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1
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Knouse KA. Breaking the rules of cell biology: Lessons from the liver's exceptional regenerative capacity. Mol Biol Cell 2025; 36:pe5. [PMID: 40408597 DOI: 10.1091/mbc.e24-06-0281] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/25/2025] Open
Abstract
The inability of most human organs to regenerate themselves after injury underlies the lifelong morbidity of numerous diseases. As we continue to seek solutions for these intractable conditions, the liver emerges as an inspiring and informative exception. The liver is the only solid organ that can completely regenerate itself. At the core of this extraordinary feat of organ physiology lie two equally exceptional features of cell biology. First, liver regeneration is driven not by stem cells, but rather by the proliferation of the liver's differentiated cells. Second, many of these liver cells are polyploid, yet still able to execute proper cell division. Understanding how liver cells maintain proliferative capacity as differentiated cells and how they execute mitosis faithfully in a polyploid state could offer powerful insights toward engineering regenerative capacity in other organs. The liver thus offers not only proof that mammalian organ regeneration is possible, but also a blueprint for achieving this long-standing goal of regenerative medicine.
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Affiliation(s)
- Kristin A Knouse
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
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2
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Lehrich BM, Delgado ER, Yasaka TM, Liu S, Cao C, Liu Y, Taheri MN, Guan X, Koeppen H, Singh S, Meadows V, Liu JJ, Singh-Varma A, Krutsenko Y, Poddar M, Hitchens TK, Foley LM, Liang B, Rialdi A, Rai RP, Patel P, Riley M, Bell A, Raeman R, Dadali T, Luke JJ, Guccione E, Ebrahimkhani MR, Lujambio A, Chen X, Maier M, Wang Y, Broom W, Tao J, Monga SP. Precision targeting of β-catenin induces tumor reprogramming and immunity in hepatocellular cancers. Nat Commun 2025; 16:5009. [PMID: 40442146 PMCID: PMC12122713 DOI: 10.1038/s41467-025-60457-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2024] [Accepted: 05/21/2025] [Indexed: 06/02/2025] Open
Abstract
First-line immune checkpoint inhibitor (ICI) combinations show responses in subsets of hepatocellular carcinoma (HCC) patients. Nearly half of HCCs are Wnt-active with mutations in CTNNB1 (encoding for β-catenin), AXIN1/2, or APC, and demonstrate heterogeneous and limited benefit to ICI due to an immune excluded tumor microenvironment. We show significant tumor responses in multiple β-catenin-mutated immunocompetent HCC models to a novel siRNA encapsulated in lipid nanoparticle targeting CTNNB1 (LNP-CTNNB1). Both single-cell and spatial transcriptomics reveal cellular and zonal reprogramming, along with activation of immune regulatory transcription factors IRF2 and POU2F1, re-engaged type I/II interferon signaling, and alterations in both innate and adaptive immunity upon β-catenin suppression with LNP-CTNNB1 at early- and advanced-stage disease. Moreover, ICI enhances response to LNP-CTNNB1 in advanced-stage disease by preventing T cell exhaustion and through formation of lymphoid aggregates (LA). In fact, expression of an LA-like gene signature prognosticates survival for patients receiving atezolizumab plus bevacizumab in the IMbrave150 phase III trial and inversely correlates with CTNNB1-mutatational status in this patient cohort. In conclusion, LNP-CTNNB1 is efficacious as monotherapy and in combination with ICI in CTNNB1-mutated HCCs through impacting tumor cell-intrinsic signaling and remodeling global immune surveillance, providing rationale for clinical investigations.
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MESH Headings
- beta Catenin/genetics
- beta Catenin/metabolism
- beta Catenin/antagonists & inhibitors
- Humans
- Liver Neoplasms/immunology
- Liver Neoplasms/genetics
- Liver Neoplasms/drug therapy
- Liver Neoplasms/pathology
- Carcinoma, Hepatocellular/immunology
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/drug therapy
- Carcinoma, Hepatocellular/pathology
- Immune Checkpoint Inhibitors/pharmacology
- Immune Checkpoint Inhibitors/therapeutic use
- Animals
- Mice
- Tumor Microenvironment/immunology
- Tumor Microenvironment/drug effects
- Tumor Microenvironment/genetics
- Antibodies, Monoclonal, Humanized/therapeutic use
- Antibodies, Monoclonal, Humanized/pharmacology
- Bevacizumab/therapeutic use
- RNA, Small Interfering/genetics
- RNA, Small Interfering/administration & dosage
- Cell Line, Tumor
- Mutation
- Nanoparticles/chemistry
- Female
- Cellular Reprogramming
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Affiliation(s)
- Brandon M Lehrich
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, USA
- Medical Scientist Training Program, University of Pittsburgh, Pittsburgh, PA, USA
| | - Evan R Delgado
- Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, USA
- Department of Pathology, Division of Experimental Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Tyler M Yasaka
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, USA
- Medical Scientist Training Program, University of Pittsburgh, Pittsburgh, PA, USA
| | - Silvia Liu
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, USA
| | - Catherine Cao
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Yuqing Liu
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Mohammad N Taheri
- Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, USA
- Department of Pathology, Division of Experimental Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA, USA
| | - Xiangnan Guan
- Translational Medicine, Genentech Inc., San Francisco, CA, USA
| | - Hartmut Koeppen
- Translational Medicine, Genentech Inc., San Francisco, CA, USA
| | - Sucha Singh
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Vik Meadows
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, USA
| | - Jia-Jun Liu
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, USA
| | - Anya Singh-Varma
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Yekaterina Krutsenko
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Minakshi Poddar
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - T Kevin Hitchens
- University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Lesley M Foley
- University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Binyong Liang
- Hepatic Surgery Center, Department of Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Alex Rialdi
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Ravi P Rai
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, USA
| | - Panari Patel
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Madeline Riley
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Aaron Bell
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, USA
| | - Reben Raeman
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, USA
| | | | - Jason J Luke
- UPMC Hillman Cancer Center and University of Pittsburgh, Pittsburgh, PA, USA
| | - Ernesto Guccione
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Mo R Ebrahimkhani
- Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, USA
- Department of Pathology, Division of Experimental Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA, USA
| | - Amaia Lujambio
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Xin Chen
- Cancer Biology Program, University of Hawaii Cancer Center, Honolulu, HI, USA
| | | | - Yulei Wang
- Translational Medicine, Genentech Inc., San Francisco, CA, USA
| | | | - Junyan Tao
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
- Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, USA.
| | - Satdarshan P Monga
- Organ Pathobiology and Therapeutics Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
- Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
- Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA, USA.
- Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
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3
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Puzzo F, Kay MA. The deLIVERed promises of gene therapy: Past, present, and future of liver-directed gene therapy. Mol Ther 2025; 33:1966-1987. [PMID: 40156191 DOI: 10.1016/j.ymthe.2025.03.041] [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: 02/11/2025] [Revised: 03/20/2025] [Accepted: 03/24/2025] [Indexed: 04/01/2025] Open
Abstract
Gene therapy has revolutionized modern medicine by offering innovative treatments for genetic and acquired diseases. The liver has been and continues as a prime target for in vivo gene therapy due to its essential biological functions, vascular access to the major target cell (hepatocytes), and relatively immunotolerant environment. Adeno-associated virus (AAV) vectors have become the cornerstone of liver-directed therapies, demonstrating remarkable success in conditions such as hemophilia A and B, with US Food and Drug Administration (FDA)-approved therapies like etranacogene dezaparvovec, Beqvez, and Roctavian marking milestones in the field. Despite these advances, challenges persist, including vector immunogenicity, species-specific barriers, and high manufacturing costs. Innovative strategies, such as capsid engineering, immune modulation, and novel delivery systems, are continuing to address these issues in expanding the scope of therapeutic applications. Some of the challenges with many new therapies result in the discordance between preclinical success and translation into humans. The advent of various genome-editing tools to repair genomic mutations or insert therapeutic DNAs into precise locations in the genome further enhances the potential for a single-dose medicine that will offer durable life-long therapeutic treatments. As advancements accelerate, liver-targeted gene therapy is poised to continue to transform the treatment landscape for both genetic and acquired disorders, for which unmet challenges remain.
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Affiliation(s)
- Francesco Puzzo
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA; Department of Genetics, Stanford University, Stanford, CA 94305, USA.
| | - Mark A Kay
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA; Department of Genetics, Stanford University, Stanford, CA 94305, USA.
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4
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Thomas H, Carlisle RC. Progress in Gene Therapy for Hereditary Tyrosinemia Type 1. Pharmaceutics 2025; 17:387. [PMID: 40143050 PMCID: PMC11945121 DOI: 10.3390/pharmaceutics17030387] [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: 02/10/2025] [Revised: 03/10/2025] [Accepted: 03/12/2025] [Indexed: 03/28/2025] Open
Abstract
Hereditary Tyrosinemia Type-1 (HT1), an inherited error of metabolism caused by a mutation in the fumarylacetoacetate hydrolase gene, is associated with liver disease, severe morbidity, and early mortality. The use of NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione) has almost eradicated the acute HT1 symptoms and childhood mortality. However, patient outcomes remain unsatisfactory due to the neurocognitive effects of NTBC and the requirement for a strict low-protein diet. Gene therapy (GT) offers a potential single-dose cure for HT1, and there is now abundant preclinical data showing how a range of vector-nucleotide payload combinations could be used with curative intent, rather than continued reliance on amelioration. Unfortunately, there have been no HT1-directed clinical trials reported, and so it is unclear which promising pre-clinical approach has the greatest chance of successful translation. Here, to fill this knowledge gap, available HT1 preclinical data and available clinical trial data pertaining to liver-directed GT for other diseases are reviewed. The aim is to establish which vector-payload combination has the most potential as a one-dose HT1 cure. Analysis provides a strong case for progressing lentiviral-based approaches into clinical trials. However, other vector-payload combinations may be more scientifically and commercially viable, but these options require additional investigation.
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Affiliation(s)
- Helen Thomas
- Department for Continuing Education, University of Oxford, Headington, Oxford OX1 3PJ, UK;
| | - Robert C. Carlisle
- Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Headington, Oxford OX3 7DL, UK
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Du Y, Yang Y, Zhang Y, Zhang F, Wu J, Yin J. Unraveling enhanced liver regeneration in ALPPS: Integrating multi-omics profiling and in vivo CRISPR in mouse models. Hepatol Commun 2025; 9:e0630. [PMID: 40048448 PMCID: PMC11888979 DOI: 10.1097/hc9.0000000000000630] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/25/2024] [Accepted: 12/06/2024] [Indexed: 03/10/2025] Open
Abstract
BACKGROUND Postoperative liver failure due to insufficient liver cell quantity and function remains a major cause of mortality following surgery. Hence, additional investigation and elucidation are required concerning suitable surgeries for promoting in vivo regeneration. METHODS We established the portal vein ligation (PVL) and associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) mouse models to compare their in vivo regeneration capacity. Then, RNA-seq and microRNA-seq were conducted on the livers from both mouse models. Weighted gene co-expression network analysis algorithm was leveraged to identify crucial gene modules. ScRNA-seq analysis was used to understand the distinctions between Signature30high hepatocytes and Signature30low hepatocytes. Moreover, in vivo, validation was performed in fumarylacetoacetate hydrolase knockout mice with gene editing using the CRISPR-cas9 system. A dual luciferase report system was carried out to further identify the regulatory mechanisms. RESULTS RNA-seq analysis revealed that ALPPS could better promote cell proliferation compared to the sham and portal vein ligation models. Moreover, a Plk1-related 30-gene signature was identified to predict the cell state. ScRNA-seq analysis confirmed that signature30high hepatocytes had stronger proliferative ability than signature30low hepatocytes. Using microRNA-seq analysis, we identified 53 microRNAs that were time-dependently reduced after ALPPS. Finally, miR-30a-3p might be able to regulate the expression of Plk1, contributing to the liver regeneration of ALPPS. CONCLUSIONS ALPPS could successfully promote liver regeneration by activating hepatocytes into a proliferative state. Moreover, a Plk1-related 30-gene signature was identified to predict the cell state of hepatocytes. miR-30a-3p might be able to regulate the expression of Plk1, contributing to the liver regeneration of ALPPS.
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Affiliation(s)
- Yuan Du
- Department of Hepatobiliary Surgery, Jiangxi Provincial People’s Hospital, The First Affiliated Hospital of Nanchang Medical College, Nanchang, Jiangxi, China
| | - YiHan Yang
- Jiangxi Provincial Key Laboratory of Respiratory Diseases, Jiangxi Institute of Respiratory Diseases, The Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, China
| | - YiPeng Zhang
- Department of General Surgery, Dalian Rehabilitation Recuperation Center of Joint Logistics Support Force of PLA, Xigang District, Dalian, China
| | - FuYang Zhang
- Department of Hepatobiliary Surgery, Jiangxi Provincial People’s Hospital, The First Affiliated Hospital of Nanchang Medical College, Nanchang, Jiangxi, China
| | - JunJun Wu
- Department of Hepatobiliary Surgery, Jiangxi Provincial People’s Hospital, The First Affiliated Hospital of Nanchang Medical College, Nanchang, Jiangxi, China
| | - JunXiang Yin
- Department of Hepatobiliary Surgery, Jiangxi Provincial People’s Hospital, The First Affiliated Hospital of Nanchang Medical College, Nanchang, Jiangxi, China
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6
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Liu M, Zhou M, Ren X, Xie Y. Establishment and application of murine models of alcoholic liver disease: A narrative review. ALCOHOL, CLINICAL & EXPERIMENTAL RESEARCH 2025; 49:271-284. [PMID: 39715699 DOI: 10.1111/acer.15520] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/02/2024] [Accepted: 11/29/2024] [Indexed: 12/25/2024]
Abstract
In recent years, there have been significant advances in pathological research on alcoholic liver disease (ALD), with suitable animal models making a significant contribution. However, the currently established animal ALD models still have some significant drawbacks, especially the inability to induce the entire human ALD lineage, which may be related to physiological differences between animals and humans. This review comprehensively summarized the most widely used experimental models of ALD, including voluntary drinking, Lieber-DeCarli, Meadows-Cook, Tsukamoto-French, NIAAA, and the "second hit" model. "Second hit" refers to an additional factor that damages the liver. There are various "second hit" models that fall into two main categories: particular diets and drugs. These models can either simulate human drinking patterns more accurately or produce varying degrees of ALD without significantly increasing animal mortality. We introduced the established method of the original models, discussed the advantages and disadvantages of the existing models from the aspects of operability and practicality, and provided existing improvement methods, hoping to provide a reference for future researchers.
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Affiliation(s)
- Mengsi Liu
- Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing International Cooperation Base for Science and Technology on NAFLD Diagnosis, Beijing, China
| | - Mingying Zhou
- Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing International Cooperation Base for Science and Technology on NAFLD Diagnosis, Beijing, China
| | - Xueyi Ren
- Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing International Cooperation Base for Science and Technology on NAFLD Diagnosis, Beijing, China
| | - Yandi Xie
- Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing International Cooperation Base for Science and Technology on NAFLD Diagnosis, Beijing, China
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7
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Shi H, Ding Y, Sun P, Lv Z, Wang C, Ma H, Lu J, Yu B, Li W, Wang C. Chemical approaches targeting the hurdles of hepatocyte transplantation: mechanisms, applications, and advances. Front Cell Dev Biol 2024; 12:1480226. [PMID: 39544361 PMCID: PMC11560891 DOI: 10.3389/fcell.2024.1480226] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2024] [Accepted: 10/21/2024] [Indexed: 11/17/2024] Open
Abstract
Hepatocyte transplantation (HTx) has been a novel cell-based therapy for severe liver diseases, as the donor livers for orthotopic liver transplantation are of great shortage. However, HTx has been confronted with two main hurdles: limited high-quality hepatocyte sources and low cell engraftment and repopulation rate. To cope with, researchers have investigated on various strategies, including small molecule drugs with unique advantages. Small molecules are promising chemical tools to modulate cell fate and function for generating high quality hepatocyte sources. In addition, endothelial barrier, immune responses, and low proliferative efficiency of donor hepatocytes mainly contributes to low cell engraftment and repopulation rate. Interfering these biological processes with small molecules is beneficial for improving cell engraftment and repopulation. In this review, we will discuss the applications and advances of small molecules in modulating cell differentiation and reprogramming for hepatocyte resources and in improving cell engraftment and repopulation as well as its underlying mechanisms.
