|
1
|
Gupta S, Sharma A, Rajakannu M, Bisevac J, Rela M, Verma RS. Small Molecule-Mediated Stage-Specific Reprogramming of MSCs to Hepatocyte-Like Cells and Hepatic Tissue for Liver Injury Treatment. Stem Cell Rev Rep 2024; 20:2215-2235. [PMID: 39259445 PMCID: PMC11554881 DOI: 10.1007/s12015-024-10771-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/04/2024] [Indexed: 09/13/2024]
Abstract
BACKGROUND Derivation of hepatocytes from stem cells has been established through various protocols involving growth factor (GF) and small molecule (SM) agents, among others. However, mesenchymal stem cell-based derivation of hepatocytes still remains expensive due to the use of a cocktail of growth factors, and a long duration of differentiation is needed, thus limiting its potential clinical application. METHODS In this study, we developed a chemically defined differentiation strategy that is exclusively based on SM and takes 14 days, while the GF-based protocol requires 23-28 days. RESULTS We optimized a stage-specific differentiation protocol for the differentiation of rat bone marrow-derived mesenchymal stem cells (MSCs) into functional hepatocyte-like cells (dHeps) that involved four stages, i.e., definitive endoderm (DE), hepatic competence (HC), hepatic specification (HS) and hepatic differentiation and growth. We further generated hepatic tissue using human decellularized liver extracellular matrix and compared it with hepatic tissue derived from the growth factor-based protocol at the transcriptional level. dHep, upon transplantation in a rat model of acute liver injury (ALI), was capable of ameliorating liver injury in rats and improving liver function and tissue damage compared to those in the ALI model. CONCLUSIONS In summary, this is the first study in which hepatocytes and hepatic tissue were derived from MSCs utilizing a stage-specific strategy by exclusively using SM as a differentiation factor.
Collapse
Affiliation(s)
- Santosh Gupta
- Stem Cell and Molecular Biology, Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, 600036, India.
- Centre for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
| | - Akriti Sharma
- Stem Cell and Molecular Biology, Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, 600036, India
| | - Muthukumarassamy Rajakannu
- The Institute of Liver Disease & Transplantation, Dr. Rela Institute & Medical Centre, Bharath Institute of Higher Education & Research, Chromepet, Tamil Nadu, India
| | - Jovana Bisevac
- Centre for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Institute of Clinical Medicine, University of Oslo, Oslo, Norway
| | - Mohamed Rela
- The Institute of Liver Disease & Transplantation, Dr. Rela Institute & Medical Centre, Bharath Institute of Higher Education & Research, Chromepet, Tamil Nadu, India
| | - Rama Shanker Verma
- Stem Cell and Molecular Biology, Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, 600036, India.
| |
Collapse
|
|
2
|
Gao Y, Gadd VL, Heim M, Grant R, Bate TSR, Esser H, Gonzalez SF, Man TY, Forbes SJ, Callanan A. Combining human liver ECM with topographically featured electrospun scaffolds for engineering hepatic microenvironment. Sci Rep 2024; 14:23192. [PMID: 39369012 PMCID: PMC11455933 DOI: 10.1038/s41598-024-73827-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2024] [Accepted: 09/20/2024] [Indexed: 10/07/2024] Open
Abstract
Liver disease cases are rapidly expanding worldwide, and transplantation remains the only effective cure for end-stage disease. There is an increasing demand for developing potential drug treatments, and regenerative therapies using in-vitro culture platforms. Human decellularized extracellular matrix (dECM) is an appealing alternative to conventional animal tissues as it contains human-specific proteins and can serve as scaffolding materials. Herein we exploit this with human donor tissue from discarded liver which was not suitable for transplant using a synergistic approach to combining biological and topographical cues in electrospun materials as an in-vitro culture platform. To realise this, we developed a methodology for incorporating human liver dECM into electrospun polycaprolactone (PCL) fibres with surface nanotopographies (230-580 nm). The hybrid scaffolds were fabricated using varying concentrations of dECM; their morphology, mechanical properties, hydrophilicity and stability were analysed. The scaffolds were validated using HepG2 and primary mouse hepatocytes, with subsequent results indicating that the modified scaffolds-maintained cell growth and influenced cell attachment, proliferation and hepatic-related gene expression. This work demonstrates a novel approach to harvesting the potential from decellularized human tissues in the form of innovative in-vitro culture platforms for liver.
Collapse
Affiliation(s)
- Yunxi Gao
- Institute for Bioengineering, School of Engineering, University of Edinburgh, Edinburgh, UK
- Foundation of Liver Research, The Roger Williams Institute of Liver Study, London, UK
| | - Victoria L Gadd
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, UK
| | - Maria Heim
- Institute for Bioengineering, School of Engineering, University of Edinburgh, Edinburgh, UK
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, UK
| | - Rhiannon Grant
- MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht, The Netherlands
| | - Thomas S R Bate
- Institute for Bioengineering, School of Engineering, University of Edinburgh, Edinburgh, UK
- Vanderbilt University Medical Center, Nashville, USA
| | - Hannah Esser
- Institute for Bioengineering, School of Engineering, University of Edinburgh, Edinburgh, UK
- Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria
| | - Sofia Ferreira Gonzalez
- Centre for Inflammation Research, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, UK
| | - Tak Yung Man
- Centre for Inflammation Research, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, UK
| | - Stuart J Forbes
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, UK
| | - Anthony Callanan
- Institute for Bioengineering, School of Engineering, University of Edinburgh, Edinburgh, UK.
| |
Collapse
|
|
3
|
Wani SI, Mir TA, Nakamura M, Tsuchiya T, Alzhrani A, Iwanaga S, Arai K, Alshehri EA, Shamma T, Obeid DA, Chinnappan R, Assiri AM, Yaqinuddin A, Vashist YK, Broering DC. A review of current state-of-the-art materiobiology and technological approaches for liver tissue engineering. BIOPRINTING 2024; 42:e00355. [DOI: 10.1016/j.bprint.2024.e00355] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/20/2025]
|
|
4
|
Xie H, Li G, Fu Y, Jiang N, Yi S, Kong X, Shi J, Yin S, Peng J, Jiang Y, Lu S, Deng H, Xie B. A two-step strategy to expand primary human hepatocytes in vitro with efficient metabolic and regenerative capacities. Stem Cell Res Ther 2024; 15:281. [PMID: 39227965 PMCID: PMC11373096 DOI: 10.1186/s13287-024-03911-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2024] [Accepted: 08/29/2024] [Indexed: 09/05/2024] Open
Abstract
BACKGROUND Primary human hepatocytes (PHHs) are highly valuable for drug-metabolism evaluation, liver disease modeling and hepatocyte transplantation. However, their availability is significantly restricted due to limited donor sources, alongside their constrained proliferation capabilities and reduced functionality when cultured in vitro. To address this challenge, we aimed to develop a novel method to efficiently expand PHHs in vitro without a loss of function. METHODS By mimicking the in vivo liver regeneration route, we developed a two-step strategy involving the de-differentiation/expansion and subsequent maturation of PHHs to generate abundant functional hepatocytes in vitro. Initially, we applied SiPer, a prediction algorithm, to identify candidate small molecules capable of activating liver regenerative transcription factors, thereby formulating a novel hepatic expansion medium to de-differentiate PHHs into proliferative human hepatic progenitor-like cells (ProHPLCs). These ProHPLCs were then re-differentiated into functionally mature hepatocytes using a new hepatocyte maturation condition. Additionally, we investigated the underlying mechanism of PHHs expansion under our new conditions. RESULTS The novel hepatic expansion medium containing hydrocortisone facilitated the de-differentiation of PHHs into ProHPLCs, which exhibited key hepatic progenitor characteristics and demonstrated a marked increase in proliferation capacity compared to cells cultivated in previously established expansion conditions. Remarkably, these subsequent matured hepatocytes rivaled PHHs in terms of transcriptome profiles, drug metabolizing activities and in vivo engraftment capabilities. Importantly, our findings suggest that the enhanced expansion of PHHs by hydrocortisone may be mediated through the PPARα signaling pathway and regenerative transcription factors. CONCLUSIONS This study presents a two-step strategy that initially induces PHHs into a proliferative state (ProHPLCs) to ensure sufficient cell quantity, followed by the maturation of ProHPLCs into fully functional hepatocytes to guarantee optimal cell quality. This approach offers a promising means of producing large numbers of seeding cells for hepatocyte-based applications.
