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Sun J, Xiong X, Lai W, Wu Z, Wang H, Yang L, Xue N, Yao Q, Song G, Zhao Y, Li L, Wang F, Fan C, Pei H. Implementing complex nucleic acid circuits in living cells. SCIENCE ADVANCES 2025; 11:eadv6512. [PMID: 40305594 PMCID: PMC12042877 DOI: 10.1126/sciadv.adv6512] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/31/2024] [Accepted: 03/26/2025] [Indexed: 05/02/2025]
Abstract
Synthetic nucleic acid-based computing has demonstrated complex computational capabilities in vitro. However, translating these circuits into living cells remains challenging because of instability and cellular interference. We introduce an allosteric strand exchange (ASE) strategy for complex intracellular computing. Leveraging conformational cooperativity to regulate strand exchange, ASE offers a modular platform for designing intracellular circuits with flexible programmability. We engineer a scalable circuit architecture based on ASE that can execute AND and OR logic and scale to an eight-input expression. We demonstrate ASE-based circuits can detect messenger RNAs with high specificity in mammalian cells via AND logic computation. The capacity of ASE-based circuits to accept messenger RNAs as inputs enables integration of endogenous cellular information for efficient multi-input information processing, demonstrated by a multi-input molecular classifier monitoring key cell reprogramming events. Reprogramming ASE-based circuit to interface with CRISPR-Cas9 enables programmable control of Cas9-targeting activity for gene editing, highlighting their potential for advancing intracellular biocomputation.
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Affiliation(s)
- Jiajia Sun
- Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, Shanghai Center of Brain-inspired Intelligent Materials and Devices, East China Normal University, Shanghai 200241, China
| | - Xiewei Xiong
- Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, Shanghai Center of Brain-inspired Intelligent Materials and Devices, East China Normal University, Shanghai 200241, China
| | - Wei Lai
- Hubei Key Laboratory of Energy Storage and Power Battery, School of Mathematics, Physics and Optoelectronic Engineering, Hubei University of Automotive Technology, Shiyan, Hubei 442002, China
| | - Zhongdong Wu
- Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, Shanghai Center of Brain-inspired Intelligent Materials and Devices, East China Normal University, Shanghai 200241, China
| | - Heming Wang
- Joint Laboratory of Biomaterials and Translational Medicine, Puheng Biomedicine Co. Ltd, Shanghai 201203, China
| | - Lei Yang
- 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 200241, China
| | - Niannian Xue
- 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 200241, China
| | - Qunyan Yao
- Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, Shanghai 200032, China
| | - Guangqi Song
- Joint Laboratory of Biomaterials and Translational Medicine, Puheng Biomedicine Co. Ltd, Shanghai 201203, China
| | - Yicheng Zhao
- Chinese Medicine Guangdong Laboratory, Hengqin, Guangdong 519031, China
| | - Li Li
- Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, Shanghai Center of Brain-inspired Intelligent Materials and Devices, East China Normal University, Shanghai 200241, China
| | - Fei Wang
- School of Chemistry and Chemical Engineering, New Cornerstone Science Laboratory, Frontiers Science Center for Transformative Molecules, National Center for Translational Medicine, Shanghai Jiao Tong University, Shanghai 200240, China
| | - Chunhai Fan
- School of Chemistry and Chemical Engineering, New Cornerstone Science Laboratory, Frontiers Science Center for Transformative Molecules, National Center for Translational Medicine, Shanghai Jiao Tong University, Shanghai 200240, China
| | - Hao Pei
- Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, Shanghai Center of Brain-inspired Intelligent Materials and Devices, East China Normal University, Shanghai 200241, China
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Zhang Y, Li L, Dong L, Cheng Y, Huang X, Xue B, Jiang C, Cao Y, Yang J. Hydrogel-Based Strategies for Liver Tissue Engineering. CHEM & BIO ENGINEERING 2024; 1:887-915. [PMID: 39975572 PMCID: PMC11835278 DOI: 10.1021/cbe.4c00079] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/14/2024] [Revised: 09/15/2024] [Accepted: 09/15/2024] [Indexed: 02/21/2025]
Abstract
The liver's role in metabolism, detoxification, and immune regulation underscores the urgency of addressing liver diseases, which claim millions of lives annually. Due to donor shortages in liver transplantation, liver tissue engineering (LTE) offers a promising alternative. Hydrogels, with their biocompatibility and ability to mimic the liver's extracellular matrix (ECM), support cell survival and function in LTE. This review analyzes recent advances in hydrogel-based strategies for LTE, including decellularized liver tissue hydrogels, natural polymer-based hydrogels, and synthetic polymer-based hydrogels. These materials are ideal for in vitro cell culture and obtaining functional hepatocytes. Hydrogels' tunable properties facilitate creating artificial liver models, such as organoids, 3D bioprinting, and liver-on-a-chip technologies. These developments demonstrate hydrogels' versatility in advancing LTE's applications, including hepatotoxicity testing, liver tissue regeneration, and treating acute liver failure. This review highlights the transformative potential of hydrogels in LTE and their implications for future research and clinical practice.
