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1
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Yi LX, Woon HR, Saw G, Zeng L, Tan EK, Zhou ZD. Induced pluripotent stem cell-related approaches to generate dopaminergic neurons for Parkinson's disease. Neural Regen Res 2025; 20:3193-3206. [PMID: 39665833 PMCID: PMC11881713 DOI: 10.4103/nrr.nrr-d-24-00771] [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: 07/15/2024] [Revised: 09/25/2024] [Accepted: 10/23/2024] [Indexed: 12/13/2024] Open
Abstract
The progressive loss of dopaminergic neurons in affected patient brains is one of the pathological features of Parkinson's disease, the second most common human neurodegenerative disease. Although the detailed pathogenesis accounting for dopaminergic neuron degeneration in Parkinson's disease is still unclear, the advancement of stem cell approaches has shown promise for Parkinson's disease research and therapy. The induced pluripotent stem cells have been commonly used to generate dopaminergic neurons, which has provided valuable insights to improve our understanding of Parkinson's disease pathogenesis and contributed to anti-Parkinson's disease therapies. The current review discusses the practical approaches and potential applications of induced pluripotent stem cell techniques for generating and differentiating dopaminergic neurons from induced pluripotent stem cells. The benefits of induced pluripotent stem cell-based research are highlighted. Various dopaminergic neuron differentiation protocols from induced pluripotent stem cells are compared. The emerging three-dimension-based brain organoid models compared with conventional two-dimensional cell culture are evaluated. Finally, limitations, challenges, and future directions of induced pluripotent stem cell-based approaches are analyzed and proposed, which will be significant to the future application of induced pluripotent stem cell-related techniques for Parkinson's disease.
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Affiliation(s)
| | | | | | - Li Zeng
- National Neuroscience Institute, Singapore
- Department of Neurology, Singapore General Hospital, Singapore
- Signature Research Program in Neuroscience and Behavioral Disorders, Duke-NUS Medical School, Singapore
| | - Eng King Tan
- National Neuroscience Institute, Singapore
- Department of Neurology, Singapore General Hospital, Singapore
- Signature Research Program in Neuroscience and Behavioral Disorders, Duke-NUS Medical School, Singapore
| | - Zhi Dong Zhou
- National Neuroscience Institute, Singapore
- Signature Research Program in Neuroscience and Behavioral Disorders, Duke-NUS Medical School, Singapore
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2
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Kim MH, Thanuthanakhun N, Kino-Oka M. A simple tool for the synchronous differentiation of human induced pluripotent stem cells into pancreatic progenitors. Biotechnol J 2024; 19:e2300364. [PMID: 37955342 DOI: 10.1002/biot.202300364] [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: 07/28/2023] [Revised: 10/01/2023] [Accepted: 11/09/2023] [Indexed: 11/14/2023]
Abstract
Efficient differentiation of human induced pluripotent stem cells (hiPSCs) into functional pancreatic cells holds great promise for diabetes research and treatment. However, a robust culture strategy for producing pancreatic progenitors with high homogeneity is lacking. Here, we established a simple differentiation strategy for generating synchronous iPSC-derived pancreatic progenitors via a two-step method of sequential cell synchronization using botulinum hemagglutinin (HA), an E-cadherin function-blocking agent. Of the various methods tested, the first-step synchronization method with HA exposure induces a synchronous switch from E- to N-cadherin and N- to E-cadherin expression by spatially controlling heterogeneous cell distribution, subsequently improving their competency for directed differentiation into definitive endodermal cells from iPSCs. The iPSC-derived definitive endodermal cells can efficiently generate PDX1+ and NKX6.1+ pancreatic progenitor cells in high yields. The PDX1+ and PDX1+ /NKX6.1+ cell densities showed 1.6- and 2.2-fold increases, respectively, compared with those from unsynchronized cultures. The intra-run and inter-run coefficient of variation were below 10%, indicating stable and robust differentiation across different cultures and runs. Our approach is a simple and efficient strategy to produce large quantities of differentiated cells with the highest homogeneity during multistage pancreatic progenitor differentiation, providing a potential tool for guided differentiation of iPSCs to functional insulin-producing cells.
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Affiliation(s)
- Mee-Hae Kim
- Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan
| | - Naruchit Thanuthanakhun
- Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan
| | - Masahiro Kino-Oka
- Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan
- Research Base for Cell Manufacturability, Osaka University, Suita, Osaka, Japan
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3
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Goh SK, Bertera S, Richardson T, Banerjee I. Repopulation of decellularized organ scaffolds with human pluripotent stem cell-derived pancreatic progenitor cells. Biomed Mater 2023; 18. [PMID: 36720168 DOI: 10.1088/1748-605x/acb7bf] [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: 08/17/2022] [Accepted: 01/31/2023] [Indexed: 02/02/2023]
Abstract
Diabetes is an emerging global epidemic that affects more that 285 million people worldwide. Engineering of endocrine pancreas tissue holds great promise for the future of diabetes therapy. Here we demonstrate the feasibility of re-engineering decellularized organ scaffolds using regenerative cell source. We differentiated human pluripotent stem cells (hPSC) toward pancreatic progenitor (PP) lineage and repopulated decellularized organ scaffolds with these hPSC-PP cells. We observed that hPSCs cultured and differentiated as aggregates are more suitable for organ repopulation than isolated single cell suspension. However, recellularization with hPSC-PP aggregates require a more extensive vascular support, which was found to be superior in decellularized liver over the decellularized pancreas scaffolds. Upon continued culture for nine days with chemical induction in the bioreactor, the seeded hPSC-PP aggregates demonstrated extensive and uniform cellular repopulation and viability throughout the thickness of the liver scaffolds. Furthermore, the decellularized liver scaffolds was supportive of the endocrine cell fate of the engrafted cells. Our novel strategy to engineer endocrine pancreas construct is expected to find potential applications in preclinical testing, drug discovery and diabetes therapy.
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Affiliation(s)
- Saik-Kia Goh
- Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, United States of America
| | - Suzanne Bertera
- Institute of Cellular Therapeutics, Allegheny Health Network, Pittsburgh, PA, United States of America
| | - Thomas Richardson
- Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA, United States of America
| | - Ipsita Banerjee
- Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA, United States of America.,Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, United States of America.,McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, United States of America
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4
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Salman IS, Al-Shammari AM, Haba MK. Direct Reprogramming of Mice Skin Fibroblasts into Insulin-Producing Cells In Vitro. Cell Reprogram 2021; 24:271-282. [PMID: 34637623 DOI: 10.1089/cell.2021.0047] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Transdifferentiation means mature cell conversion into other mature cells. Ethical issues, epigenetic failure, or teratoma development are found in cellular reprogramming strategies. Thus, new methods are needed. This study aimed to develop a new novel formula of chemical molecules and growth factors that differentiate skin fibroblasts into insulin-producing cells (IPCs). Newborn mice fibroblasts differentiated using four induction methods into IPCs to search for the best method. Fibroblasts, stem cells, and pancreatic markers were identified using an immunocytochemistry (ICC) assay. Insulin was measured using ELISA and dithizone (DTZ) assays. The skin fibroblasts were induced successfully into IPCs. The best method to obtain IPCs was indicated by measuring insulin concentration in differentiated cell supernatant from all induced cells by the four methods. The protein expression of the pancreatic markers of induced cells increased with time, as indicated by the ICC assay. OCT3/4 increased on day 9, after which the expression tended to decrease. DTZ-positive clusters were observed on day 16. Secreted insulin of differentiated cells was injected in streptozotocin-induced diabetic mice, which decreased blood glucose levels after injection. This study indicated an efficient new chemical method for transdifferentiating skin fibroblasts into functional IPCs, which is a promising method for diabetes mellitus therapy.
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Affiliation(s)
- Israa S Salman
- Department of Biology, College of Science for Women, University of Baghdad, Baghdad, Iraq
| | - Ahmed Majeed Al-Shammari
- Experimental Therapy Department, Iraqi Center of Cancer and Medical Genetic Research, Mustansiriyah University, Baghdad, Iraq
| | - Mukhtar Khamis Haba
- Department of Biology, College of Science for Women, University of Baghdad, Baghdad, Iraq
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5
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Amorim JP, Gali-Macedo A, Marcelino H, Bordeira-Carriço R, Naranjo S, Rivero-Gil S, Teixeira J, Galhardo M, Marques J, Bessa J. A Conserved Notochord Enhancer Controls Pancreas Development in Vertebrates. Cell Rep 2021; 32:107862. [PMID: 32640228 PMCID: PMC7355232 DOI: 10.1016/j.celrep.2020.107862] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2019] [Revised: 04/06/2020] [Accepted: 06/09/2020] [Indexed: 12/31/2022] Open
Abstract
The notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs β cell differentiation, compatible with the antagonistic role of Bmp in β cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs β cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas.
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Affiliation(s)
- João Pedro Amorim
- i3S (Instituto de Investigação e Inovação em Saúde), Universidade do Porto, Porto, Portugal; IBMC (Instituto de Biologia Molecular e Celular), Universidade do Porto, Porto, Portugal
| | - Ana Gali-Macedo
- i3S (Instituto de Investigação e Inovação em Saúde), Universidade do Porto, Porto, Portugal; IBMC (Instituto de Biologia Molecular e Celular), Universidade do Porto, Porto, Portugal
| | - Hugo Marcelino
- i3S (Instituto de Investigação e Inovação em Saúde), Universidade do Porto, Porto, Portugal; IBMC (Instituto de Biologia Molecular e Celular), Universidade do Porto, Porto, Portugal
| | - Renata Bordeira-Carriço
- i3S (Instituto de Investigação e Inovação em Saúde), Universidade do Porto, Porto, Portugal; IBMC (Instituto de Biologia Molecular e Celular), Universidade do Porto, Porto, Portugal
| | - Silvia Naranjo
- CABD (Centro Andaluz de Biología del Desarrollo), Universidad Pablo de Olavide, Seville, Spain
| | - Solangel Rivero-Gil
- CABD (Centro Andaluz de Biología del Desarrollo), Universidad Pablo de Olavide, Seville, Spain
| | - Joana Teixeira
- i3S (Instituto de Investigação e Inovação em Saúde), Universidade do Porto, Porto, Portugal; IBMC (Instituto de Biologia Molecular e Celular), Universidade do Porto, Porto, Portugal
| | - Mafalda Galhardo
- i3S (Instituto de Investigação e Inovação em Saúde), Universidade do Porto, Porto, Portugal; CIBIO (Centro de Investigação em Biodiversidade e Recursos Genéticos), Universidade do Porto, Vairão, Portugal
| | - Joana Marques
- i3S (Instituto de Investigação e Inovação em Saúde), Universidade do Porto, Porto, Portugal; IBMC (Instituto de Biologia Molecular e Celular), Universidade do Porto, Porto, Portugal
| | - José Bessa
- i3S (Instituto de Investigação e Inovação em Saúde), Universidade do Porto, Porto, Portugal; IBMC (Instituto de Biologia Molecular e Celular), Universidade do Porto, Porto, Portugal.
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Continuous Inhibition of Sonic Hedgehog Signaling Leads to Differentiation of Human-Induced Pluripotent Stem Cells into Functional Insulin-Producing β Cells. Stem Cells Int 2021. [DOI: 10.1155/2021/6681257] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Human-induced pluripotent stem cell- (iPSC-) derived insulin-producing cells (IPCs) can be used for islet cell transplantation into type 1 diabetic patients and as patient-specific cells for the development of novel antidiabetic drugs. However, a method is needed to generate functional IPCs from iPSCs and simplify the protocol. We compared combinations of small molecules that could induce the differentiation of cells into a definitive endoderm and preferentially into islet precursor cells. When generated using an optimal combination of small molecules, IPCs secreted insulin in response to glucose stimulation. We constructed spheroid IPCs and optimized the culture and maturation conditions. Quantitative PCR revealed that the expression of definitive endoderm-specific markers differed depending on the combination of the small molecules. The small molecule, N-[(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methylene]-4-(phenylmethyl)-1-piperazinamine, induced the differentiation of cells into functional IPCs by inhibiting Sonic hedgehog signaling. Images of the 2D culture showed that IPCs formed spheroids from day 5 and continuously secreted insulin. We developed a simple differentiation method using small molecules that produced functional IPCs that responded to glucose stimulation within a relatively short period. We posit that this method along with further refinement of the differentiation process can be applied to culture IPCs that can be used in clinical trials.
