives, scaffolds and extracellular matrix components for improvement of human pancreatic islet outcomes in vitro: A systematic review. Islets 2017; 9:73-86. [PMID: 28678625 PMCID: PMC5624286 DOI: 10.1080/19382014.2017.1335842] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/08/2017] [Revised: 05/16/2017] [Accepted: 05/24/2017] [Indexed: 01/31/2023] Open
Abstract
Pancreatic islet transplantation is an established treatment to restore insulin independence in type 1 diabetic patients. Its success rates have increased lately based on improvements in immunosuppressive therapies and on islet isolation and culture. It is known that the quality and quantity of viable transplanted islets are crucial for the achievement of insulin independence and some studies have shown that a significant number of islets are lost during culture time. Thus, in an effort to improve islet yield during culture period, researchers have tested a variety of additives in culture media as well as alternative culture devices, such as scaffolds. However, due to the use of different categories of additives or devices, it is difficult to draw a conclusion on the benefits of these strategies. Therefore, the aim of this systematic review was to summarize the results of studies that described the use of medium additives, scaffolds or extracellular matrix (ECM) components during human pancreatic islets culture. PubMed and Embase repositories were searched. Of 5083 articles retrieved, a total of 37 articles fulfilled the eligibility criteria and were included in the review. After data extraction, articles were grouped as follows: 1) "antiapoptotic/anti-inflammatory/antioxidant," 2) "hormone," 3) "sulphonylureas," 4) "serum supplements," and 5) "scaffolds or ECM components." The effects of the reviewed additives, ECM or scaffolds on islet viability, apoptosis and function (glucose-stimulated insulin secretion - GSIS) were heterogeneous, making any major conclusion hard to sustain. Overall, some "antiapoptotic/anti-inflammatory/antioxidant" additives decreased apoptosis and improved GSIS. Moreover, islet culture with ECM components or scaffolds increased GSIS. More studies are needed to define the real impact of these strategies in improving islet transplantation outcomes.
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Affiliation(s)
- Natália Emerim Lemos
- Laboratory of Human Pancreatic Islet Biology, Endocrine Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
- Postgraduation Program in Endocrinology, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Letícia de Almeida Brondani
- Laboratory of Human Pancreatic Islet Biology, Endocrine Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
- Postgraduation Program in Endocrinology, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Cristine Dieter
- Laboratory of Human Pancreatic Islet Biology, Endocrine Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
- Postgraduation Program in Endocrinology, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Jakeline Rheinheimer
- Laboratory of Human Pancreatic Islet Biology, Endocrine Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
- Postgraduation Program in Endocrinology, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Ana Paula Bouças
- Laboratory of Human Pancreatic Islet Biology, Endocrine Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
| | - Cristiane Bauermann Leitão
- Laboratory of Human Pancreatic Islet Biology, Endocrine Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
- Postgraduation Program in Endocrinology, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Daisy Crispim
- Laboratory of Human Pancreatic Islet Biology, Endocrine Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
- Postgraduation Program in Endocrinology, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Andrea Carla Bauer
- Laboratory of Human Pancreatic Islet Biology, Endocrine Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
- Postgraduation Program in Endocrinology, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
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14
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Caddeo S, Boffito M, Sartori S. Tissue Engineering Approaches in the Design of Healthy and Pathological In Vitro Tissue Models. Front Bioeng Biotechnol 2017; 5:40. [PMID: 28798911 PMCID: PMC5526851 DOI: 10.3389/fbioe.2017.00040] [Citation(s) in RCA: 161] [Impact Index Per Article: 20.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2017] [Accepted: 06/26/2017] [Indexed: 12/16/2022] Open
Abstract
In the tissue engineering (TE) paradigm, engineering and life sciences tools are combined to develop bioartificial substitutes for organs and tissues, which can in turn be applied in regenerative medicine, pharmaceutical, diagnostic, and basic research to elucidate fundamental aspects of cell functions in vivo or to identify mechanisms involved in aging processes and disease onset and progression. The complex three-dimensional (3D) microenvironment in which cells are organized in vivo allows the interaction between different cell types and between cells and the extracellular matrix, the composition of which varies as a function of the tissue, the degree of maturation, and health conditions. In this context, 3D in vitro models can more realistically reproduce a tissue or organ than two-dimensional (2D) models. Moreover, they can overcome the limitations of animal models and reduce the need for in vivo tests, according to the "3Rs" guiding principles for a more ethical research. The design of 3D engineered tissue models is currently in its development stage, showing high potential in overcoming the limitations of already available models. However, many issues are still opened, concerning the identification of the optimal scaffold-forming materials, cell source and biofabrication technology, and the best cell culture conditions (biochemical and physical cues) to finely replicate the native tissue and the surrounding environment. In the near future, 3D tissue-engineered models are expected to become useful tools in the preliminary testing and screening of drugs and therapies and in the investigation of the molecular mechanisms underpinning disease onset and progression. In this review, the application of TE principles to the design of in vitro 3D models will be surveyed, with a focus on the strengths and weaknesses of this emerging approach. In addition, a brief overview on the development of in vitro models of healthy and pathological bone, heart, pancreas, and liver will be presented.
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Affiliation(s)
- Silvia Caddeo
- Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Turin, Italy
- Department of Oral Cell Biology, Academic Center for Dentistry Amsterdam, Amsterdam, Netherlands
| | - Monica Boffito
- Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Turin, Italy
| | - Susanna Sartori
- Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Turin, Italy
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15
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Nagata N, Gu Y, Hori H, Balamurugan AN, Touma M, Kawakami Y, Wang W, Baba TT, Satake A, Nozawa M, Tabata Y, Inoue K. Evaluation of Insulin Secretion of Isolated Rat Islets Cultured in Extracellular Matrix. Cell Transplant 2017. [DOI: 10.3727/000000001783986549] [Citation(s) in RCA: 65] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Islet isolation involves enzymatic digestion of the interstitial matrix and mechanical disruption of the tissue. It is possible that a fundamental change of islet biology resulting from the loss of critical factors required for islet function or survival will occur. Extracellular matrix (ECM) is one of the most important components of the islet microenvironment. Reconstruction of the cell–matrix relationship seems to be effective for improving the loss of differentiated islet structure and function. The purpose of this study was to characterize and compare the effects of collagen gel mixture or Matrigel on β-cell function and islet cell survival. After isolation by the collagenase digestion technique, rat islets were divided and cultured with various types of collagen gel mixture. They were assessed for their glucose-stimulated insulin secretion and cell viability. Glucose-induced insulin secretion of islets cultured with collagen type I gel or a mixture of collagen type I and IV was improved after 11 days in culture. In conclusion, a type of gel composed of collagen type I and/ or type IV as an islet microenvironment is sufficient to maintain glucose responsiveness and may be useful for islet transplantation.
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Affiliation(s)
- Natsuki Nagata
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | - Yuanjun Gu
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | - Hiroshi Hori
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | - A. N. Balamurugan
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | - Maki Touma
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | - Yoshiyuki Kawakami
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | - Wenjing Wang
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | - Tomomi T. Baba
- Department of Oral Biochemistry, Nagasaki University School of Dentistry, Nagasaki, Japan
| | - Akira Satake
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | - Masumi Nozawa
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | - Yasuhiko Tabata
- Department of Biomaterials, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | - Kazutomo Inoue
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
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16
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Tchervenivanov N, Yuan S, Lipsett M, Agapitos D, Rosenberg L. Morphological and Functional Studies on Submucosal Islet Transplants in Normal and Diabetic Hamsters. Cell Transplant 2017. [DOI: 10.3727/000000002783985512] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
The long-term outcome of human islet allotransplantation is poor, and it remains to be seen if the Edmonton Protocol will make a positive impact upon the extension of posttransplant islet function. Hence, establishing an implantation site capable of sustaining islet allografts for a prolonged duration needs to be explored. In this study we investigated the submucosal space of the duodenum in Syrian golden hamsters. Following transplantation of more than 800 islets into streptozotocin (STZ)-induced diabetic hamsters, basal nonfasted blood glucose levels decreased from 403 ± 14 to 143 ± 10 mg/dl within 5 weeks posttransplantation. In these animals, in vivo islet function, as determined by intravenous glucose tolerance test (IVGTT), was similar to nondiabetic controls (K values: 1.16 ± 0.12 vs. 0.95 ± 0.06, respectively) and was significantly greater than diabetic controls (K value: 0.47 ± 0.07). Islets transplanted into the submucosal space become richly vascularized within 2 weeks, and there is minimal host inflammatory infiltrate. The β-cells of the graft remain well granulated with insulin for at least 129 days. We conclude that the submucosal space is an effective engraftment site for islets that warrants further development in a large-animal model.
