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1
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Wei H, Kappler C, Green E, Jiang H, Yeung T, Wang H. GRP94 is indispensable for definitive endoderm specification of human induced pluripotent stem cells. Mol Ther 2025:S1525-0016(25)00299-0. [PMID: 40254879 DOI: 10.1016/j.ymthe.2025.04.025] [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: 01/24/2025] [Revised: 03/24/2025] [Accepted: 04/16/2025] [Indexed: 04/22/2025] Open
Abstract
Human induced pluripotent stem cell (hiPSC)-derived insulin-producing β cell therapy shows promise in treating type 1 diabetes and potentially type 2 diabetes. Understanding the genetic factors controlling hiPSC differentiation could optimize this therapy. In this study, we investigated the role of glucose-regulated protein 94 (GRP94) in human β cell development by generating HSP90B1/GRP94 knockout (KO) hiPSCs, re-expressing GRP94 in the mutants and inducing their β cell differentiation. Our results revealed that GRP94 depletion hindered β cell generation by promoting cell death induced by endoplasmic reticulum (ER) stress and other stressors during definitive endoderm (DE) differentiation. Moreover, GRP94 deletion resulted in decreased activation of WNT/β-catenin signaling, which is critical for DE specification. Re-expression of GRP94 in GRP94 KO iPSCs partially reversed DE differentiation deficiency and alleviated cell death. These findings highlight the previously unrecognized indispensable role of GRP94 in human DE formation and consequent β cell development from hiPSCs. GRP94 mitigates ER stress-induced cell death and regulates the WNT/β-catenin signaling pathway, which is both crucial for successful β cell differentiation. These results provide new insights into the molecular mechanisms underlying β cell differentiation from hiPSCs and suggest that targeting GRP94 pathways could enhance the efficiency of hiPSC-derived insulin-producing cell therapies for diabetes treatment.
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Affiliation(s)
- Hua Wei
- Department of Surgery, Medical University of South Carolina, Charleston, SC, USA
| | - Christiana Kappler
- Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA
| | - Erica Green
- Department of Surgery, Medical University of South Carolina, Charleston, SC, USA
| | - Hanna Jiang
- Department of Surgery, Medical University of South Carolina, Charleston, SC, USA
| | - Tiffany Yeung
- Department of Surgery, Medical University of South Carolina, Charleston, SC, USA
| | - Hongjun Wang
- Department of Surgery, Medical University of South Carolina, Charleston, SC, USA; Ralph H Johnson VA Medical Center, Charleston, SC, USA.
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2
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Xu W, Yi F, Liao H, Zhu C, Zou X, Dong Y, Zhou W, Sun Z, Yin J. The Potential and Challenges of Human Pluripotent Stem Cells in the Treatment of Diabetic Nephropathy. FRONT BIOSCI-LANDMRK 2025; 30:28283. [PMID: 40302328 DOI: 10.31083/fbl28283] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2024] [Revised: 12/28/2024] [Accepted: 01/07/2025] [Indexed: 05/02/2025]
Abstract
Diabetic nephropathy (DN) is a prevalent complication of diabetes, with current treatment options offering limited effectiveness, particularly in advanced stages. Human pluripotent stem cells (hPSCs), particularly induced PSCs (iPSCs), show promising potential in the treatment of DN due to their pluripotency, capacity for differentiation into kidney-specific cells, and suitability for personalized therapies. iPSC-based personalized approaches can effectively mitigate immune rejection, a common challenge with allogeneic transplants, thus enhancing therapeutic outcomes. Clustered regularly interspaced short palindromic repeats (CRISPR) gene editing further enhances the potential of hPSCs by enabling the precise correction of disease-associated genetic defects, increasing both the safety and efficacy of therapeutic cells. In addition to direct treatment, hPSCs have proven valuable in disease modeling and drug screening, particularly for identifying and validating disease-specific targets. Kidney organoids derived from hPSCs replicate key features of DN pathology, making them useful platforms for validating therapeutic targets and assessing drug efficacy. Comparatively, both hPSCs and mesenchymal SCs (MSCs) have shown promise in improving renal function in preclinical models, with hPSCs offering broader differentiation capacity. Integration with tissue engineering technologies, such as three-dimensional bioprinting and bioengineered scaffolds, expands the regenerative potential of hPSCs by supporting the formation of functional renal structures and enhancing in vivo integration and regenerative capacity. Despite current challenges, such as tumorigenicity, genomic instability, and limited direct research, advances in gene editing, differentiation protocols, and tissue engineering promise to address these barriers. Continued optimization of these approaches will likely lead to successful clinical applications of hPSCs, potentially revolutionizing treatment options for DN.
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Affiliation(s)
- Wanyue Xu
- Nephrology Department, Hangzhou Hospital of Traditional Chinese Medicine, 310007 Hangzhou, Zhejiang, China
| | - Fangyu Yi
- Hangzhou Clinical College, Zhejiang Chinese Medical University, 310053 Hangzhou, Zhejiang, China
| | - Haiyang Liao
- Hangzhou Clinical College, Zhejiang Chinese Medical University, 310053 Hangzhou, Zhejiang, China
| | - Caifeng Zhu
- Nephrology Department, Hangzhou Hospital of Traditional Chinese Medicine, 310007 Hangzhou, Zhejiang, China
| | - Xiaodi Zou
- Department of Orthopedics, The Second Affiliated Hospital of Zhejiang Chinese Medical University, 310003 Hangzhou, Zhejiang, China
- Department of Orthopedics, The First Affiliated Hospital, Zhejiang University, 310000 Hangzhou, Zhejiang, China
| | - Yanzhao Dong
- Department of Orthopedics, The First Affiliated Hospital, Zhejiang University, 310000 Hangzhou, Zhejiang, China
| | - Weijie Zhou
- Department of Orthopedics, The First Affiliated Hospital, Zhejiang University, 310000 Hangzhou, Zhejiang, China
| | - Zexing Sun
- The First School of Clinical Medicine, Zhejiang Chinese Medical University, 310053 Hangzhou, Zhejiang, China
| | - Jiazhen Yin
- Nephrology Department, Hangzhou Hospital of Traditional Chinese Medicine, 310007 Hangzhou, Zhejiang, China
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3
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Perez-Serna AA, Guzman-Llorens D, Dos Santos RS, Marroqui L. Bcl-2 and Bcl-xL in Diabetes: Contributions to Endocrine Pancreas Viability and Function. Biomedicines 2025; 13:223. [PMID: 39857806 PMCID: PMC11760435 DOI: 10.3390/biomedicines13010223] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2024] [Revised: 01/08/2025] [Accepted: 01/15/2025] [Indexed: 01/27/2025] Open
Abstract
Diabetes is a chronic metabolic disorder whose prevalence increases every year, affecting more than 530 million adults worldwide. Type 1 (T1D) and type 2 diabetes (T2D), the most common forms of diabetes, are characterized by the loss of functional pancreatic β-cells, mostly due to apoptosis. B-cell leukemia/lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xL), two anti-apoptotic proteins belonging to the Bcl-2 family, are crucial for regulating the intrinsic pathway of apoptosis. However, over the years, they have been implicated in many other cellular processes, including intracellular Ca2+ homeostasis and the regulation of mitochondrial metabolism. Thus, understanding the biological processes in which these proteins are involved may be crucial to designing new therapeutic targets. This review summarizes the roles of Bcl-2 and Bcl-xL in apoptosis and metabolic homeostasis. It focuses on how the dysregulation of Bcl-2 and Bcl-xL affects pancreatic β-cell function and survival, and the consequences for diabetes development.
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Affiliation(s)
- Atenea A. Perez-Serna
- Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Universidad Miguel Hernández de Elche, 03202 Elche, Alicante, Spain; (A.A.P.-S.); (D.G.-L.)
- CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Instituto de Salud Carlos III, Madrid, Spain
| | - Daniel Guzman-Llorens
- Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Universidad Miguel Hernández de Elche, 03202 Elche, Alicante, Spain; (A.A.P.-S.); (D.G.-L.)
| | - Reinaldo S. Dos Santos
- Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Universidad Miguel Hernández de Elche, 03202 Elche, Alicante, Spain; (A.A.P.-S.); (D.G.-L.)
- CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Instituto de Salud Carlos III, Madrid, Spain
- Unidad de Investigación, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO), Hospital General Universitario de Elche, Camí de l’Almazara 11, 03203 Elche, Alicante, Spain
| | - Laura Marroqui
- Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Universidad Miguel Hernández de Elche, 03202 Elche, Alicante, Spain; (A.A.P.-S.); (D.G.-L.)
- CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Instituto de Salud Carlos III, Madrid, Spain
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4
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Leclerc E, Pachkov M, Morisseau L, Tokito F, Legallais C, Jellali R, Nishikawa M, Abderrahmani A, Sakai Y. Investigation of the motif activity of transcription regulators in pancreatic β-like cell subpopulations differentiated from human induced pluripotent stem cells. Mol Omics 2024. [PMID: 39494575 DOI: 10.1039/d4mo00082j] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2024]
Abstract
Pancreatic β-cells are composed of different subtypes that play a key role in the control of insulin secretion and thereby control glucose homeostasis. In vitro differentiation of human induced pluripotent stem cells (hiPSCs) into 3D spheroids leads to the generation of β-cell subtypes and thus to the development of islet-like structures. Using this cutting-edge cell model, the aim of the study was to decipher the signaling signature that underlines β-cell subtypes, with a focus on the search for the activity of motifs of important transcription regulators (TRs). The investigation was performed using data from previous single-cell sequencing analysis introduced into the integrated system for motif activity response analysis (ISMARA) of transcription regulators. We extracted the matrix of important TRs activated in the β-cell subpopulation and bi-hormonal-like β-cells. Based on these TRs and their targets, we built specific regulatory networks for main cell subpopulations. Our data confirmed the transcriptomic heterogeneity of the β-cell subtype lineage and suggested a mechanism that could account for the differentiation of β-cell subtypes during pancreas development. We do believe that our findings could be instrumental for understanding the mechanisms that affect the balance of β-cell subtypes, leading to impaired insulin secretion in type 2 diabetes.
