1
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Gilglioni EH, Bansal M, St-Pierre-Wijckmans W, Talamantes S, Kasarinaite A, Hay DC, Gurzov EN. Therapeutic potential of stem cell-derived somatic cells to treat metabolic dysfunction-associated steatotic liver disease and diabetes. Obes Rev 2025; 26:e13899. [PMID: 39861937 DOI: 10.1111/obr.13899] [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] [Received: 03/25/2024] [Revised: 10/22/2024] [Accepted: 12/04/2024] [Indexed: 01/27/2025]
Abstract
Developments in basic stem cell biology have paved the way for technology translation in human medicine. An exciting prospective use of stem cells is the ex vivo generation of hepatic and pancreatic endocrine cells for biomedical applications. This includes creating novel models 'in a dish' and developing therapeutic strategies for complex diseases, such as metabolic dysfunction-associated steatotic liver disease (MASLD) and diabetes. In this review, we explore recent advances in the generation of stem cell-derived hepatocyte-like cells and insulin-producing β-like cells. We cover the different differentiation strategies, new discoveries, and the caveats that still exist regarding their routine use. Finally, we discuss the challenges and limitations of stem cell-derived therapies as a clinical strategy to manage metabolic diseases in humans.
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Affiliation(s)
- Eduardo H Gilglioni
- Signal Transduction and Metabolism Laboratory, Université libre de Bruxelles, Brussels, Belgium
| | - Mayank Bansal
- Signal Transduction and Metabolism Laboratory, Université libre de Bruxelles, Brussels, Belgium
| | | | - Stephanie Talamantes
- Signal Transduction and Metabolism Laboratory, Université libre de Bruxelles, Brussels, Belgium
| | - Alvile Kasarinaite
- Institute for Regeneration and Repair, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK
| | - David C Hay
- Institute for Regeneration and Repair, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK
| | - Esteban N Gurzov
- Signal Transduction and Metabolism Laboratory, Université libre de Bruxelles, Brussels, Belgium
- WELBIO Department, WEL Research Institute, Wavre, Belgium
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2
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Li N, Wei R, Yuan Y, Deng M, Hu Y, Cheng CW, Yang J, Ho WI, Au KW, Tse YL, Li F, Wu X, Lau YM, Liao S, Ma S, Liu P, Ng KM, Esteban MA, Tse HF. Enhancement of hepatic differentiation from induced pluripotent stem cells by suppressing epithelial-mesenchymal transition. Hepatol Commun 2025; 9:e0702. [PMID: 40377485 PMCID: PMC12088630 DOI: 10.1097/hc9.0000000000000702] [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: 07/13/2024] [Accepted: 12/07/2024] [Indexed: 05/18/2025] Open
Abstract
BACKGROUND Induced pluripotent stem cells induced hepatocytes (iHeps) are widely used in modeling human liver diseases and as a potential cell source for replacement therapy. However, most iHeps are relatively immature and challenging to maintain for long-term in vitro culture. METHODS We optimized the differentiation protocol by addition of a combination of small molecules to inhibit epithelial-mesenchymal transition (EMT) in iHeps (iHeps EMTi), and further characterized their function both in vitro and in vivo analyses. RESULTS Inhibition of EMT extended the in vitro culture period of iHeps EMTi from day 24 to day 60. In vitro analysis revealed that, compared to control, iHeps EMTi exhibited significantly higher expression levels of hepatic functional markers and enhanced hepatocyte functions, including lipid accumulation, glycogen storage, albumin secretion, and urea acid metabolism. Moreover, the molecular profiles of iHeps EMTi are closer to those of primary human hepatocytes. In addition, the in vivo engraftment efficiency of iHeps EMTi in the chimeric mice model was also improved as compared to iHeps alone. CONCLUSIONS We established a robust protocol to generate human iHeps with improved function and capable of long-term in vitro culturing via the suppression of EMT. Moreover, those iHeps with EMT suppression have improved engraftment in human chimeric mice.
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Affiliation(s)
- Na Li
- Department of Medicine, The Cardiology Division, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
- Cardiac and Vascular Center, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China
| | - Rui Wei
- Department of Gastroenterology and Hepatology, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, China
| | - Yangyang Yuan
- Centre for Stem Cell Translational Biology, The University of Hong Kong, Hong Kong SAR, China
- School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
| | - Mingdan Deng
- Centre for Stem Cell Translational Biology, The University of Hong Kong, Hong Kong SAR, China
- School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
| | - Yang Hu
- Department of Medicine, The Cardiology Division, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
| | - Chi-Wa Cheng
- Department of Medicine, The Cardiology Division, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
| | - Jiayin Yang
- Cell Inspire Therapeutics Co., Ltd and Cell Inspire Biotechnology Co., Ltd, Shenzhen, China
- Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
| | - Wai-In Ho
- Department of Medicine, The Cardiology Division, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
| | - Ka-Wing Au
- Department of Medicine, The Cardiology Division, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
| | - Yiu-Lam Tse
- Department of Medicine, The Cardiology Division, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
| | - Fei Li
- Department of Medicine, The Cardiology Division, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
| | - Xinyi Wu
- Department of Medicine, The Cardiology Division, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
| | - Yee-Man Lau
- Department of Medicine, The Cardiology Division, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
| | - Songyan Liao
- Department of Medicine, The Cardiology Division, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
- Centre for Stem Cell Translational Biology, The University of Hong Kong, Hong Kong SAR, China
| | - Stephanie Ma
- Centre for Stem Cell Translational Biology, The University of Hong Kong, Hong Kong SAR, China
- School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
| | - Pentao Liu
- Centre for Stem Cell Translational Biology, The University of Hong Kong, Hong Kong SAR, China
- School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
| | - Kwong-Man Ng
- Department of Medicine, The Cardiology Division, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
- Centre for Stem Cell Translational Biology, The University of Hong Kong, Hong Kong SAR, China
| | - Miguel A. Esteban
- Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
- Cardiac and Vascular Center, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China
- Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
- Joint School of Life Sciences, Guangzhou Medical University and Guangzhou Institutes of Biomedicine and Health, Guangzhou, China
- 3DC STAR, Spatiotemporal Campus at BGI Shenzhen, Shenzhen, China
| | - Hung-Fat Tse
- Department of Medicine, The Cardiology Division, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
- Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
- Cardiac and Vascular Center, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China
- Centre for Stem Cell Translational Biology, The University of Hong Kong, Hong Kong SAR, China
- Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
- Advanced Biomedical Instrumentation Centre, Hong Kong Science Park, New Territories, Hong Kong
- Centre for Regenerative Medicine and Health, Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, Hong Kong SAR, China
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3
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Shimizu T, Miyoshi M, Kakinuma S, Tsuchiya J, Yamane D, Watakabe K, Mochida T, Inada K, Yamada K, Shinozaki K, Sato A, Kaneko S, Kawai-Kitahata F, Murakawa M, Nitta S, Nakagawa M, Watanabe M, Asahina Y, Okamoto R. Bile acid-FXR signaling facilitates the long-term maintenance of hepatic characteristics in human iPSC-derived organoids. Cell Rep 2025:115675. [PMID: 40367952 DOI: 10.1016/j.celrep.2025.115675] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2023] [Revised: 12/28/2024] [Accepted: 04/16/2025] [Indexed: 05/16/2025] Open
Abstract
Human induced pluripotent stem cells (iPSCs) can be differentiated into hepatocyte-like cells (iPS-Heps); however, maintaining the long-term proliferation and hepatic characteristics of iPS-Heps remains a challenge. In this study, we aimed to develop a human iPSC-derived hepatic organoid (iHO) culture system that effectively retains hepatic characteristics long term. Our original culture strategy, using bile acids and their receptor (farnesoid X receptor [FXR]) agonists, yielded human iHOs capable of long-term culture with a distinctive "grape-like" structure. Comprehensive analysis showed that these iHOs maintained hepatocyte-like phenotypes, even after multiple passages, whose gene expression profiles were consistent with those of fetal hepatocytes. In addition, the overexpression of small heterodimer partner (SHP), a downstream gene of FXR, in iHOs negatively regulated genes related to the intestine and cholangiocytes. Our data demonstrated that bile acid-FXR signaling promotes both the hepatic characteristics and proliferative potential of iHOs, offering promising potential for future applications in regenerative medicine and as a disease model.
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Affiliation(s)
- Taro Shimizu
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
| | - Masato Miyoshi
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
| | - Sei Kakinuma
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan; Department of Clinical and Diagnostic Laboratory Science, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan.
| | - Jun Tsuchiya
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
| | - Daisuke Yamane
- Department of Diseases and Infection, Tokyo Metropolitan Institute of Medical Science, Tokyo 1568506, Japan
| | - Keiya Watakabe
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
| | - Tomohiro Mochida
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
| | - Kento Inada
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
| | - Kaho Yamada
- Department of Clinical and Diagnostic Laboratory Science, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
| | - Kotomi Shinozaki
- Department of Diseases and Infection, Tokyo Metropolitan Institute of Medical Science, Tokyo 1568506, Japan
| | - Ayako Sato
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
| | - Shun Kaneko
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
| | - Fukiko Kawai-Kitahata
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
| | - Miyako Murakawa
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
| | - Sayuri Nitta
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
| | - Mina Nakagawa
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
| | - Mamoru Watanabe
- School of Medicine, Juntendo University, Tokyo 1138421, Japan
| | - Yasuhiro Asahina
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan; Division of Hepatic Medical Science, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan.
| | - Ryuichi Okamoto
- Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Science, Institute of Science Tokyo (Science Tokyo), Tokyo 1138519, Japan
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4
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Yang S, Wang L, Gao R, Li Y, Zhang D, Wang C, Liu G, Na J, Xu P, Wang X, Jia Y, Huang Y. UFMylation safeguards human hepatocyte differentiation and liver homeostasis by regulating ribosome dissociation. Cell Rep 2025; 44:115686. [PMID: 40347470 DOI: 10.1016/j.celrep.2025.115686] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2024] [Revised: 02/10/2025] [Accepted: 04/18/2025] [Indexed: 05/14/2025] Open
Abstract
Ribosomal UFMylation contributes to ribosome heterogeneity and is associated with ribosome-associated quality control at the endoplasmic reticulum. However, the specific pathophysiological functions of ribosomal UFMylation remain unknown. In this study, we systematically demonstrate the significance of UFMylation in the differentiation and maturation of hepatocytes using human embryonic stem cell-derived hepatocyte-like cells and liver bud organoids as experimental platforms. We also develop a strategy to identify UFMylated substrates and confirm that RPL26 is a substrate in the liver. Additionally, we discover that mice with the Rpl26 c.395A>G (p.K132R) mutation are more susceptible to steatosis induced by a high-fat diet. Further investigations reveal a key role of CDK5RAP3 and RPL26 UFMylation in regulating ribosome dissociation. Our findings suggest that ribosome UFMylation serves as an important safeguard for liver development and homeostasis and may represent a potential therapeutic target for nonalcoholic fatty liver disease.
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Affiliation(s)
- Shuchun Yang
- State Key Laboratory of Common Mechanism Research for Major Disease, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China; Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China
| | - Li Wang
- State Key Laboratory of Common Mechanism Research for Major Disease, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China; Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China
| | - Ran Gao
- State Key Laboratory of Common Mechanism Research for Major Disease, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China
| | - Yanchang Li
- State Key Laboratory of Medical Proteomics, Beijng Proteome Research Center, National Center for Protein Sciences (Beijing), Research Unit of Proteomics & Research and Development of New Drugs of Chinese Academy of Medical Sciences, Institute of Lifeomics, Beijing 102206, China
| | - Duo Zhang
- State Key Laboratory of Common Mechanism Research for Major Disease, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China; Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China
| | - Chenxi Wang
- State Key Laboratory of Common Mechanism Research for Major Disease, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China; Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China
| | - Guang Liu
- State Key Laboratory of Common Mechanism Research for Major Disease, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China; Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China
| | - Jie Na
- Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, China
| | - Ping Xu
- State Key Laboratory of Medical Proteomics, Beijng Proteome Research Center, National Center for Protein Sciences (Beijing), Research Unit of Proteomics & Research and Development of New Drugs of Chinese Academy of Medical Sciences, Institute of Lifeomics, Beijing 102206, China
| | - Xiaoyue Wang
- State Key Laboratory of Common Mechanism Research for Major Disease, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China
| | - Yuyan Jia
- State Key Laboratory of Common Mechanism Research for Major Disease, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China; Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China.
| | - Yue Huang
- State Key Laboratory of Common Mechanism Research for Major Disease, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China; Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China.
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5
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Martin L, Maric D, Idriss S, Delamare M, Le Roy A, Maaziz N, Caillaud A, Si-Tayeb K, Robriquet F, Lenglet M, Erceau L, Bellanné-Chantelot C, Plo I, Aral B, Garrec C, Airaud F, Gianfermi C, Antunes V, Keppner A, Vincent SM, Desfontaine A, Modé N, Laporte F, Gaignerie A, Chariau C, Leray I, Rogue C, David L, Redon R, Bézieau S, Mansour-Hendili L, Galactéros F, Maillet T, Pasquet M, Cougoul P, Nloga AM, Gardin C, Guitton C, Dubruille V, Giacobbi-Milet V, Leblanc T, Kaya Z, Semama D, James C, Carillo S, Ochmann M, Waage A, Mortier E, Maillasson M, Quéméner A, Cario H, Skoda RC, Zermati Y, Hoogewijs D, Marchand A, Girodon F, Gardie B. Identification of Hepatic-like EPO as a Cause of Polycythemia. N Engl J Med 2025; 392:1684-1697. [PMID: 40305710 DOI: 10.1056/nejmoa2414954] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 05/02/2025]
Abstract
BACKGROUND Secondary erythrocytosis often results from conditions that cause tissue hypoxia or an improper increase in erythropoietin (EPO) production. EPO, the major regulator of erythropoiesis, has a complex and tightly regulated expression during development, with a liver-to-kidney switch shortly after birth. METHODS We identified six families with erythrocytosis that was associated with circulating EPO levels within the normal range and characterized as a novel molecular and functional entity. We investigated the effect of the identified pathogenic variants using EPO promoter-driven luciferase reporter genes. Induced pluripotent stem cells (iPSCs) were generated from patient cells and differentiated into hepatocyte-like EPO-producing cells. Samples of circulating EPO from patients with hereditary erythrocytosis and from healthy newborns were analyzed by means of isoelectric focusing, and EPO activity was assessed. RESULTS Three novel variants were identified in the noncoding regions of EPO. Experiments with reporter assays and iPSC-derived hepatocyte-like cells showed that the variants targeted previously uncharacterized regulatory elements of the gene, which, when the variants were present, showed high responsiveness to hypoxia. EPO samples from all the patients showed a modified isoelectric-focusing profile, identical to hepatic EPO that is expressed in premature neonates and in patients with acquired erythrocytosis associated with liver diseases. EPO that was purified from patient plasma and umbilical-cord blood samples showed enhanced EPO receptor signaling activity in vitro, which suggests a potential gain of function linked to the liver-type glycosylation of EPO. CONCLUSIONS We found that secondary erythrocytosis can be related to variants in EPO that lead to the production of hepatic-like EPO with an atypical glycosylation pattern and increased activity. (Funded by Région des Pays de la Loire and others; ClinicalTrials.gov number, NCT03957863.).