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Affiliation(s)
- Nikolay Tchervenivanov
- Department of Surgery, McGill University and The Montreal General Hospital, Montreal, Quebec H3G 1A4, Canada
| | - Songyang Yuan
- Department of Surgery, McGill University and The Montreal General Hospital, Montreal, Quebec H3G 1A4, Canada
| | - Mark Lipsett
- Department of Surgery, McGill University and The Montreal General Hospital, Montreal, Quebec H3G 1A4, Canada
| | - Despina Agapitos
- Department of Surgery, McGill University and The Montreal General Hospital, Montreal, Quebec H3G 1A4, Canada
| | - Lawrence Rosenberg
- Department of Surgery, McGill University and The Montreal General Hospital, Montreal, Quebec H3G 1A4, Canada
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17
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Montolio M, Téllez N, Biarnés M, Soler J, Montanya E. Short-Term Culture with the Caspase Inhibitor z-VAD.fmk Reduces Beta Cell Apoptosis in Transplanted Islets and Improves the Metabolic Outcome of the Graft. Cell Transplant 2017; 14:59-65. [PMID: 15789663 DOI: 10.3727/000000005783983269] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
In the initial days after transplantation islets are particularly vulnerable and show increased apoptosis and necrosis. We have studied the effects of caspase inhibition on this early beta cell death in syngeneically transplanted islets. Streptozotocin-diabetic C57BL/6 mice were transplanted with 150 syngeneic islets, an insufficient mass to restore normoglycemia, preincubated with or without the pan-caspase inhibitor z-VAD. fmk 2 h before transplantation. Beta cell apoptosis was increased in control islets on day 3 after transplantation (0.28 ± 0.02%) compared with freshly isolated islets (0.08 ± 0.02%, p< 0.001), and was partially reduced in transplanted islets preincubated with z-VAD.fmk 200 μM (0.14 ± 0.02%, p = 0.003) or with z-VAD.fmk 500 μM (0.17 ± 0.01%, p = 0.012), but not with a lower z-VAD.fmk (100 μM) concentration. Diabetic mice transplanted with islets preincubated with z-VAD.fmk 500 μM showed an improved metabolic evolution compared with control and z-VAD.fmk 200 μM groups. The z-VAD.fmk 500 μM group showed an overall lower blood glucose after transplantation (p = 0.02), and at the end of the study blood glucose values were reduced compared with transplantation day (15.7 ± 3.6 vs. 32.5 ± 0.5 mmol/L, p = 0.001). In contrast, blood glucose was not significantly changed in control and z-VAD.fmk 200 μM groups. Four weeks after transplantation beta cell mass was higher in z-VAD.fmk 500 μM group (0.15 ± 0.02 mg) than in the control group (0.10 ± 0.02 mg) (p = 0.043). In summary, the treatment of freshly isolated islets with the caspase inhibitor z-VAD.fmk reduced the subsequent apoptosis of the islets once they were transplanted and improved the outcome of the graft.
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Affiliation(s)
- Marta Montolio
- Laboratory of Diabetes and Experimental Endocrinology, Endocrine Unit, IDIBELL-Hospital Universitari de Bellvitge, University of Barcelona, Barcelona, Spain
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18
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de Souza BM, Bouças AP, de Oliveira FDS, Reis KP, Ziegelmann P, Bauer AC, Crispim D. Effect of co-culture of mesenchymal stem/stromal cells with pancreatic islets on viability and function outcomes: a systematic review and meta-analysis. Islets 2017; 9:30-42. [PMID: 28151049 PMCID: PMC5345749 DOI: 10.1080/19382014.2017.1286434] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/11/2016] [Revised: 01/05/2017] [Accepted: 01/20/2017] [Indexed: 12/12/2022] Open
Abstract
The maintenance of viable and functional pancreatic islets is crucial for successful islet transplantation from brain-dead donors. To overcome islet quality loss during culture, some studies have co-cultured islets with mesenchymal stem/stromal cells (MSC). However, it is still uncertain if MSC-secreted factors are enough to improve islet quality or if a physical contact between MSCs and islets is needed. Therefore, we performed a systematic review and meta-analysis to clarify the effect of different culture contact systems of islets with MSCs on viability and insulin secretion outcomes. Pubmed and Embase were searched. Twenty studies fulfilled the eligibility criteria and were included in the qualitative synthesis and/or meta-analysis. For both outcomes, pooled weighted mean differences (WMD) between islet cultured alone (control group) and the co-culture condition were calculated. Viability mean was higher in islets co-cultured with MSCs compared with islet cultured alone [WMD = 18.08 (95% CI 12.59-23.57)]. The improvement in viability was higher in islets co-cultured in indirect or mixed contact with MSCs than in direct physical contact (P <0.001). Moreover, the mean of insulin stimulation index (ISI) was higher in islets from co-culture condition compared with islet cultured alone [WMD = 0.83 (95% CI 0.54-1.13)], independently of contact system. Results from the studies that were analyzed only qualitatively are in accordance with meta-analysis data. Co-culture of islets with MSCs has the potential for protecting islets from injury during culture period. Moreover, culture time appears to influence the beneficial effect of different methods of co-culture on viability and function of islets.
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Affiliation(s)
- Bianca Marmontel de Souza
- Laboratory of Human Pancreatic Islet Biology, Endocrine Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
- Postgraduate Program in Medical Sciences: Endocrinology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Ana Paula Bouças
- Laboratory of Human Pancreatic Islet Biology, Endocrine Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
- Postgraduate Program in Medical Sciences: Endocrinology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Fernanda dos Santos de Oliveira
- Laboratory of Cell Differentiation, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
| | - Karina Pires Reis
- Laboratory of Human Pancreatic Islet Biology, Endocrine Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
| | - Patrícia Ziegelmann
- Statistics Department and Post-Graduation Program in Epidemiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Andrea Carla Bauer
- Laboratory of Human Pancreatic Islet Biology, Endocrine Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
- Postgraduate Program in Medical Sciences: Endocrinology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Daisy Crispim
- Laboratory of Human Pancreatic Islet Biology, Endocrine Division, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
- Postgraduate Program in Medical Sciences: Endocrinology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
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19
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Forget A, Waibel M, Rojas-Canales DM, Chen S, Kawazoe N, Harding FJ, Loudovaris T, Coates PTH, Blencowe A, Chen G, Voelcker NH. IGF-2 coated porous collagen microwells for the culture of pancreatic islets. J Mater Chem B 2017; 5:220-225. [DOI: 10.1039/c6tb02748b] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
A new platform for the culture of pancreatic islets that improves the cell viability and quality. Paving the way for a highly efficient islet clinical transport.
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20
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Kaviani M, Azarpira N. Insight into microenvironment remodeling in pancreatic endocrine tissue engineering: Biological and biomaterial approaches. Tissue Eng Regen Med 2016; 13:475-484. [PMID: 30603429 PMCID: PMC6170842 DOI: 10.1007/s13770-016-0014-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2016] [Revised: 02/03/2016] [Accepted: 02/05/2016] [Indexed: 01/04/2023] Open
Abstract
The treatment of diabetes mellitus, as a chronic and complicated disease, is a valuable purpose. Islet transplantation can provide metabolic stability and insulin independence in type 1 diabetes patients. Diet and insulin therapy are only diabetes controllers and cannot remove all of the diabetes complications. Moreover, islet transplantation is more promising treatment than whole pancreas transplantation because of lesser invasive surgical procedure and morbidity and mortality. According to the importance of extracellular matrix for islet viability and function, microenvironment remodeling of pancreatic endocrine tissue can lead to more success in diabetes treatment by pancreatic islets. Production of bioengineered pancreas and remodeling of pancreas extracellular matrix provide essential microenvironment for re-vascularization, re-innervation and signaling cascades triggering. Therefore, islets show better viability and function in these conditions. Researchers conduct various scaffolds with different biomaterials for the improvement of islet viability, function and transplantation outcome. The attention to normal pancreas anatomy, embryology and histology is critical to understand the pancreatic Langerhans islets niche and finally to achieve efficient engineered structure. Therefore, in the present study, the status and components of the islets niche is mentioned and fundamental issues related to the tissue engineering of this structure is considered. The purpose of this review article is summarization of recent progress in the endocrine pancreas tissue engineering and biomaterials and biological aspects of it.
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Affiliation(s)
- Maryam Kaviani
- Transplant Research Center, Shiraz University of Medical Sciences, Mohamad Rasulallah Research Tower, Khalili street, Shiraz, 7193635899 Iran
| | - Negar Azarpira
- Transplant Research Center, Shiraz University of Medical Sciences, Mohamad Rasulallah Research Tower, Khalili street, Shiraz, 7193635899 Iran
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21
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Kosinová L, Cahová M, Fábryová E, Týcová I, Koblas T, Leontovyč I, Saudek F, Kříž J. Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies. PLoS One 2016; 11:e0152664. [PMID: 27035827 PMCID: PMC4817981 DOI: 10.1371/journal.pone.0152664] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2015] [Accepted: 03/17/2016] [Indexed: 12/15/2022] Open
Abstract
The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.
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Affiliation(s)
- Lucie Kosinová
- Laboratory of Pancreatic Islets, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
- First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic
| | - Monika Cahová
- Department of Metabolism and Diabetes, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
| | - Eva Fábryová
- Laboratory of Pancreatic Islets, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
| | - Irena Týcová
- Transplant Laboratory, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
| | - Tomáš Koblas
- Laboratory of Pancreatic Islets, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
| | - Ivan Leontovyč
- Laboratory of Pancreatic Islets, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
| | - František Saudek
- Laboratory of Pancreatic Islets, Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
- Department of Diabetes, Center of Diabetes, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
| | - Jan Kříž
- Department of Diabetes, Center of Diabetes, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
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22
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Ramírez-Domínguez M. Isolation of Mouse Pancreatic Islets of Langerhans. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016; 938:25-34. [DOI: 10.1007/978-3-319-39824-2_3] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
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23
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Hawthorne WJ, Williams L, Chew YV. Clinical Islet Isolation. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016; 938:89-122. [PMID: 27586424 DOI: 10.1007/978-3-319-39824-2_7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The overarching success of islet transplantation relies on the success in the laboratory to isolate the islets. This chapter focuses on the processes of human islet cell isolation and the ways to optimally provide islet cells for transplantation. The major improvements in regards to the choice of enzyme type, way the digested pancreas tissue is handled to best separate islets from the acinar and surrounding tissues, the various methods of purification of the islets, their subsequent culture and quality assurance to improve outcomes to culminate in safe and effective islet transplantation will be discussed. After decades of improvements, islet cell isolation and transplantation now clearly offer a safe, effective and feasible therapeutic treatment option for an increasing number of patients suffering from type 1 diabetes specifically for those with severe hypoglycaemic unawareness.