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Affiliation(s)
- Eric Leclerc
- CNRS IRL 2820; Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba; Meguro-ku, Tokyo, 153-8505, Japan
| | - Mikhail Pachkov
- Biozentrum, University of Basel, Spitalstrasse 41, 4056 Basel, Switzerland
- SIB Swiss Institute of Bioinformatics, Quartier Sorge - Bâtiment Amphipôle, 1015 Lausanne, Switzerland
| | - Lisa Morisseau
- CNRS UMR 7338, Laboratoire de Biomécanique et Bioingénierie, Sorbonne universités, Université de Technologies de Compiègne, France
| | - Fumiya Tokito
- Department of Chemical System Engineering, Graduate School of Engineering, the University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan
| | - Cecile Legallais
- CNRS UMR 7338, Laboratoire de Biomécanique et Bioingénierie, Sorbonne universités, Université de Technologies de Compiègne, France
| | - Rachid Jellali
- CNRS UMR 7338, Laboratoire de Biomécanique et Bioingénierie, Sorbonne universités, Université de Technologies de Compiègne, France
| | - Masaki Nishikawa
- Department of Chemical System Engineering, Graduate School of Engineering, the University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan
| | - Amar Abderrahmani
- Univ. Lille, CNRS, Centrale Lille, Univ. Polytechnique Hauts-de-France, UMR 8520, IEMN, F-59000 Lille, France
| | - Yasuyuki Sakai
- CNRS IRL 2820; Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba; Meguro-ku, Tokyo, 153-8505, Japan
- Department of Chemical System Engineering, Graduate School of Engineering, the University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan
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5
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Mathisen AF, Vacaru AM, Unger L, Lamba EM, Mardare OAM, Daian LM, Ghila L, Vacaru AM, Chera S. Molecular profiling of NOD mouse islets reveals a novel regulator of insulitis onset. Sci Rep 2024; 14:14669. [PMID: 38918575 PMCID: PMC11199597 DOI: 10.1038/s41598-024-65454-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Accepted: 06/20/2024] [Indexed: 06/27/2024] Open
Abstract
Non-obese diabetes (NOD) mice are an established, spontaneous model of type 1 diabetes in which diabetes develops through insulitis. Using next-generation sequencing, coupled with pathway analysis, the molecular fingerprint of early insulitis was mapped in a cohort of mice ranging from 4 to 12 weeks of age. The resulting dynamic timeline revealed an initial decrease in proliferative capacity followed by the emergence of an inflammatory signature between 6 and 8 weeks that increased to a regulatory plateau between 10 and 12 weeks. The inflammatory signature is identified by the activation of central immunogenic factors such as Infg, Il1b, and Tnfa, and activation of canonical inflammatory signaling. Analysis of the regulatory landscape revealed the transcription factor Atf3 as a potential novel modulator of inflammatory signaling in the NOD islets. Furthermore, the Hedgehog signaling pathway correlated with Atf3 regulation, suggesting that the two play a role in regulating islet inflammation; however, further studies are needed to establish the nature of this connection.
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Affiliation(s)
- Andreas Frøslev Mathisen
- Department of Clinical Science, Mohn Research Center for Diabetes Precision Medicine, University of Bergen, Bergen, Norway
| | - Andrei Mircea Vacaru
- BetaUpreg Research Group, Institute of Cellular Biology and Pathology "Nicolae Simionescu", Bucharest, Romania
| | - Lucas Unger
- Department of Clinical Science, Mohn Research Center for Diabetes Precision Medicine, University of Bergen, Bergen, Norway
| | - Elena Mirela Lamba
- BetaUpreg Research Group, Institute of Cellular Biology and Pathology "Nicolae Simionescu", Bucharest, Romania
| | - Oana-Ana-Maria Mardare
- BetaUpreg Research Group, Institute of Cellular Biology and Pathology "Nicolae Simionescu", Bucharest, Romania
| | - Laura Maria Daian
- BetaUpreg Research Group, Institute of Cellular Biology and Pathology "Nicolae Simionescu", Bucharest, Romania
| | - Luiza Ghila
- Department of Clinical Science, Mohn Research Center for Diabetes Precision Medicine, University of Bergen, Bergen, Norway
| | - Ana-Maria Vacaru
- BetaUpreg Research Group, Institute of Cellular Biology and Pathology "Nicolae Simionescu", Bucharest, Romania.
| | - Simona Chera
- Department of Clinical Science, Mohn Research Center for Diabetes Precision Medicine, University of Bergen, Bergen, Norway.
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6
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Wang C, Abadpour S, Aizenshtadt A, Dalmao-Fernandez A, Høyem M, Wilhelmsen I, Stokowiec J, Olsen PA, Krauss S, Chera S, Ghila L, Ræder H, Scholz H. Cell identity dynamics and insight into insulin secretagogues when employing stem cell-derived islets for disease modeling. Front Bioeng Biotechnol 2024; 12:1392575. [PMID: 38933536 PMCID: PMC11199790 DOI: 10.3389/fbioe.2024.1392575] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2024] [Accepted: 05/13/2024] [Indexed: 06/28/2024] Open
Abstract
Stem cell-derived islets (SC-islets) are not only an unlimited source for cell-based therapy of type 1 diabetes but have also emerged as an attractive material for modeling diabetes and conducting screening for treatment options. Prior to SC-islets becoming the established standard for disease modeling and drug development, it is essential to understand their response to various nutrient sources in vitro. This study demonstrates an enhanced efficiency of pancreatic endocrine cell differentiation through the incorporation of WNT signaling inhibition following the definitive endoderm stage. We have identified a tri-hormonal cell population within SC-islets, which undergoes reduction concurrent with the emergence of elevated numbers of glucagon-positive cells during extended in vitro culture. Over a 6-week period of in vitro culture, the SC-islets consistently demonstrated robust insulin secretion in response to glucose stimulation. Moreover, they manifested diverse reactivity patterns when exposed to distinct nutrient sources and exhibited deviant glycolytic metabolic characteristics in comparison to human primary islets. Although the SC-islets demonstrated an aberrant glucose metabolism trafficking, the evaluation of a potential antidiabetic drug, pyruvate kinase agonist known as TEPP46, significantly improved in vitro insulin secretion of SC-islets. Overall, this study provided cell identity dynamics investigation of SC-islets during prolonged culturing in vitro, and insights into insulin secretagogues. Associated advantages and limitations were discussed when employing SC-islets for disease modeling.
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Affiliation(s)
- Chencheng Wang
- Department of Transplant Medicine and Institute for Surgical Research, Oslo University Hospital, Oslo, Norway
- Hybrid Technology Hub, Center of Excellence, University of Oslo, Oslo, Norway
| | - Shadab Abadpour
- Department of Transplant Medicine and Institute for Surgical Research, Oslo University Hospital, Oslo, Norway
- Hybrid Technology Hub, Center of Excellence, University of Oslo, Oslo, Norway
| | | | - Andrea Dalmao-Fernandez
- Hybrid Technology Hub, Center of Excellence, University of Oslo, Oslo, Norway
- Section for Pharmacology and Pharmaceutical Biosciences, Department of Pharmacy, University of Oslo, Oslo, Norway
| | - Merete Høyem
- Department of Transplant Medicine and Institute for Surgical Research, Oslo University Hospital, Oslo, Norway
| | - Ingrid Wilhelmsen
- Hybrid Technology Hub, Center of Excellence, University of Oslo, Oslo, Norway
- Department of Immunology and Transfusion Medicine, Oslo University Hospital, Oslo, Norway
| | - Justyna Stokowiec
- Hybrid Technology Hub, Center of Excellence, University of Oslo, Oslo, Norway
| | - Petter Angell Olsen
- Hybrid Technology Hub, Center of Excellence, University of Oslo, Oslo, Norway
- Department of Immunology and Transfusion Medicine, Oslo University Hospital, Oslo, Norway
| | - Stefan Krauss
- Hybrid Technology Hub, Center of Excellence, University of Oslo, Oslo, Norway
- Department of Immunology and Transfusion Medicine, Oslo University Hospital, Oslo, Norway
| | - Simona Chera
- Department of Clinical Science, University of Bergen, Bergen, Norway
| | - Luiza Ghila
- Department of Clinical Science, University of Bergen, Bergen, Norway
| | - Helge Ræder
- Department of Clinical Science, University of Bergen, Bergen, Norway
- Department of Pediatrics, Haukeland University Hospital, Bergen, Norway
| | - Hanne Scholz
- Department of Transplant Medicine and Institute for Surgical Research, Oslo University Hospital, Oslo, Norway
- Hybrid Technology Hub, Center of Excellence, University of Oslo, Oslo, Norway
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7
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Morisseau L, Tokito F, Lucas M, Poulain S, Kim SH, Plaisance V, Pawlowski V, Legallais C, Jellali R, Sakai Y, Abderrahmani A, Leclerc E. Transcriptomic profiling analysis of the effect of palmitic acid on 3D spheroids of β-like cells derived from induced pluripotent stem cells. Gene 2024; 917:148441. [PMID: 38608795 DOI: 10.1016/j.gene.2024.148441] [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: 02/15/2024] [Accepted: 04/03/2024] [Indexed: 04/14/2024]
Abstract
Type 2 diabetes (T2D) is posing a serious public health concern with a considerable impact on human life and health expenditures worldwide. The disease develops when insulin plasma level is insufficient for coping insulin resistance, caused by the decline of pancreatic β-cell function and mass. In β-cells, the lipotoxicity exerted by saturated free fatty acids in particular palmitate (PA), which is chronically elevated in T2D, plays a major role in β-cell dysfunction and mass. However, there is a lack of human relevant in vitro model to identify the underlying mechanism through which palmitate induces β-cell failure. In this frame, we have previously developed a cutting-edge 3D spheroid model of β-like cells derived from human induced pluripotent stem cells. In the present work, we investigated the signaling pathways modified by palmitate in β-like cells derived spheroids. When compared to the 2D monolayer cultures, the transcriptome analysis (FDR set at 0.1) revealed that the 3D spheroids upregulated the pancreatic markers (such as GCG, IAPP genes), lipids metabolism and transporters (CD36, HMGSC2 genes), glucose transporter (SLC2A6). Then, the 3D spheroids are exposed to PA 0.5 mM for 72 h. The differential analysis demonstrated that 32 transcription factors and 135 target genes were mainly modulated (FDR set at 0.1) including the upregulation of lipid and carbohydrates metabolism (HMGSC2, LDHA, GLUT3), fibrin metabolism (FGG, FGB), apoptosis (CASP7). The pathway analysis using the 135 selected targets extracted the fibrin related biological process and wound healing in 3D PA treated conditions. An overall pathway gene set enrichment analysis, performed on the overall gene set (with pathway significance cutoff at 0.2), highlighted that PA perturbs the citrate cycle, FOXO signaling and Hippo signaling as observed in human islets studies. Additional RT-PCR confirmed induction of inflammatory (IGFBP1, IGFBP3) and cell growth (CCND1, Ki67) pathways by PA. All these changes were associated with unaffected glucose-stimulated insulin secretion (GSIS), suggesting that they precede the defect of insulin secretion and death induced by PA. Overall, we believe that our data demonstrate the potential of our spheroid 3D islet-like cells to investigate the pancreatic-like response to diabetogenic environment.
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Affiliation(s)
- Lisa Morisseau
- Université de Technologie de Compiègne, CNRS, Biomechanics and Bioengineering, Centre de recherche Royallieu CS 60319, 60203 Compiègne Cedex, France
| | - Fumiya Tokito
- Department of Chemical Engineering, Faculty of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
| | - Mathilde Lucas
- Univ. Lille, CNRS, Centrale Lille, Univ. Polytechnique Hauts-de-France, UMR 8520, IEMN, F-59000 Lille, France
| | - Stéphane Poulain
- Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan
| | - Soo Hyeon Kim
- Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan
| | - Valérie Plaisance
- Univ. Lille, CNRS, Centrale Lille, Univ. Polytechnique Hauts-de-France, UMR 8520, IEMN, F-59000 Lille, France
| | - Valérie Pawlowski
- Univ. Lille, CNRS, Centrale Lille, Univ. Polytechnique Hauts-de-France, UMR 8520, IEMN, F-59000 Lille, France
| | - Cécile Legallais
- Université de Technologie de Compiègne, CNRS, Biomechanics and Bioengineering, Centre de recherche Royallieu CS 60319, 60203 Compiègne Cedex, France
| | - Rachid Jellali
- Université de Technologie de Compiègne, CNRS, Biomechanics and Bioengineering, Centre de recherche Royallieu CS 60319, 60203 Compiègne Cedex, France
| | - Yasuyuki Sakai
- Department of Chemical Engineering, Faculty of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; CNRS/IIS IRL 2820, Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan
| | - Amar Abderrahmani
- Univ. Lille, CNRS, Centrale Lille, Univ. Polytechnique Hauts-de-France, UMR 8520, IEMN, F-59000 Lille, France
| | - Eric Leclerc
- CNRS/IIS IRL 2820, Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.