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Affiliation(s)
- Laurent Martin
- Laboratoire Antidopage Français, Université Paris-Saclay, Orsay, France
| | - Darko Maric
- Section of Medicine, Department of Endocrinology, Metabolism, and Cardiovascular System, University of Fribourg, Fribourg, Switzerland
- National Center of Competence in Research Kidney Control of Homeostasis (Kidney.CH), Fribourg, Switzerland
| | - Salam Idriss
- École Pratique des Hautes Études, Paris Sciences et Lettres Université, Paris
- Nantes Université, Centre Hospitalier Universitaire (CHU) Nantes, Centre National de la Recherche Scientifique (CNRS), INSERM, l'Institut du Thorax, Nantes, France
| | - Marine Delamare
- École Pratique des Hautes Études, Paris Sciences et Lettres Université, Paris
- Nantes Université, Centre Hospitalier Universitaire (CHU) Nantes, Centre National de la Recherche Scientifique (CNRS), INSERM, l'Institut du Thorax, Nantes, France
| | - Amandine Le Roy
- École Pratique des Hautes Études, Paris Sciences et Lettres Université, Paris
- Nantes Université, Centre Hospitalier Universitaire (CHU) Nantes, Centre National de la Recherche Scientifique (CNRS), INSERM, l'Institut du Thorax, Nantes, France
| | - Nada Maaziz
- Service d'Hématologie Biologique, Pôle Biologie, CHU de Dijon, Dijon, France
- Filière de Santé des Maladies Constitutionnelles Rares du Globule Rouge et de l'Erythropoïèse, Créteil, France
| | - Amandine Caillaud
- Nantes Université, Centre Hospitalier Universitaire (CHU) Nantes, Centre National de la Recherche Scientifique (CNRS), INSERM, l'Institut du Thorax, Nantes, France
| | - Karim Si-Tayeb
- Nantes Université, Centre Hospitalier Universitaire (CHU) Nantes, Centre National de la Recherche Scientifique (CNRS), INSERM, l'Institut du Thorax, Nantes, France
| | - Florence Robriquet
- École Pratique des Hautes Études, Paris Sciences et Lettres Université, Paris
- Nantes Université, Centre Hospitalier Universitaire (CHU) Nantes, Centre National de la Recherche Scientifique (CNRS), INSERM, l'Institut du Thorax, Nantes, France
| | - Marion Lenglet
- École Pratique des Hautes Études, Paris Sciences et Lettres Université, Paris
- Nantes Université, Centre Hospitalier Universitaire (CHU) Nantes, Centre National de la Recherche Scientifique (CNRS), INSERM, l'Institut du Thorax, Nantes, France
| | - Lucie Erceau
- École Pratique des Hautes Études, Paris Sciences et Lettres Université, Paris
- Nantes Université, Centre Hospitalier Universitaire (CHU) Nantes, Centre National de la Recherche Scientifique (CNRS), INSERM, l'Institut du Thorax, Nantes, France
| | - Christine Bellanné-Chantelot
- INSERM Unité Mixte de Recherche 1287, Villejuif, France
- Gustave Roussy, Villejuif, France
- Département de Génétique Médicale, Assistance Publique-Hôpitaux de Paris (AP-HP), Sorbonne Université, Hôpital Pitié-Salpêtrière, Paris
| | - Isabelle Plo
- INSERM Unité Mixte de Recherche 1287, Villejuif, France
- Gustave Roussy, Villejuif, France
- Paris-Saclay University, INSERM Unité 1287, Villejuif, France
- France Intergroup des Syndromes Myéloprolifératifs, Paris
| | - Bernard Aral
- Service d'Hématologie Biologique, Pôle Biologie, CHU de Dijon, Dijon, France
| | - Céline Garrec
- Service de Génétique Médicale, CHU de Nantes, Nantes, France
| | - Fabrice Airaud
- Service de Génétique Médicale, CHU de Nantes, Nantes, France
| | - Clara Gianfermi
- Laboratoire Antidopage Français, Université Paris-Saclay, Orsay, France
| | - Vincent Antunes
- Section of Medicine, Department of Endocrinology, Metabolism, and Cardiovascular System, University of Fribourg, Fribourg, Switzerland
- National Center of Competence in Research Kidney Control of Homeostasis (Kidney.CH), Fribourg, Switzerland
| | - Anna Keppner
- Section of Medicine, Department of Endocrinology, Metabolism, and Cardiovascular System, University of Fribourg, Fribourg, Switzerland
- National Center of Competence in Research Kidney Control of Homeostasis (Kidney.CH), Fribourg, Switzerland
| | - Sarah Mathilda Vincent
- Section of Medicine, Department of Endocrinology, Metabolism, and Cardiovascular System, University of Fribourg, Fribourg, Switzerland
- National Center of Competence in Research Kidney Control of Homeostasis (Kidney.CH), Fribourg, Switzerland
| | | | - Nina Modé
- Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine, Paris
| | - Fabien Laporte
- Nantes Université, Centre Hospitalier Universitaire (CHU) Nantes, Centre National de la Recherche Scientifique (CNRS), INSERM, l'Institut du Thorax, Nantes, France
| | - Anne Gaignerie
- Nantes Université, CHU Nantes, CNRS, INSERM, BioCore, Nantes, France
| | - Caroline Chariau
- Nantes Université, CHU Nantes, CNRS, INSERM, BioCore, Nantes, France
| | - Isabelle Leray
- Nantes Université, CHU Nantes, CNRS, INSERM, BioCore, Nantes, France
| | - Coline Rogue
- Nantes Université, CHU Nantes, CNRS, INSERM, BioCore, Nantes, France
| | - Laurent David
- Nantes Université, CHU Nantes, CNRS, INSERM, BioCore, Nantes, France
- Nantes Université, CHU Nantes, INSERM, Center for Research in Transplantation and Translational Immunology, Nantes, France
| | - Richard Redon
- Nantes Université, Centre Hospitalier Universitaire (CHU) Nantes, Centre National de la Recherche Scientifique (CNRS), INSERM, l'Institut du Thorax, Nantes, France
| | - Stéphane Bézieau
- Nantes Université, Centre Hospitalier Universitaire (CHU) Nantes, Centre National de la Recherche Scientifique (CNRS), INSERM, l'Institut du Thorax, Nantes, France
- Service de Génétique Médicale, CHU de Nantes, Nantes, France
| | - Lamisse Mansour-Hendili
- Département de Biochimie-Biologie Moléculaire, Pharmacologie, Génétique Médicale, and Red Cell Disease Referral Center, Unité des Maladies Génétiques du Globule Rouge, Université Paris-Est Créteil, AP-HP, Hôpitaux Universitaires Henri Mondor, Creteil, France
- Université Paris-Est Créteil, Mondor Institute for Biomedical Research Equipe Pirenne, Laboratoire d'Excellence GR-Ex, Creteil, France
| | - Frédéric Galactéros
- Filière de Santé des Maladies Constitutionnelles Rares du Globule Rouge et de l'Erythropoïèse, Créteil, France
- Département de Biochimie-Biologie Moléculaire, Pharmacologie, Génétique Médicale, and Red Cell Disease Referral Center, Unité des Maladies Génétiques du Globule Rouge, Université Paris-Est Créteil, AP-HP, Hôpitaux Universitaires Henri Mondor, Creteil, France
| | - Thibault Maillet
- Service de Médecine Interne, Centre Hospitalier de Mâcon, Mâcon, France
| | - Marlène Pasquet
- Department of Pediatric Hematology and Immunology Child Hospital, University Hospital of Toulouse, Toulouse, France
- Centre de Recherche en Cancérologie de Toulouse, Team Impact des Altérations Génétiques sur le Développement des Leucémies Aiguës, INSERM Unité 1037, Institut Universitaire du Cancer de Toulouse-Oncopole, Toulouse, France
| | - Pierre Cougoul
- Filière de Santé des Maladies Constitutionnelles Rares du Globule Rouge et de l'Erythropoïèse, Créteil, France
- Internal Medicine Department, Toulouse University Hospital, Toulouse, France
| | - Anne-Marie Nloga
- Hématologie Clinique, Hôpital Avicenne, AP-HP, Hôpitaux Universitaires Paris Seine Saint-Denis, Bobigny, France
- Département d'Immunologie-Hématologie, Université de Paris and Sorbonne Paris Nord, Villetaneuse, France
| | - Claude Gardin
- Hématologie Clinique, Hôpital Avicenne, AP-HP, Hôpitaux Universitaires Paris Seine Saint-Denis, Bobigny, France
- Département d'Immunologie-Hématologie, Université de Paris and Sorbonne Paris Nord, Villetaneuse, France
| | - Corinne Guitton
- Service de Pédiatrie, Hématologie Bénigne, Hôpital Bicêtre, Université Paris-Saclay, Le Kremlin-Bicêtre, France
| | | | | | - Thierry Leblanc
- Immuno-hématologie Pédiatrique, Hôpital Robert-Debré, Université Paris-Cité, Paris
| | - Zuhre Kaya
- Department of Pediatric Hematology, Gazi University Faculty of Medicine, Ankara, Turkey
| | - Denis Semama
- Réanimation Néonatale, Service de Pédiatrie, CHU Dijon, France
| | - Chloé James
- France Intergroup des Syndromes Myéloprolifératifs, Paris
- Laboratoire d'Hématologie, CHU de Bordeaux, Bordeaux, France
| | - Serge Carillo
- Laboratoire de Cytologie Clinique et Cytogénétique, Laboratoires de Biologie Médicale de Référence, Néoplasies Myéloprolifératives, CHU Caremeau, Nimes, France
| | | | - Anders Waage
- Institute of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway
- Research Department, St. Olav's Hospital, Trondheim, Norway
| | - Erwan Mortier
- Nantes Université, CHU Nantes, CNRS, INSERM, BioCore, Nantes, France
- Nantes Université, CNRS, INSERM, Centre de Recherche en Cancérologie et Immunologie Intégrée Nantes Angers, Nantes, France
- Laboratoire d'Excellence Immunotherapy-Graft-Oncology, Immuno-Onco-Greffe, Nantes, France
| | - Mike Maillasson
- Nantes Université, CHU Nantes, CNRS, INSERM, BioCore, Nantes, France
- Nantes Université, CNRS, INSERM, Centre de Recherche en Cancérologie et Immunologie Intégrée Nantes Angers, Nantes, France
- Laboratoire d'Excellence Immunotherapy-Graft-Oncology, Immuno-Onco-Greffe, Nantes, France
| | - Agnès Quéméner
- Nantes Université, CNRS, INSERM, Centre de Recherche en Cancérologie et Immunologie Intégrée Nantes Angers, Nantes, France
- Laboratoire d'Excellence Immunotherapy-Graft-Oncology, Immuno-Onco-Greffe, Nantes, France
| | - Holger Cario
- Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany
- Center for Rare Hematopoietic Disorders and Immunodeficiencies, Ulm University Medical Center, Ulm, Germany
- German Center for Child and Adolescent Health partner site Ulm, Ulm, Germany
| | - Radek C Skoda
- Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston
| | - Yaël Zermati
- Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine, Paris
- Laboratoire d'Excellence GR-Ex, Paris
| | - David Hoogewijs
- Section of Medicine, Department of Endocrinology, Metabolism, and Cardiovascular System, University of Fribourg, Fribourg, Switzerland
- National Center of Competence in Research Kidney Control of Homeostasis (Kidney.CH), Fribourg, Switzerland
| | | | - François Girodon
- Service d'Hématologie Biologique, Pôle Biologie, CHU de Dijon, Dijon, France
- Filière de Santé des Maladies Constitutionnelles Rares du Globule Rouge et de l'Erythropoïèse, Créteil, France
- France Intergroup des Syndromes Myéloprolifératifs, Paris
- Laboratoire d'Excellence GR-Ex, Paris
- INSERM Unité 1231, Université de Bourgogne, Dijon, France
| | - Betty Gardie
- École Pratique des Hautes Études, Paris Sciences et Lettres Université, Paris
- Nantes Université, Centre Hospitalier Universitaire (CHU) Nantes, Centre National de la Recherche Scientifique (CNRS), INSERM, l'Institut du Thorax, Nantes, France
- Filière de Santé des Maladies Constitutionnelles Rares du Globule Rouge et de l'Erythropoïèse, Créteil, France
- Laboratoire d'Excellence GR-Ex, Paris
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6
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Luo S, Wu F, Jin Y, Liu D. The Potential Hepatocyte Differentiation Targets and MSC Proliferation by FH1. J Cell Mol Med 2025; 29:e70601. [PMID: 40346964 PMCID: PMC12064995 DOI: 10.1111/jcmm.70601] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2024] [Revised: 03/24/2025] [Accepted: 04/30/2025] [Indexed: 05/12/2025] Open
Abstract
The main cause of acute liver failure (ALF) is hepatocellular necrosis, which induces liver repair dysfunction and leads to high mortality. In recent years, studies have increasingly shown that stem cell-derived hepatocyte-like cells (HLCs) can be used for treatment in animal models of ALF. Notably, a hepatocyte differentiation strategy based on the small-molecule compound functional hit 1 (FH1) successfully replaces HGF to promote the maturation of HLCs, but the underlying mechanism is still unclear. In this study, we used network pharmacology analysis to clarify the important role of the HGF/c-Met signalling pathway in FH1-induced hepatocyte (FH1-iHeps) differentiation. After FH1 was added to mesenchymal stem/stromal cells (MSCs), proliferation and cell cycle progression were rescued by treatment with a tyrosine kinase (c-Met) inhibitor. Additionally, c-Met signalling in MSCs was significantly increased by treatment with FH1, as shown by the increased c-Met, p-p38, p-AKT and p-ERK1/2 protein levels. FH1-iHeps efficiently improved the liver function of mice with acute liver injury and prolonged their lifespan. These data provide new insight into the mechanisms regulating the stemness properties of human umbilical cord-derived stem cells (hUC-MSCs) and reveal a previously unrecognised link between FH1 and c-Met in directing hepatocyte differentiation.
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Affiliation(s)
- Sang Luo
- Department of Beijing National Biochip Research Center Sub‐Center in Ningxia, Institute of Medical SciencesGeneral Hospital of Ningxia Medical UniversityYinchuanChina
| | - Fang Wu
- Ningxia Regional Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Regional High Incidence DiseaseYinchuanChina
| | - Yiran Jin
- Department of Beijing National Biochip Research Center Sub‐Center in Ningxia, Institute of Medical SciencesGeneral Hospital of Ningxia Medical UniversityYinchuanChina
| | - Dan Liu
- Department of Beijing National Biochip Research Center Sub‐Center in Ningxia, Institute of Medical SciencesGeneral Hospital of Ningxia Medical UniversityYinchuanChina
- Key Laboratory of Ministry of Education for Fertility Preservation and MaintenanceNingxia Medical UniversityYinchuanChina
- Department of GynecologyGeneral Hospital of Ningxia Medical UniversityYinchuanChina
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7
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Panday R, Rogy KM, Han YD, Khetani SR. Engineered microtissues to model the effects of dynamic heterotypic cell signaling on iPSC-derived human hepatocyte maturation. Acta Biomater 2025; 197:135-151. [PMID: 40089127 DOI: 10.1016/j.actbio.2025.03.020] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2024] [Revised: 01/21/2025] [Accepted: 03/12/2025] [Indexed: 03/17/2025]
Abstract
In vitro human liver models are indispensable for compound metabolism/toxicity screening, disease modeling, and regenerative medicine. While induced pluripotent stem cell-derived human hepatocyte-like cells (iHeps) mitigate the sourcing limitations with primary human hepatocytes (PHHs), their functional maturity is rate-limiting for application use. During development, immature hepatoblasts interact with different non-parenchymal cell (NPC) types, such as mesenchyme and endothelia, in a spatiotemporal manner to progress through functional maturation. Modeling such interactions in vitro is critical to elucidate the key regulators of iHep maturation. Here, we utilized high-throughput droplet microfluidics to encapsulate iHeps within monodisperse collagen I microgels (Ø ∼ 250 µm), which were coated with NPCs to generate 'microtissues' placed within microwells in multiwell plates. Embryonic fibroblasts and liver sinusoidal endothelial cells (LSECs) induced the highest level of iHep maturation over 4+ weeks of culture compared to adult hepatic stellate cells (myofibroblastic), liver portal fibroblasts, dermal fibroblasts, and human umbilical vein endothelial cells. Combining iHep microtissues in plates with Transwell inserts containing different NPC types enabled the modeling of dynamic heterotypic signaling on iHep maturation; introducing embryonic fibroblast signaling first, followed by LSECs, led to the highest iHep maturation. Unique cytokine secretion profiles were detected across the top-performing microtissue configurations; stromal-derived factor-1 alpha was validated as one factor that enhanced iHep maturation. Lastly, gene expression patterns and regulatory networks showed adult PHH-like maturation in LSEC/iHep microtissues compared to iHep-only microtissues. Overall, microtissues are useful for elucidating the microenvironmental determinants of iHep maturation and for future use in downstream applications. STATEMENT OF SIGNIFICANCE: Induced pluripotent stem cell-derived hepatocyte-like cells (iHeps) hold great promise for drug screening, disease modeling, and regenerative medicine but often exhibit immature phenotypes. We utilized high-throughput droplet microfluidics to generate 3D microtissues containing iHeps and non-parenchymal cell (NPC) types to elucidate the effects of dynamic NPC signaling on iHep maturation. We observed that iHep maturation is significantly enhanced with embryonic fibroblasts and liver sinusoidal endothelial cells (LSEC) compared to adult liver fibroblasts and non-liver endothelia; the LSEC/iHep microtissues showed adult liver-like gene expression signatures. The highest iHep maturation in microtissues was achieved when mesenchymal stimulation was introduced first, followed by LSEC stimulation. Our platform provides a robust framework to elucidate cellular and molecular mediators of iHep maturation and biomedical applications.
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Affiliation(s)
- Regeant Panday
- Department of Biomedical Engineering, University of Illinois Chicago, 851 S Morgan St, 218 SEO, Chicago, IL 60607, USA
| | - Kerry M Rogy
- Department of Biomedical Engineering, University of Illinois Chicago, 851 S Morgan St, 218 SEO, Chicago, IL 60607, USA
| | - Yong Duk Han
- Department of Biomedical Engineering, University of Illinois Chicago, 851 S Morgan St, 218 SEO, Chicago, IL 60607, USA
| | - Salman R Khetani
- Department of Biomedical Engineering, University of Illinois Chicago, 851 S Morgan St, 218 SEO, Chicago, IL 60607, USA.
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8
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Novotny LA, Kappler CS, Meissner EG. Function of Interferon Lambda Receptor 1 Variants in Stem Cell-Derived Hepatocytes with Abrogated Endogenous IFNLR1. J Interferon Cytokine Res 2025; 45:174-183. [PMID: 39929255 DOI: 10.1089/jir.2024.0262] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/02/2025] Open
Abstract
Distinct transcriptional isoforms of the interferon lambda receptor 1 (IFNLR1) are expressed in hepatocytes, but whether corresponding full-length and truncated IFNLR1 protein variants have discrete function is unclear. We quantitated IFNLR1 isoforms in liver and blood from individuals with chronic hepatitis C virus (HCV) infection before and after antiviral treatment, hypothesizing their relative expression may differentially change during resolution of virus-induced inflammation. We also expressed FLAG-tagged IFNLR1 variants in stem cell-derived hepatocytes (iHeps) with abrogated endogenous IFNLR1 to evaluate their function. IFNLR1 isoforms decreased in liver and blood during treatment of HCV, but no distinct pattern of decline was observed for any individual isoform. Expression of full-length IFNLR1 enabled lambda interferon (IFNL)-induced expression of antiviral and proinflammatory genes and augmented inhibition of hepatitis B virus (HBV) replication relative to wild-type (WT) iHeps. A noncanonical IFNLR1 variant missing part of the JAK1 binding domain enabled IFNLs to induce antiviral genes but could not support induction of proinflammatory genes or augmented HBV inhibition beyond that observed in WT iHeps with intact endogenous IFNLR1. A secreted IFNLR1 variant had no identified function in iHeps lacking endogenous IFNLR1. Although relative expression of individual IFNLR1 isoforms did not distinctly change during HCV treatment, functional studies in iHeps suggest IFNLR1 variants could function to titrate antiviral versus proinflammatory responses in hepatocytes in the context of viral hepatitis.