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Affiliation(s)
- Huanxiao Shi
- Department of Cell Biology, Naval Medical University, Shanghai, China
| | - Yi Ding
- Experimental Teaching Center, Naval Medical University, Shanghai, China
| | - Pingxin Sun
- Department of Cell Biology, Naval Medical University, Shanghai, China
| | - Zhuman Lv
- Department of Cell Biology, Naval Medical University, Shanghai, China
| | - Chunyan Wang
- Department of Cell Biology, Naval Medical University, Shanghai, China
| | - Haoxin Ma
- Department of Cell Biology, Naval Medical University, Shanghai, China
| | - Junyu Lu
- Department of Cell Biology, Naval Medical University, Shanghai, China
| | - Bing Yu
- Department of Cell Biology, Naval Medical University, Shanghai, China
| | - Wenlin Li
- Department of Cell Biology, Naval Medical University, Shanghai, China
- Shanghai Key Laboratory of Cell Engineering, Naval Medical University, Shanghai, China
| | - Chao Wang
- Department of Cell Biology, Naval Medical University, Shanghai, China
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8
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Vonada A, Grompe M. In vivo selection of hepatocytes. Hepatology 2024:01515467-990000000-01066. [PMID: 39787488 DOI: 10.1097/hep.0000000000001143] [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] [Received: 07/18/2024] [Accepted: 09/13/2024] [Indexed: 01/12/2025]
Abstract
The liver is a highly regenerative organ capable of significant proliferation and remodeling during homeostasis and injury responses. Experiments of nature in rare genetic diseases have illustrated that healthy hepatocytes may have a selective advantage, outcompete diseased cells, and result in extensive liver replacement. This observation has given rise to the concept of therapeutic liver repopulation by providing an engineered selective advantage to a subpopulation of beneficial hepatocytes. In vivo selection can greatly enhance the efficiency of both gene and cell transplantation therapies for hepatic diseases. In vivo hepatocyte selection has also enabled the expansion of human hepatocytes in animals, creating novel models of human liver disease and biology. Finally, recent work has shown that somatic mutations produce clonal expansion of injury-resistant hepatocytes in most chronic liver diseases. In this review, we will address the role of hepatocyte selection in disease pathophysiology and therapeutic strategies.
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Affiliation(s)
- Anne Vonada
- Department of Pediatrics, Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, Oregon, USA
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9
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Rivest JF, Carter S, Goupil C, Antérieux P, Cyr D, Ung RV, Dal Soglio D, Mac-Way F, Waters PJ, Paganelli M, Doyon Y. In vivo dissection of the mouse tyrosine catabolic pathway with CRISPR-Cas9 identifies modifier genes affecting hereditary tyrosinemia type 1. Genetics 2024; 228:iyae139. [PMID: 39178380 PMCID: PMC11457941 DOI: 10.1093/genetics/iyae139] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2023] [Accepted: 08/12/2024] [Indexed: 08/25/2024] Open
Abstract
Hereditary tyrosinemia type 1 is an autosomal recessive disorder caused by mutations (pathogenic variants) in fumarylacetoacetate hydrolase, an enzyme involved in tyrosine degradation. Its loss results in the accumulation of toxic metabolites that mainly affect the liver and kidneys and can lead to severe liver disease and liver cancer. Tyrosinemia type 1 has a global prevalence of approximately 1 in 100,000 births but can reach up to 1 in 1,500 births in some regions of Québec, Canada. Mutating functionally related "modifier' genes (i.e. genes that, when mutated, affect the phenotypic impacts of mutations in other genes) is an emerging strategy for treating human genetic diseases. In vivo somatic genome editing in animal models of these diseases is a powerful means to identify modifier genes and fuel treatment development. In this study, we demonstrate that mutating additional enzymes in the tyrosine catabolic pathway through liver-specific genome editing can relieve or worsen the phenotypic severity of a murine model of tyrosinemia type 1. Neonatal gene delivery using recombinant adeno-associated viral vectors expressing Staphylococcus aureus Cas9 under the control of a liver-specific promoter led to efficient gene disruption and metabolic rewiring of the pathway, with systemic effects that were distinct from the phenotypes observed in whole-body knockout models. Our work illustrates the value of using in vivo genome editing in model organisms to study the direct effects of combining pathological mutations with modifier gene mutations in isogenic settings.
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Affiliation(s)
- Jean-François Rivest
- Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Québec City, QC G1V 4G2, Canada
- Université Laval Cancer Research Centre, Québec City, QC G1V 0A6, Canada
| | - Sophie Carter
- Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Québec City, QC G1V 4G2, Canada
- Université Laval Cancer Research Centre, Québec City, QC G1V 0A6, Canada
| | - Claudia Goupil
- Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Québec City, QC G1V 4G2, Canada
- Université Laval Cancer Research Centre, Québec City, QC G1V 0A6, Canada
| | - Pénélope Antérieux
- Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Québec City, QC G1V 4G2, Canada
- Université Laval Cancer Research Centre, Québec City, QC G1V 0A6, Canada
| | - Denis Cyr
- Medical Genetics Service, Dept. Laboratory Medicine and Dept. Pediatrics, Centre Hospitalier Universitaire de Sherbrooke (CHUS), Sherbrooke, QC J1H 5N4, Canada
| | - Roth-Visal Ung
- Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Québec City, QC G1V 4G2, Canada
| | - Dorothée Dal Soglio
- Centre Hospitalier Universitaire Sainte-Justine Research Center, Université de Montréal, Montréal, QC H3T 1C5, Canada
| | - Fabrice Mac-Way
- Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Québec City, QC G1V 4G2, Canada
| | - Paula J Waters
- Medical Genetics Service, Dept. Laboratory Medicine and Dept. Pediatrics, Centre Hospitalier Universitaire de Sherbrooke (CHUS), Sherbrooke, QC J1H 5N4, Canada
| | - Massimiliano Paganelli
- Centre Hospitalier Universitaire Sainte-Justine Research Center, Université de Montréal, Montréal, QC H3T 1C5, Canada
| | - Yannick Doyon
- Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Québec City, QC G1V 4G2, Canada
- Université Laval Cancer Research Centre, Québec City, QC G1V 0A6, Canada
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10
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van Luyk ME, Krotenberg Garcia A, Lamprou M, Suijkerbuijk SJE. Cell competition in primary and metastatic colorectal cancer. Oncogenesis 2024; 13:28. [PMID: 39060237 PMCID: PMC11282291 DOI: 10.1038/s41389-024-00530-5] [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: 01/19/2024] [Revised: 07/05/2024] [Accepted: 07/16/2024] [Indexed: 07/28/2024] Open
Abstract
Adult tissues set the scene for a continuous battle between cells, where a comparison of cellular fitness results in the elimination of weaker "loser" cells. This phenomenon, named cell competition, is beneficial for tissue integrity and homeostasis. In fact, cell competition plays a crucial role in tumor suppression, through elimination of early malignant cells, as part of Epithelial Defense Against Cancer. However, it is increasingly apparent that cell competition doubles as a tumor-promoting mechanism. The comparative nature of cell competition means that mutational background, proliferation rate and polarity all factor in to determine the outcome of these processes. In this review, we explore the intricate and context-dependent involvement of cell competition in homeostasis and regeneration, as well as during initiation and progression of primary and metastasized colorectal cancer. We provide a comprehensive overview of molecular and cellular mechanisms governing cell competition and its parallels with regeneration.
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Affiliation(s)
- Merel Elise van Luyk
- Division of Developmental Biology, Institute of Biodynamics and Biocomplexity, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands
| | - Ana Krotenberg Garcia
- Division of Developmental Biology, Institute of Biodynamics and Biocomplexity, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands
| | - Maria Lamprou
- Division of Developmental Biology, Institute of Biodynamics and Biocomplexity, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands
| | - Saskia Jacoba Elisabeth Suijkerbuijk
- Division of Developmental Biology, Institute of Biodynamics and Biocomplexity, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
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11
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Miura S, Horisawa K, Iwamori T, Tsujino S, Inoue K, Karasawa S, Yamamoto J, Ohkawa Y, Sekiya S, Suzuki A. Hepatocytes differentiate into intestinal epithelial cells through a hybrid epithelial/mesenchymal cell state in culture. Nat Commun 2024; 15:3940. [PMID: 38750036 PMCID: PMC11096382 DOI: 10.1038/s41467-024-47869-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2023] [Accepted: 04/14/2024] [Indexed: 05/18/2024] Open
Abstract
Hepatocytes play important roles in the liver, but in culture, they immediately lose function and dedifferentiate into progenitor-like cells. Although this unique feature is well-known, the dynamics and mechanisms of hepatocyte dedifferentiation and the differentiation potential of dedifferentiated hepatocytes (dediHeps) require further investigation. Here, we employ a culture system specifically established for hepatic progenitor cells to study hepatocyte dedifferentiation. We found that hepatocytes dedifferentiate with a hybrid epithelial/mesenchymal phenotype, which is required for the induction and maintenance of dediHeps, and exhibit Vimentin-dependent propagation, upon inhibition of the Hippo signaling pathway. The dediHeps re-differentiate into mature hepatocytes by forming aggregates, enabling reconstitution of hepatic tissues in vivo. Moreover, dediHeps have an unexpected differentiation potential into intestinal epithelial cells that can form organoids in three-dimensional culture and reconstitute colonic epithelia after transplantation. This remarkable plasticity will be useful in the study and treatment of intestinal metaplasia and related diseases in the liver.
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Affiliation(s)
- Shizuka Miura
- Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan
| | - Kenichi Horisawa
- Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan
| | - Tokuko Iwamori
- Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan
| | - Satoshi Tsujino
- Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan
| | - Kazuya Inoue
- Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan
| | - Satsuki Karasawa
- Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan
| | - Junpei Yamamoto
- Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan
| | - Yasuyuki Ohkawa
- Division of Transcriptomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan
| | - Sayaka Sekiya
- Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan
| | - Atsushi Suzuki
- Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan.
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12
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Zhang Z, Zhang S, Wong HT, Li D, Feng B. Targeted Gene Insertion: The Cutting Edge of CRISPR Drug Development with Hemophilia as a Highlight. BioDrugs 2024; 38:369-385. [PMID: 38489061 PMCID: PMC11055778 DOI: 10.1007/s40259-024-00654-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/15/2024] [Indexed: 03/17/2024]
Abstract
The remarkable advance in gene editing technology presents unparalleled opportunities for transforming medicine and finding cures for hereditary diseases. Human trials of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9)-based therapeutics have demonstrated promising results in disrupting or deleting target sequences to treat specific diseases. However, the potential of targeted gene insertion approaches, which offer distinct advantages over disruption/deletion methods, remains largely unexplored in human trials due to intricate technical obstacles and safety concerns. This paper reviews the recent advances in preclinical studies demonstrating in vivo targeted gene insertion for therapeutic benefits, targeting somatic solid tissues through systemic delivery. With a specific emphasis on hemophilia as a prominent disease model, we highlight advancements in insertion strategies, including considerations of DNA repair pathways, targeting site selection, and donor design. Furthermore, we discuss the complex challenges and recent breakthroughs that offer valuable insights for progressing towards clinical trials.
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Affiliation(s)
- Zhenjie Zhang
- School of Biomedical Sciences, Faculty of Medicine, CUHK-GIBH CAS Joint Research Laboratory on Stem Cell and Regenerative Medicine, The Chinese University of Hong Kong, Room 105A, Lo Kwee-Seong Integrated Biomedical Sciences Building, Area 39, Shatin, NT, Hong Kong SAR, China
- Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Chinese Academy of Sciences, Hong Kong SAR, China
| | - Siqi Zhang
- School of Biomedical Sciences, Faculty of Medicine, CUHK-GIBH CAS Joint Research Laboratory on Stem Cell and Regenerative Medicine, The Chinese University of Hong Kong, Room 105A, Lo Kwee-Seong Integrated Biomedical Sciences Building, Area 39, Shatin, NT, Hong Kong SAR, China
| | - Hoi Ting Wong
- School of Biomedical Sciences, Faculty of Medicine, CUHK-GIBH CAS Joint Research Laboratory on Stem Cell and Regenerative Medicine, The Chinese University of Hong Kong, Room 105A, Lo Kwee-Seong Integrated Biomedical Sciences Building, Area 39, Shatin, NT, Hong Kong SAR, China
| | - Dali Li
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Bo Feng
- School of Biomedical Sciences, Faculty of Medicine, CUHK-GIBH CAS Joint Research Laboratory on Stem Cell and Regenerative Medicine, The Chinese University of Hong Kong, Room 105A, Lo Kwee-Seong Integrated Biomedical Sciences Building, Area 39, Shatin, NT, Hong Kong SAR, China.
- Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Chinese Academy of Sciences, Hong Kong SAR, China.
- Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.
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13
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Chuecos MA, Lagor WR. Liver directed adeno-associated viral vectors to treat metabolic disease. J Inherit Metab Dis 2024; 47:22-40. [PMID: 37254440 PMCID: PMC10687323 DOI: 10.1002/jimd.12637] [Citation(s) in RCA: 9] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/28/2023] [Revised: 05/05/2023] [Accepted: 05/25/2023] [Indexed: 06/01/2023]
Abstract
The liver is the metabolic center of the body and an ideal target for gene therapy of inherited metabolic disorders (IMDs). Adeno-associated viral (AAV) vectors can deliver transgenes to the liver with high efficiency and specificity and a favorable safety profile. Recombinant AAV vectors contain only the transgene cassette, and their payload is converted to non-integrating circular double-stranded DNA episomes, which can provide stable expression from months to years. Insights from cellular studies and preclinical animal models have provided valuable information about AAV capsid serotypes with a high liver tropism. These vectors have been applied successfully in the clinic, particularly in trials for hemophilia, resulting in the first approved liver-directed gene therapy. Lessons from ongoing clinical trials have identified key factors affecting efficacy and safety that were not readily apparent in animal models. Circumventing pre-existing neutralizing antibodies to the AAV capsid, and mitigating adaptive immune responses to transduced cells are critical to achieving therapeutic benefit. Combining the high efficiency of AAV delivery with genome editing is a promising path to achieve more precise control of gene expression. The primary safety concern for liver gene therapy with AAV continues to be the small risk of tumorigenesis from rare vector integrations. Hepatotoxicity is a key consideration in the safety of neuromuscular gene therapies which are applied at substantially higher doses. The current knowledge base and toolkit for AAV is well developed, and poised to correct some of the most severe IMDs with liver-directed gene therapy.