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7
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Akil AAS, Yassin E, Al-Maraghi A, Aliyev E, Al-Malki K, Fakhro KA. Diagnosis and treatment of type 1 diabetes at the dawn of the personalized medicine era. J Transl Med 2021; 19:137. [PMID: 33794915 PMCID: PMC8017850 DOI: 10.1186/s12967-021-02778-6] [Citation(s) in RCA: 54] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2020] [Accepted: 03/08/2021] [Indexed: 12/21/2022] Open
Abstract
Type 1 diabetes affects millions of people globally and requires careful management to avoid serious long-term complications, including heart and kidney disease, stroke, and loss of sight. The type 1 diabetes patient cohort is highly heterogeneous, with individuals presenting with disease at different stages and severities, arising from distinct etiologies, and overlaying varied genetic backgrounds. At present, the “one-size-fits-all” treatment for type 1 diabetes is exogenic insulin substitution therapy, but this approach fails to achieve optimal blood glucose control in many individuals. With advances in our understanding of early-stage diabetes development, diabetes stratification, and the role of genetics, type 1 diabetes is a promising candidate for a personalized medicine approach, which aims to apply “the right therapy at the right time, to the right patient”. In the case of type 1 diabetes, great efforts are now being focused on risk stratification for diabetes development to enable pre-clinical detection, and the application of treatments such as gene therapy, to prevent pancreatic destruction in a sub-set of patients. Alongside this, breakthroughs in stem cell therapies hold great promise for the regeneration of pancreatic tissues in some individuals. Here we review the recent initiatives in the field of personalized medicine for type 1 diabetes, including the latest discoveries in stem cell and gene therapy for the disease, and current obstacles that must be overcome before the dream of personalized medicine for all type 1 diabetes patients can be realized.
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Affiliation(s)
- Ammira Al-Shabeeb Akil
- Department of Human Genetics-Precision Medicine Program, Sidra Medicine, P.O. Box 26999, Doha, Qatar.
| | - Esraa Yassin
- Department of Human Genetics-Precision Medicine Program, Sidra Medicine, P.O. Box 26999, Doha, Qatar
| | - Aljazi Al-Maraghi
- Department of Human Genetics-Precision Medicine Program, Sidra Medicine, P.O. Box 26999, Doha, Qatar
| | - Elbay Aliyev
- Department of Human Genetics-Precision Medicine Program, Sidra Medicine, P.O. Box 26999, Doha, Qatar
| | - Khulod Al-Malki
- Department of Human Genetics-Precision Medicine Program, Sidra Medicine, P.O. Box 26999, Doha, Qatar
| | - Khalid A Fakhro
- Department of Human Genetics-Precision Medicine Program, Sidra Medicine, P.O. Box 26999, Doha, Qatar.,Department of Genetic Medicine, Weill Cornell Medicine, P.O. Box 24144, Doha, Qatar.,College of Health and Life Sciences, Hamad Bin Khalifa University, P.O. Box 34110, Doha, Qatar
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8
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Thakur G, Lee HJ, Jeon RH, Lee SL, Rho GJ. Small Molecule-Induced Pancreatic β-Like Cell Development: Mechanistic Approaches and Available Strategies. Int J Mol Sci 2020; 21:E2388. [PMID: 32235681 PMCID: PMC7178115 DOI: 10.3390/ijms21072388] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2020] [Revised: 03/26/2020] [Accepted: 03/26/2020] [Indexed: 02/06/2023] Open
Abstract
Diabetes is a metabolic disease which affects not only glucose metabolism but also lipid and protein metabolism. It encompasses two major types: type 1 and 2 diabetes. Despite the different etiologies of type 1 and 2 diabetes mellitus (T1DM and T2DM, respectively), the defining features of the two forms are insulin deficiency and resistance, respectively. Stem cell therapy is an efficient method for the treatment of diabetes, which can be achieved by differentiating pancreatic β-like cells. The consistent generation of glucose-responsive insulin releasing cells remains challenging. In this review article, we present basic concepts of pancreatic organogenesis, which intermittently provides a basis for engineering differentiation procedures, mainly based on the use of small molecules. Small molecules are more auspicious than any other growth factors, as they have unique, valuable properties like cell-permeability, as well as a nonimmunogenic nature; furthermore, they offer immense benefits in terms of generating efficient functional beta-like cells. We also summarize advances in the generation of stem cell-derived pancreatic cell lineages, especially endocrine β-like cells or islet organoids. The successful induction of stem cells depends on the quantity and quality of available stem cells and the efficient use of small molecules.
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Affiliation(s)
- Gitika Thakur
- Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea; (G.T.); (H.-J.L.); (S.-L.L.)
| | - Hyeon-Jeong Lee
- Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea; (G.T.); (H.-J.L.); (S.-L.L.)
| | - Ryoung-Hoon Jeon
- Department of Cardiovascular Medicine, Mayo Clinic, Rochester, MN 55905, USA;
| | - Sung-Lim Lee
- Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea; (G.T.); (H.-J.L.); (S.-L.L.)
| | - Gyu-Jin Rho
- Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea; (G.T.); (H.-J.L.); (S.-L.L.)
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Xu B, Fan D, Zhao Y, Li J, Wang Z, Wang J, Wang X, Guan Z, Niu B. Three-Dimensional Culture Promotes the Differentiation of Human Dental Pulp Mesenchymal Stem Cells Into Insulin-Producing Cells for Improving the Diabetes Therapy. Front Pharmacol 2020; 10:1576. [PMID: 32038250 PMCID: PMC6993085 DOI: 10.3389/fphar.2019.01576] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2019] [Accepted: 12/05/2019] [Indexed: 11/13/2022] Open
Abstract
INTRODUCTION Diabetes is a metabolic disease with a high incidence and serious harm to human health. Islet β-cell function defects can occur in the late stage of type 1 diabetes and type 2 diabetes. Studies have shown that stem cell is a promising new approach in bioengineering regenerative medicine. In the study of stem cell differentiation, three-dimensional (3D) cell culture is more capable of mimicking the microenvironment of cell growth in vivo than two-dimensional (2D) cell culture. The natural contact between cells and cells, and cells and extracellular matrix can regulate the development process and promote the formation of the artificial regenerative organs and organization. Type IV, VI collagen and laminin are the most abundant extracellular matrix components in islets. Matrigel, a basement membrane matrix biomaterial rich in laminin and collagen IV. MATERIALS AND METHODS We used Matrigel biomaterial to physically embed human dental pulp stem cells (hDPSCs) to provide vector and 3D culture conditions for cells, and we explored and compared the preparation methods and preliminary mechanisms of differentiation of hDPSCs into insulin-producing cells (IPCs) under 2D or 3D culture conditions.We first designed and screened the strategy by mimicking the critical events of pancreatogenesis in vivo, and succeeded in establishing a new method for obtaining IPCs from hDPSCs. Activin A, Noggin, and small molecule compounds were used to synergistically induce hDPSCs to differentiate into definitive endoderm-like cells, pancreatic progenitor like cells and IPCs step by step under 2D culture conditions. Then, we used Matrigel to simulate the microenvironment in vivo, induced hDPSCs to differentiate into IPCs in Matrigel, evaluated and compared the efficiency between 2D and 3D culture conditions. RESULTS The results showed that the synergistic combination of growth factors and small molecule compounds and 3D culture promoted the differentiation of hDPSCs into IPCs, significantly enhancing the release of insulin and C-peptide from IPCs. DISCUSSION Significant support is provided for obtaining a large number of functional IPCs for disease modeling and final cell therapy in regenerative medicine.
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Affiliation(s)
- Bingbing Xu
- Department of Translational Medicine, Capital Institute of Pediatrics, Beijing, China
- Graduate School of Peking Union Medical College, Beijing, China
- Knee Surgery Department of the Institute of Sports Medicine, Beijing Key Laboratory of Sports Injuries, Peking University Third Hospital, Beijing, China
| | - Daoyang Fan
- Knee Surgery Department of the Institute of Sports Medicine, Beijing Key Laboratory of Sports Injuries, Peking University Third Hospital, Beijing, China
| | - Yunshan Zhao
- Institute of General Surgery, Chinese PLA General Hospital, Beijing, China
| | - Jing Li
- Laboratory of Translational Medicine, Chinese PLA General Hospital, Beijing, China
| | - Zhendong Wang
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanxi Medical University, Taiyuan, China
| | - Jianhua Wang
- Department of Translational Medicine, Capital Institute of Pediatrics, Beijing, China
| | - Xiuwei Wang
- Department of Translational Medicine, Capital Institute of Pediatrics, Beijing, China
| | - Zhen Guan
- Department of Translational Medicine, Capital Institute of Pediatrics, Beijing, China
| | - Bo Niu
- Department of Translational Medicine, Capital Institute of Pediatrics, Beijing, China
- Graduate School of Peking Union Medical College, Beijing, China
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10
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Gao C, Peng J. A glimpse of endocrine pancreas development from single-cell analyses. J Mol Cell Biol 2019; 11:433-434. [PMID: 30500955 PMCID: PMC6604600 DOI: 10.1093/jmcb/mjy079] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2018] [Accepted: 11/29/2018] [Indexed: 11/23/2022] Open
Affiliation(s)
- Ce Gao
- MOE Key Laboratory for Molecular Animal Nutrition, College of Animal Sciences, Zhejiang University, Hangzhou, China
| | - Jinrong Peng
- MOE Key Laboratory for Molecular Animal Nutrition, College of Animal Sciences, Zhejiang University, Hangzhou, China
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11
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Small molecules and extrinsic factors promoting differentiation of stem cells into insulin-producing cells. ANNALES D'ENDOCRINOLOGIE 2019; 80:128-133. [DOI: 10.1016/j.ando.2018.11.002] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/26/2018] [Revised: 10/14/2018] [Accepted: 11/05/2018] [Indexed: 12/26/2022]
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12
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Aydin S, Sağraç D, Şahin F. Differentiation Potential of Mesenchymal Stem Cells into Pancreatic β-Cells. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1247:135-156. [PMID: 32002800 DOI: 10.1007/5584_2019_476] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Stem cells having the capability to differentiate into other type of cells and renewing themselves, gained so much importance in recent years. Investigations in stem cells revealed that mesenchymal stem cells can successfully differentiate into other type of cells like adipocytes, hepatocytes, osteocytes, neurocytes and chondrocytes. In addition, these cells can also differentiate into insulin-producing beta cells. Insulin is a crucial hormone for glucose balance of the body. Insufficiency or unavailability of insulin is called diabetes. External insulin intake, as well as pancreas or islet transplantation, is the most basic treatment of diabetes. In vivo and in vitro studies demonstrate that stem cell therapy is also used in the cure of diabetes. Differentiation process of stem cells into beta cells releasing insulin is quite complicated. There are many different reports for the differentiation of stem cells in the literature. The success of differentiation of stem cells into beta cells depends on several factors like the source of stem cells, chemicals added into the differentiation medium and the duration of differentiation protocol. Distinct studies for the differentiation of stem cells into insulin-secreting cells are available in the literature. Moreover, thanks to the superior differentiation capacity of stem cells, they are being preferred in clinical studies. Stem cells were clinically used to heal diabetic ulcer, to increase c-peptide level and insulin secretion in both type 1 and type 2 diabetes. Mesenchymal stem cells having high differentiation potential to insulin-secreting cells are encouraging vehicles for both in vivo and in vitro studies together with clinical trials for diabetes mellitus.