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Affiliation(s)
- Laura A Novotny
- Division of Infectious Diseases, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Christiana S Kappler
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Eric G Meissner
- Division of Infectious Diseases, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina, USA
- Department of Pharmacology and Immunology, Medical University of South Carolina, Charleston, South Carolina, USA
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9
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Ramandi A, Diehl AM, Sanyal AJ, de Jong YP. Experimental Models to Investigate PNPLA3 in Liver Steatosis. Liver Int 2025; 45:e70091. [PMID: 40231787 DOI: 10.1111/liv.70091] [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] [Received: 02/14/2025] [Revised: 03/26/2025] [Accepted: 03/30/2025] [Indexed: 04/16/2025]
Abstract
Patatin-like phospholipase domain-containing 3 (PNPLA3) was the first gene identified through genome-wide association studies to be linked to hepatic fat accumulation. A missense variant, encoding the PNPLA3-148M allele, has since been shown to increase the risk for the full spectrum of steatotic liver disease (SLD), from simple steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Despite extensive validation of this association and ongoing research into its pathogenic role, the precise mechanisms by which PNPLA3-148M contributes to the progression of SLD remain poorly understood. In this review, we evaluate preclinical in vitro and in vivo models used to investigate PNPLA3 and its involvement in SLD, with particular emphasis on metabolic dysfunction-associated steatotic liver disease. We assess the strengths and limitations of these models, as well as the challenges arising from species differences in PNPLA3 expression and function between human and murine systems.
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Affiliation(s)
- Alireza Ramandi
- Division of Gastroenterology and Hepatology, Weill Cornell Medicine, New York, New York, USA
| | - Anna-Mae Diehl
- Department of Medicine, Duke University, Durham, North Carolina, USA
| | - Arun J Sanyal
- Stravitz-Sanyal Institute for Liver Disease and Metabolic Health, Virginia Commonwealth University, Richmond, Virginia, USA
| | - Ype P de Jong
- Division of Gastroenterology and Hepatology, Weill Cornell Medicine, New York, New York, USA
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10
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Lim JH, Kim DH, Lee J, Jung CR, Kang HM. Transdifferentiation of Integrin Beta 1 High+ Skin Progenitor Cells Into Functional Hepatocytes. Stem Cells Int 2025; 2025:8953305. [PMID: 40313860 PMCID: PMC12043391 DOI: 10.1155/sci/8953305] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2024] [Accepted: 03/11/2025] [Indexed: 05/03/2025] Open
Abstract
A highly reproducible and functional liver model that closely resembles the human liver plays a crucial role in drug development, disease research, personalized medicine, and regenerative medicine. This study aimed to establish an in vitro liver model using skin epidermal progenitor cells (EPCs), which are easily accessible and exhibit a high proliferative capacity. Skin EPCs with high integrin beta 1 expression demonstrated multipotent differentiation potential, capable of differentiating into adipocyte- and neuron-like cells in vitro. Furthermore, when exposed to high concentrations of activin A, along with Wnt3a and BMP4, these cells efficiently differentiated into definitive endoderm, exhibiting high FOXA2 expression. Under our culture conditions, they further differentiated into functional hepatocytes. These differentiated cells exhibited high albumin secretion, CYP activity, and drug metabolism capabilities similar to those observed in vivo. In conclusion, this study highlights the potential of EPCs to differentiate into functional hepatocytes, providing a feasible and scalable source of hepatocytes for drug screening, liver disease modeling, and potential cell-based therapies.
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Affiliation(s)
- Jung Hwa Lim
- Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea
- Korea University of Science and Technology (UST), Daejeon, Republic of Korea
| | - Dae Hun Kim
- Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea
- Korea University of Science and Technology (UST), Daejeon, Republic of Korea
| | - Junhee Lee
- Korea University of Science and Technology (UST), Daejeon, Republic of Korea
- Department of Bionic Machinery, Research Institute of AI Robotics, Korea Institute of Machinery and Materials (KIMM), Daejeon 34103, Republic of Korea
| | - Cho-Rok Jung
- Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea
- Korea University of Science and Technology (UST), Daejeon, Republic of Korea
| | - Hyun Mi Kang
- Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea
- Korea University of Science and Technology (UST), Daejeon, Republic of Korea
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11
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Fukunaga I, Takebe T. In vitro liver models for toxicological research. Drug Metab Pharmacokinet 2025; 62:101478. [PMID: 40203632 DOI: 10.1016/j.dmpk.2025.101478] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2024] [Revised: 02/25/2025] [Accepted: 03/04/2025] [Indexed: 04/11/2025]
Abstract
Drug-induced liver injury (DILI) presents a major challenge not only in new drug development but also in post-marketing withdrawals and the safety of food, cosmetics, and chemicals. Experimental model organisms such as the rodents have been widely used for preclinical toxicological testing. However, the tension exists associated with the ethical and sustainable use of animals in part because animals do not necessarily inform the human-specific ADME (adsorption, dynamics, metabolism and elimination) profiling. To establish alternative models in humans, in vitro hepatic tissue models have been proposed, ranging from primary hepatocytes, immortal hepatocytes, to the development of new cell resources such as stem cell-derived hepatocytes. Given the evolving number of novel alternative methods, understanding possible combinations of cell sources and culture methods will be crucial to develop the context-of-use assays. This review primarily focuses on 3D liver organoid models for conducting. We will review the relevant cell sources, bioengineering methods, selection of training compounds, and biomarkers towards the rationale design of in vitro toxicology testing.
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Affiliation(s)
- Ichiro Fukunaga
- Center for Genomic and Regenerative Medicine, Juntendo University Graduate School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo, 113-8431, Japan.
| | - Takanori Takebe
- Human Biology Research Unit, Institute of Integrated Research, Institute of Science Tokyo, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan; Department of Genome Biology, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan; Divisions of Gastroenterology, Hepatology & Nutrition, Developmental Biology and Biomedical Informatics, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH, 45229-3039, USA; Department of Pediatrics, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH, 45229-3039, USA; Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH, 45229-3039, USA; Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Suita, Osaka, 565-0871, Japan
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12
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Kim H, Park HJ. Current hPSC-derived liver organoids for toxicity testing: Cytochrome P450 enzymes and drug metabolism. Toxicol Res 2025; 41:105-121. [PMID: 40013078 PMCID: PMC11850699 DOI: 10.1007/s43188-024-00275-8] [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: 10/13/2024] [Revised: 12/04/2024] [Accepted: 12/11/2024] [Indexed: 02/28/2025] Open
Abstract
Drug-induced hepatotoxicity is the leading cause of attrition of drug candidates and withdrawal of marketed drugs owing to safety concerns. In most hepatotoxicity cases, the parent drugs are metabolized by cytochrome P450 (CYP) enzymes, generating reactive metabolites that bind to intracellular organelles and proteins, ultimately causing hepatocellular damage. A major limitation of animal models, which are widely used for toxicity assessment, is the discrepancy in CYP-mediated drug metabolism and toxicological outcomes owing to species differences between humans and animals. Two-dimensional (2D) hepatocytes were first developed as a promising alternative model using human pluripotent stem cells (hPSCs). However, their CYP expression was similar to that of the fetal liver, and they lacked CYP-mediated hepatic metabolism. CYP expression in hPSC-derived hepatic models is closely correlated with liver maturity. Therefore, liver organoids that are more mature than hPSC-derived hepatic models and mimic the structure and physiological functions of the human liver have emerged as new alternatives. In this review, we explored the role and essentiality of CYPs in human hepatotoxicity, their expression, and epigenetic regulation in hPSC-derived hepatocytes and liver organoids, as well as the current state of liver organoid technology in terms of CYP expression and activity, drug metabolism, and toxicity. We also discussed the current challenges and future directions for the practical use of liver organoids. In conclusion, we highlight the importance of methods and metrics for accurately assessing CYP expression and activity in liver organoids to enable the development of feasible models that reproduce hepatotoxicity in humans.
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Affiliation(s)
- Hyemin Kim
- Division of Advanced Predictive Research, Korea Institute of Toxicology, Daejeon, Republic of Korea
| | - Han-Jin Park
- Division of Advanced Predictive Research, Korea Institute of Toxicology, Daejeon, Republic of Korea
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13
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Wang H, Danoy M, Gong Y, Utami T, Arakawa H, Kato Y, Nishikawa M, Sakai Y, Leclerc E. Palmitic Acid Induced a Dedifferentiation Profile at the Transcriptome Level: A Collagen Synthesis but no Triglyceride Accumulation in Hepatocyte-Like Cells Derived From Human-Induced Pluripotent Stem Cells Cultivated Inside Organ on a Chip. J Appl Toxicol 2025; 45:460-471. [PMID: 39506029 DOI: 10.1002/jat.4714] [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: 08/09/2024] [Revised: 09/24/2024] [Accepted: 10/01/2024] [Indexed: 11/08/2024]
Abstract
Nonalcoholic fatty liver disease (NAFLD) is one of the main causes of critical liver diseases leading to steatosis, steatohepatitis, fibrosis, and ultimately to liver cirrhosis and hepatic carcinoma. In this study, the effect of palmitic acid (PA), one of the most abundant dietary fatty acids, was investigated using an organ-on-a-chip (OoC) technology on hepatocyte-like cells derived from human-induced pluripotent stem cells (hiPSCs). After 1 week of hepatic maturation, followed by 1 week of exposure, the transcriptomic analysis showed lower liver transcription factor activity. It also revealed that 318 genes were differentially expressed between the control and 0.5-mM PA conditions. The 0.5-mM PA conditions were characterized by the downregulation of hepatic markers (liver transcription factors, phase I and phase II metabolism genes) of lipidic genes (metabolism and transport). In parallel, the 0.5-mM PA treatment upregulated several extracellular matrix genes (such as collagen genes). The physiopathological staining demonstrated no lipid accumulation in our model and confirmed the secretion of collagen in the 0.5-mM PA conditions. However, the production of albumin, the metabolic biotransformation by the cytochrome P450 enzymes, and the biliary acid concentrations were not altered by the PA treatments. Overall, our data illustrated the response to PA characterized by an early stage of dedifferentiation observed at the transcriptomic levels associated with a modification of the collagenic profile but without lipid accumulation. We believe that our model provides new insight of the onset of palmitic lipotoxicity in the early stage of NAFLD.
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Affiliation(s)
- Hanyuan Wang
- Department of Chemical System Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
- CNRS/IIS IRL 2820; Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, Tokyo, Japan
| | - Mathieu Danoy
- Department of Chemical System Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
| | - Ya Gong
- Department of Chemical System Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
| | - Tia Utami
- Department of Chemical System Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
| | - Hiroshi Arakawa
- Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
| | - Yukio Kato
- Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
| | - Masaki Nishikawa
- Department of Chemical System Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
| | - Yasuyuki Sakai
- Department of Chemical System Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
- CNRS/IIS IRL 2820; Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, Tokyo, Japan
| | - Eric Leclerc
- CNRS/IIS IRL 2820; Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, Tokyo, Japan
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14
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Prescott JB, Liu KJ, Lander A, Pek NMQ, Jha SK, Bokelmann M, Begur M, Koh PW, Yang H, Lim B, Red-Horse K, Weissman IL, Loh KM, Ang LT. Metabolically purified human stem cell-derived hepatocytes reveal distinct effects of Ebola and Lassa viruses. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.02.17.638665. [PMID: 40027809 PMCID: PMC11870522 DOI: 10.1101/2025.02.17.638665] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/05/2025]
Abstract
Ebola and Lassa viruses require biosafety-level-4 (BSL4) containment, infect the liver, and cause deadly hemorrhagic fevers. The cellular effects of these viruses, and whether different families of hemorrhagic-fever viruses elicit similar effects, remain fundamental questions in BSL4 virology. Here, we introduce a new metabolic selection approach to create nearly-pure hepatocytes from human pluripotent stem cells, killing non-liver cells by withholding essential nutrients. Unexpectedly, Ebola and Lassa exerted starkly different effects on human hepatocytes. Ebola infection activated the integrated stress response (ISR) and WNT pathways in hepatocytes in vitro and killed them, whereas Lassa did not. Within non-human primates, Ebola likewise infected hepatocytes and activated ISR signaling in vivo . In summary, we present a single-cell transcriptional and chromatin accessibility roadmap of human hepatocyte differentiation, purification, and viral infection.
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15
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Lekkala VKR, Shrestha S, Al Qaryoute A, Dhinoja S, Acharya P, Raheem A, Jagadeeswaran P, Lee MY. Enhanced Maturity and Functionality of Vascular Human Liver Organoids through 3D Bioprinting and Pillar Plate Culture. ACS Biomater Sci Eng 2025; 11:506-517. [PMID: 39726370 DOI: 10.1021/acsbiomaterials.4c01658] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2024]
Abstract
Liver tissues, composed of hepatocytes, cholangiocytes, stellate cells, Kupffer cells, and sinusoidal endothelial cells, are differentiated from endodermal and mesodermal germ layers. By mimicking the developmental process of the liver, various differentiation protocols have been published to generate human liver organoids (HLOs) in vitro using induced pluripotent stem cells (iPSCs). However, HLOs derived solely from the endodermal germ layer often encounter technical hurdles such as insufficient maturity and functionality, limiting their utility for disease modeling and hepatotoxicity assays. To overcome this, we separately differentiated EpCAM+ endodermal progenitor cells (EPCs) and mesoderm-derived vascular progenitor cells (VPCs) from the same human iPSC line. These cells were then mixed in a BME-2 matrix and concurrently differentiated into vascular human liver organoids (vHLOs). Remarkably, vHLOs exhibited a significantly higher maturity than vasculature-free HLOs, as demonstrated by increased coagulation factor secretion, albumin secretion, drug-metabolizing enzyme expression, and bile acid transportation. To enhance assay throughput and miniaturize vHLO culture, we 3D bioprinted expandable HLOs (eHLOs) in a BME-2 matrix on a pillar plate platform derived from EPCs and VPCs and compared them with HLOs derived from endoderm alone. Compared to HLOs cultured in a 50 μL BME-2 matrix dome in a 24-well plate, vHLOs cultured on the pillar plate exhibited superior maturity, likely due to enhanced nutrient and signaling molecule diffusion. The integration of physiologically relevant patterned liver organoids with the unique pillar plate platform enhanced the capabilities for high-throughput screening and disease modeling.
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Affiliation(s)
- Vinod Kumar Reddy Lekkala
- Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207-7102, United States
| | - Sunil Shrestha
- Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207-7102, United States
| | - Ayah Al Qaryoute
- Department of Biological Sciences, University of North Texas, Denton, Texas 76203-5017, United States
| | - Sanchi Dhinoja
- Department of Biological Sciences, University of North Texas, Denton, Texas 76203-5017, United States
| | - Prabha Acharya
- Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207-7102, United States
| | - Abida Raheem
- Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207-7102, United States
| | - Pudur Jagadeeswaran
- Department of Biological Sciences, University of North Texas, Denton, Texas 76203-5017, United States
| | - Moo-Yeal Lee
- Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207-7102, United States
- Bioprinting Laboratories Inc., Dallas, Texas 75234-7244, United States
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16
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Zhang L, Deng Y, Bai X, Wei X, Ren Y, Chen S, Deng H. Cell therapy for end-stage liver disease: Current state and clinical challenge. Chin Med J (Engl) 2024; 137:2808-2820. [PMID: 39602326 DOI: 10.1097/cm9.0000000000003332] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2024] [Indexed: 11/29/2024] Open
Abstract
ABSTRACT Liver disease involves a complex interplay of pathological processes, including inflammation, hepatocyte necrosis, and fibrosis. End-stage liver disease (ESLD), such as liver failure and decompensated cirrhosis, has a high mortality rate, and liver transplantation is the only effective treatment. However, to overcome problems such as the shortage of donor livers and complications related to immunosuppression, there is an urgent need for new treatment strategies that need to be developed for patients with ESLD. For instance, hepatocytes derived from donor livers or stem cells can be engrafted and multiplied in the liver, substituting the host hepatocytes and rebuilding the liver parenchyma. Stem cell therapy, especially mesenchymal stem cell therapy, has been widely proved to restore liver function and alleviate liver injury in patients with severe liver disease, which has contributed to the clinical application of cell therapy. In this review, we discussed the types of cells used to treat ESLD and their therapeutic mechanisms. We also summarized the progress of clinical trials around the world and provided a perspective on cell therapy.
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Affiliation(s)
- Lin Zhang
- Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
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17
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Matsuo-Takasaki M, Kambayashi S, Hemmi Y, Wakabayashi T, Shimizu T, An Y, Ito H, Takeuchi K, Ibuki M, Kawashima T, Masayasu R, Suzuki M, Kawai Y, Umekage M, Kato TM, Noguchi M, Nakade K, Nakamura Y, Nakaishi T, Nishishita N, Tsukahara M, Hayashi Y. Complete suspension culture of human induced pluripotent stem cells supplemented with suppressors of spontaneous differentiation. eLife 2024; 12:RP89724. [PMID: 39529479 PMCID: PMC11556790 DOI: 10.7554/elife.89724] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2024] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. Adding PKCβ and Wnt signaling pathway inhibitors in the suspension conditions suppressed the spontaneous differentiation of hiPSCs into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed the culture processes of hiPSCs, including the generation of hiPSCs from peripheral blood mononuclear cells with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single-cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our results demonstrate that precise control of the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.
