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Inui J, Ueyama-Toba Y, Imamura C, Nagai W, Asano R, Mizuguchi H. Two-dimensionally cultured functional hepatocytes generated from human induced pluripotent stem cell-derived hepatic organoids for pharmaceutical research. Biomaterials 2025; 318:123148. [PMID: 39904185 DOI: 10.1016/j.biomaterials.2025.123148] [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: 09/27/2024] [Revised: 01/24/2025] [Accepted: 01/26/2025] [Indexed: 02/06/2025]
Abstract
Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells (HLCs) are expected to replace primary human hepatocytes (PHHs) as a new stable source of hepatocytes for pharmaceutical research. However, HLCs have lower hepatic functions than PHHs, require a long time for differentiation and cannot be prepared in large quantities because they do not proliferate after their terminal differentiation. To overcome these problems, we here established hepatic organoids (iHOs) from HLCs. We then showed that the iHOs could proliferate approximately 105-fold by more than 3 passages and expressed most hepatic genes more highly than HLCs. In addition, to enable their widespread use for in vitro drug discovery research, we developed a two-dimensional culture protocol for iHOs. Two-dimensionally cultured iHOs (iHO-Heps) expressed most of the major hepatocyte marker genes at much higher levels than HLCs, iHOs, and even PHHs. The iHO-Heps exhibited glycogen storage capacity, the capacity to uptake and release indocyanine green (ICG), albumin and urea secretion, and the capacity for bile canaliculi formation. Importantly, the iHO-Heps had the activity of major drug-metabolizing enzymes and responded to hepatotoxic drugs, much like PHHs. Thus, iHO-Heps overcome the limitations of the current models and promise to provide robust and reproducible pharmaceutical assays.
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Affiliation(s)
- Jumpei Inui
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871, Japan.
| | - Yukiko Ueyama-Toba
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871, Japan; Laboratory of Biochemistry and Molecular Biology, School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871, Japan; Laboratory of Functional Organoid for Drug Discovery, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, 567-0085, Japan; Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Osaka, 565-0871, Japan.
| | - Chiharu Imamura
- Laboratory of Biochemistry and Molecular Biology, School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871, Japan.
| | - Wakana Nagai
- Laboratory of Biochemistry and Molecular Biology, School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871, Japan.
| | - Rei Asano
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871, Japan.
| | - Hiroyuki Mizuguchi
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871, Japan; Laboratory of Biochemistry and Molecular Biology, School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871, Japan; Laboratory of Functional Organoid for Drug Discovery, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, 567-0085, Japan; Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Osaka, 565-0871, Japan; Global Center for Medical Engineering and Informatics, Osaka University, Osaka, 565-0871, Japan; Center for Infectious Disease Education and Research (CiDER), Osaka University, Osaka, 565-0871, Japan.
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Zhou Y, Zhong Y, Lauschke VM. Evaluating the synergistic use of advanced liver models and AI for the prediction of drug-induced liver injury. Expert Opin Drug Metab Toxicol 2025; 21:563-577. [PMID: 39893552 DOI: 10.1080/17425255.2025.2461484] [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: 10/28/2024] [Accepted: 01/29/2025] [Indexed: 02/04/2025]
Abstract
INTRODUCTION Drug-induced liver injury (DILI) is a leading cause of acute liver failure. Hepatotoxicity typically occurs only in a subset of individuals after prolonged exposure and constitutes a major risk factor for the termination of drug development projects. AREAS COVERED We provide an overview of available human liver models for DILI research and discuss how they have been used to aid in early risk assessments and to mitigate the risk of project closures due to DILI in clinical stages. We summarize the different data that can be provided by such models and illustrate how these diverse data types can be interfaced with machine learning strategies to improve predictions of liver safety liabilities. EXPERT OPINION Advanced human liver models closely mimic human liver phenotypes and functions for many weeks, allowing for the recapitulation of hepatotoxicity events in vitro. Integration of the biochemical, histological, and toxicogenomic output data from these models with physicochemical compound properties using different machine learning architectures holds promise to enhance preclinical DILI predictions. However, to realize this aim, it is important to benchmark the available liver models on test sets of DILI positive and negative compounds and to carefully annotate and share the resulting data.
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Affiliation(s)
- Yitian Zhou
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
- Center for Molecular Medicine, Karolinska Institutet and University Hospital, Stockholm, Sweden
| | - Yi Zhong
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
- Center for Molecular Medicine, Karolinska Institutet and University Hospital, Stockholm, Sweden
- Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China
| | - Volker M Lauschke
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
- Center for Molecular Medicine, Karolinska Institutet and University Hospital, Stockholm, Sweden
- Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
- University of Tübingen, Tübingen, Germany
- Department of Pharmacy, The Second Xiangya Hospital, Central South University, Changsha, China
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3
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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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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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5
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Silva B, Bragança J. Induced pluripotent stem cell-derived mesenchymal stem cells for modeling and treating metabolic associated fatty liver disease and metabolic associated steatohepatitis: Challenges and opportunities. World J Stem Cells 2025; 17:99331. [DOI: 10.4252/wjsc.v17.i2.99331] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/19/2024] [Revised: 11/21/2024] [Accepted: 01/14/2025] [Indexed: 02/24/2025] Open
Abstract
The potential of induced pluripotent stem cells (iPSCs) for modeling and treating metabolic associated fatty liver disease (MAFLD) and metabolic associated steatohepatitis (MASH) is emerging. MAFLD is a growing global health concern, currently with limited treatment options. While primary mesenchymal stem cells hold promise, iPSCs offer a versatile alternative due to their ability to differentiate into various cell types, including iPSC-derived mesenchymal stem cells. However, challenges remain, including optimizing differentiation protocols, ensuring cell safety, and addressing potential tumorigenicity risks. In addition, iPSCs offer the possibility to generate complex cellular models, including three-dimensional organoid models, which are closer representations of the human disease than animal models. Those models would also be valuable for drug discovery and personalized medicine approaches. Overall, iPSCs and their derivatives offer new perspectives for advancing MAFLD/MASH research and developing novel therapeutic strategies. Further research is needed to overcome current limitations and translate this potential into effective clinical applications.
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Affiliation(s)
- Bárbara Silva
- Algarve Biomedical Center-Research Institute, University of Algarve, Faro 8005-139, Portugal
- Algarve Biomedical Center, University of Algarve, Faro 8005-139, Portugal
- Faculty of Medicine and Biomedical Sciences, University of Algarve, Faro 8005-139, Portugal
- PhD Program in Biomedical Sciences, Faculty of Medicine and Biomedical Sciences, University of Algarve, Faro 8005-139, Portugal
| | - José Bragança
- Algarve Biomedical Center, University of Algarve, Faro 8005-139, Portugal
- Faculty of Medicine and Biomedical Sciences, University of Algarve, Faro 8005-139, Portugal
- Faculty of Medicine and Biomedical Sciences, Algarve Biomedical Center-Research Institute, University of Algarve, Faro 8005-139, Portugal
- Champalimaud Research Program, Champalimaud Centre for the Unknown, Lisbon 1000-001, Portugal
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Heydari Z, Gramignoli R, Piryaei A, Zahmatkesh E, Pooyan P, Seydi H, Nussler A, Szkolnicka D, Rashidi H, Najimi M, Hay DC, Vosough M. Standard Protocols for Characterising Primary and In Vitro-Generated Human Hepatocytes. J Cell Mol Med 2025; 29:e70390. [PMID: 39910642 PMCID: PMC11798750 DOI: 10.1111/jcmm.70390] [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: 10/10/2024] [Revised: 01/13/2025] [Accepted: 01/17/2025] [Indexed: 02/07/2025] Open
Abstract
Hepatocyte-like cells (HLCs) derived from pluripotent stem cells (PSCs) or direct reprogramming are an unlimited source of human hepatocytes for biomedical applications. HLCs are used to model human diseases, develop precise drugs and establish groundbreaking regenerative cell-based therapies. Primary human hepatocytes are the gold standard for studying human liver biology and pathology. However, their widespread use is limited by their rapid dedifferentiation in vitro, reliance on transplant-rejected donor organs, poor scalability and significant batch-to-batch variations. Therefore, high-quality 'off-the-shelf' HLCs are needed to overcome those limitations. Basic stepwise differentiation protocols have been developed to generate HLCs from PSCs. To evaluate the quality of the in vitro generated products, HLCs have been phenotyped using various methods. This review discusses various biological assays and methods available for the robust evaluation of HLC quality, emphasising the importance of using 24-h cultured primary human hepatocytes (PHHs) as a reference standard for comparison.
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Affiliation(s)
- Zahra Heydari
- Department of Regenerative Medicine, Cell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
| | - Roberto Gramignoli
- Division of Pathology, Department of Laboratory MedicineKarolinska InstitutetStockholmSweden
| | - Abbas Piryaei
- Department of Biology and Anatomical Sciences, School of MedicineShahid Beheshti University of Medical SciencesTehranIran
- Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in MedicineShahid Beheshti University of Medical SciencesTehranIran
| | - Ensieh Zahmatkesh
- Department of Regenerative Medicine, Cell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
| | - Paria Pooyan
- Department of Regenerative Medicine, Cell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
| | - Homeyra Seydi
- Department of Regenerative Medicine, Cell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
| | - Andreas Nussler
- Siegfried Weller Institute for Trauma ResearchUniversity of TübingenTübingenGermany
| | - Dagmara Szkolnicka
- Centre for Regenerative Medicine, Institute for Repair and RegenerationUniversity of EdinburghEdinburghUK
| | - Hassan Rashidi
- Department of Developmental Biology and CancerUCL Great Ormond Street Institute of Child HealthLondonUK
| | - Mustapha Najimi
- Laboratory of Pediatric Hepatology and Cell TherapyInstitute of Experimental and Clinical Research, UCLouvainBrusselsBelgium
| | - David C. Hay
- Centre for Regenerative Medicine, Institute for Repair and RegenerationUniversity of EdinburghEdinburghUK
| | - Massoud Vosough
- Department of Regenerative Medicine, Cell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
- Experimental Cancer MedicineInstitution for Laboratory Medicine, Karolinska Institute HuddingeHuddingeSweden
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Guo K, van den Beucken T. Advances in drug-induced liver injury research: in vitro models, mechanisms, omics and gene modulation techniques. Cell Biosci 2024; 14:134. [PMID: 39488681 PMCID: PMC11531151 DOI: 10.1186/s13578-024-01317-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2024] [Accepted: 10/21/2024] [Indexed: 11/04/2024] Open
Abstract
Drug-induced liver injury (DILI) refers to drug-mediated damage to the structure and function of the liver, ranging from mild elevation of liver enzymes to severe hepatic insufficiency, and in some cases, progressing to liver failure. The mechanisms and clinical symptoms of DILI are diverse due to the varying combination of drugs, making clinical treatment and prevention complex. DILI has significant public health implications and is the primary reason for post-marketing drug withdrawals. The search for reliable preclinical models and validated biomarkers to predict and investigate DILI can contribute to a more comprehensive understanding of adverse effects and drug safety. In this review, we examine the progress of research on DILI, enumerate in vitro models with potential benefits, and highlight cellular molecular perturbations that may serve as biomarkers. Additionally, we discuss omics approaches frequently used to gather comprehensive datasets on molecular events in response to drug exposure. Finally, three commonly used gene modulation techniques are described, highlighting their application in identifying causal relationships in DILI. Altogether, this review provides a thorough overview of ongoing work and approaches in the field of DILI.
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Affiliation(s)
- Kaidi Guo
- Department of Toxicogenomics, GROW - Research Institute for Oncology & Reproduction, Maastricht University, Maastricht, 6200, MD, The Netherlands.
| | - Twan van den Beucken
- Department of Toxicogenomics, GROW - Research Institute for Oncology & Reproduction, Maastricht University, Maastricht, 6200, MD, The Netherlands
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Wani SI, Mir TA, Nakamura M, Tsuchiya T, Alzhrani A, Iwanaga S, Arai K, Alshehri EA, Shamma T, Obeid DA, Chinnappan R, Assiri AM, Yaqinuddin A, Vashist YK, Broering DC. A review of current state-of-the-art materiobiology and technological approaches for liver tissue engineering. BIOPRINTING 2024; 42:e00355. [DOI: 10.1016/j.bprint.2024.e00355] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/20/2025]
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9
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Kang S, Chen EC, Cifuentes H, Co JY, Cole G, Graham J, Hsia R, Kiyota T, Klein JA, Kroll KT, Nieves Lopez LM, Norona LM, Peiris H, Potla R, Romero-Lopez M, Roth JG, Tseng M, Fullerton AM, Homan KA. Complex in vitromodels positioned for impact to drug testing in pharma: a review. Biofabrication 2024; 16:042006. [PMID: 39189069 DOI: 10.1088/1758-5090/ad6933] [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: 12/22/2023] [Accepted: 07/30/2024] [Indexed: 08/28/2024]
Abstract
Recent years have seen the creation and popularization of various complexin vitromodels (CIVMs), such as organoids and organs-on-chip, as a technology with the potential to reduce animal usage in pharma while also enhancing our ability to create safe and efficacious drugs for patients. Public awareness of CIVMs has increased, in part, due to the recent passage of the FDA Modernization Act 2.0. This visibility is expected to spur deeper investment in and adoption of such models. Thus, end-users and model developers alike require a framework to both understand the readiness of current models to enter the drug development process, and to assess upcoming models for the same. This review presents such a framework for model selection based on comparative -omics data (which we term model-omics), and metrics for qualification of specific test assays that a model may support that we term context-of-use (COU) assays. We surveyed existing healthy tissue models and assays for ten drug development-critical organs of the body, and provide evaluations of readiness and suggestions for improving model-omics and COU assays for each. In whole, this review comes from a pharma perspective, and seeks to provide an evaluation of where CIVMs are poised for maximum impact in the drug development process, and a roadmap for realizing that potential.
