|
1
|
Liu K, Li L, He Y, Zhang S, You H, Wang P. Hepatic progenitor cells reprogrammed from mouse fibroblasts repopulate hepatocytes in Wilson's disease mice. Stem Cell Res Ther 2025; 16:131. [PMID: 40069754 PMCID: PMC11899129 DOI: 10.1186/s13287-025-04253-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2024] [Accepted: 02/25/2025] [Indexed: 03/14/2025] Open
Abstract
BACKGROUND Wilson's disease (WD) is a genetic disorder that impairs the excretion of copper in hepatocytes and results in excessive copper deposition in multiple organs. The replacement of disordered hepatocytes with functional hepatocytes can serve as a lifelong therapeutic strategy for the treatment of WD. The aim of this study was to determine the hepatocyte repopulation effects of fibroblast-derived hepatic progenitor cells in the treatment of WD. METHODS Induced hepatic progenitor cells (iHPCs) were generated through direct reprogramming of adult mouse fibroblasts infected with lentivirus carrying both the Foxa3 and Hnf4α genes. These iHPCs were subsequently identified and transplanted into copper-overload WD mice with the Atp7b (H1071Q) mutation via caudal vein injection. RESULTS After lentivirus infection, the fibroblasts transformed into Foxa3- and Hnf4α-overexpressing cobblestone-like cells with reduced expression of fibroblast markers and increased expression of epithelial cell and hepatic progenitor cell markers, i.e., iHPCs. Sixteen weeks after transplantation into WD mice, approximately 2% of hepatocytes were derived from iHPCs, and these iHPC-derived hepatocytes expressed a tight junction-associated protein of the bile canal, tight junction protein 1 (Zo1). There was a decrease in the serum copper concentration and relative activity of serum ceruloplasmin at weeks 4 and 8 after iHPCs transplantation compared with those of WD fed mice administered saline or fibroblasts. Furthermore, iHPC transplantation markedly reduced the proportion of CD8+ T lymphocytes and natural killer cells compared with those in fibroblast-transplanted WD mice and downregulated the transcription of the inflammatory cytokines, including tumor necrosis factor α (Tnfα), interleukin 1β (IL-1β), and IL-6, compared with those in WD mice and in fibroblast-transplanted WD mice. CONCLUSION iHPCs reprogrammed from adult fibroblasts can repopulate hepatocytes and exert therapeutic effects in WD mice, representing a potential replacement therapy for clinical application.
Collapse
Affiliation(s)
- Kai Liu
- Beijing Key Laboratory of Tolerance Induction and Organ Protection in Transplantation, Beijing Friendship Hospital, Capital Medical University, No. 95 Yong An Road, Beijing, 100050, China.
- Beijing Clinical Research Institute, Beijing, 100050, China.
| | - Li Li
- Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, China
- National Clinical Research Center for Digestive Disease, Beijing, 100069, China
| | - Yu He
- Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, China
- National Clinical Research Center for Digestive Disease, Beijing, 100069, China
| | - Song Zhang
- Beijing Clinical Research Institute, Beijing, 100050, China
| | - Hong You
- Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, China.
- National Clinical Research Center for Digestive Disease, Beijing, 100069, China.
| | - Ping Wang
- Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, China.
- National Clinical Research Center for Digestive Disease, Beijing, 100069, China.