Collapse
Affiliation(s)
- Huangfan Xie
- Laboratory of Neurological Diseases and Brain Function, The Affiliated Hospital, Southwest Medical University, Luzhou, 646000, Sichuan, China
- Institute of Epigenetics and Brain Science, Southwest Medical University, Luzhou, 646000, Sichuan, China
| | - Guangya Li
- MOE Engineering Research Center of Regenerative Medicine, School of Basic Medical Sciences, State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center and the MOE Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100191, China
| | - Yunxi Fu
- Laboratory of Neurological Diseases and Brain Function, The Affiliated Hospital, Southwest Medical University, Luzhou, 646000, Sichuan, China
- Institute of Epigenetics and Brain Science, Southwest Medical University, Luzhou, 646000, Sichuan, China
| | - Nan Jiang
- Laboratory of Neurological Diseases and Brain Function, The Affiliated Hospital, Southwest Medical University, Luzhou, 646000, Sichuan, China
| | - Simeng Yi
- Laboratory of Neurological Diseases and Brain Function, The Affiliated Hospital, Southwest Medical University, Luzhou, 646000, Sichuan, China
| | - Xi Kong
- Laboratory of Neurological Diseases and Brain Function, The Affiliated Hospital, Southwest Medical University, Luzhou, 646000, Sichuan, China
| | - Jihang Shi
- Department of Gastroenterology, The Second Medical Center of PLA General Hospital, Beijing, 100853, China
| | - Shigang Yin
- Laboratory of Neurological Diseases and Brain Function, The Affiliated Hospital, Southwest Medical University, Luzhou, 646000, Sichuan, China
- Institute of Epigenetics and Brain Science, Southwest Medical University, Luzhou, 646000, Sichuan, China
| | - Jianhua Peng
- Laboratory of Neurological Diseases and Brain Function, The Affiliated Hospital, Southwest Medical University, Luzhou, 646000, Sichuan, China
- Institute of Epigenetics and Brain Science, Southwest Medical University, Luzhou, 646000, Sichuan, China
| | - Yong Jiang
- Laboratory of Neurological Diseases and Brain Function, The Affiliated Hospital, Southwest Medical University, Luzhou, 646000, Sichuan, China
- Institute of Epigenetics and Brain Science, Southwest Medical University, Luzhou, 646000, Sichuan, China
| | - Shichun Lu
- Faculty of Hepato-Pancreato-Biliary Surgery, Key Laboratory of Digital Hepatobiliary Surgery, Institute of Hepatobiliary Surgery of Chinese PLA, Chinese PLA General Hospital, Beijing, 100853, China.
| | - Hongkui Deng
- MOE Engineering Research Center of Regenerative Medicine, School of Basic Medical Sciences, State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center and the MOE Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100191, China.
| | - Bingqing Xie
- Laboratory of Neurological Diseases and Brain Function, The Affiliated Hospital, Southwest Medical University, Luzhou, 646000, Sichuan, China.
- Institute of Epigenetics and Brain Science, Southwest Medical University, Luzhou, 646000, Sichuan, China.
| |
Collapse
|
|
5
|
Liu J, Du Y, Xiao X, Tan D, He Y, Qin L. Construction of in vitro liver-on-a-chip models and application progress. Biomed Eng Online 2024; 23:33. [PMID: 38491482 PMCID: PMC10941602 DOI: 10.1186/s12938-024-01226-y] [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: 11/23/2023] [Accepted: 02/29/2024] [Indexed: 03/18/2024] Open
Abstract
The liver is the largest internal organ of the human body. It has a complex structure and function and plays a vital role in drug metabolism. In recent decades, extensive research has aimed to develop in vitro models that can simulate liver function to demonstrate changes in the physiological and pathological environment of the liver. Animal models and in vitro cell models are common, but the data obtained from animal models lack relevance when applied to humans, while cell models have limited predictive ability for metabolism and toxicity in humans. Recent advancements in tissue engineering, biomaterials, chip technology, and 3D bioprinting have provided opportunities for further research in in vitro models. Among them, liver-on-a-Chip (LOC) technology has made significant achievements in reproducing the in vivo behavior, physiological microenvironment, and metabolism of cells and organs. In this review, we discuss the development of LOC and its research progress in liver diseases, hepatotoxicity tests, and drug screening, as well as chip combinations. First, we review the structure and the physiological function of the liver. Then, we introduce the LOC technology, including general concepts, preparation materials, and methods. Finally, we review the application of LOC in disease modeling, hepatotoxicity tests, drug screening, and chip combinations, as well as the future challenges and directions of LOC.
Collapse
Affiliation(s)
- Jie Liu
- Guizhou Engineering Research Center of Industrial Key-Technology for Dendrobium Nobile, School of Pharmacy, Zunyi Medical University, Zunyi, 563000, China
- The Second Affiliated Hospital of Zunyi Medical University, Zunyi, 563000, China
| | - Yimei Du
- Guizhou Engineering Research Center of Industrial Key-Technology for Dendrobium Nobile, School of Pharmacy, Zunyi Medical University, Zunyi, 563000, China
- Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, 563000, Guizhou, China
| | - Xinxin Xiao
- Guizhou Engineering Research Center of Industrial Key-Technology for Dendrobium Nobile, School of Pharmacy, Zunyi Medical University, Zunyi, 563000, China
| | - Daopeng Tan
- Guizhou Engineering Research Center of Industrial Key-Technology for Dendrobium Nobile, School of Pharmacy, Zunyi Medical University, Zunyi, 563000, China
- Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, 563000, Guizhou, China
| | - Yuqi He
- Guizhou Engineering Research Center of Industrial Key-Technology for Dendrobium Nobile, School of Pharmacy, Zunyi Medical University, Zunyi, 563000, China.
- Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, 563000, Guizhou, China.
| | - Lin Qin
- Guizhou Engineering Research Center of Industrial Key-Technology for Dendrobium Nobile, School of Pharmacy, Zunyi Medical University, Zunyi, 563000, China.
- Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, 563000, Guizhou, China.
| |
Collapse
|
|
6
|
Fujisaka Y, Nakagawa T, Tomoda K, Watanabe M, Matsunaga N, Tamura Y, Ikeda S, Imagawa A, Asahi M. The cytotoxicity of gefitinib on patient‑derived induced pluripotent stem cells reflects gefitinib‑induced liver injury in the clinical setting. Oncol Lett 2023; 26:520. [PMID: 37927418 PMCID: PMC10623090 DOI: 10.3892/ol.2023.14108] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2023] [Accepted: 09/22/2023] [Indexed: 11/07/2023] Open
Abstract
Gefitinib is a key drug used in the treatment of non-small cell lung cancer (NSCLC) with EGFR mutations. Gefitinib therapy is superior to conventional chemotherapy for the progression-free survival rate of patients with EGFR mutations. However, 10-26% of patients develop grade 3 or higher hepatotoxicity during gefitinib treatment; therefore, the development of preclinical tests for hepatotoxicity prior to clinical use is desirable. The present study evaluated the use of induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iPSC-heps), as a platform for preclinical test development. Patient-derived iPSCs were generated by reprogramming peripheral blood mononuclear cells obtained from two groups of gefitinib-treated patients with severe hepatotoxicity [toxicity group (T group)] or mild hepatotoxicity [no clinical toxicity group (N group)]. To examine the hepatotoxicity, the iPSCs from both T and N groups were differentiated into hepatocytes to obtain iPSC-heps. Differentiation was confirmed by measuring the expression levels of hepatocyte markers, such as albumin or α-fetoprotein, via western blotting and quantitative PCR analyses. Cytotoxicity in iPSCs and iPSC-heps after gefitinib treatment was evaluated using a lactate dehydrogenase release assay. The gefitinib-induced cytotoxicity in iPSCs from the T group was significantly higher than that from the N group, whereas there were no significant differences between the groups of iPSC-heps. These results suggested that using iPSCs in preclinical assessment may be a good indicator for the prediction of gefitinib-induced cytotoxicity in clinical use.
