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Rohban R, Martins CP, Esni F. Advanced therapy to cure diabetes: mission impossible is now possible? Front Cell Dev Biol 2024; 12:1484859. [PMID: 39629270 PMCID: PMC11611888 DOI: 10.3389/fcell.2024.1484859] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2024] [Accepted: 11/04/2024] [Indexed: 12/07/2024] Open
Abstract
Cell and Gene therapy are referred to as advanced therapies that represent overlapping fields of regenerative medicine. They have similar therapeutic goals such as to modify cellular identity, improve cell function, or fight a disease. These two therapeutic avenues, however, possess major differences. While cell therapy involves introduction of new cells, gene therapy entails introduction or modification of genes. Furthermore, the aim of cell therapy is often to replace, or repair damaged tissue, whereas gene therapy is used typically as a preventive approach. Diabetes mellitus severely affects the quality of life of afflicted individuals and has various side effects including cardiovascular, ophthalmic disorders, and neuropathy while putting enormous economic pressure on both the healthcare system and the patient. In recent years, great effort has been made to develop cutting-edge therapeutic interventions for diabetes treatment, among which cell and gene therapies stand out. This review aims to highlight various cell- and gene-based therapeutic approaches leading to the generation of new insulin-producing cells as a topmost "panacea" for treating diabetes, while deliberately avoiding a detailed molecular description of these approaches. By doing so, we aim to target readers who are new to the field and wish to get a broad helicopter overview of the historical and current trends of cell- and gene-based approaches in β-cell regeneration.
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Affiliation(s)
- Rokhsareh Rohban
- Department of Internal Medicine, Division of Hematology, Medical University of Graz, Graz, Austria
| | - Christina P. Martins
- Department of Surgery, Division of Pediatric General and Thoracic Surgery, Children’s Hospital of Pittsburgh, University of Pittsburgh Medical Center, Pittsburgh, PA, United States
| | - Farzad Esni
- Department of Surgery, Division of Pediatric General and Thoracic Surgery, Children’s Hospital of Pittsburgh, University of Pittsburgh Medical Center, Pittsburgh, PA, United States
- Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA, United States
- UPMC Hillman Cancer Center, Pittsburgh, PA, United States
- McGowan Institute for regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, United States
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Singh A, Afshan N, Singh A, Singh SK, Yadav S, Kumar M, Sarma DK, Verma V. Recent trends and advances in type 1 diabetes therapeutics: A comprehensive review. Eur J Cell Biol 2023; 102:151329. [PMID: 37295265 DOI: 10.1016/j.ejcb.2023.151329] [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: 04/05/2023] [Revised: 05/12/2023] [Accepted: 06/03/2023] [Indexed: 06/12/2023] Open
Abstract
Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of pancreatic β-cells, leading to insulin deficiency. Insulin replacement therapy is the current standard of care for T1D, but it has significant limitations. However, stem cell-based replacement therapy has the potential to restore β-cell function and achieve glycaemic control eradicating the necessity for drugs or injecting insulin externally. While significant progress has been made in preclinical studies, the clinical translation of stem cell therapy for T1D is still in its early stages. In continuation, further research is essentially required to determine the safety and efficacy of stem cell therapies and to develop strategies to prevent immune rejection of stem cell-derived β-cells. The current review highlights the current state of cellular therapies for T1D including, different types of stem cell therapies, gene therapy, immunotherapy, artificial pancreas, and cell encapsulation being investigated, and their potential for clinical translation.
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Affiliation(s)
- Akash Singh
- Stem Cell Research Centre, Department of Haematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India
| | - Noor Afshan
- Stem Cell Research Centre, Department of Haematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India
| | - Anshuman Singh
- Stem Cell Research Centre, Department of Haematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India
| | - Suraj Kumar Singh
- Stem Cell Research Centre, Department of Haematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India
| | - Sudhanshu Yadav
- Stem Cell Research Centre, Department of Haematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India
| | - Manoj Kumar
- ICMR-National Institute for Research in Environmental Health, Bhopal, India
| | | | - Vinod Verma
- Stem Cell Research Centre, Department of Haematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India.
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Challenges with Cell-based Therapies for Type 1 Diabetes Mellitus. Stem Cell Rev Rep 2022; 19:601-624. [PMID: 36434300 DOI: 10.1007/s12015-022-10482-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/13/2022] [Indexed: 11/27/2022]
Abstract
Type 1 diabetes (T1D) is a chronic, lifelong metabolic disease. It is characterised by the autoimmune-mediated loss of insulin-producing pancreatic β cells in the islets of Langerhans (β-islets), resulting in disrupted glucose homeostasis. Administration of exogenous insulin is the most common management method for T1D, but this requires lifelong reliance on insulin injections and invasive blood glucose monitoring. Replacement therapies with beta cells are being developed as an advanced curative treatment for T1D. Unfortunately, this approach is limited by the lack of donated pancreatic tissue, the difficulties in beta cell isolation and viability maintenance, the longevity of the transplanted cells in vivo, and consequently high costs. Emerging approaches to address these limitations are under intensive investigations, including the production of insulin-producing beta cells from various stem cells, and the development of bioengineered devices including nanotechnologies for improving islet transplantation efficacy without the need for recipients taking toxic anti-rejection drugs. These emerging approaches present promising prospects, while the challenges with the new techniques need to be tackled for ultimately clinical treatment of T1D. This review discussed the benefits and limitations of the cell-based therapies for beta cell replacement as potential curative treatment for T1D, and the applications of bioengineered devices including nanotechnology to overcome the challenges associated with beta cell transplantation.
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Reprogramming—Evolving Path to Functional Surrogate β-Cells. Cells 2022; 11:cells11182813. [PMID: 36139388 PMCID: PMC9496933 DOI: 10.3390/cells11182813] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2022] [Revised: 09/04/2022] [Accepted: 09/07/2022] [Indexed: 12/04/2022] Open
Abstract
Numerous cell sources are being explored to replenish functional β-cell mass since the proof-of -concept for cell therapy of diabetes was laid down by transplantation of islets. Many of these cell sources have been shown to possess a degree of plasticity permitting differentiation along new lineages into insulin-secreting β-cells. In this review, we explore emerging reprograming pathways that aim to generate bone fide insulin producing cells. We focus on small molecules and key transcriptional regulators that orchestrate phenotypic conversion and maintenance of engineered cells.
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Ahmed HH, Aglan HA, Mahmoud NS, Aly RM. Preconditioned human dental pulp stem cells with cerium and yttrium oxide nanoparticles effectively ameliorate diabetic hyperglycemia while combatting hypoxia. Tissue Cell 2021; 73:101661. [PMID: 34656024 DOI: 10.1016/j.tice.2021.101661] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2021] [Revised: 09/23/2021] [Accepted: 10/01/2021] [Indexed: 10/20/2022]
Abstract
UNLABELLED The development of efficient insulin producing cells (IPC) induction system is fundamental for the regenerative clinical applications targeting Diabetes Mellitus. This study was set to generate IPC from human dental pulp stem cells (hDPSCs) capable of surviving under hypoxic conditions in vitro and in vivo. METHODS hDPSCs were cultured in IPCs induction media augmented with Cerium or Yttrium oxide nanoparticles along with selected growth factors & cytokines. The generated IPC were subjected to hypoxic stress in vitro to evaluate the ability of the nanoparticles to combat hypoxia. Next, they were labelled and implanted into diabetic rats. Twenty eight days later, blood glucose and serum insulin levels, hepatic hexokinase and glucose-6-phosphate dehydrogenase activities were measured. Pancreatic vascular endothelial growth factor (VEGF), pancreatic duodenal homeobox1 (Pdx-1), hypoxia inducible factor 1 alpha (HIF-1α) and Caspase-3 genes expression level were evaluated. RESULTS hDPSCs were successfully differentiated into IPCs after incubation with the inductive media enriched with nanoparticles. The generated IPCs released significant amounts of insulin in response to increasing glucose concentration both in vitro & in vivo. The generated IPCs showed up-regulation in the expression levels of anti-apoptotic genes in concomitant with down-regulation in the expression levels of hypoxic, and apoptotic genes. The in vivo study confirmed the homing of PKH-26-labeled cells in pancreas of treated groups. A significant up-regulation in the expression of pancreatic VEGF and PDX-1 genes associated with significant down-regulation in the expression of pancreatic HIF-1α and caspase-3 was evident. CONCLUSION The achieved results highlight the promising role of the Cerium & Yttrium oxide nanoparticles in promoting the generation of IPCs that have the ability to combat hypoxia and govern diabetes mellitus.