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Affiliation(s)
- Mami Matsuo-Takasaki
- iPS Cell Advanced Characterization and Development Team, RIKEN BioResource Research CenterIbarakiJapan
| | - Sho Kambayashi
- Regenerative Medicine and Cell Therapy Laboratories, KANEKA CORPORATIONKobeJapan
| | - Yasuko Hemmi
- iPS Cell Advanced Characterization and Development Team, RIKEN BioResource Research CenterIbarakiJapan
| | - Tamami Wakabayashi
- iPS Cell Advanced Characterization and Development Team, RIKEN BioResource Research CenterIbarakiJapan
| | - Tomoya Shimizu
- iPS Cell Advanced Characterization and Development Team, RIKEN BioResource Research CenterIbarakiJapan
| | - Yuri An
- iPS Cell Advanced Characterization and Development Team, RIKEN BioResource Research CenterIbarakiJapan
| | - Hidenori Ito
- iPS Cell Advanced Characterization and Development Team, RIKEN BioResource Research CenterIbarakiJapan
| | - Kazuhiro Takeuchi
- Regenerative Medicine and Cell Therapy Laboratories, KANEKA CORPORATIONKobeJapan
| | - Masato Ibuki
- Regenerative Medicine and Cell Therapy Laboratories, KANEKA CORPORATIONKobeJapan
| | - Terasu Kawashima
- Regenerative Medicine and Cell Therapy Laboratories, KANEKA CORPORATIONKobeJapan
| | - Rio Masayasu
- Regenerative Medicine and Cell Therapy Laboratories, KANEKA CORPORATIONKobeJapan
| | - Manami Suzuki
- Regenerative Medicine and Cell Therapy Laboratories, KANEKA CORPORATIONKobeJapan
| | - Yoshikazu Kawai
- Regenerative Medicine and Cell Therapy Laboratories, KANEKA CORPORATIONKobeJapan
| | | | - Tomoaki M Kato
- Research and Development Center, CiRA FoundationKyotoJapan
| | - Michiya Noguchi
- Cell Engineering Division, RIKEN BioResource Research CenterIbarakiJapan
| | - Koji Nakade
- Gene Engineering Division, RIKEN BioResource Research CenterIbarakiJapan
| | - Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource Research CenterIbarakiJapan
| | - Tomoyuki Nakaishi
- Regenerative Medicine and Cell Therapy Laboratories, KANEKA CORPORATIONKobeJapan
| | - Naoki Nishishita
- Regenerative Medicine and Cell Therapy Laboratories, KANEKA CORPORATIONKobeJapan
| | | | - Yohei Hayashi
- iPS Cell Advanced Characterization and Development Team, RIKEN BioResource Research CenterIbarakiJapan
- Faculty of Medicine and School of Integrative and Global Majors, University of TsukubaIbarakiJapan
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18
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de Jong YP. Mice Engrafted with Human Liver Cells. Semin Liver Dis 2024; 44:405-415. [PMID: 39265638 PMCID: PMC11620938 DOI: 10.1055/s-0044-1790601] [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: 09/14/2024]
Abstract
Rodents are commonly employed to model human liver conditions, although species differences can restrict their translational relevance. To overcome some of these limitations, researchers have long pursued human hepatocyte transplantation into rodents. More than 20 years ago, the first primary human hepatocyte transplantations into immunodeficient mice with liver injury were able to support hepatitis B and C virus infections, as these viruses cannot replicate in murine hepatocytes. Since then, hepatocyte chimeric mouse models have transitioned into mainstream preclinical research and are now employed in a diverse array of liver conditions beyond viral hepatitis, including malaria, drug metabolism, liver-targeting gene therapy, metabolic dysfunction-associated steatotic liver disease, lipoprotein and bile acid biology, and others. Concurrently, endeavors to cotransplant other cell types and humanize immune and other nonparenchymal compartments have seen growing success. Looking ahead, several challenges remain. These include enhancing immune functionality in mice doubly humanized with hepatocytes and immune systems, efficiently creating mice with genetically altered grafts and reliably humanizing chimeric mice with renewable cell sources such as patient-specific induced pluripotent stem cells. In conclusion, hepatocyte chimeric mice have evolved into vital preclinical models that address many limitations of traditional rodent models. Continued improvements may further expand their applications.
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Affiliation(s)
- Ype P de Jong
- Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medicine, New York, New York
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York
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19
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Nijiati N, Wubuli D, Li X, Zhou Z, Julaiti M, Huang P, Hu B. The Construction of Stem Cell-Induced Hepatocyte Model and Its Application in Evaluation of Developmental Hepatotoxicity of Environmental Pollutants. Stem Cells Dev 2024; 33:575-585. [PMID: 39109950 DOI: 10.1089/scd.2024.0117] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/24/2024] Open
Abstract
Stem cells, with their ability to self-renew and differentiate into various cell types, are a unique and valuable resource for medical research and toxicological studies. The liver is the most crucial metabolic organ in the human body and serves as the primary site for the accumulation of environmental pollutants. Enrichment with environmental pollutants can disrupt the early developmental processes of the liver and have a significant impact on liver function. The liver comprises a complex array of cell types, and different environmental pollutants have varying effects on these cells. Currently, there is a lack of well-established research models that can effectively demonstrate the mechanisms by which environmental pollutants affect human liver development. The emergence of liver cells and organoids derived from stem cells offers a promising tool for investigating the impact of environmental pollutants on human health. Therefore, this study systematically reviewed the developmental processes of different types of liver cells and provided an overview of studies on the developmental toxicity of various environmental pollutants using stem cell models.
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Affiliation(s)
- Nadire Nijiati
- State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Xinjiang Key Laboratory of Molecular Biology for Endemic Diseases, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi, China
| | - Dilixiati Wubuli
- Department of Physiology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi, China
| | - Xiaobing Li
- The Third Clinical Medicine College of Xinjiang Medical University, Urumqi, China
| | - Zidong Zhou
- The Third Clinical Medicine College of Xinjiang Medical University, Urumqi, China
| | - Mulati Julaiti
- State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Xinjiang Key Laboratory of Molecular Biology for Endemic Diseases, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi, China
| | - Pengfei Huang
- State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Xinjiang Key Laboratory of Molecular Biology for Endemic Diseases, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi, China
| | - Bowen Hu
- State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Xinjiang Key Laboratory of Molecular Biology for Endemic Diseases, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi, China
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20
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Vicente P, Almeida JI, Crespo IE, Virgolini N, Isidro IA, Calleja-Cervantes ME, Rodriguez-Madoz JR, Prosper F, Alves PM, Serra M. Oxygen control in bioreactor drives high yield production of functional hiPSC-like hepatocytes for advanced liver disease modelling. Sci Rep 2024; 14:24599. [PMID: 39427033 PMCID: PMC11490613 DOI: 10.1038/s41598-024-75582-z] [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: 05/15/2024] [Accepted: 10/07/2024] [Indexed: 10/21/2024] Open
Abstract
Hepatocytes-like cells (HLC) derived from human induced pluripotent stem cells show great promise for cell-based liver therapies and disease modelling. However, their application is currently hindered by the low production yields of existing protocols. We aim to develop a bioprocess able to generate high numbers of HLC. We used stirred-tank bioreactors with a rational control of dissolved oxygen concentration (DO) for the optimization of HLC production as 3D aggregates. We evaluated the impact of controlling DO at physiological levels (4%O2) during hepatic progenitors' stage on cell proliferation and differentiation efficiency. Whole transcriptome analysis and biochemical assays were performed to provide a detailed characterization of HLC quality attributes. When DO was controlled at 4%O2 during the hepatic progenitors' stage, cells presented an upregulation of genes associated with hypoxia-inducible factor pathway and a downregulation of oxidative stress genes. This condition promoted higher HLC production (maximum cell concentration: 2 × 106 cell/mL) and improved differentiation efficiencies (80% Albumin-positive cells) when compared to the bioreactor operated under atmospheric oxygen levels (21%O2, 0.6 × 106 cell/mL, 43% Albumin positive cells). These HLC exhibited functional characteristics of hepatocytes: capacity to metabolize drugs, ability to synthesize hepatic metabolites, and inducible cytochrome P450 activity. Bioprocess robustness was confirmed with HLC derived from different donors, including a primary hyperoxaluria type 1 (PH1) patient. The generated PH1.HLC showed metabolic features of PH1 disease with higher secretion of oxalate compared with HLC generated from healthy individuals. This work reports a reproducible bioprocess, that shows the importance of controlling DO at physiological levels to increase HLC production, and the HLC capability to display PH1 disease features.
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Affiliation(s)
- Pedro Vicente
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, 2780-901, Portugal
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade de Nova de Lisboa, Av. da República, Oeiras, 2780-157, Portugal
| | - Joana I Almeida
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, 2780-901, Portugal
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade de Nova de Lisboa, Av. da República, Oeiras, 2780-157, Portugal
- Instituto de Investigación Sanitaria Aragón (IIS Aragón), Zaragoza, Spain
| | - Inês E Crespo
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, 2780-901, Portugal
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade de Nova de Lisboa, Av. da República, Oeiras, 2780-157, Portugal
| | - Nikolaus Virgolini
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, 2780-901, Portugal
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade de Nova de Lisboa, Av. da República, Oeiras, 2780-157, Portugal
| | - Inês A Isidro
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, 2780-901, Portugal
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade de Nova de Lisboa, Av. da República, Oeiras, 2780-157, Portugal
| | | | - Juan R Rodriguez-Madoz
- Hemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
- Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain
| | - Felipe Prosper
- Hemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, Pamplona, Spain
- Hematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA, Pamplona, Spain
- Cancer Center Clinica Universidad de Navarra (CCUN), Pamplona, Spain
- Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain
| | - Paula M Alves
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, 2780-901, Portugal
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade de Nova de Lisboa, Av. da República, Oeiras, 2780-157, Portugal
| | - Margarida Serra
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, 2780-901, Portugal.
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade de Nova de Lisboa, Av. da República, Oeiras, 2780-157, Portugal.
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21
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Gordeev MN, Zinovyeva AS, Petrenko EE, Lomert EV, Aksenov ND, Tomilin AN, Bakhmet EI. Embryonic Stem Cell Differentiation to Definitive Endoderm As a Model of Heterogeneity Onset During Germ Layer Specification. Acta Naturae 2024; 16:62-72. [PMID: 39877013 PMCID: PMC11771848 DOI: 10.32607/actanaturae.27510] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2024] [Accepted: 10/23/2024] [Indexed: 01/31/2025] Open
Abstract
Embryonic stem cells (ESCs) hold great promise for regenerative medicine thanks to their ability to self-renew and differentiate into somatic cells and the germline. ESCs correspond to pluripotent epiblast - the tissue from which the following three germ layers originate during embryonic gastrulation: the ectoderm, mesoderm, and endoderm. Importantly, ESCs can be induced to differentiate toward various cell types by varying culture conditions, which can be exploited for in vitro modeling of developmental processes such as gastrulation. The classical model of gastrulation postulates that mesoderm and endoderm specification is made possible through the FGF-, BMP-, Wnt-, and Nodal-signaling gradients. Hence, it can be expected that one of these signals should direct ESC differentiation towards specific germ layers. However, ESC specification appears to be more complicated, and the same signal can be interpreted differently depending on the readout. In this research, using chemically defined culture conditions, homogeneous naïve ESCs as a starting cell population, and the Foxa2 gene-driven EGFP reporter tool, we established a robust model of definitive endoderm (DE) specification. This in vitro model features formative pluripotency as an intermediate state acquired by the epiblast in vivo shortly after implantation. Despite the initially homogeneous state of the cells in the model and high Activin concentration during endodermal specification, there remains a cell subpopulation that does not reach the endodermal state. This simple model developed by us can be used to study the origins of cellular heterogeneity during germ layer specification.
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Affiliation(s)
- M. N. Gordeev
- Pluripotency Dynamics Group, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064 Russian Federation
- Laboratory of the Molecular Biology of Stem Cells, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064 Russian Federation
- Institute of Evolution, University of Haifa, Haifa, 3498838 Israel
| | - A. S. Zinovyeva
- Pluripotency Dynamics Group, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064 Russian Federation
- Laboratory of the Molecular Biology of Stem Cells, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064 Russian Federation
| | - E. E. Petrenko
- Pluripotency Dynamics Group, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064 Russian Federation
- Laboratory of the Molecular Biology of Stem Cells, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064 Russian Federation
- Faculty of Biology, Technion – Israel Institute of Technology, Haifa, 3200003 Israel
| | - E. V. Lomert
- Laboratory of Molecular Medicine, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064 Russian Federation
| | - N. D. Aksenov
- Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064 Russian Federation
| | - A. N. Tomilin
- Laboratory of the Molecular Biology of Stem Cells, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064 Russian Federation
| | - E. I. Bakhmet
- Pluripotency Dynamics Group, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064 Russian Federation
- Laboratory of the Molecular Biology of Stem Cells, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064 Russian Federation
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22
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Wang L, Koui Y, Kanegae K, Kido T, Tamura-Nakano M, Yabe S, Tai K, Nakajima Y, Kusuhara H, Sakai Y, Miyajima A, Okochi H, Tanaka M. Establishment of human induced pluripotent stem cell-derived hepatobiliary organoid with bile duct for pharmaceutical research use. Biomaterials 2024; 310:122621. [PMID: 38815455 DOI: 10.1016/j.biomaterials.2024.122621] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2024] [Revised: 04/26/2024] [Accepted: 05/19/2024] [Indexed: 06/01/2024]
Abstract
In vitro models of the human liver are promising alternatives to animal tests for drug development. Currently, primary human hepatocytes (PHHs) are preferred for pharmacokinetic and cytotoxicity tests. However, they are unable to recapitulate the flow of bile in hepatobiliary clearance owing to the lack of bile ducts, leading to the limitation of bile analysis. To address the issue, a liver organoid culture system that has a functional bile duct network is desired. In this study, we aimed to generate human iPSC-derived hepatobiliary organoids (hHBOs) consisting of hepatocytes and bile ducts. The two-step differentiation process under 2D and semi-3D culture conditions promoted the maturation of hHBOs on culture plates, in which hepatocyte clusters were covered with monolayered biliary tubes. We demonstrated that the hHBOs reproduced the flow of bile containing a fluorescent bile acid analog or medicinal drugs from hepatocytes into bile ducts via bile canaliculi. Furthermore, the hHBOs exhibited pathophysiological responses to troglitazone, such as cholestasis and cytotoxicity. Because the hHBOs can recapitulate the function of bile ducts in hepatobiliary clearance, they are suitable as a liver disease model and would be a novel in vitro platform system for pharmaceutical research use.
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Affiliation(s)
- Luyao Wang
- Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan; Laboratory of Stem Cell Regulation, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan
| | - Yuta Koui
- Laboratory of Cell Growth and Differentiation, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan
| | - Kazuko Kanegae
- Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
| | - Taketomo Kido
- Laboratory of Cell Growth and Differentiation, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan
| | - Miwa Tamura-Nakano
- Communal Laboratory, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
| | - Shigeharu Yabe
- Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
| | - Kenpei Tai
- Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
| | - Yoshiko Nakajima
- Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
| | - Hiroyuki Kusuhara
- Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
| | - Yasuyuki Sakai
- Department of Chemical System Engineering, School of Engineering, The University of Tokyo, Tokyo, Japan
| | - Atsushi Miyajima
- Laboratory of Cell Growth and Differentiation, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan
| | - Hitoshi Okochi
- Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
| | - Minoru Tanaka
- Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan; Laboratory of Stem Cell Regulation, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan.
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23
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Chi H, Qu B, Prawira A, Richardt T, Maurer L, Hu J, Fu RM, Lempp FA, Zhang Z, Grimm D, Wu X, Urban S, Dao Thi VL. An hepatitis B and D virus infection model using human pluripotent stem cell-derived hepatocytes. EMBO Rep 2024; 25:4311-4336. [PMID: 39232200 PMCID: PMC11466959 DOI: 10.1038/s44319-024-00236-0] [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: 01/23/2024] [Revised: 08/07/2024] [Accepted: 08/22/2024] [Indexed: 09/06/2024] Open
Abstract
Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study, we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg, HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV, thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection, we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients, we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions.
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Affiliation(s)
- Huanting Chi
- Schaller Research Group, Department of Infectious Diseases, Virology, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
- German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany
| | - Bingqian Qu
- Schaller Research Group, Department of Infectious Diseases, Virology, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
- Molecular Virology, Department of Infectious Diseases, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
- Division of Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany
| | - Angga Prawira
- Molecular Virology, Department of Infectious Diseases, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
| | - Talisa Richardt
- Molecular Virology, Department of Infectious Diseases, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
| | - Lars Maurer
- Schaller Research Group, Department of Infectious Diseases, Virology, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
- Department of Infectious Diseases, Virology, Section Viral Vector Technologies, University Hospital Heidelberg, Cluster of Excellence CellNetworks, BioQuant, Center for Integrative Infectious Diseases Research (CIID), Heidelberg, Germany
| | - Jungen Hu
- Schaller Research Group, Department of Infectious Diseases, Virology, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
| | - Rebecca M Fu
- Schaller Research Group, Department of Infectious Diseases, Virology, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
| | - Florian A Lempp
- Molecular Virology, Department of Infectious Diseases, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
- Humabs Biomed SA, A Subsidiary of Vir Biotechnology, Bellinzona, Switzerland
| | - Zhenfeng Zhang
- Molecular Virology, Department of Infectious Diseases, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany
- School of Public Health and Emergency Management, School of Medicine, Southern University of Science and Technology, Shenzhen, China
| | - Dirk Grimm
- German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany
- Department of Infectious Diseases, Virology, Section Viral Vector Technologies, University Hospital Heidelberg, Cluster of Excellence CellNetworks, BioQuant, Center for Integrative Infectious Diseases Research (CIID), Heidelberg, Germany
- German Center for Cardiovascular Research (DZHK), Partner Site Heidelberg, Heidelberg, Germany
| | - Xianfang Wu
- Infection Biology Program and Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA
| | - Stephan Urban
- German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany.
- Molecular Virology, Department of Infectious Diseases, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany.
| | - Viet Loan Dao Thi
- Schaller Research Group, Department of Infectious Diseases, Virology, Heidelberg University, Medical Faculty Heidelberg, Heidelberg, Germany.
- German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany.
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24
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Nakashima Y, Tsukahara M. Atelocollagen supports three-dimensional culture of human induced pluripotent stem cells. Mol Ther Methods Clin Dev 2024; 32:101302. [PMID: 39185274 PMCID: PMC11342089 DOI: 10.1016/j.omtm.2024.101302] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2024] [Accepted: 07/16/2024] [Indexed: 08/27/2024]
Abstract
As autologous induced pluripotent stem cell (iPSC) therapy requires a custom-made small-lot cell production line, and the cell production method differs significantly from the existing processes for producing allogeneic iPSC stocks for clinical use. Specifically, mass culture to produce stock is no longer necessary; instead, a series of operations from iPSC production to induction of differentiation of therapeutic cells must be performed continuously. A three-dimensional (3D) culture method using small, closed-cell manufacturing devices is suitable for autologous iPSC therapy. The use of such devices avoids the need to handle many patient-derived specimens in a single clean room; handling of cell cultures in an open system in a cell processing facility increases the risk of infection. In this study, atelocollagen beads were evaluated as a 3D biomaterial to assist 3D culture in the establishment, expansion culture, and induction of differentiation of iPSCs. It was found that iPSCs can be handled in a closed-cell device with the same ease as use of a two-dimensional (2D) culture when laminin-511 is added to the medium. In conclusion, atelocollagen beads enable 3D culture of iPSCs, and the quality of the obtained cells is at the same level as those derived from 2D culture.