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Affiliation(s)
- Yu Zhang
- National
Laboratory of Solid State Microstructures, Department of Physics, Nanjing University, Nanjing 210093, China
- Jinan
Microecological Biomedicine Shandong Laboratory, Jinan 250021, China
| | - Luofei Li
- National
Laboratory of Solid State Microstructures, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Liang Dong
- National
Laboratory of Solid State Microstructures, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Yuanqi Cheng
- National
Laboratory of Solid State Microstructures, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Xiaoyu Huang
- National
Laboratory of Solid State Microstructures, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Bin Xue
- National
Laboratory of Solid State Microstructures, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Chunping Jiang
- Jinan
Microecological Biomedicine Shandong Laboratory, Jinan 250021, China
| | - Yi Cao
- National
Laboratory of Solid State Microstructures, Department of Physics, Nanjing University, Nanjing 210093, China
- Jinan
Microecological Biomedicine Shandong Laboratory, Jinan 250021, China
| | - Jiapeng Yang
- National
Laboratory of Solid State Microstructures, Department of Physics, Nanjing University, Nanjing 210093, China
- Jinan
Microecological Biomedicine Shandong Laboratory, Jinan 250021, China
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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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Deng J, Luo K, Xu P, Jiang Q, Wang Y, Yao Y, Chen X, Cheng F, Xie D, Deng H. High-efficiency c-Myc-mediated induction of functional hepatoblasts from the human umbilical cord mesenchymal stem cells. Stem Cell Res Ther 2021; 12:375. [PMID: 34215318 PMCID: PMC8254319 DOI: 10.1186/s13287-021-02419-1] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Accepted: 05/26/2021] [Indexed: 02/08/2023] Open
Abstract
Background Direct reprogramming of human fibroblasts to hepatocyte-like cells was proposed to generate large-scale functional hepatocytes demanded by liver tissue engineering. However, the difficulty in obtaining large quantities of human fibroblasts greatly restricted the extensive implementation of this approach. Meanwhile, human umbilical cord mesenchymal stem cells (HUMSCs) are the preferred cell source for HLCs with the advantages of limited ethical concerns, easy accessibility, and propagation in vitro. However, no direct reprogramming protocol for converting HUMSCs to hepatoblast-like cells (HLCs) has been reported. Methods HLCs were successfully generated from HUMSCs by forced expression of FOXA3, HNF1A, and HNF4A (collectively as 3TFs) and c-Myc. In vitro and in vivo functional experiments were conducted to demonstrate the hepatic phenotype, characterization, and function of HUMSC-derived HLCs (HUMSC-iHeps). ChIP-seq and RNA-seq were integrated to reveal the potential molecular mechanisms underlying c-Myc-mediated reprogramming. Results We showed that c-Myc greatly improved the trans-differentiation efficiency for HLCs from HUMSCs, which remained highly efficient in reprogramming fibroblasts into HLCs, suggesting c-Myc could promote direct reprogramming and its potentially widespread applicability for generating large amounts of HLCs in vitro. Mice transplantation experiments further confirmed the therapeutic potential of HUMSC-iHeps by liver function restoration and survival prolongation. Besides, in vivo safety assessment demonstrated the low risk of the tumorigenic potential of HUMSC-iHeps. We found that c-Myc functioned predominantly at an early phase of reprogramming, and we further unraveled the regulatory network altered by c-Myc. Conclusions c-Myc enhanced reprogramming efficiency of HLCs from HUMSCs. A large scale of functional HLCs generated more conveniently from HUMSCs could benefit biomedical studies and applications of liver diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02419-1.
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Affiliation(s)
- Jie Deng
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China.,Center for Cell Lineage and Atlas (CCLA), Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, 510005, China
| | - Kai Luo
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China.,National Frontier Center of Disease Molecular Network, West China Hospital, Sichuan University, Chengdu, 610041, China
| | - Pengchao Xu
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China
| | - Qingyuan Jiang
- Department of Obstetrics, Sichuan Provincial Hospital for Women and Children, Chengdu, China
| | - Yuan Wang
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China
| | - Yunqi Yao
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China
| | - Xiaolei Chen
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China
| | - Fuyi Cheng
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China
| | - Dan Xie
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China. .,National Frontier Center of Disease Molecular Network, West China Hospital, Sichuan University, Chengdu, 610041, China.
| | - Hongxin Deng
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, People's Republic of China.