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Affiliation(s)
- Hanaa H Ahmed
- Hormones Department, National Research Centre, Giza, Egypt; Stem Cells Lab, Center of Excellence for Advanced Sciences, National Research Centre, Giza, Egypt
| | - Hadeer A Aglan
- Hormones Department, National Research Centre, Giza, Egypt; Stem Cells Lab, Center of Excellence for Advanced Sciences, National Research Centre, Giza, Egypt
| | - Nadia S Mahmoud
- Hormones Department, National Research Centre, Giza, Egypt; Stem Cells Lab, Center of Excellence for Advanced Sciences, National Research Centre, Giza, Egypt
| | - Riham M Aly
- Stem Cells Lab, Center of Excellence for Advanced Sciences, National Research Centre, Giza, Egypt; Basic Dental Science Department, National Research Centre, Giza, Egypt.
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Izeia L, Eufrasio-da-Silva T, Dolatshahi-Pirouz A, Ostrovidov S, Paolone G, Peppas NA, De Vos P, Emerich D, Orive G. Cell-laden alginate hydrogels for the treatment of diabetes. Expert Opin Drug Deliv 2021; 17:1113-1118. [PMID: 32515621 DOI: 10.1080/17425247.2020.1778667] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
INTRODUCTION Diabetes mellitus is an ever-increasing medical condition that currently suffers 1 of 11 adults who may have lifelong commitment with insulin injections. Cell-laden hydrogels releasing insulin may provide the ultimate means of correcting diabetes. Here, we provide insights of this cell-based approach including latest preclinical and clinical progress both from academia and industry. AREA COVERED The present article focuses on reviewing latest advances in cell-laden hydrogels both from the technological and biological perspective. The most relevant clinical results including clinical trials are also discussed. EXPERT OPINION Current progress in technological issues (stem cells, devices, biomaterials) have contributed cell encapsulation science to have a very relevant progress in the field of diabetes treatment.
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Affiliation(s)
- Lukin Izeia
- NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country UPV/EHU , Vitoria-Gasteiz, Spain.,Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) , Vitoria-Gasteiz, Spain
| | - Tatiane Eufrasio-da-Silva
- Department of Dentistry - Regenerative Biomaterials, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences , Nijmegen, The Netherlands
| | - Alireza Dolatshahi-Pirouz
- Department of Dentistry - Regenerative Biomaterials, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences , Nijmegen, The Netherlands.,Department of Health Technology, Institute of Biotherapeutic Engineering and Drug Targeting, Center for Intestinal Absorption and Transport of Biopharmaceuticals Technical University of Denmark , Lyngby, Denmark
| | - Serge Ostrovidov
- Center for Minimally Invasive Therapeutics (C-MIT) Department of Radiological Sciences, University of California , Los Angeles, CA, USA
| | - Giovanna Paolone
- Department of Diagnostic and Public Health, Section of Pharmacology, University of Verona , Verona, Italy
| | - Nicholas A Peppas
- Departments of Pharmaceutics, Chemical and Biomedical Engineering, The University of Texas at Austin , Austin, TX, USA
| | - Paul De Vos
- Pathology and Medical Biology Section, Immunoendocrinology, University of Groningen , Groningen, The Netherlands
| | - Dwaine Emerich
- Gloriana Therapeutics, Inc. (Formerly NsGene Inc.) , Providence, RI, USA
| | - Gorka Orive
- NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country UPV/EHU , Vitoria-Gasteiz, Spain.,Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) , Vitoria-Gasteiz, Spain.,University Institute for Regenerative Medicine and Oral Implantology - UIRMI (Upv/ehu-fundación Eduardo Anitua) , Vitoria, Spain.,The Academia, Singapore Eye Research Institute , Discovery Tower, Singapore
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Eom YS, Gwon AR, Kwak KM, Youn JY, Park H, Kim KW, Kim BJ. Notch1 Has an Important Role in β-Cell Mass Determination and Development of Diabetes. Diabetes Metab J 2021; 45:86-96. [PMID: 32174059 PMCID: PMC7850870 DOI: 10.4093/dmj.2019.0160] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/09/2019] [Accepted: 11/20/2019] [Indexed: 12/30/2022] Open
Abstract
BACKGROUND Notch signaling pathway plays an important role in regulating pancreatic endocrine and exocrine cell fate during pancreas development. Notch signaling is also expressed in adult pancreas. There are few studies on the effect of Notch on adult pancreas. Here, we investigated the role of Notch in islet mass and glucose homeostasis in adult pancreas using Notch1 antisense transgenic (NAS). METHODS Western blot analysis was performed for the liver of 8-week-old male NAS mice. We also conducted an intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test in 8-week-old male NAS mice and male C57BL/6 mice (control). Morphologic observation of pancreatic islet and β-cell was conducted in two groups. Insulin secretion capacity in islets was measured by glucose-stimulated insulin secretion (GSIS) and perifusion. RESULTS NAS mice showed higher glucose levels and lower insulin secretion in IPGTT than the control mice. There was no significant difference in insulin resistance. Total islet and β-cell masses were decreased in NAS mice. The number of large islets (≥250 µm) decreased while that of small islets (<250 µm) increased. Reduced insulin secretion was observed in GSIS and perifusion. Neurogenin3, neurogenic differentiation, and MAF bZIP transcription factor A levels increased in NAS mice. CONCLUSION Our study provides that Notch1 inhibition decreased insulin secretion and decreased islet and β-cell masses. It is thought that Notch1 inhibition suppresses islet proliferation and induces differentiation of small islets. In conclusion, Notch signaling pathway may play an important role in β-cell mass determination and diabetes.