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8
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Mueller LM, Isaacson A, Wilson H, Salowka A, Tay I, Gong M, Elbarbary NS, Raile K, Spagnoli FM. Heterozygous missense variant in GLI2 impairs human endocrine pancreas development. Nat Commun 2024; 15:2483. [PMID: 38509065 PMCID: PMC10954617 DOI: 10.1038/s41467-024-46740-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2023] [Accepted: 03/08/2024] [Indexed: 03/22/2024] Open
Abstract
Missense variants are the most common type of coding genetic variants. Their functional assessment is fundamental for defining any implication in human diseases and may also uncover genes that are essential for human organ development. Here, we apply CRISPR-Cas9 gene editing on human iPSCs to study a heterozygous missense variant in GLI2 identified in two siblings with early-onset and insulin-dependent diabetes of unknown cause. GLI2 is a primary mediator of the Hedgehog pathway, which regulates pancreatic β-cell development in mice. However, neither mutations in GLI2 nor Hedgehog dysregulation have been reported as cause or predisposition to diabetes. We establish and study a set of isogenic iPSC lines harbouring the missense variant for their ability to differentiate into pancreatic β-like cells. Interestingly, iPSCs carrying the missense variant show altered GLI2 transcriptional activity and impaired differentiation of pancreatic progenitors into endocrine cells. RNASeq and network analyses unveil a crosstalk between Hedgehog and WNT pathways, with the dysregulation of non-canonical WNT signaling in pancreatic progenitors carrying the GLI2 missense variant. Collectively, our findings underscore an essential role for GLI2 in human endocrine development and identify a gene variant that may lead to diabetes.
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Affiliation(s)
- Laura M Mueller
- Centre for Gene Therapy and Regenerative Medicine, King's College London, Great Maze Pond, London, SE1 9RT, United Kingdom
| | - Abigail Isaacson
- Centre for Gene Therapy and Regenerative Medicine, King's College London, Great Maze Pond, London, SE1 9RT, United Kingdom
| | - Heather Wilson
- Centre for Gene Therapy and Regenerative Medicine, King's College London, Great Maze Pond, London, SE1 9RT, United Kingdom
| | - Anna Salowka
- Centre for Gene Therapy and Regenerative Medicine, King's College London, Great Maze Pond, London, SE1 9RT, United Kingdom
| | - Isabel Tay
- Centre for Gene Therapy and Regenerative Medicine, King's College London, Great Maze Pond, London, SE1 9RT, United Kingdom
| | - Maolian Gong
- Department of Pediatric Endocrinology and Diabetology, Charité, Berlin, Germany
- Experimental and Clinical Research Center (ECRC), Charité Medical Faculty, Max-Delbrueck-Center for Molecular Medicine (MDC), Berlin, Germany
| | - Nancy Samir Elbarbary
- Department of Pediatrics, Diabetes and Endocrine Unit, Faculty of Medicine, Ain Shams University, Cairo, Egypt
| | - Klemens Raile
- Department of Pediatric Endocrinology and Diabetology, Charité, Berlin, Germany
- Experimental and Clinical Research Center (ECRC), Charité Medical Faculty, Max-Delbrueck-Center for Molecular Medicine (MDC), Berlin, Germany
| | - Francesca M Spagnoli
- Centre for Gene Therapy and Regenerative Medicine, King's College London, Great Maze Pond, London, SE1 9RT, United Kingdom.
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9
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Morisseau L, Tokito F, Poulain S, Plaisance V, Pawlowski V, Kim SH, Legallais C, Jellali R, Sakai Y, Abderrahmani A, Leclerc E. Generation of β-like cell subtypes from differentiated human induced pluripotent stem cells in 3D spheroids. Mol Omics 2023; 19:810-822. [PMID: 37698079 DOI: 10.1039/d3mo00050h] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/13/2023]
Abstract
Since the identification of four different pancreatic β-cell subtypes and bi-hormomal cells playing a role in the diabetes pathogenesis, the search for in vitro models that mimics such cells heterogeneity became a key priority in experimental and clinical diabetology. We investigated the potential of human induced pluripotent stem cells to lead to the development of the different β-cells subtypes in honeycomb microwell-based 3D spheroids. The glucose-stimulated insulin secretion confirmed the spheroids functionality. Then, we performed a single cell RNA sequencing of the spheroids. Using a knowledge-based analysis with a stringency on the pancreatic markers, we extracted the β-cells INS+/UCN3+ subtype (11%; β1-like cells), the INS+/ST8SIA1+/CD9- subtype (3%, β3-like cells) and INS+/CD9+/ST8SIA1-subtype (1%; β2-like cells) consistently with literature findings. We did not detect the INS+/ST8SIA1+/CD9+ cells (β4-like cells). Then, we also identified four bi-hormonal cells subpopulations including δ-like cells (INS+/SST+, 6%), γ-like cells (INS+/PPY+, 3%), α-like-cells (INS+/GCG+, 6%) and ε-like-cells (INS+/GHRL+, 2%). Using data-driven clustering, we extracted four progenitors' subpopulations (with the lower level of INS gene) that included one population highly expressing inhibin genes (INHBA+/INHBB+), one population highly expressing KCNJ3+/TPH1+, one population expressing hepatocyte-like lineage markers (HNF1A+/AFP+), and one population expressing stem-like cell pancreatic progenitor markers (SOX2+/NEUROG3+). Furthermore, among the cycling population we found a large number of REST+ cells and CD9+ cells (CD9+/SPARC+/REST+). Our data confirm that our differentiation leads to large β-cell heterogeneity, which can be used for investigating β-cells plasticity under physiological and pathophysiological conditions.
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Affiliation(s)
- Lisa Morisseau
- Biomechanics and Bioengineering UMR 7338, Université de technologie de Compiègne, CNRS, Centre de Recherche Royallieu CS 60319, Compiègne, 60203 Cedex, France
| | - Fumiya Tokito
- Department of Chemical System Engineering, Graduate School of Engineering, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan
| | - Stéphane Poulain
- Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba; Meguro-ku, Tokyo, 153-8505, Japan
| | - Valerie Plaisance
- Univ. Lille, CNRS, Centrale Lille, Univ. Polytechnique Hauts-de-France, UMR 8520, IEMN, F-59000 Lille, France
| | - Valerie Pawlowski
- Univ. Lille, CNRS, Centrale Lille, Univ. Polytechnique Hauts-de-France, UMR 8520, IEMN, F-59000 Lille, France
| | - Soo Hyeon Kim
- Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba; Meguro-ku, Tokyo, 153-8505, Japan
| | - Cécile Legallais
- Biomechanics and Bioengineering UMR 7338, Université de technologie de Compiègne, CNRS, Centre de Recherche Royallieu CS 60319, Compiègne, 60203 Cedex, France
| | - Rachid Jellali
- Biomechanics and Bioengineering UMR 7338, Université de technologie de Compiègne, CNRS, Centre de Recherche Royallieu CS 60319, Compiègne, 60203 Cedex, France
| | - Yasuyuki Sakai
- Department of Chemical System Engineering, Graduate School of Engineering, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan
- Laboratory for Integrated Micro Mechatronic Systems, CNRS/IIS IRL 2820, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba; Meguro-ku, Tokyo, 153-8505, Japan
| | - Amar Abderrahmani
- Univ. Lille, CNRS, Centrale Lille, Univ. Polytechnique Hauts-de-France, UMR 8520, IEMN, F-59000 Lille, France
| | - Eric Leclerc
- Laboratory for Integrated Micro Mechatronic Systems, CNRS/IIS IRL 2820, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba; Meguro-ku, Tokyo, 153-8505, Japan
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10
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Mathisen AF, Abadpour S, Legøy TA, Paulo JA, Ghila L, Scholz H, Chera S. Global proteomics reveals insulin abundance as a marker of human islet homeostasis alterations. Acta Physiol (Oxf) 2023; 239:e14037. [PMID: 37621186 PMCID: PMC10592125 DOI: 10.1111/apha.14037] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2023] [Revised: 08/07/2023] [Accepted: 08/10/2023] [Indexed: 08/26/2023]
Abstract
AIM The variation in quality between the human islet samples represents a major problem for research, especially when used as control material. The assays assessing the quality of human islets used in research are non-standardized and limited, with many important parameters not being consistently assessed. High-throughput studies aimed at characterizing the diversity and segregation markers among apparently functionally healthy islet preps are thus a requirement. Here, we designed a pilot study to comprehensively identify the diversity of global proteome signatures and the deviation from normal homeostasis in randomly selected human-isolated islet samples. METHODS By using Tandem Mass Tag 16-plex proteomics, we focused on the recurrently observed disparity in the detected insulin abundance between the samples, used it as a segregating parameter, and analyzed the correlated changes in the proteome signature and homeostasis by pathway analysis. RESULTS In this pilot study, we showed that insulin protein abundance is a predictor of human islet homeostasis and quality. This parameter is independent of other quality predictors within their acceptable range, thus being able to further stratify islets samples of apparent good quality. Human islets with low amounts of insulin displayed changes in their metabolic and signaling profile, especially in regard to energy homeostasis and cell identity maintenance. We further showed that xenotransplantation into diabetic hosts is not expected to improve the pre-transplantation signature, as it has a negative effect on energy balance, antioxidant activity, and islet cell identity. CONCLUSIONS Insulin protein abundance predicts significant changes in human islet homeostasis among random samples of apparently good quality.
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Affiliation(s)
- Andreas F. Mathisen
- Mohn Research Center for Diabetes Precision Medicine, Department of Clinical Science, University of Bergen, Bergen, Norway
| | - Shadab Abadpour
- Hybrid Technology Hub-Centre of Excellence, Faculty of Medicine, University of Oslo, Norway
- Institute for Surgical Research and Department of Transplant Medicine, Oslo University Hospital, Oslo, Norway
| | - Thomas Aga Legøy
- Mohn Research Center for Diabetes Precision Medicine, Department of Clinical Science, University of Bergen, Bergen, Norway
| | - Joao A. Paulo
- Department of Cell Biology, Harvard Medical School, Boston, MA, USA
| | - Luiza Ghila
- Mohn Research Center for Diabetes Precision Medicine, Department of Clinical Science, University of Bergen, Bergen, Norway
| | - Hanne Scholz
- Hybrid Technology Hub-Centre of Excellence, Faculty of Medicine, University of Oslo, Norway
- Institute for Surgical Research and Department of Transplant Medicine, Oslo University Hospital, Oslo, Norway
| | - Simona Chera
- Mohn Research Center for Diabetes Precision Medicine, Department of Clinical Science, University of Bergen, Bergen, Norway
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11
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Ali M, Kato Y, Shiraki N, Kume S. Generation of induced pluripotent stem cell-derived beta-cells in blood amino acids-like medium. Biol Open 2023; 12:287063. [PMID: 36811942 PMCID: PMC10084857 DOI: 10.1242/bio.059581] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2022] [Accepted: 02/14/2023] [Indexed: 02/24/2023] Open
Abstract
Traditional cell culture media do not accurately represent the availability of the nutrients in plasma. They usually contain a supraphysiological concentration of nutrients such as glucose, amino acids, etc. These high nutrients can alter the metabolism of cultured cells and induce metabolic phenotypes that do not reflect in vivo conditions. We demonstrate that the supraphysiological levels of nutrients interfere with endodermal differentiation. Refinement of media formulations has a potential application in maturity modulation of stem cell-derived β-cells (SC-β) generation in vitro. To address these issues, we established a defined culture system to derive SC-β-cells using a blood amino acids-like medium (BALM). Human induced pluripotent stem cells (hiPSCs) can be efficiently differentiated into the definitive endoderm, pancreatic progenitors, endocrine progenitors, and SC-β in BALM-based med. The differentiated cells secreted C-peptide in vitro in response to high glucose levels and expressed several pancreatic β-cell markers. In conclusion, amino acids at the physiological levels are sufficient for deriving functional SC-β cells.