| |
Collapse
|
|
2
|
Heydari Z, Gramignoli R, Piryaei A, Zahmatkesh E, Pooyan P, Seydi H, Nussler A, Szkolnicka D, Rashidi H, Najimi M, Hay DC, Vosough M. Standard Protocols for Characterising Primary and In Vitro-Generated Human Hepatocytes. J Cell Mol Med 2025; 29:e70390. [PMID: 39910642 PMCID: PMC11798750 DOI: 10.1111/jcmm.70390] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2024] [Revised: 01/13/2025] [Accepted: 01/17/2025] [Indexed: 02/07/2025] Open
Abstract
Hepatocyte-like cells (HLCs) derived from pluripotent stem cells (PSCs) or direct reprogramming are an unlimited source of human hepatocytes for biomedical applications. HLCs are used to model human diseases, develop precise drugs and establish groundbreaking regenerative cell-based therapies. Primary human hepatocytes are the gold standard for studying human liver biology and pathology. However, their widespread use is limited by their rapid dedifferentiation in vitro, reliance on transplant-rejected donor organs, poor scalability and significant batch-to-batch variations. Therefore, high-quality 'off-the-shelf' HLCs are needed to overcome those limitations. Basic stepwise differentiation protocols have been developed to generate HLCs from PSCs. To evaluate the quality of the in vitro generated products, HLCs have been phenotyped using various methods. This review discusses various biological assays and methods available for the robust evaluation of HLC quality, emphasising the importance of using 24-h cultured primary human hepatocytes (PHHs) as a reference standard for comparison.
Collapse
Affiliation(s)
- Zahra Heydari
- Department of Regenerative Medicine, Cell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
| | - Roberto Gramignoli
- Division of Pathology, Department of Laboratory MedicineKarolinska InstitutetStockholmSweden
| | - Abbas Piryaei
- Department of Biology and Anatomical Sciences, School of MedicineShahid Beheshti University of Medical SciencesTehranIran
- Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in MedicineShahid Beheshti University of Medical SciencesTehranIran
| | - Ensieh Zahmatkesh
- Department of Regenerative Medicine, Cell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
| | - Paria Pooyan
- Department of Regenerative Medicine, Cell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
| | - Homeyra Seydi
- Department of Regenerative Medicine, Cell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
| | - Andreas Nussler
- Siegfried Weller Institute for Trauma ResearchUniversity of TübingenTübingenGermany
| | - Dagmara Szkolnicka
- Centre for Regenerative Medicine, Institute for Repair and RegenerationUniversity of EdinburghEdinburghUK
| | - Hassan Rashidi
- Department of Developmental Biology and CancerUCL Great Ormond Street Institute of Child HealthLondonUK
| | - Mustapha Najimi
- Laboratory of Pediatric Hepatology and Cell TherapyInstitute of Experimental and Clinical Research, UCLouvainBrusselsBelgium
| | - David C. Hay
- Centre for Regenerative Medicine, Institute for Repair and RegenerationUniversity of EdinburghEdinburghUK
| | - Massoud Vosough
- Department of Regenerative Medicine, Cell Science Research CenterRoyan Institute for Stem Cell Biology and Technology, ACECRTehranIran
- Experimental Cancer MedicineInstitution for Laboratory Medicine, Karolinska Institute HuddingeHuddingeSweden
| |
Collapse
|
|
3
|
Ori C, Ansari M, Angelidis I, Olmer R, Martin U, Theis FJ, Schiller HB, Drukker M. Human pluripotent stem cell fate trajectories toward lung and hepatocyte progenitors. iScience 2023; 26:108205. [PMID: 38026193 PMCID: PMC10663741 DOI: 10.1016/j.isci.2023.108205] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2022] [Revised: 07/13/2023] [Accepted: 10/11/2023] [Indexed: 12/01/2023] Open
Abstract
In this study, we interrogate molecular mechanisms underlying the specification of lung progenitors from human pluripotent stem cells (hPSCs). We employ single-cell RNA-sequencing with high temporal precision, alongside an optimized differentiation protocol, to elucidate the transcriptional hierarchy of lung specification to chart the associated single-cell trajectories. Our findings indicate that Sonic hedgehog, TGF-β, and Notch activation are essential within an ISL1/NKX2-1 trajectory, leading to the emergence of lung progenitors during the foregut endoderm phase. Additionally, the induction of HHEX delineates an alternate trajectory at the early definitive endoderm stage, preceding the lung pathway and giving rise to a significant hepatoblast population. Intriguingly, neither KDR+ nor mesendoderm progenitors manifest as intermediate stages in the lung and hepatic lineage development. Our multistep model offers insights into lung organogenesis and provides a foundation for in-depth study of early human lung development and modeling using hPSCs.