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Affiliation(s)
- Serah Kang
- Complex in vitro Systems Group, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Eugene C Chen
- Department of Drug Metabolism and Pharmacokinetics, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Helen Cifuentes
- Complex in vitro Systems Group, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Julia Y Co
- Complex in vitro Systems Group, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Gabrielle Cole
- Investigative Toxicology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Jessica Graham
- Product Quality & Occupational Toxicology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of Americaica
| | - Rebecca Hsia
- Complex in vitro Systems Group, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Tomomi Kiyota
- Investigative Toxicology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Jessica A Klein
- Complex in vitro Systems Group, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Katharina T Kroll
- Complex in vitro Systems Group, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Lenitza M Nieves Lopez
- Complex in vitro Systems Group, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Leah M Norona
- Investigative Toxicology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Heshan Peiris
- Human Genetics, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Ratnakar Potla
- Complex in vitro Systems Group, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Monica Romero-Lopez
- Complex in vitro Systems Group, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Julien G Roth
- Complex in vitro Systems Group, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Min Tseng
- Investigative Toxicology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Aaron M Fullerton
- Investigative Toxicology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
| | - Kimberly A Homan
- Complex in vitro Systems Group, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States of America
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10
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Yuan Y, Bodke VV, Lin C, Gao S, Rehman J, Li J, Khetani SR. Long-term HBV infection of engineered cultures of induced pluripotent stem cell-derived hepatocytes. Hepatol Commun 2024; 8:e0506. [PMID: 39082962 DOI: 10.1097/hc9.0000000000000506] [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] [Received: 01/29/2024] [Accepted: 06/08/2024] [Indexed: 10/03/2024] Open
Abstract
BACKGROUND HBV infects ~257 million people and can cause hepatocellular carcinoma. Since current drugs are not curative, novel therapies are needed. HBV infects chimpanzee and human livers. However, chimpanzee studies are severely restricted and cost-prohibitive, while transgenic/chimeric mouse models that circumvent the species barrier lack natural HBV infection and disease progression. Thus, in vitro human models of HBV infection are useful in addressing the above limitations. Induced pluripotent stem cell-derived hepatocyte-like cells mitigate the supply limitations of primary human hepatocytes and the abnormal proliferation/functions of hepatoma cell lines. However, variable infection across donors, deficient drug metabolism capacity, and/or low throughput limit iHep utility for drug development. METHODS We developed an optimal pipeline using combinations of small molecules, Janus kinase inhibitor, and 3',5'-cAMP to infect iHep-containing micropatterned co-cultures (iMPCC) with stromal fibroblasts within 96-well plates with serum-derived HBV and cell culture-derived HBV (cHBV). Polyethylene glycol was necessary for cell-derived HBV but not for serum-derived HBV infection. RESULTS Unlike iHep monocultures, iMPCCs created from 3 iHep donors could sustain HBV infection for 2+ weeks. Infected iMPCCs maintained high levels of differentiated functions, including drug metabolism capacity. HBV antigen secretion and gene expression patterns in infected iMPCCs in pathways such as fatty acid metabolism and cholesterol biosynthesis were comparable to primary human hepatocyte-MPCCs. Furthermore, iMPCCs could help elucidate the effects of interferons and direct-acting antiviral drugs on the HBV lifecycle and any hepatotoxicity; iMPCC response to compounds was similar to primary human hepatocyte-MPCCs. CONCLUSIONS The iMPCC platform can enable the development of safe and efficacious drugs against HBV and ultimately help elucidate genotype-phenotype relationships in HBV pathogenesis.
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Affiliation(s)
- Yang Yuan
- Department of Biomedical Engineering, University of Illinois Chicago, Chicago, Illinois, USA
| | - Vedant V Bodke
- Department of Biomedical Engineering, University of Illinois Chicago, Chicago, Illinois, USA
| | - Christine Lin
- Department of Biomedical Engineering, University of Illinois Chicago, Chicago, Illinois, USA
| | - Shang Gao
- Department of Biomedical Engineering, University of Illinois Chicago, Chicago, Illinois, USA
| | - Jalees Rehman
- Department of Biochemistry and Molecular Genetics, University of Illinois Chicago, Chicago, Illinois, USA
| | - Jisu Li
- Liver Research Center, Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA
| | - Salman R Khetani
- Department of Biomedical Engineering, University of Illinois Chicago, Chicago, Illinois, USA
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11
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Mehta V, Karnam G, Madgula V. Liver-on-chips for drug discovery and development. Mater Today Bio 2024; 27:101143. [PMID: 39070097 PMCID: PMC11279310 DOI: 10.1016/j.mtbio.2024.101143] [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: 02/28/2024] [Revised: 06/07/2024] [Accepted: 07/01/2024] [Indexed: 07/30/2024] Open
Abstract
Recent FDA modernization act 2.0 has led to increasing industrial R&D investment in advanced in vitro 3D models such as organoids, spheroids, organ-on-chips, 3D bioprinting, and in silico approaches. Liver-related advanced in vitro models remain the prime area of interest, as liver plays a central role in drug clearance of compounds. Growing evidence indicates the importance of recapitulating the overall liver microenvironment to enhance hepatocyte maturity and culture longevity using liver-on-chips (LoC) in vitro. Hence, pharmaceutical industries have started exploring LoC assays in the two of the most challenging areas: accurate in vitro-in vivo extrapolation (IVIVE) of hepatic drug clearance and drug-induced liver injury. We examine the joint efforts of commercial chip manufacturers and pharmaceutical companies to present an up-to-date overview of the adoption of LoC technology in the drug discovery. Further, several roadblocks are identified to the rapid adoption of LoC assays in the current drug development framework. Finally, we discuss some of the underexplored application areas of LoC models, where conventional 2D hepatic models are deemed unsuitable. These include clearance prediction of metabolically stable compounds, immune-mediated drug-induced liver injury (DILI) predictions, bioavailability prediction with gut-liver systems, hepatic clearance prediction of drugs given during pregnancy, and dose adjustment studies in disease conditions. We conclude the review by discussing the importance of PBPK modeling with LoC, digital twins, and AI/ML integration with LoC.
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Affiliation(s)
- Viraj Mehta
- Organoid Technology Lab, DMPK Department, Sai Life Sciences, Hyderabad, 500078, India
| | - Guruswamy Karnam
- Organoid Technology Lab, DMPK Department, Sai Life Sciences, Hyderabad, 500078, India
| | - Vamsi Madgula
- Organoid Technology Lab, DMPK Department, Sai Life Sciences, Hyderabad, 500078, India
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12
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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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13
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Cliffe FE, Madden C, Costello P, Devitt S, Mukkunda SR, Keshava BB, Fearnhead HO, Vitkauskaite A, Dehkordi MH, Chingwaru W, Przyjalgowski M, Rebrova N, Lyons M. Mera: A scalable high throughput automated micro-physiological system. SLAS Technol 2023; 28:230-242. [PMID: 36708805 DOI: 10.1016/j.slast.2023.01.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2022] [Revised: 01/16/2023] [Accepted: 01/21/2023] [Indexed: 01/27/2023]
Abstract
There is an urgent need for scalable Microphysiological Systems (MPS's)1 that can better predict drug efficacy and toxicity at the preclinical screening stage. Here we present Mera, an automated, modular and scalable system for culturing and assaying microtissues with interconnected fluidics, inbuilt environmental control and automated image capture. The system presented has multiple possible fluidics modes. Of these the primary mode is designed so that cells may be matured into a desired microtissue type and in the secondary mode the fluid flow can be re-orientated to create a recirculating circuit composed of inter-connected channels to allow drugging or staining. We present data demonstrating the prototype system Mera using an Acetaminophen/HepG2 liver microtissue toxicity assay with Calcein AM and Ethidium Homodimer (EtHD1) viability assays. We demonstrate the functionality of the automated image capture system. The prototype microtissue culture plate wells are laid out in a 3 × 3 or 4 × 10 grid format with viability and toxicity assays demonstrated in both formats. In this paper we set the groundwork for the Mera system as a viable option for scalable microtissue culture and assay development.
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Affiliation(s)
- Finola E Cliffe
- Hooke Bio Ltd, L4A Smithstown Industrial Estate, Shannon, Co. Clare V14 XH92, Ireland
| | - Conor Madden
- Hooke Bio Ltd, L4A Smithstown Industrial Estate, Shannon, Co. Clare V14 XH92, Ireland
| | - Patrick Costello
- Hooke Bio Ltd, L4A Smithstown Industrial Estate, Shannon, Co. Clare V14 XH92, Ireland
| | - Shane Devitt
- Hooke Bio Ltd, L4A Smithstown Industrial Estate, Shannon, Co. Clare V14 XH92, Ireland
| | - Sumir Ramesh Mukkunda
- Hooke Bio Ltd, L4A Smithstown Industrial Estate, Shannon, Co. Clare V14 XH92, Ireland
| | | | - Howard O Fearnhead
- Pharmacology and Therapeutics, Biomedical Sciences, Dangan, NUI Galway, Galway, Ireland
| | - Aiste Vitkauskaite
- Pharmacology and Therapeutics, Biomedical Sciences, Dangan, NUI Galway, Galway, Ireland
| | - Mahshid H Dehkordi
- Pharmacology and Therapeutics, Biomedical Sciences, Dangan, NUI Galway, Galway, Ireland
| | - Walter Chingwaru
- Pharmacology and Therapeutics, Biomedical Sciences, Dangan, NUI Galway, Galway, Ireland
| | - Milosz Przyjalgowski
- Centre for Advanced Photonics and Process Analysis, Munster Technological University, Cork T12 P928, Ireland
| | - Natalia Rebrova
- Centre for Advanced Photonics and Process Analysis, Munster Technological University, Cork T12 P928, Ireland
| | - Mark Lyons
- Hooke Bio Ltd, L4A Smithstown Industrial Estate, Shannon, Co. Clare V14 XH92, Ireland.
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14
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Luo Q, Wang N, Que H, Mai E, Hu Y, Tan R, Gu J, Gong P. Pluripotent Stem Cell-Derived Hepatocyte-like Cells: Induction Methods and Applications. Int J Mol Sci 2023; 24:11592. [PMID: 37511351 PMCID: PMC10380504 DOI: 10.3390/ijms241411592] [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: 04/17/2023] [Revised: 07/09/2023] [Accepted: 07/12/2023] [Indexed: 07/30/2023] Open
Abstract
The development of regenerative medicine provides new options for the treatment of end-stage liver diseases. Stem cells, such as bone marrow mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells (iPSCs), are effective tools for tissue repair in regenerative medicine. iPSCs are an appropriate source of hepatocytes for the treatment of liver disease due to their unlimited multiplication capacity, their coverage of the entire range of genetics required to simulate human disease, and their evasion of ethical implications. iPSCs have the ability to gradually produce hepatocyte-like cells (HLCs) with homologous phenotypes and physiological functions. However, how to induce iPSCs to differentiate into HLCs efficiently and accurately is still a hot topic. This review describes the existing approaches for inducing the differentiation of iPSCs into HLCs, as well as some challenges faced, and summarizes various parameters for determining the quality and functionality of HLCs. Furthermore, the application of iPSCs for in vitro hepatoprotective drug screening and modeling of liver disease is discussed. In conclusion, iPSCs will be a dependable source of cells for stem-cell therapy to treat end-stage liver disease and are anticipated to facilitate individualized treatment for liver disease in the future.
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Affiliation(s)
- Qiulin Luo
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
| | - Nan Wang
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
| | - Hanyun Que
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
| | - Erziya Mai
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
| | - Yanting Hu
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
| | - Rui Tan
- College of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610032, China
| | - Jian Gu
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
| | - Puyang Gong
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
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15
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Sanchez‐Rubio A, Jayawarna V, Maxwell E, Dalby MJ, Salmeron‐Sanchez M. Keeping It Organized: Multicompartment Constructs to Mimic Tissue Heterogeneity. Adv Healthc Mater 2023; 12:e2202110. [PMID: 36938891 PMCID: PMC11469230 DOI: 10.1002/adhm.202202110] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2022] [Revised: 02/17/2023] [Indexed: 03/21/2023]
Abstract
Tissue engineering aims at replicating tissues and organs to develop applications in vivo and in vitro. In vivo, by engineering artificial constructs using functional materials and cells to provide both physiological form and function. In vitro, by engineering three-dimensional (3D) models to support drug discovery and enable understanding of fundamental biology. 3D culture constructs mimic cell-cell and cell-matrix interactions and use biomaterials seeking to increase the resemblance of engineered tissues with its in vivo homologues. Native tissues, however, include complex architectures, with compartmentalized regions of different properties containing different types of cells that can be captured by multicompartment constructs. Recent advances in fabrication technologies, such as micropatterning, microfluidics or 3D bioprinting, have enabled compartmentalized structures with defined compositions and properties that are essential in creating 3D cell-laden multiphasic complex architectures. This review focuses on advances in engineered multicompartment constructs that mimic tissue heterogeneity. It includes multiphasic 3D implantable scaffolds and in vitro models, including systems that incorporate different regions emulating in vivo tissues, highlighting the emergence and relevance of 3D bioprinting in the future of biological research and medicine.