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Affiliation(s)
- Yoshiki Nakashima
- CiRA Foundation, Research and Development Center, Nakanoshima Qross, Osaka 530-005, Japan
| | - Masayoshi Tsukahara
- CiRA Foundation, Research and Development Center, Nakanoshima Qross, Osaka 530-005, Japan
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25
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Okano M, Yasuda M, Shimomura Y, Matsuoka Y, Shirouzu Y, Fujioka T, Kyo M, Tsuji S, Kaneko K, Hitomi H. Citrin-deficient patient-derived induced pluripotent stem cells as a pathological liver model for congenital urea cycle disorders. Mol Genet Metab Rep 2024; 40:101096. [PMID: 38872960 PMCID: PMC11170474 DOI: 10.1016/j.ymgmr.2024.101096] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2024] [Revised: 05/19/2024] [Accepted: 05/21/2024] [Indexed: 06/15/2024] Open
Abstract
Citrin deficiency is a congenital secondary urea cycle disorder lacking useful disease models for effective treatment development. In this study, human induced pluripotent stem cells (iPSCs) were generated from two patients with citrin deficiency and differentiated into hepatocyte-like cells (HLCs). Citrin-deficient HLCs produced albumin and liver-specific markers but completely lacked citrin protein and expressed argininosuccinate synthase only weakly. In addition, ammonia concentrations in a medium cultured with citrin-deficient HLCs were higher than with control HLCs. Sodium pyruvate administration significantly reduced ammonia concentrations in the medium of citrin-deficient HLCs and slightly reduced ammonia in HLCs differentiated from control iPSCs, though this change was not significant. Our results suggest that sodium pyruvate may be an efficient treatment for patients with citrin deficiency. Citrin-deficient iPSCs are a pathological liver model for congenital urea cycle disorders to clarify pathogenesis and develop novel therapies.
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Affiliation(s)
- Mai Okano
- Department of iPS Stem Cell Regenerative Medicine, Kansai Medical University, Osaka, Japan
- Department of Pediatrics, Kansai Medical University, Osaka, Japan
| | - Masahiro Yasuda
- Department of iPS Stem Cell Regenerative Medicine, Kansai Medical University, Osaka, Japan
- Department of Pediatrics, Kansai Medical University, Osaka, Japan
| | - Yui Shimomura
- Department of iPS Stem Cell Regenerative Medicine, Kansai Medical University, Osaka, Japan
| | - Yoshikazu Matsuoka
- Department of iPS Stem Cell Regenerative Medicine, Kansai Medical University, Osaka, Japan
| | - Yasumasa Shirouzu
- Department of iPS Stem Cell Regenerative Medicine, Kansai Medical University, Osaka, Japan
| | - Tatsuya Fujioka
- Department of iPS Stem Cell Regenerative Medicine, Kansai Medical University, Osaka, Japan
| | - Masatoshi Kyo
- Department of Neuropsychiatry, Kansai Medical University, Osaka, Japan
| | - Shoji Tsuji
- Department of Pediatrics, Kansai Medical University, Osaka, Japan
| | - Kazunari Kaneko
- Department of Pediatrics, Kansai Medical University, Osaka, Japan
| | - Hirofumi Hitomi
- Department of iPS Stem Cell Regenerative Medicine, Kansai Medical University, Osaka, Japan
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26
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Lekkala VKR, Shrestha S, Qaryoute AA, Dhinoja S, Acharya P, Raheem A, Jagadeeswaran P, Lee MY. Enhanced Maturity and Functionality of Vascularized Human Liver Organoids through 3D Bioprinting and Pillar Plate Culture. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.08.21.608997. [PMID: 39229042 PMCID: PMC11370572 DOI: 10.1101/2024.08.21.608997] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 09/05/2024]
Abstract
Liver tissues, composed of hepatocytes, cholangiocytes, stellate cells, Kupffer cells, and sinusoidal endothelial cells, are differentiated from endodermal and mesodermal germ layers. By mimicking the developmental process of the liver, various differentiation protocols have been published to generate human liver organoids (HLOs) in vitro using induced pluripotent stem cells (iPSCs). However, HLOs derived solely from the endodermal germ layer often encounter technical hurdles, such as insufficient maturity and functionality, limiting their utility for disease modeling and hepatotoxicity assays. To overcome this, we separately differentiated EpCAM+ endodermal progenitor cells (EPCs) and mesoderm-derived vascular progenitor cells (VPCs) from the same human iPSC line. These cells were then mixed in BME-2 matrix and concurrently differentiated into vascular human liver organoids (vHLOs). Remarkably, vHLOs exhibited significantly higher maturity than vasculature-free HLOs, as demonstrated by increased coagulation factor secretion, albumin secretion, drug-metabolizing enzyme (DME) expression, and bile acid transportation. To enhance assay throughput and miniaturize vHLO culture, we 3D bioprinted expandable HLOs (eHLOs) in BME-2 matrix on a pillar plate platform derived from EPCs and VPCs and compared with HLOs derived from endoderm alone. Compared to HLOs cultured in a 50 μL BME-2 matrix dome in a 24-well plate, vHLOs cultured on the pillar plate exhibited superior maturity, likely due to enhanced nutrient and signaling molecule diffusion. The integration of physiologically relevant patterned liver organoids with the unique pillar plate platform enhanced the capabilities for high-throughput screening and disease modeling.
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Affiliation(s)
| | - Sunil Shrestha
- Department of Biomedical Engineering, University of North Texas, Denton, Texas, USA
| | - Ayah Al Qaryoute
- Department of Biological Sciences, University of North Texas, Denton, TX, USA
| | - Sanchi Dhinoja
- Department of Biological Sciences, University of North Texas, Denton, TX, USA
| | - Prabha Acharya
- Department of Biomedical Engineering, University of North Texas, Denton, Texas, USA
| | - Abida Raheem
- Department of Biomedical Engineering, University of North Texas, Denton, Texas, USA
| | - Pudur Jagadeeswaran
- Department of Biological Sciences, University of North Texas, Denton, TX, USA
| | - Moo-Yeal Lee
- Department of Biomedical Engineering, University of North Texas, Denton, Texas, USA
- Bioprinting Laboratories Inc., Dallas, Texas, USA
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27
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Doueiry C, Kappler CS, Martinez-Morant C, Duncan SA. A PNPLA3-Deficient iPSC-Derived Hepatocyte Screen Identifies Pathways to Potentially Reduce Steatosis in Metabolic Dysfunction-Associated Fatty Liver Disease. Int J Mol Sci 2024; 25:7277. [PMID: 39000384 PMCID: PMC11242544 DOI: 10.3390/ijms25137277] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2024] [Revised: 06/25/2024] [Accepted: 06/29/2024] [Indexed: 07/16/2024] Open
Abstract
The incidence of nonalcoholic fatty liver disease (NAFLD), or metabolic dysfunction-associated fatty liver disease (MAFLD), is increasing in adults and children. Unfortunately, effective pharmacological treatments remain unavailable. Single nucleotide polymorphisms (SNPs) in the patatin-like phospholipase domain-containing protein (PNPLA3 I148M) have the most significant genetic association with the disease at all stages of its progression. A roadblock to identifying potential treatments for PNPLA3-induced NAFLD is the lack of a human cell platform that recapitulates the PNPLA3 I148M-mediated onset of lipid accumulation. Hepatocyte-like cells were generated from PNPLA3-/- and PNPLA3I148M/M-induced pluripotent stem cells (iPSCs). Lipid levels were measured by staining with BODIPY 493/503 and were found to increase in PNPLA3 variant iPSC-derived hepatocytes. A small-molecule screen identified multiple compounds that target Src/PI3K/Akt signaling and could eradicate lipid accumulation in these cells. We found that drugs currently in clinical trials for cancer treatment that target the same pathways also reduced lipid accumulation in PNPLA3 variant cells.
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Affiliation(s)
- Caren Doueiry
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, USA; (C.D.); (C.M.-M.)
- Medical Scientist Training Program, Medical University of South Carolina, Charleston, SC 29425, USA
| | - Christiana S. Kappler
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, USA; (C.D.); (C.M.-M.)
| | - Carla Martinez-Morant
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, USA; (C.D.); (C.M.-M.)
| | - Stephen A. Duncan
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, USA; (C.D.); (C.M.-M.)
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28
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Roudaut M, Caillaud A, Souguir Z, Bray L, Girardeau A, Rimbert A, Croyal M, Lambert G, Patitucci M, Delpouve G, Vandenhaute É, Le May C, Maubon N, Cariou B, Si‐Tayeb K. Human induced pluripotent stem cells-derived liver organoids grown on a Biomimesys® hyaluronic acid-based hydroscaffold as a new model for studying human lipoprotein metabolism. Bioeng Transl Med 2024; 9:e10659. [PMID: 39036087 PMCID: PMC11256179 DOI: 10.1002/btm2.10659] [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: 07/27/2023] [Revised: 01/29/2024] [Accepted: 02/12/2024] [Indexed: 07/23/2024] Open
Abstract
The liver plays a key role in the metabolism of lipoproteins, controlling both production and catabolism. To accelerate the development of new lipid-lowering therapies in humans, it is essential to have a relevant in vitro study model available. The current hepatocyte-like cells (HLCs) models derived from hiPSC can be used to model many genetically driven diseases but require further improvement to better recapitulate the complexity of liver functions. Here, we aimed to improve the maturation of HLCs using a three-dimensional (3D) approach using Biomimesys®, a hyaluronic acid-based hydroscaffold in which hiPSCs may directly form aggregates and differentiate toward a functional liver organoid model. After a 28-day differentiation 3D protocol, we showed that many hepatic genes were upregulated in the 3D model (liver organoids) in comparison with the 2D model (HLCs). Liver organoids, grown on Biomimesys®, exhibited an autonomous cell organization, were composed of different cell types and displayed enhanced cytochromes P450 activities compared to HLCs. Regarding the functional capacities of these organoids, we showed that they were able to accumulate lipids (hepatic steatosis), internalize low-density lipoprotein and secrete apolipoprotein B. Interestingly, we showed for the first time that this model was also able to produce apolipoprotein (a), the apolipoprotein (a) specific of Lp(a). This innovative hiPSC-derived liver organoid model may serve as a relevant model for studying human lipopoprotein metabolism, including Lp(a).
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Affiliation(s)
- Meryl Roudaut
- Nantes Université, CHU Nantes, CNRS, Inserm, l'institut du thoraxNantesFrance
- HCS PharmaLilleFrance
| | - Amandine Caillaud
- Nantes Université, CHU Nantes, CNRS, Inserm, l'institut du thoraxNantesFrance
| | | | - Lise Bray
- Nantes Université, CHU Nantes, CNRS, Inserm, l'institut du thoraxNantesFrance
| | - Aurore Girardeau
- Nantes Université, CHU Nantes, CNRS, Inserm, l'institut du thoraxNantesFrance
| | - Antoine Rimbert
- Nantes Université, CHU Nantes, CNRS, Inserm, l'institut du thoraxNantesFrance
| | - Mikaël Croyal
- Nantes Université, CHU Nantes, CNRS, Inserm, l'institut du thoraxNantesFrance
- CRNH‐Ouest Mass Spectrometry Core FacilityNantesFrance
| | - Gilles Lambert
- Inserm, UMR 1188 Diabète Athérothrombose Thérapies Réunion Océan Indien (DéTROI)Université de La RéunionSaint‐Denisde La RéunionFrance
| | - Murielle Patitucci
- Nantes Université, CHU Nantes, CNRS, Inserm, l'institut du thoraxNantesFrance
| | | | | | - Cédric Le May
- Nantes Université, CHU Nantes, CNRS, Inserm, l'institut du thoraxNantesFrance
| | | | - Bertrand Cariou
- Nantes Université, CHU Nantes, CNRS, Inserm, l'institut du thoraxNantesFrance
| | - Karim Si‐Tayeb
- Nantes Université, CHU Nantes, CNRS, Inserm, l'institut du thoraxNantesFrance
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29
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Liu R, Liu Y, Zhang W, Zhang G, Zhang Z, Huang L, Tang N, Wang K. PCK1 attenuates tumor stemness via activating the Hippo signaling pathway in hepatocellular carcinoma. Genes Dis 2024; 11:101114. [PMID: 38560500 PMCID: PMC10978540 DOI: 10.1016/j.gendis.2023.101114] [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: 08/09/2022] [Revised: 06/19/2023] [Accepted: 08/04/2023] [Indexed: 04/04/2024] Open
Abstract
Liver cancer stem cells were found to rely on glycolysis as the preferred metabolic program. Phosphoenolpyruvate carboxylase 1 (PCK1), a gluconeogenic metabolic enzyme, is down-regulated in hepatocellular carcinoma and is closely related to poor prognosis. The oncogenesis and progression of tumors are closely related to cancer stem cells. It is not completely clear whether the PCK1 deficiency increases the stemness of hepatoma cells and promotes the oncogenesis of hepatocellular carcinoma. Herein, the results showed that PCK1 inhibited the self-renewal property of hepatoma cells, reduced the mRNA level of cancer stem cell markers, and inhibited tumorigenesis. Moreover, PCK1 increased the sensitivity of hepatocellular carcinoma cells to sorafenib. Furthermore, we found that PCK1 activated the Hippo pathway by enhancing the phosphorylation of YAP and inhibiting its nuclear translocation. Verteporfin reduced the stemness of hepatoma cells and promoted the pro-apoptotic effect of sorafenib. Thus, combined treatment with verteporfin and sorafenib may be a potential anti-tumor strategy in hepatocellular carcinoma.
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Affiliation(s)
- Rui Liu
- Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
| | - Yi Liu
- Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
| | - Wenlu Zhang
- Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
| | - Guiji Zhang
- Department of Clinical Laboratory, Sichuan Clinical Research Center for Cancer, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, Affiliated Cancer Hospital of University of Electronic Science and Technology of China, Chengdu, Sichuan 610041, China
| | - Zhirong Zhang
- Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
| | - Luyi Huang
- Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
| | - Ni Tang
- Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
| | - Kai Wang
- Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
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30
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Deng B, Ma Y, Huang J, He R, Luo M, Mao L, Zhang E, Zhao Y, Wang X, Wang Q, Pang M, Mao Y, Yang H, Liu L, Huang P. Revitalizing liver function in mice with liver failure through transplantation of 3D-bioprinted liver with expanded primary hepatocytes. SCIENCE ADVANCES 2024; 10:eado1550. [PMID: 38848358 PMCID: PMC11160470 DOI: 10.1126/sciadv.ado1550] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/19/2024] [Accepted: 05/02/2024] [Indexed: 06/09/2024]
Abstract
The utilization of three-dimensional (3D) bioprinting technology to create a transplantable bioartificial liver emerges as a promising remedy for the scarcity of liver donors. This study outlines our strategy for constructing a 3D-bioprinted liver, using in vitro-expanded primary hepatocytes recognized for their safety and enhanced functional robustness as hepatic cell sources for bioartificial liver construction. In addition, we have developed bioink biomaterials with mechanical and rheological properties, as well as printing capabilities, tailored for 3D bioprinting. Upon heterotopic transplantation into the mesentery of tyrosinemia or 90% hepatectomy mice, our 3D-bioprinted liver effectively restored lost liver functions, consequently extending the life span of mice afflicted with liver injuries. Notably, the inclusion of an artificial blood vessel in our 3D-bioprinted liver allowed for biomolecule exchange with host blood vessels, demonstrating, in principle, the rapid integration of the bioartificial liver into the host vascular system. This model underscores the therapeutic potential of transplantation for the treatment of liver failure diseases.
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Affiliation(s)
- Bo Deng
- State Key Laboratory of Advanced Medical Materials and Devices, Engineering Research Center of Pulmonary and Critical Care Medicine Technology and Device (Ministry of Education), Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Tianjin Institutes of Health Science, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin 300192, China
| | - Yue Ma
- State Key Laboratory of Advanced Medical Materials and Devices, Engineering Research Center of Pulmonary and Critical Care Medicine Technology and Device (Ministry of Education), Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Tianjin Institutes of Health Science, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin 300192, China
| | - Jialyu Huang
- Center for Reproductive Medicine, Jiangxi Maternal and Child Health Hospital, Jiangxi Branch of National Clinical Research Center for Obstetrics and Gynecology, Nanchang Medical College, Nanchang, China
| | - Runbang He
- State Key Laboratory of Advanced Medical Materials and Devices, Engineering Research Center of Pulmonary and Critical Care Medicine Technology and Device (Ministry of Education), Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Tianjin Institutes of Health Science, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin 300192, China
| | - Miaomiao Luo
- State Key Laboratory of Advanced Medical Materials and Devices, Engineering Research Center of Pulmonary and Critical Care Medicine Technology and Device (Ministry of Education), Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Tianjin Institutes of Health Science, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin 300192, China
| | - Lina Mao
- State Key Laboratory of Advanced Medical Materials and Devices, Engineering Research Center of Pulmonary and Critical Care Medicine Technology and Device (Ministry of Education), Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Tianjin Institutes of Health Science, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin 300192, China
| | - Enhua Zhang
- State Key Laboratory of Advanced Medical Materials and Devices, Engineering Research Center of Pulmonary and Critical Care Medicine Technology and Device (Ministry of Education), Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Tianjin Institutes of Health Science, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin 300192, China
| | - Yuanyuan Zhao
- State Key Laboratory of Advanced Medical Materials and Devices, Engineering Research Center of Pulmonary and Critical Care Medicine Technology and Device (Ministry of Education), Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Tianjin Institutes of Health Science, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin 300192, China
| | - Xiaoli Wang
- State Key Laboratory of Advanced Medical Materials and Devices, Engineering Research Center of Pulmonary and Critical Care Medicine Technology and Device (Ministry of Education), Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Tianjin Institutes of Health Science, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin 300192, China
| | - Qiangsong Wang
- State Key Laboratory of Advanced Medical Materials and Devices, Engineering Research Center of Pulmonary and Critical Care Medicine Technology and Device (Ministry of Education), Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Tianjin Institutes of Health Science, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin 300192, China
| | - Mingchang Pang
- Department of Liver Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Yilei Mao
- Department of Liver Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Huayu Yang
- Department of Liver Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Lanxia Liu
- State Key Laboratory of Advanced Medical Materials and Devices, Engineering Research Center of Pulmonary and Critical Care Medicine Technology and Device (Ministry of Education), Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Tianjin Institutes of Health Science, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin 300192, China
| | - Pengyu Huang
- State Key Laboratory of Advanced Medical Materials and Devices, Engineering Research Center of Pulmonary and Critical Care Medicine Technology and Device (Ministry of Education), Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Tianjin Institutes of Health Science, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin 300192, China
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31
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Okumura A, Aoshima K, Tanimizu N. Generation of in vivo-like multicellular liver organoids by mimicking developmental processes: A review. Regen Ther 2024; 26:219-234. [PMID: 38903867 PMCID: PMC11186971 DOI: 10.1016/j.reth.2024.05.020] [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: 05/01/2024] [Revised: 05/24/2024] [Accepted: 05/30/2024] [Indexed: 06/22/2024] Open
Abstract
Liver is involved in metabolic reactions, ammonia detoxification, and immunity. Multicellular liver tissue cultures are more desirable for drug screening, disease modeling, and researching transplantation therapy, than hepatocytes monocultures. Hepatocytes monocultures are not stable for long. Further, hepatocyte-like cells induced from pluripotent stem cells and in vivo hepatocytes are functionally dissimilar. Organoid technology circumvents these issues by generating functional ex vivo liver tissue from intrinsic liver progenitor cells and extrinsic stem cells, including pluripotent stem cells. To function as in vivo liver tissue, the liver organoid cells must be arranged precisely in the 3-dimensional space, closely mimicking in vivo liver tissue. Moreover, for long term functioning, liver organoids must be appropriately vascularized and in contact with neighboring epithelial tissues (e.g., bile canaliculi and intrahepatic bile duct, or intrahepatic and extrahepatic bile ducts). Recent discoveries in liver developmental biology allows one to successfully induce liver component cells and generate organoids. Thus, here, in this review, we summarize the current state of knowledge on liver development with a focus on its application in generating different liver organoids. We also cover the future prospects in creating (functionally and structurally) in vivo-like liver organoids using the current knowledge on liver development.