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Affiliation(s)
- Marwa Ali
- School of Life Science and Technology, Tokyo Institute of Technology, 4259-B-25 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
| | - Yusuke Kato
- School of Life Science and Technology, Tokyo Institute of Technology, 4259-B-25 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
| | - Nobuaki Shiraki
- School of Life Science and Technology, Tokyo Institute of Technology, 4259-B-25 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
| | - Shoen Kume
- School of Life Science and Technology, Tokyo Institute of Technology, 4259-B-25 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
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12
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Giallongo S, Lo Re O, Resnick I, Raffaele M, Vinciguerra M. Gene Editing and Human iPSCs in Cardiovascular and Metabolic Diseases. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2023; 1396:275-298. [DOI: 10.1007/978-981-19-5642-3_18] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
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13
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The type 1 diabetes gene TYK2 regulates β-cell development and its responses to interferon-α. Nat Commun 2022; 13:6363. [PMID: 36289205 PMCID: PMC9606380 DOI: 10.1038/s41467-022-34069-z] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2022] [Accepted: 10/13/2022] [Indexed: 01/05/2023] Open
Abstract
Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of insulin producing pancreatic β-cells. One of the genes associated with T1D is TYK2, which encodes a Janus kinase with critical roles in type-Ι interferon (IFN-Ι) mediated intracellular signalling. To study the role of TYK2 in β-cell development and response to IFNα, we generated TYK2 knockout human iPSCs and directed them into the pancreatic endocrine lineage. Here we show that loss of TYK2 compromises the emergence of endocrine precursors by regulating KRAS expression, while mature stem cell-islets (SC-islets) function is not affected. In the SC-islets, the loss or inhibition of TYK2 prevents IFNα-induced antigen processing and presentation, including MHC Class Ι and Class ΙΙ expression, enhancing their survival against CD8+ T-cell cytotoxicity. These results identify an unsuspected role for TYK2 in β-cell development and support TYK2 inhibition in adult β-cells as a potent therapeutic target to halt T1D progression.
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14
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Szymczak F, Alvelos MI, Marín-Cañas S, Castela Â, Demine S, Colli ML, Op de Beeck A, Thomaidou S, Marselli L, Zaldumbide A, Marchetti P, Eizirik DL. Transcription and splicing regulation by NLRC5 shape the interferon response in human pancreatic β cells. SCIENCE ADVANCES 2022; 8:eabn5732. [PMID: 36103539 PMCID: PMC9473574 DOI: 10.1126/sciadv.abn5732] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/07/2021] [Accepted: 07/27/2022] [Indexed: 06/15/2023]
Abstract
IFNα is a key regulator of the dialogue between pancreatic β cells and the immune system in early type 1 diabetes (T1D). IFNα up-regulates HLA class I expression in human β cells, fostering autoantigen presentation to the immune system. We observed by bulk and single-cell RNA sequencing that exposure of human induced pluripotent-derived islet-like cells to IFNα induces expression of HLA class I and of other genes involved in antigen presentation, including the transcriptional activator NLRC5. We next evaluated the global role of NLRC5 in human insulin-producing EndoC-βH1 and human islet cells by RNA sequencing and targeted gene/protein determination. NLRC5 regulates expression of HLA class I, antigen presentation-related genes, and chemokines. NLRC5 also mediates the effects of IFNα on alternative splicing, a generator of β cell neoantigens, suggesting that it is a central player of the effects of IFNα on β cells that contribute to trigger and amplify autoimmunity in T1D.
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Affiliation(s)
- Florian Szymczak
- ULB Center for Diabetes Research, Medical Faculty, Université Libre De Bruxelles (ULB), Brussels, Belgium
- Interuniversity Institute of Bioinformatics in Brussels, Université Libre de Bruxelles-Vrije Universiteit Brussel, Brussels, Belgium
| | - Maria Inês Alvelos
- ULB Center for Diabetes Research, Medical Faculty, Université Libre De Bruxelles (ULB), Brussels, Belgium
| | - Sandra Marín-Cañas
- ULB Center for Diabetes Research, Medical Faculty, Université Libre De Bruxelles (ULB), Brussels, Belgium
| | - Ângela Castela
- ULB Center for Diabetes Research, Medical Faculty, Université Libre De Bruxelles (ULB), Brussels, Belgium
| | - Stéphane Demine
- Indiana Biosciences Research Institute, Indianapolis, IN, USA
| | - Maikel Luis Colli
- ULB Center for Diabetes Research, Medical Faculty, Université Libre De Bruxelles (ULB), Brussels, Belgium
| | - Anne Op de Beeck
- ULB Center for Diabetes Research, Medical Faculty, Université Libre De Bruxelles (ULB), Brussels, Belgium
| | - Sofia Thomaidou
- Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands
| | - Lorella Marselli
- Department of Clinical and Experimental Medicine, Islet Cell Laboratory, University of Pisa, Pisa, Italy
| | - Arnaud Zaldumbide
- Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands
| | - Piero Marchetti
- Department of Clinical and Experimental Medicine, Islet Cell Laboratory, University of Pisa, Pisa, Italy
| | - Décio L. Eizirik
- ULB Center for Diabetes Research, Medical Faculty, Université Libre De Bruxelles (ULB), Brussels, Belgium
- Welbio, Medical Faculty, Université Libre De Bruxelles (ULB), Brussels, Belgium
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15
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Fantuzzi F, Toivonen S, Schiavo AA, Chae H, Tariq M, Sawatani T, Pachera N, Cai Y, Vinci C, Virgilio E, Ladriere L, Suleiman M, Marchetti P, Jonas JC, Gilon P, Eizirik DL, Igoillo-Esteve M, Cnop M. In depth functional characterization of human induced pluripotent stem cell-derived beta cells in vitro and in vivo. Front Cell Dev Biol 2022; 10:967765. [PMID: 36060810 PMCID: PMC9428245 DOI: 10.3389/fcell.2022.967765] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2022] [Accepted: 07/06/2022] [Indexed: 01/05/2023] Open
Abstract
In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro. Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo, beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes.
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Affiliation(s)
- Federica Fantuzzi
- ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium,Endocrinology and Metabolism, Department of Medicine and Surgery, University of Parma, Parma, Italy,*Correspondence: Miriam Cnop, ; Federica Fantuzzi,
| | - Sanna Toivonen
- ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium
| | - Andrea Alex Schiavo
- ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium
| | - Heeyoung Chae
- Institut de Recherche Expérimentale et Clinique, Pôle d’Endocrinologie, Diabète et Nutrition, Université Catholique de Louvain, Brussels, Belgium
| | - Mohammad Tariq
- Institut de Recherche Expérimentale et Clinique, Pôle d’Endocrinologie, Diabète et Nutrition, Université Catholique de Louvain, Brussels, Belgium
| | - Toshiaki Sawatani
- ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium
| | - Nathalie Pachera
- ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium
| | - Ying Cai
- ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium
| | - Chiara Vinci
- ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium
| | - Enrico Virgilio
- ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium
| | - Laurence Ladriere
- ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium
| | - Mara Suleiman
- Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy
| | - Piero Marchetti
- Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy
| | - Jean-Christophe Jonas
- Institut de Recherche Expérimentale et Clinique, Pôle d’Endocrinologie, Diabète et Nutrition, Université Catholique de Louvain, Brussels, Belgium
| | - Patrick Gilon
- Institut de Recherche Expérimentale et Clinique, Pôle d’Endocrinologie, Diabète et Nutrition, Université Catholique de Louvain, Brussels, Belgium
| | - Décio L. Eizirik
- ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium
| | | | - Miriam Cnop
- ULB Center for Diabetes Research, Université Libre de Bruxelles, Brussels, Belgium,Division of Endocrinology, Erasmus Hospital, Université Libre de Bruxelles, Brussels, Belgium,*Correspondence: Miriam Cnop, ; Federica Fantuzzi,
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16
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Balboa D, Barsby T, Lithovius V, Saarimäki-Vire J, Omar-Hmeadi M, Dyachok O, Montaser H, Lund PE, Yang M, Ibrahim H, Näätänen A, Chandra V, Vihinen H, Jokitalo E, Kvist J, Ustinov J, Nieminen AI, Kuuluvainen E, Hietakangas V, Katajisto P, Lau J, Carlsson PO, Barg S, Tengholm A, Otonkoski T. Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells. Nat Biotechnol 2022; 40:1042-1055. [PMID: 35241836 PMCID: PMC9287162 DOI: 10.1038/s41587-022-01219-z] [Citation(s) in RCA: 198] [Impact Index Per Article: 66.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2021] [Accepted: 01/11/2022] [Indexed: 12/19/2022]
Abstract
Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes. Pancreatic islets derived from stem cells are benchmarked against primary cells.
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Affiliation(s)
- Diego Balboa
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland.,Bioinformatics and Genomics Program, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain.,Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain
| | - Tom Barsby
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Väinö Lithovius
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Jonna Saarimäki-Vire
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | | | - Oleg Dyachok
- Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
| | - Hossam Montaser
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Per-Eric Lund
- Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
| | - Mingyu Yang
- Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
| | - Hazem Ibrahim
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Anna Näätänen
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Vikash Chandra
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Helena Vihinen
- Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki, Helsinki, Finland
| | - Eija Jokitalo
- Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki, Helsinki, Finland
| | - Jouni Kvist
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Jarkko Ustinov
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Anni I Nieminen
- Metabolomics Unit, Institute for Molecular Medicine Finland, Helsinki, Finland
| | - Emilia Kuuluvainen
- Institute of Biotechnology, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland
| | - Ville Hietakangas
- Institute of Biotechnology, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland.,Molecular and Integrative Bioscience Research Program, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
| | - Pekka Katajisto
- Institute of Biotechnology, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland.,Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
| | - Joey Lau
- Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
| | - Per-Ola Carlsson
- Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
| | - Sebastian Barg
- Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
| | - Anders Tengholm
- Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
| | - Timo Otonkoski
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland. .,Children's Hospital, Helsinki University Hospital and University of Helsinki, Helsinki, Finland.
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17
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Dabi YT, Degechisa ST. Genome Editing and Human Pluripotent Stem Cell Technologies for in vitro Monogenic Diabetes Modeling. Diabetes Metab Syndr Obes 2022; 15:1785-1797. [PMID: 35719247 PMCID: PMC9199525 DOI: 10.2147/dmso.s366967] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Accepted: 06/08/2022] [Indexed: 12/01/2022] Open
Abstract
Diabetes is a metabolic disease characterized by chronic hyperglycemia. Polygenic diabetes, which encompasses type-1 and type-2 diabetes, is the most prevalent kind of diabetes and is caused by a combination of different genetic and environmental factors, whereas rare phenotype monogenic diabetes is caused by a single gene mutation. Monogenic diabetes includes Neonatal diabetes mellitus and Maturity-onset diabetes of the young. The majority of our current knowledge about the pathogenesis of diabetes stems from studies done on animal models. However, the genetic difference between these creatures and humans makes it difficult to mimic human clinical pathophysiology, limiting their value in modeling key aspects of human disease. Human pluripotent stem cell technologies combined with genome editing techniques have been shown to be better alternatives for creating in vitro models that can provide crucial knowledge about disease etiology. This review paper addresses genome editing and human pluripotent stem cell technologies for in vitro monogenic diabetes modeling.