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Affiliation(s)
- Marcel A. Chuecos
- Department of Integrative Physiology, Baylor College of Medicine, Houston, TX USA
- Translational Biology and Molecular Medicine Program, Baylor College of Medicine, Houston, TX USA
| | - William R. Lagor
- Department of Integrative Physiology, Baylor College of Medicine, Houston, TX USA
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14
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Martinez M, Harding CO, Schwank G, Thöny B. State-of-the-art 2023 on gene therapy for phenylketonuria. J Inherit Metab Dis 2024; 47:80-92. [PMID: 37401651 PMCID: PMC10764640 DOI: 10.1002/jimd.12651] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/30/2023] [Revised: 06/13/2023] [Accepted: 06/30/2023] [Indexed: 07/05/2023]
Abstract
Phenylketonuria (PKU) or hyperphenylalaninemia is considered a paradigm for an inherited (metabolic) liver defect and is, based on murine models that replicate all human pathology, an exemplar model for experimental studies on liver gene therapy. Variants in the PAH gene that lead to hyperphenylalaninemia are never fatal (although devastating if untreated), newborn screening has been available for two generations, and dietary treatment has been considered for a long time as therapeutic and satisfactory. However, significant shortcomings of contemporary dietary treatment of PKU remain. A long list of various gene therapeutic experimental approaches using the classical model for human PKU, the homozygous enu2/2 mouse, witnesses the value of this model to develop treatment for a genetic liver defect. The list of experiments for proof of principle includes recombinant viral (AdV, AAV, and LV) and non-viral (naked DNA or LNP-mRNA) vector delivery methods, combined with gene addition, genome, gene or base editing, and gene insertion or replacement. In addition, a list of current and planned clinical trials for PKU gene therapy is included. This review summarizes, compares, and evaluates the various approaches for the sake of scientific understanding and efficacy testing that may eventually pave the way for safe and efficient human application.
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Affiliation(s)
- Michael Martinez
- Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA
| | - Cary O. Harding
- Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA
| | - Gerald Schwank
- Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland
| | - Beat Thöny
- Division of Metabolism, University Children’s Hospital Zurich and Children’s Research Centre, Zurich, Switzerland
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15
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Mitaka T, Ichinohe N, Tanimizu N. "Small Hepatocytes" in the Liver. Cells 2023; 12:2718. [PMID: 38067145 PMCID: PMC10705974 DOI: 10.3390/cells12232718] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2023] [Revised: 11/17/2023] [Accepted: 11/21/2023] [Indexed: 12/18/2023] Open
Abstract
Mature hepatocytes (MHs) in an adult rodent liver are categorized into the following three subpopulations based on their proliferative capability: type I cells (MH-I), which are committed progenitor cells that possess a high growth capability and basal hepatocytic functions; type II cells (MH-II), which possess a limited proliferative capability; and type III cells (MH-III), which lose the ability to divide (replicative senescence) and reach the final differentiated state. These subpopulations may explain the liver's development and growth after birth. Generally, small-sized hepatocytes emerge in mammal livers. The cells are characterized by being morphologically identical to hepatocytes except for their size, which is substantially smaller than that of ordinary MHs. We initially discovered small hepatocytes (SHs) in the primary culture of rat hepatocytes. We believe that SHs are derived from MH-I and play a role as hepatocytic progenitors to supply MHs. The population of MH-I (SHs) is distributed in the whole lobules, a part of which possesses a self-renewal capability, and decreases with age. Conversely, injured livers of experimental models and clinical cases showed the emergence of SHs. Studies demonstrate the involvement of SHs in liver regeneration. SHs that appeared in the injured livers are not a pure population but a mixture of two distinct origins, MH-derived and hepatic-stem-cell-derived cells. The predominant cell-derived SHs depend on the proliferative capability of the remaining MHs after the injury. This review will focus on the SHs that appeared in the liver and discuss the significance of SHs in liver regeneration.
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Affiliation(s)
- Toshihiro Mitaka
- Department of Tissue Development and Regeneration, Institute of Regenerative Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan; (N.I.); (N.T.)
| | - Norihisa Ichinohe
- Department of Tissue Development and Regeneration, Institute of Regenerative Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan; (N.I.); (N.T.)
| | - Naoki Tanimizu
- Department of Tissue Development and Regeneration, Institute of Regenerative Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan; (N.I.); (N.T.)
- Division of Regenerative Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan
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16
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Iakovleva V, Wuestefeld A, Ong ABL, Gao R, Kaya NA, Lee MY, Zhai W, Tam WL, Dan YY, Wuestefeld T. Mfap4: a promising target for enhanced liver regeneration and chronic liver disease treatment. NPJ Regen Med 2023; 8:63. [PMID: 37935709 PMCID: PMC10630300 DOI: 10.1038/s41536-023-00337-9] [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: 03/27/2023] [Accepted: 10/11/2023] [Indexed: 11/09/2023] Open
Abstract
The liver has a remarkable regenerative capacity. Nevertheless, under chronic liver-damaging conditions, this capacity becomes exhausted, allowing the accumulation of fibrotic tissue and leading to end-stage liver disease. Enhancing the endogenous regenerative capacity by targeting regeneration breaks is an innovative therapeutic approach. We set up an in vivo functional genetic screen to identify such regeneration breaks. As the top hit, we identified Microfibril associated protein 4 (Mfap4). Knockdown of Mfap4 in hepatocytes enhances cell proliferation, accelerates liver regeneration, and attenuates chronic liver disease by reducing liver fibrosis. Targeting Mfap4 modulates several liver regeneration-related pathways including mTOR. Our research opens the way to siRNA-based therapeutics to enhance hepatocyte-based liver regeneration.
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Affiliation(s)
- Viktoriia Iakovleva
- Laboratory of In Vivo Genetics and Gene Therapy, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Singapore, 138672, Republic of Singapore
- Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Republic of Singapore
- Cancer Science Institute of Singapore, National University of Singapore, Singapore, 117599, Republic of Singapore
| | - Anna Wuestefeld
- Laboratory of In Vivo Genetics and Gene Therapy, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Singapore, 138672, Republic of Singapore
| | - Agnes Bee Leng Ong
- Laboratory of In Vivo Genetics and Gene Therapy, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Singapore, 138672, Republic of Singapore
| | - Rong Gao
- Laboratory of In Vivo Genetics and Gene Therapy, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Singapore, 138672, Republic of Singapore
| | - Neslihan Arife Kaya
- Laboratory of Translational Cancer Biology, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Singapore, 138672, Republic of Singapore
| | - May Yin Lee
- Laboratory of Translational Cancer Biology, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Singapore, 138672, Republic of Singapore
| | - Weiwei Zhai
- Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, 100101, Beijing, China
- Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, 650223, China
| | - Wai Leong Tam
- Laboratory of Translational Cancer Biology, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Singapore, 138672, Republic of Singapore
- Cancer Science Institute of Singapore, National University of Singapore, Singapore, 117599, Republic of Singapore
| | - Yock Young Dan
- Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Republic of Singapore
- Division of Gastroenterology and Hepatology, National University Health System, Singapore, 119074, Republic of Singapore
| | - Torsten Wuestefeld
- Laboratory of In Vivo Genetics and Gene Therapy, Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Singapore, 138672, Republic of Singapore.
- School of Biological Science, Nanyang University of Singapore, Singapore, 637551, Republic of Singapore.
- National Cancer Centre, Singapore, 169610, Republic of Singapore.
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17
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Prochownik EV, Wang H. Lessons in aging from Myc knockout mouse models. Front Cell Dev Biol 2023; 11:1244321. [PMID: 37621775 PMCID: PMC10446843 DOI: 10.3389/fcell.2023.1244321] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2023] [Accepted: 07/31/2023] [Indexed: 08/26/2023] Open
Abstract
Despite MYC being among the most intensively studied oncogenes, its role in normal development has not been determined as Myc-/- mice do not survival beyond mid-gestation. Myc ± mice live longer than their wild-type counterparts and are slower to accumulate many age-related phenotypes. However, Myc haplo-insufficiency likely conceals other important phenotypes as many high-affinity Myc targets genes continue to be regulated normally. By delaying Myc inactivation until after birth it has recently been possible to study the consequences of its near-complete total body loss and thus to infer its normal function. Against expectation, these "MycKO" mice lived significantly longer than control wild-type mice but manifested a marked premature aging phenotype. This seemingly paradoxical behavior was potentially explained by a >3-fold lower lifetime incidence of cancer, normally the most common cause of death in mice and often Myc-driven. Myc loss accelerated the accumulation of numerous "Aging Hallmarks", including the loss of mitochondrial and ribosomal structural and functional integrity, the generation of reactive oxygen species, the acquisition of genotoxic damage, the detrimental rewiring of metabolism and the onset of senescence. In both mice and humans, normal aging in many tissues was accompaniued by the downregulation of Myc and the loss of Myc target gene regulation. Unlike most mouse models of premature aging, which are based on monogenic disorders of DNA damage recognition and repair, the MycKO mouse model directly impacts most Aging Hallmarks and may therefore more faithfully replicate the normal aging process of both mice and humans. It further establishes that the strong association between aging and cancer can be genetically separated and is maintained by a single gene.
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Affiliation(s)
- Edward V. Prochownik
- Division of Hematology/Oncology, UPMC Children’s Hospital of Pittsburgh, Pittsburgh, PA, United States
- The Department of Microbiology and Molecular Genetics, UPMC, Pittsburgh, PA, United States
- The Hillman Cancer Center of UPMC, Pittsburgh, PA, United States
- The Pittsburgh Liver Research Center, UPMC, Pittsburgh, PA, United States
| | - Huabo Wang
- Division of Hematology/Oncology, UPMC Children’s Hospital of Pittsburgh, Pittsburgh, PA, United States
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18
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Jiang M, Guo R, Ai Y, Wang G, Tang P, Jia X, He B, Yuan Q, Xie X. Small molecule drugs promote repopulation of transplanted hepatocytes by stimulating cell dedifferentiation. JHEP Rep 2023; 5:100670. [PMID: 36873420 PMCID: PMC9976449 DOI: 10.1016/j.jhepr.2023.100670] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2022] [Revised: 12/17/2022] [Accepted: 12/21/2022] [Indexed: 01/15/2023] Open
Abstract
Background & Aims Hepatocyte transplantation has emerged as a possible treatment option for end-stage liver disease. However, an important obstacle to therapeutic success is the low level of engraftment and proliferation of transplanted hepatocytes, which do not survive long enough to exert therapeutic effects. Thus, we aimed to explore the mechanisms of hepatocyte proliferation in vivo and find a way to promote the growth of transplanted hepatocytes. Methods Hepatocyte transplantation was performed in Fah -/- mice to explore the mechanisms of hepatocyte proliferation in vivo. Guided by in vivo regeneration mechanisms, we identified compounds that promote hepatocyte proliferation in vitro. The in vivo effects of these compounds on transplanted hepatocytes were then evaluated. Results The transplanted mature hepatocytes were found to dedifferentiate into hepatic progenitor cells (HPCs), which proliferate and then convert back to a mature state at the completion of liver repopulation. The combination of two small molecules Y-27632 (Y, ROCK inhibitor) and CHIR99021 (C, Wnt agonist) could convert mouse primary hepatocytes into HPCs, which could be passaged for more than 30 passages in vitro. Moreover, YC could stimulate the proliferation of transplanted hepatocytes in Fah -/- livers by promoting their conversion into HPCs. Netarsudil (N) and LY2090314 (L), two clinically used drugs which target the same pathways as YC, could also promote hepatocyte proliferation in vitro and in vivo, by facilitating HPC conversion. Conclusions Our work suggests drugs promoting hepatocyte dedifferentiation may facilitate the growth of transplanted hepatocytes in vivo and may facilitate the application of hepatocyte therapy. Impact and implications Hepatocyte transplantation may be a treatment option for patients with end-stage liver disease. However, one important obstacle to hepatocyte therapy is the low level of engraftment and proliferation of the transplanted hepatocytes. Herein, we show that small molecule compounds which promote hepatocyte proliferation in vitro by facilitating dedifferentiation, could promote the growth of transplanted hepatocytes in vivo and may facilitate the application of hepatocyte therapy.
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Key Words
- (i)HPCs, (induced) hepatic progenitor cells
- A, A-83-01
- ALP, alkaline phosphatase
- ALT, alanine aminotransferase
- AST, aspartate aminotransferase
- C, CHIR99021
- DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine
- Dedifferentiation
- HMM, hepatic maturation medium
- Hepatocyte expansion
- Hepatocyte progenitor cells
- Hepatocyte transplantation
- L, LY2090314
- N, netarsudil
- NTBC, 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclo-hexanedione
- PHx, partial hepatectomy
- RT-PCR, reverse-transcription PCR
- Small molecule compounds
- Y, Y27632
- iMHs, induced mature hepatocytes
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Affiliation(s)
- Mengmeng Jiang
- School of Life Science and Technology, Shanghai Tech University, Shanghai 201210, China.,CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.,University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
| | - Ren Guo
- CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
| | - Yan Ai
- School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, China
| | - Gang Wang
- Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, China
| | - Peilan Tang
- School of Pharmaceutical Science, Nanchang University, Nanchang 330006, PR China
| | - Xiaohui Jia
- CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.,University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
| | - Bingqing He
- School of Life Science and Technology, Shanghai Tech University, Shanghai 201210, China.,CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.,University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
| | - Qianting Yuan
- CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
| | - Xin Xie
- School of Life Science and Technology, Shanghai Tech University, Shanghai 201210, China.,CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.,University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China.,School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, China.,State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
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19
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Chen Y, Chen L, Wu X, Zhao Y, Wang Y, Jiang D, Liu X, Zhou T, Li S, Wei Y, Liu Y, Hu C, Zhou B, Qin J, Ying H, Ding Q. Acute liver steatosis translationally controls the epigenetic regulator MIER1 to promote liver regeneration in a study with male mice. Nat Commun 2023; 14:1521. [PMID: 36934083 PMCID: PMC10024732 DOI: 10.1038/s41467-023-37247-9] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2022] [Accepted: 03/07/2023] [Indexed: 03/20/2023] Open
Abstract
The early phase lipid accumulation is essential for liver regeneration. However, whether this acute lipid accumulation can serve as signals to direct liver regeneration rather than simply providing building blocks for cell proliferation remains unclear. Through in vivo CRISPR screening, we identify MIER1 (mesoderm induction early response 1) as a key epigenetic regulator that bridges the acute lipid accumulation and cell cycle gene expression during liver regeneration in male animals. Physiologically, liver acute lipid accumulation induces the phosphorylation of EIF2S1(eukaryotic translation initiation factor 2), which consequently attenuated Mier1 translation. MIER1 downregulation in turn promotes cell cycle gene expression and regeneration through chromatin remodeling. Importantly, the lipids-EIF2S1-MIER1 pathway is impaired in animals with chronic liver steatosis; whereas MIER1 depletion significantly improves regeneration in these animals. Taken together, our studies identify an epigenetic mechanism by which the early phase lipid redistribution from adipose tissue to liver during regeneration impacts hepatocyte proliferation, and suggest a potential strategy to boost liver regeneration.
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Affiliation(s)
- Yanhao Chen
- CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China.
| | - Lanlan Chen
- CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China
| | - Xiaoshan Wu
- CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China
- School of Pharmacy, East China University of Science and Technology, Shanghai, 200237, P. R. China
| | - Yongxu Zhao
- CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China
| | - Yuchen Wang
- CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China
| | - Dacheng Jiang
- CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China
| | - Xiaojian Liu
- CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China
| | - Tingting Zhou
- CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China
| | - Shuang Li
- CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China
| | - Yuda Wei
- Department of Clinical Laboratory, Linyi People's Hospital, Xuzhou Medical University, Xuzhou, Shandong, 276000, P. R. China
| | - Yan Liu
- CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China
| | - Cheng Hu
- Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, 200233, China
| | - Ben Zhou
- CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China
| | - Jun Qin
- CAS Key Laboratory of Tissue Microenvironment and Tumor, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China
| | - Hao Ying
- CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China
| | - Qiurong Ding
- CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, P. R. China.
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, 100101, Beijing, P. R. China.