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Affiliation(s)
| | - Vineetha Jayawarna
- Centre for the Cellular MicroenvironmentUniversity of GlasgowGlasgowG11 6EWUK
| | - Emily Maxwell
- Centre for the Cellular MicroenvironmentUniversity of GlasgowGlasgowG11 6EWUK
| | - Matthew J. Dalby
- Centre for the Cellular MicroenvironmentUniversity of GlasgowGlasgowG11 6EWUK
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16
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Liu JS, Madruga LYC, Yuan Y, Kipper MJ, Khetani SR. Decellularized Liver Nanofibers Enhance and Stabilize the Long-Term Functions of Primary Human Hepatocytes In Vitro. Adv Healthc Mater 2023; 12:e2202302. [PMID: 36947401 PMCID: PMC11469040 DOI: 10.1002/adhm.202202302] [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: 09/08/2022] [Revised: 03/07/2023] [Indexed: 03/23/2023]
Abstract
Owing to significant differences across species in liver functions, in vitro human liver models are used for screening the metabolism and toxicity of compounds, modeling diseases, and cell-based therapies. However, the extracellular matrix (ECM) scaffold used for such models often does not mimic either the complex composition or the nanofibrous topography of native liver ECM. Thus, here novel methods are developed to electrospin decellularized porcine liver ECM (PLECM) and collagen I into nano- and microfibers (≈200-1000 nm) without synthetic polymer blends. Primary human hepatocytes (PHHs) on nanofibers in monoculture or in coculture with nonparenchymal cells (3T3-J2 embryonic fibroblasts or primary human liver endothelial cells) display higher albumin secretion, urea synthesis, and cytochrome-P450 1A2, 2A6, 2C9, and 3A4 enzyme activities than on conventionally adsorbed ECM controls. PHH functions are highest on the collagen/PLECM blended nanofibers (up to 34-fold higher CYP3A4 activity relative to adsorbed ECM) for nearly 7 weeks in the presence of the fibroblasts. In conclusion, it is shown for the first time that ECM composition and topography synergize to enhance and stabilize PHH functions for several weeks in vitro. The nanofiber platform can prove useful for the above applications and to elucidate cell-ECM interactions in the human liver.
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Affiliation(s)
- Jennifer S. Liu
- Department of Biomedical EngineeringUniversity of Illinois at ChicagoChicagoIL60607USA
| | - Liszt Y. C. Madruga
- Department of Chemical & Biological EngineeringColorado State UniversityFort CollinsCO80523‐1370USA
| | - Yang Yuan
- Department of Biomedical EngineeringUniversity of Illinois at ChicagoChicagoIL60607USA
| | - Matt J. Kipper
- Department of Chemical & Biological EngineeringColorado State UniversityFort CollinsCO80523‐1370USA
| | - Salman R. Khetani
- Department of Biomedical EngineeringUniversity of Illinois at ChicagoChicagoIL60607USA
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17
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Ietto G, Iori V, Gritti M, Inversini D, Costantino A, Izunza Barba S, Jiang ZG, Carcano G, Dalla Gasperina D, Pettinato G. Multicellular Liver Organoids: Generation and Importance of Diverse Specialized Cellular Components. Cells 2023; 12:1429. [PMID: 37408262 PMCID: PMC10217024 DOI: 10.3390/cells12101429] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2023] [Revised: 05/11/2023] [Accepted: 05/17/2023] [Indexed: 07/07/2023] Open
Abstract
Over 40,000 patients in the United States are estimated to suffer from end-stage liver disease and acute hepatic failure, for which liver transplantation is the only available therapy. Human primary hepatocytes (HPH) have not been employed as a therapeutic tool due to the difficulty in growing and expanding them in vitro, their sensitivity to cold temperatures, and tendency to dedifferentiate following two-dimensional culture. The differentiation of human-induced pluripotent stem cells (hiPSCs) into liver organoids (LO) has emerged as a potential alternative to orthotropic liver transplantation (OLT). However, several factors limit the efficiency of liver differentiation from hiPSCs, including a low proportion of differentiated cells capable of reaching a mature phenotype, the poor reproducibility of existing differentiation protocols, and insufficient long-term viability in vitro and in vivo. This review will analyze various methodologies being developed to improve hepatic differentiation from hiPSCs into liver organoids, paying particular attention to the use of endothelial cells as supportive cells for their further maturation. Here, we demonstrate why differentiated liver organoids can be used as a research tool for drug testing and disease modeling, or employed as a bridge for liver transplantation following liver failure.
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Affiliation(s)
- Giuseppe Ietto
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Valentina Iori
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Mattia Gritti
- Department of General Surgery, Humanitas Clinical and Research Center, Rozzano, 20089 Milan, Italy
| | - Davide Inversini
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Angelita Costantino
- Department of Drug and Health Sciences, University of Catania, 95124 Catania, Italy;
| | - Sofia Izunza Barba
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
| | - Z. Gordon Jiang
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
| | - Giulio Carcano
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Daniela Dalla Gasperina
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
- Department of Infectious Diseases, ASST-Sette Laghi, 21100 Varese, Italy
| | - Giuseppe Pettinato
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
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18
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Yuan Y, Cotton K, Samarasekera D, Khetani SR. Engineered Platforms for Maturing Pluripotent Stem Cell-Derived Liver Cells for Disease Modeling. Cell Mol Gastroenterol Hepatol 2023; 15:1147-1160. [PMID: 36738860 PMCID: PMC10034210 DOI: 10.1016/j.jcmgh.2023.01.013] [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: 11/08/2022] [Revised: 01/25/2023] [Accepted: 01/26/2023] [Indexed: 02/06/2023]
Abstract
Several liver diseases (eg, hepatitis B/C viruses, alcoholic/nonalcoholic fatty liver, malaria, monogenic diseases, and drug-induced liver injury) significantly impact global mortality and morbidity. Species-specific differences in liver functions limit the use of animals to fully elucidate/predict human outcomes; therefore, in vitro human liver models are used for basic and translational research to complement animal studies. However, primary human liver cells are in short supply and display donor-to-donor variability in viability/quality. In contrast, human hepatocyte-like cells (HLCs) differentiated from induced pluripotent stem cells and embryonic stem cells are a near infinite cell resource that retains the patient/donor's genetic background; however, conventional protocols yield immature phenotypes. HLC maturation can be significantly improved using advanced techniques, such as protein micropatterning to precisely control cell-cell interactions, controlled sized spheroids, organoids with multiple cell types and layers, 3-dimensional bioprinting to spatially control cell populations, microfluidic devices for automated nutrient exchange and to induce liver zonation via soluble factor gradients, and synthetic biology to genetically modify the HLCs to accelerate and enhance maturation. Here, we present design features and characterization for representative advanced HLC maturation platforms and then discuss HLC use for modeling various liver diseases. Lastly, we discuss desirable advances to move this field forward. We anticipate that with continued advances in this space, pluripotent stem cell-derived liver models will provide human-relevant data much earlier in preclinical drug development and reduce animal usage, help elucidate liver disease mechanisms for the discovery of efficacious and safe therapeutics, and be useful as cell-based therapies for patients suffering from end-stage liver failure.
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Affiliation(s)
- Yang Yuan
- Department of Biomedical Engineering, University of Illinois at Chicago, Chicago, Illinois
| | - Kristen Cotton
- Department of Biomedical Engineering, University of Illinois at Chicago, Chicago, Illinois
| | - Dinithi Samarasekera
- Department of Biomedical Engineering, University of Illinois at Chicago, Chicago, Illinois
| | - Salman R Khetani
- Department of Biomedical Engineering, University of Illinois at Chicago, Chicago, Illinois.
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19
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Grimaldi C, Ibraghimov A, Kiessling A, Rattel B, Ji C, Fuller CL, Brennan FR, Regenass-Lechner F, Shenton J, Price KD, Piché MS, Steeves MA, Prell R, Dudal S, Kronenberg S, Freebern W, Blanset D. Current nonclinical approaches for immune assessments of immuno-oncology biotherapeutics. Drug Discov Today 2023; 28:103440. [PMID: 36375739 DOI: 10.1016/j.drudis.2022.103440] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Revised: 08/30/2022] [Accepted: 11/08/2022] [Indexed: 11/13/2022]
Abstract
Harnessing the immune system to kill tumors has been revolutionary and, as a result, has had an enormous benefit for patients in extending life and resulting in effective cures in some. However, activation of the immune system can come at the cost of undesirable adverse events such as cytokine release syndrome, immune-related adverse events, on-target/off-tumor toxicity, neurotoxicity and tumor lysis syndrome, which are safety risks that can be challenging to assess non-clinically. This article provides a review of the biology and mechanisms that can result in immune-mediated adverse effects and describes industry approaches using in vitro and in vivo models to aid in the nonclinical safety risk assessments for immune-oncology modalities. Challenges and limitations of knowledge and models are also discussed.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | | | | | | | | | - Sherri Dudal
- Roche Pharmaceutical Research and Early Development, United States
| | - Sven Kronenberg
- Roche Pharmaceutical Research and Early Development, United States
| | | | - Diann Blanset
- Boehringer Ingelheim Pharmaceuticals, Inc., United States.
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20
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Deguchi S, Takayama K. State-of-the-art liver disease research using liver-on-a-chip. Inflamm Regen 2022; 42:62. [PMID: 36494740 PMCID: PMC9733013 DOI: 10.1186/s41232-022-00248-0] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2022] [Accepted: 11/30/2022] [Indexed: 12/13/2022] Open
Abstract
To understand disease pathophysiologies, models that recapitulate human functions are necessary. In vitro models that consist of human cells are preferred to ones using animal cells, because organ functions can vary from species to species. However, conventional in vitro models do not recapitulate human organ functions well. Organ-on-a-chip technology provides a reliable in vitro model of the functional units of human organs. Organ-on-a-chip technology uses microfluidic devices and their accessories to impart organ functions to human cells. Using microfluidic devices, we can co-culture multiple cell types that compose human organs. Moreover, we can culture human cells under physiologically relevant stresses, such as mechanical and shear stresses. Current organ-on-a-chip technology can reproduce the functions of several organs including the liver. Because it is difficult to maintain the function of human hepatocytes, which are the gold standard of in vitro liver models, under conventional culture conditions, the application of liver-on-a-chips to liver disease research is expected. This review introduces the current status and future prospects of liver-on-a-chips in liver disease research.
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Affiliation(s)
- Sayaka Deguchi
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507 Japan
- Department of Medical Science, Graduate School of Medicine, Kyoto University, Kyoto, 606-8507 Japan
| | - Kazuo Takayama
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507 Japan
- AMED-CREST, Japan Agency for Medical Research and Development (AMED), Tokyo, 100-0004 Japan
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21
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Modulation of human iPSC-derived hepatocyte phenotype via extracellular matrix microarrays. Acta Biomater 2022; 153:216-230. [PMID: 36115650 PMCID: PMC9869484 DOI: 10.1016/j.actbio.2022.09.013] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2022] [Revised: 08/28/2022] [Accepted: 09/07/2022] [Indexed: 01/26/2023]
Abstract
In vitro human liver models are essential for drug screening, disease modeling, and cell-based therapies. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHeps) mitigate sourcing limitations of primary human hepatocytes (PHHs) and enable precision medicine; however, current protocols yield iHeps with very low differentiated functions. The composition and stiffness of liver's extracellular matrix (ECM) cooperatively regulate hepatic phenotype in vivo, but such effects on iHeps remain unelucidated. Here, we utilized ECM microarrays and high content imaging to assess human iHep attachment and functions on ten major liver ECM proteins in single and two-way combinations robotically spotted onto polyacrylamide gels of liver-like stiffnesses; microarray findings were validated using hydrogel-conjugated multiwell plates. Collagen-IV supported higher iHep attachment than collagen-I over 2 weeks on 1 kPa, while laminin and its combinations with collagen-III, fibronectin, tenascin C, or hyaluronic acid led to both high iHep attachment and differentiated functions; laminin and its combination with tenascin or fibronectin led to similar albumin expression in iHeps and PHHs. Additionally, several collagen-IV-, laminin-, fibronectin-, and collagen-V-containing combinations on 1 kPa led to similar or higher CYP3A4 staining in iHeps than PHHs. Lastly, collagen-I or -III mixed with laminin, collagen-IV mixed with lumican, and collagen-V mixed with fibronectin led to high and stable functional output (albumin/urea secretions; CYP1A2/2C9/3A4 activities) in iHep cultures versus declining PHH numbers/functions for 3 weeks within multiwell plates containing 1 kPa hydrogels. Ultimately, these platforms can help elucidate ECM's role in liver diseases and serve as building blocks of engineered tissues for applications. STATEMENT OF SIGNIFICANCE: We utilized high-throughput extracellular matrix (ECM) microarrays and high content imaging to assess the attachment and differentiated functions of iPSC-derived human hepatocyte-like cells (iHep) on major liver ECM protein combinations spotted onto polyacrylamide gels of liver-like stiffnesses. We observed that iHep responses are regulated in unexpected ways via the cooperation between ECM stiffness and protein composition. Using this approach, we induced mature functions in iHeps on substrates of physiological stiffness and select ECM coatings at higher levels over 3+ weeks than analogous primary human hepatocyte cultures, which is useful for building platforms for drug screening, disease modeling, and regenerative medicine.