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Affiliation(s)
- Safa Aydin
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, İstanbul, Turkey.
| | - Derya Sağraç
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, İstanbul, Turkey
| | - Fikrettin Şahin
- Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, İstanbul, Turkey
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13
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Solis MA, Moreno Velásquez I, Correa R, Huang LLH. Stem cells as a potential therapy for diabetes mellitus: a call-to-action in Latin America. Diabetol Metab Syndr 2019; 11:20. [PMID: 30820250 PMCID: PMC6380040 DOI: 10.1186/s13098-019-0415-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/07/2018] [Accepted: 02/13/2019] [Indexed: 02/06/2023] Open
Abstract
Latin America is a fast-growing region that currently faces unique challenges in the treatment of all forms of diabetes mellitus. The burden of this disease will be even greater in the coming years due, in part, to the large proportion of young adults living in urban areas and engaging in unhealthy lifestyles. Unfortunately, the national health systems in Latin-American countries are unprepared and urgently need to reorganize their health care services to achieve diabetic therapeutic goals. Stem cell research is attracting increasing attention as a promising and fast-growing field in Latin America. As future healthcare systems will include the development of regenerative medicine through stem cell research, Latin America is urged to issue a call-to-action on stem cell research. Increased efforts are required in studies focused on stem cells for the treatment of diabetes. In this review, we aim to inform physicians, researchers, patients and funding sources about the advances in stem cell research for possible future applications in diabetes mellitus. Emerging studies are demonstrating the potential of stem cells for β cell differentiation and pancreatic regeneration. The major economic burden implicated in patients with diabetes complications suggests that stem cell research may relieve diabetic complications. Closer attention should be paid to stem cell research in the future as an alternative treatment for diabetes mellitus.
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Affiliation(s)
| | | | - Ricardo Correa
- Department of Medicine, Warren Alpert School of Medicine, Brown University, Rhode Island, USA
- Department of Medicine, University of Arizona College of Medicine, Phoenix, AZ USA
| | - Lynn L. H. Huang
- Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan, Taiwan
- Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan
- Research Center of Excellence in Regenerative Medicine, National Cheng Kung University, Tainan, Taiwan
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Hashemitabar M, Heidari E. Redefining the signaling pathways from pluripotency to pancreas development: In vitro β-cell differentiation. J Cell Physiol 2018; 234:7811-7827. [PMID: 30480819 DOI: 10.1002/jcp.27736] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2018] [Accepted: 10/22/2018] [Indexed: 02/06/2023]
Abstract
Pancreatic β-cells are destroyed by the immune system, in type 1 diabetes (T1D) and are impaired by glucose insensitivity in type 2 diabetes (T2D). Islet-cells transplantation is a promising therapeutic approach based on in vitro differentiation of pluripotent stem cells (PSCs) to insulin-producing cells (IPCs). According to evolutionary stages in β-cell development, there are several distinct checkpoints; each one has a unique characteristic, including definitive endoderm (DE), primitive gut (PG), posterior foregut (PF), pancreatic epithelium (PE), endocrine precursor (EP), and immature β-cells up to functional β-cells. A better understanding of the gene regulatory networks (GRN) and associated transcription factors in each specific developmental stage, guarantees the achievement of the next successful checkpoints and ensures an efficient β-cell differentiation procedure. The new findings in signaling pathways, related to the development of the pancreas are discussed here, including Wnt, Activin/Nodal, FGF, BMP, retinoic acid (RA), sonic hedgehog (Shh), Notch, and downstream regulators, required for β-cell commitment. We also summarized different approaches in the IPCs protocol to conceptually define a standardized system, leading to the creation of a reproducible method for β-cell differentiation. To normalize blood glucose level in diabetic mice, the replacement therapy in the early differentiation stage, such as EP stages was associated with better outcome when compared with the fully differentiated β-cells' graft.
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Affiliation(s)
- Mahmoud Hashemitabar
- Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.,Department of Anatomy and Embryology, Faculty of Medicine, Joundishapur University of Medical Sciences, Ahvaz, Iran
| | - Elham Heidari
- Department of Anatomy and Embryology, Faculty of Medicine, Joundishapur University of Medical Sciences, Ahvaz, Iran
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15
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Treatment with specific soluble factors promotes the functional maturation of transcription factor-mediated, pancreatic transdifferentiated cells. PLoS One 2018; 13:e0197175. [PMID: 29768476 PMCID: PMC5955553 DOI: 10.1371/journal.pone.0197175] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2017] [Accepted: 04/28/2018] [Indexed: 12/19/2022] Open
Abstract
Pancreatic lineage-specific transcription factors (TFs) display instructive roles in converting adult cells to endocrine pancreatic cells through a process known as transdifferentiation. However, little is known about potential factors capable of accelerating transdifferentiation following transduction to achieve the functional maturation of transdifferentiated cells. In this study, we demonstrated, using adult liver-derived progenitor cells, that soluble factors utilized in pancreatic differentiation protocols of pluripotent stem cells promote functional maturation of TFs-mediated transdifferentiated cells. Treatment with an N2 supplement in combination with three soluble factors (glucagon-like peptide-1 [GLP-1] receptor agonist, notch inhibitor, and transforming growth factor-β [TGF-β] inhibitor) enhanced liver-to-pancreas transdifferentiation based on the following findings: i) the incidence of c-peptide-positive cells increased by approximately 1.2-fold after the aforementioned treatment; ii) the c-peptide expression level in the treated cells increased by approximately 12-fold as compared with the level in the untreated cells; iii) the treated cells secreted insulin in a glucose-dependent manner, whereas the untreated cells did not; and iv) transplantation of treated-transdifferentiated cells into streptozotocin-induced immunodeficient diabetic mice led to the amelioration of hyperglycemia. These results suggest that treatment with specific soluble factors promotes the functional maturation of transdifferentiated cells. Our findings could facilitate the development of new modalities for cell-replacement therapy for patients with diabetes.
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16
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Memon B, Karam M, Al-Khawaga S, Abdelalim EM. Enhanced differentiation of human pluripotent stem cells into pancreatic progenitors co-expressing PDX1 and NKX6.1. Stem Cell Res Ther 2018; 9:15. [PMID: 29361979 PMCID: PMC5781269 DOI: 10.1186/s13287-017-0759-z] [Citation(s) in RCA: 57] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2017] [Revised: 12/19/2017] [Accepted: 12/20/2017] [Indexed: 01/19/2023] Open
Abstract
Background Pancreatic progenitors (PPs) co-expressing the two transcription factors (TFs) PDX1 and NKX6.1 are recognized as the indispensable precursors of functional pancreatic β cells. Here, we aimed to establish an efficient protocol for maximizing generation of PDX1+/NKX6.1+ PPs from human pluripotent stem cells (hPSCs). Methods In order to enhance the PDX1+/NKX6.1+ population, we manipulated in vitro culture conditions during differentiation by dissociating densely formed endodermal cells and re-plating them at different densities. These dissociated cells were subjected to an augmented duration of retinoid and fibroblast growth factor (FGF)10 signaling to induce higher PDX1 and NKX6.1 expression. Results Our optimized protocol dramatically increased the expression of NKX6.1, leading to an increase in the proportion of PDX1+/NKX6.1+ progenitors (~90%) in monolayer, higher than the previously published protocols, as well as upregulated key TFs controlling pancreatic development. The improved efficiency of pancreatic differentiation was complemented by an inhibited hepatic specification and an increased proliferation of NKX6.1+ cells. Interestingly, we were able to enrich a novel PDX1–/NKX6.1+ population by manipulating the re-plating density; these oriented themselves in three-dimensional clusters. Further differentiation validated the ability of our PDX1+/NKX6.1+ progenitors to generate NGN3+ endocrine progenitors. Conclusions We provide a novel technique that facilitates appropriate cellular rearrangement in monolayer culture to yield a high proportion of PDX1+/NKX6.1+ PPs with an elevated self-replicating capacity, thereby aiding scalable production of functional β cells from hPSCs in vitro. Our innovative method also enriches a novel NKX6.1+/PDX1– population, with characteristics of proposed endocrine precursors, allowing further studies on deciphering routes to β-cell development. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0759-z) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Bushra Memon
- Diabetes Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar
| | - Manale Karam
- Cancer Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar
| | - Sara Al-Khawaga
- Diabetes Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar
| | - Essam M Abdelalim
- Diabetes Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha, Qatar.
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17
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Loo LSW, Lau HH, Jasmen JB, Lim CS, Teo AKK. An arduous journey from human pluripotent stem cells to functional pancreatic β cells. Diabetes Obes Metab 2018; 20:3-13. [PMID: 28474496 DOI: 10.1111/dom.12996] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/02/2017] [Revised: 04/29/2017] [Accepted: 05/01/2017] [Indexed: 12/11/2022]
Abstract
Type 1 and type 2 diabetes are caused by a destruction and decrease in the number of functional insulin-producing β cells, respectively; therefore, the generation of functional β cells from human embryonic stem cells and human induced pluripotent stem cells, collectively known as human pluripotent stem cells (hPSCs), for potential cell replacement therapy and disease modelling is an intensely investigated area. Recent scientific breakthroughs enabled derivation of large quantities of human pancreatic β-like cells in vitro, although with varied glucose-stimulated insulin secretion kinetics. In the present review, we comprehensively summarize, compare and critically analyze the intricacies of these developing technologies, including differentiation platforms, robustness of protocols, and methodologies used to characterize hPSC-derived β-like cells. We also discuss experimental issues that need to be resolved before these β-like cells can be used clinically.
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Affiliation(s)
- Larry Sai Weng Loo
- Programme in Stem Cell, Regenerative Medicine and Ageing, Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, Singapore, Singapore
- School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
| | - Hwee Hui Lau
- Programme in Stem Cell, Regenerative Medicine and Ageing, Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, Singapore, Singapore
| | - Joanita Binte Jasmen
- Programme in Stem Cell, Regenerative Medicine and Ageing, Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, Singapore, Singapore
| | - Chang Siang Lim
- Programme in Stem Cell, Regenerative Medicine and Ageing, Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, Singapore, Singapore
| | - Adrian Kee Keong Teo
- Programme in Stem Cell, Regenerative Medicine and Ageing, Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, Singapore, Singapore
- School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
- Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
- Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
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18
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Petersen MB, Gonçalves CA, Kim YH, Grapin-Botton A. Recapitulating and Deciphering Human Pancreas Development From Human Pluripotent Stem Cells in a Dish. Curr Top Dev Biol 2018; 129:143-190. [DOI: 10.1016/bs.ctdb.2018.02.009] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
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19
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Shirouzu Y, Yanai G, Yang KC, Sumi S. Effects of Activin in Embryoid Bodies Expressing Fibroblast Growth Factor 5. Cell Reprogram 2017; 18:171-86. [PMID: 27253628 DOI: 10.1089/cell.2015.0074] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Nodal/activin signaling is indispensable for embryonic development. We examined what activin does to the embryoid bodies (EBs) produced from mouse embryonic stem cells (mESCs) expressing an epiblast marker. The EBs were produced by culturing mESCs by the hanging drop method for 24 hours. The resulting EBs were transferred onto gelatin-coated dishes and allowed to further differentiate. The 24-hour EBs showed a stronger expression of fibroblast growth factor (FGF)5 and Brachyury (specific to the epiblast) in comparison with mESCs. Treating the transferred EBs with activin A maintained transcript levels of FGF5 and Oct4, while inhibiting definitive endoderm differentiation. The activin A treatment reversed the endoderm differentiation induced by retinoic acid (RA), while the inhibition of nodal/activin signaling promoted RA-induced endoderm differentiation. Inhibition of nodal/activin signaling in EBs, including epiblast-like cells, promotes differentiation into the endoderm, facilitating the transition from the pluripotent state to specification of the endoderm.