Collapse
Affiliation(s)
- Yasuhito Fujisaka
- Department of Medical Oncology, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Takatoshi Nakagawa
- Department of Pharmacology, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Kiichiro Tomoda
- Department of Pharmacology, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Marina Watanabe
- Department of Pharmacology, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Ninso Matsunaga
- Department of Internal Medicine (I), Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Yosuke Tamura
- Department of Internal Medicine (I), Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Soichiro Ikeda
- Department of Internal Medicine (I), Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Akihisa Imagawa
- Department of Internal Medicine (I), Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| | - Michio Asahi
- Department of Pharmacology, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka 569-0801, Japan
| |
Collapse
|
|
7
|
Luo Q, Wang N, Que H, Mai E, Hu Y, Tan R, Gu J, Gong P. Pluripotent Stem Cell-Derived Hepatocyte-like Cells: Induction Methods and Applications. Int J Mol Sci 2023; 24:11592. [PMID: 37511351 PMCID: PMC10380504 DOI: 10.3390/ijms241411592] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Revised: 07/09/2023] [Accepted: 07/12/2023] [Indexed: 07/30/2023] Open
Abstract
The development of regenerative medicine provides new options for the treatment of end-stage liver diseases. Stem cells, such as bone marrow mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells (iPSCs), are effective tools for tissue repair in regenerative medicine. iPSCs are an appropriate source of hepatocytes for the treatment of liver disease due to their unlimited multiplication capacity, their coverage of the entire range of genetics required to simulate human disease, and their evasion of ethical implications. iPSCs have the ability to gradually produce hepatocyte-like cells (HLCs) with homologous phenotypes and physiological functions. However, how to induce iPSCs to differentiate into HLCs efficiently and accurately is still a hot topic. This review describes the existing approaches for inducing the differentiation of iPSCs into HLCs, as well as some challenges faced, and summarizes various parameters for determining the quality and functionality of HLCs. Furthermore, the application of iPSCs for in vitro hepatoprotective drug screening and modeling of liver disease is discussed. In conclusion, iPSCs will be a dependable source of cells for stem-cell therapy to treat end-stage liver disease and are anticipated to facilitate individualized treatment for liver disease in the future.
Collapse
Affiliation(s)
- Qiulin Luo
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
| | - Nan Wang
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
| | - Hanyun Que
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
| | - Erziya Mai
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
| | - Yanting Hu
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
| | - Rui Tan
- College of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610032, China
| | - Jian Gu
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
| | - Puyang Gong
- College of Pharmacy, Southwest Minzu University, Chengdu 610225, China
| |
Collapse
|
|
8
|
Kasarinaite A, Sinton M, Saunders PTK, Hay DC. The Influence of Sex Hormones in Liver Function and Disease. Cells 2023; 12:1604. [PMID: 37371074 PMCID: PMC10296738 DOI: 10.3390/cells12121604] [Citation(s) in RCA: 25] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2023] [Revised: 06/09/2023] [Accepted: 06/09/2023] [Indexed: 06/29/2023] Open
Abstract
The liver performs a multitude of bodily functions, whilst retaining the ability to regenerate damaged tissue. In this review, we discuss sex steroid biology, regulation of mammalian liver physiology and the development of new model systems to improve our understanding of liver biology in health and disease. A major risk factor for the development of liver disease is hepatic fibrosis. Key drivers of this process are metabolic dysfunction and pathologic activation of the immune system. Although non-alcoholic fatty liver disease (NAFLD) is largely regarded as benign, it does progress to non-alcoholic steatohepatitis in a subset of patients, increasing their risk of developing cirrhosis and hepatocellular carcinoma. NAFLD susceptibility varies across the population, with obesity and insulin resistance playing a strong role in the disease development. Additionally, sex and age have been identified as important risk factors. In addition to the regulation of liver biochemistry, sex hormones also regulate the immune system, with sexual dimorphism described for both innate and adaptive immune responses. Therefore, sex differences in liver metabolism, immunity and their interplay are important factors to consider when designing, studying and developing therapeutic strategies to treat human liver disease. The purpose of this review is to provide the reader with a general overview of sex steroid biology and their regulation of mammalian liver physiology.
Collapse
Affiliation(s)
- Alvile Kasarinaite
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, The University of Edinburgh, Edinburgh BioQuarter, Edinburgh EH16 4UU, UK
| | - Matthew Sinton
- School of Biodiversity, One Health, and Veterinary Medicine, University of Glasgow, Glasgow G61 1QH, UK
- Wellcome Centre for Integrative Parasitology, University of Glasgow, Glasgow G12 9TA, UK
| | - Philippa T. K. Saunders
- Centre for Inflammation Research, Institute for Regeneration and Repair, The University of Edinburgh, Edinburgh BioQuarter, Edinburgh EH16 4UU, UK
| | - David C. Hay
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, The University of Edinburgh, Edinburgh BioQuarter, Edinburgh EH16 4UU, UK
| |
Collapse
|
|
9
|
Tsai HHD, House JS, Wright FA, Chiu WA, Rusyn I. A tiered testing strategy based on in vitro phenotypic and transcriptomic data for selecting representative petroleum UVCBs for toxicity evaluation in vivo. Toxicol Sci 2023; 193:219-233. [PMID: 37079747 PMCID: PMC10230285 DOI: 10.1093/toxsci/kfad041] [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] [Indexed: 04/22/2023] Open
Abstract
Hazard evaluation of substances of "unknown or variable composition, complex reaction products and biological materials" (UVCBs) remains a major challenge in regulatory science because their chemical composition is difficult to ascertain. Petroleum substances are representative UVCBs and human cell-based data have been previously used to substantiate their groupings for regulatory submissions. We hypothesized that a combination of phenotypic and transcriptomic data could be integrated to make decisions as to selection of group-representative worst-case petroleum UVCBs for subsequent toxicity evaluation in vivo. We used data obtained from 141 substances from 16 manufacturing categories previously tested in 6 human cell types (induced pluripotent stem cell [iPSC]-derived hepatocytes, cardiomyocytes, neurons, and endothelial cells, and MCF7 and A375 cell lines). Benchmark doses for gene-substance combinations were calculated, and both transcriptomic and phenotype-derived points of departure (PODs) were obtained. Correlation analysis and machine learning were used to assess associations between phenotypic and transcriptional PODs and to determine the most informative cell types and assays, thus representing a cost-effective integrated testing strategy. We found that 2 cell types-iPSC-derived-hepatocytes and -cardiomyocytes-contributed the most informative and protective PODs and may be used to inform selection of representative petroleum UVCBs for further toxicity evaluation in vivo. Overall, although the use of new approach methodologies to prioritize UVCBs has not been widely adopted, our study proposes a tiered testing strategy based on iPSC-derived hepatocytes and cardiomyocytes to inform selection of representative worst-case petroleum UVCBs from each manufacturing category for further toxicity evaluation in vivo.