Collapse
Affiliation(s)
- Chaido Ori
- Institute of Stem Cell Research, Helmholtz Munich, Neuherberg, Munich, Germany
| | - Meshal Ansari
- Comprehensive Pneumology Center Munich (CPC-M), Institute of Lung Health and Immunity (LHI), Helmholtz Munich, Member of the German Center for Lung Research (DZL), Munich, Germany
- Department of Computational Health, Institute of Computational Biology, Helmholtz Munich, Munich, Germany
| | - Ilias Angelidis
- Comprehensive Pneumology Center Munich (CPC-M), Institute of Lung Health and Immunity (LHI), Helmholtz Munich, Member of the German Center for Lung Research (DZL), Munich, Germany
| | - Ruth Olmer
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany
- Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), Hannover Medical School, 30625 Hannover, Germany
- REBIRTH-Research Center for Translational and Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Ulrich Martin
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany
- Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), Hannover Medical School, 30625 Hannover, Germany
- REBIRTH-Research Center for Translational and Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany
| | - Fabian J. Theis
- Department of Computational Health, Institute of Computational Biology, Helmholtz Munich, Munich, Germany
- TUM School of Life Sciences, Technical University of Munich, Munich, Germany
| | - Herbert B. Schiller
- Comprehensive Pneumology Center Munich (CPC-M), Institute of Lung Health and Immunity (LHI), Helmholtz Munich, Member of the German Center for Lung Research (DZL), Munich, Germany
- Institute of Experimental Pneumology, LMU University Hospital, Ludwig-Maximilians University, Munich, Germany
| | - Micha Drukker
- Institute of Stem Cell Research, Helmholtz Munich, Neuherberg, Munich, Germany
- Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, the Netherlands
| |
Collapse
|
|
4
|
Afzal Z, Huguet EL. Bioengineering liver tissue by repopulation of decellularised scaffolds. World J Hepatol 2023; 15:151-179. [PMID: 36926238 PMCID: PMC10011915 DOI: 10.4254/wjh.v15.i2.151] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/26/2022] [Revised: 11/22/2022] [Accepted: 02/15/2023] [Indexed: 02/24/2023] Open
Abstract
Liver transplantation is the only curative therapy for end stage liver disease, but is limited by the organ shortage, and is associated with the adverse consequences of immunosuppression. Repopulation of decellularised whole organ scaffolds with appropriate cells of recipient origin offers a theoretically attractive solution, allowing reliable and timely organ sourcing without the need for immunosuppression. Decellularisation methodologies vary widely but seek to address the conflicting objectives of removing the cellular component of tissues whilst keeping the 3D structure of the extra-cellular matrix intact, as well as retaining the instructive cell fate determining biochemicals contained therein. Liver scaffold recellularisation has progressed from small rodent in vitro studies to large animal in vivo perfusion models, using a wide range of cell types including primary cells, cell lines, foetal stem cells, and induced pluripotent stem cells. Within these models, a limited but measurable degree of physiologically significant hepatocyte function has been reported with demonstrable ammonia metabolism in vivo. Biliary repopulation and function have been restricted by challenges relating to the culture and propagations of cholangiocytes, though advances in organoid culture may help address this. Hepatic vasculature repopulation has enabled sustainable blood perfusion in vivo, but with cell types that would limit clinical applications, and which have not been shown to have the specific functions of liver sinusoidal endothelial cells. Minority cell groups such as Kupffer cells and stellate cells have not been repopulated. Bioengineering by repopulation of decellularised scaffolds has significantly progressed, but there remain significant experimental challenges to be addressed before therapeutic applications may be envisaged.