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Ge JY, Zheng YW, Liu LP, Isoda H, Oda T. Impelling force and current challenges by chemicals in somatic cell reprogramming and expansion beyond hepatocytes. World J Stem Cells 2019; 11:650-665. [PMID: 31616541 PMCID: PMC6789182 DOI: 10.4252/wjsc.v11.i9.650] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/02/2019] [Revised: 07/07/2019] [Accepted: 08/21/2019] [Indexed: 02/06/2023] Open
Abstract
In the field of regenerative medicine, generating numerous transplantable functional cells in the laboratory setting on a large scale is a major challenge. However, the in vitro maintenance and expansion of terminally differentiated cells are challenging because of the lack of specific environmental and intercellular signal stimulations, markedly hindering their therapeutic application. Remarkably, the generation of stem/progenitor cells or functional cells with effective proliferative potential is markedly in demand for disease modeling, cell-based transplantation, and drug discovery. Despite the potent genetic manipulation of transcription factors, integration-free chemically defined approaches for the conversion of somatic cell fate have garnered considerable attention in recent years. This review aims to summarize the progress thus far and discuss the advantages, limitations, and challenges of the impact of full chemicals on the stepwise reprogramming of pluripotency, direct lineage conversion, and direct lineage expansion on somatic cells. Owing to the current chemical-mediated induction, reprogrammed pluripotent stem cells with reproducibility difficulties, and direct lineage converted cells with marked functional deficiency, it is imperative to generate the desired cell types directly by chemically inducing their potent proliferation ability through a lineage-committed progenitor state, while upholding the maturation and engraftment capacity posttransplantation in vivo. Together with the comprehensive understanding of the mechanism of chemical drives, as well as the elucidation of specificity and commonalities, the precise manipulation of the expansion for diverse functional cell types could broaden the available cell sources and enhance the cellular function for clinical application in future.
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Affiliation(s)
- Jian-Yun Ge
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
| | - Yun-Wen Zheng
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
- Institute of Regenerative Medicine and Affiliated Hospital, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
- Department of Regenerative Medicine, School of Medicine, Yokohama City University, Yokohama 236-0004, Japan.
| | - Li-Ping Liu
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
- Institute of Regenerative Medicine and Affiliated Hospital, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
| | - Hiroko Isoda
- Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
| | - Tatsuya Oda
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
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Sun L, Wang Y, Cen J, Ma X, Cui L, Qiu Z, Zhang Z, Li H, Yang RZ, Wang C, Chen X, Wang L, Ye Y, Zhang H, Pan G, Kang JS, Ji Y, Zheng YW, Zheng S, Hui L. Modelling liver cancer initiation with organoids derived from directly reprogrammed human hepatocytes. Nat Cell Biol 2019; 21:1015-1026. [PMID: 31332348 DOI: 10.1038/s41556-019-0359-5] [Citation(s) in RCA: 99] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2018] [Accepted: 06/11/2019] [Indexed: 12/24/2022]
Abstract
Human liver cancers, including hepatocellular carcinomas and intra-hepatic cholangiocarcinomas, are often diagnosed late with poor prognosis. A better understanding of cancer initiation could provide potential preventive therapies and increase survival. Models for studying human liver cancer initiation are largely missing. Here, using directly reprogrammed human hepatocytes (hiHeps) and inactivation of p53 and RB, we established organoids possessing liver architecture and function. HiHep organoids were genetically engineered to model the initial alterations in human liver cancers. Bona fide hepatocellular carcinomas were developed by overexpressing c-Myc. Excessive mitochondrion-endoplasmic reticulum coupling induced by c-Myc facilitated hepatocellular carcinoma initiation and seemed to be a target of preventive treatment. Furthermore, through the analysis of human intra-hepatic cholangiocarcinoma-enriched mutations, we demonstrate that the RAS-induced lineage conversion from hepatocytes to intra-hepatic cholangiocarcinoma cells can be prevented by the combined inhibition of Notch and JAK-STAT. Together, hiHep organoids represent a system that can be genetically manipulated to model cancer initiation and identify potential preventive therapies.
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Affiliation(s)
- Lulu Sun
- State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Yuqing Wang
- Department of Pediatric Surgery, Children's Hospital of Fudan University, Shanghai, China
| | - Jin Cen
- State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Xiaolong Ma
- State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Lei Cui
- State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Zhixin Qiu
- State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Zhengtao Zhang
- State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Hong Li
- Key Lab of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
| | - Run-Zhou Yang
- Shanghai Institute for Nutrition and Health, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Chenhua Wang
- State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Xiaotao Chen
- State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Le Wang
- Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
| | - Yao Ye
- Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Haibin Zhang
- Fifth Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China
| | - Guoyu Pan
- Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
| | - Jian-Sheng Kang
- The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Yuan Ji
- Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, China.
| | - Yun-Wen Zheng
- Faculty of Medicine, University of Tsukuba, Tsukuba-shi, Japan.
- School of Medicine, Yokohama City University, Yokohama, Japan.
- Institute of Regenerative Medicine, Jiangsu University, Zhenjiang, China.
| | - Shan Zheng
- Department of Pediatric Surgery, Children's Hospital of Fudan University, Shanghai, China.
| | - Lijian Hui
- State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.
- School of Life Science and Technology, Shanghai Tech University, Shanghai, China.
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China.
- Bio-Research Innovation Center, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Suzhou, China.