Collapse
Affiliation(s)
- Han-Hsuan Doris Tsai
- Interdisciplinary Faculty of Toxicology, College Station, Texas 77843, USA
- Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843, USA
| | - John S House
- National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA
| | - Fred A Wright
- Interdisciplinary Faculty of Toxicology, College Station, Texas 77843, USA
- Department of Statistics and Bioinformatics Research Center, North Carolina State University, Raleigh, North Carolina 27603, USA
- Department of Biological Sciences and Bioinformatics Research Center, North Carolina State University, Raleigh, North Carolina 27603, USA
| | - Weihsueh A Chiu
- Interdisciplinary Faculty of Toxicology, College Station, Texas 77843, USA
- Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843, USA
| | - Ivan Rusyn
- Interdisciplinary Faculty of Toxicology, College Station, Texas 77843, USA
- Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843, USA
| |
Collapse
|
|
10
|
Kato Y, Lim AY, Sakolish C, Valdiviezo A, Moyer HL, Hewitt P, Bajaj P, Han G, Rusyn I. Analysis of reproducibility and robustness of OrganoPlate® 2-lane 96, a liver microphysiological system for studies of pharmacokinetics and toxicological assessment of drugs. Toxicol In Vitro 2022; 85:105464. [PMID: 36057418 PMCID: PMC10015056 DOI: 10.1016/j.tiv.2022.105464] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2022] [Revised: 07/26/2022] [Accepted: 08/26/2022] [Indexed: 02/06/2023]
Abstract
Establishing the functionality, reproducibility, robustness, and reliability of microphysiological systems is a critical need for adoption of these technologies. A high throughput microphysiological system for liver studies was recently proposed in which induced pluripotent stem cell-derived hepatocytes (iHeps) and non-parenchymal cells (endothelial cells and THP-1 cells differentiated with phorbol 12-myristate 13-acetate into macrophage-like cells) were co-cultured in OrganoPlate® 2-lane 96 devices. The goal of this study was to evaluate this platform using additional cell types and conditions and characterize its utility and reproducibility. Primary human hepatocytes or iHeps, with and without non-parenchymal cells, were cultured for up to 17 days. Image-based cell viability, albumin and urea secretion into culture media, CYP3A4 activity and drug metabolism were assessed. The iHeps co-cultured with non-parenchymal cells demonstrated stable cell viability and function up to 17 days; however, variability was appreciable both within and among studies. The iHeps in monoculture did not form clusters and lost viability and function over time. The primary human hepatocytes in monoculture also exhibited low cell viability and hepatic function. Metabolism of various drugs was most efficient when iHeps were co-cultured with non-parenchymal cells. Overall, we found that the OrganoPlate® 2-lane 96 device, when used with iHeps and non-parenchymal cells, is a functional liver microphysiological model; however, the high-throughput nature of this model is somewhat dampened by the need for replicates to compensate for high variability.
Collapse
Affiliation(s)
- Yuki Kato
- Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA; Laboratory for Drug Discovery and Development, Shionogi Pharmaceutical Research Center, Shionogi & Co., Ltd., Osaka 561-0825, Japan
| | - Alicia Y Lim
- Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA
| | - Courtney Sakolish
- Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA
| | - Alan Valdiviezo
- Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA
| | - Haley L Moyer
- Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA
| | - Philip Hewitt
- Chemical and Preclinical Safety, Merck Healthcare KGaA, 64293 Darmstadt, Germany
| | - Piyush Bajaj
- Global Investigative Toxicology, Preclinical Safety, Sanofi USA, MA 01701, USA
| | - Gang Han
- Department of Epidemiology and Biostatistics, Texas A&M University, College Station, TX 77843, USA
| | - Ivan Rusyn
- Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA.
| |
Collapse
|
|
11
|
Tsukamoto M, Kimura K, Yoshida T, Sugiura K, Hatoya S. Canine induced pluripotent stem cells efficiently differentiate into definitive endoderm in 3D cell culture conditions using high-dose activin A. Regen Ther 2022; 21:502-510. [DOI: 10.1016/j.reth.2022.10.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2022] [Revised: 09/09/2022] [Accepted: 10/08/2022] [Indexed: 11/06/2022] Open
|
|
12
|
Valdiviezo A, Kato Y, Baker ES, Chiu WA, Rusyn I. Evaluation of Metabolism of a Defined Pesticide Mixture through Multiple In Vitro Liver Models. TOXICS 2022; 10:566. [PMID: 36287846 PMCID: PMC9609317 DOI: 10.3390/toxics10100566] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/18/2022] [Revised: 09/16/2022] [Accepted: 09/23/2022] [Indexed: 06/16/2023]
Abstract
The evaluation of exposure to multiple contaminants in a mixture presents a number of challenges. For example, the characterization of chemical metabolism in a mixture setting remains a research area with critical knowledge gaps. Studies of chemical metabolism typically utilize suspension cultures of primary human hepatocytes; however, this model is not suitable for studies of more extended exposures and donor-to-donor variability in a metabolic capacity is unavoidable. To address this issue, we utilized several in vitro models based on human-induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep) to characterize the metabolism of an equimolar (1 or 5 µM) mixture of 20 pesticides. We used iHep suspensions and 2D sandwich cultures, and a microphysiological system OrganoPlate® 2-lane 96 (MimetasTM) that also included endothelial cells and THP-1 cell-derived macrophages. When cell culture media were evaluated using gas and liquid chromatography coupled to tandem mass spectrometry methods, we found that the parent molecule concentrations diminished, consistent with metabolic activity. This effect was most pronounced in iHep suspensions with a 1 µM mixture, and was lowest in OrganoPlate® 2-lane 96 for both mixtures. Additionally, we used ion mobility spectrometry-mass spectrometry (IMS-MS) to screen for metabolite formation in these cultures. These analyses revealed the presence of five primary metabolites that allowed for a more comprehensive evaluation of chemical metabolism in vitro. These findings suggest that iHep-based suspension assays maintain higher metabolic activity compared to 2D sandwich and OrganoPlate® 2-lane 96 model. Moreover, this study illustrates that IMS-MS can characterize in vitro metabolite formation following exposure to mixtures of environmental contaminants.
Collapse
Affiliation(s)
- Alan Valdiviezo
- Interdisciplinary Faculty of Toxicology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA
- Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA
| | - Yuki Kato
- Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA
- Laboratory for Drug Discovery and Development, Shionogi Pharmaceutical Research Center, Shionogi & Co., Ltd., Osaka 561-0825, Japan
| | - Erin S. Baker
- Interdisciplinary Faculty of Toxicology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA
- Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA
| | - Weihsueh A. Chiu
- Interdisciplinary Faculty of Toxicology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA
- Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA
| | - Ivan Rusyn
- Interdisciplinary Faculty of Toxicology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA
- Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA
| |
Collapse
|
|
13
|
Duff C, Baruteau J. Modelling urea cycle disorders using iPSCs. NPJ Regen Med 2022; 7:56. [PMID: 36163209 PMCID: PMC9513077 DOI: 10.1038/s41536-022-00252-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Accepted: 08/10/2022] [Indexed: 11/23/2022] Open
Abstract
The urea cycle is a liver-based pathway enabling disposal of nitrogen waste. Urea cycle disorders (UCDs) are inherited metabolic diseases caused by deficiency of enzymes or transporters involved in the urea cycle and have a prevalence of 1:35,000 live births. Patients present recurrent acute hyperammonaemia, which causes high rate of death and neurological sequelae. Long-term therapy relies on a protein-restricted diet and ammonia scavenger drugs. Currently, liver transplantation is the only cure. Hence, high unmet needs require the identification of effective methods to model these diseases to generate innovative therapeutics. Advances in both induced pluripotent stem cells (iPSCs) and genome editing technologies have provided an invaluable opportunity to model patient-specific phenotypes in vitro by creating patients' avatar models, to investigate the pathophysiology, uncover novel therapeutic targets and provide a platform for drug discovery. This review summarises the progress made thus far in generating 2- and 3-dimensional iPSCs models for UCDs, the challenges encountered and how iPSCs offer future avenues for innovation in developing the next-generation of therapies for UCDs.