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Affiliation(s)
- Young Sil Eom
- Department of Internal Medicine, Gachon University Gil Medical Center, Gachon University College of Medicine, Incheon, Korea
| | - A-Ryeong Gwon
- Department of Internal Medicine, Gachon University Gil Medical Center, Incheon, Korea
| | - Kyung Min Kwak
- Department of Internal Medicine, Gachon University Gil Medical Center, Incheon, Korea
| | - Jin-Young Youn
- Institute of Clinical Medicine, Gachon University Gil Medical Center, Incheon, Korea
| | - Heekyoung Park
- Department of Internal Medicine, Gachon University Gil Medical Center, Incheon, Korea
| | - Kwang-Won Kim
- Department of Internal Medicine, Gachon University Gil Medical Center, Incheon, Korea
| | - Byung-Joon Kim
- Department of Internal Medicine, Gachon University Gil Medical Center, Gachon University College of Medicine, Incheon, Korea
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Chemically defined and xenogeneic-free differentiation of human pluripotent stem cells into definitive endoderm in 3D culture. Sci Rep 2019; 9:996. [PMID: 30700818 PMCID: PMC6353891 DOI: 10.1038/s41598-018-37650-z] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2018] [Accepted: 12/10/2018] [Indexed: 12/18/2022] Open
Abstract
In vitro differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm (DE) represents a key step towards somatic cells of lung, liver and pancreas. For future clinical applications, mass production of differentiated cells at chemically defined conditions and free of xenogeneic substances is envisioned. In this study we adapted our previously published two-dimensional (2D) DE induction protocol to three-dimensional (3D) static suspension culture in the absence of the xenogeneic extracellular matrix Matrigel. Next, fetal calf serum and bovine serum albumin present in the standard medium were replaced by a custom-made and xeno-free B-27. This yielded in a chemically defined and xenogeneic-free 3D culture protocol for differentiation of hPSCs into DE at efficiencies similar to standard 2D conditions. This novel protocol successfully worked with different hPSC lines including hESCs and hiPSCs maintained in two different stem cell media prior to differentiation. DE cells obtained by our novel BSA-free 3D protocol could be further differentiated into PDX1- or NKX6.1-expressing pancreatic progenitor cells. Notably, upon DE differentiation, we also identified a CXCR4+/NCAM+/EpCAMlow cell population with reduced DE marker gene expression. These CXCR4+/NCAM+/EpCAMlow cells emerge as a result of Wnt/beta-catenin hyperactivation via elevated CHIR-99021 concentrations and likely represent misspecified DE.
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Rattananinsruang P, Dechsukhum C, Leeanansaksiri W. Establishment of Insulin-Producing Cells From Human Embryonic Stem Cells Underhypoxic Condition for Cell Based Therapy. Front Cell Dev Biol 2018; 6:49. [PMID: 29868580 PMCID: PMC5962719 DOI: 10.3389/fcell.2018.00049] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2018] [Accepted: 04/16/2018] [Indexed: 12/27/2022] Open
Abstract
Diabetes mellitus (DM) is a group of diseases characterized by abnormally high levels of glucose in the blood stream. In developing a potential therapy for diabetic patients, pancreatic cells transplantation has drawn great attention. However, the hinder of cell transplantation for diabetes treatment is insufficient sources of insulin-producing cells. Therefore, new cell based therapy need to be developed. In this regard, human embryonic stem cells (hESCs) may serve as good candidates for this based on their capability of differentiation into various cell types. In this study, we designed a new differentiation protocol that can generate hESC-derived insulin-producing cells (hES-DIPCs) in a hypoxic condition. We also emphasized on the induction of definitive endoderm during embryoid bodies (EBs) formation. After induction of hESCs differentiation into insulin-producing cells (IPCs), the cells obtained from the cultures exhibited pancreas-related genes such as Pdx1, Ngn3, Nkx6.1, GLUT2, and insulin. These cells also showed positive for DTZ-stained cellular clusters and contained ability of insulin secretion in a glucose-dependent manner. After achievement to generated functional hES-DIPCs in vitro, some of the hES-DIPCs were then encapsulated named encapsulated hES-DIPCs. The data showed that the encapsulated cells could possess the function of insulin secretion in a time-dependent manner. The hES-DIPCs and encapsulated hES-DIPCs were then separately transplanted into STZ-induced diabetic mice. The findings showed the significant blood glucose levels regulation capacity and declination of IL-1β concentration in all transplanted mice. These results indicated that both hES-DIPCs and encapsulated hES-DIPCs contained the ability to sustain hyperglycemia condition as well as decrease inflammatory cytokine level in vivo. The findings of this study may apply for generation of a large number of hES-DIPCs in vitro. In addition, the implication of this work is therapeutic value in type I diabetes treatment in the future. The application for type II diabetes treatment remain to be investigated.
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Affiliation(s)
- Piyaporn Rattananinsruang
- School of Preclinic, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand
| | - Chavaboon Dechsukhum
- School of Pathology, Institute of Medicine, Suranaree University of Technology, Nakhon Ratchasima, Thailand
| | - Wilairat Leeanansaksiri
- School of Preclinic, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand
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Berezin AE. New Trends in Stem Cell Transplantation in Diabetes Mellitus Type I and Type II. ACTA ACUST UNITED AC 2017. [DOI: 10.1007/978-3-319-55687-1_3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
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Diekmann U, Davenport C, Kresse J, Naujok O. Purification of Definitive Endoderm Generated from Pluripotent Stem Cells by Magnetic Cell Sorting. ACTA ACUST UNITED AC 2017; 40:1D.9.1-1D.9.17. [DOI: 10.1002/cpsc.22] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Affiliation(s)
- Ulf Diekmann
- Institute of Clinical Biochemistry, Hannover Medical School Hannover Germany
| | - Claudia Davenport
- Institute of Clinical Biochemistry, Hannover Medical School Hannover Germany
| | - Jasmin Kresse
- Institute of Clinical Biochemistry, Hannover Medical School Hannover Germany
| | - Ortwin Naujok
- Institute of Clinical Biochemistry, Hannover Medical School Hannover Germany
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12
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Islet-like organoids derived from human pluripotent stem cells efficiently function in the glucose responsiveness in vitro and in vivo. Sci Rep 2016; 6:35145. [PMID: 27731367 PMCID: PMC5059670 DOI: 10.1038/srep35145] [Citation(s) in RCA: 72] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2016] [Accepted: 09/26/2016] [Indexed: 12/30/2022] Open
Abstract
Insulin secretion is elaborately modulated in pancreatic ß cells within islets of three-dimensional (3D) structures. Using human pluripotent stem cells (hPSCs) to develop islet-like structures with insulin-producing ß cells for the treatment of diabetes is challenging. Here, we report that pancreatic islet-like clusters derived from hESCs are functionally capable of glucose-responsive insulin secretion as well as therapeutic effects. Pancreatic hormone-expressing endocrine cells (ECs) were differentiated from hESCs using a step-wise protocol. The hESC-derived ECs expressed pancreatic endocrine hormones, such as insulin, somatostatin, and pancreatic polypeptide. Notably, dissociated ECs autonomously aggregated to form islet-like, 3D structures of consistent sizes (100–150 μm in diameter). These EC clusters (ECCs) enhanced insulin secretion in response to glucose stimulus and potassium channel inhibition in vitro. Furthermore, ß cell-deficient mice transplanted with ECCs survived for more than 40 d while retaining a normal blood glucose level to some extent. The expression of pancreatic endocrine hormones was observed in tissues transplanted with ECCs. In addition, ECCs could be generated from human induced pluripotent stem cells. These results suggest that hPSC-derived, islet-like clusters may be alternative therapeutic cell sources for treating diabetes.
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Davenport C, Diekmann U, Naujok O. A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells. J Vis Exp 2016. [PMID: 26966833 DOI: 10.3791/53655] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.