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20
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Shafritz DA, Ebrahimkhani MR, Oertel M. Therapeutic Cell Repopulation of the Liver: From Fetal Rat Cells to Synthetic Human Tissues. Cells 2023; 12:529. [PMID: 36831196 PMCID: PMC9954009 DOI: 10.3390/cells12040529] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2022] [Revised: 01/20/2023] [Accepted: 01/26/2023] [Indexed: 02/10/2023] Open
Abstract
Progenitor cells isolated from the fetal liver can provide a unique cell source to generate new healthy tissue mass. Almost 20 years ago, it was demonstrated that rat fetal liver cells repopulate the normal host liver environment via a mechanism akin to cell competition. Activin A, which is produced by hepatocytes, was identified as an important player during cell competition. Because of reduced activin receptor expression, highly proliferative fetal liver stem/progenitor cells are resistant to activin A and therefore exhibit a growth advantage compared to hepatocytes. As a result, transplanted fetal liver cells are capable of repopulating normal livers. Important for cell-based therapies, hepatic stem/progenitor cells containing repopulation potential can be separated from fetal hematopoietic cells using the cell surface marker δ-like 1 (Dlk-1). In livers with advanced fibrosis, fetal epithelial stem/progenitor cells differentiate into functional hepatic cells and out-compete injured endogenous hepatocytes, which cause anti-fibrotic effects. Although fetal liver cells efficiently repopulate the liver, they will likely not be used for human cell transplantation. Thus, utilizing the underlying mechanism of repopulation and developed methods to produce similar growth-advantaged cells in vitro, e.g., human induced pluripotent stem cells (iPSCs), this approach has great potential for developing novel cell-based therapies in patients with liver disease. The present review gives a brief overview of the classic cell transplantation models and various cell sources studied as donor cell candidates. The advantages of fetal liver-derived stem/progenitor cells are discussed, as well as the mechanism of liver repopulation. Moreover, this article reviews the potential of in vitro developed synthetic human fetal livers from iPSCs and their therapeutic benefits.
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Affiliation(s)
- David A. Shafritz
- Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Mo R. Ebrahimkhani
- Division of Experimental Pathology, Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA
- Pittsburgh Liver Research Center (PLRC), University of Pittsburgh, Pittsburgh, PA 15213, USA
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA
| | - Michael Oertel
- Division of Experimental Pathology, Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA
- Pittsburgh Liver Research Center (PLRC), University of Pittsburgh, Pittsburgh, PA 15213, USA
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA
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21
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Wang J, Huang X, Zheng D, Li Q, Mei M, Bao S. PRMT5 determines the pattern of polyploidization and prevents liver from cirrhosis and carcinogenesis. J Genet Genomics 2023; 50:87-98. [PMID: 35500745 DOI: 10.1016/j.jgg.2022.04.008] [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: 03/27/2022] [Revised: 04/12/2022] [Accepted: 04/12/2022] [Indexed: 11/25/2022]
Abstract
Human hepatocellular carcinoma (HCC) occurs almost exclusively in cirrhotic livers. Here, we report that hepatic loss of protein arginine methyltransferase 5 (PRMT5) in mice is sufficient to cause cirrhosis and HCC in a clinically relevant way. Furthermore, pathological polyploidization induced by hepatic loss of PRMT5 promotes liver cirrhosis and hepatic tumorigenesis in aged liver. The loss of PRMT5 leads to hyper-accumulation of P21 and endoreplication-dependent formation of pathological mono-nuclear polyploid hepatocytes. PRMT5 and symmetric dimethylation at histone H4 arginine 3 (H4R3me2s) directly associate with chromatin of P21 to suppress its transcription. More importantly, loss of P21 rescues the pathological mono-nuclear polyploidy and prevents PRMT5-deficiency-induced liver cirrhosis and HCC. Thus, our results indicate that PRMT5-mediated symmetric dimethylation at histone H4 arginine 3 (H4R3me2s) is crucial for preventing pathological polyploidization, liver cirrhosis and tumorigenesis in mouse liver.
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Affiliation(s)
- Jincheng Wang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
| | - Xiang Huang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; School of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Daoshan Zheng
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; School of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Qiuling Li
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
| | - Mei Mei
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
| | - Shilai Bao
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China; School of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China.
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22
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IL6 supports long-term expansion of hepatocytes in vitro. Nat Commun 2022; 13:7345. [PMID: 36446858 PMCID: PMC9708838 DOI: 10.1038/s41467-022-35167-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2022] [Accepted: 11/21/2022] [Indexed: 11/30/2022] Open
Abstract
Hepatocytes are very difficult to expand in vitro. A few studies have demonstrated that chemical cocktails with growth factors or Wnt ligands can support long-term expansion of hepatocytes via dedifferentiation. However, the culture conditions are complex, and clonal expansion of hepatic progenitors with full differentiation capacity are rarely reported. Here, we discover IL6, combined with EGF and HGF, promotes long-term expansion (>30 passages in ~150 days with theoretical expansion of ~1035 times) of primary mouse hepatocytes in vitro in simple 2D culture, by converting hepatocytes into induced hepatic progenitor cells (iHPCs), which maintain the capacity of differentiation into hepatocytes. IL6 also supports the establishment of single hepatocyte-derived iHPC clones. The summation of the downstream STAT3, ERK and AKT pathways induces a number of transcription factors which support rapid growth. This physiological and simple way may provide ideas for culturing previously difficult-to-culture cell types and support their future applications.
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23
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Guo J, Wang S, Gao Q. Can next-generation humanized mice that reconstituted with both functional human immune system and hepatocytes model the progression of viral hepatitis to hepatocarcinogenesis? Front Med (Lausanne) 2022; 9:1002260. [PMID: 36213658 PMCID: PMC9537463 DOI: 10.3389/fmed.2022.1002260] [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: 07/25/2022] [Accepted: 09/08/2022] [Indexed: 11/30/2022] Open
Abstract
Hepatitis B virus (HBV) and Hepatitis C virus (HCV) chronic infections cause liver immunopathological diseases such as hepatitis, fibrosis, cirrhosis, and hepatocellular carcinomas, which are difficult to treat and continue to be major health problems globally. Due to the species-specific hepato-tropism of HBV and HCV, conventional rodent models are limited in their utility for studying the infection and associated liver immunopathogenesis. Humanized mice reconstituted with both functional human immune system and hepatocytes (HIS-HuHEP mice) have been extremely instrumental for in vivo studies of HBV or HCV infection and human-specific aspects of the progression of liver immunopathogenesis. However, none of the current HIS-HuHEP mice can model the progression of viral hepatitis to hepatocarcinogenesis which may be a notorious result of HBV or HCV chronic infection in patients, suggesting that they were functionally compromised and that there is still significant space to improve and establish next-generation of HIS-HuHEP mice with more sophisticated functions. In this review, we first summarize the principal requirements to establish HIS-HuHEP mice. We then discuss the respective protocols for current HIS-HuHEP mice and their applications, as well as their advantages and disadvantages. We also raise perspectives for further improving and establishing next-generation HIS-HuHEP mice.
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Affiliation(s)
- Jinglong Guo
- Department of Cardiovascular Disease, The First Hospital of Jilin University, Changchun, China
| | - Siyue Wang
- Graduate Program in Immunology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, United States
| | - Qi Gao
- Department of Cardiovascular Disease, The First Hospital of Jilin University, Changchun, China
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24
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Roy N, Alencastro F, Roseman BA, Wilson SR, Delgado ER, May MC, Bhushan B, Bello FM, Jurczak MJ, Shiva S, Locker J, Gingras S, Duncan AW. Dysregulation of Lipid and Glucose Homeostasis in Hepatocyte-Specific SLC25A34 Knockout Mice. THE AMERICAN JOURNAL OF PATHOLOGY 2022; 192:1259-1281. [PMID: 35718058 PMCID: PMC9472157 DOI: 10.1016/j.ajpath.2022.06.002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Revised: 05/18/2022] [Accepted: 06/08/2022] [Indexed: 10/18/2022]
Abstract
Nonalcoholic fatty liver disease (NAFLD) is an epidemic affecting 30% of the US population. It is characterized by insulin resistance, and by defective lipid metabolism and mitochondrial dysfunction in the liver. SLC25A34 is a major repressive target of miR-122, a miR that has a central role in NAFLD and liver cancer. However, little is known about the function of SLC25A34. To investigate SLC25A34 in vitro, mitochondrial respiration and bioenergetics were examined using hepatocytes depleted of Slc25a34 or overexpressing Slc25a34. To test the function of SLC25A34 in vivo, a hepatocyte-specific knockout mouse was generated, and loss of SLC25A34 was assessed in mice maintained on a chow diet and a fast-food diet (FFD), a model for NAFLD. Hepatocytes depleted of Slc25a34 displayed increased mitochondrial biogenesis, lipid synthesis, and ADP/ATP ratio; Slc25a34 overexpression had the opposite effect. In the knockout model on chow diet, SLC25A34 loss modestly affected liver function (altered glucose metabolism was the most pronounced defect). RNA-sequencing revealed changes in metabolic processes, especially fatty acid metabolism. After 2 months on FFD, knockouts had a more severe phenotype, with increased lipid content and impaired glucose tolerance, which was attenuated after longer FFD feeding (6 months). This work thus presents a novel model for studying SLC25A34 in vivo in which SLC25A34 plays a role in mitochondrial respiration and bioenergetics during NAFLD.
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Affiliation(s)
- Nairita Roy
- Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Pittsburgh Liver Research Center, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Frances Alencastro
- Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Pittsburgh Liver Research Center, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Bayley A Roseman
- Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Pittsburgh Liver Research Center, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Sierra R Wilson
- Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Pittsburgh Liver Research Center, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Evan R Delgado
- Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Pittsburgh Liver Research Center, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Meredith C May
- Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Pittsburgh Liver Research Center, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Bharat Bhushan
- Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Pittsburgh Liver Research Center, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Fiona M Bello
- Pittsburgh Liver Research Center, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Medicine, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Michael J Jurczak
- Pittsburgh Liver Research Center, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Medicine, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Sruti Shiva
- Departments of Pharmacology and Chemical Biology, Vascular Medicine Institute, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Joseph Locker
- Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Pittsburgh Liver Research Center, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Sebastien Gingras
- Department of Immunology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Andrew W Duncan
- Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Pittsburgh Liver Research Center, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Bioengineering, School of Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania.
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25
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Nicolas CT, VanLith CJ, Hickey RD, Du Z, Hillin LG, Guthman RM, Cao WJ, Haugo B, Lillegard A, Roy D, Bhagwate A, O'Brien D, Kocher JP, Kaiser RA, Russell SJ, Lillegard JB. In vivo lentiviral vector gene therapy to cure hereditary tyrosinemia type 1 and prevent development of precancerous and cancerous lesions. Nat Commun 2022; 13:5012. [PMID: 36008405 PMCID: PMC9411607 DOI: 10.1038/s41467-022-32576-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2021] [Accepted: 08/08/2022] [Indexed: 11/23/2022] Open
Abstract
Conventional therapy for hereditary tyrosinemia type-1 (HT1) with 2-(2-nitro-4-trifluoromethylbenzoyl)−1,3-cyclohexanedione (NTBC) delays and in some cases fails to prevent disease progression to liver fibrosis, liver failure, and activation of tumorigenic pathways. Here we demonstrate cure of HT1 by direct, in vivo administration of a therapeutic lentiviral vector targeting the expression of a human fumarylacetoacetate hydrolase (FAH) transgene in the porcine model of HT1. This therapy is well tolerated and provides stable long-term expression of FAH in pigs with HT1. Genomic integration displays a benign profile, with subsequent fibrosis and tumorigenicity gene expression patterns similar to wild-type animals as compared to NTBC-treated or diseased untreated animals. Indeed, the phenotypic and genomic data following in vivo lentiviral vector administration demonstrate comparative superiority over other therapies including ex vivo cell therapy and therefore support clinical application of this approach. Hereditary tyrosinemia type 1 (HT1) is an inborn error of metabolism caused by a deficiency in fumarylacetoacetate hydrolase (FAH). Here, the authors show in an animal model that HT1 can be treated via in vivo portal vein administration of a lentiviral vector carrying the human FAH transgene.
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Affiliation(s)
- Clara T Nicolas
- Department of Surgery, Mayo Clinic, Rochester, MN, USA.,Faculty of Medicine, University of Barcelona, Barcelona, Spain.,Department of Surgery, University of Alabama Birmingham, Birmingham, AL, USA
| | | | - Raymond D Hickey
- Department of Surgery, Mayo Clinic, Rochester, MN, USA.,Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA
| | - Zeji Du
- Department of Surgery, Mayo Clinic, Rochester, MN, USA
| | - Lori G Hillin
- Department of Surgery, Mayo Clinic, Rochester, MN, USA
| | - Rebekah M Guthman
- Department of Surgery, Mayo Clinic, Rochester, MN, USA.,Medical College of Wisconsin, Wausau, WI, USA
| | - William J Cao
- Department of Surgery, Mayo Clinic, Rochester, MN, USA
| | | | | | - Diya Roy
- Department of Surgery, Mayo Clinic, Rochester, MN, USA
| | - Aditya Bhagwate
- Department of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN, USA
| | - Daniel O'Brien
- Department of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN, USA
| | - Jean-Pierre Kocher
- Department of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN, USA
| | - Robert A Kaiser
- Department of Surgery, Mayo Clinic, Rochester, MN, USA.,Midwest Fetal Care Center, Children's Hospitals and Clinics of Minnesota, Minneapolis, MN, USA
| | | | - Joseph B Lillegard
- Department of Surgery, Mayo Clinic, Rochester, MN, USA. .,Midwest Fetal Care Center, Children's Hospitals and Clinics of Minnesota, Minneapolis, MN, USA. .,Pediatric Surgical Associates, Minneapolis, MN, USA.
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26
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Liver Regeneration by Hematopoietic Stem Cells: Have We Reached the End of the Road? Cells 2022; 11:cells11152312. [PMID: 35954155 PMCID: PMC9367594 DOI: 10.3390/cells11152312] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Revised: 07/22/2022] [Accepted: 07/22/2022] [Indexed: 02/01/2023] Open
Abstract
The liver is the organ with the highest regenerative capacity in the human body. However, various insults, including viral infections, alcohol or drug abuse, and metabolic overload, may cause chronic inflammation and fibrosis, leading to irreversible liver dysfunction. Despite advances in surgery and pharmacological treatments, liver diseases remain a leading cause of death worldwide. To address the shortage of donor liver organs for orthotopic liver transplantation, cell therapy in liver disease has emerged as a promising regenerative treatment. Sources include primary hepatocytes or functional hepatocytes generated from the reprogramming of induced pluripotent stem cells (iPSC). Different types of stem cells have also been employed for transplantation to trigger regeneration, including hematopoietic stem cells (HSCs), mesenchymal stromal cells (MSCs), endothelial progenitor cells (EPCs) as well as adult and fetal liver progenitor cells. HSCs, usually defined by the expression of CD34 and CD133, and MSCs, defined by the expression of CD105, CD73, and CD90, are attractive sources due to their autologous nature, ease of isolation and cryopreservation. The present review focuses on the use of bone marrow HSCs for liver regeneration, presenting evidence for an ongoing crosstalk between the hematopoietic and the hepatic system. This relationship commences during embryogenesis when the fetal liver emerges as the crossroads between the two systems converging the presence of different origins of cells (mesoderm and endoderm) in the same organ. Ample evidence indicates that the fetal liver supports the maturation and expansion of HSCs during development but also later on in life. Moreover, the fact that the adult liver remains one of the few sites for extramedullary hematopoiesis—albeit pathological—suggests that this relationship between the two systems is ongoing. Can, however, the hematopoietic system offer similar support to the liver? The majority of clinical studies using hematopoietic cell transplantation in patients with liver disease report favourable observations. The underlying mechanism—whether paracrine, fusion or transdifferentiation or a combination of the three—remains to be confirmed.