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Affiliation(s)
- Yasumasa Shirouzu
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University , Kyoto, Japan
| | - Goichi Yanai
- Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University , Kyoto, Japan
| | - Kai-Chiang Yang
- 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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20
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ROCKII inhibition promotes the maturation of human pancreatic beta-like cells. Nat Commun 2017; 8:298. [PMID: 28824164 PMCID: PMC5563509 DOI: 10.1038/s41467-017-00129-y] [Citation(s) in RCA: 61] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2017] [Accepted: 06/01/2017] [Indexed: 01/05/2023] Open
Abstract
Diabetes is linked to loss of pancreatic beta-cells. Pluripotent stem cells offer a valuable source of human beta-cells for basic studies of their biology and translational applications. However, the signalling pathways that regulate beta-cell development and functional maturation are not fully understood. Here we report a high content chemical screen, revealing that H1152, a ROCK inhibitor, promotes the robust generation of insulin-expressing cells from multiple hPSC lines. The insulin expressing cells obtained after H1152 treatment show increased expression of mature beta cell markers and improved glucose stimulated insulin secretion. Moreover, the H1152-treated beta-like cells show enhanced glucose stimulated insulin secretion and increased capacity to maintain glucose homeostasis after transplantation. Conditional gene knockdown reveals that inhibition of ROCKII promotes the generation and maturation of glucose-responding cells. This study provides a strategy to promote human beta-cell maturation and identifies an unexpected role for the ROCKII pathway in the development and maturation of beta-like cells.Our incomplete understanding of how pancreatic beta cells form limits the generation of beta-like cells from human pluripotent stem cells (hPSC). Here, the authors identify a ROCKII inhibitor H1152 as increasing insulin secreting cells from hPSCs and improving beta-cell maturation on transplantation in vivo.
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21
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Chen ACH, Lee YL, Fong SW, Wong CCY, Ng EHY, Yeung WSB. Hyperglycemia impedes definitive endoderm differentiation of human embryonic stem cells by modulating histone methylation patterns. Cell Tissue Res 2017; 368:563-578. [PMID: 28283910 DOI: 10.1007/s00441-017-2583-2] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2016] [Accepted: 01/27/2017] [Indexed: 12/25/2022]
Abstract
Exposure to maternal diabetes during fetal growth is a risk factor for the development of type II diabetes (T2D) in later life. Discovery of the mechanisms involved in this association should provide valuable background for therapeutic treatments. Early embryogenesis involves epigenetic changes including histone modifications. The bivalent histone methylation marks H3K4me3 and H3K27me3 are important for regulating key developmental genes during early fetal pancreas specification. We hypothesized that maternal hyperglycemia disrupted early pancreas development through changes in histone bivalency. A human embryonic stem cell line (VAL3) was used as the cell model for studying the effects of hyperglycemia upon differentiation into definitive endoderm (DE), an early stage of the pancreatic lineage. Hyperglycemic conditions significantly down-regulated the expression levels of DE markers SOX17, FOXA2, CXCR4 and EOMES during differentiation. This was associated with retention of the repressive histone methylation mark H3K27me3 on their promoters under hyperglycemic conditions. The disruption of histone methylation patterns was observed as early as the mesendoderm stage, with Wnt/β-catenin signaling being suppressed during hyperglycemia. Treatment with Wnt/β-catenin signaling activator CHIR-99021 restored the expression levels and chromatin methylation status of DE markers, even in a hyperglycemic environment. The disruption of DE development was also found in mouse embryos at day 7.5 post coitum from diabetic mothers. Furthermore, disruption of DE differentiation in VAL3 cells led to subsequent impairment in pancreatic progenitor formation. Thus, early exposure to hyperglycemic conditions hinders DE development with a possible relationship to the later impairment of pancreas specification.
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Affiliation(s)
- A C H Chen
- Department of Obstetrics and Gynaecology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, People's Republic of China
| | - Y L Lee
- Department of Obstetrics and Gynaecology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, People's Republic of China.
- Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, The University of Hong Kong, Shenzhen, People's Republic of China.
- Center for Reproduction, Development and Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, People's Republic of China.
- Department of Obstetrics and Gynaecology, LKS Faculty of Medicine, The University of Hong Kong, Room 747, 21 Sassoon Road, Hong Kong, SAR, People's Republic of China.
| | - S W Fong
- Department of Obstetrics and Gynaecology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, People's Republic of China
| | - C C Y Wong
- Department of Obstetrics and Gynaecology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, People's Republic of China
| | - E H Y Ng
- Department of Obstetrics and Gynaecology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, People's Republic of China
- Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, The University of Hong Kong, Shenzhen, People's Republic of China
- Center for Reproduction, Development and Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, People's Republic of China
| | - W S B Yeung
- Department of Obstetrics and Gynaecology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, People's Republic of China
- Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, The University of Hong Kong, Shenzhen, People's Republic of China
- Center for Reproduction, Development and Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, People's Republic of China
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22
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Kuo YC, Liu YC, Rajesh R. Pancreatic differentiation of induced pluripotent stem cells in activin A-grafted gelatin-poly(lactide-co-glycolide) nanoparticle scaffolds with induction of LY294002 and retinoic acid. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2017; 77:384-393. [PMID: 28532044 DOI: 10.1016/j.msec.2017.03.265] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/18/2017] [Accepted: 03/28/2017] [Indexed: 01/09/2023]
Abstract
The differentiation of induced pluripotent stem cells (iPSCs) in biomaterial scaffolds is an emerging area for biomedical applications. This study proposes, for the first time, the production of pancreatic cells from iPSCs in gelatin-poly(lactide-co-glycolide) nanoparticle (PLGA NP) scaffolds. The porosity and swelling ratio of the scaffolds decreased with increases in gelatin and PLGA NP concentrations. The adhesion efficiency of iPSCs in gelatin-PLGA NP scaffolds was found to be higher at 6.7% (w/w) PLGA NP. A 3-step induction of iPSCs was used to differentiate into pancreatic islet cells using activin A, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), and retinoic acid (RA). The ability of iPSCs to differentiate into pancreatic islet cells in a scaffold was demonstrated by immunofluorescence staining and flow-cytometry analysis. The results indicate that the concentration of activin A, LY294002, and RA plays a decisive role in the differentiation of iPSCs into pancreatic cells. Activin A and LY294002 induce the iPSCs into endoderm and RA induces endoderm into islet cells. A maximum insulin secretion by glucose stimulation was obtained with a higher concentration (2μM) of RA. The use of activin A-grafted gelatin-PLGA NP scaffolds induced by LY294002 and RA can be a promising approach to developing pancreatic islet cells from iPSCs.
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Affiliation(s)
- Yung-Chih Kuo
- Department of Chemical Engineering, National Chung Cheng University, Chia-Yi 62102, Taiwan, Republic of China.
| | - Yu-Chuan Liu
- Department of Chemical Engineering, National Chung Cheng University, Chia-Yi 62102, Taiwan, Republic of China
| | - Rajendiran Rajesh
- Department of Chemical Engineering, National Chung Cheng University, Chia-Yi 62102, Taiwan, Republic of China
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23
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Kaitsuka T, Kobayashi K, Otsuka W, Kubo T, Hakim F, Wei FY, Shiraki N, Kume S, Tomizawa K. Erythropoietin facilitates definitive endodermal differentiation of mouse embryonic stem cells via activation of ERK signaling. Am J Physiol Cell Physiol 2017; 312:C573-C582. [PMID: 28298334 DOI: 10.1152/ajpcell.00071.2016] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2016] [Revised: 03/06/2017] [Accepted: 03/06/2017] [Indexed: 01/07/2023]
Abstract
Artificially generated pancreatic β-cells from pluripotent stem cells are expected for cell replacement therapy for type 1 diabetes. Several strategies are adopted to direct pluripotent stem cells toward pancreatic differentiation. However, a standard differentiation method for clinical application has not been established. It is important to develop more effective and safer methods for generating pancreatic β-cells without toxic or mutagenic chemicals. In the present study, we screened several endogenous factors involved in organ development to identify the factor, which induced the efficiency of pancreatic differentiation and found that treatment with erythropoietin (EPO) facilitated the differentiation of mouse embryonic stem cells (ESCs) into definitive endoderm. At an early stage of differentiation, EPO treatment significantly increased Sox17 gene expression, as a marker of the definitive endoderm. Contrary to the canonical function of EPO, it did not affect the levels of phosphorylated JAK2 and STAT5, but stimulated the phosphorylation of ERK1/2 and Akt. The MEK inhibitor U0126 significantly inhibited EPO-induced Sox17 expression. The differentiation of ESCs into definitive endoderm is an important step for the differentiation into pancreatic and other endodermal lineages. This study suggests a possible role of EPO in embryonic endodermal development and a new agent for directing the differentiation into endodermal lineages like pancreatic β-cells.
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Affiliation(s)
- Taku Kaitsuka
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Kohei Kobayashi
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Wakako Otsuka
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Takuya Kubo
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Farzana Hakim
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Fan-Yan Wei
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Nobuaki Shiraki
- Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan; and.,Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
| | - Shoen Kume
- Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan; and.,Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
| | - Kazuhito Tomizawa
- Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan;
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24
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Wang W, Jin S, Ye K. Development of Islet Organoids from H9 Human Embryonic Stem Cells in Biomimetic 3D Scaffolds. Stem Cells Dev 2017; 26:394-404. [PMID: 27960594 DOI: 10.1089/scd.2016.0115] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Success in the differentiating human embryonic stem cells (hESCs) into insulin-secreting β cells raises new hopes for diabetes treatment. In this work, we demonstrated the feasibility of developing islet organoids from hESCs within biomimetic 3D scaffolds. We showed that such a 3D microenvironment is critical to the generation of pancreatic endoderm and endocrine from hESCs. The organoids formed consisted of pancreatic α, β, δ, and pancreatic polypeptide (PP) cells. A high-level co-expression of PDX1, NKX6.1, and NGN3 in these cells suggests the characteristics of pancreatic β cells. More importantly, most insulin-secreting cells generated did not express glucagon, somatostatin, or PP. The expression of mature β cell marker genes such as Pdx1, Ngn3, Insulin, MafA, and Glut2 was detected in these 3D-induced cell clusters. A high-level expression of C-peptide confirmed the de novo endogenous insulin production in these 3D induced cells. Insulin-secretory granules, an indication of β cell maturity, were detected in these cells as well. Glucose challenging experiments suggested that these cells are sensitive to glucose levels due to their elevated maturity. Exposing the cells to a high concentration of glucose induced a sharp increase in insulin secretion.
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Affiliation(s)
- Weiwei Wang
- 1 Department of Biomedical Engineering, College of Engineering, University of Arkansas , Fayetteville, Arkansas
| | - Sha Jin
- 1 Department of Biomedical Engineering, College of Engineering, University of Arkansas , Fayetteville, Arkansas.,2 Department of Biomedical Engineering, Center of Biomanufacturing for Regenerative Medicine, Watson School of Engineering and Applied Science, Binghamton University, State University of New York (SUNY) , Binghamton, New York
| | - Kaiming Ye
- 1 Department of Biomedical Engineering, College of Engineering, University of Arkansas , Fayetteville, Arkansas.,2 Department of Biomedical Engineering, Center of Biomanufacturing for Regenerative Medicine, Watson School of Engineering and Applied Science, Binghamton University, State University of New York (SUNY) , Binghamton, New York
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Mu X, Ren L, Yan H, Zhang X, Xu T, Wei A, Jiang J. Enhanced differentiation of human amniotic fluid-derived stem cells into insulin-producing cells in vitro. J Diabetes Investig 2017; 8:34-43. [PMID: 27240324 PMCID: PMC5217909 DOI: 10.1111/jdi.12544] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/07/2016] [Revised: 04/19/2016] [Accepted: 05/02/2016] [Indexed: 01/14/2023] Open
Abstract
AIMS/INTRODUCTION To investigate the ability of human amniotic fluid stem cells (hAFSCs) to differentiate into insulin-producing cells. MATERIALS AND METHODS hAFSCs were induced to differentiate into pancreatic cells by a multistep protocol. The expressions of pancreas-related genes and proteins, including pancreatic and duodenal homeobox-1, insulin, and glucose transporter 2, were detected by polymerase chain reaction and immunofluorescence. Insulin secreted from differentiated cells was tested by enzyme-linked immunosorbent assay. RESULTS hAFSCs were successfully isolated from amniotic fluid that expressed the pluripotent markers of embryonic stem cells, such as Oct3/4, and mesenchymal stem cells, such as integrin β-1 and ecto-5'-nucleotidase. Here, we first obtained the hAFSCs that expressed pluripotent marker stage-specific embryonic antigen 1. Real-time polymerase chain reaction analysis showed that pancreatic and duodenal homeobox-1, paired box gene 4 and paired box gene 6 were expressed in the early phase of induction, and then stably expressed in the differentiated cells. The pancreas-related genes, such as insulin, glucokinase, glucose transporter 2 and Nkx6.1, were expressed in the differentiated cells. Immunofluorescence showed that these differentiated cells co-expressed insulin, C-peptide, and pancreatic and duodenal homeobox-1. Insulin was released in response to glucose stimulation in a manner similar to that of adult human islets. CONCLUSIONS The present study showed that hAFSCs, under selective culture conditions, could differentiate into islet-like insulin-producing cells, which might be used as a potential source for transplantation in patients with type 1 diabetes mellitus.