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Affiliation(s)
- Yosef Tsegaye Dabi
- Department of Medical Biochemistry, School of Medicine, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia
- Department of Medical Laboratory Science, Wollega University, Nekemte, Ethiopia
- Correspondence: Yosef Tsegaye Dabi, Email
| | - Sisay Teka Degechisa
- Department of Medical Biochemistry, School of Medicine, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia
- Department of Medical Laboratory Sciences, College of Medicine and Health Sciences, Arba Minch University, Arba Minch, Ethiopia
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18
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Houeiss P, Boitard C, Luce S. Preclinical Models to Evaluate the Human Response to Autoantigen and Antigen-Specific Immunotherapy in Human Type 1 Diabetes. Front Endocrinol (Lausanne) 2022; 13:883000. [PMID: 35498419 PMCID: PMC9044628 DOI: 10.3389/fendo.2022.883000] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/24/2022] [Accepted: 03/14/2022] [Indexed: 11/13/2022] Open
Abstract
Type 1 Diabetes (T1D) is an autoimmune disease that results from the destruction of pancreatic islet β-cells by auto-reactive T cells. The clinical management of T1D faces the lack of fully predictive biomarkers in its preclinical stage and of antigen-specific therapies to induce or re-induce immune tolerance to β-cell autoantigens and prevent its development. From a therapeutic standpoint, preclinical models of T1D have fallen short of directly translating into humans. To circumvent this limitation, preclinical models are being optimized to allow defining autoantigen epitopes that are presented to T cells and directly apply to the human. In this review, we propose to make a point on the latest available models such as humanized immunodeficient NOD mice models and HLA and autoantigen transgenic mice and their application in the context of T1D.
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Affiliation(s)
- Pamela Houeiss
- Laboratory Immunology of Diabetes, Cochin Institute, Department Endocrinology, Metabolism and Diabetologia (EMD), Institut Nationale de la Santé et de la Recherche Médicale, Unité 1016 (INSERMU1016), Paris, France
- Medical Faculty, Paris University, Paris, France
| | - Christian Boitard
- Laboratory Immunology of Diabetes, Cochin Institute, Department Endocrinology, Metabolism and Diabetologia (EMD), Institut Nationale de la Santé et de la Recherche Médicale, Unité 1016 (INSERMU1016), Paris, France
- Medical Faculty, Paris University, Paris, France
| | - Sandrine Luce
- Laboratory Immunology of Diabetes, Cochin Institute, Department Endocrinology, Metabolism and Diabetologia (EMD), Institut Nationale de la Santé et de la Recherche Médicale, Unité 1016 (INSERMU1016), Paris, France
- Medical Faculty, Paris University, Paris, France
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Abstract
This review focuses on the human pancreatic islet-including its structure, cell composition, development, function, and dysfunction. After providing a historical timeline of key discoveries about human islets over the past century, we describe new research approaches and technologies that are being used to study human islets and how these are providing insight into human islet physiology and pathophysiology. We also describe changes or adaptations in human islets in response to physiologic challenges such as pregnancy, aging, and insulin resistance and discuss islet changes in human diabetes of many forms. We outline current and future interventions being developed to protect, restore, or replace human islets. The review also highlights unresolved questions about human islets and proposes areas where additional research on human islets is needed.
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Affiliation(s)
- John T Walker
- Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Diane C Saunders
- Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - Marcela Brissova
- Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Alvin C Powers
- Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
- Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA
- VA Tennessee Valley Healthcare System, Nashville, Tennessee, USA
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20
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Burgos JI, Vallier L, Rodríguez-Seguí SA. Monogenic Diabetes Modeling: In Vitro Pancreatic Differentiation From Human Pluripotent Stem Cells Gains Momentum. Front Endocrinol (Lausanne) 2021; 12:692596. [PMID: 34295307 PMCID: PMC8290520 DOI: 10.3389/fendo.2021.692596] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/08/2021] [Accepted: 06/15/2021] [Indexed: 12/14/2022] Open
Abstract
The occurrence of diabetes mellitus is characterized by pancreatic β cell loss and chronic hyperglycemia. While Type 1 and Type 2 diabetes are the most common types, rarer forms involve mutations affecting a single gene. This characteristic has made monogenic diabetes an interesting disease group to model in vitro using human pluripotent stem cells (hPSCs). By altering the genotype of the original hPSCs or by deriving human induced pluripotent stem cells (hiPSCs) from patients with monogenic diabetes, changes in the outcome of the in vitro differentiation protocol can be analyzed in detail to infer the regulatory mechanisms affected by the disease-associated genes. This approach has been so far applied to a diversity of genes/diseases and uncovered new mechanisms. The focus of the present review is to discuss the latest findings obtained by modeling monogenic diabetes using hPSC-derived pancreatic cells generated in vitro. We will specifically focus on the interpretation of these studies, the advantages and limitations of the models used, and the future perspectives for improvement.
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Affiliation(s)
- Juan Ignacio Burgos
- Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires and Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), CONICET-Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina
| | - Ludovic Vallier
- Wellcome-Medical Research Council Cambridge Stem Cell Institute and Department of Surgery, University of Cambridge, Cambridge, United Kingdom
| | - Santiago A. Rodríguez-Seguí
- Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires and Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), CONICET-Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina
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21
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George MN, Leavens KF, Gadue P. Genome Editing Human Pluripotent Stem Cells to Model β-Cell Disease and Unmask Novel Genetic Modifiers. Front Endocrinol (Lausanne) 2021; 12:682625. [PMID: 34149620 PMCID: PMC8206553 DOI: 10.3389/fendo.2021.682625] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/18/2021] [Accepted: 05/13/2021] [Indexed: 01/21/2023] Open
Abstract
A mechanistic understanding of the genetic basis of complex diseases such as diabetes mellitus remain elusive due in large part to the activity of genetic disease modifiers that impact the penetrance and/or presentation of disease phenotypes. In the face of such complexity, rare forms of diabetes that result from single-gene mutations (monogenic diabetes) can be used to model the contribution of individual genetic factors to pancreatic β-cell dysfunction and the breakdown of glucose homeostasis. Here we review the contribution of protein coding and non-protein coding genetic disease modifiers to the pathogenesis of diabetes subtypes, as well as how recent technological advances in the generation, differentiation, and genome editing of human pluripotent stem cells (hPSC) enable the development of cell-based disease models. Finally, we describe a disease modifier discovery platform that utilizes these technologies to identify novel genetic modifiers using induced pluripotent stem cells (iPSC) derived from patients with monogenic diabetes caused by heterozygous mutations.
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Affiliation(s)
- Matthew N. George
- Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA, United States
| | - Karla F. Leavens
- Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA, United States
- Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States
- Division of Endocrinology and Diabetes, The Children’s Hospital of Philadelphia, Philadelphia, PA, United States
| | - Paul Gadue
- Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA, United States
- Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, United States
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22
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Tafaleng EN, Malizio MR, Fox IJ, Soto-Gutierrez A. Synthetic human livers for modeling metabolic diseases. Curr Opin Gastroenterol 2021; 37:224-230. [PMID: 33769378 PMCID: PMC8223234 DOI: 10.1097/mog.0000000000000726] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
PURPOSE OF REVIEW In this review, we will explore recent advances in human induced pluripotent stem cell (iPSC)-based modeling of metabolic liver disease and biofabrication of synthetic human liver tissue while also discussing the emerging concept of synthetic biology to generate more physiologically relevant liver disease models. RECENT FINDING iPSC-based platforms have facilitated the study of underlying cellular mechanisms and potential therapeutic strategies for a number of metabolic liver diseases. Concurrently, rapid progress in biofabrication and gene editing technologies have led to the generation of human hepatic tissue that more closely mimic the complexity of the liver. SUMMARY iPSC-based liver tissue is rapidly becoming available for modeling liver physiology due to its ability to recapitulate the complex three-dimensional architecture of the liver and recapitulate interactions between the different cell types and their surroundings. These mini livers have also been used to recapitulate liver disease pathways using the tools of synthetic biology, such as gene editing, to control gene circuits. Further development in this field will undoubtedly bolster future investigations not only in disease modeling and basic research, but also in personalized medicine and autologous transplantation.
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Affiliation(s)
- Edgar N. Tafaleng
- Department of Surgery, University of Pittsburgh School of Medicine, Pennsylvania, USA
| | - Michelle R. Malizio
- Department of Pathology, University of Pittsburgh School of Medicine, Pennsylvania, USA
| | - Ira J. Fox
- Department of Surgery, University of Pittsburgh School of Medicine, Pennsylvania, USA
- Pittsburgh Liver Research Center, University of Pittsburgh, Pennsylvania, USA
- McGowan Institute for Regenerative Medicine, Pittsburgh, Pennsylvania, USA
| | - Alejandro Soto-Gutierrez
- Department of Pathology, University of Pittsburgh School of Medicine, Pennsylvania, USA
- Pittsburgh Liver Research Center, University of Pittsburgh, Pennsylvania, USA
- McGowan Institute for Regenerative Medicine, Pittsburgh, Pennsylvania, USA
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23
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Chronically Elevated Exogenous Glucose Elicits Antipodal Effects on the Proteome Signature of Differentiating Human iPSC-Derived Pancreatic Progenitors. Int J Mol Sci 2021; 22:ijms22073698. [PMID: 33918250 PMCID: PMC8038174 DOI: 10.3390/ijms22073698] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2021] [Revised: 03/22/2021] [Accepted: 03/27/2021] [Indexed: 12/14/2022] Open
Abstract
The past decade revealed that cell identity changes, such as dedifferentiation or transdifferentiation, accompany the insulin-producing β-cell decay in most diabetes conditions. Mapping and controlling the mechanisms governing these processes is, thus, extremely valuable for managing the disease progression. Extracellular glucose is known to influence cell identity by impacting the redox balance. Here, we use global proteomics and pathway analysis to map the response of differentiating human pancreatic progenitors to chronically increased in vitro glucose levels. We show that exogenous high glucose levels impact different protein subsets in a concentration-dependent manner. In contrast, regardless of concentration, glucose elicits an antipodal effect on the proteome landscape, inducing both beneficial and detrimental changes in regard to achieving the desired islet cell fingerprint. Furthermore, we identified that only a subgroup of these effects and pathways are regulated by changes in redox balance. Our study highlights a complex effect of exogenous glucose on differentiating pancreas progenitors characterized by a distinct proteome signature.
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24
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Joshi K, Cameron F, Tiwari S, Mannering SI, Elefanty AG, Stanley EG. Modeling Type 1 Diabetes Using Pluripotent Stem Cell Technology. Front Endocrinol (Lausanne) 2021; 12:635662. [PMID: 33868170 PMCID: PMC8047192 DOI: 10.3389/fendo.2021.635662] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Accepted: 03/03/2021] [Indexed: 12/26/2022] Open
Abstract
Induced pluripotent stem cell (iPSC) technology is increasingly being used to create in vitro models of monogenic human disorders. This is possible because, by and large, the phenotypic consequences of such genetic variants are often confined to a specific and known cell type, and the genetic variants themselves can be clearly identified and controlled for using a standardized genetic background. In contrast, complex conditions such as autoimmune Type 1 diabetes (T1D) have a polygenic inheritance and are subject to diverse environmental influences. Moreover, the potential cell types thought to contribute to disease progression are many and varied. Furthermore, as HLA matching is critical for cell-cell interactions in disease pathogenesis, any model that seeks to test the involvement of particular cell types must take this restriction into account. As such, creation of an in vitro model of T1D will require a system that is cognizant of genetic background and enables the interaction of cells representing multiple lineages to be examined in the context of the relevant environmental disease triggers. In addition, as many of the lineages critical to the development of T1D cannot be easily generated from iPSCs, such models will likely require combinations of cell types derived from in vitro and in vivo sources. In this review we imagine what an ideal in vitro model of T1D might look like and discuss how the required elements could be feasibly assembled using existing technologies. We also examine recent advances towards this goal and discuss potential uses of this technology in contributing to our understanding of the mechanisms underlying this autoimmune condition.