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22
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Valdiviezo A, Brown GE, Michell AR, Trinconi CM, Bodke VV, Khetani SR, Luo YS, Chiu WA, Rusyn I. Reanalysis of Trichloroethylene and Tetrachloroethylene Metabolism to Glutathione Conjugates Using Human, Rat, and Mouse Liver in Vitro Models to Improve Precision in Risk Characterization. ENVIRONMENTAL HEALTH PERSPECTIVES 2022; 130:117009. [PMID: 36445294 PMCID: PMC9707501 DOI: 10.1289/ehp12006] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/15/2022] [Revised: 10/16/2022] [Accepted: 11/15/2022] [Indexed: 06/16/2023]
Abstract
BACKGROUND Both trichloroethylene (TCE) and tetrachloroethylene (PCE) are high-priority chemicals subject to numerous human health risk evaluations by a range of agencies. Metabolism of TCE and PCE determines their ultimate toxicity; important uncertainties exist in quantitative characterization of metabolism to genotoxic moieties through glutathione (GSH) conjugation and species differences therein. OBJECTIVES This study aimed to address these uncertainties using novel in vitro liver models, interspecies comparison, and a sensitive assay for quantification of GSH conjugates of TCE and PCE, S-(1,2-dichlorovinyl)glutathione (DCVG) and S-(1,2,2-trichlorovinyl) glutathione (TCVG), respectively. METHODS Liver in vitro models used herein were suspension, 2-D culture, and micropatterned coculture (MPCC) with primary human, rat, and mouse hepatocytes, as well as human induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep). RESULTS We found that, although efficiency of metabolism varied among models, consistent with known differences in their metabolic capacity, formation rates of DCVG and TCVG generally followed the patterns human ≥ rat ≥ mouse , and primary hepatocytes > iHep . Data derived from MPCC were most consistent with estimates from physiologically based pharmacokinetic models calibrated to in vivo data. DISCUSSION For TCE, the new data provided additional empirical support for inclusion of GSH conjugation-mediated kidney effects as critical for the derivation of noncancer toxicity values. For PCE, the data reduced previous uncertainties regarding the extent of TCVG formation in humans; this information was used to update several candidate kidney-specific noncancer toxicity values. Overall, MPCC-derived data provided physiologically relevant estimates of GSH-mediated metabolism of TCE and PCE to reduce uncertainties in interspecies extrapolation that constrained previous risk evaluations, thereby increasing the precision of risk characterizations of these high-priority toxicants. https://doi.org/10.1289/EHP12006.
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Affiliation(s)
- Alan Valdiviezo
- Interdisciplinary Faculty of Toxicology, Texas A&M University, College Station, Texas, USA
- Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA
| | - Grace E. Brown
- Department of Biomedical Engineering, University of Illinois Chicago, Illinois, USA
| | - Ashlin R. Michell
- Department of Biomedical Engineering, University of Illinois Chicago, Illinois, USA
| | | | - Vedant V. Bodke
- Department of Biomedical Engineering, University of Illinois Chicago, Illinois, USA
| | - Salman R. Khetani
- Department of Biomedical Engineering, University of Illinois Chicago, Illinois, USA
| | - Yu-Syuan Luo
- Interdisciplinary Faculty of Toxicology, Texas A&M University, College Station, Texas, USA
- Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA
| | - Weihsueh A. Chiu
- Interdisciplinary Faculty of Toxicology, Texas A&M University, College Station, Texas, USA
- Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA
| | - Ivan Rusyn
- Interdisciplinary Faculty of Toxicology, Texas A&M University, College Station, Texas, USA
- Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA
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23
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Wesley BT, Ross ADB, Muraro D, Miao Z, Saxton S, Tomaz RA, Morell CM, Ridley K, Zacharis ED, Petrus-Reurer S, Kraiczy J, Mahbubani KT, Brown S, Garcia-Bernardo J, Alsinet C, Gaffney D, Horsfall D, Tysoe OC, Botting RA, Stephenson E, Popescu DM, MacParland S, Bader G, McGilvray ID, Ortmann D, Sampaziotis F, Saeb-Parsy K, Haniffa M, Stevens KR, Zilbauer M, Teichmann SA, Vallier L. Single-cell atlas of human liver development reveals pathways directing hepatic cell fates. Nat Cell Biol 2022; 24:1487-1498. [PMID: 36109670 PMCID: PMC7617064 DOI: 10.1038/s41556-022-00989-7] [Citation(s) in RCA: 63] [Impact Index Per Article: 21.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2020] [Accepted: 07/29/2022] [Indexed: 12/14/2022]
Abstract
The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances have been hampered by the lack of knowledge concerning human hepatic development. Here, we addressed this limitation by describing the developmental trajectories of different cell types that make up the human liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing essential roles during organogenesis. We utilized this information to derive bipotential hepatoblast organoids and then exploited this model system to validate the importance of signalling pathways in hepatocyte and cholangiocyte specification. Further insights into hepatic maturation also enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a platform to investigate the basic mechanisms directing human liver development and to produce cell types for clinical applications.
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Affiliation(s)
- Brandon T Wesley
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Alexander D B Ross
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
- Department of Paediatrics, University of Cambridge, Cambridge, UK
| | - Daniele Muraro
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
- Wellcome Sanger Institute, Hinxton, UK
| | - Zhichao Miao
- Wellcome Sanger Institute, Hinxton, UK
- European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Cambridge, UK
| | - Sarah Saxton
- Departments of Bioengineering and Pathology, University of Washington, Seattle, WA, USA
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA
| | - Rute A Tomaz
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Carola M Morell
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Katherine Ridley
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Paediatrics, University of Cambridge, Cambridge, UK
| | - Ekaterini D Zacharis
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Sandra Petrus-Reurer
- Department of Surgery, University of Cambridge, Cambridge, UK
- NIHR Cambridge Biomedical Research Centre, Cambridge, UK
| | - Judith Kraiczy
- Department of Paediatrics, University of Cambridge, Cambridge, UK
| | | | - Stephanie Brown
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | | | | | | | - Dave Horsfall
- Digital Institute, Newcastle University, Newcastle upon Tyne, UK
| | - Olivia C Tysoe
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Rachel A Botting
- Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK
| | - Emily Stephenson
- Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK
| | | | | | - Gary Bader
- University of Toronto, Toronto, Ontario, Canada
| | - Ian D McGilvray
- Multi-Organ Transplant Program, Toronto General Hospital Research Institute, Toronto, Ontario, Canada
| | - Daniel Ortmann
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Fotios Sampaziotis
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Surgery, University of Cambridge, Cambridge, UK
| | - Kourosh Saeb-Parsy
- Department of Surgery, University of Cambridge, Cambridge, UK
- NIHR Cambridge Biomedical Research Centre, Cambridge, UK
| | - Muzlifah Haniffa
- Wellcome Sanger Institute, Hinxton, UK
- Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK
- Department of Dermatology and NIHR Newcastle Biomedical Research Centre, Newcastle Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK
| | - Kelly R Stevens
- Departments of Bioengineering and Pathology, University of Washington, Seattle, WA, USA
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA
| | - Matthias Zilbauer
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
- Department of Paediatrics, University of Cambridge, Cambridge, UK
| | - Sarah A Teichmann
- Wellcome Sanger Institute, Hinxton, UK
- Theory of Condensed Matter Group, Cavendish Laboratory, University of Cambridge, Cambridge, UK
| | - Ludovic Vallier
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.
- Department of Surgery, University of Cambridge, Cambridge, UK.
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McDuffie D, Barr D, Agarwal A, Thomas E. Physiologically relevant microsystems to study viral infection in the human liver. Front Microbiol 2022; 13:999366. [PMID: 36246284 PMCID: PMC9555087 DOI: 10.3389/fmicb.2022.999366] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2022] [Accepted: 08/29/2022] [Indexed: 11/13/2022] Open
Abstract
Viral hepatitis is a leading cause of liver disease and mortality. Infection can occur acutely or chronically, but the mechanisms that govern the clearance of virus or lack thereof are poorly understood and merit further investigation. Though cures for viral hepatitis have been developed, they are expensive, not readily accessible in vulnerable populations and some patients may remain at an increased risk of developing hepatocellular carcinoma (HCC) even after viral clearance. To sustain infection in vitro, hepatocytes must be fully mature and remain in a differentiated state. However, primary hepatocytes rapidly dedifferentiate in conventional 2D in vitro platforms. Physiologically relevant or physiomimetic microsystems, are increasingly popular alternatives to traditional two-dimensional (2D) monocultures for in vitro studies. Physiomimetic systems reconstruct and incorporate elements of the native cellular microenvironment to improve biologic functionality in vitro. Multiple elements contribute to these models including ancillary tissue architecture, cell co-cultures, matrix proteins, chemical gradients and mechanical forces that contribute to increased viability, longevity and physiologic function for the tissue of interest. These microsystems are used in a wide variety of applications to study biological phenomena. Here, we explore the use of physiomimetic microsystems as tools for studying viral hepatitis infection in the liver and how the design of these platforms is tailored for enhanced investigation of the viral lifecycle when compared to conventional 2D cell culture models. Although liver-based physiomimetic microsystems are typically applied in the context of drug studies, the platforms developed for drug discovery purposes offer a solid foundation to support studies on viral hepatitis. Physiomimetic platforms may help prolong hepatocyte functionality in order to sustain chronic viral hepatitis infection in vitro for studying virus-host interactions for prolonged periods.
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Affiliation(s)
- Dennis McDuffie
- Department of Biomedical Engineering, University of Miami, Coral Gables, FL, United States
| | - David Barr
- Department of Pathology and Laboratory Medicine, University of Miami Miller School of Medicine, Miami, FL, United States
| | - Ashutosh Agarwal
- Department of Biomedical Engineering, University of Miami, Coral Gables, FL, United States
- Desai Sethi Urology Institute, University of Miami Miller School of Medicine, Miami, FL, United States
- Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL, United States
| | - Emmanuel Thomas
- Department of Biomedical Engineering, University of Miami, Coral Gables, FL, United States
- Department of Pathology and Laboratory Medicine, University of Miami Miller School of Medicine, Miami, FL, United States
- Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL, United States
- Schiff Center for Liver Diseases, University of Miami Miller School of Medicine, Miami, FL, United States
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25
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Construction of a culture protocol for functional bile canaliculi formation to apply human iPS cell-derived hepatocytes for cholestasis evaluation. Sci Rep 2022; 12:15192. [PMID: 36071090 PMCID: PMC9452549 DOI: 10.1038/s41598-022-19469-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2022] [Accepted: 08/30/2022] [Indexed: 11/08/2022] Open
Abstract
Cholestatic toxicity causes the failure of pharmaceutical agents during drug development and, thus, should be identified at an early stage of drug discovery and development. The formation of functional bile canaliculi in human hepatocytes is required for in vitro cholestasis toxicity tests conducted during the early stage of drug development. In this study, we investigated the culture conditions required for the formation of bile canaliculi using human-induced pluripotent stem cell-derived hepatocytes (hiPSC-Heps). When hiPSC-Heps were sandwich-cultured under the condition we established, extended bile canaliculi were formed on the whole well surfaces. Biliary efflux transporters were localized in the formed bile canaliculi structures which had junctional complexes. After the model substrates of the biliary efflux transporters were taken up into cells, their subsequent excretion into the bile canaliculi was observed and was found to be impeded by each inhibitor of the biliary efflux transporter. These findings suggest that bile canaliculi have transporter-specific bile excretion abilities. We will continue to study the application of this culture protocol to cell-based cholestasis assay system. As a result, the culture protocol could lead to a highly predictable, robust cell-based cholestasis assay system because it forms functional bile canaliculi reproducibly and efficiently.
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26
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Laemmle A, Poms M, Hsu B, Borsuk M, Rüfenacht V, Robinson J, Sadowski MC, Nuoffer J, Häberle J, Willenbring H. Aquaporin 9 induction in human iPSC-derived hepatocytes facilitates modeling of ornithine transcarbamylase deficiency. Hepatology 2022; 76:646-659. [PMID: 34786702 PMCID: PMC9295321 DOI: 10.1002/hep.32247] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/11/2021] [Revised: 10/30/2021] [Accepted: 11/14/2021] [Indexed: 12/16/2022]
Abstract
BACKGROUND AND AIMS Patient-derived human-induced pluripotent stem cells (hiPSCs) differentiated into hepatocytes (hiPSC-Heps) have facilitated the study of rare genetic liver diseases. Here, we aimed to establish an in vitro liver disease model of the urea cycle disorder ornithine transcarbamylase deficiency (OTCD) using patient-derived hiPSC-Heps. APPROACH AND RESULTS Before modeling OTCD, we addressed the question of why hiPSC-Heps generally secrete less urea than adult primary human hepatocytes (PHHs). Because hiPSC-Heps are not completely differentiated and maintain some characteristics of fetal PHHs, we compared gene-expression levels in human fetal and adult liver tissue to identify genes responsible for reduced urea secretion in hiPSC-Heps. We found lack of aquaporin 9 (AQP9) expression in fetal liver tissue as well as in hiPSC-Heps, and showed that forced expression of AQP9 in hiPSC-Heps restores urea secretion and normalizes the response to ammonia challenge by increasing ureagenesis. Furthermore, we proved functional ureagenesis by challenging AQP9-expressing hiPSC-Heps with ammonium chloride labeled with the stable isotope [15 N] (15 NH4 Cl) and by assessing enrichment of [15 N]-labeled urea. Finally, using hiPSC-Heps derived from patients with OTCD, we generated a liver disease model that recapitulates the hepatic manifestation of the human disease. Restoring OTC expression-together with AQP9-was effective in fully correcting OTC activity and normalizing ureagenesis as assessed by 15 NH4 Cl stable-isotope challenge. CONCLUSION Our results identify a critical role for AQP9 in functional urea metabolism and establish the feasibility of in vitro modeling of OTCD with hiPSC-Heps. By facilitating studies of OTCD genotype/phenotype correlation and drug screens, our model has potential for improving the therapy of OTCD.