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Affiliation(s)
- Xu‐Peng Mu
- Department of Central LaboratoryChina‐Japan Union Hospital of Jilin UniversityChangchunChina
| | - Li‐Qun Ren
- College of PharmacyJilin UniversityChangchunChina
| | - Hao‐Wei Yan
- College of PharmacyJilin UniversityChangchunChina
| | | | - Tian‐Min Xu
- The Second Affiliated Hospital of Jilin UniversityChangchunChina
| | - An‐Hui Wei
- College of PharmacyJilin UniversityChangchunChina
| | - Jin‐Lan Jiang
- Department of Central LaboratoryChina‐Japan Union Hospital of Jilin UniversityChangchunChina
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26
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Ghosh D, Mehta N, Patil A, Sengupta J. Ethical issues in biomedical use of human embryonic stem cells (hESCs). ACTA ACUST UNITED AC 2016. [DOI: 10.1016/j.jrhm.2016.09.002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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Lee S, Jeong S, Lee C, Oh J, Kim SC. Mesenchymal Stem Cells Derived from Human Exocrine Pancreas Spontaneously Express Pancreas Progenitor-Cell Markers in a Cell-Passage-Dependent Manner. Stem Cells Int 2016; 2016:2142646. [PMID: 27630717 PMCID: PMC5007373 DOI: 10.1155/2016/2142646] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2016] [Accepted: 07/21/2016] [Indexed: 12/31/2022] Open
Abstract
Mesenchymal stem cells (MSCs) derived from bone marrow, adipose tissue, and most connective tissues have been recognized as promising sources for cell-based therapies. MSCs have also been detected in human pancreatic tissue, including endocrine and exocrine cells. These adult human pancreas-derived MSCs have generated a great deal of interest owing to their potential use in the differentiation of insulin-producing cells for diabetes treatment. In the present study, we isolated MSCs from the adult human exocrine pancreas to determine whether isolated MSCs have the potential to differentiate into pancreatic endocrine cells and, therefore, whether they can be used in stem cell-based therapies. Pancreatic tissue was digested by collagenase and an enriched exocrine-cell fraction was obtained by density-gradient separation. Crude exocrine cells were methodically cultured in suspension and then in adherent culture. We expanded the human pancreatic exocrine-derived MSCs (hpMSCs) by cell passaging in culture and confirmed by flow cytometry that >90% expressed human classic surface markers of MSCs. Interestingly, these cells expressed pancreatic transcription factors, such as Pdx1, Ngn3, and MafA, similar to pancreatic progenitor cells. These results indicated that hpMSCs can be used for the differentiation of pancreatic endocrine cells and may be used in type 1 diabetes treatment.
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Affiliation(s)
- Song Lee
- Laboratory of Stem Cell Biology and Cell Therapy, Asan Institute for Life Science, Asan Medical Center, Seoul 05505, Republic of Korea
- Department of Surgery, University of Ulsan College of Medicine and Asan Medical Center, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea
| | - Seonghee Jeong
- Laboratory of Stem Cell Biology and Cell Therapy, Asan Institute for Life Science, Asan Medical Center, Seoul 05505, Republic of Korea
| | - Chanmi Lee
- Laboratory of Stem Cell Biology and Cell Therapy, Asan Institute for Life Science, Asan Medical Center, Seoul 05505, Republic of Korea
| | - Jooyun Oh
- Laboratory of Stem Cell Biology and Cell Therapy, Asan Institute for Life Science, Asan Medical Center, Seoul 05505, Republic of Korea
| | - Song-Cheol Kim
- Laboratory of Stem Cell Biology and Cell Therapy, Asan Institute for Life Science, Asan Medical Center, Seoul 05505, Republic of Korea
- Department of Surgery, University of Ulsan College of Medicine and Asan Medical Center, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea
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Nazareth EJP, Rahman N, Yin T, Zandstra PW. A Multi-Lineage Screen Reveals mTORC1 Inhibition Enhances Human Pluripotent Stem Cell Mesendoderm and Blood Progenitor Production. Stem Cell Reports 2016; 6:679-691. [PMID: 27132889 PMCID: PMC4939733 DOI: 10.1016/j.stemcr.2016.04.003] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2015] [Revised: 04/04/2016] [Accepted: 04/04/2016] [Indexed: 01/27/2023] Open
Abstract
Human pluripotent stem cells (hPSCs) exist in heterogeneous micro-environments with multiple subpopulations, convoluting fate-regulation analysis. We patterned hPSCs into engineered micro-environments and screened responses to 400 small-molecule kinase inhibitors, measuring yield and purity outputs of undifferentiated, neuroectoderm, mesendoderm, and extra-embryonic populations. Enrichment analysis revealed mammalian target of rapamycin (mTOR) inhibition as a strong inducer of mesendoderm. Dose responses of mTOR inhibitors such as rapamycin synergized with Bone Morphogenetic protein 4 (BMP4) and activin A to enhance the yield and purity of BRACHYURY-expressing cells. Mechanistically, small interfering RNA knockdown of RAPTOR, a component of mTOR complex 1, phenocopied the mesendoderm-enhancing effects of rapamycin. Functional analysis during mesoderm and endoderm differentiation revealed that mTOR inhibition increased the output of hemogenic endothelial cells 3-fold, with a concomitant enhancement of blood colony-forming cells. These data demonstrate the power of our multi-lineage screening approach and identify mTOR signaling as a node in hPSC differentiation to mesendoderm and its derivatives.
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Affiliation(s)
| | - Nafees Rahman
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada; Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada
| | - Ting Yin
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada
| | - Peter William Zandstra
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada; Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada; Medicine by Design, University of Toronto, Toronto, ON M5S 3G9, Canada.
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PTF1a Activity in Enriched Posterior Foregut Endoderm, but Not Definitive Endoderm, Leads to Enhanced Pancreatic Differentiation in an In Vitro Mouse ESC-Based Model. Stem Cells Int 2016; 2016:6939438. [PMID: 27066080 PMCID: PMC4811216 DOI: 10.1155/2016/6939438] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2015] [Revised: 11/07/2015] [Accepted: 11/26/2015] [Indexed: 01/22/2023] Open
Abstract
Transcription factors are tools repetitively used by the embryo to generate a variety of lineages. Hence, their context of activation is an important determinant of their ability to specifically trigger certain cell fates, but not others. The context is also consequential when considering directing differentiation of embryonic stem cells (ESCs). In this study, we sought to assess the context of pancreatic transcription factor 1a (PTF1a) activation in reference to its propancreatic effects in mouse ESCs (mESCs). We hypothesized that an enriched endodermal population would respond to PTF1a and trigger the pancreatic program more effectively than a spontaneously differentiated population. Using an in vitro model of pancreas development that we recently established, we found that inducing PTF1a in highly enriched definitive endoderm did not promote pancreatic differentiation but induction in more differentiated endoderm, specifically posterior foregut endoderm, did form pancreatic progenitors. These progenitors never underwent terminal differentiation to endocrine or acinar phenotype. However, a short 3D culture period, prior to PTF1a induction, led to the generation of monohormonal insulin(+) cells and amylase-expressing cells. Our findings suggest that enriched posterior foregut endoderm is competent to respond to PTF1a's propancreatic activity; but a 3D culture environment is essential for terminal differentiation of pancreatic progenitors.
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Kawser Hossain M, Abdal Dayem A, Han J, Kumar Saha S, Yang GM, Choi HY, Cho SG. Recent Advances in Disease Modeling and Drug Discovery for Diabetes Mellitus Using Induced Pluripotent Stem Cells. Int J Mol Sci 2016; 17:256. [PMID: 26907255 PMCID: PMC4783985 DOI: 10.3390/ijms17020256] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2016] [Revised: 02/05/2016] [Accepted: 02/15/2016] [Indexed: 02/07/2023] Open
Abstract
Diabetes mellitus (DM) is a widespread metabolic disease with a progressive incidence of morbidity and mortality worldwide. Despite extensive research, treatment options for diabetic patients remains limited. Although significant challenges remain, induced pluripotent stem cells (iPSCs) have the capacity to differentiate into any cell type, including insulin-secreting pancreatic β cells, highlighting its potential as a treatment option for DM. Several iPSC lines have recently been derived from both diabetic and healthy donors. Using different reprogramming techniques, iPSCs were differentiated into insulin-secreting pancreatic βcells. Furthermore, diabetes patient-derived iPSCs (DiPSCs) are increasingly being used as a platform to perform cell-based drug screening in order to develop DiPSC-based cell therapies against DM. Toxicity and teratogenicity assays based on iPSC-derived cells can also provide additional information on safety before advancing drugs to clinical trials. In this review, we summarize recent advances in the development of techniques for differentiation of iPSCs or DiPSCs into insulin-secreting pancreatic β cells, their applications in drug screening, and their role in complementing and replacing animal testing in clinical use. Advances in iPSC technologies will provide new knowledge needed to develop patient-specific iPSC-based diabetic therapies.
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Affiliation(s)
- Mohammed Kawser Hossain
- Department of Animal Biotechnology, Animal Resources Research Center, and Incurable Disease Animal Model and Stem Cell Institute (IDASI), Konkuk University, Gwangjin-gu, Seoul 05029, Korea.
| | - Ahmed Abdal Dayem
- Department of Animal Biotechnology, Animal Resources Research Center, and Incurable Disease Animal Model and Stem Cell Institute (IDASI), Konkuk University, Gwangjin-gu, Seoul 05029, Korea.
| | - Jihae Han
- Department of Animal Biotechnology, Animal Resources Research Center, and Incurable Disease Animal Model and Stem Cell Institute (IDASI), Konkuk University, Gwangjin-gu, Seoul 05029, Korea.
| | - Subbroto Kumar Saha
- Department of Animal Biotechnology, Animal Resources Research Center, and Incurable Disease Animal Model and Stem Cell Institute (IDASI), Konkuk University, Gwangjin-gu, Seoul 05029, Korea.
| | - Gwang-Mo Yang
- Department of Animal Biotechnology, Animal Resources Research Center, and Incurable Disease Animal Model and Stem Cell Institute (IDASI), Konkuk University, Gwangjin-gu, Seoul 05029, Korea.
| | - Hye Yeon Choi
- Department of Animal Biotechnology, Animal Resources Research Center, and Incurable Disease Animal Model and Stem Cell Institute (IDASI), Konkuk University, Gwangjin-gu, Seoul 05029, Korea.
| | - Ssang-Goo Cho
- Department of Animal Biotechnology, Animal Resources Research Center, and Incurable Disease Animal Model and Stem Cell Institute (IDASI), Konkuk University, Gwangjin-gu, Seoul 05029, Korea.
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Abdelalim EM, Emara MM. Pluripotent Stem Cell-Derived Pancreatic β Cells: From In Vitro Maturation to Clinical Application. RECENT ADVANCES IN STEM CELLS 2016. [DOI: 10.1007/978-3-319-33270-3_6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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Rostovskaya M, Bredenkamp N, Smith A. Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells. Philos Trans R Soc Lond B Biol Sci 2015; 370:20140365. [PMID: 26416676 PMCID: PMC4633994 DOI: 10.1098/rstb.2014.0365] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/10/2015] [Indexed: 12/12/2022] Open
Abstract
Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.