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A serum-free medium suitable for maintaining cell morphology and liver-specific function in induced human hepatocytes. Cytotechnology 2019; 71:329-344. [PMID: 30603919 DOI: 10.1007/s10616-018-0289-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2018] [Accepted: 12/05/2018] [Indexed: 12/15/2022] Open
Abstract
hiHep is a new type of hepatocyte-like cell that is predicted to be a potential unlimited source of hepatocytes for a bioartificial liver. However, hiHep cannot currently be used in clinical settings because serum must be added during the culture process. Thus, a defined medium is required. Because serum is complex, an efficient statistical approach based on the Plackett-Burman design was used. In this manner, an original medium and several significant cell growth factors were identified. These factors include insulin, VH, and VE, and the original medium was optimized based on these significant factors. Additionally, hiHep liver-specific functions and metabolism in the optimized serum-free medium were measured. Results showed that hiHep functions, such as glycogen storage, albumin secretion, and urea production, were well maintained in our optimized serum-free medium. In summary, we created a chemically defined, serum-free medium in which cell growth, proliferation, metabolism, and function were well maintained. This medium has the potential to support the clinical use of hiHep.
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Zhang K, Zhang L, Liu W, Ma X, Cen J, Sun Z, Wang C, Feng S, Zhang Z, Yue L, Sun L, Zhu Z, Chen X, Feng A, Wu J, Jiang Z, Li P, Cheng X, Gao D, Peng L, Hui L. In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity. Cell Stem Cell 2018; 23:806-819.e4. [PMID: 30416071 DOI: 10.1016/j.stem.2018.10.018] [Citation(s) in RCA: 148] [Impact Index Per Article: 21.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2018] [Revised: 09/17/2018] [Accepted: 10/12/2018] [Indexed: 12/17/2022]
Abstract
Transplantation of human hepatocytes (HHs) holds significant potential for treating liver diseases. However, the supply of transplantable HHs is severely constrained by limited donor availability and compromised capacity for in vitro expansion. In response to chronic injury, some HHs are reprogrammed into proliferative cells that express both hepatocyte and progenitor markers, suggesting exploitable strategies for expanding HHs in vitro. Here, we report defined medium conditions that allow 10,000-fold expansion of HHs. These proliferating HHs are bi-phenotypic, partially retaining hepatic features while gaining expression of progenitor-associated genes. Importantly, these cells engraft into injured mouse liver at a level comparable to primary HHs, and they undergo maturation following transplantation in vivo or differentiation in vitro. Thus, this study provides a protocol that enables large-scale expansion of transplantable HHs, which could be further developed for modeling and treating human liver disease.
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Affiliation(s)
- Kun Zhang
- Key Laboratory of Arrhythmias, Ministry of Education, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China; State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Ludi Zhang
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.
| | - Wenming Liu
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Xiaolong Ma
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Jin Cen
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Zhen Sun
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; School of Life Science and Technology, Shanghai Tech University, Shanghai 201210, China
| | - Chenhua Wang
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Sisi Feng
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Zhengtao Zhang
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Liyun Yue
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Lulu Sun
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Zhenfeng Zhu
- Department of Integrative Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Xiaotao Chen
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Anqi Feng
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Jiaying Wu
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Zhiwu Jiang
- Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Peng Li
- Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Xin Cheng
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Dong Gao
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Luying Peng
- Key Laboratory of Arrhythmias, Ministry of Education, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China.
| | - Lijian Hui
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; School of Life Science and Technology, Shanghai Tech University, Shanghai 201210, China; Stem Cell and Regenerative Medicine Innovation Academy, Beijing 100101, China.
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9
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Zhang L, Guan Z, Ye JS, Yin YF, Stoltz JF, de Isla N. Research progress in liver tissue engineering. Biomed Mater Eng 2017; 28:S113-S119. [PMID: 28372286 DOI: 10.3233/bme-171632] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Liver transplantation is the definitive treatment for patients with end-stage liver diseases (ESLD). However, it is hampered by shortage of liver donor. Liver tissue engineering, aiming at fabricating new livers in vitro, provides a potential resolution for donor shortage. Three elements need to be considered in liver tissue engineering: seeding cell resources, scaffolds and bioreactors. Studies have shown potential cell sources as hepatocytes, hepatic cell line, mesenchymal stem cells and others. They need scaffolds with perfect biocompatiblity, suitable micro-structure and appropriate degradation rate, which are essential charateristics for cell attachment, proliferation and secretion in forming extracellular matrix. The most promising scaffolds in research include decellularized whole liver, collagens and biocompatible plastic. The development and function of cells in scaffold need a microenvironment which can provide them with oxygen, nutrition, growth factors, et al. Bioreactor is expected to fulfill these requirements by mimicking the living condition in vivo. Although there is great progress in these three domains, a large gap stays still between their researches and applications. Herein, we summarized the recent development in these three major fields which are indispensable in liver tissue engineering.