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Affiliation(s)
- Alexander Laemmle
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell ResearchUniversity of California San FranciscoSan FranciscoCaliforniaUSA,Department of PediatricsUniversity Children's HospitalBernSwitzerland,University Institute of Clinical ChemistryUniversity of BernBernSwitzerland
| | - Martin Poms
- Division of Clinical Chemistry and BiochemistryUniversity Children’s Hospital ZurichZurichSwitzerland
| | - Bernadette Hsu
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell ResearchUniversity of California San FranciscoSan FranciscoCaliforniaUSA
| | - Mariia Borsuk
- University Institute of Clinical ChemistryUniversity of BernBernSwitzerland
| | - Véronique Rüfenacht
- Division of Metabolism and Children`s Research CenterUniversity Children’s HospitalZurichSwitzerland
| | - Joshua Robinson
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell ResearchUniversity of California San FranciscoSan FranciscoCaliforniaUSA,Center for Reproductive SciencesUniversity of California San FranciscoSan FranciscoCaliforniaUSA,Department of Obstetrics, Gynecology, and Reproductive SciencesUniversity of California San FranciscoSan FranciscoCaliforniaUSA,Department of PediatricsMedical GeneticsUniversity of California San FranciscoSan FranciscoCaliforniaUSA
| | | | - Jean‐Marc Nuoffer
- Department of PediatricsUniversity Children's HospitalBernSwitzerland,University Institute of Clinical ChemistryUniversity of BernBernSwitzerland
| | - Johannes Häberle
- Division of Metabolism and Children`s Research CenterUniversity Children’s HospitalZurichSwitzerland,Zurich Center for Integrative Human PhysiologyUniversity of ZurichZurichSwitzerland
| | - Holger Willenbring
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell ResearchUniversity of California San FranciscoSan FranciscoCaliforniaUSA,Department of SurgeryDivision of Transplant SurgeryUniversity of California San FranciscoSan FranciscoCaliforniaUSA,Liver CenterUniversity of California San FranciscoSan FranciscoCaliforniaUSA
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27
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Raggi C, Selleri S, M'Callum MA, Paganelli M. Generation of Complex Syngeneic Liver Organoids from Induced Pluripotent Stem Cells to Model Human Liver Pathophysiology. Curr Protoc 2022; 2:e389. [PMID: 35263041 DOI: 10.1002/cpz1.389] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
The study of human liver pathophysiology has been hampered for decades by the lack of easily accessible, robust, and representative in vitro models. The discovery of induced pluripotent stem cells (iPSCs)-which can be generated from patients' somatic cells, engineered to harbor specific mutations, and differentiated into hepatocyte-like cells-opened the way to more meaningful modeling of liver development and disease. Nevertheless, representative modeling of many complex liver conditions requires the recreation of the interplay between hepatocytes and nonparenchymal liver cells. Here we describe protocols we developed to generate and characterize complex human liver organoids composed of iPSC-derived hepatic, endothelial, and mesenchymal cells. With all cell types derived from the same iPSC population, such organoids reproduce the liver niche, allowing for the study of liver development and the modeling of complex inflammatory and fibrotic conditions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Differentiation of human iPSCs into hepatic progenitor cells (hepatoblasts) Basic Protocol 2: Differentiation of human iPSCs into endothelial progenitor cells Support Protocol 1: Characterization of iPSC-derived endothelial progenitor cells Basic Protocol 3: Differentiation of human iPSCs into mesenchymal progenitor cells Support Protocol 2: Characterization of iPSC-derived mesenchymal progenitor cells Basic Protocol 4: Generation of complex syngeneic liver organoids.
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Affiliation(s)
- Claudia Raggi
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine Research Centre, Montreal, Canada
- Morphocell Technologies, Inc., Montreal, Canada
| | - Silvia Selleri
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine Research Centre, Montreal, Canada
| | - Marie-Agnes M'Callum
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine Research Centre, Montreal, Canada
| | - Massimiliano Paganelli
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine Research Centre, Montreal, Canada
- Pediatric Hepatology, CHU Sainte-Justine, Montreal, Canada
- Department of Pediatrics, Faculty of Medicine, University of Montréal, Montreal, Canada
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28
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Raggi C, M'Callum MA, Pham QT, Gaub P, Selleri S, Baratang NV, Mangahas CL, Cagnone G, Reversade B, Joyal JS, Paganelli M. Leveraging interacting signaling pathways to robustly improve the quality and yield of human pluripotent stem cell-derived hepatoblasts and hepatocytes. Stem Cell Reports 2022; 17:584-598. [PMID: 35120625 PMCID: PMC9039749 DOI: 10.1016/j.stemcr.2022.01.003] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2020] [Revised: 01/04/2022] [Accepted: 01/05/2022] [Indexed: 12/24/2022] Open
Abstract
Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs) have shown great potential as an alternative to primary human hepatocytes (PHHs) for in vitro modeling. Several differentiation protocols have been described to direct PSCs toward the hepatic fate. Here, by leveraging recent knowledge of the signaling pathways involved in liver development, we describe a robust, scalable protocol that allowed us to consistently generate high-quality bipotent human hepatoblasts and HLCs from both embryonic stem cells and induced PSC (iPSCs). Although not yet fully mature, such HLCs were more similar to adult PHHs than were cells obtained with previously described protocols, showing good potential as a physiologically representative alternative to PHHs for in vitro modeling. PSC-derived hepatoblasts effectively generated with this protocol could differentiate into mature hepatocytes and cholangiocytes within syngeneic liver organoids, thus opening the way for representative human 3D in vitro modeling of liver development and pathophysiology.
We generated human hepatoblasts and hepatocyte-like cells (HLCs) from pluripotent stem cells Timed action on Wnt/β-catenin and TGFβ pathways improved maturity and yield of HLCs Hepatoblasts matured into hepatocytes and bile ducts within complex liver organoids The protocol is robust and showed potential for scalability and drug testing
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Affiliation(s)
- Claudia Raggi
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine, Montreal, QC, Canada; Morphocell Technologies Inc., Montreal, QC, Canada
| | - Marie-Agnès M'Callum
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine, Montreal, QC, Canada
| | - Quang Toan Pham
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine, Montreal, QC, Canada
| | - Perrine Gaub
- CHU Sainte-Justine Research Center, Montreal, QC, Canada; Morphocell Technologies Inc., Montreal, QC, Canada
| | - Silvia Selleri
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine, Montreal, QC, Canada
| | | | - Chenicka Lyn Mangahas
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine, Montreal, QC, Canada
| | - Gaël Cagnone
- CHU Sainte-Justine Research Center, Montreal, QC, Canada
| | - Bruno Reversade
- Institute of Molecular and Cell Biology and Institute of Medical Biology, A(∗)STAR, Singapore, Singapore
| | - Jean-Sébastien Joyal
- CHU Sainte-Justine Research Center, Montreal, QC, Canada; Department of Pediatrics, Université de Montréal, Montreal, QC, Canada
| | - Massimiliano Paganelli
- Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine, Montreal, QC, Canada; Department of Pediatrics, Université de Montréal, Montreal, QC, Canada; Morphocell Technologies Inc., Montreal, QC, Canada; Pediatric Hepatology, CHU Sainte-Justine, Montreal, QC, Canada.
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29
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Alvarez-Dominguez JR, Melton DA. Cell maturation: Hallmarks, triggers, and manipulation. Cell 2022; 185:235-249. [PMID: 34995481 PMCID: PMC8792364 DOI: 10.1016/j.cell.2021.12.012] [Citation(s) in RCA: 45] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2021] [Revised: 12/03/2021] [Accepted: 12/10/2021] [Indexed: 02/06/2023]
Abstract
How cells become specialized, or "mature," is important for cell and developmental biology. While maturity is usually deemed a terminal fate, it may be more helpful to consider maturation not as a switch but as a dynamic continuum of adaptive phenotypic states set by genetic and environment programing. The hallmarks of maturity comprise changes in anatomy (form, gene circuitry, and interconnectivity) and physiology (function, rhythms, and proliferation) that confer adaptive behavior. We discuss efforts to harness their chemical (nutrients, oxygen, and growth factors) and physical (mechanical, spatial, and electrical) triggers in vitro and in vivo and how maturation strategies may support disease research and regenerative medicine.
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Affiliation(s)
- Juan R. Alvarez-Dominguez
- Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA
| | - Douglas A. Melton
- Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
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30
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Wang X, Guo C, Guo L, Wang M, Liu M, Song Y, Jiao H, Wei X, Zhao Z, Kaplan DL. Radially Aligned Porous Silk Fibroin Scaffolds as Functional Templates for Engineering Human Biomimetic Hepatic Lobules. ACS APPLIED MATERIALS & INTERFACES 2022; 14:201-213. [PMID: 34929079 DOI: 10.1021/acsami.1c18215] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Bioengineering functional hepatic tissue constructs that physiologically replicate the human native liver tissue in vitro is sought for clinical research and drug discovery. However, the intricate architecture and specific biofunctionality possessed by the native liver tissue remain challenging to mimic in vitro. In the present study, a versatile strategy to fabricate lobular-like silk protein scaffolds with radially aligned lamellar sheets, interconnected channels, and a converging central cavity was designed and implemented. A proof-of-concept study to bioengineer biomimetic hepatic lobules was conducted through coculturing human hepatocytes and primary endothelial cells on these lobular-like scaffolds. Relatively long-term viability of hepatocyte/endothelial cells was found along with cell alignment and organization in vitro. The hepatocytes showed special epithelial polarity and bile duct formation, similar to the liver plate, while the aligned endothelial cells generated endothelial networks, similar to natural hepatic sinuses. This endowed the three-dimensional (3D) tissue constructs with the capability to recapitulate hepatic-like parenchymal-mesenchymal growth patterns in vitro. More importantly, the cocultured hepatocytes outperformed monocultures or monolayer cultures, displaying significantly enhanced hepatocyte functions, including functional gene expression, albumin (ALB) secretion, urea synthesis, and metabolic activity. Thus, this functional unit with a biomimetic phenotype provides a novel technology for bioengineering biomimetic hepatic lobules in vitro, with potential utility as a building block for bioartificial liver (BAL) engineering or as a robust tool for drug metabolism investigation.
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Affiliation(s)
- Xiuli Wang
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
- Biomedical Engineering Department, Tufts University, Medford, Massachusetts 02155, United States
| | - Chengchen Guo
- Biomedical Engineering Department, Tufts University, Medford, Massachusetts 02155, United States
- School of Engineering, Westlake University, Hangzhou, Zhejiang 310023, China
| | - Lina Guo
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - Mingqi Wang
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - Ming Liu
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - Yizhe Song
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - Hui Jiao
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - Xiaoqing Wei
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - Zinan Zhao
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - David L Kaplan
- Biomedical Engineering Department, Tufts University, Medford, Massachusetts 02155, United States
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Youhanna S, Kemas AM, Preiss L, Zhou Y, Shen JX, Cakal SD, Paqualini FS, Goparaju SK, Shafagh RZ, Lind JU, Sellgren CM, Lauschke VM. Organotypic and Microphysiological Human Tissue Models for Drug Discovery and Development-Current State-of-the-Art and Future Perspectives. Pharmacol Rev 2022; 74:141-206. [PMID: 35017176 DOI: 10.1124/pharmrev.120.000238] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2020] [Accepted: 10/12/2021] [Indexed: 12/11/2022] Open
Abstract
The number of successful drug development projects has been stagnant for decades despite major breakthroughs in chemistry, molecular biology, and genetics. Unreliable target identification and poor translatability of preclinical models have been identified as major causes of failure. To improve predictions of clinical efficacy and safety, interest has shifted to three-dimensional culture methods in which human cells can retain many physiologically and functionally relevant phenotypes for extended periods of time. Here, we review the state of the art of available organotypic culture techniques and critically review emerging models of human tissues with key importance for pharmacokinetics, pharmacodynamics, and toxicity. In addition, developments in bioprinting and microfluidic multiorgan cultures to emulate systemic drug disposition are summarized. We close by highlighting important trends regarding the fabrication of organotypic culture platforms and the choice of platform material to limit drug absorption and polymer leaching while supporting the phenotypic maintenance of cultured cells and allowing for scalable device fabrication. We conclude that organotypic and microphysiological human tissue models constitute promising systems to promote drug discovery and development by facilitating drug target identification and improving the preclinical evaluation of drug toxicity and pharmacokinetics. There is, however, a critical need for further validation, benchmarking, and consolidation efforts ideally conducted in intersectoral multicenter settings to accelerate acceptance of these novel models as reliable tools for translational pharmacology and toxicology. SIGNIFICANCE STATEMENT: Organotypic and microphysiological culture of human cells has emerged as a promising tool for preclinical drug discovery and development that might be able to narrow the translation gap. This review discusses recent technological and methodological advancements and the use of these systems for hit discovery and the evaluation of toxicity, clearance, and absorption of lead compounds.