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Affiliation(s)
- Maria Rostovskaya
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Nicholas Bredenkamp
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
| | - Austin Smith
- Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
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Tse HM, Kozlovskaya V, Kharlampieva E, Hunter CS. Minireview: Directed Differentiation and Encapsulation of Islet β-Cells-Recent Advances and Future Considerations. Mol Endocrinol 2015; 29:1388-99. [PMID: 26340406 DOI: 10.1210/me.2015-1085] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Diabetes mellitus has rapidly become a 21st century epidemic with the promise to create vast economic and health burdens, if left unchecked. The 2 major forms of diabetes arise from unique causes, with outcomes being an absolute (type 1) or relative (type 2) loss of functional pancreatic islet β-cell mass. Currently, patients rely on exogenous insulin and/or other pharmacologies that restore glucose homeostasis. Although these therapies have prolonged countless lives over the decades, the striking increases in both type 1 and type 2 diabetic diagnoses worldwide suggest a need for improved treatments. To this end, islet biologists are developing cell-based therapies by which a patient's lost insulin-producing β-cell mass is replenished. Pancreatic or islet transplantation from cadaveric donors into diabetic patients has been successful, yet the functional islet demand far surpasses supply. Thus, the field has been striving toward transplantation of renewable in vitro-derived β-cells that can restore euglycemia. Challenges have been numerous, but progress over the past decade has generated much excitement. In this review we will summarize recent findings that have placed us closer than ever to β-cell replacement therapies. With the promise of cell-based diabetes therapies on the horizon, we will also provide an overview of cellular encapsulation technologies that will deliver critical protection of newly implanted cells.
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Affiliation(s)
- Hubert M Tse
- Department of Microbiology and the Comprehensive Diabetes Center (H.M.T.) and Departments of Chemistry (V.K., E.K.) and Medicine, Division of Endocrinology Diabetes and Metabolism, and Comprehensive Diabetes Center (C.S.H.), University of Alabama at Birmingham, Birmingham, Alabama 35294
| | - Veronika Kozlovskaya
- Department of Microbiology and the Comprehensive Diabetes Center (H.M.T.) and Departments of Chemistry (V.K., E.K.) and Medicine, Division of Endocrinology Diabetes and Metabolism, and Comprehensive Diabetes Center (C.S.H.), University of Alabama at Birmingham, Birmingham, Alabama 35294
| | - Eugenia Kharlampieva
- Department of Microbiology and the Comprehensive Diabetes Center (H.M.T.) and Departments of Chemistry (V.K., E.K.) and Medicine, Division of Endocrinology Diabetes and Metabolism, and Comprehensive Diabetes Center (C.S.H.), University of Alabama at Birmingham, Birmingham, Alabama 35294
| | - Chad S Hunter
- Department of Microbiology and the Comprehensive Diabetes Center (H.M.T.) and Departments of Chemistry (V.K., E.K.) and Medicine, Division of Endocrinology Diabetes and Metabolism, and Comprehensive Diabetes Center (C.S.H.), University of Alabama at Birmingham, Birmingham, Alabama 35294
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Sahraneshin Samani F, Ebrahimi M, Zandieh T, Khoshchehreh R, Baghaban Eslaminejad M, Aghdami N, Baharvand H. In Vitro Differentiation of Human Umbilical Cord Blood CD133(+)Cells into Insulin Producing Cells in Co-Culture with Rat Pancreatic Mesenchymal Stem Cells. CELL JOURNAL 2015. [PMID: 26199900 PMCID: PMC4503835 DOI: 10.22074/cellj.2016.3717] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Objective Pancreatic stroma plays an important role in the induction of pancreatic cells
by the use of close range signaling. In this respect, we presume that pancreatic mesenchymal cells (PMCs) as a fundamental factor of the stromal niche may have an effective
role in differentiation of umbilical cord blood cluster of differentiation 133+ (UCB-CD133+)
cells into newly-formed β-cells in vitro.
Materials and Methods This study is an experimental research. The UCB-CD133+cells
were purified by magnetic activated cell sorting (MACS) and differentiated into insulin
producing cells (IPCs) in co-culture, both directly and indirectly with rat PMCs. Immunocytochemistry and enzyme linked immune sorbent assay (ELISA) were used to determine
expression and production of insulin and C-peptide at the protein level.
Results Our results demonstrated that UCB-CD133+differentiated into IPCs. Cells in
islet-like clusters with (out) co-cultured with rat pancreatic stromal cells produced insulin
and C-peptide and released them into the culture medium at the end of the induction protocol. However they did not respond well to glucose challenges.
Conclusion Rat PMCs possibly affect differentiation of UCB-CD133+cells into IPCs by
increasing the number of immature β-cells.
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Affiliation(s)
- Fazel Sahraneshin Samani
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran ; Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran
| | - Marzieh Ebrahimi
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran ; Department of Regenerative Biomedicine at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Tahereh Zandieh
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Reyhaneh Khoshchehreh
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran ; Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran
| | - Mohamadreza Baghaban Eslaminejad
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Nasser Aghdami
- Department of Regenerative Biomedicine at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
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35
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Toyoda T, Mae SI, Tanaka H, Kondo Y, Funato M, Hosokawa Y, Sudo T, Kawaguchi Y, Osafune K. Cell aggregation optimizes the differentiation of human ESCs and iPSCs into pancreatic bud-like progenitor cells. Stem Cell Res 2015; 14:185-97. [DOI: 10.1016/j.scr.2015.01.007] [Citation(s) in RCA: 77] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/15/2014] [Revised: 12/28/2014] [Accepted: 01/19/2015] [Indexed: 01/22/2023] Open
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Ikonomou L, Kotton DN. Derivation of Endodermal Progenitors From Pluripotent Stem Cells. J Cell Physiol 2015; 230:246-58. [PMID: 25160562 PMCID: PMC4344429 DOI: 10.1002/jcp.24771] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2014] [Accepted: 08/22/2014] [Indexed: 01/18/2023]
Abstract
Stem and progenitor cells play important roles in organogenesis during development and in tissue homeostasis and response to injury postnatally. As the regenerative capacity of many human tissues is limited, cell replacement therapies hold great promise for human disease management. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are prime candidates for the derivation of unlimited quantities of clinically relevant cell types through development of directed differentiation protocols, that is, the recapitulation of developmental milestones in in vitro cell culture. Tissue-specific progenitors, including progenitors of endodermal origin, are important intermediates in such protocols since they give rise to all mature parenchymal cells. In this review, we focus on the in vivo biology of embryonic endodermal progenitors in terms of key transcription factors and signaling pathways. We critically review the emerging literature aiming to apply this basic knowledge to achieve the efficient and reproducible in vitro derivation of endodermal progenitors such as pancreas, liver and lung precursor cells.
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Affiliation(s)
- Laertis Ikonomou
- Center for Regenerative Medicine, Boston University and Boston
Medical Center, Boston, MA, USA
- Boston University Pulmonary Center, Boston University School of
Medicine, Boston, MA, USA
| | - Darrell N. Kotton
- Center for Regenerative Medicine, Boston University and Boston
Medical Center, Boston, MA, USA
- Boston University Pulmonary Center, Boston University School of
Medicine, Boston, MA, USA
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Abdelalim EM, Emara MM. Advances and challenges in the differentiation of pluripotent stem cells into pancreatic β cells. World J Stem Cells 2015; 7:174-181. [PMID: 25621117 PMCID: PMC4300928 DOI: 10.4252/wjsc.v7.i1.174] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/28/2014] [Revised: 09/16/2014] [Accepted: 09/19/2014] [Indexed: 02/06/2023] Open
Abstract
Pluripotent stem cells (PSCs) are able to differentiate into several cell types, including pancreatic β cells. Differentiation of pancreatic β cells depends on certain transcription factors, which function in a coordinated way during pancreas development. The existing protocols for in vitro differentiation produce pancreatic β cells, which are not highly responsive to glucose stimulation except after their transplantation into immune-compromised mice and allowing several weeks for further differentiation to ensure the maturation of these cells in vivo. Thus, although the substantial improvement that has been made for the differentiation of induced PSCs and embryonic stem cells toward pancreatic β cells, several challenges still hindering their full generation. Here, we summarize recent advances in the differentiation of PSCs into pancreatic β cells and discuss the challenges facing their differentiation as well as the different applications of these potential PSC-derived β cells.
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Abdelalim EM, Bonnefond A, Bennaceur-Griscelli A, Froguel P. Pluripotent stem cells as a potential tool for disease modelling and cell therapy in diabetes. Stem Cell Rev Rep 2014; 10:327-37. [PMID: 24577791 DOI: 10.1007/s12015-014-9503-6] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Diabetes mellitus is the most prevailing disease with progressive incidence worldwide. To date, the pathogenesis of diabetes is far to be understood, and there is no permanent treatment available for diabetes. One of the promising approaches to understand and cure diabetes is to use pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PCSs (iPSCs). ESCs and iPSCs have a great potential to differentiate into all cell types, and they have a high ability to differentiate into insulin-secreting β cells. Obtaining PSCs genetically identical to the patient presenting with diabetes has been a longstanding dream for the in vitro modeling of disease and ultimately cell therapy. For several years, somatic cell nuclear transfer (SCNT) was the method of choice to generate patient-specific ESC lines. However, this technology faces ethical and practical concerns. Interestingly, the recently established iPSC technology overcomes the major problems of other stem cell types including the lack of ethical concern and no risk of immune rejection. Several iPSC lines have been recently generated from patients with different types of diabetes, and most of these cell lines are able to differentiate into insulin-secreting β cells. In this review, we summarize recent advances in the differentiation of pancreatic β cells from PSCs, and describe the challenges for their clinical use in diabetes cell therapy. Furthermore, we discuss the potential use of patient-specific PSCs as an in vitro model, providing new insights into the pathophysiology of diabetes.
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Affiliation(s)
- Essam M Abdelalim
- Qatar Biomedical Research Institute, Qatar Foundation, Education City, 5825, Doha, Qatar,
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Kumar SS, Alarfaj AA, Munusamy MA, Singh AJAR, Peng IC, Priya SP, Hamat RA, Higuchi A. Recent developments in β-cell differentiation of pluripotent stem cells induced by small and large molecules. Int J Mol Sci 2014; 15:23418-47. [PMID: 25526563 PMCID: PMC4284775 DOI: 10.3390/ijms151223418] [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: 10/29/2014] [Revised: 12/03/2014] [Accepted: 12/08/2014] [Indexed: 12/21/2022] Open
Abstract
Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold promise as novel therapeutic tools for diabetes treatment because of their self-renewal capacity and ability to differentiate into beta (β)-cells. Small and large molecules play important roles in each stage of β-cell differentiation from both hESCs and hiPSCs. The small and large molecules that are described in this review have significantly advanced efforts to cure diabetic disease. Lately, effective protocols have been implemented to induce hESCs and human mesenchymal stem cells (hMSCs) to differentiate into functional β-cells. Several small molecules, proteins, and growth factors promote pancreatic differentiation from hESCs and hMSCs. These small molecules (e.g., cyclopamine, wortmannin, retinoic acid, and sodium butyrate) and large molecules (e.g. activin A, betacellulin, bone morphogentic protein (BMP4), epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), noggin, transforming growth factor (TGF-α), and WNT3A) are thought to contribute from the initial stages of definitive endoderm formation to the final stages of maturation of functional endocrine cells. We discuss the importance of such small and large molecules in uniquely optimized protocols of β-cell differentiation from stem cells. A global understanding of various small and large molecules and their functions will help to establish an efficient protocol for β-cell differentiation.