Collapse
Affiliation(s)
- Claire Duff
- Genetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College London, London, UK
| | - Julien Baruteau
- Genetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College London, London, UK.
- National Institute of Health Research Great Ormond Street Biomedical Research Centre, London, UK.
- Metabolic Medicine Department, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK.
| |
Collapse
|
|
14
|
Scheffschick A, Babel J, Sperling S, Nerusch J, Herzog N, Seehofer D, Damm G. Primary-like Human Hepatocytes Genetically Engineered to Obtain Proliferation Competence as a Capable Application for Energy Metabolism Experiments in In Vitro Oncologic Liver Models. BIOLOGY 2022; 11:biology11081195. [PMID: 36009822 PMCID: PMC9405410 DOI: 10.3390/biology11081195] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/08/2022] [Revised: 07/21/2022] [Accepted: 08/03/2022] [Indexed: 11/17/2022]
Abstract
Simple Summary Fatty liver disease is an increasing health concern in Westernized countries. A fatty liver can lead to hepatocellular carcinoma (HCC), a type of liver cancer arising from hepatocytes, the major cells of the liver. How HCC may develop from the fatty liver is not known, and good cellular systems to investigate this are lacking. Recently, hepatocytes that can multiply continuously have been generated and suggested for hepatocyte research. In this study, we compared these continuously multiplying human hepatocytes to normal human hepatocytes and liver cancer cells, both within the state of fatty liver or not. We identified that these multiplying hepatocytes displayed many similarities to the liver cancer cells in terms of energy metabolism and concluded that these hepatocytes could be a pre-cancer model for liver cancer research and would be a valuable tool for HCC research. Abstract Non-alcoholic fatty liver disease (NAFLD), characterized by lipid accumulation in the liver, is the most common cause of liver diseases in Western countries. NAFLD is a major risk factor for developing hepatocellular carcinoma (HCC); however, in vitro evaluation of hepatic cancerogenesis fails due to a lack of liver models displaying a proliferation of hepatocytes. Originally designed to overcome primary human hepatocyte (PHH) shortages, upcyte hepatocytes were engineered to obtain continuous proliferation and, therefore, could be a suitable tool for HCC research. We generated upcyte hepatocytes, termed HepaFH3 cells, and compared their metabolic characteristics to HepG2 hepatoma cells and PHHs isolated from resected livers. For displaying NAFLD-related HCCs, we induced steatosis in all liver models. Lipid accumulation, lipotoxicity and energy metabolism were characterized using biochemical assays and Western blot analysis. We showed that proliferating HepaFH3 cells resemble HepG2, both showing a higher glucose uptake rate, lactate levels and metabolic rate compared to PHHs. Confluent HepaFH3 cells displayed some similarities to PHHs, including higher levels of the transaminases AST and ALT compared to proliferating HepaFH3 cells. We recommend proliferating HepaFH3 cells as a pre-malignant cellular model for HCC research, while confluent HepaFH3 cells could serve as PHH surrogates for energy metabolism studies.
Collapse
Affiliation(s)
- Andrea Scheffschick
- Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, 04103 Leipzig, Germany
- Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, 04103 Leipzig, Germany
| | - Jonas Babel
- Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, 04103 Leipzig, Germany
| | - Sebastian Sperling
- Department of General, Visceral and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany
| | - Julia Nerusch
- Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, 04103 Leipzig, Germany
- Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, 04103 Leipzig, Germany
| | - Natalie Herzog
- Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, 01968 Senftenberg, Germany
| | - Daniel Seehofer
- Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, 04103 Leipzig, Germany
- Department of General, Visceral and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany
| | - Georg Damm
- Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, 04103 Leipzig, Germany
- Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, 04103 Leipzig, Germany
- Department of General, Visceral and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany
- Correspondence: ; Tel.: +49-341-97-39656
| |
Collapse
|
|
15
|
Zhu L, Li HD, Xu JJ, Li JJ, Cheng M, Meng XM, Huang C, Li J. Advancements in the Alcohol-Associated Liver Disease Model. Biomolecules 2022; 12:biom12081035. [PMID: 36008929 PMCID: PMC9406170 DOI: 10.3390/biom12081035] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2022] [Revised: 07/22/2022] [Accepted: 07/25/2022] [Indexed: 02/06/2023] Open
Abstract
Alcohol-associated liver disease (ALD) is an intricate disease that results in a broad spectrum of liver damage. The presentation of ALD can include simple steatosis, steatohepatitis, liver fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC). Effective prevention and treatment strategies are urgently required for ALD patients. In previous decades, numerous rodent models were established to investigate the mechanisms of alcohol-associated liver disease and explore therapeutic targets. This review provides a summary of the latest developments in rodent models, including those that involve EtOH administration, which will help us to understand the characteristics and causes of ALD at different stages. In addition, we discuss the pathogenesis of ALD and summarize the existing in vitro models. We analyse the pros and cons of these models and their translational relevance and summarize the insights that have been gained regarding the mechanisms of alcoholic liver injury.
Collapse
Affiliation(s)
| | | | | | | | | | - Xiao-Ming Meng
- Correspondence: (X.-M.M.); (C.H.); (J.L.); Tel.: +86-551-65161001 (J.L.); Fax: +86-551-65161001 (J.L.)
| | - Cheng Huang
- Correspondence: (X.-M.M.); (C.H.); (J.L.); Tel.: +86-551-65161001 (J.L.); Fax: +86-551-65161001 (J.L.)
| | - Jun Li
- Correspondence: (X.-M.M.); (C.H.); (J.L.); Tel.: +86-551-65161001 (J.L.); Fax: +86-551-65161001 (J.L.)
| |
Collapse
|
|
16
|
Tricot T, Verfaillie CM, Kumar M. Current Status and Challenges of Human Induced Pluripotent Stem Cell-Derived Liver Models in Drug Discovery. Cells 2022; 11:442. [PMID: 35159250 PMCID: PMC8834601 DOI: 10.3390/cells11030442] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2021] [Revised: 01/13/2022] [Accepted: 01/24/2022] [Indexed: 02/08/2023] Open
Abstract
The pharmaceutical industry is in high need of efficient and relevant in vitro liver models, which can be incorporated in their drug discovery pipelines to identify potential drugs and their toxicity profiles. Current liver models often rely on cancer cell lines or primary cells, which both have major limitations. However, the development of human induced pluripotent stem cells (hiPSCs) has created a new opportunity for liver disease modeling, drug discovery and liver toxicity research. hiPSCs can be differentiated to any cell of interest, which makes them good candidates for disease modeling and drug discovery. Moreover, hiPSCs, unlike primary cells, can be easily genome-edited, allowing the creation of reporter lines or isogenic controls for patient-derived hiPSCs. Unfortunately, even though liver progeny from hiPSCs has characteristics similar to their in vivo counterparts, the differentiation of iPSCs to fully mature progeny remains highly challenging and is a major obstacle for the full exploitation of these models by pharmaceutical industries. In this review, we discuss current liver-cell differentiation protocols and in vitro iPSC-based liver models that could be used for disease modeling and drug discovery. Furthermore, we will discuss the challenges that still need to be overcome to allow for the successful implementation of these models into pharmaceutical drug discovery platforms.