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Affiliation(s)
- Lei Zhang
- BRC, First Hospital of Kun Ming (Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Zheng Guan
- BRC, First Hospital of Kun Ming (Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Jun-Song Ye
- BRC, First Hospital of Kun Ming (Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Yan-Feng Yin
- BRC, First Hospital of Kun Ming (Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Jean-François Stoltz
- Lorraine University and CNRS UNR 7365, Medical college, Vandoeuvre-lès-Nancy, France.,CHRU Nancy, Unité Therapie Cellulaire et Tissulaire, Vandoeuvre-lès-Nancy, France
| | - Natalia de Isla
- Lorraine University and CNRS UNR 7365, Medical college, Vandoeuvre-lès-Nancy, France
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10
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11
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Abstract
A decade ago mainstream molecular biologists regarded it impossible or biologically ill-motivated to understand the dynamics of complex biological phenomena, such as cancer genesis and progression, from a network perspective. Indeed, there are numerical difficulties even for those who were determined to explore along this direction. Undeterred, seven years ago a group of Chinese scientists started a program aiming to obtain quantitative connections between tumors and network dynamics. Many interesting results have been obtained. In this paper we wish to test such idea from a different angle: the connection between a normal biological process and the network dynamics. We have taken early myelopoiesis as our biological model. A standard roadmap for the cell-fate diversification during hematopoiesis has already been well established experimentally, yet little was known for its underpinning dynamical mechanisms. Compounding this difficulty there were additional experimental challenges, such as the seemingly conflicting hematopoietic roadmaps and the cell-fate inter-conversion events. With early myeloid cell-fate determination in mind, we constructed a core molecular endogenous network from well-documented gene regulation and signal transduction knowledge. Turning the network into a set of dynamical equations, we found computationally several structurally robust states. Those states nicely correspond to known cell phenotypes. We also found the states connecting those stable states. They reveal the developmental routes-how one stable state would most likely turn into another stable state. Such interconnected network among stable states enabled a natural organization of cell-fates into a multi-stable state landscape. Accordingly, both the myeloid cell phenotypes and the standard roadmap were explained mechanistically in a straightforward manner. Furthermore, recent challenging observations were also explained naturally. Moreover, the landscape visually enables a prediction of a pool of additional cell states and developmental routes, including the non-sequential and cross-branch transitions, which are testable by future experiments. In summary, the endogenous network dynamics provide an integrated quantitative framework to understand the heterogeneity and lineage commitment in myeloid progenitors.
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12
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Ji S, Zhu L, Gao Y, Zhang X, Yan Y, Cen J, Li R, Zeng R, Liao L, Hou C, Gao Y, Gao S, Wei G, Hui L. Baf60b-mediated ATM-p53 activation blocks cell identity conversion by sensing chromatin opening. Cell Res 2017; 27:642-656. [PMID: 28303890 PMCID: PMC5520852 DOI: 10.1038/cr.2017.36] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2017] [Revised: 02/07/2017] [Accepted: 02/10/2017] [Indexed: 12/13/2022] Open
Abstract
Lineage conversion by expression of lineage-specific transcription factors is a process of epigenetic remodeling that has low efficiency. The mechanism by which a cell resists lineage conversion is largely unknown. Using hepatic-specific transcription factors Foxa3, Hnf1α and Gata4 (3TF) to induce hepatic conversion in mouse fibroblasts, we showed that 3TF induced strong activation of the ATM-p53 pathway, which led to proliferation arrest and cell death, and it further prevented hepatic conversion. Notably, ATM activation, independent of DNA damage, responded to chromatin opening during hepatic conversion. By characterizing the early molecular events during hepatic conversion, we found that Baf60b, a member of the SWI/SNF chromatin remodeling complex, links chromatin opening to ATM activation by facilitating ATM recruitment to the open chromatin regions of a panel of hepatic gene loci. These findings shed light on cellular responses to lineage conversion by revealing a function of the ATM-p53 pathway in sensing chromatin opening.
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Affiliation(s)
- Shuyi Ji
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Linying Zhu
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yimeng Gao
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Xiaoran Zhang
- CAS Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yupeng Yan
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Jin Cen
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Rongxia Li
- Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Rong Zeng
- Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Lujian Liao
- Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Chunhui Hou
- Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong 518055, China
| | - Yawei Gao
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Shaorong Gao
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Gang Wei
- CAS Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Lijian Hui
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.,School of Life Science and Technology, Shanghai Tech University, 100 Haike Road, Shanghai 201210, China
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13
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Katayama H, Yasuchika K, Miyauchi Y, Kojima H, Yamaoka R, Kawai T, Yukie Yoshitoshi E, Ogiso S, Kita S, Yasuda K, Sasaki N, Fukumitsu K, Komori J, Ishii T, Uemoto S. Generation of non-viral, transgene-free hepatocyte like cells with piggyBac transposon. Sci Rep 2017; 7:44498. [PMID: 28295042 PMCID: PMC5353749 DOI: 10.1038/srep44498] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2016] [Accepted: 02/08/2017] [Indexed: 12/12/2022] Open
Abstract
Somatic cells can be reprogrammed to induced hepatocyte-like cells (iHeps) by overexpressing certain defined factors in direct reprogramming techniques. Of the various methods to deliver genes into cells, typically used genome-integrating viral vectors are associated with integration-related adverse events such as mutagenesis, whereas non-integrating viral vectors have low efficiency, making viral vectors unsuitable for clinical application. Therefore, we focused on developing a transposon system to establish a non-viral reprogramming method. Transposons are unique DNA elements that can be integrated into and removed from chromosomes. PiggyBac, a type of transposon, has high transduction efficiency and cargo capacity, and the integrated transgene can be precisely excised in the presence of transposase. This feature enables the piggyBac vector to achieve efficient transgene expression and a transgene-free state, thus making it a promising method for cell reprogramming. Here, we attempted to utilize the piggyBac transposon system to generate iHeps by integrating a transgene consisting of Hnf4a and Foxa3, and successfully obtained functional iHeps. We then demonstrated removal of the transgene to obtain transgene-free iHeps, which still maintained hepatocyte functions. This non-viral, transgene-free reprogramming method using the piggyBac vector may facilitate clinical applications of iHeps in upcoming cell therapy.