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Affiliation(s)
- Wayne J Hawthorne
- National Pancreas and Islet Transplant Laboratories, The Westmead Institute for Medical Research, Westmead, NSW, 2145, Australia. .,Department of Surgery, Westmead Clinical School, Westmead Hospital, University of Sydney, Westmead, NSW, 2145, Australia.
| | - Lindy Williams
- National Pancreas and Islet Transplant Laboratories, The Westmead Institute for Medical Research, Westmead, NSW, 2145, Australia
| | - Yi Vee Chew
- National Pancreas and Islet Transplant Laboratories, The Westmead Institute for Medical Research, Westmead, NSW, 2145, Australia
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24
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Uzunalli G, Tumtas Y, Delibasi T, Yasa O, Mercan S, Guler MO, Tekinay AB. Improving pancreatic islet in vitro functionality and transplantation efficiency by using heparin mimetic peptide nanofiber gels. Acta Biomater 2015; 22:8-18. [PMID: 25931015 DOI: 10.1016/j.actbio.2015.04.032] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2014] [Revised: 04/17/2015] [Accepted: 04/21/2015] [Indexed: 11/29/2022]
Abstract
Pancreatic islet transplantation is a promising treatment for type 1 diabetes. However, viability and functionality of the islets after transplantation are limited due to loss of integrity and destruction of blood vessel networks. Thus, it is important to provide a proper mechanically and biologically supportive environment for enhancing both in vitro islet culture and transplantation efficiency. Here, we demonstrate that heparin mimetic peptide amphiphile (HM-PA) nanofibrous network is a promising platform for these purposes. The islets cultured with peptide nanofiber gel containing growth factors exhibited a similar glucose stimulation index as that of the freshly isolated islets even after 7 days. After transplantation of islets to STZ-induced diabetic rats, 28 day-long monitoring displayed that islets that were transplanted in HM-PA nanofiber gels maintained better blood glucose levels at normal levels compared to the only islet transplantation group. In addition, intraperitoneal glucose tolerance test revealed that animals that were transplanted with islets within peptide gels showed a similar pattern with the healthy control group. Histological assessment showed that islets transplanted within peptide nanofiber gels demonstrated better islet integrity due to increased blood vessel density. This work demonstrates that using the HM-PA nanofiber gel platform enhances the islets function and islet transplantation efficiency both in vitro and in vivo.
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Affiliation(s)
- Gozde Uzunalli
- Institute of Materials Science and Nanotechnology, National Nanotechnology Research Center (UNAM), Bilkent University, Ankara 06800, Turkey
| | - Yasin Tumtas
- Institute of Materials Science and Nanotechnology, National Nanotechnology Research Center (UNAM), Bilkent University, Ankara 06800, Turkey
| | - Tuncay Delibasi
- Pancreas Islet Cell Research Center, Ankara Diskapi Yildirim Beyazit Training and Research Hospital Etlik Polyclinic, Department of Endocrinology and Metabolism, Ankara 06800, Turkey; Hacettepe University, School of Medicine, Department of Endocrinology, Ankara 06100, Turkey.
| | - Oncay Yasa
- Institute of Materials Science and Nanotechnology, National Nanotechnology Research Center (UNAM), Bilkent University, Ankara 06800, Turkey
| | - Sercan Mercan
- Pancreas Islet Cell Research Center, Ankara Diskapi Yildirim Beyazit Training and Research Hospital Etlik Polyclinic, Department of Endocrinology and Metabolism, Ankara 06800, Turkey; Gazi University, Faculty of Science, Department of Chemistry, Ankara 06560, Turkey
| | - Mustafa O Guler
- Institute of Materials Science and Nanotechnology, National Nanotechnology Research Center (UNAM), Bilkent University, Ankara 06800, Turkey.
| | - Ayse B Tekinay
- Institute of Materials Science and Nanotechnology, National Nanotechnology Research Center (UNAM), Bilkent University, Ankara 06800, Turkey.
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Dadheech N, Srivastava A, Paranjape N, Gupta S, Dave A, Shah GM, Bhonde RR, Gupta S. Swertisin an Anti-Diabetic Compound Facilitate Islet Neogenesis from Pancreatic Stem/Progenitor Cells via p-38 MAP Kinase-SMAD Pathway: An In-Vitro and In-Vivo Study. PLoS One 2015; 10:e0128244. [PMID: 26047129 PMCID: PMC4457488 DOI: 10.1371/journal.pone.0128244] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2014] [Accepted: 04/23/2015] [Indexed: 12/12/2022] Open
Abstract
Transplanting islets serves best option for restoring lost beta cell mass and function. Small bio-chemical agents do have the potential to generate new islets mass, however lack of understanding about mechanistic action of these small molecules eventually restricts their use in cell-based therapies for diabetes. We recently reported "Swertisin" as a novel islet differentiation inducer, generating new beta cells mass more effectively. Henceforth, in the present study we attempted to investigate the molecular signals that Swertisin generate for promoting differentiation of pancreatic progenitors into islet cells. To begin with, both human pancreatic progenitors (PANC-1 cells) and primary cultured mouse intra-islet progenitor cells (mIPC) were used and tested for Swertisin induced islet neogenesis mechanism, by monitoring immunoblot profile of key transcription factors in time dependent manner. We observed Swertisin follow Activin-A mediated MEPK-TKK pathway involving role of p38 MAPK via activating Neurogenin-3 (Ngn-3) and Smad Proteins cascade. This MAP Kinase intervention in differentiation of cells was confirmed using strong pharmacological inhibitor of p38 MAPK (SB203580), which effectively abrogated this process. We further confirmed this mechanism in-vivo in partial pancreatectomised (PPx) mice model, where we could show Swertisin exerted potential increase in insulin transcript levels with persistent down-regulation of progenitor markers like Nestin, Ngn-3 and Pancreatic Duodenal Homeobox Gene-1 (PDX-1) expression, within three days post PPx. With detailed molecular investigations here in, we first time report the molecular mode of action of Swertisin for islet neogenesis mediated through MAP Kinase (MEPK-TKK) pathway involving Ngn-3 and Smad transcriptional regulation. These findings held importance for developing Swertisin as potent pharmacological drug candidate for effective and endogenous differentiation of islets in cell based therapy for diabetes.
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Affiliation(s)
- Nidheesh Dadheech
- Molecular Endocrinology and Stem Cell Research Lab, Department of Biochemistry, Faculty of Science, The M S University of Baroda, Vadodara, Gujarat, India
| | - Abhay Srivastava
- Molecular Endocrinology and Stem Cell Research Lab, Department of Biochemistry, Faculty of Science, The M S University of Baroda, Vadodara, Gujarat, India
| | - Neha Paranjape
- Hislope College of Biotechnology, Nagpur, Maharashtra, India
| | - Shivika Gupta
- Hislope College of Biotechnology, Nagpur, Maharashtra, India
| | - Arpita Dave
- Molecular Endocrinology and Stem Cell Research Lab, Department of Biochemistry, Faculty of Science, The M S University of Baroda, Vadodara, Gujarat, India
| | - Girish M. Shah
- Skin Cancer Research Laboratory, Centre de Recherche du CHUL, CHUQ, Univerisity Laval, Quebec City, Quebec, Canada
| | - Ramesh R. Bhonde
- Manipal Hospital and Regenerative Medicine Centre, Manipal Hospital, Manipal, Karnataka, India
| | - Sarita Gupta
- Molecular Endocrinology and Stem Cell Research Lab, Department of Biochemistry, Faculty of Science, The M S University of Baroda, Vadodara, Gujarat, India
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Truchan NA, Brar HK, Gallagher SJ, Neuman JC, Kimple ME. A single-islet microplate assay to measure mouse and human islet insulin secretion. Islets 2015; 7:e1076607. [PMID: 26452321 PMCID: PMC4708880 DOI: 10.1080/19382014.2015.1076607] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/22/2022] Open
Abstract
One complication to comparing β-cell function among islet preparations, whether from genetically identical or diverse animals or human organ donors, is the number of islets required per assay. Islet numbers can be limiting, meaning that fewer conditions can be tested; other islet measurements must be excluded; or islets must be pooled from multiple animals/donors for each experiment. Furthermore, pooling islets negates the possibility of performing single-islet comparisons. Our aim was to validate a 96-well plate-based single islet insulin secretion assay that would be as robust as previously published methods to quantify glucose-stimulated insulin secretion from mouse and human islets. First, we tested our new assay using mouse islets, showing robust stimulation of insulin secretion 24 or 48 h after islet isolation. Next, we utilized the assay to quantify mouse islet function on an individual islet basis, measurements that would not be possible with the standard pooled islet assay methods. Next, we validated our new assay using human islets obtained from the Integrated Islet Distribution Program (IIDP). Human islets are known to have widely varying insulin secretion capacity, and using our new assay we reveal biologically relevant factors that are significantly correlated with human islet function, whether displayed as maximal insulin secretion response or fold-stimulation of insulin secretion. Overall, our results suggest this new microplate assay will be a useful tool for many laboratories, expert or not in islet techniques, to be able to precisely quantify islet insulin secretion from their models of interest.