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Affiliation(s)
| | - Ulf Diekmann
- Institute of Clinical Biochemistry, Hannover Medical School
| | - Ortwin Naujok
- Institute of Clinical Biochemistry, Hannover Medical School;
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Abouzaripour M, Pasbakhsh P, Atlasi N, Shahverdi AH, Mahmoudi R, Kashani IR. In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells. CELL JOURNAL 2016; 17:701-10. [PMID: 26862529 PMCID: PMC4746420 DOI: 10.22074/cellj.2016.3842] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/07/2014] [Accepted: 02/02/2015] [Indexed: 01/08/2023]
Abstract
OBJECTIVE Bone marrow has recently been recognized as a novel source of stem cells for the treatment of wide range of diseases. A number of studies on murine bone mar- row have shown a homogenous population of rare stage-specific embryonic antigen 1 (SSEA-1) positive cells that express markers of pluripotent stem cells. This study focuses on SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiate them into insulin-secreting cells (ISCs) in order to investigate their differentiation potential for future use in cell therapy. MATERIALS AND METHODS This study is an experimental research. Mouse SSEA-1 positive cells were isolated by Magnetic-activated cell sorting (MACS) followed by characteriza- tion with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs with specific differentiation media. In order to evaluate differentiation quality and analysis, dithizone (DTZ) staining was use, followed by reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and insulin secretion assay. Statistical results were analyzed by one-way ANOVA. RESULTS The results achieved in this study reveal that mouse bone marrow contains a population of SSEA-1 positive cells that expresses pluripotent stem cells markers such as SSEA-1, octamer-binding transcription factor 4 (OCT-4) detected by immunocytochem- istry and C-X-C chemokine receptor type 4 (CXCR4) and stem cell antigen-1 (SCA-1) detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCs cell clusters as evidenced by their DTZ positive staining and expression of genes such as Pdx1 (pancreatic transcription factors), Ngn3 (endocrine progenitor marker), Insulin1 and Insulin2 (pancreaticβ-cell markers). Additionally, our results demonstrate expression of Pdx1 and Glut2 protein and insulin secretion in response to a glucose challenge in the differentiated cells. CONCLUSION Our study clearly demonstrates the potential of SSEA-1 positive cells to differentiate into insulin secreting cells in defined culture conditions for clinical ap- plications.
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Affiliation(s)
- Morteza Abouzaripour
- Department of Anatomical Sciences, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Parichehr Pasbakhsh
- Department of Anatomical Sciences, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Nader Atlasi
- Department of Anatomical Sciences, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Abdol Hossein Shahverdi
- Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Medicine, ACECR, Tehran, Iran
| | - Reza Mahmoudi
- Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran
| | - Iraj Ragerdi Kashani
- Department of Anatomical Sciences, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
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Bâlici Ş, Şuşman S, Rusu D, Nicula GZ, Soriţău O, Rusu M, Biris AS, Matei H. Differentiation of stem cells into insulin-producing cells under the influence of nanostructural polyoxometalates. J Appl Toxicol 2015; 36:373-84. [DOI: 10.1002/jat.3218] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2015] [Revised: 07/01/2015] [Accepted: 07/01/2015] [Indexed: 01/29/2023]
Affiliation(s)
- Ştefana Bâlici
- Department of Cell and Molecular Biology, Faculty of Medicine; “Iuliu Haţieganu” University of Medicine and Pharmacy; Cluj-Napoca România
- Department of Inorganic Chemistry, Faculty of Chemistry and Chemical Engineering; “Babeş-Bolyai” University; Cluj-Napoca România
| | - Sergiu Şuşman
- Department of Morphological Sciences, Faculty of Medicine; “Iuliu Haţieganu” University of Medicine and Pharmacy; Cluj-Napoca România
- Imogen Research Centre - Department of Pathology; Cluj-Napoca România
- Radiotherapy, Tumor and Radiobiology Laboratory; The Oncology Institute “Prof. Dr. Ion Chiricuţă”; Cluj-Napoca România
| | - Dan Rusu
- Department of Physical-Chemistry, Faculty of Pharmacy; “Iuliu Haţieganu” University of Medicine and Pharmacy; Cluj-Napoca România
| | - Gheorghe Zsolt Nicula
- Department of Cell and Molecular Biology, Faculty of Medicine; “Iuliu Haţieganu” University of Medicine and Pharmacy; Cluj-Napoca România
| | - Olga Soriţău
- Radiotherapy, Tumor and Radiobiology Laboratory; The Oncology Institute “Prof. Dr. Ion Chiricuţă”; Cluj-Napoca România
| | - Mariana Rusu
- Department of Inorganic Chemistry, Faculty of Chemistry and Chemical Engineering; “Babeş-Bolyai” University; Cluj-Napoca România
| | - Alexandru S. Biris
- Center for Integrative Nanotechnology Sciences; University of Arkansas at Little Rock; Little Rock AR USA
| | - Horea Matei
- Department of Cell and Molecular Biology, Faculty of Medicine; “Iuliu Haţieganu” University of Medicine and Pharmacy; Cluj-Napoca România
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17
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The generation of definitive endoderm from human embryonic stem cells is initially independent from activin A but requires canonical Wnt-signaling. Stem Cell Rev Rep 2015; 10:480-93. [PMID: 24913278 DOI: 10.1007/s12015-014-9509-0] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
The activation of the TGF-beta pathway by activin A directs ES cells into the definitive endoderm germ layer. However, there is evidence that activin A/TGF-beta is not solely responsible for differentiation into definitive endoderm. GSK3beta inhibition has recently been shown to generate definitive endoderm-like cells from human ES cells via activation of the canonical Wnt-pathway. The GSK3beta inhibitor CHIR-99021 has been reported to generate mesoderm from human iPS cells. Thus, the specific role of the GSK3beta inhibitor CHIR-99021 was analyzed during the differentiation of human ES cells and compared against a classic endoderm differentiation protocol. At high concentrations of CHIR-99021, the cells were directed towards mesodermal cell fates, while low concentrations permitted mesodermal and endodermal differentiation. Finally, the analyses revealed that GSK3beta inhibition rapidly directed human ES cells into a primitive streak-like cell type independently from the TGF-beta pathway with mesoderm and endoderm differentiation potential. Addition of low activin A concentrations effectively differentiated these primitive streak-like cells into definitive endoderm. Thus, the in vitro differentiation of human ES cells into definitive endoderm is initially independent from the activin A/TGF-beta pathway but requires high canonical Wnt-signaling activity.
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18
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Diekmann U, Lenzen S, Naujok O. A Reliable and Efficient Protocol for Human Pluripotent Stem Cell Differentiation into the Definitive Endoderm Based on Dispersed Single Cells. Stem Cells Dev 2015; 24:190-204. [DOI: 10.1089/scd.2014.0143] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Affiliation(s)
- Ulf Diekmann
- Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany
| | - Sigurd Lenzen
- Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany
| | - Ortwin Naujok
- Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany
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19
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Diekmann U, Naujok O. Generation and Purification of Definitive Endoderm Cells Generated from Pluripotent Stem Cells. Methods Mol Biol 2015; 1341:157-72. [PMID: 25762297 DOI: 10.1007/7651_2015_220] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Differentiation of pluripotent stem cells into cells of the definitive endoderm requires an in vitro gastrulation event. Differentiated somatic cells derived from this germ layer may then be used for cell replacement therapies of degenerative diseases of the liver, lung, and pancreas. Here we describe an endoderm differentiation protocol, which initiates the differentiation from a defined cell number of dispersed single cells and reliably yields in >70-80 % endoderm-committed cells in a short 5-day treatment regimen.
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Affiliation(s)
- Ulf Diekmann
- Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany
| | - Ortwin Naujok
- Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany.
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20
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Berezin AE. Diabetes mellitus and cellular replacement therapy: Expected clinical potential and perspectives. World J Diabetes 2014; 5:777-786. [PMID: 25512780 PMCID: PMC4265864 DOI: 10.4239/wjd.v5.i6.777] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/29/2014] [Revised: 07/16/2014] [Accepted: 09/23/2014] [Indexed: 02/05/2023] Open
Abstract
Diabetes mellitus (DM) is the most prevailing disease with progressive incidence worldwide. Despite contemporary treatment type one DM and type two DM are frequently associated with long-term major microvascular and macrovascular complications. Currently restoration of failing β-cell function, regulation of metabolic processes with stem cell transplantation is discussed as complements to contemporary DM therapy regimens. The present review is considered paradigm of the regenerative care and the possibly effects of cell therapy in DM. Reprogramming stem cells, bone marrow-derived mononuclear cells; lineage-specified progenitor cells are considered for regenerative strategy in DM. Finally, perspective component of stem cell replacement in DM is discussed.