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Affiliation(s)
- Hokahiro Katayama
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Kentaro Yasuchika
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Yuya Miyauchi
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Hidenobu Kojima
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Ryoya Yamaoka
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Takayuki Kawai
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Elena Yukie Yoshitoshi
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.,Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Satoshi Ogiso
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Sadahiko Kita
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Katsutaro Yasuda
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.,Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Naoya Sasaki
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Ken Fukumitsu
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Junji Komori
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Takamichi Ishii
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Shinji Uemoto
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
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14
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Zhang L, Shao Y, Li L, Tian F, Cen J, Chen X, Hu D, Zhou Y, Xie W, Zheng Y, Ji Y, Liu M, Li D, Hui L. Efficient liver repopulation of transplanted hepatocyte prevents cirrhosis in a rat model of hereditary tyrosinemia type I. Sci Rep 2016; 6:31460. [PMID: 27510266 PMCID: PMC4980609 DOI: 10.1038/srep31460] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2016] [Accepted: 07/18/2016] [Indexed: 12/15/2022] Open
Abstract
Hereditary tyrosinemia type I (HT1) is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase (Fah). Fah-deficient mice and pigs are phenotypically analogous to human HT1, but do not recapitulate all the chronic features of the human disorder, especially liver fibrosis and cirrhosis. Rats as an important model organism for biomedical research have many advantages over other animal models. Genome engineering in rats is limited till the availability of new gene editing technologies. Using the recently developed CRISPR/Cas9 technique, we generated Fah(-/-) rats. The Fah(-/-) rats faithfully represented major phenotypic and biochemical manifestations of human HT1, including hypertyrosinemia, liver failure, and renal tubular damage. More importantly, the Fah(-/-) rats developed remarkable liver fibrosis and cirrhosis, which have not been observed in Fah mutant mice or pigs. Transplantation of wild-type hepatocytes rescued the Fah(-/-) rats from impending death. Moreover, the highly efficient repopulation of hepatocytes in Fah(-/-) livers prevented the progression of liver fibrosis to cirrhosis and in turn restored liver architecture. These results indicate that Fah(-/-) rats may be used as an animal model of HT1 with liver cirrhosis. Furthermore, Fah(-/-) rats may be used as a tool in studying hepatocyte transplantation and a bioreactor for the expansion of hepatocytes.
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Affiliation(s)
- Ludi Zhang
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academic of Sciences, Shanghai, China
| | - Yanjiao Shao
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Lu Li
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academic of Sciences, Shanghai, China
| | - Feng Tian
- Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Jin Cen
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academic of Sciences, Shanghai, China
| | - Xiaotao Chen
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academic of Sciences, Shanghai, China
| | - Dan Hu
- State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai, China
| | - Yan Zhou
- State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai, China
| | - Weifen Xie
- Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China
| | - Yunwen Zheng
- Department of Advanced Gastroenterological Surgical Science and Technology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
| | - Yuan Ji
- Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Mingyao Liu
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Dali Li
- Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Lijian Hui
- State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academic of Sciences, Shanghai, China
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15
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Fu Y, Deng J, Jiang Q, Wang Y, Zhang Y, Yao Y, Cheng F, Chen X, Xu F, Huang M, Yang Y, Zhang S, Yu D, Zhao RC, Wei Y, Deng H. Rapid generation of functional hepatocyte-like cells from human adipose-derived stem cells. Stem Cell Res Ther 2016; 7:105. [PMID: 27495937 PMCID: PMC4974756 DOI: 10.1186/s13287-016-0364-6] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2016] [Revised: 06/24/2016] [Accepted: 07/13/2016] [Indexed: 02/05/2023] Open
Abstract
BACKGROUND Liver disease is a major cause of death worldwide. Orthotropic liver transplantation (OLT) represents the only effective treatment for patients with liver failure, but the increasing demand for organs is unfortunately so great that its application is limited. Hepatocyte transplantation is a promising alternative to OLT for the treatment of some liver-based metabolic disorders or acute liver failure. Unfortunately, the lack of donor livers also makes it difficult to obtain enough viable hepatocytes for hepatocyte-based therapies. Currently, a fundamental solution to this key problem is still lacking. Here we show a novel non-transgenic protocol that facilitates the rapid generation of functional induced hepatocytes (iHeps) from human adipose-derived stem cells (hADSCs), providing a source of available cells for autologous hepatocytes to treat liver disease. METHODS We used collagenase digestion to isolate hADSCs. The surface marker was detected by flow cytometry. The multipotential differentiation potency was detected by induction into adipocytes, osteocytes, and chondrocytes. Passage 3-7 hADSCs were induced into iHeps using an induction culture system composed of small molecule compounds and cell factors. RESULTS Primary cultured hADSCs presented a fusiform or polygon appearance that became fibroblast-like after passage 3. More than 95 % of the cells expressed the mesenchymal cell markers CD29, CD44, CD166, CD105, and CD90. hADSCs possessed multipotential differentiation towards adipocytes, osteocytes, and chondrocytes. We rapidly induced hADSCs into iHeps within 10 days in vitro; the cellular morphology changed from fusiform to close-connected cubiform, which was similar to hepatocytes. After induction, most of the iHeps co-expressed albumin and alpha-1 antitrypsin; they also expressed mature hepatocyte special genes and achieved the basic functions of hepatocyte. Moreover, iHep transplantation could improve the liver function of acute liver-injured NPG mice and prolong life. CONCLUSIONS We isolated highly purified hADSCs and rapidly induced them into functional hepatocyte-like cells within 10 days. These results provide a source of available cells for autologous hepatocytes to treat liver disease.