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Affiliation(s)
- Nathan A Truchan
- Department of Medicine; Division of Endocrinology, Diabetes & Metabolism; University of Wisconsin-Madison; Madison, WI USA
- William S Middleton Memorial Veterans Hospital; Madison, WI USA
| | - Harpreet K Brar
- Department of Medicine; Division of Endocrinology, Diabetes & Metabolism; University of Wisconsin-Madison; Madison, WI USA
- William S Middleton Memorial Veterans Hospital; Madison, WI USA
| | - Shannon J Gallagher
- Department of Medicine; Division of Endocrinology, Diabetes & Metabolism; University of Wisconsin-Madison; Madison, WI USA
- William S Middleton Memorial Veterans Hospital; Madison, WI USA
| | - Joshua C Neuman
- Interdisciplinary Graduate Program in Nutritional Sciences; University of Wisconsin-Madison; Madison, WI USA
- William S Middleton Memorial Veterans Hospital; Madison, WI USA
| | - Michelle E Kimple
- Department of Medicine; Division of Endocrinology, Diabetes & Metabolism; University of Wisconsin-Madison; Madison, WI USA
- Interdisciplinary Graduate Program in Nutritional Sciences; University of Wisconsin-Madison; Madison, WI USA
- William S Middleton Memorial Veterans Hospital; Madison, WI USA
- Correspondence to: Michelle E Kimple;
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Mansouri A, Esmaeili F, Nejatpour A, Houshmand F, Shabani L, Ebrahimie E. Differentiation of P19 embryonal carcinoma stem cells into insulin-producing cells promoted by pancreas-conditioned medium. J Tissue Eng Regen Med 2014; 10:600-12. [DOI: 10.1002/term.1927] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2013] [Revised: 04/25/2014] [Accepted: 05/05/2014] [Indexed: 12/27/2022]
Affiliation(s)
- Akram Mansouri
- Department of Biology, Faculty of Basic Sciences; Shahrekord University; Iran
| | - Fariba Esmaeili
- Research Institute of Biotechnology; Shahrekord University; Iran
- Department of Biology, Faculty of Basic Sciences; University of Isfahan; Iran
| | | | - Fariba Houshmand
- Department of Physiology, Faculty of Medical Sciences; Shahrekord University of Medical Sciences; Iran
| | - Leila Shabani
- Department of Biology, Faculty of Basic Sciences; Shahrekord University; Iran
- Research Institute of Biotechnology; Shahrekord University; Iran
| | - Esmaeil Ebrahimie
- Institute of Biotechnology; Shiraz University; Shiraz Iran
- School of Molecular and Biomedical Science; The University of Adelaide; Adelaide Australia
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Abdelli S, Papas KK, Mueller KR, Murtaugh MP, Hering BJ, Bonny C. Regulation of the JNK3 signaling pathway during islet isolation: JNK3 and c-fos as new markers of islet quality for transplantation. PLoS One 2014; 9:e99796. [PMID: 24983249 PMCID: PMC4077704 DOI: 10.1371/journal.pone.0099796] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2014] [Accepted: 05/19/2014] [Indexed: 12/22/2022] Open
Abstract
Stress conditions generated throughout pancreatic islet processing initiate the activation of pro-inflammatory pathways and beta-cell destruction. Our goal is to identify relevant and preferably beta-specific markers to assess the activation of beta-cell stress and apoptotic mechanisms, and therefore the general quality of the islet preparation prior to transplantation. Protein expression and activation were analyzed by Western blotting and kinase assays. ATP measurements were performed by a luminescence-based assay. Oxygen consumption rate (OCR) was measured based on standard protocols using fiber optic sensors. Total RNA was used for gene expression analyzes. Our results indicate that pancreas digestion initiates a potent stress response in the islets by activating two stress kinases, c-Jun N-terminal Kinase (JNK) and p38. JNK1 protein levels remained unchanged between different islet preparations and following culture. In contrast, levels of JNK3 increased after islet culture, but varied markedly, with a subset of preparations bearing low JNK3 expression. The observed changes in JNK3 protein content strongly correlated with OCR measurements as determined by the Spearman's rank correlation coefficient rho in the matching islet samples, while inversely correlating with c-fos mRNA expression . In conclusion, pancreas digestion recruits JNK and p38 kinases that are known to participate to beta-cell apoptosis. Concomitantly, the islet isolation alters JNK3 and c-fos expression, both strongly correlating with OCR. Thus, a comparative analysis of JNK3 and c-fos expression before and after culture may provide for novel markers to assess islet quality prior to transplantation. JNK3 has the advantage over all other proposed markers to be islet-specific, and thus to provide for a marker independent of non-beta cell contamination.
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Affiliation(s)
- Saida Abdelli
- Departement of Medical Genetics, Centre Hospitalier Universitaire Vaudois (CHUV) and University of Lausanne, Lausanne, Switzerland
| | - Klearchos K. Papas
- Department of Surgery, University of Arizona, Institute for Cellular Transplantation, Tucson, Arizona, United States of America
| | - Kate R. Mueller
- Department of Surgery, University of Arizona, Institute for Cellular Transplantation, Tucson, Arizona, United States of America
| | - Mike P. Murtaugh
- Department of Veterinary and Biomedical Sciences, St. Paul, University of Minnesota, Minneapolis, Minnesota, United States of America
| | - Bernhard J. Hering
- Department of Surgery, Schulze Diabetes Institute, University of Minnesota, Minneapolis, Minnesota, United States of America
| | - Christophe Bonny
- Departement of Medical Genetics, Centre Hospitalier Universitaire Vaudois (CHUV) and University of Lausanne, Lausanne, Switzerland
- * E-mail:
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29
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Hughes A, Rojas-Canales D, Drogemuller C, Voelcker NH, Grey ST, Coates PTH. IGF2: an endocrine hormone to improve islet transplant survival. J Endocrinol 2014; 221:R41-8. [PMID: 24883437 DOI: 10.1530/joe-13-0557] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
In the week following pancreatic islet transplantation, up to 50% of transplanted islets are lost due to apoptotic cell death triggered by hypoxic and pro-inflammatory cytokine-mediated cell stress. Thus, therapeutic approaches designed to protect islet cells from apoptosis could significantly improve islet transplant success. IGF2 is an anti-apoptotic endocrine protein that inhibits apoptotic cell death through the mitochondrial (intrinsic pathway) or via antagonising activation of pro-inflammatory cytokine signalling (extrinsic pathway), in doing so IGF2 has emerged as a promising therapeutic molecule to improve islet survival in the immediate post-transplant period. The development of novel biomaterials coated with IGF2 is a promising strategy to achieve this. This review examines the mechanisms mediating islet cell apoptosis in the peri- and post-transplant period and aims to identify the utility of IGF2 to promote islet survival and enhance long-term insulin independence rates within the setting of clinical islet transplantation.
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Wang X, Chang F, Bai Y, Chen F, Zhang J, Chen L. Bisphenol A enhances kisspeptin neurons in anteroventral periventricular nucleus of female mice. J Endocrinol 2014; 221:201-13. [PMID: 24532816 DOI: 10.1530/joe-13-0475] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Bisphenol-A (BPA), an environmental estrogen, adversely affects female reproductive health. However, the underlying mechanisms remain largely unknown. We found that oral administration (p.o.) of BPA (20 μg/kg) to adult female mice at proestrus, but not at estrus or diestrus, significantly increased the levels of plasma E₂, LH and FSH, and Gnrh mRNA within 6 h. The administration of BPA at proestrus, but not at diestrus, could elevate the levels of Kiss1 mRNA and kisspeptin protein in anteroventral periventricular nucleus (AVPV) within 6 h. In contrast, the level of Kiss1 mRNA in arcuate nucleus (ARC) was hardly altered by BPA administration. In addition, at proestrus, a single injection (i.c.v.) of BPA dose-dependently enhanced the AVPV-kisspeptin expression within 6 h, this was sensitive to E₂ depletion by ovariectomy and an estrogen receptor α (ERα) antagonist. Similarly, the injection of BPA (i.c.v.) at proestrus could elevate the levels of plasma E₂, LH, and Gnrh mRNA within 6 h in a dose-dependent manner, which was blocked by antagonists of GPR54 or ERα. Injection of BPA (i.c.v.) at proestrus failed to alter the timing and peak concentration of LH-surge generation. In ovariectomized mice, the application of E₂ induced a dose-dependent increase in the AVPV-Kiss1 mRNA level, indicating 'E₂-induced positive feedback', which was enhanced by BPA injection (i.c.v.). The levels of Erα (Esr1) and Erβ (Esr2) mRNAs in AVPV and ARC did not differ significantly between vehicle-and BPA-treated groups. This study provides in vivo evidence that exposure of adult female mice to a low dose of BPA disrupts the hypothalamic-pituitary-gonadal reproductive endocrine system through enhancing AVPV-kisspeptin expression and release.