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Affiliation(s)
- S Suresh Kumar
- Department of Medical Microbiology and Parasitology, Universities Putra Malaysia, Serdang 43400, Selangor, Malaysia.
| | - Abdullah A Alarfaj
- Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
| | - Murugan A Munusamy
- Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
| | - A J A Ranjith Singh
- Department of Bioscience, Jacintha Peter College of Arts and Sciences, Ayakudi, Tenkasi, Tamilnadu 627852, India.
| | - I-Chia Peng
- Department of Chemical and Materials Engineering, National Central University, No. 300, Jhongda RD., Jhongli, Taoyuan 32001, Taiwan.
| | - Sivan Padma Priya
- Department of Basic Science and Department of Surgical Sciences, Ajman University of Science and Technology-Fujairah Campus, P.O. Box 9520, Al Fujairah, United Arab Emirates.
| | - Rukman Awang Hamat
- Department of Medical Microbiology and Parasitology, Universities Putra Malaysia, Serdang 43400, Selangor, Malaysia.
| | - Akon Higuchi
- Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
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Rezania A, Bruin JE, Xu J, Narayan K, Fox JK, O'Neil JJ, Kieffer TJ. Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo. Stem Cells 2014; 31:2432-42. [PMID: 23897760 DOI: 10.1002/stem.1489] [Citation(s) in RCA: 193] [Impact Index Per Article: 17.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2012] [Revised: 06/09/2013] [Accepted: 07/01/2013] [Indexed: 12/24/2022]
Abstract
Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes. We previously described a differentiation protocol to generate pancreatic progenitor cells from hESCs, composed of mainly pancreatic endoderm (PDX1/NKX6.1-positive), endocrine precursors (NKX2.2/synaptophysin-positive, hormone/NKX6.1-negative), and polyhormonal cells (insulin/glucagon-positive, NKX6.1-negative). However, the relative contributions of NKX6.1-negative versus NKX6.1-positive cell fractions to the maturation of functional β-cells remained unclear. To address this question, we generated two distinct pancreatic progenitor cell populations using modified differentiation protocols. Prior to transplant, both populations contained a high proportion of PDX1-expressing cells (~85%-90%) but were distinguished by their relatively high (~80%) or low (~25%) expression of NKX6.1. NKX6.1-high and NKX6.1-low progenitor populations were transplanted subcutaneously within macroencapsulation devices into diabetic mice. Mice transplanted with NKX6.1-low cells remained hyperglycemic throughout the 5-month post-transplant period whereas diabetes was reversed in NKX6.1-high recipients within 3 months. Fasting human C-peptide levels were similar between groups throughout the study, but only NKX6.1-high grafts displayed robust meal-, glucose- and arginine-responsive insulin secretion as early as 3 months post-transplant. NKX6.1-low recipients displayed elevated fasting glucagon levels. Theracyte devices from both groups contained almost exclusively pancreatic endocrine tissue, but NKX6.1-high grafts contained a greater proportion of insulin-positive and somatostatin-positive cells, whereas NKX6.1-low grafts contained mainly glucagon-expressing cells. Insulin-positive cells in NKX6.1-high, but not NKX6.1-low grafts expressed nuclear MAFA. Collectively, this study demonstrates that a pancreatic endoderm-enriched population can mature into highly functional β-cells with only a minor contribution from the endocrine subpopulation.
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Affiliation(s)
- Alireza Rezania
- BetaLogics Venture, Janssen R & D LLC, Raritan, New Jersey, USA
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Tsuji N, Ninov N, Delawary M, Osman S, Roh AS, Gut P, Stainier DYR. Whole organism high content screening identifies stimulators of pancreatic beta-cell proliferation. PLoS One 2014; 9:e104112. [PMID: 25117518 PMCID: PMC4130527 DOI: 10.1371/journal.pone.0104112] [Citation(s) in RCA: 64] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2014] [Accepted: 07/04/2014] [Indexed: 12/21/2022] Open
Abstract
Inducing beta-cell mass expansion in diabetic patients with the aim to restore glucose homeostasis is a promising therapeutic strategy. Although several in vitro studies have been carried out to identify modulators of beta-cell mass expansion, restoring endogenous beta-cell mass in vivo has yet to be achieved. To identify potential stimulators of beta-cell replication in vivo, we established transgenic zebrafish lines that monitor and allow the quantification of cell proliferation by using the fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. Using these new reagents, we performed an unbiased chemical screen, and identified 20 small molecules that markedly increased beta-cell proliferation in vivo. Importantly, these structurally distinct molecules, which include clinically-approved drugs, modulate three specific signaling pathways: serotonin, retinoic acid and glucocorticoids, showing the high sensitivity and robustness of our screen. Notably, two drug classes, retinoic acid and glucocorticoids, also promoted beta-cell regeneration after beta-cell ablation. Thus, this study establishes a proof of principle for a high-throughput small molecule-screen for beta-cell proliferation in vivo, and identified compounds that stimulate beta-cell proliferation and regeneration.
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Affiliation(s)
- Naoki Tsuji
- Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, the Diabetes Center, Institute for Regeneration Medicine and Liver Center, University of California San Francisco, San Francisco, California, United States of America
| | - Nikolay Ninov
- Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, the Diabetes Center, Institute for Regeneration Medicine and Liver Center, University of California San Francisco, San Francisco, California, United States of America
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
- DFG Research Center for Regenerative Therapies Dresden, Technische Universität Dresden, Dresden, Germany
- Paul Langerhans Institute Dresden, German Center for Diabetes Research, Dresden, Germany
| | - Mina Delawary
- Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, the Diabetes Center, Institute for Regeneration Medicine and Liver Center, University of California San Francisco, San Francisco, California, United States of America
| | - Sahar Osman
- Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, the Diabetes Center, Institute for Regeneration Medicine and Liver Center, University of California San Francisco, San Francisco, California, United States of America
| | - Alex S. Roh
- Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, the Diabetes Center, Institute for Regeneration Medicine and Liver Center, University of California San Francisco, San Francisco, California, United States of America
| | - Philipp Gut
- Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, the Diabetes Center, Institute for Regeneration Medicine and Liver Center, University of California San Francisco, San Francisco, California, United States of America
| | - Didier Y. R. Stainier
- Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, the Diabetes Center, Institute for Regeneration Medicine and Liver Center, University of California San Francisco, San Francisco, California, United States of America
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
- * E-mail:
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Shahjalal HM, Shiraki N, Sakano D, Kikawa K, Ogaki S, Baba H, Kume K, Kume S. Generation of insulin-producing β-like cells from human iPS cells in a defined and completely xeno-free culture system. J Mol Cell Biol 2014; 6:394-408. [PMID: 24970864 DOI: 10.1093/jmcb/mju029] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
Human induced pluripotent stem (hiPS) cells are considered a potential source for the generation of insulin-producing pancreatic β-cells because of their differentiation capacity. In this study, we have developed a five-step xeno-free culture system to efficiently differentiate hiPS cells into insulin-producing cells in vitro. We found that a high NOGGIN concentration is crucial for specifically inducing the differentiation first into pancreatic and duodenal homeobox-1 (PDX1)-positive pancreatic progenitors and then into neurogenin 3 (NGN3)-expressing pancreatic endocrine progenitors, while suppressing the differentiation into hepatic or intestinal cells. We also found that a combination of 3-isobutyl-1-methylxanthine (IBMX), exendin-4, and nicotinamide was important for the differentiation into insulin single-positive cells that expressed various pancreatic β-cell markers. Most notably, the differentiated cells contained endogenous C-peptide pools that were released in response to various insulin secretagogues and high levels of glucose. Therefore, our results demonstrate the feasibility of generating hiPS-derived pancreatic β-cells under xeno-free conditions and highlight their potential to treat patients with type 1 diabetes.
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Affiliation(s)
- Hussain Md Shahjalal
- Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Chuo-Ku, Kumamoto 860-0811, Japan Global-Center of Excellence (G-COE), Kumamoto University, Honjo 2-2-1, Chuo-ku, Kumamoto 860-0811, Japan
| | - Nobuaki Shiraki
- Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Chuo-Ku, Kumamoto 860-0811, Japan
| | - Daisuke Sakano
- Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Chuo-Ku, Kumamoto 860-0811, Japan Global-Center of Excellence (G-COE), Kumamoto University, Honjo 2-2-1, Chuo-ku, Kumamoto 860-0811, Japan
| | - Kazuhide Kikawa
- Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Chuo-Ku, Kumamoto 860-0811, Japan Global-Center of Excellence (G-COE), Kumamoto University, Honjo 2-2-1, Chuo-ku, Kumamoto 860-0811, Japan Department of Pediatrics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Chuo-Ku, Kumamoto 860-8556, Japan
| | - Soichiro Ogaki
- Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Chuo-Ku, Kumamoto 860-0811, Japan
| | - Hideo Baba
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Chuo-Ku, Kumamoto 860-8556, Japan
| | - Kazuhiko Kume
- Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe Street, Mizuho, Nagoya 467-8603, Japan
| | - Shoen Kume
- Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Chuo-Ku, Kumamoto 860-0811, Japan Global-Center of Excellence (G-COE), Kumamoto University, Honjo 2-2-1, Chuo-ku, Kumamoto 860-0811, Japan Program for Leading Graduate Schools 'HIGO (Health Life Science; Interdisciplinary and Glocal Oriented) Program,' Kumamoto University, Honjo 2-2-1, Chuo-ku, Kumamoto 860-0811, Japan
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van der Meulen T, Huising MO. Maturation of stem cell-derived beta-cells guided by the expression of urocortin 3. Rev Diabet Stud 2014; 11:115-32. [PMID: 25148370 DOI: 10.1900/rds.2014.11.115] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Type 1 diabetes (T1D) is a devastating disease precipitated by an autoimmune response directed at the insulin-producing beta-cells of the pancreas for which no cure exists. Stem cell-derived beta-cells show great promise for a cure as they have the potential to supply unlimited numbers of cells that could be derived from a patient's own cells, thus eliminating the need for immunosuppression. Current in vitro protocols for the differentiation of stem cell-derived beta-cells can successfully generate pancreatic endoderm cells. In diabetic rodents, such cells can differentiate further along the beta-cell lineage until they are eventually capable of restoring normoglycemia. While these observations demonstrate that stem cell-derived pancreatic endoderm has the potential to differentiate into mature, glucose-responsive beta-cells, the signals that direct differentiation and maturation from pancreatic endoderm onwards remain poorly understood. In this review, we analyze the sequence of events that culminates in the formation of beta-cells during embryonic development. and summarize how current protocols to generate beta-cells have sought to capitalize on this ontogenic template. We place particular emphasis on the current challenges and opportunities which occur in the later stages of beta-cell differentiation and maturation of transplantable stem cell-derived beta-cells. Another focus is on the question how the use of recently identified maturation markers such as urocortin 3 can be instrumental in guiding these efforts.
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Affiliation(s)
- Talitha van der Meulen
- The Salk Institute for Biological Studies, Clayton Laboratories for Peptide Biology, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
| | - Mark O Huising
- The Salk Institute for Biological Studies, Clayton Laboratories for Peptide Biology, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
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Wang Y, Lanzoni G, Carpino G, Cui CB, Dominguez-Bendala J, Wauthier E, Cardinale V, Oikawa T, Pileggi A, Gerber D, Furth ME, Alvaro D, Gaudio E, Inverardi L, Reid LM. Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible life-long pancreatic organogenesis. Stem Cells 2014; 31:1966-79. [PMID: 23847135 DOI: 10.1002/stem.1460] [Citation(s) in RCA: 80] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2012] [Revised: 09/19/2013] [Accepted: 09/25/2012] [Indexed: 12/13/2022]
Abstract
Peribiliary glands (PBGs) in bile duct walls, and pancreatic duct glands (PDGs) associated with pancreatic ducts, in humans of all ages, contain a continuous, ramifying network of cells in overlapping maturational lineages. We show that proximal (PBGs)-to-distal (PDGs) maturational lineages start near the duodenum with cells expressing markers of pluripotency (NANOG, OCT4, and SOX2), proliferation (Ki67), self-replication (SALL4), and early hepato-pancreatic commitment (SOX9, SOX17, PDX1, and LGR5), transitioning to PDG cells with no expression of pluripotency or self-replication markers, maintenance of pancreatic genes (PDX1), and expression of markers of pancreatic endocrine maturation (NGN3, MUC6, and insulin). Radial-axis lineages start in PBGs near the ducts' fibromuscular layers with stem cells and end at the ducts' lumens with cells devoid of stem cell traits and positive for pancreatic endocrine genes. Biliary tree-derived cells behaved as stem cells in culture under expansion conditions, culture plastic and serum-free Kubota's Medium, proliferating for months as undifferentiated cells, whereas pancreas-derived cells underwent only approximately 8-10 divisions, then partially differentiated towards an islet fate. Biliary tree-derived cells proved precursors of pancreas' committed progenitors. Both could be driven by three-dimensional conditions, islet-derived matrix components and a serum-free, hormonally defined medium for an islet fate (HDM-P), to form spheroids with ultrastructural, electrophysiological and functional characteristics of neoislets, including glucose regulatability. Implantation of these neoislets into epididymal fat pads of immunocompromised mice, chemically rendered diabetic, resulted in secretion of human C-peptide, regulatable by glucose, and able to alleviate hyperglycemia in hosts. The biliary tree-derived stem cells and their connections to pancreatic committed progenitors constitute a biological framework for life-long pancreatic organogenesis.