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21
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Sawangmake C, Nowwarote N, Pavasant P, Chansiripornchai P, Osathanon T. A feasibility study of an in vitro differentiation potential toward insulin-producing cells by dental tissue-derived mesenchymal stem cells. Biochem Biophys Res Commun 2014; 452:581-7. [PMID: 25181343 DOI: 10.1016/j.bbrc.2014.08.121] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2014] [Accepted: 08/22/2014] [Indexed: 12/30/2022]
Abstract
Dental tissue-derived mesenchymal stem cells have been proposed as an alternative source for mesenchymal stem cells. Here, we investigated the differentiation ability toward insulin producing cells (IPCs) of human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs). These cells expressed mesenchymal stem cell surface markers and were able to differentiate toward osteogenic and adipogenic lineages. Upon 3 step-IPCs induction, hDPSCs exhibited more colony number than hPDLSCs. The mRNA upregulation of pancreatic endoderm/islet markers was noted. However, the significant increase was noted only for PDX-1, NGN-3, and INSULIN mRNA expression of hDPSCs. The hDPSCs-derived IPCs expressed PRO-INSULIN and released C-PEPTIDE upon glucose stimulation in dose-dependent manner. After IPCs induction, the Notch target, HES-1 and HEY-1, mRNA expression was markedly noted. Notch inhibition during the last induction step or throughout the protocol disturbed the ability of C-PEPTIDE release upon glucose stimulation. The results suggested that hDPSCs had better differentiation potential toward IPCs than hPDLSCs. In addition, the Notch signalling might involve in the differentiation regulation of hDPSCs into IPCs.
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Affiliation(s)
- Chenphop Sawangmake
- Department of Pharmacology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand; Graduate Program in Veterinary Bioscience, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand; Research Unit of Mineralized Tissue, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand
| | - Nunthawan Nowwarote
- Research Unit of Mineralized Tissue, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand
| | - Prasit Pavasant
- Research Unit of Mineralized Tissue, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand; Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand
| | - Piyarat Chansiripornchai
- Department of Pharmacology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
| | - Thanaphum Osathanon
- Research Unit of Mineralized Tissue, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand; Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand.
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22
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Wu C, Liu F, Li P, Zhao G, Lan S, Jiang W, Meng X, Tian L, Li G, Li Y, Liu JY. Engineered hair follicle mesenchymal stem cells overexpressing controlled-release insulin reverse hyperglycemia in mice with type L diabetes. Cell Transplant 2014; 24:891-907. [PMID: 24835482 DOI: 10.3727/096368914x681919] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Genetically engineered stem cells that overexpress genes encoding therapeutic products can be exploited to correct metabolic disorders by repairing and regenerating diseased organs or restoring their function. Hair follicles are readily accessible and serve as a rich source of autologous stem cells for cell-based gene therapy. Here we isolated mesenchymal stem cells from human hair follicles (HF-MSCs) and engineered them to overexpress the human insulin gene and release human insulin in a time- and dose-dependent manner in response to rapamycin. The engineered HF-MSCs retained their characteristic cell surface markers and retained their potential to differentiate into adipocytes and osteoblasts. When mice with streptozotocin-induced type 1 diabetes were engrafted with these engineered HF-MSCs, these cells expressed and released a dose of human insulin, dramatically reversed hyperglycemia, and significantly reduced death rate. Moreover, the engineered HF-MSCs did not form detectable tumors throughout the 120-day animal tests in our experiment. Our results show that HF-MSCs can be used to safely and efficiently express therapeutic transgenes and therefore show promise for cell-based gene therapy of human disease.
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Affiliation(s)
- Chunling Wu
- The Key Laboratory of Pathobiology, Ministry of Education, Norman Bethune College of Medicine, Jilin University, Changchun, Jilin, P.R. China
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23
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Chmielowiec J, Borowiak M. In vitro differentiation and expansion of human pluripotent stem cell-derived pancreatic progenitors. Rev Diabet Stud 2014; 11:19-34. [PMID: 25148365 DOI: 10.1900/rds.2014.11.19] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Recent progress in understanding stem cell biology has been remarkable, especially in deciphering signals that support differentiation towards tissue-specific lineages. This achievement positions us firmly at the beginning of an era of patient-specific regenerative medicine and human disease modeling. It will be necessary to equip the progress in this era with a reliable source of self-renewing progenitor cells that differentiate into functional target cells. The generation of pancreatic progenitors that mature in vivo into functional beta-cells has raised the hope for new therapeutic options in diabetes, but key challenges still remain including the production of sufficient numbers of cells for research and transplantation. Recent approaches to this problem have shown that the presence of organ- and stage-specific mesenchyme improves the generation of progenitors, from endoderm to endocrine cells. Alternatively, utilization of three-dimensional culture may improve the efficiency and yield of directed differentiation. Here, we review the current knowledge of pancreatic directed differentiation and ex vivo expansion of pancreatic progenitors, including recent advances in differentiation strategies for the generation of pancreatic progenitors, and we discuss persistent challenges which will need to be overcome before personalized cell-based therapy becomes a practical strategy.
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Affiliation(s)
- Jolanta Chmielowiec
- Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
| | - Malgorzata Borowiak
- Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
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24
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Massumi M, Hoveizi E, Baktash P, Hooti A, Ghazizadeh L, Nadri S, Pourasgari F, Hajarizadeh A, Soleimani M, Nabiuni M, Khorramizadeh MR. Efficient programming of human eye conjunctiva-derived induced pluripotent stem (ECiPS) cells into definitive endoderm-like cells. Exp Cell Res 2014; 322:51-61. [DOI: 10.1016/j.yexcr.2014.01.006] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2013] [Revised: 12/15/2013] [Accepted: 01/06/2014] [Indexed: 01/08/2023]
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25
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Sarker MMH, Zhou M, Rameshwar P, Hanover JA. Functions and roles of proteins: diabetes as a paradigm. PROGRESS IN BIOPHYSICS AND MOLECULAR BIOLOGY 2014; 114:2-7. [PMID: 24239502 PMCID: PMC10483990 DOI: 10.1016/j.pbiomolbio.2013.11.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/15/2013] [Accepted: 11/04/2013] [Indexed: 01/01/2023]
Abstract
Molecular and cellular biology has moved towards complete and accurate knowledge of how molecules behave in space and time. Protein is considered as the primary group of molecules responsible for mediating most physiological processes. Changes in the levels of proteins may lead to the altered function and are responsible for many diseases. This review provides a partial molecular explanation of biological force-ratio generation that may act to split protein into branches, and shows molecular functional divergence. Developing a non-reductionist theory of the cellular function in medicine is clearly not sufficient. Finding effective parameters of the models by characterizing molecular interactions becomes necessary. Protein interactivity and stability provides a basis for an integrated understanding of pathologies such diabetes. One example of how a mechanistic analysis of such physiological processes can be of value is the time-delay between mRNA and translation that can act as a fork allowing a slowdown in gene expression.
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Affiliation(s)
- Md Mosharrof Hossain Sarker
- Dept of Electrical and Computer Engineering, New Jersey Institute of Technology (NJIT), Newark, NJ 07102, USA.
| | - MengChu Zhou
- Dept of Electrical and Computer Engineering, New Jersey Institute of Technology (NJIT), Newark, NJ 07102, USA.
| | - Pranela Rameshwar
- Dept of Medicine-Hematology/Oncology, Graduate School of Biomedical Science, Rutgers-New Jersey Medical School, NJ 07103, USA.
| | - John A Hanover
- Laboratory of Cellular and Molecular Biology, NIDDK, National Institutes of Health (NIH), Bethesda, MD 20892-0851, USA.