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Affiliation(s)
- Xiaoli Wang
- State Key Laboratory of Reproductive Medicine Department of Physiology, Nanjing Medical University, Hanzhong Road 140, Nanjing 210029, China MOE-Shanghai Key Laboratory of Children's Environmental Health, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
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31
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Aida K, Saitoh S, Nishida Y, Yokota S, Ohno S, Mao X, Akiyama D, Tanaka S, Awata T, Shimada A, Oikawa Y, Shimura H, Furuya F, Takizawa S, Ichijo M, Ichijo S, Itakura J, Fujii H, Hashiguchi A, Takasawa S, Endo T, Kobayashi T. Distinct cell clusters touching islet cells induce islet cell replication in association with over-expression of Regenerating Gene (REG) protein in fulminant type 1 diabetes. PLoS One 2014; 9:e95110. [PMID: 24759849 PMCID: PMC3997392 DOI: 10.1371/journal.pone.0095110] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2013] [Accepted: 03/23/2014] [Indexed: 01/09/2023] Open
Abstract
BACKGROUND Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs), extracellular matrix (ECM), and possible cell clusters, are unclear. PROCEDURES The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans. RESULT Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG) Iα protein. The vesicles containing REG Iα protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG Iα protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells. CONCLUSION The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG Iα protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Iα in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells.
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Affiliation(s)
- Kaoru Aida
- Department of Internal Medicine III, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Sei Saitoh
- Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Yoriko Nishida
- Department of Nursing, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Sadanori Yokota
- Section of Functional Morphology, Faculty of Pharmaceutical Sciences, Nagasaki International University, Saseho, Nagasaki, Japan
| | - Shinichi Ohno
- Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Xiayang Mao
- Department of Computer Science, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Daiichiro Akiyama
- Department of Internal Medicine III, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Shoichiro Tanaka
- Department of Internal Medicine III, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Takuya Awata
- Division of Endocrinology and Diabetes, Department of Medicine, Saitama Medical School, Moroyama, Saitama, Japan
| | - Akira Shimada
- Department of Internal Medicine, Saiseikai Central Hospital, Tokyo, Japan
| | - Youichi Oikawa
- Department of Internal Medicine, Saiseikai Central Hospital, Tokyo, Japan
| | - Hiroki Shimura
- Department of Laboratory Medicine, Fukushima Medical University, Fukushima, Fukushima, Japan
| | - Fumihiko Furuya
- Department of Internal Medicine III, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Soichi Takizawa
- Department of Internal Medicine III, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Masashi Ichijo
- Department of Internal Medicine III, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Sayaka Ichijo
- Department of Internal Medicine III, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Jun Itakura
- Department of Surgery I, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Hideki Fujii
- Department of Surgery I, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Akinori Hashiguchi
- Department of Pathology, Keio University School of Medicine, Tokyo, Japan
| | - Shin Takasawa
- Department of Biochemistry, Nara Medical University, Kashihara, Wakayama, Japan
| | - Toyoshi Endo
- Department of Internal Medicine III, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
| | - Tetsuro Kobayashi
- Department of Internal Medicine III, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
- * E-mail:
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Yamada S, Shimada M, Utsunomiya T, Ikemoto T, Saito Y, Morine Y, Imura S, Mori H, Arakawa Y, Kanamoto M, Iwahashi S. Trophic effect of adipose tissue-derived stem cells on porcine islet cells. J Surg Res 2013; 187:667-72. [PMID: 24238974 DOI: 10.1016/j.jss.2013.10.031] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2013] [Revised: 10/11/2013] [Accepted: 10/17/2013] [Indexed: 01/06/2023]
Abstract
BACKGROUND Adipose tissue-derived stem cells (ADSCs), which are widely known as multipotent progenitor cells, release several cytokines that support cell survival and repair. The aim of this study was to investigate whether ADSC-secreted molecules could induce a trophic effect in pancreatic islet culture conditions in vitro. MATERIALS AND METHODS We cocultured porcine islet cells with ADSCs using a transwell system for 48 h and evaluated the viability of islet cells. We also determined the concentration levels of cytokines and insulin in the supernatant of the culture medium. We used anti-vascular endothelial growth factor (VEGF) and anti-interleukin (IL)-6 receptor antibodies to investigate the effect of VEGF and IL-6 on islet cells. RESULTS ADSCs improved the viability of islet cells in the absence of cell-cell contact (P < 0.05). VEGF and IL-6 levels in the culture medium increased when islet cells were cocultured with ADSCs (P < 0.05). Furthermore, inhibition of VEGF decreased the viability of islet cells (P < 0.05); however, inhibition of IL-6 did not affect islet cell viability. CONCLUSIONS These results suggested that trophic factors, particularly VEGF, secreted by human ADSCs enhanced the survival and function of porcine islet cells.
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Affiliation(s)
- Shinichiro Yamada
- Department of Digestive and Transplant Surgery, The University of Tokushima, Tokushima City, Tokushima, Japan
| | - Mitsuo Shimada
- Department of Digestive and Transplant Surgery, The University of Tokushima, Tokushima City, Tokushima, Japan.
| | - Tohru Utsunomiya
- Department of Digestive and Transplant Surgery, The University of Tokushima, Tokushima City, Tokushima, Japan
| | - Tetsuya Ikemoto
- Department of Digestive and Transplant Surgery, The University of Tokushima, Tokushima City, Tokushima, Japan
| | - Yu Saito
- Department of Digestive and Transplant Surgery, The University of Tokushima, Tokushima City, Tokushima, Japan
| | - Yuji Morine
- Department of Digestive and Transplant Surgery, The University of Tokushima, Tokushima City, Tokushima, Japan
| | - Satoru Imura
- Department of Digestive and Transplant Surgery, The University of Tokushima, Tokushima City, Tokushima, Japan
| | - Hiroki Mori
- Department of Digestive and Transplant Surgery, The University of Tokushima, Tokushima City, Tokushima, Japan
| | - Yusuke Arakawa
- Department of Digestive and Transplant Surgery, The University of Tokushima, Tokushima City, Tokushima, Japan
| | - Mami Kanamoto
- Department of Digestive and Transplant Surgery, The University of Tokushima, Tokushima City, Tokushima, Japan
| | - Shuichi Iwahashi
- Department of Digestive and Transplant Surgery, The University of Tokushima, Tokushima City, Tokushima, Japan
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Li Z, Sun H, Zhang J, Zhang H, Meng F, Cui Z. Development of in vitro 3D TissueFlex® islet model for diabetic drug efficacy testing. PLoS One 2013; 8:e72612. [PMID: 23977329 PMCID: PMC3744493 DOI: 10.1371/journal.pone.0072612] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2013] [Accepted: 07/12/2013] [Indexed: 11/19/2022] Open
Abstract
Increasing individuals diagnosed with type II diabetes pose a strong demand for the development of more effective anti-diabetic drugs. However, expensive, ethically controversial animal-based screening for anti-diabetic compounds is not always predictive of the human response. The use of in vitro cell-based models in research presents obviously ethical and cost advantages over in vivo models. This study was to develop an in vitro three-dimensional (3D) perfused culture model of islets (Islet TF) for maintaining viability and functionality longer for diabetic drug efficacy tests. Briefly fresh isolated rat islets were encapsulated in ultrapure alginate and the encapsulated islets were cultured in TissueFlex(®), a multiple, parallel perfused microbioreactor system for 7 days. The encapsulated islets cultured statically in cell culture plates (3D static) and islets cultured in suspension (2D) were used as the comparisons. In this study we demonstrate for the first time that Islet TF model can maintain the in vitro islet viability, and more importantly, the elevated functionality in terms of insulin release and dynamic responses over a 7-day culture period. The Islet TF displays a high sensitivity in responding to drugs and drug dosages over conventional 2D and 3D static models. Actual drug administration in clinics could be simulated using the developed Islet TF model, and the patterns of insulin release response to the tested drugs were in agreement with the data obtained in vivo. Islet TF could be a more predictive in vitro model for routine short- and long-term anti-diabetic drug efficacy testing.
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Affiliation(s)
- Zhaohui Li
- Institute of Biomedical Engineering, University of Oxford, Oxford, United Kingdom
| | - He Sun
- Tianjin Weikai Bioeng Ltd, Tianjin, China
| | | | | | - Fanyu Meng
- Tianjin Weikai Bioeng Ltd, Tianjin, China
| | - Zhanfeng Cui
- Institute of Biomedical Engineering, University of Oxford, Oxford, United Kingdom
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Yanai G, Hayashi T, Zhi Q, Yang KC, Shirouzu Y, Shimabukuro T, Hiura A, Inoue K, Sumi S. Electrofusion of mesenchymal stem cells and islet cells for diabetes therapy: a rat model. PLoS One 2013; 8:e64499. [PMID: 23724055 PMCID: PMC3665804 DOI: 10.1371/journal.pone.0064499] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2012] [Accepted: 04/14/2013] [Indexed: 01/22/2023] Open
Abstract
Islet transplantation is a minimally invasive treatment for severe diabetes. However, it often requires multiple donors to accomplish insulin-independence and the long-term results are not yet satisfying. Therefore, novel ways to overcome these problems have been explored. Isolated islets are fragile and susceptible to pro-apoptotic factors and poorly proliferative. In contrast, mesenchymal stem cells (MSCs) are highly proliferative, anti-apoptotic and pluripotent to differentiate toward various cell types, promote angiogenesis and modulate inflammation, thereby studied as an enhancer of islet function and engraftment. Electrofusion is an efficient method of cell fusion and nuclear reprogramming occurs in hybrid cells between different cell types. Therefore, we hypothesized that electrofusion between MSC and islet cells may yield robust islet cells for diabetes therapy. We establish a method of electrofusion between dispersed islet cells and MSCs in rats. The fusion cells maintained glucose-responsive insulin release for 20 days in vitro. Renal subcapsular transplantation of fusion cells prepared from suboptimal islet mass (1,000 islets) that did not correct hyperglycemia even if co-transplanted with MSCs, caused slow but consistent lowering of blood glucose with significant weight gain within the observation period in streptozotocin-induced diabetic rats. In the fusion cells between rat islet cells and mouse MSCs, RT-PCR showed new expression of both rat MSC-related genes and mouse β-cell-related genes, indicating bidirectional reprogramming of both β-cell and MSCs nuclei. Moreover, decreased caspase3 expression and new expression of Ki-67 in the islet cell nuclei suggested alleviated apoptosis and gain of proliferative capability, respectively. These results show that electrofusion between MSCs and islet cells yield special cells with β-cell function and robustness of MSCs and seems feasible for novel therapeutic strategy for diabetes mellitus.