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Affiliation(s)
- Kriti Joshi
- Department of Endocrinology and Metabolism, All India Institute of Medical Sciences Rishikesh, Uttarakhand, India
- Department of Molecular Medicine & Biotechnology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India
- Department of Cell Biology, Murdoch Children’s Research Institute, Parkville, Vic, Australia
| | - Fergus Cameron
- Department of Cell Biology, Murdoch Children’s Research Institute, Parkville, Vic, Australia
- Department of Endocrinology and Diabetes, The Royal Children’s Hospital, Parkville, Vic, Australia
- Department of Paediatrics, University of Melbourne, Parkville, Vic, Australia
| | - Swasti Tiwari
- Department of Molecular Medicine & Biotechnology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India
| | - Stuart I. Mannering
- Immunology and Diabetes Unit, St. Vincent’s Institute of Medical Research, Fitzroy, Vic, Australia
| | - Andrew G. Elefanty
- Department of Cell Biology, Murdoch Children’s Research Institute, Parkville, Vic, Australia
- Department of Paediatrics, University of Melbourne, Parkville, Vic, Australia
- Department of Anatomy and Developmental Biology, Monash University, Clayton, Vic, Australia
| | - Edouard G. Stanley
- Department of Cell Biology, Murdoch Children’s Research Institute, Parkville, Vic, Australia
- Department of Paediatrics, University of Melbourne, Parkville, Vic, Australia
- Department of Anatomy and Developmental Biology, Monash University, Clayton, Vic, Australia
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25
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James EC, Tomaskovic-Crook E, Crook JM. Bioengineering Clinically Relevant Cardiomyocytes and Cardiac Tissues from Pluripotent Stem Cells. Int J Mol Sci 2021; 22:ijms22063005. [PMID: 33809429 PMCID: PMC8001925 DOI: 10.3390/ijms22063005] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2021] [Revised: 03/11/2021] [Accepted: 03/12/2021] [Indexed: 12/23/2022] Open
Abstract
The regenerative capacity of cardiomyocytes is insufficient to functionally recover damaged tissue, and as such, ischaemic heart disease forms the largest proportion of cardiovascular associated deaths. Human-induced pluripotent stem cells (hiPSCs) have enormous potential for developing patient specific cardiomyocytes for modelling heart disease, patient-based cardiac toxicity testing and potentially replacement therapy. However, traditional protocols for hiPSC-derived cardiomyocytes yield mixed populations of atrial, ventricular and nodal-like cells with immature cardiac properties. New insights gleaned from embryonic heart development have progressed the precise production of subtype-specific hiPSC-derived cardiomyocytes; however, their physiological immaturity severely limits their utility as model systems and their use for drug screening and cell therapy. The long-entrenched challenges in this field are being addressed by innovative bioengingeering technologies that incorporate biophysical, biochemical and more recently biomimetic electrical cues, with the latter having the potential to be used to both direct hiPSC differentiation and augment maturation and the function of derived cardiomyocytes and cardiac tissues by mimicking endogenous electric fields.
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Affiliation(s)
- Emma Claire James
- ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, AIIM Facility, University of Wollongong, Wollongong 2500, Australia;
| | - Eva Tomaskovic-Crook
- ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, AIIM Facility, University of Wollongong, Wollongong 2500, Australia;
- Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong 2500, Australia
- Correspondence: (E.T.-C.); (J.M.C.)
| | - Jeremy Micah Crook
- ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, AIIM Facility, University of Wollongong, Wollongong 2500, Australia;
- Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong 2500, Australia
- Department of Surgery, St Vincent’s Hospital, The University of Melbourne, Fitzroy 3065, Australia
- Correspondence: (E.T.-C.); (J.M.C.)
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26
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Essaouiba A, Jellali R, Shinohara M, Scheidecker B, Legallais C, Sakai Y, Leclerc E. Analysis of the behavior of 2D monolayers and 3D spheroid human pancreatic beta cells derived from induced pluripotent stem cells in a microfluidic environment. J Biotechnol 2021; 330:45-56. [PMID: 33617908 DOI: 10.1016/j.jbiotec.2021.02.009] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2020] [Revised: 02/01/2021] [Accepted: 02/16/2021] [Indexed: 12/17/2022]
Abstract
The limited availability of primary human β-cells/islets and their quality (due to donor diversity) restrict the development of in vitro models for diabetes research. Human induced pluripotent stem cells (hiPSCs) may be a promising cell-source for diabetes studies, anti-diabetic drug screening and personalized therapies. However, achieving levels of maturity/functionality that are comparable to the in vivo situation and islets rebuilt from iPSCs is still challenging. Here, we compare and discuss two strategies for culturing human pancreatic β-cells derived from hiPSCs in microfluidic biochips. First, we confirmed that the protocol in conventional Petri 2D monolayer led to insulin, PDX1 and MAFA positive staining, to C-Peptide productive cells, and to tissue responsive to high/low glucose and GLP1 stimulation. This protocol and its subsequent modifications (including extracellular matrix coating, cell adhesion time, cell inoculation density, flow rate) was not successful in the 2D biochip culture. We proposed a second strategy using 3D spheroids created from honeycomb static cultures. Spheroids in static experiments carried out over 14 days demonstrated that they expressed high levels of β-cell markers (INS mRNA) and higher α-cell markers (GCG mRNA and glucagon positive staining), when compared to Petri 2D cultures. Furthermore, the 3D spheroids were specifically able to secrete insulin in response to both high/low glucose stimulation and GLP1 exposure. The spheroids were successfully inoculated into biochips and maintained for 10 days in perfusion. The 3D biochip cultures increased mRNA levels of GCG and maintained high levels of β-cell markers and responsiveness to both high/low glucose and GLP1 stimulation. Finally, C-peptide and insulin secretion were higher in biochips when compared to static spheroids. These results illustrate the promising potential for hiPSCs derived β-cells and their spheroid-based pancreas-on-chip model for pancreatic disease/diabetes modeling and anti-diabetic drug screening.
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Affiliation(s)
- Amal Essaouiba
- Université de technologie de Compiègne, CNRS, Biomechanics and Bioengineering, Centre de recherche Royallieu, CS 60319, 60203, Compiègne Cedex, France; CNRS UMI 2820, Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan
| | - Rachid Jellali
- Université de technologie de Compiègne, CNRS, Biomechanics and Bioengineering, Centre de recherche Royallieu, CS 60319, 60203, Compiègne Cedex, France.
| | - Marie Shinohara
- Department of Chemical Engineering, Faculty of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan
| | - Benedikt Scheidecker
- Department of Chemical Engineering, Faculty of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan
| | - Cécile Legallais
- Université de technologie de Compiègne, CNRS, Biomechanics and Bioengineering, Centre de recherche Royallieu, CS 60319, 60203, Compiègne Cedex, France
| | - Yasuyuki Sakai
- CNRS UMI 2820, Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan; Department of Chemical Engineering, Faculty of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan
| | - Eric Leclerc
- Université de technologie de Compiègne, CNRS, Biomechanics and Bioengineering, Centre de recherche Royallieu, CS 60319, 60203, Compiègne Cedex, France; CNRS UMI 2820, Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan.
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27
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Lewis PL, Wells JM. Engineering-inspired approaches to study β-cell function and diabetes. Stem Cells 2021; 39:522-535. [PMID: 33497522 DOI: 10.1002/stem.3340] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2020] [Accepted: 01/13/2021] [Indexed: 12/21/2022]
Abstract
Strategies to mitigate the pathologies from diabetes range from simply administering insulin to prescribing complex drug/biologic regimens combined with lifestyle changes. There is a substantial effort to better understand β-cell physiology during diabetes pathogenesis as a means to develop improved therapies. The convergence of multiple fields ranging from developmental biology to microfluidic engineering has led to the development of new experimental systems to better study complex aspects of diabetes and β-cell biology. Here we discuss the available insulin-secreting cell types used in research, ranging from primary human β-cells, to cell lines, to pluripotent stem cell-derived β-like cells. Each of these sources possess inherent strengths and weaknesses pertinent to specific applications, especially in the context of engineered platforms. We then outline how insulin-expressing cells have been used in engineered platforms and how recent advances allow for better mimicry of in vivo conditions. Chief among these conditions are β-cell interactions with other endocrine organs. This facet is beginning to be thoroughly addressed by the organ-on-a-chip community, but holds enormous potential in the development of novel diabetes therapeutics. Furthermore, high throughput strategies focused on studying β-cell biology, improving β-cell differentiation, or proliferation have led to enormous contributions in the field and will no doubt be instrumental in bringing new diabetes therapeutics to the clinic.
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Affiliation(s)
- Phillip L Lewis
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA
| | - James M Wells
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.,Division of Endocrinology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.,Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA
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28
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MacFarlane EM, Bruin JE. Human Pluripotent Stem Cells: A Unique Tool for Toxicity Testing in Pancreatic Progenitor and Endocrine Cells. Front Endocrinol (Lausanne) 2021; 11:604998. [PMID: 33542706 PMCID: PMC7851047 DOI: 10.3389/fendo.2020.604998] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/11/2020] [Accepted: 11/27/2020] [Indexed: 12/18/2022] Open
Abstract
Diabetes prevalence is increasing worldwide, and epidemiological studies report an association between diabetes incidence and environmental pollutant exposure. There are >84,000 chemicals in commerce, many of which are released into the environment without a clear understanding of potential adverse health consequences. While in vivo rodent studies remain an important tool for testing chemical toxicity systemically, we urgently need high-throughput screening platforms in biologically relevant models to efficiently prioritize chemicals for in depth toxicity analysis. Given the increasing global burden of obesity and diabetes, identifying chemicals that disrupt metabolism should be a high priority. Pancreatic endocrine cells are key regulators of systemic metabolism, yet often overlooked as a target tissue in toxicology studies. Immortalized β-cell lines and primary human, porcine, and rodent islets are widely used for studying the endocrine pancreas in vitro, but each have important limitations in terms of scalability, lifespan, and/or biological relevance. Human pluripotent stem cell (hPSC) culture is a powerful tool for in vitro toxicity testing that addresses many of the limitations with other β-cell models. Current in vitro differentiation protocols can efficiently generate glucose-responsive insulin-secreting β-like cells that are not fully mature, but still valuable for high-throughput toxicity screening in vitro. Furthermore, hPSCs can be applied as a model of developing pancreatic endocrine cells to screen for chemicals that influence endocrine cell formation during critical windows of differentiation. Given their versatility, we recommend using hPSCs to identify potential β-cell toxins, which can then be prioritized as chemicals of concern for metabolic disruption.