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Affiliation(s)
- Yanli Fu
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
| | - Jie Deng
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
| | - Qingyuan Jiang
- Department of Obstetrics, Sichuan Provincial Hospital For Women and Children, Chengdu, People’s Republic of China
| | - Yuan Wang
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
| | - Yujing Zhang
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
| | - Yunqi Yao
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
| | - Fuyi Cheng
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
| | - Xiaolei Chen
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
| | - Fen Xu
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
| | - Meijuan Huang
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
- Department of Thoracic Oncology, Cancer Center, West China Hospital, Sichuan University, Chengdu, People’s Republic of China
| | - Yang Yang
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
| | - Shuang Zhang
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
| | - Dechao Yu
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
| | - Robert Chunhua Zhao
- Center of Excellence in Tissue Engineering, Key Laboratory of Beijing, Institute of Basic Medical Sciences and School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China
| | - Yuquan Wei
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
| | - Hongxin Deng
- State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041 People’s Republic of China
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16
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Kapere Ochieng J, Schilders K, Kool H, Buscop-van Kempen M, Boerema-De Munck A, Grosveld F, Wijnen R, Tibboel D, Rottier RJ. Differentiated type II pneumocytes can be reprogrammed by ectopic Sox2 expression. PLoS One 2014; 9:e107248. [PMID: 25210856 PMCID: PMC4161395 DOI: 10.1371/journal.pone.0107248] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2014] [Accepted: 08/12/2014] [Indexed: 12/24/2022] Open
Abstract
The adult lung contains several distinct stem cells, although their properties and full potential are still being sorted out. We previously showed that ectopic Sox2 expression in the developing lung manipulated the fate of differentiating cells. Here, we addressed the question whether fully differentiated cells could be redirected towards another cell type. Therefore, we used transgenic mice to express an inducible Sox2 construct in type II pneumocytes, which are situated in the distal, respiratory areas of the lung. Within three days after the induction of the transgene, the type II cells start to proliferate and form clusters of cuboidal cells. Prolonged Sox2 expression resulted in the reversal of the type II cell towards a more embryonic, precursor-like cell, being positive for the stem cell markers Sca1 and Ssea1. Moreover, the cells started to co-express Spc and Cc10, characteristics of bronchioalveolar stem cells. We demonstrated that Sox2 directly regulates the expression of Sca1. Subsequently, these cells expressed Trp63, a marker for basal cells of the trachea. So, we show that the expression of one transcription factor in fully differentiated, distal lung cells changes their fate towards proximal cells through intermediate cell types. This may have implications for regenerative medicine, and repair of diseased and damaged lungs.
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Affiliation(s)
- Joshua Kapere Ochieng
- Department of Pediatric Surgery of the Erasmus MC-Sophia Children's Hospital, Rotterdam, the Netherlands
| | - Kim Schilders
- Department of Pediatric Surgery of the Erasmus MC-Sophia Children's Hospital, Rotterdam, the Netherlands
| | - Heleen Kool
- Department of Pediatric Surgery of the Erasmus MC-Sophia Children's Hospital, Rotterdam, the Netherlands
| | - Marjon Buscop-van Kempen
- Department of Pediatric Surgery of the Erasmus MC-Sophia Children's Hospital, Rotterdam, the Netherlands
| | - Anne Boerema-De Munck
- Department of Pediatric Surgery of the Erasmus MC-Sophia Children's Hospital, Rotterdam, the Netherlands
| | - Frank Grosveld
- Department of Cell Biology of the Erasmus MC, Rotterdam, the Netherlands
| | - Rene Wijnen
- Department of Pediatric Surgery of the Erasmus MC-Sophia Children's Hospital, Rotterdam, the Netherlands
| | - Dick Tibboel
- Department of Pediatric Surgery of the Erasmus MC-Sophia Children's Hospital, Rotterdam, the Netherlands
| | - Robbert J. Rottier
- Department of Pediatric Surgery of the Erasmus MC-Sophia Children's Hospital, Rotterdam, the Netherlands
- Department of Cell Biology of the Erasmus MC, Rotterdam, the Netherlands
- * E-mail:
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17
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Krueger W, Boelsterli UA, Rasmussen TP. Stem Cell Strategies to Evaluate Idiosyncratic Drug-induced Liver Injury. J Clin Transl Hepatol 2014; 2:143-52. [PMID: 26355943 PMCID: PMC4521249 DOI: 10.14218/jcth.2014.00012] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/04/2014] [Revised: 05/13/2014] [Accepted: 06/07/2014] [Indexed: 12/14/2022] Open
Abstract
The host-dependent nature of idiosyncratic drug-induced liver injury (iDILI) suggests that rare genetic polymorphisms may contribute to the disease. Indeed, a few mutations in key genes have already been identified using conventional human genetics approaches. Over 50 commonly used drugs can precipitate iDILI, making this a substantial medical problem. Only recently have human induced pluripotent stem cells been used as a research tool to discover novel iDILI genes and to study the mechanisms of iDILI in vitro. Here we review the current state of stem cell use in the investigation of iDILI, with a special focus on genetics. In addition, the concerns and difficulties associated with genetics and animal model research are discussed. We then present the features of patient-specific pluripotent stem cells (which may be derived from iDILI patients themselves), and explain why these cells may be of great utility. A variety of recent approaches to produce hepatocyte-like cells from pluripotent cells and the associated advantages and limitations of such cells are discussed. Future directions for the use of stem cell science to investigate iDILI include novel ways to identify new iDILI genes, a consideration of epigenetic impacts on iDILI, and the development of new and improved strategies for the production of hepatocytes from human pluripotent cells.