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Affiliation(s)
- Ayumu Okumura
- Division of Regenerative Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-0071, Japan
| | - Kenji Aoshima
- Division of Regenerative Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-0071, Japan
| | - Naoki Tanimizu
- Division of Regenerative Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-0071, Japan
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32
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Caddeo A, Maurotti S, Kovooru L, Romeo S. 3D culture models to study pathophysiology of steatotic liver disease. Atherosclerosis 2024; 393:117544. [PMID: 38677899 DOI: 10.1016/j.atherosclerosis.2024.117544] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/21/2023] [Revised: 03/19/2024] [Accepted: 04/10/2024] [Indexed: 04/29/2024]
Abstract
Steatotic liver disease (SLD) refers to a spectrum of diseases caused by hepatic lipid accumulation. SLD has emerged as the leading cause of chronic liver disease worldwide. Despite this burden and many years, understanding the pathophysiology of this disease is challenging due to the inaccessibility to human liver specimens. Therefore, cell-based in vitro systems are widely used as models to investigate the pathophysiology of SLD. Culturing hepatic cells in monolayers causes the loss of their hepatocyte-specific phenotype and, consequently, tissue-specific function and architecture. Hence, three-dimensional (3D) culture models allow cells to mimic the in vivo microenvironment and spatial organization of the liver unit. The utilization of 3D in vitro models minimizes the drawbacks of two-dimensional (2D) cultures and aligns with the 3Rs principles to alleviate the number of in vivo experiments. This article provides an overview of liver 3D models highlighting advantages and limitations, and culminates by discussing their applications in pharmaceutical and biomedical research.
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Affiliation(s)
- Andrea Caddeo
- Department of Biomedical Sciences, Unit of Oncology and Molecular Pathology, University of Cagliari, Cagliari, Italy.
| | - Samantha Maurotti
- Department of Clinical and Experimental Medicine, University Magna Graecia, Catanzaro, Italy
| | - Lohitesh Kovooru
- Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, Wallenberg Laboratory, University of Gothenburg, Gothenburg, Sweden
| | - Stefano Romeo
- Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, Wallenberg Laboratory, University of Gothenburg, Gothenburg, Sweden; Department of Cardiology, Sahlgrenska University Hospital, Gothenburg, Sweden; Clinical Nutrition Unit, Department of Medical and Surgical Sciences, University Magna Graecia, Catanzaro, Italy.
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33
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Liu Y, Zhou Y, Ahodantin J, Jin Y, Zhu J, Sun Z, Wu X, Su L, Yang Y. Generation and characterization of mature hepatocyte organoids for liver metabolic studies. J Cell Sci 2024; 137:jcs261961. [PMID: 38700490 PMCID: PMC11166457 DOI: 10.1242/jcs.261961] [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: 01/16/2024] [Accepted: 04/12/2024] [Indexed: 05/05/2024] Open
Abstract
Hepatocyte organoids (HOs) generated in vitro are powerful tools for liver regeneration. However, previously reported HOs have mostly been fetal in nature with low expression levels of metabolic genes characteristic of adult liver functions, hampering their application in studies of metabolic regulation and therapeutic testing for liver disorders. Here, we report development of novel culture conditions that combine optimized levels of triiodothyronine (T3) with the removal of growth factors to enable successful generation of mature hepatocyte organoids (MHOs) of both mouse and human origin with metabolic functions characteristic of adult livers. We show that the MHOs can be used to study various metabolic functions including bile and urea production, zonal metabolic gene expression, and metabolic alterations in both alcoholic liver disease and non-alcoholic fatty liver disease, as well as hepatocyte proliferation, injury and cell fate changes. Notably, MHOs derived from human fetal hepatocytes also show improved hepatitis B virus infection. Therefore, these MHOs provide a powerful in vitro model for studies of human liver physiology and diseases. The human MHOs are potentially also a robust research tool for therapeutic development.
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Affiliation(s)
- Yuchen Liu
- Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Ave, Boston, MA 02115, USA
| | - Yaxing Zhou
- Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Ave, Boston, MA 02115, USA
| | - James Ahodantin
- Division of Virology, Pathogenesis, and Cancer, Institute of Human Virology, Departments of Pharmacology and Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
| | - Yu Jin
- Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Ave, Boston, MA 02115, USA
| | - Juanjuan Zhu
- Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Ave, Boston, MA 02115, USA
| | - Zhonghe Sun
- Cancer Research Technology Program, Frederick National Laboratory for Cancer, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA
| | - Xiaolin Wu
- Cancer Research Technology Program, Frederick National Laboratory for Cancer, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA
| | - Lishan Su
- Division of Virology, Pathogenesis, and Cancer, Institute of Human Virology, Departments of Pharmacology and Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
| | - Yingzi Yang
- Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Ave, Boston, MA 02115, USA
- Harvard Stem Cell Institute, Program in Gastrointestinal Malignancies, Dana-Farber/Harvard Cancer Center, 188 Longwood Ave, Boston, MA 02115, USA
- Program in Gastrointestinal Malignancies, Dana-Farber/Harvard Cancer Center, 188 Longwood Ave, Boston, MA 02115, USA
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Yanagihara K, Hayashi Y, Liu Y, Yamaguchi T, Hemmi Y, Kokunugi M, Yamada KU, Fukumoto K, Suga M, Terada S, Nikawa H, Kawabata K, Furue M. Trisomy 12 compromises the mesendodermal differentiation propensity of human pluripotent stem cells. In Vitro Cell Dev Biol Anim 2024; 60:521-534. [PMID: 38169039 PMCID: PMC11126453 DOI: 10.1007/s11626-023-00824-9] [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: 08/12/2023] [Accepted: 09/08/2023] [Indexed: 01/05/2024]
Abstract
Trisomy 12 is one of the most frequent chromosomal abnormalities in cultured human pluripotent stem cells (hPSCs). Although potential oncogenic properties and augmented cell cycle caused by trisomy 12 have been reported, the consequences of trisomy 12 in terms of cell differentiation, which is the basis for regenerative medicine, drug development, and developmental biology studies, have not yet been investigated. Here, we report that trisomy 12 compromises the mesendodermal differentiation of hPSCs. We identified sublines of hPSCs carrying trisomy 12 after their prolonged culture. Transcriptome analysis revealed that these hPSC sublines carried abnormal gene expression patterns in specific signaling pathways in addition to cancer-related cell cycle pathways. These hPSC sublines showed a lower propensity for mesendodermal differentiation in embryoid bodies cultured in a serum-free medium. BMP4-induced exit from the self-renewal state was impaired in the trisomy 12 hPSC sublines, with less upregulation of key transcription factor gene expression. As a consequence, the differentiation efficiency of hematopoietic and hepatic lineages was also impaired in the trisomy 12 hPSC sublines. We reveal that trisomy 12 disrupts the genome-wide expression patterns that are required for proper mesendodermal differentiation.
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Affiliation(s)
- Kana Yanagihara
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Yohei Hayashi
- iPS Cell Advanced Characterization and Development Team, RIKEN Bioresource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan.
| | - Yujung Liu
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Tomoko Yamaguchi
- Laboratory of Cell Model for Drug Discovery, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Yasuko Hemmi
- iPS Cell Advanced Characterization and Development Team, RIKEN Bioresource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan
| | - Minako Kokunugi
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
- Department of Oral Biology & Engineering Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Kozue Uchio Yamada
- Laboratory of Animal Models for Human Diseases, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Ken Fukumoto
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
- Department of Applied Chemistry and Biotechnology, University of Fukui, Fukui City, 3-9-1 Bunkyo, Fukui, 910-8507, Japan
| | - Mika Suga
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Satoshi Terada
- Department of Applied Chemistry and Biotechnology, University of Fukui, Fukui City, 3-9-1 Bunkyo, Fukui, 910-8507, Japan
| | - Hiroki Nikawa
- Department of Oral Biology & Engineering Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Kenji Kawabata
- Laboratory of Cell Model for Drug Discovery, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Miho Furue
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan.
- Cel-MiM, Ltd., Tokyo, Japan.
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35
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Acun A, Fan L, Oganesyan R, Uygun KM, Yeh H, Yarmush ML, Uygun BE. Effect of Donor Age and Liver Steatosis on Potential of Decellularized Liver Matrices to be used as a Platform for iPSC-Hepatocyte Culture. Adv Healthc Mater 2024; 13:e2302943. [PMID: 38266310 PMCID: PMC11102338 DOI: 10.1002/adhm.202302943] [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: 09/03/2023] [Revised: 11/13/2023] [Indexed: 01/26/2024]
Abstract
Decellularization of discarded whole livers and their recellularization with patient-specific induced pluripotent stem cells (iPSCs) to develop a functional organ is a promising approach to increasing the donor pool. The effect of extracellular matrix (ECM) of marginal livers on iPSC-hepatocyte differentiation and function has not been shown. To test the effect of donor liver ECM age and steatosis, young and old, as well as no, low, and high steatosis livers, are decellularized. All livers are decellularized successfully. High steatosis livers have fat remaining on the ECM after decellularization. Old donor liver ECM induces lower marker expression in early differentiation stages, compared to young liver ECM, while this difference is closed at later stages and do not affect iPSC-hepatocyte function significantly. High steatosis levels of liver ECM lead to higher albumin mRNA expression and secretion while at later stages of differentiation expression of major cytochrome (CYP) 450 enzymes is highest in low steatosis liver ECM. Both primary human hepatocytes and iPSC-hepatocytes show an increase in fat metabolism marker expression with increasing steatosis levels most likely induced by excess fat remaining on the ECM. Overall, removal of excess fat from liver ECM may be needed for inducing proper hepatic function after recellularization.
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Affiliation(s)
- Aylin Acun
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA
- Shriners Children’s, Boston, Boston, MA, 02114, USA
- Department of Biomedical Engineering, Widener University, Chester, PA, 19013, USA
| | - Letao Fan
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA
- Shriners Children’s, Boston, Boston, MA, 02114, USA
| | - Ruben Oganesyan
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA
- Shriners Children’s, Boston, Boston, MA, 02114, USA
| | - Korkut M. Uygun
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA
- Shriners Children’s, Boston, Boston, MA, 02114, USA
| | - Heidi Yeh
- Shriners Children’s, Boston, Boston, MA, 02114, USA
| | - Martin L. Yarmush
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA
- Shriners Children’s, Boston, Boston, MA, 02114, USA
- Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, 08854, USA
| | - Basak E. Uygun
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA
- Shriners Children’s, Boston, Boston, MA, 02114, USA
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36
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Carpentier A. Cell Culture Models for Hepatitis B and D Viruses Infection: Old Challenges, New Developments and Future Strategies. Viruses 2024; 16:716. [PMID: 38793598 PMCID: PMC11125795 DOI: 10.3390/v16050716] [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: 04/11/2024] [Revised: 04/23/2024] [Accepted: 04/29/2024] [Indexed: 05/26/2024] Open
Abstract
Chronic Hepatitis B and D Virus (HBV and HDV) co-infection is responsible for the most severe form of viral Hepatitis, the Hepatitis Delta. Despite an efficient vaccine against HBV, the HBV/HDV infection remains a global health burden. Notably, no efficient curative treatment exists against any of these viruses. While physiologically distinct, HBV and HDV life cycles are closely linked. HDV is a deficient virus that relies on HBV to fulfil is viral cycle. As a result, the cellular response to HDV also influences HBV replication. In vitro studying of HBV and HDV infection and co-infection rely on various cell culture models that differ greatly in terms of biological relevance and amenability to classical virology experiments. Here, we review the various cell culture models available to scientists to decipher HBV and HDV virology and host-pathogen interactions. We discuss their relevance and how they may help address the remaining questions, with one objective in mind: the development of new therapeutic approaches allowing viral clearance in patients.
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Affiliation(s)
- Arnaud Carpentier
- Institute for Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a Joint Venture between Hannover Medical School (MHH) and Helmholtz Centre for Infection Research (HZI), Feodor-Lynen-Strasse 7, 30625 Hannover, Germany;
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany
- Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, Germany
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37
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Jabri A, Khan J, Taftafa B, Alsharif M, Mhannayeh A, Chinnappan R, Alzhrani A, Kazmi S, Mir MS, Alsaud AW, Yaqinuddin A, Assiri AM, AlKattan K, Vashist YK, Broering DC, Mir TA. Bioengineered Organoids Offer New Possibilities for Liver Cancer Studies: A Review of Key Milestones and Challenges. Bioengineering (Basel) 2024; 11:346. [PMID: 38671768 PMCID: PMC11048289 DOI: 10.3390/bioengineering11040346] [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: 02/26/2024] [Revised: 03/25/2024] [Accepted: 03/27/2024] [Indexed: 04/28/2024] Open
Abstract
Hepatic cancer is widely regarded as the leading cause of cancer-related mortality worldwide. Despite recent advances in treatment options, the prognosis of liver cancer remains poor. Therefore, there is an urgent need to develop more representative in vitro models of liver cancer for pathophysiology and drug screening studies. Fortunately, an exciting new development for generating liver models in recent years has been the advent of organoid technology. Organoid models hold huge potential as an in vitro research tool because they can recapitulate the spatial architecture of primary liver cancers and maintain the molecular and functional variations of the native tissue counterparts during long-term culture in vitro. This review provides a comprehensive overview and discussion of the establishment and application of liver organoid models in vitro. Bioengineering strategies used to construct organoid models are also discussed. In addition, the clinical potential and other relevant applications of liver organoid models in different functional states are explored. In the end, this review discusses current limitations and future prospects to encourage further development.
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Affiliation(s)
- Abdullah Jabri
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia (R.C.); (A.W.A.); (K.A.)
| | - Jibran Khan
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia (R.C.); (A.W.A.); (K.A.)
| | - Bader Taftafa
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia (R.C.); (A.W.A.); (K.A.)
| | - Mohamed Alsharif
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia (R.C.); (A.W.A.); (K.A.)
| | - Abdulaziz Mhannayeh
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia (R.C.); (A.W.A.); (K.A.)
| | - Raja Chinnappan
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia (R.C.); (A.W.A.); (K.A.)
- Tissue/Organ Bioengineering and BioMEMS Lab, Organ Transplant Centre of Excellence (TR&I Dpt), King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia
| | - Alaa Alzhrani
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia (R.C.); (A.W.A.); (K.A.)
- Tissue/Organ Bioengineering and BioMEMS Lab, Organ Transplant Centre of Excellence (TR&I Dpt), King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia
- Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21423, Saudi Arabia
| | - Shadab Kazmi
- Tissue/Organ Bioengineering and BioMEMS Lab, Organ Transplant Centre of Excellence (TR&I Dpt), King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia
- Pathology and laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Mohammad Shabab Mir
- School of Pharmacy, Desh Bhagat University, Mandi Gobindgarh 147301, Punjab, India;
| | - Aljohara Waleed Alsaud
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia (R.C.); (A.W.A.); (K.A.)
| | - Ahmed Yaqinuddin
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia (R.C.); (A.W.A.); (K.A.)
| | - Abdullah M. Assiri
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia (R.C.); (A.W.A.); (K.A.)
- Tissue/Organ Bioengineering and BioMEMS Lab, Organ Transplant Centre of Excellence (TR&I Dpt), King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia
| | - Khaled AlKattan
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia (R.C.); (A.W.A.); (K.A.)
- Tissue/Organ Bioengineering and BioMEMS Lab, Organ Transplant Centre of Excellence (TR&I Dpt), King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia
| | - Yogesh K. Vashist
- Tissue/Organ Bioengineering and BioMEMS Lab, Organ Transplant Centre of Excellence (TR&I Dpt), King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia
| | - Dieter C. Broering
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia (R.C.); (A.W.A.); (K.A.)
- Tissue/Organ Bioengineering and BioMEMS Lab, Organ Transplant Centre of Excellence (TR&I Dpt), King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia
| | - Tanveer Ahmad Mir
- College of Medicine, Alfaisal University, Riyadh 11211, Saudi Arabia (R.C.); (A.W.A.); (K.A.)