Collapse
Affiliation(s)
- Zeeshan Afzal
- Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Centre; Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
| | - Emmanuel Laurent Huguet
- Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Centre; Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
| |
Collapse
|
|
5
|
Tomofuji K, Fukumitsu K, Kondo J, Horie H, Makino K, Wakama S, Ito T, Oshima Y, Ogiso S, Ishii T, Inoue M, Hatano E. Liver ductal organoids reconstruct intrahepatic biliary trees in decellularized liver grafts. Biomaterials 2022; 287:121614. [PMID: 35688027 DOI: 10.1016/j.biomaterials.2022.121614] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2021] [Revised: 05/14/2022] [Accepted: 05/30/2022] [Indexed: 12/13/2022]
Abstract
Three-dimensional scaffolds decellularized from native organs are a promising technique to establish engineered liver grafts and overcome the current shortage of donor organs. However, limited sources of bile duct cells and inappropriate cell distribution in bioengineered liver grafts have hindered their practical application. Organoid technology is anticipated to be an excellent tool for the advancement of regenerative medicine. In the present study, we reconstructed intrahepatic bile ducts in a rat decellularized liver graft by recellularization with liver ductal organoids. Using an ex vivo perfusion culture system, we demonstrated the biliary characteristics of repopulated mouse liver organoids, which maintained bile duct markers and reconstructed biliary tree-like networks with luminal structures. We also established a method for the co-recellularization with engineered bile ducts and primary hepatocytes, revealing the appropriate cell distribution to mimic the native liver. We then utilized this model in human organoids to demonstrate the reconstructed bile ducts. Our results show that liver ductal organoids are a potential cell source for bile ducts from bioengineered liver grafts using three-dimensional scaffolds.
Collapse
Affiliation(s)
- Katsuhiro Tomofuji
- Department of Clinical Bio-resource Research and Development, Graduate School of Medicine, Kyoto University, 46-29, Shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan; Department of Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Ken Fukumitsu
- Department of Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
| | - Jumpei Kondo
- Department of Clinical Bio-resource Research and Development, Graduate School of Medicine, Kyoto University, 46-29, Shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
| | - Hiroshi Horie
- Department of Clinical Bio-resource Research and Development, Graduate School of Medicine, Kyoto University, 46-29, Shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan; Department of Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Kenta Makino
- Department of Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Satoshi Wakama
- Department of Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Takashi Ito
- Department of Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Yu Oshima
- Department of Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Satoshi Ogiso
- Department of Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Takamichi Ishii
- Department of Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Masahiro Inoue
- Department of Clinical Bio-resource Research and Development, Graduate School of Medicine, Kyoto University, 46-29, Shimoadachi-cho, Sakyo-ku, Kyoto, 606-8501, Japan
| | - Etsuro Hatano
- Department of Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| |
Collapse
|
|
6
|
Rombaut M, Boeckmans J, Rodrigues RM, van Grunsven LA, Vanhaecke T, De Kock J. Direct reprogramming of somatic cells into induced hepatocytes: Cracking the Enigma code. J Hepatol 2021; 75:690-705. [PMID: 33989701 DOI: 10.1016/j.jhep.2021.04.048] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/12/2021] [Revised: 04/29/2021] [Accepted: 04/30/2021] [Indexed: 01/10/2023]
Abstract
There is an unmet need for functional primary human hepatocytes to support the pharmaceutical and (bio)medical demand. The unique discovery, a decade ago, that somatic cells can be drawn out of their apparent biological lockdown to reacquire a pluripotent state has revealed a completely new avenue of possibilities for generating surrogate human hepatocytes. Since then, the number of papers reporting the direct conversion of somatic cells into induced hepatocytes (iHeps) has burgeoned. A hepatic cell fate can be established via the ectopic expression of native liver-enriched transcription factors in somatic cells, thereby bypassing the need for an intermediate (pluripotent) stem cell state. That said, understanding and eventually controlling the processes that give rise to functional iHeps remains challenging. In this review, we provide an overview of the state-of-the-art reprogramming cocktails and techniques, as well as their corresponding conversion efficiencies. Special attention is paid to the role of liver-enriched transcription factors as hepatogenic reprogramming tools and small molecules as facilitators of hepatic transdifferentiation. To conclude, we formulate recommendations to optimise, standardise and enrich the in vitro production of iHeps to reach clinical standards, and propose minimal criteria for their characterisation.