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Affiliation(s)
- Goichi Yanai
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | | | - Qi Zhi
- Department of Histology and Embryology, School of Medicine, Nankai University, Tianjin, China
| | - Kai-Chiang Yang
- School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan
| | - Yasumasa Shirouzu
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | | | - Akihito Hiura
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
| | | | - Shoichiro Sumi
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
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Insulin-Like growth factor-II (IGF-II) prevents proinflammatory cytokine-induced apoptosis and significantly improves islet survival after transplantation. Transplantation 2013; 95:671-8. [PMID: 23364485 DOI: 10.1097/tp.0b013e31827fa453] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND The early loss of functional islet mass (50-70%) due to apoptosis after clinical transplantation contributes to islet allograft failure. Insulin-like growth factor (IGF)-II is an antiapoptotic protein that is highly expressed in β-cells during development but rapidly decreases in postnatal life. METHODS We used an adenoviral (Ad) vector to overexpress IGF-II in isolated rat islets and investigated its antiapoptotic action against exogenous cytokines interleukin-1β- and interferon-γ-induced islet cell death in vitro. Using an immunocompromised marginal mass islet transplant model, the ability of Ad-IGF-II-transduced rat islets to restore euglycemia in nonobese diabetic/severe combined immunodeficient diabetic recipients was assessed. RESULTS Ad-IGF-II transduction did not affect islet viability or function. Ad-IGF-II cytokine-treated islets exhibited decreased cell death (40% ± 2.8%) versus Ad-GFP and untransduced control islets (63.2% ± 2.5% and 53.6% ± 2.3%, respectively). Ad-IGF-II overexpression during cytokine treatment resulted in a marked reduction in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells (8.3% ± 1.4%) versus Ad-GFP control (41% ± 4.2%) and untransduced control islets (46.5% ± 6.2%). Western blot analysis confirmed that IGF-II inhibits apoptosis via activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. Transplantation of IGF-II overexpressing islets under the kidney capsule of diabetic mice restored euglycemia in 77.8% of recipients compared with 18.2% and 47.5% of Ad-GFP and untransduced control islet recipients, respectively (P<0.05, log-rank [Mantel-Cox] test). CONCLUSIONS Antiapoptotic IGF-II decreases apoptosis in vitro and significantly improved islet transplant outcomes in vivo. Antiapoptotic gene transfer is a potentially powerful tool to improve islet survival after transplantation.
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Kimura Y, Okitsu T, Xibao L, Teramae H, Okonogi A, Toyoda K, Uemoto S, Fukushima M. Improved hypothermic short-term storage of isolated mouse islets by adding serum to preservation solutions. Islets 2013; 5:45-52. [PMID: 23552019 PMCID: PMC3655792 DOI: 10.4161/isl.24025] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Preserving isolated islets at low temperature appears attractive because it can keep islet quantity comparable to freshly isolated islets. In this study, we evaluated the effect of serum as an additive to preservation solutions on islet quality after short-term hypothermic storage. Isolated mouse islets were preserved at 4°C in University of Wisconsin solution (UW) alone, UW with serum, M-Kyoto solution (MK) alone or MK with serum. We then assessed islet quantity, morphology, viability and function in vitro as well as in vivo. Islet quantity after storage in all four solutions was well maintained for up to 120 h. However, islets functioned for different duration; glucose-stimulated insulin release assay revealed that the duration was 72 h when islets were stored in UW with serum and MK with serum, but only 24 h in UW alone, and the islet function disappeared immediately in MK alone. Viability assay confirmed that more than 70% islet cells survived for up to 48 h when islets are preserved in UW with serum and MK with serum, but the viability decreased rapidly in UW alone and MK alone. In in vivo bioassays using 48-h preserved isogeneic islets, all recipient mice restored normal blood glucose concentrations by transplants preserved in UW with serum or MK with serum, whereas 33.3% recipients and no recipient restored diabetes by transplants preserved in UW alone and in MK alone respectively. Adding serum to both UW and MK improves their capability to store isolated islets by maintaining islet functional viability.
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Affiliation(s)
- Yasuko Kimura
- Translational Research Informatics Center; Foundation for Biomedical Research and Innovation; Kobe, Japan
| | - Teru Okitsu
- Institute of Industrial Science; University of Tokyo; Tokyo, Japan
- Correspondence to: Teru Okitsu,
| | - Liu Xibao
- Department of Endocrinology; the First Hospital of Hebei Medical University; ShiJiaZhuang, China
| | - Hiroki Teramae
- Faculty of Teacher Education; Shumei University ; Yachiyo, Chiba, Japan
| | | | - Kentaro Toyoda
- Department of Diabetes and Clinical Nutrition; Graduate School of Medicine; Kyoto University; Kyoto, Japan
| | - Shinji Uemoto
- Department of Surgery; Division of Hepato-Pancreato-Biliary Surgery and Transplantation; Kyoto University Hospital; Kyoto, Japan
| | - Masanori Fukushima
- Translational Research Informatics Center; Foundation for Biomedical Research and Innovation; Kobe, Japan
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Fraker CA, Cechin S, Álvarez-Cubela S, Echeverri F, Bernal A, Poo R, Ricordi C, Inverardi L, Domínguez-Bendala J. A physiological pattern of oxygenation using perfluorocarbon-based culture devices maximizes pancreatic islet viability and enhances β-cell function. Cell Transplant 2012; 22:1723-33. [PMID: 23068091 DOI: 10.3727/096368912x657873] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Conventional culture vessels are not designed for physiological oxygen (O2) delivery. Both hyperoxia and hypoxia-commonly observed when culturing cells in regular plasticware-have been linked to reduced cellular function and death. Pancreatic islets, used for the clinical treatment of diabetes, are especially sensitive to sub- and supraphysiological O2 concentrations. A result of current culture standards is that a high percentage of islet preparations are never transplanted because of cell death and loss of function in the 24-48 h postisolation. Here, we describe a new culture system designed to provide quasiphysiological oxygenation to islets in culture. The use of dishes where islets rest atop a perfluorocarbon (PFC)-based membrane, coupled with a careful adjustment of environmental O2 concentration to target the islet physiological pO2 range, resulted in dramatic gains in viability and function. These observations underline the importance of approximating culture conditions as closely as possible to those of the native microenvironment, and fill a widely acknowledged gap in our ability to preserve islet functionality in vitro. As stem cell-derived insulin-producing cells are likely to suffer from the same limitations as those observed in real islets, our findings are especially timely in the context of current efforts to define renewable sources for transplantation.
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Affiliation(s)
- Chris A Fraker
- Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, USA
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Webb MA, Chen JJ, Illouz SC, Pollard CA, Dennison B, West KP, James RFL, Dennison AR. The impact of potential islet precursor cells on islet autotransplantation outcomes. Cell Transplant 2012; 22:1041-51. [PMID: 23007077 DOI: 10.3727/096368912x655046] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Islet autotransplant patients represent excellent subjects to assess the posttransplant impact of islet precursors, as chronic pancreatitis (CP) causes an elevation of ductal cells, pancreatic precursors cells, and hormone-positive acinar cells. The relationship between these cell types and autograft outcomes should be more apparent than would be the case in the context of an allograft program with confounding immunological variables. To improve diabetic control following total pancreatectomy for CP, nonpurified islets were autotransplanted into the liver. Pancreas specimens were recovered from 23 patients and stained for antigens including: insulin, glucagon, cytokeratin 19, cytokeratin 7, and PDX-1. In line with previous reports, the prevalence of ductal cells, non-islet endocrine cells and non-islet PDX-1-expressing cells was significantly higher in CP glands compared with normal pancreata. When correlating follow-up data (i.e., fasting and stimulated C-peptide/glucose levels and HbA1c%) with pancreas immunoreactivity, high levels of ductal cells, non-islet PDX-1-positive cells, and non-islet glucagon-positive cells were associated with superior outcomes, detectable up to 2 years posttransplant. To conclude, the acinar parenchyma and ductal epithelium of the CP pancreas show an upregulation of both endocrine and pre-endocrine cell types, which appear to have a positive effect on islet graft outcomes in autotransplantation setting.
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Affiliation(s)
- M A Webb
- Department of Hepatobiliary Surgery, University Hospitals of Leicester, NHS Trust, Leicester General Hospital, Leicester, UK.
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Chhoun JM, Voltzke KJ, Firpo MT. From cell culture to a cure: pancreatic β-cell replacement strategies for diabetes mellitus. Regen Med 2012; 7:685-95. [DOI: 10.2217/rme.12.39] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Numerous advances have been made in pancreatic β-cell replacement therapies for diabetes mellitus. While these therapies provide a positive impact and possible cure for the individual recipient, access is limited by availability of donor tissues. The derivation of pluripotent stem cells using efficient differentiation technologies has resulted in the generation of insulin-producing cells with characteristics similar to islet β-cells. Experimental transplantation studies have shown that these cells are capable of reducing hyperglycemia in short-term assays. Novel methodologies that facilitate the neogenesis of β-cells from endogenous hepatic or pancreatic tissue sources are also being investigated as a β-cell replacement strategy. Further research is necessary to protect these transplanted or regenerated cells from diabetic autoimmune pathology.