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26
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Diekmann U, Elsner M, Fiedler J, Thum T, Lenzen S, Naujok O. MicroRNA target sites as genetic tools to enhance promoter-reporter specificity for the purification of pancreatic progenitor cells from differentiated embryonic stem cells. Stem Cell Rev Rep 2013; 9:555-68. [PMID: 23111459 DOI: 10.1007/s12015-012-9416-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Pluripotent cells hold great promise for cell replacement therapies in regenerative medicine. All known protocols for directed in vitro differentiation of pluripotent cells did not yield pure populations complicating the characterization of the derived cells. In addition, the risk of tumor formation due to residual undifferentiated cells is a serious unresolved problem. In the present study the tissue-specific mouse Pdx1 promoter was used to control the expression of the reporter gene GFP2 in mouse ES cells in order to purify them via FACS during in vitro differentiation. The background fluorescence of transduced ES cells hampered the purification of Pdx1-positive cells due to a contaminating population of partially undifferentiated cells. MicroRNAs (mir) are important regulators of gene expression and were used to enhance promoter specificity during differentiation towards pancreatic progenitor cells. The mouse mmu-mir-294 was found to be mainly expressed during pluripotency, whereas the expression of the mir-302 cluster was increased during early differentiation. Integration of a microRNA target site for the mmu-mir-294 into the lentiviral vector reduced the background fluorescence specifically during pluripotency and permitted re-occurrence of GFP2 expression upon differentiation. A combination of the microRNA target site with the Pdx1 promoter fragment allowed the purification of pancreatic progenitors from differentiated ES cells. This population reflected an early pancreatic progenitor population without other contaminating cell lineages. In conclusion, microRNA target sites are efficient regulatory elements to control transgene expression and to enhance tissue specificity as presented in this study facilitating the sorting and purification of Pdx1-positive pancreatic progenitor cells.
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Affiliation(s)
- Ulf Diekmann
- Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany
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27
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Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation. Proc Natl Acad Sci U S A 2013; 110:12691-6. [PMID: 23852729 DOI: 10.1073/pnas.1304507110] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Developmental biology is challenged to reveal the function of numerous candidate genes implicated by recent genome-scale studies as regulators of organ development and diseases. Recapitulating organogenesis from purified progenitor cells that can be genetically manipulated would provide powerful opportunities to dissect such gene functions. Here we describe systems for reconstructing pancreas development, including islet β-cell and α-cell differentiation, from single fetal progenitor cells. A strict requirement for native genetic regulators of in vivo pancreas development, such as Ngn3, Arx, and Pax4, revealed the authenticity of differentiation programs in vitro. Efficient genetic screens permitted by this system revealed that Prdm16 is required for pancreatic islet development in vivo. Discovering the function of genes regulating pancreas development with our system should enrich strategies for regenerating islets for treating diabetes mellitus.
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28
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Wang Q, Wang H, Sun Y, Li SW, Donelan W, Chang LJ, Jin S, Terada N, Cheng H, Reeves WH, Yang LJ. The reprogrammed pancreatic progenitor-like intermediate state of hepatic cells is more susceptible to pancreatic beta cell differentiation. J Cell Sci 2013; 126:3638-48. [PMID: 23750005 DOI: 10.1242/jcs.124925] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) hold great promise for cell therapy. However, their low efficiency of lineage-specific differentiation and tumorigenesis severely hinder clinical translation. We hypothesized that reprogramming of somatic cells into lineage-specific progenitor cells might allow for large-scale expansion, avoiding the tumorigenesis inherent with iPSCs and simultaneously facilitating lineage-specific differentiation. Here we aimed at reprogramming rat hepatic WB cells, using four Yamanaka factors, into pancreatic progenitor cells (PPCs) or intermediate (IM) cells that have characteristics of PPCs. IM clones were selected based on their specific morphology and alkaline phosphatase activity and stably passaged under defined culture conditions. IM cells did not have iPSC properties, could be stably expanded in large quantity, and expressed all 14 genes that are used to define the PPC developmental stage. Directed differentiation of IM and WB cells by Pdx1-Ngn3-MafA (PNM) into pancreatic beta-like cells revealed that the IM cells are more susceptible to directed beta cell differentiation because of their open chromatin configuration, as demonstrated by expression of key pancreatic beta cell genes, secretion of insulin in response to glucose stimulation, and easy access to exogenous PNM proteins at the rat insulin 1 and Pdx1 promoters. This notion that IM cells are superior to their parental cells is further supported by the epigenetic demonstration of accessibility of Pdx1 and insulin 1 promoters. In conclusion, we have developed a strategy to derive and expand PPC cells from hepatic WB cells using conventional cell reprogramming. This proof-of-principal study may offer a novel, safe and effective way to generate autologous pancreatic beta cells for cell therapy of diabetes.
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Affiliation(s)
- Qiwei Wang
- Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, College of Medicine, Gainesville, Florida 32610, USA
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29
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Lin HT, Otsu M, Nakauchi H. Stem cell therapy: an exercise in patience and prudence. Philos Trans R Soc Lond B Biol Sci 2013; 368:20110334. [PMID: 23166396 DOI: 10.1098/rstb.2011.0334] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
In recent times, the epigenetic study of pluripotency based on cellular reprogramming techniques led to the creation of induced pluripotent stem cells. It has come to represent the forefront of a new wave of alternative therapeutic approaches in the field of stem cell therapy. Progress in drug development has saved countless lives, but there are numerous intractable diseases where curative treatment cannot be achieved through pharmacological intervention alone. Consequently, there has been an unfortunate rise in incidences of organ failures, degenerative disorders and cancers, hence novel therapeutic interventions are required. Stem cells have unique self-renewal and multilineage differentiation capabilities that could be harnessed for therapeutic purposes. Although a number of mature differentiated cells have been characterized in vitro, few have been demonstrated to function in a physiologically relevant context. Despite fervent levels of enthusiasm in the field, the reality is that other than the employment of haematopoietic stem cells, many other therapies have yet to be thoroughly proven for their therapeutic benefit and safety in application. This review shall focus on a discussion regarding the current status of stem cell therapy, the issues surrounding it and its future prospects with a general background on the regulatory networks underlying pluripotency.
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Affiliation(s)
- Huan-Ting Lin
- Center for Stem Cell Biology and Regenerative Medicine, IMSUT, 4-6-1 Shirokanedai Minato-ku, Tokyo, 108-8639, Japan
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30
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Chen AE, Borowiak M, Sherwood RI, Kweudjeu A, Melton DA. Functional evaluation of ES cell-derived endodermal populations reveals differences between Nodal and Activin A-guided differentiation. Development 2013; 140:675-86. [PMID: 23293299 DOI: 10.1242/dev.085431] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Embryonic stem (ES) cells hold great promise with respect to their potential to be differentiated into desired cell types. Of interest are organs derived from the definitive endoderm, such as the pancreas and liver, and animal studies have revealed an essential role for Nodal in development of the definitive endoderm. Activin A is a related TGFβ member that acts through many of the same downstream signaling effectors as Nodal and is thought to mimic Nodal activity. Detailed characterization of ES cell-derived endodermal cell types by gene expression analysis in vitro and functional analysis in vivo reveal that, despite their similarity in gene expression, Nodal and Activin-derived endodermal cells exhibit a distinct difference in functional competence following transplantation into the developing mouse embryo. Pdx1-expressing cells arising from the respective endoderm populations exhibit extended differences in their competence to mature into insulin/c-peptide-expressing cells in vivo. Our findings underscore the importance of functional cell-type evaluation during stepwise differentiation of stem cells.