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27
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Du Y, Zhang W, Qiu H, Xiao C, Shi J, Reid LM, He Z. Mouse Models of Liver Parenchyma Injuries and Regeneration. Front Cell Dev Biol 2022; 10:903740. [PMID: 35721478 PMCID: PMC9198899 DOI: 10.3389/fcell.2022.903740] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2022] [Accepted: 04/11/2022] [Indexed: 12/11/2022] Open
Abstract
Mice have genetic and physiological similarities with humans and a well-characterized genetic background that is easy to manipulate. Murine models have become the most favored, robust mammalian systems for experimental analyses of biological processes and disease conditions due to their low cost, rapid reproduction, a wealth of mouse strains with defined genetic conditions (both native ones as well as ones established experimentally), and high reproducibility with respect to that which can be done in experimental studies. In this review, we focus on murine models for liver, an organ with renown regenerative capacity and the organ most central to systemic, complex metabolic and physiological functions for mammalian hosts. Establishment of murine models has been achieved for all aspects of studies of normal liver, liver diseases, liver injuries, and regenerative repair mechanisms. We summarize key information on current mouse systems that partially model facets of clinical scenarios, particularly those associated with drug-induced acute or chronic liver injuries, dietary related, non-alcoholic liver disease (NAFLD), hepatitis virus infectious chronic liver diseases, and autoimmune hepatitis (AIH). In addition, we also include mouse models that are suitable for studying liver cancers (e.g., hepatocellular carcinomas), the aging process (senescence, apoptosis), and various types of liver injuries and regenerative processes associated with them.
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Affiliation(s)
- Yuan Du
- Department of General Surgery, Ji’an Hospital, Shanghai East Hospital, School of Medicine, Tongji University, Ji’an, China
- Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China
- The First Affiliated Hospital of Nanchang University, Nanchang, China
| | - Wencheng Zhang
- Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China
- Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai, China
- Shanghai Engineering Research Center of Stem Cells Translational Medicine, Shanghai, China
| | - Hua Qiu
- Department of General Surgery, Ji’an Hospital, Shanghai East Hospital, School of Medicine, Tongji University, Ji’an, China
- Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China
- The First Affiliated Hospital of Nanchang University, Nanchang, China
| | - Canjun Xiao
- Department of General Surgery, Ji’an Hospital, Shanghai East Hospital, School of Medicine, Tongji University, Ji’an, China
| | - Jun Shi
- Department of General Surgery, Ji’an Hospital, Shanghai East Hospital, School of Medicine, Tongji University, Ji’an, China
- The First Affiliated Hospital of Nanchang University, Nanchang, China
- *Correspondence: Zhiying He, ; Lola M. Reid, , ; Jun Shi,
| | - Lola M. Reid
- Departments of Cell Biology and Physiology, Program in Molecular Biology and Biotechnology, UNC School of Medicine, Chapel Hill, NC, United States
- *Correspondence: Zhiying He, ; Lola M. Reid, , ; Jun Shi,
| | - Zhiying He
- Department of General Surgery, Ji’an Hospital, Shanghai East Hospital, School of Medicine, Tongji University, Ji’an, China
- Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China
- Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai, China
- Shanghai Engineering Research Center of Stem Cells Translational Medicine, Shanghai, China
- *Correspondence: Zhiying He, ; Lola M. Reid, , ; Jun Shi,
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28
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Kopasz AG, Pusztai DZ, Karkas R, Hudoba L, Abdullah KSA, Imre G, Pankotai-Bodó G, Migh E, Nagy A, Kriston A, Germán P, Drubi AB, Molnár A, Fekete I, Dani VÉ, Ocsovszki I, Puskás LG, Horváth P, Sükösd F, Mátés L. A versatile transposon-based technology to generate loss- and gain-of-function phenotypes in the mouse liver. BMC Biol 2022; 20:74. [PMID: 35361222 PMCID: PMC8974095 DOI: 10.1186/s12915-022-01262-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2021] [Accepted: 02/22/2022] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Understanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice. RESULTS We exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model. CONCLUSIONS We developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.
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Affiliation(s)
| | - Dávid Zsolt Pusztai
- grid.481815.1Institute of Genetics, Biological Research Centre, Szeged, Hungary ,grid.9008.10000 0001 1016 9625Doctoral School of Biology, University of Szeged, Szeged, Hungary
| | - Réka Karkas
- grid.481815.1Institute of Genetics, Biological Research Centre, Szeged, Hungary ,grid.9008.10000 0001 1016 9625Doctoral School of Multidisciplinary Medical Sciences, University of Szeged, Szeged, Hungary
| | - Liza Hudoba
- grid.481815.1Institute of Genetics, Biological Research Centre, Szeged, Hungary
| | - Khaldoon Sadiq Ahmed Abdullah
- grid.481815.1Institute of Genetics, Biological Research Centre, Szeged, Hungary ,grid.9008.10000 0001 1016 9625Doctoral School of Multidisciplinary Medical Sciences, University of Szeged, Szeged, Hungary
| | - Gergely Imre
- grid.481815.1Institute of Genetics, Biological Research Centre, Szeged, Hungary ,grid.9008.10000 0001 1016 9625Doctoral School of Biology, University of Szeged, Szeged, Hungary
| | | | - Ede Migh
- grid.481814.00000 0004 0479 9817Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, Hungary
| | - Andrea Nagy
- grid.481815.1Institute of Genetics, Biological Research Centre, Szeged, Hungary
| | - András Kriston
- grid.481814.00000 0004 0479 9817Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, Hungary
| | - Péter Germán
- grid.481815.1Institute of Genetics, Biological Research Centre, Szeged, Hungary
| | - Andrea Bakné Drubi
- grid.481815.1Institute of Genetics, Biological Research Centre, Szeged, Hungary ,grid.9008.10000 0001 1016 9625Doctoral School of Biology, University of Szeged, Szeged, Hungary
| | - Anna Molnár
- grid.481815.1Institute of Genetics, Biological Research Centre, Szeged, Hungary
| | - Ildikó Fekete
- grid.481815.1Institute of Genetics, Biological Research Centre, Szeged, Hungary
| | - Virág Éva Dani
- grid.481815.1Institute of Genetics, Biological Research Centre, Szeged, Hungary
| | - Imre Ocsovszki
- grid.9008.10000 0001 1016 9625Department of Biochemistry, University of Szeged, Szeged, Hungary
| | - László Géza Puskás
- grid.481815.1Institute of Genetics, Biological Research Centre, Szeged, Hungary
| | - Péter Horváth
- grid.481814.00000 0004 0479 9817Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, Hungary ,grid.452494.a0000 0004 0409 5350Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland
| | - Farkas Sükösd
- grid.9008.10000 0001 1016 9625Institute of Pathology, University of Szeged, Szeged, Hungary
| | - Lajos Mátés
- Institute of Genetics, Biological Research Centre, Szeged, Hungary.
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29
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Ren J, Yu D, Wang J, Xu K, Xu Y, Sun R, An P, Li C, Feng G, Zhang Y, Dai X, Zhao H, Wang Z, Han Z, Zhu H, Ding Y, You X, Liu X, Wu M, Luo L, Li Z, Yang YG, Hu Z, Wei HJ, Ge L, Hai T, Li W. Generation of immunodeficient pig with hereditary tyrosinemia type 1 and their preliminary application for humanized liver. Cell Biosci 2022; 12:26. [PMID: 35255981 PMCID: PMC8900390 DOI: 10.1186/s13578-022-00760-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2021] [Accepted: 02/08/2022] [Indexed: 01/17/2023] Open
Abstract
Background Mice with humanized livers are important models to study drug toxicology testing, development of hepatitis virus treatments, and hepatocyte transplantation therapy. However, the huge difference between mouse and human in size and anatomy limited the application of humanized mice in investigating human diseases. Therefore, it is urgent to construct humanized livers in pigs to precisely investigate hepatocyte regeneration and human hepatocyte therapy. CRISPR/Cas9 system and somatic cell cloning technology were used to generate two pig models with FAH deficiency and exhibiting severe immunodeficiency (FAH/RAG1 and FAH/RAG1/IL2RG deficiency). Human primary hepatocytes were then successfully transplanted into the FG pig model and constructed two pigs with human liver. Results The constructed FAH/RAG1/IL2RG triple-knockout pig models were characterized by chronic liver injury and severe immunodeficiency. Importantly, the FG pigs transplanted with primary human hepatocytes produced human albumin in a time dependent manner as early as 1 week after transplantation. Furthermore, the colonization of human hepatocytes was confirmed by immunochemistry staining. Conclusions We successfully generated pig models with severe immunodeficiency that could construct human liver tissues. Supplementary Information The online version contains supplementary material available at 10.1186/s13578-022-00760-3.
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Affiliation(s)
- Jilong Ren
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China.,Institute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing, 100193, China
| | - Dawei Yu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China. .,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China. .,Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101, China.
| | - Jing Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China.,University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Kai Xu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China.,Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101, China
| | - Yanan Xu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China.,University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Renren Sun
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, First Hospital, Jilin University, Changchun, 130062, China
| | - Peipei An
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, First Hospital, Jilin University, Changchun, 130062, China
| | - Chongyang Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China
| | - Guihai Feng
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China
| | - Ying Zhang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China
| | - Xiangpeng Dai
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, First Hospital, Jilin University, Changchun, 130062, China
| | - Hongye Zhao
- State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming, 650201, China
| | - Zhengzhu Wang
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, First Hospital, Jilin University, Changchun, 130062, China
| | - Zhiqiang Han
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China.,Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101, China
| | - Haibo Zhu
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, First Hospital, Jilin University, Changchun, 130062, China.,Center of Reproductive Medicine and Center of Prenatal Diagnosis, First Hospital, Jilin University, Changchun, 130021, China
| | - Yuchun Ding
- Chongqing Academy of Animal Sciences, Chongqing, 402460, China.,Key Laboratory of Pig Industry Sciences, Ministry of Agriculture, Chongqing, 402460, China.,Chongqing Key Laboratory of Pig Industry Sciences, Chongqing, 402460, China.,Technical Engineering Center for the Development and Utilization of Medical Animal Resources, Chongqing, 402460, China
| | - Xiaoyan You
- Chongqing Academy of Animal Sciences, Chongqing, 402460, China.,Key Laboratory of Pig Industry Sciences, Ministry of Agriculture, Chongqing, 402460, China.,Chongqing Key Laboratory of Pig Industry Sciences, Chongqing, 402460, China.,Technical Engineering Center for the Development and Utilization of Medical Animal Resources, Chongqing, 402460, China
| | - Xueqin Liu
- Chongqing Academy of Animal Sciences, Chongqing, 402460, China.,Key Laboratory of Pig Industry Sciences, Ministry of Agriculture, Chongqing, 402460, China.,Chongqing Key Laboratory of Pig Industry Sciences, Chongqing, 402460, China.,Technical Engineering Center for the Development and Utilization of Medical Animal Resources, Chongqing, 402460, China
| | - Meng Wu
- Chongqing Academy of Animal Sciences, Chongqing, 402460, China.,Key Laboratory of Pig Industry Sciences, Ministry of Agriculture, Chongqing, 402460, China.,Chongqing Key Laboratory of Pig Industry Sciences, Chongqing, 402460, China.,Technical Engineering Center for the Development and Utilization of Medical Animal Resources, Chongqing, 402460, China
| | - Lin Luo
- Chongqing Academy of Animal Sciences, Chongqing, 402460, China.,Key Laboratory of Pig Industry Sciences, Ministry of Agriculture, Chongqing, 402460, China.,Chongqing Key Laboratory of Pig Industry Sciences, Chongqing, 402460, China.,Technical Engineering Center for the Development and Utilization of Medical Animal Resources, Chongqing, 402460, China
| | - Ziyi Li
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, First Hospital, Jilin University, Changchun, 130062, China
| | - Yong-Guang Yang
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, First Hospital, Jilin University, Changchun, 130062, China
| | - Zheng Hu
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, First Hospital, Jilin University, Changchun, 130062, China.
| | - Hong-Jiang Wei
- State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming, 650201, China.
| | - Liangpeng Ge
- Chongqing Academy of Animal Sciences, Chongqing, 402460, China. .,Key Laboratory of Pig Industry Sciences, Ministry of Agriculture, Chongqing, 402460, China. .,Chongqing Key Laboratory of Pig Industry Sciences, Chongqing, 402460, China. .,Technical Engineering Center for the Development and Utilization of Medical Animal Resources, Chongqing, 402460, China.
| | - Tang Hai
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China. .,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China. .,Beijing Farm Animal Research Center, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.
| | - Wei Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China. .,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China. .,Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101, China. .,University of Chinese Academy of Sciences, Beijing, 100049, China.
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30
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In vivo CRISPR screening identifies BAZ2 chromatin remodelers as druggable regulators of mammalian liver regeneration. Cell Stem Cell 2022; 29:372-385.e8. [PMID: 35090595 PMCID: PMC8897233 DOI: 10.1016/j.stem.2022.01.001] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2020] [Revised: 09/17/2021] [Accepted: 12/30/2021] [Indexed: 12/17/2022]
Abstract
Identifying new pathways that regulate mammalian regeneration is challenging due to the paucity of in vivo screening approaches. We employed pooled CRISPR knockout and activation screening in the regenerating liver to evaluate 165 chromatin regulatory proteins. Both screens identified the imitation-SWI chromatin remodeling components Baz2a and Baz2b, not previously implicated in regeneration. In vivo sgRNA, siRNA, and knockout strategies against either paralog confirmed increased regeneration. Distinct BAZ2-specific bromodomain inhibitors, GSK2801 and BAZ2-ICR, resulted in accelerated liver healing after diverse injuries. Inhibitor-treated mice also exhibited improved healing in an inflammatory bowel disease model, suggesting multi-tissue applicability. Transcriptomics on regenerating livers showed increases in ribosomal and cell cycle mRNAs. Surprisingly, CRISPRa screening to define mechanisms showed that overproducing Rpl10a or Rpl24 was sufficient to drive regeneration, whereas Rpl24 haploinsufficiency was rate limiting for BAZ2 inhibition-mediated regeneration. The discovery of regenerative roles for imitation-SWI components provides immediate strategies to enhance tissue repair.
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31
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Larson EL, Joo DJ, Nelson ED, Amiot BP, Aravalli RN, Nyberg SL. Fumarylacetoacetate hydrolase gene as a knockout target for hepatic chimerism and donor liver production. Stem Cell Reports 2021; 16:2577-2588. [PMID: 34678209 PMCID: PMC8581169 DOI: 10.1016/j.stemcr.2021.09.018] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2021] [Revised: 09/22/2021] [Accepted: 09/23/2021] [Indexed: 11/15/2022] Open
Abstract
A reliable source of human hepatocytes and transplantable livers is needed. Interspecies embryo complementation, which involves implanting donor human stem cells into early morula/blastocyst stage animal embryos, is an emerging solution to the shortage of transplantable livers. We review proposed mutations in the recipient embryo to disable hepatogenesis, and discuss the advantages of using fumarylacetoacetate hydrolase knockouts and other genetic modifications to disable hepatogenesis. Interspecies blastocyst complementation using porcine recipients for primate donors has been achieved, although percentages of chimerism remain persistently low. Recent investigation into the dynamic transcriptomes of pigs and primates have created new opportunities to intimately match the stage of developing animal embryos with one of the many varieties of human induced pluripotent stem cell. We discuss techniques for decreasing donor cell apoptosis, targeting donor tissue to endodermal structures to avoid neural or germline chimerism, and decreasing the immunogenicity of chimeric organs by generating donor endothelium.