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Affiliation(s)
- Jennifer M Chhoun
- University of Minnesota, Department of Medicine, 2001 6th Street SE, Minneapolis, MN 55455, USA
| | - Kristin J Voltzke
- University of Minnesota, Department of Medicine, 2001 6th Street SE, Minneapolis, MN 55455, USA
| | - Meri T Firpo
- University of Minnesota, Department of Medicine, 2001 6th Street SE, Minneapolis, MN 55455, USA
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40
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Kim JS, Lim JH, Nam HY, Lim HJ, Shin JS, Shin JY, Ryu JH, Kim K, Kwon IC, Jin SM, Kim HR, Kim SJ, Park CG. In situ application of hydrogel-type fibrin-islet composite optimized for rapid glycemic control by subcutaneous xenogeneic porcine islet transplantation. J Control Release 2012; 162:382-90. [PMID: 22820449 DOI: 10.1016/j.jconrel.2012.07.018] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2012] [Accepted: 07/12/2012] [Indexed: 11/15/2022]
Abstract
Maximum engraftment of transplanted islets is essential for the clinical application of a subcutaneous site. Significant barriers to the current approaches are associated with their low effectiveness, complexity and unproven biosafety. Here, we evaluated and optimized a fibrin-islet composite for effective glycemic control in a subcutaneous site whose environment is highly hypoxic due to low vascularization potential. In the setting of xenogeneic porcine islet transplantation into the subcutaneous space of a diabetic mouse, the in vivo islet functions were greatly affected by the concentrations of fibrinogen and thrombin. The optimized hydrogel-type fibrin remarkably reduced the marginal islet mass to approximately one tenth that of islets without fibrin. This marginal islet mass was comparable to that in the setting of the subcapsular space of the kidney, which is a highly vascularized organ. Highly vascularized structures were generated inside and on the outer surface of the grafts. A hydrogel-type fibrin-islet composite established early diabetic control within an average of 3.4days after the transplantation. In the mechanistic studies, fibrin promoted local angiogenesis, enhanced islet viability and prevented fragmentation of islets into single cells. In conclusion, in situ application of hydrogel-type fibrin-islet composite may be a promising modality in the clinical success of subcutaneous islet transplantation.
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Affiliation(s)
- Jung-Sik Kim
- Department of Microbiology and Immunology, Seoul National University College of Medicine, 103 Daehak-Ro, Jongno-Gu, Seoul 110-799, South Korea
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Enhanced function of pancreatic islets co-encapsulated with ECM proteins and mesenchymal stromal cells in a silk hydrogel. Biomaterials 2012; 33:6691-7. [PMID: 22766242 DOI: 10.1016/j.biomaterials.2012.06.015] [Citation(s) in RCA: 115] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2012] [Accepted: 06/14/2012] [Indexed: 01/06/2023]
Abstract
Pancreatic islet encapsulation within biosynthetic materials has had limited clinical success due to loss of islet function and cell death. As an alternative encapsulation material, a silk-based scaffold was developed to reestablish the islet microenvironment lost during cell isolation. Islets were encapsulated with ECM proteins (laminin and collagen IV) and mesenchymal stromal cells (MSCs), known to have immunomodulatory properties or to enhance islet cell graft survival and function. After a 7 day in vitro encapsulation, islets remained viable and maintained insulin secretion in response to glucose stimulation. Islets encapsulated with collagen IV, or laminin had increased insulin secretion at day 2 and day 7, respectively. A 3.2-fold synergistic improvement in islet insulin secretion was observed when islets were co-encapsulated with MSCs and ECM proteins. Furthermore, encapsulated islets had increased gene expression of functional genes; insulin I, insulin II, glucagon, somatostatin, and PDX-1, and lower expression of the de-differentiation genes cytokeratin 19 and vimentin compared to non-encapsulated cells. This work demonstrates that encapsulation in silk with both MSCs and ECM proteins enhances islet function and with further development may have potential as a suitable platform for islet delivery in vivo.
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Webb MA, Dennison AR, James RF. The potential benefit of non-purified islets preparations for islet transplantation. Biotechnol Genet Eng Rev 2012; 28:101-14. [PMID: 22616483 DOI: 10.5661/bger-28-101] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Since the advent of islet transplantation, there has been a significant emphasis on the importance of islet purity despite an inevitable associated loss of islet mass during the purification process. One of the key elements of the 'Edmonton Protocol' for islet transplantation published in 2000 was an emphasis on the need for sequential transplants of highly purified islets (averaging 24% beta cell purity) and the close correlation between the numbers of islets transplanted and the success of the procedure. However, the emphasis on islet purity may warrant further consideration as auto transplantation of non-purified islets currently provides the most successful insulin independence rates within the field of islet transplantation. While the role of auto and allo immunity could contribute to the differences in the success rates it is clear that within the clinical setting, significant acinar and ductal contamination is well tolerated. However, one could go further and hypothesize that extra-insular tissue including acinar tissue, ductal tissue, peri-pancreatic lymph nodes and vascular tissue actually confer an advantage to islet survival/function and may even contribute to the insulin secreting capacity of the graft post transplant. As such this review will assess the influence of extra-insular pancreatic tissue on the results of islet transplantation based on published evidence and will also explore the possibility that non-islet pancreatic cells are capable of differentiating into a beta cell phenotype in vivo contributing to an ongoing regeneration of endocrine mass during the period following transplantation.
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Affiliation(s)
- M'Balu A Webb
- Department of Hepatobiliary Surgery, University Hospitals of Leicester, NHS Trust, Leicester General Hospital, Gwendolen Road, Leicester LE5 4PW, UK.
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Estil les E, Téllez N, Escoriza J, Montanya E. Increased β-cell replication and β-cell mass regeneration in syngeneically transplanted rat islets overexpressing insulin-like growth factor II. Cell Transplant 2012; 21:2119-29. [PMID: 22507193 DOI: 10.3727/096368912x638955] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Insulin-like growth factor II (IGF2) is a growth-promoting peptide that increases β-cell proliferation and survival. The aim of the study was to determine the effect of IGF2 overexpression on β-cell mass in transplanted islets. Islets infected with adenovirus encoding for IGF2 (Ad-IGF2 group), for luciferase (Ad-Luc control group), or with uninfected islets (control group) were syngeneically transplanted to streptozotocin-diabetic Lewis rats. Eight hundred islets, a minimal mass model to restore normoglycemia, or 500 islets, a clearly insufficient mass, were transplanted. Rats transplanted with 800 Ad-IGF2 islets showed a better metabolic evolution than control groups. As expected, rats transplanted with 500 Ad-IGF2 or control islets maintained similar hyperglycemia throughout the study, ensuring comparable metabolic conditions among both groups. β-Cell replication was higher in Ad-IGF2 group than in control group on days 3 [1.45% (IQR: 0.26) vs. 0.58% (IQR: 0.18), p = 0.006], 10 [1.58% (IQR: 1.40) vs. 0.90% (IQR: 0.61), p = 0.035], and 28 [1.35% (IQR: 0.35) vs. 0.64% (IQR: 0.28), p = 0.004] after transplantation. β-Cell mass was similarly reduced on day 3 after transplantation in Ad-IGF2 and control group [0.36 mg (IQR: 0.26) vs. 0.38 mg (IQR: 0.19)], it increased on day 10, and on day 28 it was higher in Ad-IGF2 than in control group [0.63 mg (IQR: 0.38) vs. 0.42 mg (IQR: 0.31), p = 0.008]. Apoptosis was similarly increased in Ad-IGF2 and control islets after transplantation. No differences in insulin secretion were found between Ad-IGF2 and uninfected control islets. In summary, IGF2 overexpression in transplanted islets increased β-cell replication, induced the regeneration of the transplanted β-cell mass, and had a beneficial effect on the metabolic outcome reducing the β-cell mass needed to achieve normoglycemia.
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Affiliation(s)
- Elisabet Estil les
- Laboratory of Diabetes and Experimental Endocrinology, Department of Clinical Sciences, IDIBELL-University of Barcelona, Barcelona, Spain
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Abstract
OBJECTIVES We recently reported that mitomycin C (MMC) treatment and subsequent culture of islets significantly prolongs graft survival in allotransplantation and xenotransplantation models. The present study was performed to determine the changes in morphology and signal transduction in pancreatic islets after MMC treatment. METHODS Freshly isolated rat islets were treated with 10 μg/mL MMC for 30 minutes and then cultured for up to 3 days. The samples were processed for immunohistologic studies and electron microscopic examination at various times after treatment. A DNA fragmentation assay was performed to detect apoptotic cell death. Western blotting was performed to determine the effects of MMC on signal transduction. RESULTS As early as 4 hours after culture, the islets showed central damage; most cells were necrotic and stained with anti-high mobility group box 1 antibody, and a few were apoptotic. The ratio of the damaged area to the whole area was significantly decreased after MMC treatment. Western blotting showed that MMC treatment increased the levels of activated forms of p53 and p21, whereas levels of the activated forms of Akt and caspase-3 were unchanged. CONCLUSIONS Mitomycin C treatment protects islets from the progression of central damage during culture. The p53-p21 pathway might be involved in these effects. ABBREVIATIONS MMC - mitomycin C, HMGB1 - high mobility group box 1.