- Tissue/Organ Bioengineering and BioMEMS Lab, Organ Transplant Centre of Excellence (TR&I Dpt), King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia
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38
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Utami T, Danoy M, Khadim RR, Tokito F, Arakawa H, Kato Y, Kido T, Miyajima A, Nishikawa M, Sakai Y. A highly efficient cell culture method using oxygen-permeable PDMS-based honeycomb microwells produces functional liver organoids from human induced pluripotent stem cell-derived carboxypeptidase M liver progenitor cells. Biotechnol Bioeng 2024; 121:1178-1190. [PMID: 38184815 DOI: 10.1002/bit.28640] [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: 04/20/2023] [Revised: 10/19/2023] [Accepted: 12/10/2023] [Indexed: 01/08/2024]
Abstract
Recent advancements in bioengineering have introduced potential alternatives to liver transplantation via the development of self-assembled liver organoids, derived from human-induced pluripotent stem cells (hiPSCs). However, the limited maturity of the tissue makes it challenging to implement this technology on a large scale in clinical settings. In this study, we developed a highly efficient method for generating functional liver organoids from hiPSC-derived carboxypeptidase M liver progenitor cells (CPM+ LPCs), using a microwell structure, and enhanced maturation through direct oxygenation in oxygen-permeable culture plates. We compared the morphology, gene expression profile, and function of the liver organoid with those of cells cultured under conventional conditions using either monolayer or spheroid culture systems. Our results revealed that liver organoids generated using polydimethylsiloxane-based honeycomb microwells significantly exhibited enhanced albumin secretion, hepatic marker expression, and cytochrome P450-mediated metabolism. Additionally, the oxygenated organoids consisted of both hepatocytes and cholangiocytes, which showed increased expression of bile transporter-related genes as well as enhanced bile transport function. Oxygen-permeable polydimethylsiloxane membranes may offer an efficient approach to generating highly mature liver organoids consisting of diverse cell populations.
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Affiliation(s)
- Tia Utami
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
| | - Mathieu Danoy
- Department of Chemical System Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
| | - Rubina Rahaman Khadim
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
| | - Fumiya Tokito
- Department of Chemical System Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
| | - Hiroshi Arakawa
- Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
| | - Yukio Kato
- Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
| | - Taketomo Kido
- Laboratory of Stem Cell Therapy, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan
| | - Atsushi Miyajima
- Laboratory of Stem Cell Therapy, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan
| | - Masaki Nishikawa
- Department of Chemical System Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
| | - Yasuyuki Sakai
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
- Department of Chemical System Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
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39
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Feng L, Wang Y, Fu Y, Li T, He G. Stem Cell-Based Strategies: The Future Direction of Bioartificial Liver Development. Stem Cell Rev Rep 2024; 20:601-616. [PMID: 38170319 DOI: 10.1007/s12015-023-10672-5] [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] [Accepted: 12/21/2023] [Indexed: 01/05/2024]
Abstract
Acute liver failure (ALF) results from severe liver damage or end-stage liver disease. It is extremely fatal and causes serious health and economic burdens worldwide. Once ALF occurs, liver transplantation (LT) is the only definitive and recommended treatment; however, LT is limited by the scarcity of liver grafts. Consequently, the clinical use of bioartificial liver (BAL) has been proposed as a treatment strategy for ALF. Human primary hepatocytes are an ideal cell source for these methods. However, their high demand and superior viability prevent their widespread use. Hence, finding alternatives that meet the seed cell quality and quantity requirements is imperative. Stem cells with self-renewing, immunogenic, and differentiative capacities are potential cell sources. MSCs and its secretomes encompass a spectrum of beneficial properties, such as anti-inflammatory, immunomodulatory, anti-ROS (reactive oxygen species), anti-apoptotic, pro-metabolomic, anti-fibrogenesis, and pro-regenerative attributes. This review focused on the recent status and future directions of stem cell-based strategies in BAL for ALF. Additionally, we discussed the opportunities and challenges associated with promoting such strategies for clinical applications.
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Affiliation(s)
- Lei Feng
- Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, Guangdong, China.
- Department of Hepatobiliary Surgery, The Affiliated Hospital of Guizhou Medical University, Guiyang, 550000, Guizhou, China.
| | - Yi Wang
- Shanxi Cancer Hospital/Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences/Cancer Hospital Affiliated to Shanxi Medical University, Taiyuan, 030013, Shanxi, China
| | - Yu Fu
- Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, Guangdong, China
| | - Ting Li
- Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, Guangdong, China.
- Department of Hepatobiliary Surgery, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510140, Guangdong, China.
| | - Guolin He
- Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, Guangdong, China.
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40
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Gandhi N, Wills L, Akers K, Su Y, Niccum P, Murali TM, Rajagopalan P. Comparative transcriptomic and phenotypic analysis of induced pluripotent stem cell hepatocyte-like cells and primary human hepatocytes. Cell Tissue Res 2024; 396:119-139. [PMID: 38369646 DOI: 10.1007/s00441-024-03868-9] [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: 02/13/2023] [Accepted: 01/22/2024] [Indexed: 02/20/2024]
Abstract
Primary human hepatocytes (PHHs) are used extensively for in vitro liver cultures to study hepatic functions. However, limited availability and invasive retrieval prevent their widespread use. Induced pluripotent stem cells exhibit significant potential since they can be obtained non-invasively and differentiated into hepatic lineages, such as hepatocyte-like cells (iHLCs). However, there are concerns about their fetal phenotypic characteristics and their hepatic functions compared to PHHs in culture. Therefore, we performed an RNA-sequencing (RNA-seq) analysis to understand pathways that are either up- or downregulated in each cell type. Analysis of the RNA-seq data showed an upregulation in the bile secretion pathway where genes such as AQP9 and UGT1A1 were higher expressed in PHHs compared to iHLCs by 455- and 15-fold, respectively. Upon immunostaining, bile canaliculi were shown to be present in PHHs. The TCA cycle in PHHs was upregulated compared to iHLCs. Cellular analysis showed a 2-2.5-fold increase in normalized urea production in PHHs compared to iHLCs. In addition, drug metabolism pathways, including cytochrome P450 (CYP450) and UDP-glucuronosyltransferase enzymes, were upregulated in PHHs compared to iHLCs. Of note, CYP2E1 gene expression was significantly higher (21,810-fold) in PHHs. Acetaminophen and ethanol were administered to PHH and iHLC cultures to investigate differences in biotransformation. CYP450 activity of baseline and toxicant-treated samples was significantly higher in PHHs compared to iHLCs. Our analysis revealed that iHLCs have substantial differences from PHHs in critical hepatic functions. These results have highlighted the differences in gene expression and hepatic functions between PHHs and iHLCs to motivate future investigation.
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Affiliation(s)
- Neeti Gandhi
- Department of Chemical Engineering, Virginia Tech, 333 Kelly Hall, Blacksburg, VA, 24061, USA
| | - Lauren Wills
- School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, USA
| | - Kyle Akers
- Genetics, Bioinformatics, and Computational Biology Ph.D. Program, Virginia Tech, Blacksburg, VA, USA
| | - Yiqi Su
- Department of Computer Science, Virginia Tech, Blacksburg, VA, USA
| | - Parker Niccum
- Genetics, Bioinformatics, and Computational Biology Ph.D. Program, Virginia Tech, Blacksburg, VA, USA
| | - T M Murali
- Department of Computer Science, Virginia Tech, Blacksburg, VA, USA
| | - Padmavathy Rajagopalan
- Department of Chemical Engineering, Virginia Tech, 333 Kelly Hall, Blacksburg, VA, 24061, USA.
- School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, USA.
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41
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Scheidecker B, Poulain S, Sugimoto M, Kido T, Kawanishi T, Miyajima A, Kim SH, Arakawa H, Kato Y, Nishikawa M, Danoy M, Sakai Y, Leclerc E. Dynamic, IPSC-derived hepatic tissue tri-culture system for the evaluation of liver physiology in vitro. Biofabrication 2024; 16:025037. [PMID: 38447229 DOI: 10.1088/1758-5090/ad30c5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2023] [Accepted: 03/06/2024] [Indexed: 03/08/2024]
Abstract
Availability of hepatic tissue for the investigation of metabolic processes is severely limited. While primary hepatocytes or animal models are widely used in pharmacological applications, a change in methodology towards more sustainable and ethical assays is highly desirable. Stem cell derived hepatic cells are generally regarded as a viable alternative for the above model systems, if current limitations in functionality and maturation can be overcome. By combining microfluidic organ-on-a-chip technology with individually differentiated, multicellular hepatic tissue fractions, we aim to improve overall functionality of hepatocyte-like cells, as well as evaluate cellular composition and interactions with non-parenchymal cell populations towards the formation of mature liver tissue. Utilizing a multi-omic approach, we show the improved maturation profiles of hepatocyte-like cells maintained in a dynamic microenvironment compared to standard tissue culture setups without continuous perfusion. In order to evaluate the resulting tissue, we employ single cell sequencing to distinguish formed subpopulations and spatial localization. While cellular input was strictly defined based on established differentiation protocols of parenchyma, endothelial and stellate cell fractions, resulting hepatic tissue was shown to comprise a complex mixture of epithelial and non-parenchymal fractions with specific local enrichment of phenotypes along the microchannel. Following this approach, we show the importance of passive, paracrine developmental processes in tissue formation. Using such complex tissue models is a crucial first step to develop stem cell-derivedin vitrosystems that can compare functionally with currently used pharmacological and toxicological applications.
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Affiliation(s)
- Benedikt Scheidecker
- CNRS UMI 2820, Institute of Industrial Science, University of Tokyo, 153-8505 Tokyo, Japan
| | - Stéphane Poulain
- Institute of Industrial Science, University of Tokyo, 153-8505 Tokyo, Japan
| | - Masahiro Sugimoto
- Institute for Advanced Biosciences, Keio University, 997-0035 Yamagata, Japan
- Institute of Medical Science, Tokyo Medical University, 160-8402 Tokyo, Japan
| | - Taketomo Kido
- Institute for Quantitative Biosciences, University of Tokyo, 113-0032 Tokyo, Japan
| | - Takumi Kawanishi
- School of Pharmaceutical Sciences, Kanazawa University, 920-1102 Kanazawa, Japan
| | - Atsushi Miyajima
- Institute for Quantitative Biosciences, University of Tokyo, 113-0032 Tokyo, Japan
| | - Soo Hyeon Kim
- Institute of Industrial Science, University of Tokyo, 153-8505 Tokyo, Japan
| | - Hiroshi Arakawa
- School of Pharmaceutical Sciences, Kanazawa University, 920-1102 Kanazawa, Japan
| | - Yukio Kato
- School of Pharmaceutical Sciences, Kanazawa University, 920-1102 Kanazawa, Japan
| | - Masaki Nishikawa
- Department of Chemical System Engineering, University of Tokyo, 113-8654 Tokyo, Japan
| | - Mathieu Danoy
- Department of Chemical System Engineering, University of Tokyo, 113-8654 Tokyo, Japan
| | - Yasuyuki Sakai
- Department of Chemical System Engineering, University of Tokyo, 113-8654 Tokyo, Japan
| | - Eric Leclerc
- CNRS UMI 2820, Institute of Industrial Science, University of Tokyo, 153-8505 Tokyo, Japan
- CNRS UMR 7338, Laboratoire de Biomécanique et Bioingénierie, Université de Technologies de Compiègne, 60203 Compiègne, France
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Chui JS, Izuel‐Idoype T, Qualizza A, de Almeida RP, Piessens L, van der Veer BK, Vanmarcke G, Malesa A, Athanasouli P, Boon R, Vriens J, van Grunsven L, Koh KP, Verfaillie CM, Lluis F. Osmolar Modulation Drives Reversible Cell Cycle Exit and Human Pluripotent Cell Differentiation via NF-κВ and WNT Signaling. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2307554. [PMID: 38037844 PMCID: PMC10870039 DOI: 10.1002/advs.202307554] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/10/2023] [Indexed: 12/02/2023]
Abstract
Terminally differentiated cells are commonly regarded as the most stable cell state in adult organisms, characterized by growth arrest while fulfilling their specialized functions. A better understanding of the mechanisms involved in promoting cell cycle exit will improve the ability to differentiate pluripotent cells into mature tissues for both pharmacological and therapeutic use. Here, it demonstrates that a hyperosmolar environment enforces a protective p53-independent quiescent state in immature hepatoma cells and in pluripotent stem cell-derived models of human hepatocytes and endothelial cells. Prolonged culture in hyperosmolar conditions stimulates changes in gene expression promoting functional cell maturation. Interestingly, hyperosmolar conditions do not only trigger growth arrest and cellular maturation but are also necessary to maintain this maturated state, as switching back to plasma osmolarity reverses the changes in expression of maturation and proliferative markers. Transcriptome analysis revealed sequential stages of osmolarity-regulated growth arrest followed by cell maturation, mediated by activation of NF-κВ, and repression of WNT signaling, respectively. This study reveals that a modulated increase in osmolarity serves as a biochemical signal to promote long-term growth arrest and cellular maturation into different lineages, providing a practical method to generate differentiated hiPSCs that resemble their mature counterpart more closely.
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Affiliation(s)
- Jonathan Sai‐Hong Chui
- KU LeuvenDepartment of Development and RegenerationStem Cell InstituteHerestraat 49Leuven3000Belgium
| | - Teresa Izuel‐Idoype
- KU LeuvenDepartment of Development and RegenerationStem Cell InstituteHerestraat 49Leuven3000Belgium
| | - Alessandra Qualizza
- KU LeuvenDepartment of Development and RegenerationStem Cell InstituteHerestraat 49Leuven3000Belgium
| | - Rita Pires de Almeida
- KU LeuvenDepartment of Development and RegenerationStem Cell InstituteHerestraat 49Leuven3000Belgium
| | - Lindsey Piessens
- KU LeuvenDepartment of Development and RegenerationStem Cell InstituteHerestraat 49Leuven3000Belgium
| | - Bernard K. van der Veer
- KU LeuvenDepartment of Development and RegenerationStem Cell InstituteHerestraat 49Leuven3000Belgium
| | - Gert Vanmarcke
- KU LeuvenDepartment of Development and RegenerationStem Cell InstituteHerestraat 49Leuven3000Belgium
| | - Aneta Malesa
- KU LeuvenDepartment of Development and RegenerationStem Cell InstituteHerestraat 49Leuven3000Belgium
| | - Paraskevi Athanasouli
- KU LeuvenDepartment of Development and RegenerationStem Cell InstituteHerestraat 49Leuven3000Belgium
| | - Ruben Boon
- KU LeuvenDepartment of Development and RegenerationStem Cell InstituteHerestraat 49Leuven3000Belgium
| | - Joris Vriens
- Laboratory of Endometrium, Endometriosis and Reproductive MedicineDepartment of Development and RegenerationKU LeuvenHerestraat 49Leuven3000Belgium
| | - Leo van Grunsven
- Liver Cell Biology Research GroupVrije Universiteit BrusselLaarbeeklaan 103Brussels1090Belgium
| | - Kian Peng Koh
- KU LeuvenDepartment of Development and RegenerationStem Cell InstituteHerestraat 49Leuven3000Belgium
| | - Catherine M. Verfaillie
- KU LeuvenDepartment of Development and RegenerationStem Cell InstituteHerestraat 49Leuven3000Belgium
| | - Frederic Lluis
- KU LeuvenDepartment of Development and RegenerationStem Cell InstituteHerestraat 49Leuven3000Belgium
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43
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Lv Y, Rao Z, Liu L, Jia J, Wu C, Xu J, Du Y, Liu Y, Liu B, Shi J, Li G, Zhao D, Deng H. The efficient generation of functional human hepatocytes from chemically induced pluripotent stem cells. Cell Prolif 2024; 57:e13540. [PMID: 37814474 PMCID: PMC10849784 DOI: 10.1111/cpr.13540] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2023] [Revised: 08/08/2023] [Accepted: 08/21/2023] [Indexed: 10/11/2023] Open
Abstract
Derivation of human hepatocytes from pluripotent stem cells in vitro has important applications including cell therapy and drug discovery. However, the differentiation of pluripotent stem cells into hepatocytes in vitro was not well recapitulated the development of liver. Here, we developed a differentiation protocol by mimicking the two-stage development of hepatoblasts, which permits the efficient generation of hepatic progenitor cells from chemically induced pluripotent stem cells (hCiPSCs). Single-cell RNA sequencing (scRNA-seq) indicates the similarity between hepatoblasts differentiated in vitro and in vivo. Moreover, hCiPSC-derived hepatic progenitor cells can further differentiate into hepatocytes that are similar to primary human hepatocytes with respect to gene expression and key hepatic functions. Our results demonstrate the feasibility of generating hepatic progenitor cells and hepatocytes from hCiPSCs with high efficiency and set the foundation for broad translational applications of hCiPSC-derived hepatocytes.