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Affiliation(s)
| | - Jennifer E. Bruin
- Department of Biology & Institute of Biochemistry, Carleton University, Ottawa, ON, Canada
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29
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Braverman-Gross C, Benvenisty N. Modeling Maturity Onset Diabetes of the Young in Pluripotent Stem Cells: Challenges and Achievements. Front Endocrinol (Lausanne) 2021; 12:622940. [PMID: 33692757 PMCID: PMC7937923 DOI: 10.3389/fendo.2021.622940] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/29/2020] [Accepted: 01/06/2021] [Indexed: 12/17/2022] Open
Abstract
Maturity onset diabetes of the young (MODY), is a group of monogenic diabetes disorders. Rodent models for MODY do not fully recapitulate the human phenotypes, calling for models generated in human cells. Human pluripotent stem cells (hPSCs), capable of differentiation towards pancreatic cells, possess a great opportunity to model MODY disorders in vitro. Here, we review the models for MODY diseases in hPSCs to date and the molecular lessons learnt from them. We also discuss the limitations and challenges that these types of models are still facing.
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30
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Akolpoglu MB, Inceoglu Y, Bozuyuk U, Sousa AR, Oliveira MB, Mano JF, Kizilel S. Recent advances in the design of implantable insulin secreting heterocellular islet organoids. Biomaterials 2020; 269:120627. [PMID: 33401104 DOI: 10.1016/j.biomaterials.2020.120627] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2020] [Revised: 12/14/2020] [Accepted: 12/18/2020] [Indexed: 12/11/2022]
Abstract
Islet transplantation has proved one of the most remarkable transmissions from an experimental curiosity into a routine clinical application for the treatment of type I diabetes (T1D). Current efforts for taking this technology one-step further are now focusing on overcoming islet donor shortage, engraftment, prolonged islet availability, post-transplant vascularization, and coming up with new strategies to eliminate lifelong immunosuppression. To this end, insulin secreting 3D cell clusters composed of different types of cells, also referred as heterocellular islet organoids, spheroids, or pseudoislets, have been engineered to overcome the challenges encountered by the current islet transplantation protocols. β-cells or native islets are accompanied by helper cells, also referred to as accessory cells, to generate a cell cluster that is not only able to accurately secrete insulin in response to glucose, but also superior in terms of other key features (e.g. maintaining a vasculature, longer durability in vivo and not necessitating immunosuppression after transplantation). Over the past decade, numerous 3D cell culture techniques have been integrated to create an engineered heterocellular islet organoid that addresses current obstacles. Here, we first discuss the different cell types used to prepare heterocellular organoids for islet transplantation and their contribution to the organoids design. We then introduce various cell culture techniques that are incorporated to prepare a fully functional and insulin secreting organoids with select features. Finally, we discuss the challenges and present a future outlook for improving clinical outcomes of islet transplantation.
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Affiliation(s)
- M Birgul Akolpoglu
- Chemical and Biological Engineering, Koc University, Sariyer, 34450, Istanbul, Turkey
| | - Yasemin Inceoglu
- Chemical and Biological Engineering, Koc University, Sariyer, 34450, Istanbul, Turkey
| | - Ugur Bozuyuk
- Chemical and Biological Engineering, Koc University, Sariyer, 34450, Istanbul, Turkey
| | - Ana Rita Sousa
- Department of Chemistry, CICECO - Aveiro Institute of Materials. University of Aveiro. Campus Universitário de Santiago. 3810-193 Aveiro. Portugal
| | - Mariana B Oliveira
- Department of Chemistry, CICECO - Aveiro Institute of Materials. University of Aveiro. Campus Universitário de Santiago. 3810-193 Aveiro. Portugal.
| | - João F Mano
- Department of Chemistry, CICECO - Aveiro Institute of Materials. University of Aveiro. Campus Universitário de Santiago. 3810-193 Aveiro. Portugal
| | - Seda Kizilel
- Chemical and Biological Engineering, Koc University, Sariyer, 34450, Istanbul, Turkey.
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31
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iPSCs-laden GDF8-grafted aldehyde hyaluronic acid-polyacrylamide inverted colloidal crystal constructs with controlled release of CHIR99021 and retinoic acid to generate insulin-producing cells. J Taiwan Inst Chem Eng 2020. [DOI: 10.1016/j.jtice.2020.11.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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32
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Hu M, Cherkaoui I, Misra S, Rutter GA. Functional Genomics in Pancreatic β Cells: Recent Advances in Gene Deletion and Genome Editing Technologies for Diabetes Research. Front Endocrinol (Lausanne) 2020; 11:576632. [PMID: 33162936 PMCID: PMC7580382 DOI: 10.3389/fendo.2020.576632] [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] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Accepted: 09/17/2020] [Indexed: 12/13/2022] Open
Abstract
The inheritance of variants that lead to coding changes in, or the mis-expression of, genes critical to pancreatic beta cell function can lead to alterations in insulin secretion and increase the risk of both type 1 and type 2 diabetes. Recently developed clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene editing tools provide a powerful means of understanding the impact of identified variants on cell function, growth, and survival and might ultimately provide a means, most likely after the transplantation of genetically "corrected" cells, of treating the disease. Here, we review some of the disease-associated genes and variants whose roles have been probed up to now. Next, we survey recent exciting developments in CRISPR/Cas9 technology and their possible exploitation for β cell functional genomics. Finally, we will provide a perspective as to how CRISPR/Cas9 technology may find clinical application in patients with diabetes.
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Affiliation(s)
- Ming Hu
- Section of Cell Biology and Functional Genomics, Faculty of Medicine, Imperial College London, London, United Kingdom
| | - Ines Cherkaoui
- Section of Cell Biology and Functional Genomics, Faculty of Medicine, Imperial College London, London, United Kingdom
| | - Shivani Misra
- Metabolic Medicine, Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, London, United Kingdom
| | - Guy A. Rutter
- Section of Cell Biology and Functional Genomics, Faculty of Medicine, Imperial College London, London, United Kingdom
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Panova AV, Klementieva NV, Sycheva AV, Goliusova DV, Khokhlov NV, Zubkova NA, Tiulpakov AN, Kiselev SL. Generation of an induced pluripotent stem cell line MNDINSi001-A from a patient with neonatal diabetes caused by a heterozygous INS mutation. Stem Cell Res 2020; 47:101929. [PMID: 32739878 DOI: 10.1016/j.scr.2020.101929] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/11/2020] [Revised: 07/01/2020] [Accepted: 07/20/2020] [Indexed: 02/07/2023] Open
Abstract
Insulin gene (INS) mutations prove to be the second most common cause of permanent neonatal diabetes. Here, we report the generation of iPSC line from a patient, heterozygous for the intronic INS mutation that presumably leads to aberrant splicing. Dermal fibroblasts were reprogrammed using non-integrating RNA-based vector. Derivation and expansion of iPSCs were performed under feeder-free culture conditions. The iPSC line expressed pluripotency markers, had normal karyotype, could differentiate into three germ layers in vitro and retained the disease mutation. This line can be a powerful tool for modeling of diabetes and cell replacement therapy as well.
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Affiliation(s)
- Alexandra V Panova
- Endocrinology Research Centre, Moscow, Russia; Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
| | | | | | - Daria V Goliusova
- Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
| | | | | | | | - Sergey L Kiselev
- Endocrinology Research Centre, Moscow, Russia; Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
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Gintant G, Traebert M. The roles of human induced pluripotent stem cell-derived cardiomyocytes in drug discovery: managing in vitro safety study expectations. Expert Opin Drug Discov 2020; 15:719-729. [PMID: 32129680 DOI: 10.1080/17460441.2020.1736549] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
INTRODUCTION Human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) preparations are increasingly employed in in vitro cardiac safety studies to support candidate drug selection and regulatory submissions. The value of hiPSC-CM-based approaches depends on their ability to recapitulate the cellular mechanisms responsible for cardiotoxicity as well as overall assay characteristics (thus defining model performance). Different expectations at different drug development stages define the utility of these human-derived models. AREAS COVERED Herein, the authors review the importance of understanding the functional characteristics of the evolving spectrum of simpler (2D) and more complex (co-cultures, 3D constructs, and engineered tissues) human-derived cardiac preparations, and how their performance may be evaluated based on analytical sensitivity, variability, and reproducibility in order to correctly match preparations with expectations of different safety assays. The need for consensus clinical examples of electrophysiologic, contractile, and structural cardiotoxicities essential for benchmarking human-derived models is also discussed. EXPERT OPINION It is helpful (but not essential) that hiPSC-CMs preparations fully recapitulate pharmacological responses of native adult human ventricular myocytes when evaluating cardiotoxicity in vitro. Further calibration and model standardization (aligning concordance with clinical findings) are necessary to understand the role of hiPSC-CMs in guiding cardiotoxicity assessments in early drug discovery efforts.
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Affiliation(s)
- Gary Gintant
- Department of Integrative Pharmacology (ZR13), AP-9A-LL, AbbVie Inc. , North Chicago, IL, USA
| | - Martin Traebert
- Novartis Institutes for Biomedical Research , Safety Pharmacology, Basel, Switzerland
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Mattis KK, Gloyn AL. From Genetic Association to Molecular Mechanisms for Islet-cell Dysfunction in Type 2 Diabetes. J Mol Biol 2020; 432:1551-1578. [PMID: 31945378 DOI: 10.1016/j.jmb.2019.12.045] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2019] [Revised: 12/15/2019] [Accepted: 12/17/2019] [Indexed: 12/30/2022]
Abstract
Genome-wide association studies (GWAS) have identified over 400 signals robustly associated with risk for type 2 diabetes (T2D). At the vast majority of these loci, the lead single nucleotide polymorphisms (SNPs) reside in noncoding regions of the genome, which hampers biological inference and translation of genetic discoveries into disease mechanisms. The study of these T2D risk variants in normoglycemic individuals has revealed that a significant proportion are exerting their disease risk through islet-cell dysfunction. The central role of the islet is also demonstrated by numerous studies, which have shown an enrichment of these signals in islet-specific epigenomic annotations. In recent years the emergence of authentic human beta-cell lines, and advances in genome-editing technologies coupled with improved protocols differentiating human pluripotent stem cells into beta-like cells has opened up new opportunities for T2D disease modeling. Here we review the current understanding on the genetic basis of T2D focusing on approaches, which have facilitated the identification of causal variants and their effector transcripts in human islets. We will present examples of functional studies based on animal and conventional cellular systems and highlight the potential of novel stem cell-based T2D disease models.
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Affiliation(s)
- Katia K Mattis
- Oxford Centre for Diabetes Endocrinology & Metabolism, University of Oxford, UK
| | - Anna L Gloyn
- Oxford Centre for Diabetes Endocrinology & Metabolism, University of Oxford, UK; Wellcome Trust Centre for Human Genetics, University of Oxford, UK; National Institute of Health Research, Biomedical Research Centre, Churchill Hospital, Headington, Oxford, UK.
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In Vivo and In Vitro Models of Diabetes: A Focus on Pregnancy. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2020; 1307:553-576. [PMID: 32504388 DOI: 10.1007/5584_2020_536] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Diabetes in pregnancy is associated with an increased risk of poor outcomes, both for the mother and her offspring. Although clinical and epidemiological studies are invaluable to assess these outcomes and the effectiveness of potential treatments, there are certain ethical and practical limitations to what can be assessed in human studies.Thus, both in vivo and in vitro models can aid us in the understanding of the mechanisms behind these complications and, in the long run, towards their prevention and treatment. This review summarizes the existing animal and cell models used to mimic diabetes, with a specific focus on the intrauterine environment. Summary of this review.