Collapse
Affiliation(s)
| | | | - Manoj Kumar
- Stem Cell Institute, KU Leuven, 3000 Leuven, Belgium; (T.T.); (C.M.V.)
| |
Collapse
|
|
17
|
Xu H, Wu L, Yuan G, Liang X, Liu X, Li Z, Chen N, Farzaneh M. MicroRNAs: Crucial Players in the Differentiation of Human Pluripotent and Multipotent Stem Cells into Functional Hepatocyte-Like Cells. Curr Stem Cell Res Ther 2021; 17:734-740. [PMID: 34615452 DOI: 10.2174/1574888x16666211006102039] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2021] [Revised: 06/19/2021] [Accepted: 06/24/2021] [Indexed: 11/22/2022]
Abstract
Hepatic disease negatively impacts liver function and metabolism. Primary human hepatocytes are the gold standard for the prediction and successful treatment of liver disease. However, the sources of hepatocytes for drug toxicity testing and disease modeling are limited. To overcome this issue, pluripotent stem cells (PSCs) have emerged as an alternative strategy for liver disease therapy. Human PSCs, including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can self-renew and give rise to all cells of the body. Human PSCs are attractive cell sources for regenerative medicine, tissue engineering, drug discovery, and developmental studies. Several recent studies have shown that mesenchymal stem cells (MSCs) can also differentiate (or trans-differentiate) into hepatocytes. Differentiation of human PSCs and MSCs into functional hepatocyte-like cells (HLCs) opens new strategies to study genetic diseases, hepatotoxicity, infection of hepatotropic viruses, and analyze hepatic biology. Numerous in vitro and in vivo differentiation protocols have been established to obtain human PSCs/MSCs-derived HLCs and mimic their characteristics. It was recently discovered that microRNAs (miRNAs) play a critical role in controlling the ectopic expression of transcription factors and governing the hepatocyte differentiation of human PSCs and MSCs. In this review, we focused on the role of miRNAs in the differentiation of human PSCs and MSCs into hepatocytes.
Collapse
Affiliation(s)
- Hao Xu
- Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong. China
| | - Liying Wu
- Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong. China
| | - Guojia Yuan
- Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong. China
| | - Xiaolu Liang
- Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong. China
| | - Xiaoguang Liu
- Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong. China
| | - Zuobiao Li
- Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong. China
| | - Nianping Chen
- Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong. China
| | - Maryam Farzaneh
- Fertility, Infertility and Perinatology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz. Iran
| |
Collapse
|
|
18
|
Faccioli LAP, Kocas-Kilicarslan ZN, Diaz-Aragon R, Motomura T, Amirneni S, Malizio MR, Coard MC, Frau C, Haep N, Florentino RM, Ostrowska A. Human Hepatocytes Isolated from Explanted Livers: A Powerful Tool to Understand End-stage Liver Disease and Drug Screening. Organogenesis 2021; 17:117-125. [PMID: 35114888 DOI: 10.1080/15476278.2021.1992216] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022] Open
Abstract
The use of primary human hepatocytes has been hampered by limited availability of adequate numbers of fresh and viable cells due to the ongoing shortage of liver donors. Thus, there is no surplus of healthy organs from which freshly isolated cells can be prepared when needed. However, primary hepatocytes can be successfully isolated from explanted liver specimens obtained from patients receiving orthotopic liver transplantation for decompensated liver cirrhosis or for metabolic liver disease without end-stage liver disease and are a valuable resource for the pharmaceutical industry research. This review focuses on the isolation, characterization and cryopreservation of hepatocytes derived from therapeutically resected livers with various hepatic diseases.
Collapse
Affiliation(s)
- Lanuza A P Faccioli
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | | | - Ricardo Diaz-Aragon
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Takashi Motomura
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Sriram Amirneni
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Michelle R Malizio
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Michael C Coard
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Carla Frau
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Nils Haep
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Rodrigo M Florentino
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.,Department of Surgery, Children's Hospital of Pittsburgh of Upmc, Pittsburgh, Pennsylvania, USA
| | - Alina Ostrowska
- Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.,Department of Surgery, Children's Hospital of Pittsburgh of Upmc, Pittsburgh, Pennsylvania, USA
| |
Collapse
|
|
19
|
Polidoro MA, Ferrari E, Marzorati S, Lleo A, Rasponi M. Experimental liver models: From cell culture techniques to microfluidic organs-on-chip. Liver Int 2021; 41:1744-1761. [PMID: 33966344 DOI: 10.1111/liv.14942] [Citation(s) in RCA: 39] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2021] [Revised: 05/02/2021] [Accepted: 05/03/2021] [Indexed: 12/12/2022]
Abstract
The liver is one of the most studied organs of the human body owing to its central role in xenobiotic and drug metabolism. In recent decades, extensive research has aimed at developing in vitro liver models able to mimic liver functions to study pathophysiological clues in high-throughput and reproducible environments. Two-dimensional (2D) models have been widely used in screening potential toxic compounds but have failed to accurately reproduce the three-dimensionality (3D) of the liver milieu. To overcome these limitations, improved 3D culture techniques have been developed to recapitulate the hepatic native microenvironment. These models focus on reproducing the liver architecture, representing both parenchymal and nonparenchymal cells, as well as cell interactions. More recently, Liver-on-Chip (LoC) models have been developed with the aim of providing physiological fluid flow and thus achieving essential hepatic functions. Given their unprecedented ability to recapitulate critical features of the liver cellular environments, LoC have been extensively adopted in pathophysiological modelling and currently represent a promising tool for tissue engineering and drug screening applications. In this review, we discuss the evolution of experimental liver models, from the ancient 2D hepatocyte models, widely used for liver toxicity screening, to 3D and LoC culture strategies adopted for mirroring a more physiological microenvironment for the study of liver diseases.
Collapse
Affiliation(s)
- Michela Anna Polidoro
- Hepatobiliary Immunopathology Laboratory, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy
| | - Erika Ferrari
- Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy
| | - Simona Marzorati
- Hepatobiliary Immunopathology Laboratory, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy
| | - Ana Lleo
- Department of Biomedical Sciences, Humanitas University, Pieve Emanuele, Milan, Italy.,Division of Internal Medicine and Hepatology, Department of Gastroenterology, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy
| | - Marco Rasponi
- Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy
| |
Collapse
|
|
20
|
Arez F, Rodrigues AF, Brito C, Alves PM. Bioengineered Liver Cell Models of Hepatotropic Infections. Viruses 2021; 13:773. [PMID: 33925701 PMCID: PMC8146083 DOI: 10.3390/v13050773] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2021] [Revised: 04/22/2021] [Accepted: 04/23/2021] [Indexed: 02/07/2023] Open
Abstract
Hepatitis viruses and liver-stage malaria are within the liver infections causing higher morbidity and mortality rates worldwide. The highly restricted tropism of the major human hepatotropic pathogens-namely, the human hepatitis B and C viruses and the Plasmodium falciparum and Plasmodium vivax parasites-has hampered the development of disease models. These models are crucial for uncovering the molecular mechanisms underlying the biology of infection and governing host-pathogen interaction, as well as for fostering drug development. Bioengineered cell models better recapitulate the human liver microenvironment and extend hepatocyte viability and phenotype in vitro, when compared with conventional two-dimensional cell models. In this article, we review the bioengineering tools employed in the development of hepatic cell models for studying infection, with an emphasis on 3D cell culture strategies, and discuss how those tools contributed to the level of recapitulation attained in the different model layouts. Examples of host-pathogen interactions uncovered by engineered liver models and their usefulness in drug development are also presented. Finally, we address the current bottlenecks, trends, and prospect toward cell models' reliability, robustness, and reproducibility.