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Affiliation(s)
- Winfried Krueger
- Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT, USA
| | - Urs A. Boelsterli
- Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT, USA
| | - Theodore P. Rasmussen
- Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT, USA
- Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA
- University of Connecticut Stem Cell Institute, Storrs/Farmington, CT, USA
- Correspondence to: Theodore P. Rasmussen, Department of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Road, U-3092, Storrs, CT 06269, USA. Tel: +86-486-8339, Fax: +86-486-5792. E-mail:
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19
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Huang P, Zhang L, Gao Y, He Z, Yao D, Wu Z, Cen J, Chen X, Liu C, Hu Y, Lai D, Hu Z, Chen L, Zhang Y, Cheng X, Ma X, Pan G, Wang X, Hui L. Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. Cell Stem Cell 2014; 14:370-84. [PMID: 24582927 DOI: 10.1016/j.stem.2014.01.003] [Citation(s) in RCA: 407] [Impact Index Per Article: 37.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2013] [Revised: 11/07/2013] [Accepted: 01/02/2014] [Indexed: 12/25/2022]
Abstract
The generation of large numbers of functional human hepatocytes for cell-based approaches to liver disease is an important and unmet goal. Direct reprogramming of fibroblasts to hepatic lineages could offer a solution to this problem but so far has only been achieved with mouse cells. Here, we generated human induced hepatocytes (hiHeps) from fibroblasts by lentiviral expression of FOXA3, HNF1A, and HNF4A. hiHeps express hepatic gene programs, can be expanded in vitro, and display functions characteristic of mature hepatocytes, including cytochrome P450 enzyme activity and biliary drug clearance. Upon transplantation into mice with concanavalin-A-induced acute liver failure and fatal metabolic liver disease due to fumarylacetoacetate dehydrolase (Fah) deficiency, hiHeps restore the liver function and prolong survival. Collectively, our results demonstrate successful lineage conversion of nonhepatic human cells into mature hepatocytes with potential for biomedical and pharmaceutical applications.
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Affiliation(s)
- Pengyu Huang
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Ludi Zhang
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yimeng Gao
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Zhiying He
- Department of Cell Biology, Second Military Medical University, Shanghai 200433, China
| | - Dan Yao
- Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
| | - Zhitao Wu
- Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
| | - Jin Cen
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Xiaotao Chen
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Changcheng Liu
- Department of Cell Biology, Second Military Medical University, Shanghai 200433, China
| | - Yiping Hu
- Department of Cell Biology, Second Military Medical University, Shanghai 200433, China
| | - Dongmei Lai
- The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200030, China
| | - Zhenlei Hu
- Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China
| | - Li Chen
- Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
| | - Ying Zhang
- Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
| | - Xin Cheng
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Xiaojun Ma
- Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
| | - Guoyu Pan
- Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
| | - Xin Wang
- Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010021, China; Department of Laboratory Medicine and Pathology, Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA; Hepatoscience Incorporation, 4062 Fabian Way, Palo Alto, CA 94303, USA
| | - Lijian Hui
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
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Zhang S, Wang XJ. Promote Connections of Young Computational Biologists in China. GENOMICS, PROTEOMICS & BIOINFORMATICS 2013; 11:253-6. [PMID: 23835348 PMCID: PMC4357815 DOI: 10.1016/j.gpb.2013.07.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/21/2013] [Revised: 06/23/2013] [Accepted: 07/01/2013] [Indexed: 02/01/2023]
Affiliation(s)
- Shihua Zhang
- National Center for Mathematics and Interdisciplinary Sciences, Academy of Mathematics and Systems Science, Chinese Academy of Sciences, Beijing 100190, China
- Corresponding authors.
| | - Xiu-Jie Wang
- Center for Molecular Systems Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
- Corresponding authors.
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