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Affiliation(s)
- Ellen L Larson
- Department of Surgery, Division of Transplant Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA
| | - Dong Jin Joo
- Department of Surgery, Division of Transplantation, Yonsei University College of Medicine, Seoul, South Korea
| | - Erek D Nelson
- Department of Surgery, Mayo Clinic, Rochester, MN, USA
| | - Bruce P Amiot
- Department of Surgery, Division of Transplant Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA
| | - Rajagopal N Aravalli
- Department of Electrical and Computer Engineering, College of Science and Engineering, University of Minnesota, Minneapolis, MN, USA
| | - Scott L Nyberg
- Department of Surgery, Division of Transplant Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
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32
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Liang R, Lin YH, Zhu H. Genetic and Cellular Contributions to Liver Regeneration. Cold Spring Harb Perspect Biol 2021; 14:a040832. [PMID: 34750173 PMCID: PMC9438780 DOI: 10.1101/cshperspect.a040832] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
The regenerative capabilities of the liver represent a paradigm for understanding tissue repair in solid organs. Regeneration after partial hepatectomy in rodent models is well understood, while regeneration in the context of clinically relevant chronic injuries is less studied. Given the growing incidence of fatty liver disease, cirrhosis, and liver cancer, interest in liver regeneration is increasing. Here, we will review the principles, genetics, and cell biology underlying liver regeneration, as well as new approaches being used to study heterogeneity in liver tissue maintenance and repair.
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Affiliation(s)
- Roger Liang
- Children's Research Institute, Departments of Pediatrics and Internal Medicine, Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA
| | - Yu-Hsuan Lin
- Children's Research Institute, Departments of Pediatrics and Internal Medicine, Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA
| | - Hao Zhu
- Children's Research Institute, Departments of Pediatrics and Internal Medicine, Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA
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33
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Zhang L, Ge J, Zheng Y, Sun Z, Wang C, Peng Z, Wu B, Fang M, Furuya K, Ma X, Shao Y, Ohkohchi N, Oda T, Fan J, Pan G, Li D, Hui L. Survival-Assured Liver Injury Preconditioning (SALIC) Enables Robust Expansion of Human Hepatocytes in Fah -/- Rag2 -/- IL2rg -/- Rats. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2021; 8:e2101188. [PMID: 34382351 PMCID: PMC8498896 DOI: 10.1002/advs.202101188] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 03/23/2021] [Revised: 06/13/2021] [Indexed: 06/13/2023]
Abstract
Although liver-humanized animals are desirable tools for drug development and expansion of human hepatocytes in large quantities, their development is restricted to mice. In animals larger than mice, a precondition for efficient liver humanization remains preliminary because of different xeno-repopulation kinetics in livers of larger sizes. Since rats are ten times larger than mice and widely used in pharmacological studies, liver-humanized rats are more preferable. Here, Fah-/- Rag2-/- IL2rg-/- (FRG) rats are generated by CRISPR/Cas9, showing accelerated liver failure and lagged liver xeno-repopulation compared to FRG mice. A survival-assured liver injury preconditioning (SALIC) protocol, which consists of retrorsine pretreatment and cycling 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) administration by defined concentrations and time intervals, is developed to reduce the mortality of FRG rats and induce a regenerative microenvironment for xeno-repopulation. Human hepatocyte repopulation is boosted to 31 ± 4% in rat livers at 7 months after transplantation, equivalent to approximately a 1200-fold expansion. Human liver features of transcriptome and zonation are reproduced in humanized rats. Remarkably, they provide sufficient samples for the pharmacokinetic profiling of human-specific metabolites. This model is thus preferred for pharmacological studies and human hepatocyte production. SALIC may also be informative to hepatocyte transplantation in other large-sized species.
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Affiliation(s)
- Ludi Zhang
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of SciencesUniversity of Chinese Academy of ScienceShanghai200031China
| | - Jian‐Yun Ge
- Department of Gastrointestinal and Hepato‐Biliary‐Pancreatic Surgery, Faculty of MedicineUniversity of TsukubaTsukubaIbaraki305‐8575Japan
- Guangdong Provincial Key Laboratory of Large Animal Models for BiomedicineSchool of Biotechnology and Heath SciencesWuyi UniversityJiangmenGuangdong529020China
| | - Yun‐Wen Zheng
- Department of Gastrointestinal and Hepato‐Biliary‐Pancreatic Surgery, Faculty of MedicineUniversity of TsukubaTsukubaIbaraki305‐8575Japan
- Guangdong Provincial Key Laboratory of Large Animal Models for BiomedicineSchool of Biotechnology and Heath SciencesWuyi UniversityJiangmenGuangdong529020China
- Institute of Regenerative MedicineAffiliated Hospital of Jiangsu UniversityJiangsu UniversityZhenjiangJiangsu212001China
- Yokohama City University School of MedicineYokohamaKanagawa234‐0006Japan
| | - Zhen Sun
- School of Life Science and TechnologyShanghaiTech UniversityShanghai201210China
| | - Chenhua Wang
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of SciencesUniversity of Chinese Academy of ScienceShanghai200031China
| | - Zhaoliang Peng
- Shanghai Institute of Materia MedicaChinese Academy of SciencesShanghai201203China
| | - Baihua Wu
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of SciencesUniversity of Chinese Academy of ScienceShanghai200031China
| | - Mei Fang
- Institute of Regenerative MedicineAffiliated Hospital of Jiangsu UniversityJiangsu UniversityZhenjiangJiangsu212001China
| | - Kinji Furuya
- Department of Gastrointestinal and Hepato‐Biliary‐Pancreatic Surgery, Faculty of MedicineUniversity of TsukubaTsukubaIbaraki305‐8575Japan
| | - Xiaolong Ma
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of SciencesUniversity of Chinese Academy of ScienceShanghai200031China
| | - Yanjiao Shao
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life SciencesEast China Normal UniversityShanghai200241China
| | - Nobuhiro Ohkohchi
- Department of Gastrointestinal and Hepato‐Biliary‐Pancreatic Surgery, Faculty of MedicineUniversity of TsukubaTsukubaIbaraki305‐8575Japan
| | - Tatsuya Oda
- Department of Gastrointestinal and Hepato‐Biliary‐Pancreatic Surgery, Faculty of MedicineUniversity of TsukubaTsukubaIbaraki305‐8575Japan
| | - Jianglin Fan
- Guangdong Provincial Key Laboratory of Large Animal Models for BiomedicineSchool of Biotechnology and Heath SciencesWuyi UniversityJiangmenGuangdong529020China
- Department of Molecular Pathology, Faculty of MedicineInterdisciplinary Graduate School of MedicineUniversity of YamanashiShimokatoYamanashi409‐3898Japan
| | - Guoyu Pan
- Shanghai Institute of Materia MedicaChinese Academy of SciencesShanghai201203China
| | - Dali Li
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life SciencesEast China Normal UniversityShanghai200241China
| | - Lijian Hui
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of SciencesUniversity of Chinese Academy of ScienceShanghai200031China
- School of Life Science and TechnologyShanghaiTech UniversityShanghai201210China
- School of Life Science, Hangzhou Institute for Advanced StudyUniversity of Chinese Academy of SciencesHangzhou310024China
- Institute for Stem Cell and RegenerationChinese Academy of SciencesBeijing100101China
- Bio‐Research Innovation CenterShanghai Institute of Biochemistry and Cell BiologySuzhouJiangsu215121China
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34
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Peng WC, Kraaier LJ, Kluiver TA. Hepatocyte organoids and cell transplantation: What the future holds. Exp Mol Med 2021; 53:1512-1528. [PMID: 34663941 PMCID: PMC8568948 DOI: 10.1038/s12276-021-00579-x] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2020] [Revised: 01/11/2021] [Accepted: 01/14/2021] [Indexed: 12/29/2022] Open
Abstract
Historically, primary hepatocytes have been difficult to expand or maintain in vitro. In this review, we will focus on recent advances in establishing hepatocyte organoids and their potential applications in regenerative medicine. First, we provide a background on the renewal of hepatocytes in the homeostatic as well as the injured liver. Next, we describe strategies for establishing primary hepatocyte organoids derived from either adult or fetal liver based on insights from signaling pathways regulating hepatocyte renewal in vivo. The characteristics of these organoids will be described herein. Notably, hepatocyte organoids can adopt either a proliferative or a metabolic state, depending on the culture conditions. Furthermore, the metabolic gene expression profile can be modulated based on the principles that govern liver zonation. Finally, we discuss the suitability of cell replacement therapy to treat different types of liver diseases and the current state of cell transplantation of in vitro-expanded hepatocytes in mouse models. In addition, we provide insights into how the regenerative microenvironment in the injured host liver may facilitate donor hepatocyte repopulation. In summary, transplantation of in vitro-expanded hepatocytes holds great potential for large-scale clinical application to treat liver diseases.
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Affiliation(s)
- Weng Chuan Peng
- Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS, Utrecht, The Netherlands.
| | - Lianne J Kraaier
- Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS, Utrecht, The Netherlands
| | - Thomas A Kluiver
- Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS, Utrecht, The Netherlands
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35
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Maestro S, Weber ND, Zabaleta N, Aldabe R, Gonzalez-Aseguinolaza G. Novel vectors and approaches for gene therapy in liver diseases. JHEP Rep 2021; 3:100300. [PMID: 34159305 PMCID: PMC8203845 DOI: 10.1016/j.jhepr.2021.100300] [Citation(s) in RCA: 71] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/21/2021] [Revised: 03/23/2021] [Accepted: 04/18/2021] [Indexed: 12/13/2022] Open
Abstract
Gene therapy is becoming an increasingly valuable tool to treat many genetic diseases with no or limited treatment options. This is the case for hundreds of monogenic metabolic disorders of hepatic origin, for which liver transplantation remains the only cure. Furthermore, the liver contains 10-15% of the body's total blood volume, making it ideal for use as a factory to secrete proteins into the circulation. In recent decades, an expanding toolbox has become available for liver-directed gene delivery. Although viral vectors have long been the preferred approach to target hepatocytes, an increasing number of non-viral vectors are emerging as highly efficient vehicles for the delivery of genetic material. Herein, we review advances in gene delivery vectors targeting the liver and more specifically hepatocytes, covering strategies based on gene addition and gene editing, as well as the exciting results obtained with the use of RNA as a therapeutic molecule. Moreover, we will briefly summarise some of the limitations of current liver-directed gene therapy approaches and potential ways of overcoming them.
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Key Words
- AAT, α1-antitrypsin
- AAV, adeno-associated virus
- AHP, acute hepatic porphyrias
- AIP, acute intermittent porphyria
- ALAS1, aminolevulic synthase 1
- APCs, antigen-presenting cells
- ASGCT, American Society of Gene and Cell Therapy
- ASGPR, asialoglycoprotein receptor
- ASOs, antisense oligonucleotides
- Ad, adenovirus
- CBS, cystathionine β-synthase
- CN, Crigel-Najjar
- CRISPR, clustered regularly interspaced short palindromic repeats
- CRISPR/Cas9, CRISPR associated protein 9
- DSBs, double-strand breaks
- ERT, enzyme replacement therapy
- FH, familial hypercholesterolemia
- FSP27, fat-specific protein 27
- GO, glycolate oxidase
- GSD1a, glycogen storage disorder 1a
- GT, gene therapy
- GUSB, β-glucuronidase
- GalNAc, N-acetyl-D-galactosamine
- HDAd, helper-dependent adenovirus
- HDR, homology-directed repair
- HT, hereditary tyrosinemia
- HemA/B, haemophilia A/B
- IDS, iduronate 2-sulfatase
- IDUA, α-L-iduronidase
- IMLD, inherited metabolic liver diseases
- ITR, inverted terminal repetition
- LDH, lactate dehydrogenase
- LDLR, low-density lipoprotein receptor
- LNP, Lipid nanoparticles
- LTR, long terminal repeat
- LV, lentivirus
- MMA, methylmalonic acidemia
- MPR, metabolic pathway reprograming
- MPS type I, MPSI
- MPS type VII, MPSVII
- MPS, mucopolysaccharidosis
- NASH, non-alcoholic steatohepatitis
- NHEJ, non-homologous end joining
- NHPs, non-human primates
- Non-viral vectors
- OLT, orthotopic liver transplantation
- OTC, ornithine transcarbamylase
- PA, propionic acidemia
- PB, piggyBac
- PCSK9, proprotein convertase subtilisin/kexin type 9
- PEG, polyethylene glycol
- PEI, polyethyleneimine
- PFIC3, progressive familial cholestasis type 3
- PH1, Primary hyperoxaluria type 1
- PKU, phenylketonuria
- RV, retrovirus
- S/MAR, scaffold matrix attachment regions
- SB, Sleeping Beauty
- SRT, substrate reduction therapy
- STK25, serine/threonine protein kinase 25
- TALEN, transcription activator-like effector nucleases
- TTR, transthyretin
- UCD, urea cycle disorders
- VLDLR, very-low-density lipoprotein receptor
- WD, Wilson’s disease
- ZFN, zinc finger nucleases
- apoB/E, apolipoprotein B/E
- dCas9, dead Cas9
- efficacy
- gene addition
- gene editing
- gene silencing
- hepatocytes
- immune response
- lncRNA, long non-coding RNA
- miRNAs, microRNAs
- siRNA, small-interfering RNA
- toxicity
- viral vectors
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Affiliation(s)
- Sheila Maestro
- Gene Therapy Area, Foundation for Applied Medical Research, University of Navarra, IdisNA, Pamplona, Spain
| | | | - Nerea Zabaleta
- Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, MA, USA
| | - Rafael Aldabe
- Gene Therapy Area, Foundation for Applied Medical Research, University of Navarra, IdisNA, Pamplona, Spain
- Corresponding authors. Address: CIMA, Universidad de Navarra. Av. Pio XII 55 31008 Pamplona. Spain
| | - Gloria Gonzalez-Aseguinolaza
- Gene Therapy Area, Foundation for Applied Medical Research, University of Navarra, IdisNA, Pamplona, Spain
- Vivet Therapeutics, Pamplona, Spain
- Corresponding authors. Address: CIMA, Universidad de Navarra. Av. Pio XII 55 31008 Pamplona. Spain
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Chronowski C, Akhanov V, Chan D, Catic A, Finegold M, Sahin E. Fructose Causes Liver Damage, Polyploidy, and Dysplasia in the Setting of Short Telomeres and p53 Loss. Metabolites 2021; 11:metabo11060394. [PMID: 34204343 PMCID: PMC8234056 DOI: 10.3390/metabo11060394] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2021] [Revised: 06/09/2021] [Accepted: 06/10/2021] [Indexed: 01/01/2023] Open
Abstract
Studies in humans and model systems have established an important role of short telomeres in predisposing to liver fibrosis through pathways that are incompletely understood. Recent studies have shown that telomere dysfunction impairs cellular metabolism, but whether and how these metabolic alterations contribute to liver fibrosis is not well understood. Here, we investigated whether short telomeres change the hepatic response to metabolic stress induced by fructose, a sugar that is highly implicated in non-alcoholic fatty liver disease. We find that telomere shortening in telomerase knockout mice (TKO) imparts a pronounced susceptibility to fructose as reflected in the activation of p53, increased apoptosis, and senescence, despite lower hepatic fat accumulation in TKO mice compared to wild type mice with long telomeres. The decreased fat accumulation in TKO is mediated by p53 and deletion of p53 normalizes hepatic fat content but also causes polyploidy, polynuclearization, dysplasia, cell death, and liver damage. Together, these studies suggest that liver tissue with short telomers are highly susceptible to fructose and respond with p53 activation and liver damage that is further exacerbated when p53 is lost resulting in dysplastic changes.