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Affiliation(s)
- Yunfang Wang
- Department of Cell Biology and Physiology, Program in Molecular Biology and Biotechnology, Lineberger Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA
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Role of BMP signaling in pancreatic progenitor differentiation from human embryonic stem cells. Stem Cell Rev Rep 2014; 9:569-77. [PMID: 23468018 DOI: 10.1007/s12015-013-9435-6] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Transplantation of pancreatic progenitors derived from human embryonic stem cells (hESCs) is a promising way to treat diabetes. Strategies to obtain the required cell mass would rely on the up scaling of current differentiation protocols, or the proliferation of committed progenitors. We aimed at finding conditions that maintain a proliferating pancreatic progenitor pool and we assessed the role of BMP4 signaling in this process. hESCs were differentiated into PDX1 positive pancreatic progenitor stage following our established protocol with few modifications, and then the progenitor cells were passaged in a defined proliferation medium (PM). During passage, the effect of BMP4 signaling on the differentiation and proliferation of pancreatic progenitors was examined by RT-PCR and immunofluorescence analysis. We found that PDX1 positive pancreatic progenitors proliferated and gained NKX6.1 expression in the PM, whereas they failed to express NKX6.1 if BMP signaling was inhibited with Noggin. In this latter condition, part of the progenitors rather generated pro-endocrine cells denoted by NGN3 and synaptophysin expression. On the contrary, addition of BMP4 to the PM promoted the early derivation of PDX1 and NKX6.1 coexpressing pancreatic progenitors. Our findings are in line with mouse pancreas development, and indicate that BMP4 signaling is required for the derivation and maintenance of hESC-derived PDX1+NKX6.1+ pancreatic progenitors. These results are instructive for guiding the development of an efficient pancreas differentiation protocol in view of diabetes cell replacement therapy.
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46
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Takeuchi H, Nakatsuji N, Suemori H. Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture. Sci Rep 2014; 4:4488. [PMID: 24671046 PMCID: PMC3967149 DOI: 10.1038/srep04488] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2014] [Accepted: 03/10/2014] [Indexed: 12/11/2022] Open
Abstract
Insulin-producing cells (IPCs) derived from human pluripotent stem cells (hPSCs) may be useful in cell therapy and drug discovery for diabetes. Here, we examined various growth factors and small molecules including those previously reported to develop a robust differentiation method for induction of mature IPCs from hPSCs. We established a protocol that induced PDX1-positive pancreatic progenitor cells at high efficiency, and further induced mature IPCs by treatment with forskolin, dexamethasone, Alk5 inhibitor II and nicotinamide in 3D culture. The cells that differentiated into INSULIN-positive and C-PEPTIDE-positive cells secreted insulin in response to glucose stimulation, indicating a functional IPC phenotype. We also found that this method was applicable to different types of hPSCs.
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Affiliation(s)
- Hiroki Takeuchi
- Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
| | - Norio Nakatsuji
- 1] Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Ushinomiya-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan [2] Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
| | - Hirofumi Suemori
- Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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Pandian GN, Taniguchi J, Sugiyama H. Cellular reprogramming for pancreatic β-cell regeneration: clinical potential of small molecule control. Clin Transl Med 2014; 3:6. [PMID: 24679123 PMCID: PMC3984496 DOI: 10.1186/2001-1326-3-6] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2014] [Accepted: 03/17/2014] [Indexed: 12/14/2022] Open
Abstract
Recent scientific breakthroughs in stem cell biology suggest that a sustainable treatment approach to cure diabetes mellitus (DM) can be achieved in the near future. However, the transplantation complexities and the difficulty in obtaining the stem cells from adult cells of pancreas, liver, bone morrow and other cells is a major concern. The epoch-making strategy of transcription-factor based cellular reprogramming suggest that these barriers could be overcome, and it is possible to reprogram any cells into functional β cells. Contemporary biological and analytical techniques help us to predict the key transcription factors needed for β-cell regeneration. These β cell-specific transcription factors could be modulated with diverse reprogramming protocols. Among cellular reprogramming strategies, small molecule approach gets proclaimed to have better clinical prospects because it does not involve genetic manipulation. Several small molecules targeting certain epigenetic enzymes and/or signaling pathways have been successful in helping to induce pancreatic β-cell specification. Recently, a synthetic DNA-based small molecule triggered targeted transcriptional activation of pancreas-related genes to suggest the possibility of achieving desired cellular phenotype in a precise mode. Here, we give a brief overview of treating DM by regenerating pancreatic β-cells from various cell sources. Through a comprehensive overview of the available transcription factors, small molecules and reprogramming strategies available for pancreatic β-cell regeneration, this review compiles the current progress made towards the generation of clinically relevant insulin-producing β-cells.
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Affiliation(s)
| | | | - Hiroshi Sugiyama
- Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Sakyo, Kyoto 606-8502, Japan.
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Bose B, Katikireddy KR, Shenoy PS. Regenerative medicine for diabetes: differentiation of human pluripotent stem cells into functional β-cells in vitro and their proposed journey to clinical translation. VITAMINS AND HORMONES 2014; 95:223-48. [PMID: 24559920 DOI: 10.1016/b978-0-12-800174-5.00009-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
Abstract
Diabetes is a group of metabolic diseases, rising globally at an alarming rate. Type 1 (juvenile diabetes) is the autoimmune version of diabetes where the pancreas is unable to produce insulin, whereas type 2 (adult onset diabetes) is caused due to insulin resistance of the cells. In either of the cases, elevated blood glucose levels are observed which leads to progressive comorbidity like renal failure, cardiovascular disease, retinopathy, etc. Metformin, sulphonyl urea group of drugs, as well as insulin injections are the available therapies. In advanced cases of diabetes, the drug alone or drug in combination with insulin injections are not able to maintain a steady level of blood glucose. Moreover, frequent insulin injections are rather cumbersome for the patient. So, regenerative medicine could be a permanent solution for fighting diabetes. Islet transplantation has been tried with a limited amount of success on a large population of diabetics because of the shortage of cadaveric pancreas. Therefore, the best proposed alternative is regenerative medicine involving human pluripotent stem cell (hPSC)-derived beta islet transplantation which can be obtained in large quantities. Efficient protocols for in vitro differentiation of hPSC into a large number of sustained insulin-producing beta cells for transplantation will be considered to be a giant leap to address global rise in diabetic cases. Although most of the protocols mimic in vivo pancreatic development in humans, considerable amount of lacuna persists for near-perfect differentiation strategies. Moreover, beta islets differentiated from hPSC have not yet been successfully translated under clinical scenario.
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Affiliation(s)
- Bipasha Bose
- Nanyang Technological University, School of Biological Sciences, NTU Lab Location @ Level 2 Singapore Institute for Clinical Sciences, Brenner Centre for Molecular Medicine, Singapore, Singapore.
| | | | - P Sudheer Shenoy
- Nanyang Technological University, School of Biological Sciences, NTU Lab Location @ Level 2 Singapore Institute for Clinical Sciences, Brenner Centre for Molecular Medicine, Singapore, Singapore
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49
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Gage BK, Webber TD, Kieffer TJ. Initial cell seeding density influences pancreatic endocrine development during in vitro differentiation of human embryonic stem cells. PLoS One 2013; 8:e82076. [PMID: 24324748 PMCID: PMC3852888 DOI: 10.1371/journal.pone.0082076] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2013] [Accepted: 10/26/2013] [Indexed: 11/19/2022] Open
Abstract
Human embryonic stem cells (hESCs) have the ability to form cells derived from all three germ layers, and as such have received significant attention as a possible source for insulin-secreting pancreatic beta-cells for diabetes treatment. While considerable advances have been made in generating hESC-derived insulin-producing cells, to date in vitro-derived glucose-responsive beta-cells have remained an elusive goal. With the objective of increasing the in vitro formation of pancreatic endocrine cells, we examined the effect of varying initial cell seeding density from 1.3 x 10(4) cells/cm(2) to 5.3 x 10(4) cells/cm(2) followed by a 21-day pancreatic endocrine differentiation protocol. Low density-seeded cells were found to be biased toward the G2/M phases of the cell cycle and failed to efficiently differentiate into SOX17-CXCR4 co-positive definitive endoderm cells leaving increased numbers of OCT4 positive cells in day 4 cultures. Moderate density cultures effectively formed definitive endoderm and progressed to express PDX1 in approximately 20% of the culture. High density cultures contained approximately double the numbers of PDX1 positive pancreatic progenitor cells and also showed increased expression of MNX1, PTF1a, NGN3, ARX, and PAX4 compared to cultures seeded at moderate density. The cultures seeded at high density displayed increased formation of polyhormonal pancreatic endocrine cell populations co-expressing insulin, glucagon and somatostatin. The maturation process giving rise to these endocrine cell populations followed the expected cascade of pancreatic progenitor marker (PDX1 and MNX1) expression, followed by pancreatic endocrine specification marker expression (BRN4, PAX4, ARX, NEUROD1, NKX6.1 and NKX2.2) and then pancreatic hormone expression (insulin, glucagon and somatostatin). Taken together these data suggest that initial cell seeding density plays an important role in both germ layer specification and pancreatic progenitor commitment, which precedes pancreatic endocrine cell formation. This work highlights the need to examine standard culture variables such as seeding density when optimizing hESC differentiation protocols.
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Affiliation(s)
- Blair K. Gage
- Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada
| | - Travis D. Webber
- Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada
| | - Timothy J. Kieffer
- Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada
- Department of Surgery, University of British Columbia, Vancouver, British Columbia, Canada
- * E-mail:
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50
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Nazareth EJP, Ostblom JEE, Lücker PB, Shukla S, Alvarez MM, Oh SKW, Yin T, Zandstra PW. High-throughput fingerprinting of human pluripotent stem cell fate responses and lineage bias. Nat Methods 2013; 10:1225-31. [PMID: 24141495 PMCID: PMC5061564 DOI: 10.1038/nmeth.2684] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2013] [Accepted: 08/30/2013] [Indexed: 12/22/2022]
Abstract
Populations of cells create local environments that lead to emergent heterogeneity. This is particularly evident in human pluripotent stem cells (hPSCs) where microenvironmental heterogeneity limits cell fate control. We have developed a high-throughput platform to screen hPSCs in configurable micro-environments, enabling the optimization of colony size, cell density, and additional parameters for rapid and robust cell fate responses. Single-cell protein expression profiling revealed that Oct4 and Sox2 co-staining discriminate pluripotent, neuroectoderm, primitive streak, and extraembryonic cell fates, allowing dose responses of 27 developmental factors to simultaneously delineate lineage-specific concentration optima. This platform also enabled quantification of endogenous signaling pathway activation and differentiation bias (fingerprinting). Short-term (48 h) fingerprinting is predictive of definitive endoderm induction efficiency across 12 cell lines and was used a priori to rescue long-term (>18 day) differentiation of a cell line reticent to cardiac induction. These findings facilitate high-throughput hPSC-based screening and quantification of lineage induction bias.
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Affiliation(s)
- Emanuel J P Nazareth
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
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