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Affiliation(s)
- Alice E Chen
- Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
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31
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Diekmann U, Naujok O, Blasczyk R, Müller T. Embryonic stem cells of the non-human primate Callithrix jacchus can be differentiated into definitive endoderm by Activin-A but not IDE-1/2. J Tissue Eng Regen Med 2013; 9:473-9. [PMID: 23418163 DOI: 10.1002/term.1709] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2012] [Revised: 11/01/2012] [Accepted: 12/20/2012] [Indexed: 12/14/2022]
Abstract
Pluripotent stem cells hold great promise for regenerative medicine, due to their unlimited self-renewal potential and the ability to differentiate into all somatic cell types. Differences between the rodent disease models and the situation in humans can be narrowed down with non-human primate models. The common marmoset monkey (Callithrix jacchus) is an interesting model for biomedical research because these animals are easy to breed, get relatively old (≤ 13 years), are small in size, are relatively cost-effective and have a high genetic proximity to the human. In particular, diseases of the liver and pancreas are interesting for cell replacement therapies but the in vitro differentiation of ESCs into the definitive endoderm germ layer is still a demanding task. Membrane-permeable, chemically defined small molecules can possibly replace recombinant growth factors used in most directed differentiation protocols. However, the potent small molecules IDE-1 and IDE-2 were not able to induce definitive endoderm-like cells when ESCs from the common marmoset were treated with these compounds, whereas the recombinant growth factor Activin A could force the differentiation into this lineage. Our results indicate that ESCs from the common marmoset are less sensitive or even insensitive to these small molecules. Thus, differences between the species of human ESCs and ESCs of this non-human primate might be a useful model to further evaluate the exact mode of action of these compounds.
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Affiliation(s)
- Ulf Diekmann
- Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany; Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany
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32
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Naujok O, Lenzen S. A critical re-evaluation of CD24-positivity of human embryonic stem cells differentiated into pancreatic progenitors. Stem Cell Rev Rep 2012; 8:779-91. [PMID: 22529013 DOI: 10.1007/s12015-012-9362-y] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Differentiation of embryonic stem cells (ESCs) into insulin-producing cells for cell replacement therapy of diabetes mellitus comprises the stepwise recapitulation of in vivo developmental stages of pancreatic organogenesis in an in vitro differentiation protocol. The chemical compounds IDE-1 and (-)-indolactam-V can be used to direct mouse and human ESCs through these stages to form definitive endoderm via an intermediate mesendodermal stage and finally into pancreatic endoderm. Cells of the pancreatic endoderm express the PDX1 transcription factor and contribute to all pancreatic cell types upon further in vitro or in vivo differentiation. Even though this differentiation approach is highly effective and reproducible, it generates heterogeneous populations containing PDX1-expressing pancreatic progenitors amongst other cell types. Thus, a technique to separate PDX1-expressing cells from this mixture is very desirable. Recently it has been reported that PDX1-positive pancreatic progenitors, derived from human embryonic stem cells, express the surface marker CD24. Therefore were subjected mouse and human ESCs to a small molecule differentiation approach and the expression of the surface marker CD24 was monitored in undifferentiated cells, cells committed to the definitive endoderm and cells reminiscent of the pancreatic endoderm. We observed that both mouse and human ESCs expressed CD24 in the pluripotent state, during the whole process of endoderm formation and upon further differentiation towards pancreatic endoderm. Thus CD24 is not a suitable cell surface marker for identification of PDX1-positive progenitor cells.
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Affiliation(s)
- Ortwin Naujok
- Institute of Clinical Biochemistry, Hannover Medical School, 30625 Hannover, Germany
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33
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Stanekzai J, Isenovic ER, Mousa SA. Treatment options for diabetes: potential role of stem cells. Diabetes Res Clin Pract 2012; 98:361-8. [PMID: 23020931 DOI: 10.1016/j.diabres.2012.09.010] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/03/2012] [Revised: 06/27/2012] [Accepted: 09/04/2012] [Indexed: 01/09/2023]
Abstract
There are diseases and injuries in which a patient's cells or tissues are destroyed that can only be adequately corrected by tissue or organ transplants. Stem cells may be able to generate new tissue and even cure diseases for which there is no adequate therapy. Type 1 diabetes (T1DM), an insulin-dependent diabetes, is a chronic disease affecting genetically predisposed individuals, in which insulin-secreting beta (β)-cells within pancreatic islets of Langerhans are selectively and irreversibly destroyed by autoimmune assault. Type 2 diabetes (T2DM) is characterized by a gradual decrease in insulin sensitivity in peripheral tissues and the liver (insulin resistance), followed by a gradual decline in β-cell function and insulin secretion. Successful replacing of damaged β-cells has shown considerable potential in treating T1DM, but lack of adequate donors is a barrier. The literature suggests that embryonic and adult stem cells are promising alternatives in long-term treatment of diabetes. However, any successful strategy should address both the need for β-cell replacement and controlling the autoimmune response to cells that express insulin. This review summarizes the current knowledge of options and the potential of stem cell transplantation in diabetes treatment.
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Affiliation(s)
- Jamil Stanekzai
- Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, 1 Discovery Drive, Rensselaer, NY 12144, USA
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Harsha S, Attimard M, Khan TA, Nair AB, Aldhubiab BE, Sangi S, Shariff A. Design and formulation of mucoadhesive microspheres of sitagliptin. J Microencapsul 2012. [DOI: 10.3109/02652048.2012.720722] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
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Islet β-Cell Mass Preservation and Regeneration in Diabetes Mellitus: Four Factors with Potential Therapeutic Interest. J Transplant 2012; 2012:230870. [PMID: 22919462 PMCID: PMC3420151 DOI: 10.1155/2012/230870] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2012] [Revised: 07/05/2012] [Accepted: 07/06/2012] [Indexed: 12/20/2022] Open
Abstract
Islet β-cell replacement and regeneration are two promising approaches for the treatment of Type 1 Diabetes Mellitus. Indeed, the success of islet transplantation in normalizing blood glucose in diabetic patients has provided the proof of principle that cell replacement can be employed as a safe and efficacious treatment. Nonetheless, shortage of organ donors has hampered expansion of this approach. Alternative sources of insulin-producing cells are mandatory to fill this gap. Although great advances have been achieved in generating surrogate β-cells from stem cells, current protocols have yet to produce functionally mature insulin-secreting cells. Recently, the concept of islet regeneration in which new β-cells are formed from either residual β-cell proliferation or transdifferentiation of other endocrine islet cells has gained much interest as an attractive therapeutic alternative to restore β-cell mass. Complementary approaches to cell replacement and regeneration could aim at enhancing β-cell survival and function. Herein, we discuss the value of Hepatocyte Growth Factor (HGF), Glucose-Dependent Insulinotropic Peptide (GIP), Paired box gene 4 (Pax4) and Liver Receptor Homolog-1 (LRH-1) as key players for β-cell replacement and regeneration therapies. These factors convey β-cell protection and enhanced function as well as facilitating proliferation and transdifferentiation of other pancreatic cell types to β-cells, under stressful conditions.