Collapse
Affiliation(s)
- Matthias Rombaut
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium.
| | - Joost Boeckmans
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium
| | - Robim M Rodrigues
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium
| | - Leo A van Grunsven
- Liver Cell Biology Research Group, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium
| | - Tamara Vanhaecke
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium
| | - Joery De Kock
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium.
| |
Collapse
|
|
7
|
Tao M, Ao T, Mao X, Yan X, Javed R, Hou W, Wang Y, Sun C, Lin S, Yu T, Ao Q. Sterilization and disinfection methods for decellularized matrix materials: Review, consideration and proposal. Bioact Mater 2021; 6:2927-2945. [PMID: 33732964 PMCID: PMC7930362 DOI: 10.1016/j.bioactmat.2021.02.010] [Citation(s) in RCA: 54] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2020] [Revised: 02/08/2021] [Accepted: 02/11/2021] [Indexed: 02/08/2023] Open
Abstract
Sterilization is the process of killing all microorganisms, while disinfection is the process of killing or removing all kinds of pathogenic microorganisms except bacterial spores. Biomaterials involved in cell experiments, animal experiments, and clinical applications need to be in the aseptic state, but their physical and chemical properties as well as biological activities can be affected by sterilization or disinfection. Decellularized matrix (dECM) is the low immunogenicity material obtained by removing cells from tissues, which retains many inherent components in tissues such as proteins and proteoglycans. But there are few studies concerning the effects of sterilization or disinfection on dECM, and the systematic introduction of sterilization or disinfection for dECM is even less. Therefore, this review systematically introduces and analyzes the mechanism, advantages, disadvantages, and applications of various sterilization and disinfection methods, discusses the factors influencing the selection of sterilization and disinfection methods, summarizes the sterilization and disinfection methods for various common dECM, and finally proposes a graphical route for selecting an appropriate sterilization or disinfection method for dECM and a technical route for validating the selected method, so as to provide the reference and basis for choosing more appropriate sterilization or disinfection methods of various dECM.
Asepsis is the prerequisite for the experiment and application of biomaterials. Sterilization or disinfection affects physic-chemical properties of biomaterials. Mechanism, advantages and disadvantages of sterilization or disinfection methods. Factors influencing the selection of sterilization or disinfection methods. Selection of sterilization or disinfection methods for decellularized matrix.
Collapse
Affiliation(s)
- Meihan Tao
- Department of Tissue Engineering, China Medical University, Shenyang, China
| | - Tianrang Ao
- Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Xiaoyan Mao
- Department of Tissue Engineering, China Medical University, Shenyang, China
| | - Xinzhu Yan
- Department of Tissue Engineering, China Medical University, Shenyang, China
| | - Rabia Javed
- Department of Tissue Engineering, China Medical University, Shenyang, China
| | - Weijian Hou
- Department of Tissue Engineering, China Medical University, Shenyang, China
| | - Yang Wang
- Department of Tissue Engineering, China Medical University, Shenyang, China
| | - Cong Sun
- Department of Tissue Engineering, China Medical University, Shenyang, China
| | - Shuang Lin
- Department of Tissue Engineering, China Medical University, Shenyang, China
| | - Tianhao Yu
- The VIP Department, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang, China
| | - Qiang Ao
- Department of Tissue Engineering, China Medical University, Shenyang, China.,Department of Developmental Cell Biology, Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, China.,Institute of Regulatory Science for Medical Device, National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, China
| |
Collapse
|
|
8
|
Schneeberger K, Sánchez‐Romero N, Ye S, van Steenbeek FG, Oosterhoff LA, Pla Palacin I, Chen C, van Wolferen ME, van Tienderen G, Lieshout R, Colemonts‐Vroninks H, Schene I, Hoekstra R, Verstegen MM, van der Laan LJ, Penning LC, Fuchs SA, Clevers H, De Kock J, Baptista PM, Spee B. Large-Scale Production of LGR5-Positive Bipotential Human Liver Stem Cells. Hepatology 2020; 72:257-270. [PMID: 31715015 PMCID: PMC7496924 DOI: 10.1002/hep.31037] [Citation(s) in RCA: 96] [Impact Index Per Article: 19.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/19/2019] [Accepted: 11/07/2019] [Indexed: 12/16/2022]
Abstract
BACKGROUND AND AIMS The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. APPROACH AND RESULTS Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after 2 weeks, compared with 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up-regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. CONCLUSIONS We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.