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Valenti MT, Serena M, Carbonare LD, Zipeto D. CRISPR/Cas system: An emerging technology in stem cell research. World J Stem Cells 2019; 11:937-956. [PMID: 31768221 PMCID: PMC6851009 DOI: 10.4252/wjsc.v11.i11.937] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/23/2019] [Revised: 08/12/2019] [Accepted: 09/11/2019] [Indexed: 02/06/2023] Open
Abstract
The identification of new and even more precise technologies for modifying and manipulating the genome has been a challenge since the discovery of the DNA double helix. The ability to modify selectively specific genes provides a powerful tool for characterizing gene functions, performing gene therapy, correcting specific genetic mutations, eradicating diseases, engineering cells and organisms to achieve new and different functions and obtaining transgenic animals as models for studying specific diseases. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has recently revolutionized genome engineering. The application of this new technology to stem cell research allows disease models to be developed to explore new therapeutic tools. The possibility of translating new systems of molecular knowledge to clinical research is particularly appealing for addressing degenerative diseases. In this review, we describe several applications of CRISPR/Cas9 to stem cells related to degenerative diseases. In addition, we address the challenges and future perspectives regarding the use of CRISPR/Cas9 as an important technology in the medical sciences.
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Affiliation(s)
- Maria Teresa Valenti
- Department of Medicine, Section of Internal Medicine D, University of Verona, Verona 37134, Italy.
| | - Michela Serena
- Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
| | - Luca Dalle Carbonare
- Department of Medicine, Section of Internal Medicine D, University of Verona, Verona 37134, Italy
| | - Donato Zipeto
- Department of Neurosciences, Biomedicine and Movement Sciences, Laboratory of Molecular Biology, Verona 37134, Italy
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Reprogrammed Cells Display Distinct Proteomic Signatures Associated with Colony Morphology Variability. Stem Cells Int 2019; 2019:8036035. [PMID: 31827534 PMCID: PMC6885794 DOI: 10.1155/2019/8036035] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2019] [Revised: 08/15/2019] [Accepted: 09/10/2019] [Indexed: 01/14/2023] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) are of high interest because they can be differentiated into a vast range of different cell types. Ideally, reprogrammed cells should sustain long-term culturing in an undifferentiated state. However, some reprogrammed cell lines represent an unstable state by spontaneously differentiating and changing their cellular phenotype and colony morphology. This phenomenon is not fully understood, and no method is available to predict it reliably. In this study, we analyzed and compared the proteome landscape of 20 reprogrammed cell lines classified as stable and unstable based on long-term colony morphology. We identified distinct proteomic signatures associated with stable colony morphology and with unstable colony morphology, although the typical pluripotency markers (POU5F1, SOX2) were present with both morphologies. Notably, epithelial to mesenchymal transition (EMT) protein markers were associated with unstable colony morphology, and the transforming growth factor beta (TGFB) signalling pathway was predicted as one of the main regulator pathways involved in this process. Furthermore, we identified specific proteins that separated the stable from the unstable state. Finally, we assessed both spontaneous embryonic body (EB) formation and directed differentiation and showed that reprogrammed lines with an unstable colony morphology had reduced differentiation capacity. To conclude, we found that different defined patterns of colony morphology in reprogrammed cells were associated with distinct proteomic profiles and different outcomes in differentiation capacity.
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Abstract
BACKGROUND Current therapeutic strategies for type 1 (T1DM) and type 2 diabetes mellitus (T2DM) rely on increasing or substituting endogenous insulin secretion in combination with lifestyle changes. β-cell regeneration, a process whereby new β-cells arise from progenitors, self-renewal or transdifferentiation, has the potential to become a viable route to insulin self-sufficiency. Current regeneration strategies capture many of the transcriptomic and protein features of native β-cells, generating cells capable of glucose-dependent insulin secretion in vitro and alleviation of hyperglycemia in vivo. However, whether novel β-cells display appreciable heterogeneity remains poorly understood, with potential consequences for long-term functional robustness. SCOPE OF REVIEW The review brings together crucial discoveries in the β-cell regeneration field with state-of-the-art knowledge regarding β-cell heterogeneity. Aspects that might aid production of longer-lasting and more plastic regenerated β-cells are highlighted and discussed. MAJOR CONCLUSIONS Different β-cell regeneration approaches result in a similar outcome: glucose-sensitive, insulin-positive cells that mimic the native β-cell phenotype but which lack normal plasticity. The β-cell subpopulations identified to date expand our understanding of β-cell survival, proliferation and function, signposting the direction for future regeneration strategies. Therefore, regenerated β-cells should exhibit stimulus-dependent differences in gene and protein expression, as well as establish a functional network with different β-cells, all while coexisting with other cell types on a three-dimensional platform.
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Affiliation(s)
- Daniela Nasteska
- Institute of Metabolism and Systems Research (IMSR), Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, Birmingham, UK; Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, UK
| | - Katrina Viloria
- Institute of Metabolism and Systems Research (IMSR), Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, Birmingham, UK; Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, UK
| | - Lewis Everett
- Institute of Metabolism and Systems Research (IMSR), Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, Birmingham, UK; Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, UK
| | - David J Hodson
- Institute of Metabolism and Systems Research (IMSR), Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, Birmingham, UK; Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, UK.
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Balboa D, Prasad RB, Groop L, Otonkoski T. Genome editing of human pancreatic beta cell models: problems, possibilities and outlook. Diabetologia 2019; 62:1329-1336. [PMID: 31161346 PMCID: PMC6647170 DOI: 10.1007/s00125-019-4908-z] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/18/2019] [Accepted: 04/25/2019] [Indexed: 12/14/2022]
Abstract
Understanding the molecular mechanisms behind beta cell dysfunction is essential for the development of effective and specific approaches for diabetes care and prevention. Physiological human beta cell models are needed for this work. We review the possibilities and limitations of currently available human beta cell models and how they can be dramatically enhanced using genome-editing technologies. In addition to the gold standard, primary isolated islets, other models now include immortalised human beta cell lines and pluripotent stem cell-derived islet-like cells. The scarcity of human primary islet samples limits their use, but valuable gene expression and functional data from large collections of human islets have been made available to the scientific community. The possibilities for studying beta cell physiology using immortalised human beta cell lines and stem cell-derived islets are rapidly evolving. However, the functional immaturity of these cells is still a significant limitation. CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) has enabled precise engineering of specific genetic variants, targeted transcriptional modulation and genome-wide genetic screening. These approaches can now be exploited to gain understanding of the mechanisms behind coding and non-coding diabetes-associated genetic variants, allowing more precise evaluation of their contribution to diabetes pathogenesis. Despite all the progress, genome editing in primary pancreatic islets remains difficult to achieve, an important limitation requiring further technological development.
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Affiliation(s)
- Diego Balboa
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, PO Box 63, (Haartmaninkatu 8), FI-00014, Helsinki, Finland
- Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain
| | - Rashmi B Prasad
- Genomics, Diabetes and Endocrinology, Lund University Diabetes Centre, CRC, Malmö, Sweden
| | - Leif Groop
- Genomics, Diabetes and Endocrinology, Lund University Diabetes Centre, CRC, Malmö, Sweden
- Institute of Molecular Medicine, Centre of Excellence in Complex Disease Genetics, University of Helsinki, Helsinki, Finland
| | - Timo Otonkoski
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, PO Box 63, (Haartmaninkatu 8), FI-00014, Helsinki, Finland.
- Children's Hospital, Helsinki University Hospital and University of Helsinki, Helsinki, Finland.
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Short-Term Protocols to Obtain Insulin-Producing Cells from Rat Adipose Tissue: Signaling Pathways and In Vivo Effect. Int J Mol Sci 2019; 20:ijms20102458. [PMID: 31109026 PMCID: PMC6566438 DOI: 10.3390/ijms20102458] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2019] [Revised: 05/09/2019] [Accepted: 05/13/2019] [Indexed: 02/06/2023] Open
Abstract
Studies using mesenchymal stromal cells (MSCs) as a source of insulin-secreting cells (IPCs) are a promising path in the pursuit for diabetes therapy. Here, we investigate three short-term differentiation protocols in order to generate IPCs from autologous adipose-derived stromal cells (ADSCs) with an expressive insulin-secreting profile in vitro and in vivo, as well as the signaling pathways involved in the chosen differentiation protocols. We extracted and cultured ADSCs and differentiated them into IPCs, using three different protocols with different inductors. Afterwards, the secretory profile was analyzed and IPCs differentiated in exendin-4/activin A medium, which presented the best secretory profile, was implanted in the kidney subcapsular region of diabetic rats. All protocols induced the differentiation, but media supplemented with exendin-4/activin A or resveratrol induced the expression and secretion of insulin more efficiently, and only the exendin-4/activin-A-supplemented medium generated an insulin secretion profile more like β-cells, in response to glucose. The PI3K/Akt pathway seems to play a negative role in IPC differentiation; however, the differentiation of ADSCs with exendin-4/activin A positively modulated the p38/MAPK pathway. Resveratrol medium activated the Jak/STAT3 pathway and generated IPCs apparently less sensitive to insulin and insulin-like receptors. Finally, the implant of IPCs with the best secretory behavior caused a decrease in hyperglycemia after one-week implantation in diabetic rats. Our data provide further information regarding the generation of IPCs from ADSCs and strengthen evidence to support the use of MSCs in regenerative medicine, specially the use of exendin-4/activin A to produce rapid and effectively IPCs with significant in vivo effects.
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Navarro-Tableros V, Gomez Y, Brizzi MF, Camussi G. Generation of Human Stem Cell-Derived Pancreatic Organoids (POs) for Regenerative Medicine. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1212:179-220. [PMID: 31025308 DOI: 10.1007/5584_2019_340] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Insulin-dependent diabetes mellitus or type 1 diabetes mellitus (T1DM) is an auto-immune condition characterized by the loss of pancreatic β-cells. The curative approach for highly selected patients is the pancreas or the pancreatic islet transplantation. Nevertheless, these options are limited by a growing shortage of donor organs and by the requirement of immunosuppression.Xenotransplantation of porcine islets has been extensively investigated. Nevertheless, the strong xenoimmunity and the risk of transmission of porcine endogenous retroviruses, have limited their application in clinic. Generation of β-like cells from stem cells is one of the most promising strategies in regenerative medicine. Embryonic, and more recently, adult stem cells are currently the most promising cell sources exploited to generate functional β-cells in vitro. A number of studies demonstrated that stem cells could generate functional pancreatic organoids (POs), able to restore normoglycemia when implanted in different preclinical diabetic models. Nevertheless, a gradual loss of function and cell dead are commonly detected when POs are transplanted in immunocompetent animals. So far, the main issue to be solved is the post-transplanted islet loss, due to the host immune attack. To avoid this hurdle, nanotechnology has provided a number of polymers currently under investigation for islet micro and macro-encapsulation. These new approaches, besides conferring PO immune protection, are able to supply oxygen and nutrients and to preserve PO morphology and long-term viability.Herein, we summarize the current knowledge on bioengineered POs and the stem cell differentiation platforms. We also discuss the in vitro strategies used to generate functional POs, and the protocols currently used to confer immune-protection against the host immune attack (micro- and macro-encapsulation). In addition, the most relevant ongoing clinical trials, and the most relevant hurdles met to move towards clinical application are revised.
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Affiliation(s)
- Victor Navarro-Tableros
- 2i3T Società per la gestione dell'incubatore di imprese e per il trasferimento tecnologico Scarl, University of Turin, Turin, Italy
| | - Yonathan Gomez
- Department of Medical Sciences, University of Turin, Turin, Italy
| | | | - Giovanni Camussi
- Department of Medical Sciences, University of Turin, Turin, Italy.
- Fondazione per la Ricerca Biomedica-ONLUS, Turin, Italy.
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