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Affiliation(s)
- Sonia Youhanna
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Aurino M Kemas
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Lena Preiss
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Yitian Zhou
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Joanne X Shen
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Selgin D Cakal
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Francesco S Paqualini
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Sravan K Goparaju
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Reza Zandi Shafagh
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Johan Ulrik Lind
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Carl M Sellgren
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Volker M Lauschke
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
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Xie Y, Yao J, Jin W, Ren L, Li X. Induction and Maturation of Hepatocyte-Like Cells In Vitro: Focus on Technological Advances and Challenges. Front Cell Dev Biol 2021; 9:765980. [PMID: 34901010 PMCID: PMC8662991 DOI: 10.3389/fcell.2021.765980] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2021] [Accepted: 11/08/2021] [Indexed: 12/17/2022] Open
Abstract
Limited by the poor proliferation and restricted sources of adult hepatocytes, there is an urgent need to find substitutes for proliferation and cultivation of mature hepatocytes in vitro for use in disease treatment, drug approval, and toxicity testing. Hepatocyte-like cells (HLCs), which originate from undifferentiated stem cells or modified adult cells, are considered good candidates because of their advantages in terms of cell source and in vitro expansion ability. However, the majority of induced HLCs are in an immature state, and their degree of differentiation is heterogeneous, diminishing their usability in basic research and limiting their clinical application. Therefore, various methods have been developed to promote the maturation of HLCs, including chemical approaches, alteration of cell culture systems, and genetic manipulation, to meet the needs of in vivo transplantation and in vitro model establishment. This review proposes different cell types for the induction of HLCs, and provide a comprehensive overview of various techniques to promote the generation and maturation of HLCs in vitro.
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Affiliation(s)
- Ye Xie
- The First Clinical Medical College, Lanzhou University, Lanzhou, China
| | - Jia Yao
- The First Clinical Medical College, Lanzhou University, Lanzhou, China.,Key Laboratory of Biotherapy and Regenerative Medicine of Gansu Province, Lanzhou, China
| | - Weilin Jin
- The First Clinical Medical College, Lanzhou University, Lanzhou, China.,Institute of Cancer Neuroscience, The First Hospital of Lanzhou University, Lanzhou, China.,The Medical Frontier Innovation Research Center, The First Hospital of Lanzhou University, Lanzhou, China
| | - Longfei Ren
- The First Clinical Medical College, Lanzhou University, Lanzhou, China.,The Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, China
| | - Xun Li
- The First Clinical Medical College, Lanzhou University, Lanzhou, China.,Key Laboratory of Biotherapy and Regenerative Medicine of Gansu Province, Lanzhou, China.,The Medical Frontier Innovation Research Center, The First Hospital of Lanzhou University, Lanzhou, China.,The Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, China.,Hepatopancreatobiliary Surgery Institute of Gansu Province, Lanzhou, China
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33
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Segovia-Zafra A, Di Zeo-Sánchez DE, López-Gómez C, Pérez-Valdés Z, García-Fuentes E, Andrade RJ, Lucena MI, Villanueva-Paz M. Preclinical models of idiosyncratic drug-induced liver injury (iDILI): Moving towards prediction. Acta Pharm Sin B 2021; 11:3685-3726. [PMID: 35024301 PMCID: PMC8727925 DOI: 10.1016/j.apsb.2021.11.013] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2021] [Revised: 11/07/2021] [Accepted: 11/10/2021] [Indexed: 02/08/2023] Open
Abstract
Idiosyncratic drug-induced liver injury (iDILI) encompasses the unexpected harms that prescription and non-prescription drugs, herbal and dietary supplements can cause to the liver. iDILI remains a major public health problem and a major cause of drug attrition. Given the lack of biomarkers for iDILI prediction, diagnosis and prognosis, searching new models to predict and study mechanisms of iDILI is necessary. One of the major limitations of iDILI preclinical assessment has been the lack of correlation between the markers of hepatotoxicity in animal toxicological studies and clinically significant iDILI. Thus, major advances in the understanding of iDILI susceptibility and pathogenesis have come from the study of well-phenotyped iDILI patients. However, there are many gaps for explaining all the complexity of iDILI susceptibility and mechanisms. Therefore, there is a need to optimize preclinical human in vitro models to reduce the risk of iDILI during drug development. Here, the current experimental models and the future directions in iDILI modelling are thoroughly discussed, focusing on the human cellular models available to study the pathophysiological mechanisms of the disease and the most used in vivo animal iDILI models. We also comment about in silico approaches and the increasing relevance of patient-derived cellular models.
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Affiliation(s)
- Antonio Segovia-Zafra
- Unidad de Gestión Clínica de Gastroenterología, Servicio de Farmacología Clínica, Instituto de Investigación Biomédica de Málaga-IBIMA, Hospital Universitario Virgen de la Victoria, Universidad de Málaga, Málaga 29071, Spain
- Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Madrid 28029, Spain
| | - Daniel E. Di Zeo-Sánchez
- Unidad de Gestión Clínica de Gastroenterología, Servicio de Farmacología Clínica, Instituto de Investigación Biomédica de Málaga-IBIMA, Hospital Universitario Virgen de la Victoria, Universidad de Málaga, Málaga 29071, Spain
| | - Carlos López-Gómez
- Unidad de Gestión Clínica de Aparato Digestivo, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Universitario Virgen de la Victoria, Málaga 29010, Spain
| | - Zeus Pérez-Valdés
- Unidad de Gestión Clínica de Gastroenterología, Servicio de Farmacología Clínica, Instituto de Investigación Biomédica de Málaga-IBIMA, Hospital Universitario Virgen de la Victoria, Universidad de Málaga, Málaga 29071, Spain
| | - Eduardo García-Fuentes
- Unidad de Gestión Clínica de Aparato Digestivo, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Universitario Virgen de la Victoria, Málaga 29010, Spain
| | - Raúl J. Andrade
- Unidad de Gestión Clínica de Gastroenterología, Servicio de Farmacología Clínica, Instituto de Investigación Biomédica de Málaga-IBIMA, Hospital Universitario Virgen de la Victoria, Universidad de Málaga, Málaga 29071, Spain
- Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Madrid 28029, Spain
| | - M. Isabel Lucena
- Unidad de Gestión Clínica de Gastroenterología, Servicio de Farmacología Clínica, Instituto de Investigación Biomédica de Málaga-IBIMA, Hospital Universitario Virgen de la Victoria, Universidad de Málaga, Málaga 29071, Spain
- Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Madrid 28029, Spain
- Platform ISCIII de Ensayos Clínicos, UICEC-IBIMA, Málaga 29071, Spain
| | - Marina Villanueva-Paz
- Unidad de Gestión Clínica de Gastroenterología, Servicio de Farmacología Clínica, Instituto de Investigación Biomédica de Málaga-IBIMA, Hospital Universitario Virgen de la Victoria, Universidad de Málaga, Málaga 29071, Spain
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34
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Monckton CP, Brougham-Cook A, Kaylan KB, Underhill GH, Khetani SR. Elucidating Extracellular Matrix and Stiffness Control of Primary Human Hepatocyte Phenotype Via Cell Microarrays. ADVANCED MATERIALS INTERFACES 2021; 8:2101284. [PMID: 35111564 PMCID: PMC8803000 DOI: 10.1002/admi.202101284] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Indexed: 05/30/2023]
Abstract
How the liver's extracellular matrix (ECM) protein composition and stiffness cooperatively regulate primary human hepatocyte (PHH) phenotype is unelucidated. Here, we utilize protein microarrays and high content imaging with single-cell resolution to assess PHH attachment/functions on 10 major liver ECM proteins in single and two-way combinations robotically spotted onto polyacrylamide gels of 1 kPa or 25 kPa stiffness. Albumin, cytochrome-P450 3A4 (CYP3A4), and hepatocyte nuclear factor alpha (HNF4α) positively correlate with each other and cell density on both stiffnesses. The 25 kPa stiffness supports higher average albumin and HNF4α expression after 14 days, while ECM protein composition significantly modulates PHH functions across both stiffnesses. Unlike previous rodent data, PHH functions are highest only when collagen-IV or fibronectin are mixed with specific proteins, whereas non-collagenous proteins without mixed collagens downregulate functions. Combination of collagen-IV and hyaluronic acid retains high CYP3A4 on 1 kPa, whereas collagens-IV and -V better retain HNF4α on 25 kPa over 14 days. Adapting ECM conditions to 96-well plates containing conjugated hydrogels reveals novel regulation of other functions (urea, CYP1A2/2A6/2C9) and drug-mediated CYP induction by the ECM protein composition/stiffness. This high-throughput pipeline can be adapted to elucidate ECM's role in liver diseases and facilitate optimization of engineered tissues.
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Affiliation(s)
- Chase P Monckton
- Department of Biomedical Engineering, University of Illinois at Chicago, 851 South Morgan Street, Chicago, Illinois, 60607, USA
| | - Aidan Brougham-Cook
- Department of Bioengineering, University of Illinois at Urbana-Champaign, 2112 Everitt Laboratory, 1406 West Green Street, Urbana, Illinois, 61801, USA
| | - Kerim B Kaylan
- Department of Bioengineering, University of Illinois at Urbana-Champaign, 2112 Everitt Laboratory, 1406 West Green Street, Urbana, Illinois, 61801, USA
| | - Gregory H Underhill
- Department of Bioengineering, University of Illinois at Urbana-Champaign, 2112 Everitt Laboratory, 1406 West Green Street, Urbana, Illinois, 61801, USA
| | - Salman R Khetani
- Department of Biomedical Engineering, University of Illinois at Chicago, 851 South Morgan Street, Chicago, Illinois, 60607, USA
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35
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Ware BR, Liu JS, Monckton CP, Ballinger KR, Khetani SR. Micropatterned Coculture With 3T3-J2 Fibroblasts Enhances Hepatic Functions and Drug Screening Utility of HepaRG Cells. Toxicol Sci 2021; 181:90-104. [PMID: 33590212 DOI: 10.1093/toxsci/kfab018] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Human liver models are useful for assessing compound metabolism/toxicity; however, primary human hepatocyte (PHH) lots are limited and highly variable in quality/viability. In contrast, cell lines, such as HepaRG, are cheaper and more reproducible surrogates for initial compound screening; however, hepatic functions and sensitivity for drug outcomes need improvement. Here, we show that HepaRGs cocultured with murine embryonic 3T3-J2 fibroblasts, previously shown to induce PHH functions, could address such limitations. We either micropatterned HepaRGs or seeded them "randomly" onto collagen-coated plates before 3T3-J2 coculture. Micropatterned cocultures (HepaRG-MPCCs) secreted 2- to 4-fold more albumin and displayed more stable cytochrome P450 activities than HepaRG conventional confluent monocultures (HepaRG-CCs) and HepaRG micropatterned hepatocytes (HepaRG-MPHs) for 4 weeks, even when excluding dimethyl sulfoxide from the medium. Furthermore, HepaRG-MPCCs had the most albumin-only positive cells (hepatic), lowest cytokeratin 19 (CK19)-only positive cells (cholangiocytic), and highest mean albumin intensity per cell than HepaRG random cocultures and monocultures; however, 80%-84% of HepaRGs remained bipotential (albumin+/CK19+) across all models. The 3T3-J2s also induced higher albumin in HepaRG spheroids than HepaRG-only spheroids. Additionally, although rifampin induced CYP3A4 in HepaRG-MPCCs and HepaRG-CCs, only HepaRG-MPCCs showed the dual omeprazole-mediated CYP1A2/3A4 induction as with PHHs. Lastly, when treated for 6 days with 47 drugs and evaluated for albumin and ATP to make binary hepatotoxicity calls, HepaRG-MPCCs displayed a sensitivity of 54% and specificity of 100% (70%/100% in PHH-MPCCs), whereas HepaRG-CCs misclassified several hepatotoxins. Ultimately, HepaRG-MPCCs could be a more cost-effective and reproducible model than PHHs for executing a tier 1 compound screen.
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Affiliation(s)
- Brenton R Ware
- School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado 80523, USA.,Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois 60607, USA
| | - Jennifer S Liu
- Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois 60607, USA
| | - Chase P Monckton
- Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois 60607, USA
| | - Kimberly R Ballinger
- School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado 80523, USA.,Department of Mechanical Engineering, Colorado State University, Fort Collins, Colorado 80523, USA
| | - Salman R Khetani
- School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado 80523, USA.,Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois 60607, USA.,Department of Mechanical Engineering, Colorado State University, Fort Collins, Colorado 80523, USA
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36
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Cao D, Ge JY, Wang Y, Oda T, Zheng YW. Hepatitis B virus infection modeling using multi-cellular organoids derived from human induced pluripotent stem cells. World J Gastroenterol 2021; 27:4784-4801. [PMID: 34447226 PMCID: PMC8371505 DOI: 10.3748/wjg.v27.i29.4784] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Revised: 04/30/2021] [Accepted: 07/15/2021] [Indexed: 02/06/2023] Open
Abstract
Chronic infection with hepatitis B virus (HBV) remains a global health concern despite the availability of vaccines. To date, the development of effective treatments has been severely hampered by the lack of reliable, reproducible, and scalable in vitro modeling systems that precisely recapitulate the virus life cycle and represent virus-host interactions. With the progressive understanding of liver organogenesis mechanisms, the development of human induced pluripotent stem cell (iPSC)-derived hepatic sources and stromal cellular compositions provides novel strategies for personalized modeling and treatment of liver disease. Further, advancements in three-dimensional culture of self-organized liver-like organoids considerably promote in vitro modeling of intact human liver tissue, in terms of both hepatic function and other physiological characteristics. Combined with our experiences in the investigation of HBV infections using liver organoids, we have summarized the advances in modeling reported thus far and discussed the limitations and ongoing challenges in the application of liver organoids, particularly those with multi-cellular components derived from human iPSCs. This review provides general guidelines for establishing clinical-grade iPSC-derived multi-cellular organoids in modeling personalized hepatitis virus infection and other liver diseases, as well as drug testing and transplantation therapy.