Collapse
Affiliation(s)
- Kerstin Schneeberger
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary MedicineUtrecht UniversityUtrechtthe Netherlands
| | | | - Shicheng Ye
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary MedicineUtrecht UniversityUtrechtthe Netherlands
| | - Frank G. van Steenbeek
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary MedicineUtrecht UniversityUtrechtthe Netherlands
| | - Loes A. Oosterhoff
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary MedicineUtrecht UniversityUtrechtthe Netherlands
| | - Iris Pla Palacin
- Instituto de Investigación Sanitaria Aragón (IIS Aragón)ZaragozaSpain
| | - Chen Chen
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary MedicineUtrecht UniversityUtrechtthe Netherlands,Hubrecht InstituteRoyal Netherlands Academy of Arts and Sciences and University Medical Center UtrechtUtrechtthe Netherlands
| | - Monique E. van Wolferen
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary MedicineUtrecht UniversityUtrechtthe Netherlands
| | - Gilles van Tienderen
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary MedicineUtrecht UniversityUtrechtthe Netherlands
| | - Ruby Lieshout
- Department of SurgeryErasmus MC‐University Medical CenterRotterdamthe Netherlands
| | - Haaike Colemonts‐Vroninks
- Department of In Vitro Toxicology and Dermato‐cosmetologyFaculty of Medicine and PharmacyVrije Universiteit BrusselBrusselsBelgium
| | - Imre Schene
- Division of Pediatric GastroenterologyWilhelmina Children's HospitalUniversity Medical Center UtrechtUtrechtthe Netherlands
| | - Ruurdtje Hoekstra
- Tytgat Institute for Liver and Intestinal ResearchGastroenterology and MetabolismAcademic Medical CenterUniversity of AmsterdamAmsterdamthe Netherlands,Surgical LaboratoryDepartment of SurgeryAcademic Medical CenterUniversity of AmsterdamAmsterdamthe Netherlands
| | | | | | - Louis C. Penning
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary MedicineUtrecht UniversityUtrechtthe Netherlands
| | - Sabine A. Fuchs
- Division of Pediatric GastroenterologyWilhelmina Children's HospitalUniversity Medical Center UtrechtUtrechtthe Netherlands
| | - Hans Clevers
- Hubrecht InstituteRoyal Netherlands Academy of Arts and Sciences and University Medical Center UtrechtUtrechtthe Netherlands,Cancer Genomics NetherlandsUniversity Medical Center UtrechtUtrechtthe Netherlands,Princess Máxima CenterUtrechtthe Netherlands
| | - Joery De Kock
- Department of In Vitro Toxicology and Dermato‐cosmetologyFaculty of Medicine and PharmacyVrije Universiteit BrusselBrusselsBelgium
| | - Pedro M. Baptista
- Instituto de Investigación Sanitaria Aragón (IIS Aragón)ZaragozaSpain,Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas (CIBERehd)MadridSpain,Fundación ARAIDZaragozaSpain,Instituto de Investigación Sanitaria de la Fundación Jiménez DíazMadridSpain,Department of Biomedical and Aerospace EngineeringUniversidad Carlos III de MadridMadridSpain
| | - Bart Spee
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary MedicineUtrecht UniversityUtrechtthe Netherlands
| |
Collapse
|
|
9
|
Baptista PM, Penning LC. Transplantable Liver Organoids, Too Many Cell Types to Choose: a Need for Scientific Self-Organization. CURRENT TRANSPLANTATION REPORTS 2020. [DOI: 10.1007/s40472-020-00266-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Abstract
Purpose of Review
Liver stem cells have been proposed as alternatives or additions for whole liver transplantations to accommodate the donor liver shortage. Various sources of liver stem cells have been described in experimental animal studies. Here we aim to compare the various studies.