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Affiliation(s)
- Christopher Chronowski
- Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA; (C.C.); (V.A.); (A.C.)
| | - Viktor Akhanov
- Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA; (C.C.); (V.A.); (A.C.)
| | - Doug Chan
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA;
| | - Andre Catic
- Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA; (C.C.); (V.A.); (A.C.)
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA;
| | - Milton Finegold
- Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA;
| | - Ergün Sahin
- Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA; (C.C.); (V.A.); (A.C.)
- Department of Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
- Correspondence: ; Tel.: +1-713-798-6685; Fax: +1-713-798-4146
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Hsu YC, Yu IS, Tsai YF, Wu YM, Chen YT, Sheu JC, Lin SW. A Preconditioning Strategy to Augment Retention and Engraftment Rate of Donor Cells During Hepatocyte Transplantation. Transplantation 2021; 105:785-795. [PMID: 32976366 DOI: 10.1097/tp.0000000000003461] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Hepatocyte transplantation has been extensively investigated as an alternative to orthotopic liver transplantation. However, its application in routine clinical practice has been restricted because of low initial engraftment and subsequent repopulation. METHODS Using mice as a model, we have developed a minimally invasive and nontoxic preconditioning strategy based on preadministration of antibodies against hepsin to increase donor hepatocyte retention and engraftment rate. RESULTS Liver sinusoid diameters decreased significantly with antihepsin pretreatment, and graft cell numbers increased nearly 2-fold in the recipients' liver parenchyma for 20 days after hepatocyte transplantation. Postoperative complications such as hepatic ischemia injury or apparent immune cell accumulation were not observed in recipients. In a hemophilia B mouse model, antihepsin preconditioning enhanced the expression and clotting activity of coagulation factor IX (FIX) to nearly 2-fold that of immunoglobulin G-treated controls and maintained higher plasma FIX clotting activity relative to the prophylactic range for 50 days after hepatocyte transplantation. Antihepsin pretreatment combined with adeno-associated virus-transduced donor hepatocytes expressing human FIX-Triple, a hyperfunctional FIX variant, resulted in plasma FIX levels similar to those associated with mild hemophilia, which protected hemophilia B mice from major bleeding episodes for 50 days after transplantation. Furthermore, antihepsin pretreatment and repeated transplantation resulted in extending the therapeutic period by 30 days relative to the immunoglobulin G control. CONCLUSIONS Thus, this antihepsin strategy improved the therapeutic effect of hepatocyte transplantation in mice with tremendous safety and minimal invasion. Taken together, we suggest that preconditioning with antihepsin may have clinical applications for liver cell therapy.
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Affiliation(s)
- Yu-Chen Hsu
- Liver Disease Prevention and Treatment Research Foundation, Taipei, Taiwan (R.O.C.)
| | - I-Shing Yu
- Laboratory Animal Center, College of Medicine, National Taiwan University, Taipei, Taiwan (R.O.C.)
| | - Yu-Fei Tsai
- Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan (R.O.C.)
| | - Yao-Ming Wu
- Department of Surgery, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan (R.O.C.)
| | - You-Tzung Chen
- Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University, Taipei, Taiwan (R.O.C.)
- Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan (R.O.C.)
| | - Jin-Chuan Sheu
- Liver Disease Prevention and Treatment Research Foundation, Taipei, Taiwan (R.O.C.)
- Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan (R.O.C.)
| | - Shu-Wha Lin
- Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan (R.O.C.)
- Department of Laboratory Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan (R.O.C.)
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38
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Abstract
Liver cancer typically arises after years of inflammatory insults to hepatocytes. These cells can change their ploidy state during health and disease. Whilst polyploidy may offer some protection, new research shows it may also promote the formation of liver tumours.
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Genome editing in the human liver: Progress and translational considerations. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021; 182:257-288. [PMID: 34175044 DOI: 10.1016/bs.pmbts.2021.01.030] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Liver-targeted genome editing offers the prospect of life-long therapeutic benefit following a single treatment and is set to rapidly supplant conventional gene addition approaches. Combining progress in liver-targeted gene delivery with genome editing technology, makes this not only feasible but realistically achievable in the near term. However, important challenges remain to be addressed. These include achieving therapeutic levels of editing, particularly in vivo, avoidance of off-target effects on the genome and the potential impact of pre-existing immunity to bacteria-derived nucleases, when used to improve editing rates. In this chapter, we outline the unique features of the liver that make it an attractive target for genome editing, the impact of liver biology on therapeutic efficacy, and disease specific challenges, including whether the approach targets a cell autonomous or non-cell autonomous disease. We also discuss strategies that have been used successfully to achieve genome editing outcomes in the liver and address translational considerations as genome editing technology moves into the clinic.
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40
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Yang H, Sun L, Pang Y, Hu D, Xu H, Mao S, Peng W, Wang Y, Xu Y, Zheng YC, Du S, Zhao H, Chi T, Lu X, Sang X, Zhong S, Wang X, Zhang H, Huang P, Sun W, Mao Y. Three-dimensional bioprinted hepatorganoids prolong survival of mice with liver failure. Gut 2021; 70:567-574. [PMID: 32434830 PMCID: PMC7873413 DOI: 10.1136/gutjnl-2019-319960] [Citation(s) in RCA: 115] [Impact Index Per Article: 28.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/28/2019] [Revised: 04/28/2020] [Accepted: 05/06/2020] [Indexed: 12/20/2022]
Abstract
OBJECTIVE Shortage of organ donors, a critical challenge for treatment of end-stage organ failure, has motivated the development of alternative strategies to generate organs in vitro. Here, we aim to describe the hepatorganoids, which is a liver tissue model generated by three-dimensional (3D) bioprinting of HepaRG cells and investigate its liver functions in vitro and in vivo. DESIGN 3D bioprinted hepatorganoids (3DP-HOs) were constructed using HepaRG cells and bioink, according to specific 3D printing procedures. Liver functions of 3DP-HOs were detected after 7 days of differentiation in vitro, which were later transplanted into Fah-deficient mice. The in vivo liver functions of 3DP-HOs were evaluated by survival time and liver damage of mice, human liver function markers and human-specific debrisoquine metabolite production. RESULTS 3DP-HOs broadly acquired liver functions, such as ALBUMIN secretion, drug metabolism and glycogen storage after 7 days of differentiation. After transplantation into abdominal cavity of Fah-/-Rag2-/- mouse model of liver injury, 3DP-HOs further matured and displayed increased synthesis of liver-specific proteins. Particularly, the mice acquired human-specific drug metabolism activities. Functional vascular systems were also formed in transplanted 3DP-HOs, further enhancing the material transport and liver functions of 3DP-HOs. Most importantly, transplantation of 3DP-HOs significantly improved the survival of mice. CONCLUSIONS Our results demonstrated a comprehensive proof of principle, which indicated that 3DP-HO model of liver tissues possessed in vivo hepatic functions and alleviated liver failure after transplantation, suggesting that 3D bioprinting could be used to generate human liver tissues as the alternative transplantation donors for treatment of liver diseases.
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Affiliation(s)
- Huayu Yang
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Beijing, China
| | - Lejia Sun
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Beijing, China
| | - Yuan Pang
- Biomanufacturing Center, Department of Mechanical Engineering, Tsinghua University, Beijing, China,Biomanufacturing and Rapid Forming Technology Key Laboratory of Beijing, Tsinghua University, Beijing, China,Overseas Expertise Introduction Center for Discipline Innovation, Tsinghua University, Beijing, China
| | - Dandan Hu
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Beijing, China,Department of Hepatobiliary Surgery, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China
| | - Haifeng Xu
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Beijing, China
| | - Shuangshuang Mao
- Biomanufacturing Center, Department of Mechanical Engineering, Tsinghua University, Beijing, China,Biomanufacturing and Rapid Forming Technology Key Laboratory of Beijing, Tsinghua University, Beijing, China,Overseas Expertise Introduction Center for Discipline Innovation, Tsinghua University, Beijing, China
| | - Wenbo Peng
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
| | - Yanan Wang
- State Key Laboratory of Medical Molecular Biology, Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China
| | - Yiyao Xu
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Beijing, China
| | - Yong-Chang Zheng
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Beijing, China
| | - Shunda Du
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Beijing, China
| | - Haitao Zhao
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Beijing, China
| | - Tianyi Chi
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Beijing, China
| | - Xin Lu
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Beijing, China
| | - Xinting Sang
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Beijing, China
| | - Shouxian Zhong
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Beijing, China
| | - Xin Wang
- Research Center for Laboratory Animal Science, Inner Mongolia University, Hohhot, Inner Mongolia, China,Hepatoscience Section, Cell Lab Tech Inc, Sunnyvale, California, USA,Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, USA
| | - Hongbing Zhang
- State Key Laboratory of Medical Molecular Biology, Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China
| | - Pengyu Huang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China .,Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China
| | - Wei Sun
- Biomanufacturing Center, Department of Mechanical Engineering, Tsinghua University, Beijing, China .,Biomanufacturing and Rapid Forming Technology Key Laboratory of Beijing, Tsinghua University, Beijing, China.,Overseas Expertise Introduction Center for Discipline Innovation, Tsinghua University, Beijing, China.,Department of Mechanical Engineering, Drexel University, Philadelphia, Pennsylvania, USA
| | - Yilei Mao
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Beijing, China
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41
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Lai F, Wee CYY, Chen Q. Establishment of Humanized Mice for the Study of HBV. Front Immunol 2021; 12:638447. [PMID: 33679796 PMCID: PMC7933441 DOI: 10.3389/fimmu.2021.638447] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2020] [Accepted: 02/03/2021] [Indexed: 12/28/2022] Open
Abstract
Viral hepatitis particularly Hepatitis B Virus (HBV) is still an ongoing health issue worldwide. Despite the vast technological advancements in research and development, only HBV vaccines, typically given during early years, are currently available as a preventive measure against acquiring the disease from a secondary source. In general, HBV can be cleared naturally by the human immune system if detected at low levels early. However, long term circulation of HBV in the peripheral blood may be detrimental to the human liver, specifically targeting human hepatocytes for cccDNA integration which inevitably supports HBV life cycle for the purpose of reinfection in healthy cells. Although there is some success in using nucleoside analogs or polyclonal antibodies targeting HBV surface antigens (HBsAg) in patients with acute or chronic HBV+ (CHB), majority of them would either respond only partially or succumb to the disease entirely unless they undergo liver transplants from a fully matched healthy donor and even so may not necessarily guarantee a 100% chance of survival. Indeed, in vitro/ex vivo cultures and various transgenic animal models have already provided us with a good understanding of HBV but they primarily lack human specificity or virus-host interactions in the presence of human immune surveillance. Therefore, the demand of utilizing humanized mice has increased over the last decade as a pre-clinical platform for investigating human-specific immune responses against HBV as well as identifying potential immunotherapeutic strategies in eradicating the virus. Basically, this review covers some of the recent developments and key advantages of humanized mouse models over other conventional transgenic mice platforms.
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Affiliation(s)
- Fritz Lai
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
- Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
| | - Cherry Yong Yi Wee
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
| | - Qingfeng Chen
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
- Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
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42
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Matsumoto T, Wakefield L, Peters A, Peto M, Spellman P, Grompe M. Proliferative polyploid cells give rise to tumors via ploidy reduction. Nat Commun 2021; 12:646. [PMID: 33510149 PMCID: PMC7843634 DOI: 10.1038/s41467-021-20916-y] [Citation(s) in RCA: 58] [Impact Index Per Article: 14.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2020] [Accepted: 12/14/2020] [Indexed: 01/18/2023] Open
Abstract
Polyploidy is a hallmark of cancer, and closely related to chromosomal instability involved in cancer progression. Importantly, polyploid cells also exist in some normal tissues. Polyploid hepatocytes proliferate and dynamically reduce their ploidy during liver regeneration. This raises the question whether proliferating polyploids are prone to cancer via chromosome missegregation during mitosis and/or ploidy reduction. Conversely polyploids could be resistant to tumor development due to their redundant genomes. Therefore, the tumor-initiation risk of physiologic polyploidy and ploidy reduction is still unclear. Using in vivo lineage tracing we here show that polyploid hepatocytes readily form liver tumors via frequent ploidy reduction. Polyploid hepatocytes give rise to regenerative nodules with chromosome aberrations, which are enhanced by ploidy reduction. Although polyploidy should theoretically prevent tumor suppressor loss, the high frequency of ploidy reduction negates this protection. Importantly, polyploid hepatocytes that undergo multiple rounds of cell division become predominantly mononucleated and are resistant to ploidy reduction. Our results suggest that ploidy reduction is an early step in the initiation of carcinogenesis from polyploid hepatocytes.
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Affiliation(s)
- Tomonori Matsumoto
- Department of Pediatrics, Oregon Health and Science University, Portland, OR, USA.
| | - Leslie Wakefield
- Department of Pediatrics, Oregon Health and Science University, Portland, OR, USA
| | - Alexander Peters
- Department of Pediatrics, Oregon Health and Science University, Portland, OR, USA
| | - Myron Peto
- Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA
| | - Paul Spellman
- Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA
| | - Markus Grompe
- Department of Pediatrics, Oregon Health and Science University, Portland, OR, USA.
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43
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Michalopoulos GK, Bhushan B. Liver regeneration: biological and pathological mechanisms and implications. Nat Rev Gastroenterol Hepatol 2021; 18:40-55. [PMID: 32764740 DOI: 10.1038/s41575-020-0342-4] [Citation(s) in RCA: 553] [Impact Index Per Article: 138.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 06/24/2020] [Indexed: 02/08/2023]
Abstract
The liver is the only solid organ that uses regenerative mechanisms to ensure that the liver-to-bodyweight ratio is always at 100% of what is required for body homeostasis. Other solid organs (such as the lungs, kidneys and pancreas) adjust to tissue loss but do not return to 100% of normal. The current state of knowledge of the regenerative pathways that underlie this 'hepatostat' will be presented in this Review. Liver regeneration from acute injury is always beneficial and has been extensively studied. Experimental models that involve partial hepatectomy or chemical injury have revealed extracellular and intracellular signalling pathways that are used to return the liver to equivalent size and weight to those prior to injury. On the other hand, chronic loss of hepatocytes, which can occur in chronic liver disease of any aetiology, often has adverse consequences, including fibrosis, cirrhosis and liver neoplasia. The regenerative activities of hepatocytes and cholangiocytes are typically characterized by phenotypic fidelity. However, when regeneration of one of the two cell types fails, hepatocytes and cholangiocytes function as facultative stem cells and transdifferentiate into each other to restore normal liver structure. Liver recolonization models have demonstrated that hepatocytes have an unlimited regenerative capacity. However, in normal liver, cell turnover is very slow. All zones of the resting liver lobules have been equally implicated in the maintenance of hepatocyte and cholangiocyte populations in normal liver.
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Affiliation(s)
- George K Michalopoulos
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
| | - Bharat Bhushan
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
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Fontes P, Komori J, Lopez R, Marsh W, Lagasse E. Development of Ectopic Livers by Hepatocyte Transplantation Into Swine Lymph Nodes. Liver Transpl 2020; 26:1629-1643. [PMID: 32810371 PMCID: PMC7756213 DOI: 10.1002/lt.25872] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/25/2020] [Revised: 08/01/2020] [Accepted: 08/09/2020] [Indexed: 12/16/2022]
Abstract
Orthotopic liver transplantation continues to be the only effective therapy for patients with end-stage liver disease. Unfortunately, many of these patients are not considered transplant candidates, lacking effective therapeutic options that would address both the irreversible progression of their hepatic failure and the control of their portal hypertension. In this prospective study, a swine model was exploited to induce subacute liver failure. Autologous hepatocytes, isolated from the left hepatic lobe, were transplanted into the mesenteric lymph nodes (LNs) by direct cell injection. At 30-60 days after transplantation, hepatocyte engraftment in LNs was successfully identified in all transplanted animals with the degree of ectopic liver mass detected being proportional to the induced native liver injury. These ectopic livers developed within the LNs showed remarkable histologic features of swine hepatic lobules, including the formation of sinusoids and bile ducts. On the basis of our previous tyrosinemic mouse model and the present pig models of induced subacute liver failure, the generation of auxiliary liver tissue using the LNs as hepatocyte engraftment sites represents a potential therapeutic approach to supplement declining hepatic function in the treatment of liver disease.