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Affiliation(s)
- Di Cao
- Institute of Regenerative Medicine and Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
| | - Jian-Yun Ge
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Ibaraki, Japan
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, and School of Biotechnology and Heath Sciences, Wuyi University, Jiangmen 529020, Guangdong Province, China
| | - Yun Wang
- Institute of Regenerative Medicine and Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
| | - Tatsuya Oda
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Ibaraki, Japan
| | - Yun-Wen Zheng
- Institute of Regenerative Medicine and Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Ibaraki, Japan
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, and School of Biotechnology and Heath Sciences, Wuyi University, Jiangmen 529020, Guangdong Province, China
- School of Medicine, Yokohama City University, Yokohama 234-0006, Kanagawa, Japan
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Kukla DA, Khetani SR. Bioengineered Liver Models for Investigating Disease Pathogenesis and Regenerative Medicine. Semin Liver Dis 2021; 41:368-392. [PMID: 34139785 DOI: 10.1055/s-0041-1731016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Owing to species-specific differences in liver pathways, in vitro human liver models are utilized for elucidating mechanisms underlying disease pathogenesis, drug development, and regenerative medicine. To mitigate limitations with de-differentiated cultures, bioengineers have developed advanced techniques/platforms, including micropatterned cocultures, spheroids/organoids, bioprinting, and microfluidic devices, for perfusing cell cultures and liver slices. Such techniques improve mature functions and culture lifetime of primary and stem-cell human liver cells. Furthermore, bioengineered liver models display several features of liver diseases including infections with pathogens (e.g., malaria, hepatitis C/B viruses, Zika, dengue, yellow fever), alcoholic/nonalcoholic fatty liver disease, and cancer. Here, we discuss features of bioengineered human liver models, their uses for modeling aforementioned diseases, and how such models are being augmented/adapted for fabricating implantable human liver tissues for clinical therapy. Ultimately, continued advances in bioengineered human liver models have the potential to aid the development of novel, safe, and efficacious therapies for liver disease.
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Affiliation(s)
- David A Kukla
- Deparment of Bioengineering, University of Illinois at Chicago, Chicago, Illinois
| | - Salman R Khetani
- Deparment of Bioengineering, University of Illinois at Chicago, Chicago, Illinois
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38
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Pasqua M, Di Gesù R, Chinnici CM, Conaldi PG, Francipane MG. Generation of Hepatobiliary Cell Lineages from Human Induced Pluripotent Stem Cells: Applications in Disease Modeling and Drug Screening. Int J Mol Sci 2021; 22:8227. [PMID: 34360991 PMCID: PMC8348238 DOI: 10.3390/ijms22158227] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2021] [Revised: 07/28/2021] [Accepted: 07/28/2021] [Indexed: 12/13/2022] Open
Abstract
The possibility to reproduce key tissue functions in vitro from induced pluripotent stem cells (iPSCs) is offering an incredible opportunity to gain better insight into biological mechanisms underlying development and disease, and a tool for the rapid screening of drug candidates. This review attempts to summarize recent strategies for specification of iPSCs towards hepatobiliary lineages -hepatocytes and cholangiocytes-and their use as platforms for disease modeling and drug testing. The application of different tissue-engineering methods to promote accurate and reliable readouts is discussed. Space is given to open questions, including to what extent these novel systems can be informative. Potential pathways for improvement are finally suggested.
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Affiliation(s)
- Mattia Pasqua
- Fondazione Ri.MED, 90133 Palermo, Italy; (M.P.); (R.D.G.); (C.M.C.)
| | - Roberto Di Gesù
- Fondazione Ri.MED, 90133 Palermo, Italy; (M.P.); (R.D.G.); (C.M.C.)
| | - Cinzia Maria Chinnici
- Fondazione Ri.MED, 90133 Palermo, Italy; (M.P.); (R.D.G.); (C.M.C.)
- Dipartimento della Ricerca, IRCCS ISMETT, 90127 Palermo, Italy;
| | | | - Maria Giovanna Francipane
- Fondazione Ri.MED, 90133 Palermo, Italy; (M.P.); (R.D.G.); (C.M.C.)
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA
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Cordero-Espinoza L, Dowbaj AM, Kohler TN, Strauss B, Sarlidou O, Belenguer G, Pacini C, Martins NP, Dobie R, Wilson-Kanamori JR, Butler R, Prior N, Serup P, Jug F, Henderson NC, Hollfelder F, Huch M. Dynamic cell contacts between periportal mesenchyme and ductal epithelium act as a rheostat for liver cell proliferation. Cell Stem Cell 2021; 28:1907-1921.e8. [PMID: 34343491 PMCID: PMC8577825 DOI: 10.1016/j.stem.2021.07.002] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2019] [Revised: 05/19/2021] [Accepted: 07/09/2021] [Indexed: 02/06/2023]
Abstract
In the liver, ductal cells rarely proliferate during homeostasis but do so transiently after tissue injury. These cells can be expanded as organoids that recapitulate several of the cell-autonomous mechanisms of regeneration but lack the stromal interactions of the native tissue. Here, using organoid co-cultures that recapitulate the ductal-to-mesenchymal cell architecture of the portal tract, we demonstrate that a subpopulation of mouse periportal mesenchymal cells exerts dual control on proliferation of the epithelium. Ductal cell proliferation is either induced and sustained or, conversely, completely abolished, depending on the number of direct mesenchymal cell contacts, through a mechanism mediated, at least in part, by Notch signaling. Our findings expand the concept of the cellular niche in epithelial tissues, whereby not only soluble factors but also cell-cell contacts are the key regulatory cues involved in the control of cellular behaviors, suggesting a critical role for cell-cell contacts during regeneration.
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Affiliation(s)
- Lucía Cordero-Espinoza
- Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge CB2 1QN, UK; Wellcome Trust-Medical Research Council Stem Cell Institute, Cambridge CB2 1QR, UK; Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3DY, UK
| | - Anna M Dowbaj
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden 01307, Germany
| | - Timo N Kohler
- Wellcome Trust-Medical Research Council Stem Cell Institute, Cambridge CB2 1QR, UK; Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK
| | - Bernhard Strauss
- Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge CB2 1QN, UK
| | - Olga Sarlidou
- Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge CB2 1QN, UK
| | - German Belenguer
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden 01307, Germany
| | - Clare Pacini
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UK
| | - Nuno P Martins
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden 01307, Germany
| | - Ross Dobie
- Centre for Inflammation Research, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, UK
| | - John R Wilson-Kanamori
- Centre for Inflammation Research, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, UK
| | - Richard Butler
- Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge CB2 1QN, UK
| | - Nicole Prior
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden 01307, Germany
| | - Palle Serup
- Novo Nordisk Foundation Center for Stem Cell Biology (DanStem), University of Copenhagen, Copenhagen 2200, Denmark
| | - Florian Jug
- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden 01307, Germany
| | - Neil C Henderson
- Centre for Inflammation Research, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, UK; MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - Florian Hollfelder
- Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK
| | - Meritxell Huch
- Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge CB2 1QN, UK; Wellcome Trust-Medical Research Council Stem Cell Institute, Cambridge CB2 1QR, UK; Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3DY, UK; Max Planck Institute of Molecular Cell Biology and Genetics, Dresden 01307, Germany.
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Monckton CP, Brown GE, Khetani SR. Latest impact of engineered human liver platforms on drug development. APL Bioeng 2021; 5:031506. [PMID: 34286173 PMCID: PMC8286174 DOI: 10.1063/5.0051765] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2021] [Accepted: 06/21/2021] [Indexed: 01/07/2023] Open
Abstract
Drug-induced liver injury (DILI) is a leading cause of drug attrition, which is partly due to differences between preclinical animals and humans in metabolic pathways. Therefore, in vitro human liver models are utilized in biopharmaceutical practice to mitigate DILI risk and assess related mechanisms of drug transport and metabolism. However, liver cells lose phenotypic functions within 1–3 days in two-dimensional monocultures on collagen-coated polystyrene/glass, which precludes their use to model the chronic effects of drugs and disease stimuli. To mitigate such a limitation, bioengineers have adapted tools from the semiconductor industry and additive manufacturing to precisely control the microenvironment of liver cells. Such tools have led to the fabrication of advanced two-dimensional and three-dimensional human liver platforms for different throughput needs and assay endpoints (e.g., micropatterned cocultures, spheroids, organoids, bioprinted tissues, and microfluidic devices); such platforms have significantly enhanced liver functions closer to physiologic levels and improved functional lifetime to >4 weeks, which has translated to higher sensitivity for predicting drug outcomes and enabling modeling of diseased phenotypes for novel drug discovery. Here, we focus on commercialized engineered liver platforms and case studies from the biopharmaceutical industry showcasing their impact on drug development. We also discuss emerging multi-organ microfluidic devices containing a liver compartment that allow modeling of inter-tissue crosstalk following drug exposure. Finally, we end with key requirements for engineered liver platforms to become routine fixtures in the biopharmaceutical industry toward reducing animal usage and providing patients with safe and efficacious drugs with unprecedented speed and reduced cost.
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Affiliation(s)
- Chase P Monckton
- Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois 60607, USA
| | - Grace E Brown
- Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois 60607, USA
| | - Salman R Khetani
- Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois 60607, USA
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41
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Ferrari E, Ugolini GS, Piutti C, Marzorati S, Rasponi M. Plasma-enhanced protein patterning in a microfluidic compartmentalized platform for multi-organs-on-chip: a liver-tumor model. Biomed Mater 2021; 16. [PMID: 34030149 DOI: 10.1088/1748-605x/ac0454] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2020] [Accepted: 05/24/2021] [Indexed: 11/12/2022]
Abstract
A microfluidic technique is presented for micropatterning protein domains and cell cultures within permanently bonded organs-on-chip devices. This method is based on the use of polydimethylsiloxane layers coupled with the plasma ablation technique for selective protein removal. We show how this technique can be employed to generate a multi-organin vitromodel directly within a microscale platform suitable for pharmacokinetic-based drug screening. We miniaturized a liver model based on micropatterned co-cultures in dual-compartment microfluidic devices. The cytotoxic effect of liver-metabolized Tegafur on colon cancer cell line was assessed using two microfluidic devices where microgrooves and valves systems are used to model drug diffusion between culture compartments. The platforms can reproduce the metabolism of Tegafur in the liver, thus killing colon cancer cells. The proposed plasma-enhanced microfluidic protein patterning method thus successfully combines the ability to generate precise cell micropatterning with the intrinsic advantages of microfluidics in cell biology.
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Affiliation(s)
- Erika Ferrari
- Politecnico di Milano, Department of Electronics, Information and Bioengineering, Via Golgi 39, Milano 20133, Italy
| | - Giovanni Stefano Ugolini
- Politecnico di Milano, Department of Electronics, Information and Bioengineering, Via Golgi 39, Milano 20133, Italy
| | - Claudia Piutti
- Accelera Srl, Viale Pasteur 10, 20014 Nerviano, MI, Italy
| | | | - Marco Rasponi
- Politecnico di Milano, Department of Electronics, Information and Bioengineering, Via Golgi 39, Milano 20133, Italy
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42
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Davidson MD, Khetani SR. Intermittent Starvation Extends the Functional Lifetime of Primary Human Hepatocyte Cultures. Toxicol Sci 2021; 174:266-277. [PMID: 31977024 DOI: 10.1093/toxsci/kfaa003] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Primary human hepatocyte (PHH) cultures have become indispensable to mitigate the risk of adverse drug reactions in human patients. In contrast to dedifferentiating monocultures, coculture with nonparenchymal cells maintains PHH functions for 2-4 weeks. However, because the functional lifespan of PHHs in vivo is 200-400 days, it is desirable to further prolong PHH functions in vitro toward modeling chronic drug exposure and disease progression. Fasting has benefits on the longevity of organisms and the health of tissues such as the liver. We hypothesized that a culturing protocol that mimics dynamic fasting/starvation could activate starvation pathways and prolong PHH functional lifetime. To mimic starvation, serum and hormones were intermittently removed from the culture medium of micropatterned cocultures (MPCCs) containing PHHs organized onto collagen domains and surrounded by 3T3-J2 murine fibroblasts. A weekly 2-day starvation optimally prolonged PHH functional lifetime for 6+ weeks in MPCCs versus a decline after 3 weeks in nonstarved controls. The 2-day starvation also enhanced the functions of PHH monocultures for 2 weeks, suggesting direct effects on PHHs. In MPCCs, starvation activated 5' adenosine monophosphate-activated protein kinase (AMPK) and restricted fibroblast overgrowth onto PHH islands, thereby maintaining hepatic polarity. The effects of starvation on MPCCs were partially recapitulated by activating AMPK using metformin or growth arresting fibroblasts via mitomycin-C. Lastly, starved MPCCs demonstrated lower false positives for drug toxicity tests and higher drug-induced cytochrome-P450 activities versus nonstarved controls even after 5 weeks. In conclusion, intermittent serum/hormone starvation extends PHH functional lifetime toward enabling clinically relevant drug screening.