Collapse
MESH Headings
- Animals
- Bioengineering/methods
- Cell Culture Techniques
- Disease Models, Animal
- Disease Susceptibility
- Drug Discovery
- Hepatitis/drug therapy
- Hepatitis/etiology
- Hepatitis/metabolism
- Hepatitis/pathology
- Hepatitis, Viral, Human/etiology
- Hepatitis, Viral, Human/metabolism
- Hepatitis, Viral, Human/pathology
- Hepatocytes/metabolism
- Hepatocytes/parasitology
- Hepatocytes/virology
- Host-Pathogen Interactions
- Humans
- Liver/metabolism
- Liver/parasitology
- Liver/virology
- Liver Diseases, Parasitic/etiology
- Liver Diseases, Parasitic/metabolism
- Liver Diseases, Parasitic/pathology
Collapse
Affiliation(s)
- Francisca Arez
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; (F.A.); (A.F.R.); (C.B.)
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
| | - Ana F. Rodrigues
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; (F.A.); (A.F.R.); (C.B.)
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
| | - Catarina Brito
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; (F.A.); (A.F.R.); (C.B.)
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
- The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Av. da República, 2780-157 Oeiras, Portugal
| | - Paula M. Alves
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; (F.A.); (A.F.R.); (C.B.)
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
| |
Collapse
|
|
21
|
Elsayed AK, Vimalraj S, Nandakumar M, Abdelalim EM. Insulin resistance in diabetes: The promise of using induced pluripotent stem cell technology. World J Stem Cells 2021; 13:221-235. [PMID: 33815671 PMCID: PMC8006014 DOI: 10.4252/wjsc.v13.i3.221] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/18/2020] [Revised: 02/07/2021] [Accepted: 03/11/2021] [Indexed: 02/06/2023] Open
Abstract
Insulin resistance (IR) is associated with several metabolic disorders, including type 2 diabetes (T2D). The development of IR in insulin target tissues involves genetic and acquired factors. Persons at genetic risk for T2D tend to develop IR several years before glucose intolerance. Several rodent models for both IR and T2D are being used to study the disease pathogenesis; however, these models cannot recapitulate all the aspects of this complex disorder as seen in each individual. Human pluripotent stem cells (hPSCs) can overcome the hurdles faced with the classical mouse models for studying IR. Human induced pluripotent stem cells (hiPSCs) can be generated from the somatic cells of the patients without the need to destroy a human embryo. Therefore, patient-specific hiPSCs can generate cells genetically identical to IR individuals, which can help in distinguishing between genetic and acquired defects in insulin sensitivity. Combining the technologies of genome editing and hiPSCs may provide important information about the genetic factors underlying the development of different forms of IR. Further studies are required to fill the gaps in understanding the pathogenesis of IR and diabetes. In this review, we summarize the factors involved in the development of IR in the insulin-target tissues leading to diabetes. Also, we highlight the use of hPSCs to understand the mechanisms underlying the development of IR.
Collapse
Affiliation(s)
- Ahmed K Elsayed
- Diabetes Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha 34110, Qatar
| | | | - Manjula Nandakumar
- Diabetes Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha 34110, Qatar
| | - Essam M Abdelalim
- Diabetes Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, Doha 34110, Qatar
- College of Health and Life Sciences, Hamad Bin Khalifa University, Qatar Foundation, Doha 34110, Qatar
| |
Collapse
|
|
22
|
Iwado S, Abe S, Oshimura M, Kazuki Y, Nakajima Y. Bioluminescence Measurement of Time-Dependent Dynamic Changes of CYP-Mediated Cytotoxicity in CYP-Expressing Luminescent HepG2 Cells. Int J Mol Sci 2021; 22:ijms22062843. [PMID: 33799598 PMCID: PMC7999318 DOI: 10.3390/ijms22062843] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2021] [Revised: 03/05/2021] [Accepted: 03/08/2021] [Indexed: 12/26/2022] Open
Abstract
We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC50) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC50 value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC50 values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.
Collapse
Affiliation(s)
- Satoru Iwado
- Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago 683-8503, Tottori, Japan; (S.I.); (S.A.); (M.O.)
| | - Satoshi Abe
- Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago 683-8503, Tottori, Japan; (S.I.); (S.A.); (M.O.)
| | - Mitsuo Oshimura
- Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago 683-8503, Tottori, Japan; (S.I.); (S.A.); (M.O.)
| | - Yasuhiro Kazuki
- Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago 683-8503, Tottori, Japan; (S.I.); (S.A.); (M.O.)
- Division of Genome and Cellular Functions, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago 683-8503, Tottori, Japan
- Correspondence: (Y.K.); (Y.N.); Tel.: +81-859-38-6219 (Y.K.); +81-87-869-3525 (Y.N.)
| | - Yoshihiro Nakajima
- Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago 683-8503, Tottori, Japan; (S.I.); (S.A.); (M.O.)
- Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu 761-0395, Kagawa, Japan
- Correspondence: (Y.K.); (Y.N.); Tel.: +81-859-38-6219 (Y.K.); +81-87-869-3525 (Y.N.)
| |
Collapse
|
|
23
|
Ali M, Payne SL. Biomaterial-based cell delivery strategies to promote liver regeneration. Biomater Res 2021; 25:5. [PMID: 33632335 PMCID: PMC7905561 DOI: 10.1186/s40824-021-00206-w] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2020] [Accepted: 02/05/2021] [Indexed: 02/08/2023] Open
Abstract
Chronic liver disease and cirrhosis is a widespread and untreatable condition that leads to lifelong impairment and eventual death. The scarcity of liver transplantation options requires the development of new strategies to attenuate disease progression and reestablish liver function by promoting regeneration. Biomaterials are becoming an increasingly promising option to both culture and deliver cells to support in vivo viability and long-term function. There is a wide variety of both natural and synthetic biomaterials that are becoming established as delivery vehicles with their own unique advantages and disadvantages for liver regeneration. We review the latest developments in cell transplantation strategies to promote liver regeneration, with a focus on the use of both natural and synthetic biomaterials for cell culture and delivery. We conclude that future work will need to refine the use of these biomaterials and combine them with novel strategies that recapitulate liver organization and function in order to translate this strategy to clinical use.
Collapse
Affiliation(s)
- Maqsood Ali
- Department of Regenerative Medicine, College of Medicine, Soonchunhyang University, Cheonan, South Korea
| | - Samantha L Payne
- Department of Biomedical Engineering, School of Engineering, Tufts University, Medford, MA, 02155, USA.
| |
Collapse
|
|
24
|
Tao L, Fang SY, Zhao L, He TC, He Y, Bi Y. Indocyanine Green Uptake and Periodic Acid-Schiff Staining Method for Function Detection of Liver Cells are Affected by Different Cell Confluence. Cytotechnology 2021; 73:159-167. [PMID: 33927473 DOI: 10.1007/s10616-021-00453-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2020] [Accepted: 01/17/2021] [Indexed: 12/21/2022] Open
Abstract
Hepatic stem cell transplantation has been demonstrated as an effective alternative therapy for the end-stage liver failure patients. Therefore, the functional detection of hepatic stem cell is essentially required. The present study confirmed that adenovirus BMP9 (Ad-BMP9) could increase the ALB-Gluc activity of HP14-19 hepatic progenitor cells, the expression of specific hepatic markers ALB, TAT, UGT1A were up-regulated while the hepatic stem cell markers DLK, AFP were down-regulated, and the number of positive Periodic acid-Schiff (PAS) stained cells were significantly higher than those in control group. However, the indocyanine green (ICG) uptake failed to be detectable in induced hepatocytes, which was inconsistent. By using another cell line LC14d, we found out that positive ICG uptake cells were located in the area of low cell density, while positive PAS stained cells were mainly concentrated in the area where cells were overlapped, indicating that different cell confluence might affect the outcomes of ICG uptake and PAS staining. A manual wound healing of Ad-BMP9 induced HP14-19 cells was made, the crawling cells were stained positive for ICG but not for PAS. Therefore, our finding may provide evidence for better application of PAS staining and ICG uptake assay in functional detection of mature hepatocytes. Supplementary Information The online version contains supplementary material available at 10.1007/s10616-021-00453-8.