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Affiliation(s)
- Paulo Fontes
- WVU MedicineDepartment of SurgerySchool of MedicineWest Virginia UniversityMorgantownWV,LyGenesis, Inc.PittsburghPA
| | - Junji Komori
- McGowan Institute for Regenerative MedicineDepartment of PathologySchool of MedicineUniversity of PittsburghPittsburghPA,Department of SurgeryTakamatsu Red Cross HospitalKagawaJapan
| | - Roberto Lopez
- WVU MedicineDepartment of SurgerySchool of MedicineWest Virginia UniversityMorgantownWV,LyGenesis, Inc.PittsburghPA
| | - Wallis Marsh
- WVU MedicineDepartment of SurgerySchool of MedicineWest Virginia UniversityMorgantownWV
| | - Eric Lagasse
- LyGenesis, Inc.PittsburghPA,McGowan Institute for Regenerative MedicineDepartment of PathologySchool of MedicineUniversity of PittsburghPittsburghPA
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45
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Salminen AT, Allahyari Z, Gholizadeh S, McCloskey MC, Ajalik R, Cottle RN, Gaborski TR, McGrath JL. In vitro Studies of Transendothelial Migration for Biological and Drug Discovery. FRONTIERS IN MEDICAL TECHNOLOGY 2020; 2:600616. [PMID: 35047883 PMCID: PMC8757899 DOI: 10.3389/fmedt.2020.600616] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2020] [Accepted: 10/20/2020] [Indexed: 12/13/2022] Open
Abstract
Inflammatory diseases and cancer metastases lack concrete pharmaceuticals for their effective treatment despite great strides in advancing our understanding of disease progression. One feature of these disease pathogeneses that remains to be fully explored, both biologically and pharmaceutically, is the passage of cancer and immune cells from the blood to the underlying tissue in the process of extravasation. Regardless of migratory cell type, all steps in extravasation involve molecular interactions that serve as a rich landscape of targets for pharmaceutical inhibition or promotion. Transendothelial migration (TEM), or the migration of the cell through the vascular endothelium, is a particularly promising area of interest as it constitutes the final and most involved step in the extravasation cascade. While in vivo models of cancer metastasis and inflammatory diseases have contributed to our current understanding of TEM, the knowledge surrounding this phenomenon would be significantly lacking without the use of in vitro platforms. In addition to the ease of use, low cost, and high controllability, in vitro platforms permit the use of human cell lines to represent certain features of disease pathology better, as seen in the clinic. These benefits over traditional pre-clinical models for efficacy and toxicity testing are especially important in the modern pursuit of novel drug candidates. Here, we review the cellular and molecular events involved in leukocyte and cancer cell extravasation, with a keen focus on TEM, as discovered by seminal and progressive in vitro platforms. In vitro studies of TEM, specifically, showcase the great experimental progress at the lab bench and highlight the historical success of in vitro platforms for biological discovery. This success shows the potential for applying these platforms for pharmaceutical compound screening. In addition to immune and cancer cell TEM, we discuss the promise of hepatocyte transplantation, a process in which systemically delivered hepatocytes must transmigrate across the liver sinusoidal endothelium to successfully engraft and restore liver function. Lastly, we concisely summarize the evolving field of porous membranes for the study of TEM.
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Affiliation(s)
- Alec T. Salminen
- Biomedical Engineering, University of Rochester, Rochester, NY, United States
| | - Zahra Allahyari
- Biomedical Engineering, Rochester Institute of Technology, Rochester, NY, United States
| | - Shayan Gholizadeh
- Biomedical Engineering, Rochester Institute of Technology, Rochester, NY, United States
| | - Molly C. McCloskey
- Biomedical Engineering, University of Rochester, Rochester, NY, United States
| | - Raquel Ajalik
- Biomedical Engineering, University of Rochester, Rochester, NY, United States
| | - Renee N. Cottle
- Bioengineering, Clemson University, Clemson, SC, United States
| | - Thomas R. Gaborski
- Biomedical Engineering, University of Rochester, Rochester, NY, United States
- Biomedical Engineering, Rochester Institute of Technology, Rochester, NY, United States
| | - James L. McGrath
- Biomedical Engineering, University of Rochester, Rochester, NY, United States
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Nitzahn M, Lipshutz GS. CPS1: Looking at an ancient enzyme in a modern light. Mol Genet Metab 2020; 131:289-298. [PMID: 33317798 PMCID: PMC7738762 DOI: 10.1016/j.ymgme.2020.10.003] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/17/2020] [Revised: 10/02/2020] [Accepted: 10/03/2020] [Indexed: 02/06/2023]
Abstract
The mammalian urea cycle (UC) is responsible for siphoning catabolic waste nitrogen into urea for excretion. Disruptions of the functions of any of the enzymes or transporters lead to elevated ammonia and neurological injury. Carbamoyl phosphate synthetase 1 (CPS1) is the first and rate-limiting UC enzyme responsible for the direct incorporation of ammonia into UC intermediates. Symptoms in CPS1 deficiency are typically the most severe of all UC disorders, and current clinical management is insufficient to prevent the associated morbidities and high mortality. With recent advances in basic and translational studies of CPS1, appreciation for this enzyme's essential role in the UC has been broadened to include systemic metabolic regulation during homeostasis and disease. Here, we review recent advances in CPS1 biology and contextualize them around the role of CPS1 in health and disease.
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Affiliation(s)
- Matthew Nitzahn
- Molecular Biology Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA; Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
| | - Gerald S Lipshutz
- Molecular Biology Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA; Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA; Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA; Psychiatry, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA; Intellectual and Developmental Disabilities Research Center, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA; Semel Institute for Neuroscience, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
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47
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Yu B, Li H, Chen J, He Z, Sun H, Yang G, Shang C, Wang X, Li C, Chen Y, Hu Y. Extensively expanded murine-induced hepatic stem cells maintain high-efficient hepatic differentiation potential for repopulation of injured livers. Liver Int 2020; 40:2293-2304. [PMID: 32394491 DOI: 10.1111/liv.14509] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/20/2019] [Revised: 04/10/2020] [Accepted: 05/04/2020] [Indexed: 12/28/2022]
Abstract
BACKGROUND & AIM Shortage of donor hepatocytes limits hepatocyte transplantation for clinical application. Induced hepatic stem cells (iHepSCs) have capacities of self-renewal and bipotential differentiations. Here, we investigated whether iHepSCs could be extensively expanded, and whether they could differentiate into sufficient functional hepatocytes as donors for transplantation therapy after their extensive expansions. METHODS Murine extensively expanded iHepSCs (50-55 passages) were induced to differentiate into iHepSC-Heps under a chemically defined condition. iHepSC-Heps were proved for carrying morphological hepatocyte characters and hepatocytic functions including low-density lipoprotein uptake, glycogen storage, CLF secretion, ICG uptake and release, Alb secretion, urea synthesis and metabolism-relative gene expressions respectively. Next, both iHepSCs and iHepSC-Heps were transplanted into Fah-/- mice respectively. Both liver repopulation and alleviation of liver function were compared between two transplantation groups. RESULTS Murine iHepSCs still maintained the capacities of self-renewal and bipotential differentiations after extensive expansion. The efficiency for the functional hepatocyte differentiation from extensively expanded iHepSCs reached to 72.64%. Transplantations of both extensively expanded iHepSCs and iHepSC-Heps resulted in liver engraftment in Fah-/- mice. Survival rate of Fah-/- mice recipients and level of liver repopulation were 50% and 20.32 ± 4.58% respectively in iHepSC-Heps group, while 33% and 10.4 ± 4.3% in iHepSCs group. CONCLUSIONS Extensively expanded iHepSCs can efficiently differentiate into hepatocytes in chemical defined medium. Transplantation of iHepSC-Heps was more effective and more efficient than transplantation of iHepSCs in Fah-/- mice. Our results suggested an innovative system to obtain sufficient hepatocytes through hepatic differentiation of iHepSCs generated by lineage reprogramming.
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Affiliation(s)
- Bing Yu
- Department of Cell Biology, Center for Stem Cell and Medicine, Navy Medical University (Second Military Medical University), Shanghai, P. R. China.,Department of Hepatic Surgery V, Eastern Hepatobiliary Surgery Hospital, Navy Medical University (Second Military Medical University), Shanghai, P.R. China
| | - Hengyu Li
- Department of General Surgery IV, Changhai Hospital, Navy Medical University (Second Military Medical University), Shanghai, P.R. China
| | - Jie Chen
- Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-sen University, Guangzhou, P.R. China
| | - Zhiying He
- Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, P.R. China
| | - Haixiang Sun
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion (Fudan University), Ministry of Education, Shanghai, China
| | - Guangshun Yang
- Department of Hepatic Surgery V, Eastern Hepatobiliary Surgery Hospital, Navy Medical University (Second Military Medical University), Shanghai, P.R. China
| | - Changzhen Shang
- Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-sen University, Guangzhou, P.R. China
| | - Xin Wang
- Research Center for Laboratory Animal Science, Inner Mongolia University, Huhhot, P.R. China.,Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA.,Hepatoscience Section, Cell Lab Tech Incorporation, Sunnyvale, CA, USA
| | - Chuanjiang Li
- Division of Hepatobiliopancreatic Surgery, Department of General Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, P. R. China
| | - Yajin Chen
- Department of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-sen University, Guangzhou, P.R. China
| | - Yiping Hu
- Department of Cell Biology, Center for Stem Cell and Medicine, Navy Medical University (Second Military Medical University), Shanghai, P. R. China
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48
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Thompson WS, Mondal G, Vanlith CJ, Kaiser RA, Lillegard JB. The future of gene-targeted therapy for hereditary tyrosinemia type 1 as a lead indication among the inborn errors of metabolism. Expert Opin Orphan Drugs 2020; 8:245-256. [PMID: 33224636 PMCID: PMC7676758 DOI: 10.1080/21678707.2020.1791082] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Introduction Inborn errors of metabolism (IEMs) often result from single-gene mutations and collectively cause liver dysfunction in neonates leading to chronic liver and systemic disease. Current treatments for many IEMs are limited to maintenance therapies that may still require orthotropic liver transplantation. Gene therapies offer a potentially superior approach by correcting or replacing defective genes with functional isoforms; however, they face unique challenges from complexities presented by individual diseases and their diverse etiology, presentation, and pathophysiology. Furthermore, immune responses, off-target gene disruption, and tumorigenesis are major concerns that need to be addressed before clinical application of gene therapy. Areas covered The current treatments for IEMs are reviewed as well as the advances in, and barriers to, gene therapy for IEMs. Attention is then given to ex vivo and in vivo gene therapy approaches for hereditary tyrosinemia type 1 (HT1). Of all IEMs, HT1 is particularly amenable to gene therapy because of a selective growth advantage conferred to corrected cells, thereby lowering the initial transduction threshold for phenotypic relevance. Expert opinion It is proposed that not only is HT1 a safe indication for gene therapy, its unique characteristics position it to be an ideal IEM to develop for clinical investigation.
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Affiliation(s)
| | - Gourish Mondal
- Department of Surgery, Research Scientist, Mayo Clinic, Rochester, MN, USA
| | | | - Robert A Kaiser
- Department of Surgery, Research Scientist, Mayo Clinic, Rochester, MN, USA.,Midwest Fetal Care Center, Childrens Hospital of Minnesota, MN, USA
| | - Joseph B Lillegard
- Midwest Fetal Care Center, Childrens Hospital of Minnesota, MN, USA.,Assistant Professor of Surgery, Mayo Clinic, Rochester, MN, USA
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49
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Trevisan M, Masi G, Palù G. Genome editing technologies to treat rare liver diseases. Transl Gastroenterol Hepatol 2020; 5:23. [PMID: 32258527 DOI: 10.21037/tgh.2019.10.10] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/31/2019] [Accepted: 10/18/2019] [Indexed: 12/13/2022] Open
Abstract
Liver has a central role in protein and lipid metabolism, and diseases involving hepatocytes have often repercussions on multiple organs and systems. Hepatic disorders are frequently characterized by production of defective or non-functional proteins, and traditional gene therapy approaches have been attempted for years to restore adequate protein levels through delivery of transgenes. Recently, many different genome editing platforms have been developed aimed at correcting at DNA level the defects underlying the diseases. In this Review we discuss the latest applications of these tools applied to develop therapeutic strategies for rare liver disorders, in particular updating the literature with the most recent strategies relying on base editors technology.
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Affiliation(s)
- Marta Trevisan
- Department of Molecular Medicine, University of Padova, Padova, Italy
| | - Giulia Masi
- Department of Molecular Medicine, University of Padova, Padova, Italy
| | - Giorgio Palù
- Department of Molecular Medicine, University of Padova, Padova, Italy
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50
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Stahl EC, Delgado ER, Alencastro F, LoPresti ST, Wilkinson PD, Roy N, Haschak MJ, Skillen CD, Monga SP, Duncan AW, Brown BN. Inflammation and Ectopic Fat Deposition in the Aging Murine Liver Is Influenced by CCR2. THE AMERICAN JOURNAL OF PATHOLOGY 2020; 190:372-387. [PMID: 31843499 PMCID: PMC7013280 DOI: 10.1016/j.ajpath.2019.10.016] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/30/2019] [Revised: 10/02/2019] [Accepted: 10/22/2019] [Indexed: 02/08/2023]
Abstract
Aging is associated with inflammation and metabolic syndrome, which manifests in the liver as nonalcoholic fatty liver disease (NAFLD). NAFLD can range in severity from steatosis to fibrotic steatohepatitis and is a major cause of hepatic morbidity. However, the pathogenesis of NAFLD in naturally aged animals is unclear. Herein, we performed a comprehensive study of lipid content and inflammatory signature of livers in 19-month-old aged female mice. These animals exhibited increased body and liver weight, hepatic triglycerides, and inflammatory gene expression compared with 3-month-old young controls. The aged mice also had a significant increase in F4/80+ hepatic macrophages, which coexpressed CD11b, suggesting a circulating monocyte origin. A global knockout of the receptor for monocyte chemoattractant protein (CCR2) prevented excess steatosis and inflammation in aging livers but did not reduce the number of CD11b+ macrophages, suggesting changes in macrophage accumulation precede or are independent from chemokine (C-C motif) ligand-CCR2 signaling in the development of age-related NAFLD. RNA sequencing further elucidated complex changes in inflammatory and metabolic gene expression in the aging liver. In conclusion, we report a previously unknown accumulation of CD11b+ macrophages in aged livers with robust inflammatory and metabolic transcriptomic changes. A better understanding of the hallmarks of aging in the liver will be crucial in the development of preventive measures and treatments for end-stage liver disease in elderly patients.
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Affiliation(s)
- Elizabeth C Stahl
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Evan R Delgado
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Frances Alencastro
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Samuel T LoPresti
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Bioengineering Department, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Patrick D Wilkinson
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Nairita Roy
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Martin J Haschak
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Bioengineering Department, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Clint D Skillen
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Satdarshan P Monga
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Andrew W Duncan
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pathology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania.
| | - Bryan N Brown
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Bioengineering Department, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania; Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania.
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