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Affiliation(s)
- Matthew D Davidson
- School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado.,Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois
| | - Salman R Khetani
- School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado.,Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois.,Department of Mechanical Engineering, Colorado State University, Fort Collins, Colorado
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43
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Lee-Montiel FT, Laemmle A, Charwat V, Dumont L, Lee CS, Huebsch N, Okochi H, Hancock MJ, Siemons B, Boggess SC, Goswami I, Miller EW, Willenbring H, Healy KE. Integrated Isogenic Human Induced Pluripotent Stem Cell-Based Liver and Heart Microphysiological Systems Predict Unsafe Drug-Drug Interaction. Front Pharmacol 2021; 12:667010. [PMID: 34025426 PMCID: PMC8138446 DOI: 10.3389/fphar.2021.667010] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2021] [Accepted: 04/14/2021] [Indexed: 12/14/2022] Open
Abstract
Three-dimensional (3D) microphysiological systems (MPSs) mimicking human organ function in vitro are an emerging alternative to conventional monolayer cell culture and animal models for drug development. Human induced pluripotent stem cells (hiPSCs) have the potential to capture the diversity of human genetics and provide an unlimited supply of cells. Combining hiPSCs with microfluidics technology in MPSs offers new perspectives for drug development. Here, the integration of a newly developed liver MPS with a cardiac MPS—both created with the same hiPSC line—to study drug–drug interaction (DDI) is reported. As a prominent example of clinically relevant DDI, the interaction of the arrhythmogenic gastroprokinetic cisapride with the fungicide ketoconazole was investigated. As seen in patients, metabolic conversion of cisapride to non-arrhythmogenic norcisapride in the liver MPS by the cytochrome P450 enzyme CYP3A4 was inhibited by ketoconazole, leading to arrhythmia in the cardiac MPS. These results establish integration of hiPSC-based liver and cardiac MPSs to facilitate screening for DDI, and thus drug efficacy and toxicity, isogenic in the same genetic background.
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Affiliation(s)
- Felipe T Lee-Montiel
- Departments of Bioengineering, and Materials Science & Engineering, University of California Berkeley, Berkeley, CA, United States
| | - Alexander Laemmle
- Department of Surgery, Division of Transplant Surgery, Liver Center and Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA, United States.,Institute of Clinical Chemistry and Department of Pediatrics, Inselspital, University Hospital Bern, Bern, Switzerland
| | - Verena Charwat
- Departments of Bioengineering, and Materials Science & Engineering, University of California Berkeley, Berkeley, CA, United States
| | - Laure Dumont
- Department of Surgery, Division of Transplant Surgery, Liver Center and Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA, United States
| | - Caleb S Lee
- Departments of Bioengineering, and Materials Science & Engineering, University of California Berkeley, Berkeley, CA, United States
| | - Nathaniel Huebsch
- Departments of Bioengineering, and Materials Science & Engineering, University of California Berkeley, Berkeley, CA, United States
| | - Hideaki Okochi
- Department of Bioengineering and Therapeutic Sciences, Schools of Pharmacy and Medicine, University of California San Francisco, San Francisco, CA, United States
| | | | - Brian Siemons
- Departments of Bioengineering, and Materials Science & Engineering, University of California Berkeley, Berkeley, CA, United States
| | - Steven C Boggess
- Department of Chemistry, University of California Berkeley, Berkeley, CA, United States
| | - Ishan Goswami
- Departments of Bioengineering, and Materials Science & Engineering, University of California Berkeley, Berkeley, CA, United States
| | - Evan W Miller
- Departments of Chemistry and Molecular & Cell Biology, and Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, CA, United States
| | - Holger Willenbring
- Department of Surgery, Division of Transplant Surgery, Liver Center and Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA, United States
| | - Kevin E Healy
- Departments of Bioengineering, and Materials Science & Engineering, University of California Berkeley, Berkeley, CA, United States
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Gough A, Soto-Gutierrez A, Vernetti L, Ebrahimkhani MR, Stern AM, Taylor DL. Human biomimetic liver microphysiology systems in drug development and precision medicine. Nat Rev Gastroenterol Hepatol 2021; 18:252-268. [PMID: 33335282 PMCID: PMC9106093 DOI: 10.1038/s41575-020-00386-1] [Citation(s) in RCA: 64] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 11/02/2020] [Indexed: 02/07/2023]
Abstract
Microphysiology systems (MPS), also called organs-on-chips and tissue chips, are miniaturized functional units of organs constructed with multiple cell types under a variety of physical and biochemical environmental cues that complement animal models as part of a new paradigm of drug discovery and development. Biomimetic human liver MPS have evolved from simpler 2D cell models, spheroids and organoids to address the increasing need to understand patient-specific mechanisms of complex and rare diseases, the response to therapeutic treatments, and the absorption, distribution, metabolism, excretion and toxicity of potential therapeutics. The parallel development and application of transdisciplinary technologies, including microfluidic devices, bioprinting, engineered matrix materials, defined physiological and pathophysiological media, patient-derived primary cells, and pluripotent stem cells as well as synthetic biology to engineer cell genes and functions, have created the potential to produce patient-specific, biomimetic MPS for detailed mechanistic studies. It is projected that success in the development and maturation of patient-derived MPS with known genotypes and fully matured adult phenotypes will lead to advanced applications in precision medicine. In this Review, we examine human biomimetic liver MPS that are designed to recapitulate the liver acinus structure and functions to enhance our knowledge of the mechanisms of disease progression and of the absorption, distribution, metabolism, excretion and toxicity of therapeutic candidates and drugs as well as to evaluate their mechanisms of action and their application in precision medicine and preclinical trials.
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Affiliation(s)
- Albert Gough
- University of Pittsburgh Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA, USA
- Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, USA
- Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
| | - Alejandro Soto-Gutierrez
- University of Pittsburgh Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA, USA
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA
- McGowan Institute of Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
- Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
| | - Lawrence Vernetti
- University of Pittsburgh Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA, USA
- Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, USA
- Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
| | - Mo R Ebrahimkhani
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA
- McGowan Institute of Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
- Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
| | - Andrew M Stern
- University of Pittsburgh Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA, USA
- Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, USA
| | - D Lansing Taylor
- University of Pittsburgh Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA, USA.
- Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, USA.
- Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA.
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Khetani SR. Pluripotent Stem Cell-Derived Human Liver Organoids Enter the Realm of High-Throughput Drug Screening. Gastroenterology 2021; 160:653-655. [PMID: 33307027 DOI: 10.1053/j.gastro.2020.12.005] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/01/2020] [Indexed: 12/21/2022]
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Bircsak KM, DeBiasio R, Miedel M, Alsebahi A, Reddinger R, Saleh A, Shun T, Vernetti LA, Gough A. A 3D microfluidic liver model for high throughput compound toxicity screening in the OrganoPlate®. Toxicology 2021; 450:152667. [PMID: 33359578 DOI: 10.1016/j.tox.2020.152667] [Citation(s) in RCA: 108] [Impact Index Per Article: 27.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2020] [Revised: 12/11/2020] [Accepted: 12/15/2020] [Indexed: 12/18/2022]
Abstract
We report the development, automation and validation of a 3D, microfluidic liver-on-a-chip for high throughput hepatotoxicity screening, the OrganoPlate LiverTox™. The model is comprised of aggregates of induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep) seeded in an extracellular matrix in the organ channel and co-cultured with endothelial cells and THP-1 monoblasts differentiated to macrophages seeded in the vascular channel of the 96 well Mimetas OrganoPlate 2-lane. A key component of high throughput screening is automation and we report a protocol to seed, dose, collect and replenish media and add assay reagents in the OrganoPlate 2-lane using a standard laboratory liquid handling robot. A combination of secretome measurements and image-based analysis was used to demonstrate stable 15 day cell viability, albumin and urea secretion. Over the same time-period, CYP3A4 activity increased and alpha-fetoprotein secretion decreased suggesting further maturation of the iHeps. Troglitazone, a clinical hepatotoxin, was chosen as a control compound for validation studies. Albumin, urea, hepatocyte nuclear size and viability staining provided Robust Z'factors > 0.2 in plates treated 72 h with 180 μM troglitazone compared with a vehicle control. The viability assay provided the most robust statistic for a Robust Z' factor = 0.6. A small library of 159 compounds with known liver effects was added to the OrganoPlate LiverTox model for 72 h at 50 μM and the Toxicological Prioritization scores were calculated. A follow up dose-response evaluation of select hits revealed the albumin assay to be the most sensitive in calculating TC50 values. This platform provides a robust, novel model which can be used for high throughput hepatotoxicity screening.
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Affiliation(s)
| | - Richard DeBiasio
- Drug Discovery Institute and Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, 15260, USA
| | - Mark Miedel
- Drug Discovery Institute and Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, 15260, USA
| | | | | | | | - Tongying Shun
- Drug Discovery Institute and Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, 15260, USA
| | - Lawrence A Vernetti
- Drug Discovery Institute and Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, 15260, USA
| | - Albert Gough
- Drug Discovery Institute and Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, 15260, USA.
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47
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Huang D, Zhang X, Fu X, Zu Y, Sun W, Zhao Y. Liver spheroids on chips as emerging platforms for drug screening. ENGINEERED REGENERATION 2021. [DOI: 10.1016/j.engreg.2021.10.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
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48
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Thompson WL, Takebe T. Human liver model systems in a dish. Dev Growth Differ 2021; 63:47-58. [PMID: 33423319 PMCID: PMC7940568 DOI: 10.1111/dgd.12708] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2020] [Revised: 01/05/2021] [Accepted: 01/06/2021] [Indexed: 12/13/2022]
Abstract
The human adult liver has a multi-cellular structure consisting of large lobes subdivided into lobules containing portal triads and hepatic cords lined by specialized blood vessels. Vital hepatic functions include filtering blood, metabolizing drugs, and production of bile and blood plasma proteins like albumin, among many other functions, which are generally dependent on the location or zone in which the hepatocyte resides in the liver. Due to the liver's intricate structure, there are many challenges to design differentiation protocols to generate more mature functional hepatocytes from human stem cells and maintain the long-term viability and functionality of primary hepatocytes. To this end, recent advancements in three-dimensional (3D) stem cell culture have accelerated the generation of a human miniature liver system, also known as liver organoids, with polarized epithelial cells, supportive cell types and extra-cellular matrix deposition by translating knowledge gained in studies of animal organogenesis and regeneration. To facilitate the efforts to study human development and disease using in vitro hepatic models, a thorough understanding of state-of-art protocols and underlying rationales is essential. Here, we review rapidly evolving 3D liver models, mainly focusing on organoid models differentiated from human cells.
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Affiliation(s)
- Wendy L. Thompson
- Division of Gastroenterology, Hepatology & Nutrition, Developmental Biology, Center for Stem Cell and Organoid Medicine (CuSTOM). Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229-3039, USA
| | - Takanori Takebe
- Division of Gastroenterology, Hepatology & Nutrition, Developmental Biology, Center for Stem Cell and Organoid Medicine (CuSTOM). Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229-3039, USA
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA
- Institute of Research, Tokyo Medical and Dental University, Tokyo, Japan
- Communication Design Center, Advanced Medical Research Center, Yokohama City University Graduate School of Medicine, Japan
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49
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Bove G, Mehnert AK, Dao Thi VL. iPSCs for modeling hepatotropic pathogen infections. IPSCS FOR STUDYING INFECTIOUS DISEASES 2021:149-213. [DOI: 10.1016/b978-0-12-823808-0.00013-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2025]
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50
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Gurevich I, Burton SA, Munn C, Ohshima M, Goedland ME, Czysz K, Rajesh D. iPSC-derived hepatocytes generated from NASH donors provide a valuable platform for disease modeling and drug discovery. Biol Open 2020; 9:bio055087. [PMID: 33268331 PMCID: PMC7758638 DOI: 10.1242/bio.055087] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Accepted: 11/16/2020] [Indexed: 12/17/2022] Open
Abstract
Non-alcoholic fatty liver disease (NAFLD) affects 30-40% of adults and 10% of children in the US. About 20% of people with NAFLD develop non-alcoholic steatohepatitis (NASH), which may lead to cirrhosis and liver cancer, and is projected to be a leading cause of liver transplantation in the near future. Human induced pluripotent stem cells (iPSC) from NASH patients are useful for generating a large number of hepatocytes for NASH modeling applications and identification of potential drug targets. We developed a novel defined in vitro differentiation process to generate cryopreservable hepatocytes using an iPSC panel of NASH donors and apparently healthy normal (AHN) controls. iPSC-derived hepatocytes displayed stage specific phenotypic markers, hepatocyte morphology, with bile canaliculi. Importantly, both fresh and cryopreserved definitive endoderm and hepatoblasts successfully differentiated to pure and functional hepatocytes with increased CYP3A4 activity in response to rifampicin and lipid accumulation upon fatty acid (FA) treatment. End-stage hepatocytes integrated into three-dimensional (3D) liver organoids and demonstrated increased levels of albumin secretion compared to aggregates consisting of hepatocytes alone. End-stage hepatocytes derived from NASH donors demonstrated spontaneous lipidosis without FA supplementation, recapitulating a feature of NASH hepatocytes in vivo Cryopreserved hepatocytes generated by this protocol across multiple donors will provide a critical cell source to facilitate the fundamental understanding of NAFLD/NASH biology and potential high throughput screening applications for preclinical evaluation of therapeutic targets.
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Affiliation(s)
- Igor Gurevich
- Life Science R&D Division, FUJIFILM Cellular Dynamics, Inc., 525 Science Drive, Madison, WI 53711, USA
| | - Sarah A Burton
- Life Science R&D Division, FUJIFILM Cellular Dynamics, Inc., 525 Science Drive, Madison, WI 53711, USA
| | - Christie Munn
- Life Science R&D Division, FUJIFILM Cellular Dynamics, Inc., 525 Science Drive, Madison, WI 53711, USA
| | - Makiko Ohshima
- Life Science R&D Division, FUJIFILM Cellular Dynamics, Inc., 525 Science Drive, Madison, WI 53711, USA
| | - Madelyn E Goedland
- Life Science R&D Division, FUJIFILM Cellular Dynamics, Inc., 525 Science Drive, Madison, WI 53711, USA
| | - Katherine Czysz
- Life Science R&D Division, FUJIFILM Cellular Dynamics, Inc., 525 Science Drive, Madison, WI 53711, USA
| | - Deepika Rajesh
- Life Science R&D Division, FUJIFILM Cellular Dynamics, Inc., 525 Science Drive, Madison, WI 53711, USA
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