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Loganathan G, Dawra RK, Pugazhenthi S, Guo Z, Soltani SM, Wiseman A, Sanders MA, papas KK, Kumaravel V, Saluja AK, Sutherland DE, Hering BJ, Balamurugan AN. Insulin degradation by acinar cell proteases creates a dysfunctional environment for human islets before/after transplantation: benefits of α-1 antitrypsin treatment. Transplantation 2011; 92:1222-30. [PMID: 22089666 PMCID: PMC3587768 DOI: 10.1097/tp.0b013e318237585c] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Pancreatic acinar cells are commonly cotransplanted along with islets during auto- and allotransplantations. The aims of this study were to identify how acinar cell proteases cause human islet cell loss before and after transplantation of impure islet preparations and to prevent islet loss and improve function with supplementation of α-1 antitrypsin (A1AT). METHODS Acinar cell protease activity, insulin levels, and percent islet loss were measured after culture of pure and impure clinical islet preparations. The effect of proteases on ultrastructure of islets and β-cell insulin granules were examined by transmission electron microscopy. The number of insulin granules and insulin-labeled immunogold particles were counted. The in vivo effect of proteases on islet function was studied by transplanting acinar cells adjacent to islet grafts in diabetic mice. The effects of A1AT culture supplementation on protease activity, insulin levels, and islet function were assessed in pure and impure islets. RESULTS Islet loss after culture was significantly higher in impure relative to pure preparations (30% vs. 14%, P<0.04). Lower islet purity was associated with increased protease activity and decreased insulin levels in culture supernatants. Reduced β-cell insulin granules and insulin degradation by proteases were confirmed by transmission electron microscopy. Transplantations in mice showed delayed islet graft function when acinar cells were transplanted adjacent to the islets under the kidney capsule. Supplementation of A1AT to impure islet cultures maintained islet cell mass, restored insulin levels, and preserved islet functional integrity. CONCLUSION Culture of impure human islet fractions in the presence of A1AT prevents insulin degradation and improves islet recovery.
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Affiliation(s)
| | - Rajinder K. Dawra
- Department of Surgery, Basic and Translational Research, University of Minnesota, Minneapolis, MN.
| | | | - Zhiguang Guo
- Sanford Project, Sanford Health/University of South Dakota, Sioux Falls, SD.
| | - Sajjad M. Soltani
- Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN.
| | | | | | - Klearchos K. papas
- Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN.
| | - V. Kumaravel
- Alpha Hospital and Research Center, Institute of Diabetes and Endocrinology, Madurai-9, Tamilnadu, India.
| | - Ashok K. Saluja
- Department of Surgery, Basic and Translational Research, University of Minnesota, Minneapolis, MN.
| | - David E.R. Sutherland
- Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN.
| | - Bernhard J. Hering
- Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN.
| | - A. N. Balamurugan
- Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN.
- Corresponding author: Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN, 55455, USA. Phone: 651-253-0656, Fax: 612-626-5855
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Abstract
OBJECTIVES We aimed to develop an accurate and reproducible method to quantify transplanted islets and monitor their functional status in vivo. To support this aim, we investigated the cytotoxic effect of Resovist on islet function and survival. METHODS The average pixel number for a single Resovist-labeled islet was measured. To determine Resovist cytotoxicity, DNA fragmentation, adenosine diphosphate-adenosine triphosphate ratio, ion channel activity, and in vivo islet function were evaluated. To quantitatively monitor the fate of islet transplant, we transplanted Resovist-labeled islets into syngeneic C57BL/6 mice for magnetic resonance imaging analysis. RESULTS The average pixel volume for a medium-sized islet (100-150 μm in diameter) was determined from the contrast signal void of magnetic resonance image. Toxicological analysis showed that Resovist did not affect islet at concentrations up to 40 times the labeling dose. In the quantitative analysis, the number of contrast spots did not correlated with the number of transplanted islets, whereas our newly adopted measure showed a significant correlation. CONCLUSIONS Islet transplant survival may be safely and accurately monitored using magnetic resonance imaging with the Resovist. We found in this study that pixel number may correlate more closely than the number of contrast spots with the number of islets transplanted.
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Cell-cell communication mimicry with poly(ethylene glycol) hydrogels for enhancing beta-cell function. Proc Natl Acad Sci U S A 2011; 108:6380-5. [PMID: 21464290 DOI: 10.1073/pnas.1014026108] [Citation(s) in RCA: 129] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
A biomimetic hydrogel platform was designed to signal encapsulated cells using immobilized cell-cell communication cues, with a focus on enhancing the survival and function of encapsulated pancreatic β-cells to treat type 1 diabetes. When MIN6 cells, a pancreatic β-cell line, were encapsulated in poly(ethylene glycol) (PEG) hydrogels, their survival and glucose responsiveness to insulin were highly dependent on the cell-packing density. A minimum packing density of 10(7) cells/mL was necessary to maintain the survival of encapsulated β-cells without the addition of material functionalities (e.g., cell adhesion ligands). While single cell suspensions can improve diffusion-limited mass transfer, direct cell-cell interactions are limited. Thus, thiolated EphA5-Fc receptor and ephrinA5-Fc ligand were conjugated into PEG hydrogels via a thiol-acrylate photopolymerization to render an otherwise inert PEG hydrogel bioactive. The biomimetic hydrogels presented here can provide crucial cell-cell communication signals for dispersed β-cells and improve their survival and proliferation. Together with the cell-adhesive peptide RGDS, the immobilized fusion proteins (EphA5-Fc and ephrinA5-Fc) synergistically increased the survival of both MIN6 β-cells and dissociated islet cells, both at a very low cell-packing density (< 2 × 10(6) cells/mL). This unique gel platform demonstrates new strategies for tailoring biomimetic environments to enhance the encapsulation of cells that require cell-cell contact to survive and function.
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Jourdan G, Dusseault J, Benhamou PY, Rosenberg L, Hallé JP. Co-encapsulation of bioengineered IGF-II-producing cells and pancreatic islets: effect on beta-cell survival. Gene Ther 2011; 18:539-45. [DOI: 10.1038/gt.2010.166] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
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Daoud J, Rosenberg L, Tabrizian M. Pancreatic Islet Culture and Preservation Strategies: Advances, Challenges, and Future Outlook. Cell Transplant 2010; 19:1523-35. [DOI: 10.3727/096368910x515872] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Postisolation islet survival is a critical step for achieving successful and efficient islet transplantation. This involves the optimization of islet culture in order to prolong survival and functionality in vitro. Many studies have focused on different strategies to culture pancreatic islets in vitro through manipulation of culture media, surface modified substrates, and the use of various techniques such as encapsulation, embedding, scaffold, and bioreactor culture strategies. This review aims to present and discuss the different methodologies employed to optimize pancreatic islet culture in vitro as well as address their respective advantages and drawbacks.
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Affiliation(s)
- Jamal Daoud
- Department of Biomedical Engineering, Faculty of Medicine, McGill University, Montreal, QC, Canada
| | - Lawrence Rosenberg
- Department of Surgery, Faculty of Medicine, McGill University, Montreal, QC, Canada
| | - Maryam Tabrizian
- Department of Biomedical Engineering, Faculty of Medicine, McGill University, Montreal, QC, Canada
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Trophic molecules derived from human mesenchymal stem cells enhance survival, function, and angiogenesis of isolated islets after transplantation. Transplantation 2010; 89:694-701. [PMID: 20125064 DOI: 10.1097/tp.0b013e3181c7dc99] [Citation(s) in RCA: 215] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
BACKGROUND Mesenchymal stem cells (MSCs), also known as multipotent progenitor cells, release several factors that support cell survival and enhance wound healing. We hypothesized that MSC-secreted molecules would induce a trophic effect in pancreatic islet culture conditions. METHODS Pancreatic islets were co-cultured with MSCs, and ADP/ATP ratios, glucose stimulated insulin secretion (GSIS), and DNA fragmentation were evaluated to measure islet quality and viability in vitro. The induction of signal molecules related to the control of survival, function, and angiogenesis was also analyzed. Cell quality assays, DNA fragmentation assays, and islet transplantation into streptozotocin-induced diabetic mice were performed using MSC-conditioned medium (CM)-cultured islets. Furthermore, we identified soluble molecules within MSC-CM. RESULTS Islets co-cultured with MSCs demonstrated lower ADP/ATP ratios, and higher GSIS indexes and viability. Furthermore, co-cultured islets revealed higher levels of anti-apoptotic signal molecules (X-linked inhibitor of apoptosis protein, Bcl-xL, Bcl-2, and heat shock protein-32) and demonstrated increased vascular endothelial growth factor receptor 2 and Tie-2 mRNA expression and increased levels of phosphorylated Tie-2 and focal adhesion kinase protein. Islets cultured in MSC-CM demonstrated lower ADP/ATP ratios, less apoptosis, and a higher GSIS indexes. Diabetic mice that received islet transplants (200 islet equivalent) cultured in MSC-CM for 48 hr demonstrated significantly lower blood glucose levels and enhanced blood vessel formation. In addition, interleukin-6, interleukin-8, vascular endothelial growth factor-A, hepatocyte growth factor, and transforming growth factor-beta were detected at significant levels in MSC-CM. CONCLUSIONS These results suggest that the trophic factors secreted by human MSCs enhance islet survival and function after transplantation.
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