Recent Findings
Irrespective of the experimental design, the percentage of long-lasting survival and functional recovery of transplanted cells is generally very low. An exception to this are the proliferating hepatocytes transplanted into Fah(-/-) Rag2−/−IL2rg−/− mice; here 4-month post-transplantation around 65% repopulation was observed, and 11/14 mice survived in contrast to zero survival in sham-treated animals.
Summary
Taking the different cellular sources for the organoids, the different maturation status of the transplanted cells, and the variable animal models into account, a paper-to-paper comparison is compromised. This lack of objective comparison restricts the translation of these model studies into clinical practice.
Collapse
|
|
10
|
HORISAWA K, SUZUKI A. Direct cell-fate conversion of somatic cells: Toward regenerative medicine and industries. PROCEEDINGS OF THE JAPAN ACADEMY. SERIES B, PHYSICAL AND BIOLOGICAL SCIENCES 2020; 96:131-158. [PMID: 32281550 PMCID: PMC7247973 DOI: 10.2183/pjab.96.012] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/07/2023]
Abstract
Cells of multicellular organisms have diverse characteristics despite having the same genetic identity. The distinctive phenotype of each cell is determined by molecular mechanisms such as epigenetic changes that occur throughout the lifetime of an individual. Recently, technologies that enable modification of the fate of somatic cells have been developed, and the number of studies using these technologies has increased drastically in the last decade. Various cell types, including neuronal cells, cardiomyocytes, and hepatocytes, have been generated using these technologies. Although most direct reprogramming methods employ forced transduction of a defined sets of transcription factors to reprogram cells in a manner similar to induced pluripotent cell technology, many other strategies, such as methods utilizing chemical compounds and microRNAs to change the fate of somatic cells, have also been developed. In this review, we summarize transcription factor-based reprogramming and various other reprogramming methods. Additionally, we describe the various industrial applications of direct reprogramming technologies.
Collapse
Affiliation(s)
- Kenichi HORISAWA
- Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
| | - Atsushi SUZUKI
- Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
- Correspondence should be addressed: A. Suzuki, Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan (e-mail: )
| |
Collapse
|
|
11
|
Oct4 and Hnf4α-induced hepatic stem cells ameliorate chronic liver injury in liver fibrosis model. PLoS One 2019; 14:e0221085. [PMID: 31404112 PMCID: PMC6690533 DOI: 10.1371/journal.pone.0221085] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2019] [Accepted: 07/30/2019] [Indexed: 12/20/2022] Open
Abstract
Direct conversion from fibroblasts to generate hepatocyte like-cells (iHeps) bypassing the pluripotent state has been described in previous reports as an attractive method acquiring hepatocytes for cell-based therapy. The limited proliferation of iHeps, however, has hampered it uses in cell-based therapy. Since hepatic stem cells (HepSCs) possess self-renewal and bipotency with the capacity to differentiate into both hepatocytes and cholangiocytes, they have therapeutic potential for treating liver disease. Here, we investigated the therapeutic effects of induced HepSCs (iHepSCs) on a carbon tetrachloride (CCl4)-induced liver fibrosis model. We demonstrate that Oct4 and Hnf4a are sufficient to convert fibroblasts into expandable iHepSCs. Hepatocyte-like cells derived from iHepSCs (iHepSC-HEPs) exhibit the typical morphology of hepatocytes and hepatic functions, including glycogen storage, low-density lipoprotein (LDL) uptake, Indocyanine green (ICG) detoxification, drug metabolism, urea production, and albumin secretion. iHepSCs-derived cholangiocyte-like cells (iHepSC-CLCs) expressed cholangiocyte-specific markers and formed cysts and tubule-like structures with apical-basal polarity and secretory function in three-dimensional culture condition. Furthermore, iHepSCs showed anti-inflammatory and anti-fibrotic effects in CCl4-induced liver fibrosis. This study demonstrates that Oct4 and Hnf4α-induced HepSCs show typical hepatic and biliary functionality in vitro. It also presents the therapeutic effect of iHepSCs in liver fibrosis. Therefore, directly converting iHepSCs from somatic cells may facilitate the development of patient-specific cell-based therapy for chronic liver damage.