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Affiliation(s)
- Yun Lv
- School of Basic Medical Sciences, MOE Engineering Research Center of Regenerative Medicine, State Key Laboratory of Natural and Biomimetic DrugsPeking University Health Science Center and the MOE Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking‐Tsinghua Center for Life Sciences, Peking UniversityBeijingChina
| | - Ziyan Rao
- Department of Biomedical Informatics, School of Basic Medical SciencesPeking University Health Science CenterBeijingChina
- State Key Laboratory of Vascular Homeostasis and RemodelingPeking UniversityBeijingChina
| | - Lulu Liu
- Peking University‐Tsinghua University‐National Institute of Biological Science Joint Graduate Program, Academy for Advanced Interdisciplinary Studies, Peking UniversityBeijingChina
| | - Jun Jia
- Changping LaboratoryBeijingChina
| | - Chenyang Wu
- Department of Biomedical Informatics, School of Basic Medical SciencesPeking University Health Science CenterBeijingChina
- State Key Laboratory of Vascular Homeostasis and RemodelingPeking UniversityBeijingChina
| | - Jun Xu
- Department of Cell Biology, School of Basic Medical SciencesPeking University Stem Cell Research Center, Peking University Health Science Center, Peking UniversityBeijingChina
| | - Yuanyuan Du
- School of Basic Medical Sciences, MOE Engineering Research Center of Regenerative Medicine, State Key Laboratory of Natural and Biomimetic DrugsPeking University Health Science Center and the MOE Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking‐Tsinghua Center for Life Sciences, Peking UniversityBeijingChina
| | - Yinan Liu
- Department of Cell Biology, School of Basic Medical SciencesPeking University Stem Cell Research Center, Peking University Health Science Center, Peking UniversityBeijingChina
| | - Bei Liu
- School of Basic Medical Sciences, MOE Engineering Research Center of Regenerative Medicine, State Key Laboratory of Natural and Biomimetic DrugsPeking University Health Science Center and the MOE Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking‐Tsinghua Center for Life Sciences, Peking UniversityBeijingChina
| | - Jihang Shi
- Department of Hepatobiliary Surgery, First Medical Center of Chinese PLA General HospitalBeijingChina
| | - Guangya Li
- Ministry of Education (MOE) Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking‐Tsinghua Center for Life SciencesPeking UniversityBeijingChina
| | - Dongyu Zhao
- Department of Biomedical Informatics, School of Basic Medical SciencesPeking University Health Science CenterBeijingChina
- State Key Laboratory of Vascular Homeostasis and RemodelingPeking UniversityBeijingChina
| | - Hongkui Deng
- School of Basic Medical Sciences, MOE Engineering Research Center of Regenerative Medicine, State Key Laboratory of Natural and Biomimetic DrugsPeking University Health Science Center and the MOE Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking‐Tsinghua Center for Life Sciences, Peking UniversityBeijingChina
- Changping LaboratoryBeijingChina
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44
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Beltran AS. Novel Approaches to Studying SLC13A5 Disease. Metabolites 2024; 14:84. [PMID: 38392976 PMCID: PMC10890222 DOI: 10.3390/metabo14020084] [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: 11/18/2023] [Revised: 01/17/2024] [Accepted: 01/18/2024] [Indexed: 02/25/2024] Open
Abstract
The role of the sodium citrate transporter (NaCT) SLC13A5 is multifaceted and context-dependent. While aberrant dysfunction leads to neonatal epilepsy, its therapeutic inhibition protects against metabolic disease. Notably, insights regarding the cellular and molecular mechanisms underlying these phenomena are limited due to the intricacy and complexity of the latent human physiology, which is poorly captured by existing animal models. This review explores innovative technologies aimed at bridging such a knowledge gap. First, I provide an overview of SLC13A5 variants in the context of human disease and the specific cell types where the expression of the transporter has been observed. Next, I discuss current technologies for generating patient-specific induced pluripotent stem cells (iPSCs) and their inherent advantages and limitations, followed by a summary of the methods for differentiating iPSCs into neurons, hepatocytes, and organoids. Finally, I explore the relevance of these cellular models as platforms for delving into the intricate molecular and cellular mechanisms underlying SLC13A5-related disorders.
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Affiliation(s)
- Adriana S Beltran
- Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA
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45
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Xu Y, Wang Y, Qi R, Li K, Wang X, Li X, Shi B. Role of connexin 32 in the directional differentiation of induced pluripotent stem cells into hepatocytes. Int J Med Sci 2024; 21:508-518. [PMID: 38250613 PMCID: PMC10797672 DOI: 10.7150/ijms.83973] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/02/2023] [Accepted: 11/17/2023] [Indexed: 01/23/2024] Open
Abstract
This study aimed to explore the role of connexin 32 (Cx32) in the directional differentiation of induced pluripotent stem cells (iPSCs) into hepatocytes. Urine-derived epithelial cells were collected from the fresh urine of a healthy donor and transducted with reprogramming plasmid mixture to generate iPSCs. The iPSCs were then directionally differentiated into hepatocytes. During the differentiation, the upregulated and downregulated groups were treated with vitamin K2 (VK2) and 2-aminoethoxyboronate diphenylester (2-APB) to increase and inhibit Cx32 expression, respectively. The control group was not treated with the regulatory factor. Expression of Cx32 and hepatocyte-specific markers, including AFP, hepatocyte nuclear factor 4α (HNF-4α), albumin (ALB) and cytokeratin 18 (CK18) were detected. It indicated that Cx32 expression was not observed in iPSCs, but gradually increased during the process of hepatic differentiation from iPSCs. Upregulation of Cx32 expression by VK2 treatment promoted hepatocyte maturation and enhanced the expression of the aforementioned hepatic specific markers, whereas downregulation of Cx32 expression by 2-APB treatment had the opposite effects. In conclusion, urine-derived iPSCs could be directionally differentiated into hepatocytes. Up-regulation of Cx32 improves the efficiency and maturity of differentiation of iPSCs into hepatocytes, and Cx32 may be a promoting factor during the process of hepatic differentiation from iPSCs.
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Affiliation(s)
- Yan Xu
- Department of General Surgery, Tongji Hospital, School of Medicine, Tongji University, Shanghai, 200065, China
| | - Yufeng Wang
- Department of General Surgery, Tongji Hospital, School of Medicine, Tongji University, Shanghai, 200065, China
| | - Ran Qi
- Department of General Surgery, Tongji Hospital, School of Medicine, Tongji University, Shanghai, 200065, China
| | - Kun Li
- Department of General Surgery, Tongji Hospital, School of Medicine, Tongji University, Shanghai, 200065, China
| | - Xiuyan Wang
- Department of Ultrasonography, Tongji Hospital, School of Medicine, Tongji University, Shanghai, 200065, China
| | - Xinbo Li
- Ophthalmology, Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, 97239, USA
| | - Baomin Shi
- Department of General Surgery, Tongji Hospital, School of Medicine, Tongji University, Shanghai, 200065, China
- Department of General Surgery, Xinhua Hospital, School of medicine, Shanghai Jiaotong University, 200025, China
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46
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Huang H, Karanth SS, Guan Y, Freeman S, Soron R, Godovich DS, Guan J, Ye K, Jin S. Oxygenated Scaffolds for Pancreatic Endocrine Differentiation from Induced Pluripotent Stem Cells. Adv Healthc Mater 2024; 13:e2302275. [PMID: 37885129 PMCID: PMC11578060 DOI: 10.1002/adhm.202302275] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2023] [Revised: 10/06/2023] [Indexed: 10/28/2023]
Abstract
A 3D microenvironment is known to endorse pancreatic islet development from human induced pluripotent stem cells (iPSCs). However, oxygen supply becomes a limiting factor in a scaffold culture. In this study, oxygen-releasing biomaterials are fabricated and an oxygenated scaffold culture platform is developed to offer a better oxygen supply during 3D iPSC pancreatic differentiation. It is found that the oxygenation does not alter the scaffold's mechanical properties. The in situ oxygenation improves oxygen tension within the scaffolds. The unique 3D differentiation system enables the generation of islet organoids with enhanced expression of islet signature genes and proteins. Additionally, it is discovered that the oxygenation at the early stage of differentiation has more profound impacts on islet development from iPSCs. More C-peptide+ /MAFA+ β and glucagon+ /MAFB+ α cells formed in the iPSC-derived islet organoids generated under oxygenated conditions, suggesting enhanced maturation of the organoids. Furthermore, the oxygenated 3D cultures improve islet organoids' sensitivity to glucose for insulin secretion. It is herein demonstrated that the oxygenated scaffold culture empowers iPSC islet differentiation to generate clinically relevant tissues for diabetes research and treatment.
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Affiliation(s)
- Hui Huang
- Department of Biomedical Engineering, Thomas J. Watson College of Engineering and Applied Sciences, State University of New York (SUNY) at Binghamton, New York, 13902, USA
| | - Soujanya S Karanth
- Department of Biomedical Engineering, Thomas J. Watson College of Engineering and Applied Sciences, State University of New York (SUNY) at Binghamton, New York, 13902, USA
| | - Ya Guan
- Department of Mechanical Engineering and Materials Science, Washington University in St. Louis, St. Louis, MO, 63130, USA
| | - Sebastian Freeman
- Department of Biomedical Engineering, Thomas J. Watson College of Engineering and Applied Sciences, State University of New York (SUNY) at Binghamton, New York, 13902, USA
| | - Ryan Soron
- Department of Biomedical Engineering, Thomas J. Watson College of Engineering and Applied Sciences, State University of New York (SUNY) at Binghamton, New York, 13902, USA
| | - David S Godovich
- Department of Biomedical Engineering, Thomas J. Watson College of Engineering and Applied Sciences, State University of New York (SUNY) at Binghamton, New York, 13902, USA
| | - Jianjun Guan
- Department of Mechanical Engineering and Materials Science, Washington University in St. Louis, St. Louis, MO, 63130, USA
| | - Kaiming Ye
- Department of Biomedical Engineering, Thomas J. Watson College of Engineering and Applied Sciences, State University of New York (SUNY) at Binghamton, New York, 13902, USA
- Center of Biomanufacturing for Regenerative Medicine, State University of New York (SUNY) at Binghamton, New York, 13902, USA
| | - Sha Jin
- Department of Biomedical Engineering, Thomas J. Watson College of Engineering and Applied Sciences, State University of New York (SUNY) at Binghamton, New York, 13902, USA
- Center of Biomanufacturing for Regenerative Medicine, State University of New York (SUNY) at Binghamton, New York, 13902, USA
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47
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Novotny LA, Evans JG, Guo H, Kappler CS, Meissner EG. Interferon lambda receptor-1 isoforms differentially influence gene expression and HBV replication in stem cell-derived hepatocytes. Antiviral Res 2024; 221:105779. [PMID: 38070830 PMCID: PMC10872352 DOI: 10.1016/j.antiviral.2023.105779] [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: 09/01/2023] [Revised: 11/02/2023] [Accepted: 12/04/2023] [Indexed: 12/28/2023]
Abstract
BACKGROUND In the tolerogenic liver, inadequate or ineffective interferon signaling fails to clear chronic HBV infection. Lambda IFNs (IFNL) bind the interferon lambda receptor-1 (IFNLR1) which dimerizes with IL10RB to induce transcription of antiviral interferon-stimulated genes (ISG). IFNLR1 is expressed on hepatocytes, but low expression may limit the strength and antiviral efficacy of IFNL signaling. Three IFNLR1 transcriptional variants are detected in hepatocytes whose role in regulation of IFNL signaling is unclear: a full-length and signaling-capable form (isoform 1), a form that lacks a portion of the intracellular JAK1 binding domain (isoform 2), and a secreted form (isoform 3), the latter two predicted to be signaling defective. We hypothesized that altering expression of IFNLR1 isoforms would differentially impact the hepatocellular response to IFNLs and HBV replication. METHODS Induced pluripotent stem-cell derived hepatocytes (iHeps) engineered to contain FLAG-tagged, doxycycline-inducible IFNLR1 isoform constructs were HBV-infected then treated with IFNL3 followed by assessment of gene expression, HBV replication, and cellular viability. RESULTS Minimal overexpression of IFNLR1 isoform 1 markedly augmented ISG expression, induced de novo proinflammatory gene expression, and enhanced inhibition of HBV replication after IFNL treatment without adversely affecting cell viability. In contrast, overexpression of IFNLR1 isoform 2 or 3 partially augmented IFNL-induced ISG expression but did not support proinflammatory gene expression and minimally impacted HBV replication. CONCLUSIONS IFNLR1 isoforms differentially influence IFNL-induced gene expression and HBV replication in hepatocytes. Regulated IFNLR1 expression in vivo could limit the capacity of this pathway to counteract HBV replication.
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Affiliation(s)
- Laura A Novotny
- Division of Infectious Diseases, Department of Medicine, Medical University of South Carolina, Charleston, SC, USA
| | - J Grayson Evans
- Division of Infectious Diseases, Department of Medicine, Medical University of South Carolina, Charleston, SC, USA
| | - Haitao Guo
- Department of Microbiology and Molecular Genetics, Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Christiana S Kappler
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA
| | - Eric G Meissner
- Division of Infectious Diseases, Department of Medicine, Medical University of South Carolina, Charleston, SC, USA; Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, USA.
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48
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Baert L, Rudy S, Pellisson M, Doll T, Rocchetti R, Kaiser M, Mäser P, Müller M. Induced pluripotent stem cell-derived human macrophages as an infection model for Leishmania donovani. PLoS Negl Trop Dis 2024; 18:e0011559. [PMID: 38166146 PMCID: PMC10786377 DOI: 10.1371/journal.pntd.0011559] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2023] [Revised: 01/12/2024] [Accepted: 12/19/2023] [Indexed: 01/04/2024] Open
Abstract
The parasite Leishmania donovani is one of the species causing visceral leishmaniasis in humans, a deadly infection claiming up to 40,000 lives each year. The current drugs for leishmaniasis treatment have severe drawbacks and there is an urgent need to find new anti-leishmanial compounds. However, the search for drug candidates is complicated by the intracellular lifestyle of Leishmania. Here, we investigate the use of human induced pluripotent stem cell (iPS)-derived macrophages (iMACs) as host cells for L. donovani. iMACs obtained through embryoid body differentiation were infected with L. donovani promastigotes, and high-content imaging techniques were used to optimize the iMACs seeding density and multiplicity of infection, allowing us to reach infection rates up to 70% five days after infection. IC50 values obtained for miltefosine and amphotericin B using the infected iMACs or mouse peritoneal macrophages as host cells were comparable and in agreement with the literature, showing the potential of iMACs as an infection model for drug screening.
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Affiliation(s)
- Lore Baert
- Swiss Tropical and Public Health Institute (SwissTPH), Basel, Switzerland
- University of Basel, Basel, Switzerland
| | - Serena Rudy
- Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland
| | - Mélanie Pellisson
- Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland
| | - Thierry Doll
- Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland
| | - Romina Rocchetti
- Swiss Tropical and Public Health Institute (SwissTPH), Basel, Switzerland
- University of Basel, Basel, Switzerland
| | - Marcel Kaiser
- Swiss Tropical and Public Health Institute (SwissTPH), Basel, Switzerland
- University of Basel, Basel, Switzerland
| | - Pascal Mäser
- Swiss Tropical and Public Health Institute (SwissTPH), Basel, Switzerland
- University of Basel, Basel, Switzerland
| | - Matthias Müller
- Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland
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49
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Hidalgo-Álvarez J, Salas-Lucia F, Vera Cruz D, Fonseca TL, Bianco AC. Localized T3 production modifies the transcriptome and promotes the hepatocyte-like lineage in iPSC-derived hepatic organoids. JCI Insight 2023; 8:e173780. [PMID: 37856222 PMCID: PMC10795825 DOI: 10.1172/jci.insight.173780] [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: 07/10/2023] [Accepted: 10/17/2023] [Indexed: 10/21/2023] Open
Abstract
Thyroid hormone (TH) levels are low during development, and the deiodinases control TH signaling through tissue-specific activation or inactivation of TH. Here, we studied human induced pluripotent stem cell-derived (iPSC-derived) hepatic organoids and identified a robust induction of DIO2 expression (the deiodinase that activates T4 to T3) that occurs in hepatoblasts. The surge in DIO2-T3 (the deiodinase that activates thyroxine [T4] to triiodothyronine [T3]) persists until the hepatoblasts differentiate into hepatocyte- or cholangiocyte-like cells, neither of which expresses DIO2. Preventing the induction of the DIO2-T3 signaling modified the expression of key transcription factors, decreased the number of hepatocyte-like cells by ~60%, and increased the number of cholangiocyte-like cells by ~55% without affecting the growth or the size of the mature liver organoid. Physiological levels of T3 could not fully restore the transition from hepatoblasts to mature cells. This indicates that the timed surge in DIO2-T3 signaling critically determines the fate of developing human hepatoblasts and the transcriptome of the maturing hepatocytes, with physiological and clinical implications for how the liver handles energy substrates.
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Affiliation(s)
| | | | - Diana Vera Cruz
- Center for Research Informatics, The University of Chicago, Chicago, Illinois, USA
| | - Tatiana L. Fonseca
- Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, and
| | - Antonio C. Bianco
- Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, and
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50
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Fujisaka Y, Nakagawa T, Tomoda K, Watanabe M, Matsunaga N, Tamura Y, Ikeda S, Imagawa A, Asahi M. The cytotoxicity of gefitinib on patient‑derived induced pluripotent stem cells reflects gefitinib‑induced liver injury in the clinical setting. Oncol Lett 2023; 26:520. [PMID: 37927418 PMCID: PMC10623090 DOI: 10.3892/ol.2023.14108] [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: 06/05/2023] [Accepted: 09/22/2023] [Indexed: 11/07/2023] Open
Abstract
Gefitinib is a key drug used in the treatment of non-small cell lung cancer (NSCLC) with EGFR mutations. Gefitinib therapy is superior to conventional chemotherapy for the progression-free survival rate of patients with EGFR mutations. However, 10-26% of patients develop grade 3 or higher hepatotoxicity during gefitinib treatment; therefore, the development of preclinical tests for hepatotoxicity prior to clinical use is desirable. The present study evaluated the use of induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iPSC-heps), as a platform for preclinical test development. Patient-derived iPSCs were generated by reprogramming peripheral blood mononuclear cells obtained from two groups of gefitinib-treated patients with severe hepatotoxicity [toxicity group (T group)] or mild hepatotoxicity [no clinical toxicity group (N group)]. To examine the hepatotoxicity, the iPSCs from both T and N groups were differentiated into hepatocytes to obtain iPSC-heps. Differentiation was confirmed by measuring the expression levels of hepatocyte markers, such as albumin or α-fetoprotein, via western blotting and quantitative PCR analyses. Cytotoxicity in iPSCs and iPSC-heps after gefitinib treatment was evaluated using a lactate dehydrogenase release assay. The gefitinib-induced cytotoxicity in iPSCs from the T group was significantly higher than that from the N group, whereas there were no significant differences between the groups of iPSC-heps. These results suggested that using iPSCs in preclinical assessment may be a good indicator for the prediction of gefitinib-induced cytotoxicity in clinical use.
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Affiliation(s)
- Yasuhito Fujisaka
- Department of Medical Oncology, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Takatoshi Nakagawa
- Department of Pharmacology, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Kiichiro Tomoda
- Department of Pharmacology, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Marina Watanabe
- Department of Pharmacology, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Ninso Matsunaga
- Department of Internal Medicine (I), Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Yosuke Tamura
- Department of Internal Medicine (I), Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Soichiro Ikeda
- Department of Internal Medicine (I), Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Akihisa Imagawa
- Department of Internal Medicine (I), Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Michio Asahi
- Department of Pharmacology, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
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