Collapse
Affiliation(s)
- Li Tao
- Department of Pediatric Research Institute of Children's Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, People's Republic of China
| | - Shu-Yu Fang
- Department of Pediatric Research Institute of Children's Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, People's Republic of China
| | - Li Zhao
- Department of Pediatric Research Institute of Children's Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, People's Republic of China
| | - Tong-Chuan He
- Molecular Oncology Laboratory, The University of Chicago Medical Center, Chicago, IL 60637 USA
| | - Yun He
- Department of Pediatric Research Institute of Children's Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, People's Republic of China.,Stem Cell Biology and Therapy Laboratory, The Children's Hospital of Chongqing Medical University, Chongqing, People's Republic of China
| | - Yang Bi
- Department of Pediatric Research Institute of Children's Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, People's Republic of China.,Department of Pediatric Surgery, The Children's Hospital of Chongqing Medical University, Chongqing, People's Republic of China
| |
Collapse
|
|
25
|
Dame K, Ribeiro AJ. Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects. Exp Biol Med (Maywood) 2020; 246:317-331. [PMID: 32938227 PMCID: PMC7859673 DOI: 10.1177/1535370220959598] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Hepatic and cardiac drug adverse effects are among the leading causes of attrition in drug development programs, in part due to predictive failures of current animal or in vitro models. Hepatocytes and cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) hold promise for predicting clinical drug effects, given their human-specific properties and their ability to harbor genetically determined characteristics that underlie inter-individual variations in drug response. Currently, the fetal-like properties and heterogeneity of hepatocytes and cardiomyocytes differentiated from iPSCs make them physiologically different from their counterparts isolated from primary tissues and limit their use for predicting clinical drug effects. To address this hurdle, there have been ongoing advances in differentiation and maturation protocols to improve the quality and use of iPSC-differentiated lineages. Among these are in vitro hepatic and cardiac cellular microsystems that can further enhance the physiology of cultured cells, can be used to better predict drug adverse effects, and investigate drug metabolism, pharmacokinetics, and pharmacodynamics to facilitate successful drug development. In this article, we discuss how cellular microsystems can establish microenvironments for these applications and propose how they could be used for potentially controlling the differentiation of hepatocytes or cardiomyocytes. The physiological relevance of cells is enhanced in cellular microsystems by simulating properties of tissue microenvironments, such as structural dimensionality, media flow, microfluidic control of media composition, and co-cultures with interacting cell types. Recent studies demonstrated that these properties also affect iPSC differentiations and we further elaborate on how they could control differentiation efficiency in microengineered devices. In summary, we describe recent advances in the field of cellular microsystems that can control the differentiation and maturation of hepatocytes and cardiomyocytes for drug evaluation. We also propose how future research with iPSCs within engineered microenvironments could enable their differentiation for scalable evaluations of drug effects.
Collapse
Affiliation(s)
- Keri Dame
- Division of Applied Regulatory Science, Office of Clinical Pharmacology, Office of Translation Sciences, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD 20993, USA
| | - Alexandre Js Ribeiro
- Division of Applied Regulatory Science, Office of Clinical Pharmacology, Office of Translation Sciences, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD 20993, USA
| |
Collapse
|
|
26
|
Mun SJ, Hong YH, Ahn HS, Ryu JS, Chung KS, Son MJ. Long-Term Expansion of Functional Human Pluripotent Stem Cell-Derived Hepatic Organoids. Int J Stem Cells 2020; 13:279-286. [PMID: 32323516 PMCID: PMC7378903 DOI: 10.15283/ijsc20060] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2020] [Accepted: 04/12/2020] [Indexed: 12/16/2022] Open
Abstract
A human cell-based liver model capable of long-term expansion and mature hepatic function is a fundamental requirement for pre-clinical drug development. We previously established self-renewing and functionally mature human pluripotent stem cell-derived liver organoids as an alternate to primary human hepatocytes. In this study, we tested long-term prolonged culture of organoids to increase their maturity. Organoid growing at the edge of Matrigel started to deteriorate two weeks after culturing, and the expression levels of the functional mature hepatocyte marker ALB were decreased at four weeks of culture. Replating the organoids weekly at a 1：2 ratio in fresh Matrigel, resulted in healthier morphology with a thicker layer compared to organoids maintained on the same Matrigel and significantly increased ALB expression until three weeks, although, it decreased sharply at four weeks. The levels of the fetal hepatocyte marker AFP were considerably increased in long-term cultures of organoids. Therefore, we performed serial passaging of organoids, whereby they were mechanically split weekly at a 1：3∼1：5 ratio in fresh Matrigel. The organoids expanded so far over passage 55, or 1 year, without growth retardation and maintained a normal karyotype after long-term cryopreservation. Differentiation potentials were maintained or increased after long-term passaging, while AFP expression considerably decreased after passaging. Therefore, these data demonstrate that organoids can be exponentially expanded by serial passaging, while maintaining long-term functional maturation potential. Thus, hepatic organoids can be a practical and renewable cell source for human cell-based and personalized 3D liver models.
Collapse
Affiliation(s)
- Seon Ju Mun
- Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea.,Department of Functional Genomics, Korea University of Science & Technology (UST), Daejeon, Korea
| | - Yeon-Hwa Hong
- Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea.,Department of Functional Genomics, Korea University of Science & Technology (UST), Daejeon, Korea
| | - Hyo-Suk Ahn
- Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea
| | - Jae-Sung Ryu
- Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea
| | - Kyung-Sook Chung
- Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea.,Department of Functional Genomics, Korea University of Science & Technology (UST), Daejeon, Korea.,Biomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea
| | - Myung Jin Son
- Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea.,Department of Functional Genomics, Korea University of Science & Technology (UST), Daejeon, Korea
| |
Collapse
|
|
27
|
Corbett JL, Duncan SA. iPSC-Derived Hepatocytes as a Platform for Disease Modeling and Drug Discovery. Front Med (Lausanne) 2019; 6:265. [PMID: 31803747 PMCID: PMC6873655 DOI: 10.3389/fmed.2019.00265] [Citation(s) in RCA: 89] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2019] [Accepted: 10/30/2019] [Indexed: 12/15/2022] Open
Abstract
The liver is one of the largest organs in the body and is responsible for a diverse repertoire of metabolic processes. Such processes include the secretion of serum proteins, carbohydrate and lipid metabolism, bile acid and urea synthesis, detoxification of drugs and metabolic waste products, and vitamin and carbohydrate storage. Currently, liver disease is one of the most prevalent causes of mortality in the USA with congenital liver defects contributing to a significant proportion of these deaths. Historically the study of liver disease has been hampered by a shortage of organ donors, the subsequent scarcity of healthy tissue, and the failure of animal models to fully recapitulate human liver function. In vitro culture of hepatocytes has also proven difficult because primary hepatocytes rapidly de-differentiate in culture. Recent advances in stem cell technology have facilitated the generation of induced pluripotent stem cells (iPSCs) from various somatic cell types from patients. Such cells can be differentiated to a liver cell fate, essentially providing a limitless supply of cells with hepatocyte characteristics that can mimic the pathophysiology of liver disease. Furthermore, development of the CRISPR-Cas9 system, as well as advancement of miniaturized differentiation platforms has facilitated the development of high throughput models for the investigation of hepatocyte differentiation and drug discovery. In this review, we will explore the latest advances in iPSC-based disease modeling and drug screening platforms and examine how this technology is being used to identify new pharmacological interventions, and to advance our understanding of liver development and mechanisms of disease. We will cover how iPSC technology is being used to develop predictive models for rare diseases and how information gained from large in vitro screening experiments can be used to directly inform clinical investigation.
Collapse
Affiliation(s)
| | - Stephen A. Duncan
- Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, United States
| |
Collapse
|