Collapse
|
|
12
|
Chen C, Pla‐Palacín I, Baptista PM, Shang P, Oosterhoff LA, van Wolferen ME, Penning LC, Geijsen N, Spee B. Hepatocyte-like cells generated by direct reprogramming from murine somatic cells can repopulate decellularized livers. Biotechnol Bioeng 2018; 115:2807-2816. [PMID: 29959867 PMCID: PMC6221165 DOI: 10.1002/bit.26784] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2018] [Revised: 05/08/2018] [Accepted: 06/26/2018] [Indexed: 12/30/2022]
Abstract
Direct reprogramming represents an easy technique to generate induced hepatocytes (iHeps) from somatic cells. However, current protocols are accompanied by several drawbacks as iHeps are heterogenous and lack fully mature phenotypes of primary hepatocytes. Here, we established a polycistronic expression system to induce the direct reprogramming of mouse embryonic fibroblasts towards hepatocytes. The resulting iHeps are homogenous and display key properties of primary hepatocytes, such as expression of hepatocyte markers, albumin secretion, and presence of liver transaminases. iHeps also possess the capacity to repopulate decellularized liver tissue and exhibit enhanced hepatic maturation. As such, we present a novel strategy to generate homogenous and functional iHeps for applications in tissue engineering and cell therapy.
Collapse
Affiliation(s)
- Chen Chen
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary Medicine, Utrecht UniversityUtrechtThe Netherlands
- Hubrecht Institute‐KNAW and University Medical Centre UtrechtUtrechtThe Netherlands
| | - Iris Pla‐Palacín
- Instituto de Investigación Sanitaria de Aragón (IIS Aragón)ZaragozaSpain
| | - Pedro M. Baptista
- Instituto de Investigación Sanitaria de Aragón (IIS Aragón)ZaragozaSpain
- Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas (CIBERehd)MadridSpain
- Fundación ARAIDZaragozaSpain
- Instituto de Investigación Sanitaria de la Fundación Jiménez DíazMadridSpain
- Department of Biomedical and Aerospace EngineeringUniversidad Carlos III de MadridMadridSpain
| | - Peng Shang
- Hubrecht Institute‐KNAW and University Medical Centre UtrechtUtrechtThe Netherlands
| | - Loes A. Oosterhoff
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary Medicine, Utrecht UniversityUtrechtThe Netherlands
| | - Monique E. van Wolferen
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary Medicine, Utrecht UniversityUtrechtThe Netherlands
| | - Louis C. Penning
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary Medicine, Utrecht UniversityUtrechtThe Netherlands
| | - Niels Geijsen
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary Medicine, Utrecht UniversityUtrechtThe Netherlands
- Hubrecht Institute‐KNAW and University Medical Centre UtrechtUtrechtThe Netherlands
| | - Bart Spee
- Department of Clinical Sciences of Companion AnimalsFaculty of Veterinary Medicine, Utrecht UniversityUtrechtThe Netherlands
| |
Collapse
|