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Agarwal SK, Jothi R. Genome-wide characterization of menin-dependent H3K4me3 reveals a specific role for menin in the regulation of genes implicated in MEN1-like tumors. PLoS One 2012; 7:e37952. [PMID: 22666422 PMCID: PMC3364203 DOI: 10.1371/journal.pone.0037952] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2012] [Accepted: 04/30/2012] [Indexed: 01/07/2023] Open
Abstract
Inactivating mutations in the MEN1 gene predisposing to the multiple endocrine neoplasia type 1 (MEN1) syndrome can also cause sporadic pancreatic endocrine tumors. MEN1 encodes menin, a subunit of MLL1/MLL2-containing histone methyltransferase complexes that trimethylate histone H3 at lysine 4 (H3K4me3). The importance of menin-dependent H3K4me3 in normal and transformed pancreatic endocrine cells is unclear. To study the role of menin-dependent H3K4me3, we performed in vitro differentiation of wild-type as well as menin-null mouse embryonic stem cells (mESCs) into pancreatic islet-like endocrine cells (PILECs). Gene expression analysis and genome-wide H3K4me3 ChIP-Seq profiling in wild-type and menin-null mESCs and PILECs revealed menin-dependent H3K4me3 at the imprinted Dlk1-Meg3 locus in mESCs, and all four Hox loci in differentiated PILECs. Specific and significant loss of H3K4me3 and gene expression was observed for genes within the imprinted Dlk1-Meg3 locus in menin-null mESCs and the Hox loci in menin-null PILECs. Given that the reduced expression of genes within the DLK1-MEG3 locus and the HOX loci is associated with MEN1-like sporadic tumors, our data suggests a possible role for menin-dependent H3K4me3 at these genes in the initiation and progression of sporadic pancreatic endocrine tumors. Furthermore, our investigation also demonstrates that menin-null mESCs can be differentiated in vitro into islet-like endocrine cells, underscoring the utility of menin-null mESC-derived specialized cell types for genome-wide high-throughput studies.
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Affiliation(s)
- Sunita K. Agarwal
- Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
- * E-mail: (SKA); (RJ)
| | - Raja Jothi
- Systems Biology Section, Biostatistics Branch, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America
- * E-mail: (SKA); (RJ)
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Karadimos MJ, Kapoor A, El Khattabi I, Sharma A. β-cell preservation and regeneration for diabetes treatment: where are we now? ACTA ACUST UNITED AC 2012; 2:213-222. [PMID: 23049620 DOI: 10.2217/dmt.12.21] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Over the last decade, our knowledge of β-cell biology has expanded with the use of new scientific techniques and strategies. Growth factors, hormones and small molecules have been shown to enhance β-cell proliferation and function. Stem cell technology and research into the developmental biology of the pancreas have yielded new methods for in vivo and in vitro regeneration of β cells from stem cells and endogenous progenitors as well as transdifferentiation of non-β cells. Novel pharmacological approaches have been developed to preserve and enhance β-cell function. Strategies to increase expression of insulin gene transcription factors in dysfunctional and immature β cells have ameliorated these impairments. Hence, we suggest that strategies to minimize β-cell loss and to increase their function and regeneration will ultimately lead to therapy for both Type 1 and 2 diabetes.
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Affiliation(s)
- Michael J Karadimos
- Section of Islet Cell & Regenerative Biology, Joslin Diabetes Center, Boston, MA 02215, USA ; Department of Medicine, Harvard Medical School, Boston, MA 02215, USA
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Plentz RR, Palagani V, Wiedemann A, Diekmann U, Glage S, Naujok O, Jörns A, Müller T. Islet microarchitecture and glucose transporter expression of the pancreas of the marmoset monkey display similarities to the human. Islets 2012; 4:123-9. [PMID: 22627676 DOI: 10.4161/isl.19254] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
The common marmoset New World monkey (Callithrix jacchus), is a primate model with great potential for scientific research, including research on diabetes. However, in opposite to Rhesus and Java monkeys (Macaca mulatta and Macaca fascicularis) little is known about the marmosets islet microarchitecture, glucose transporter and pancreatic marker gene expression. In this work we analyze differences and similarities in size, shape, cellular composition and intra-islet topography between the common marmoset and the human endocrine pancreas. Different sized, circular and a-circular shaped islets of the common marmoset and human display α-cells in the whole islet organ leading to a ribbon-like islet type. The number of islets was significantly higher in the common marmoset compared with humans. However, the area of insulin-producing cells was significantly higher in the human pancreas. Intra-islet distribution pattern of δ- and β-cells was similar in both species. The morphology of the exocrine pancreas regarding acinar and ductal cells was quite similar as confirmed by ultrastructural analysis. Additionally the ultrastructure of secretory granules from α-, δ- and β-cells of human and non-human primate pancreas showed the same characteristics. Molecular analysis showed the presence of endocrine pancreatic marker genes like PMCA2, NCX1, SUR1, KIR6.2, MAFA, NGN3 and PDX1 also expressed in the human. For the first time we could show presence of Glut 5 and 9 transporters in addition to the low abundance transporter Glut2 and the highly expressed Glut1 glucose transporter. We propose that Callithrix jacchus displays a new animal model for diabetes research and regenerative medicine.
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Affiliation(s)
- Ruben R Plentz
- Department of Gastroenterology, Hannover Medical School, Hannover, Germany
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Reversal of diabetes through gene therapy of diabetic rats by hepatic insulin expression via lentiviral transduction. Mol Ther 2012; 20:918-26. [PMID: 22354377 DOI: 10.1038/mt.2012.8] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
Due to shortage of donor tissue a cure for type 1 diabetes by pancreas organ or islet transplantation is an option only for very few patients. Gene therapy is an alternative approach to cure the disease. Insulin generation in non-endocrine cells through genetic engineering is a promising therapeutic concept to achieve insulin independence in patients with diabetes. In the present study furin-cleavable human insulin was expressed in the liver of autoimmune-diabetic IDDM rats (LEW.1AR1/Ztm-iddm) and streptozotocin-diabetic rats after portal vein injection of INS-lentivirus. Within 5-7 days after the virus injection of 7 × 10(9) INS-lentiviral particles the blood glucose concentrations were normalized in the treated animals. This glucose lowering effect remained stable for the 1 year observation period. Human C-peptide as a marker for hepatic release of human insulin was in the range of 50-100 pmol/ml serum. Immunofluorescence staining of liver tissue was positive for insulin showing no signs of transdifferentiation into pancreatic β-cells. This study shows that the diabetic state can be efficiently reversed by insulin release from non-endocrine cells through a somatic gene therapy approach.
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Lysy PA, Weir GC, Bonner-Weir S. Concise review: pancreas regeneration: recent advances and perspectives. Stem Cells Transl Med 2012. [PMID: 23197762 DOI: 10.5966/sctm.2011-0025] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The replacement of functional pancreatic β-cells is seen as an attractive potential therapy for diabetes, because diabetes results from an inadequate β-cell mass. Inducing replication of the remaining β-cells and new islet formation from progenitors within the pancreas (neogenesis) are the most direct ways to increase the β-cell mass. Stimulation of both replication and neogenesis have been reported in rodents, but their clinical significance must still be shown. Because human islet transplantation is limited by the scarcity of donors and graft failure within a few years, efforts have recently concentrated on the use of stem cells to replace the deficient β-cells. Currently, embryonic stem cells and induced pluripotent stem cells achieve high levels of β-cell differentiation, but their clinical use is still hampered by ethical issues and/or the risk of developing tumors after transplantation. Pancreatic epithelial cells (duct, acinar, or α-cells) represent an appealing alternative to stem cells because they demonstrate β-cell differentiation capacities. Yet translation of such capacity to human cells after significant in vitro expansion has yet to be achieved. Besides providing new β-cells, cell therapy also has to address the question on how to protect the transplanted cells from destruction by the immune system via either allo- or autoimmunity. Encouraging developments have been made in encapsulation and immunomodulation techniques, but many challenges still remain. Herein, we discuss recent advances in the search for β-cell replacement therapies, current strategies for circumventing the immune system, and mandatory steps for new techniques to be translated from bench to clinics.
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Affiliation(s)
- Philippe A Lysy
- Joslin Diabetes Center, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts, USA
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