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Kawakatsu-Hatada Y, Murata S, Mori A, Kimura K, Taniguchi H. Serous Membrane Detachment with Ultrasonic Homogenizer Improves Engraftment of Fetal Liver to Liver Surface in a Rat Model of Cirrhosis. Int J Mol Sci 2021; 22:11589. [PMID: 34769019 PMCID: PMC8584093 DOI: 10.3390/ijms222111589] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2021] [Revised: 10/23/2021] [Accepted: 10/24/2021] [Indexed: 11/16/2022] Open
Abstract
Liver transplantation is the most effective treatment for end-stage cirrhosis. However, due to serious donor shortages, new treatments to replace liver transplantation are sorely needed. Recent studies have focused on novel therapeutic methods using hepatocytes and induced pluripotent stem cells, we try hard to develop methods for transplanting these cells to the liver surface. In the present study, we evaluated several methods for their efficiency in the detachment of serous membrane covering the liver surface for transplantation to the liver surface. The liver surface of dipeptidyl peptidase IV (DPPIV)-deficient rats in a cirrhosis model was detached by various methods, and then fetal livers from DPPIV-positive rats were transplanted. We found that the engraftment rate and area as well as the liver function were improved in rats undergoing transplantation following serous membrane detachment with an ultrasonic homogenizer, which mimics the Cavitron Ultrasonic Surgical Aspirator® (CUSA), compared with no detachment. Furthermore, the bleeding amount was lower with the ultrasonic homogenizer method than with the needle and electric scalpel methods. These findings provide evidence that transplantation to the liver surface with serous membrane detachment using CUSA might contribute to the development of new treatments for cirrhosis using cells or tissues.
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Affiliation(s)
- Yumi Kawakatsu-Hatada
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan; (Y.K.-H.); (A.M.); (K.K.); (H.T.)
| | - Soichiro Murata
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan; (Y.K.-H.); (A.M.); (K.K.); (H.T.)
- Division of Regenerative Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
| | - Akihiro Mori
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan; (Y.K.-H.); (A.M.); (K.K.); (H.T.)
| | - Kodai Kimura
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan; (Y.K.-H.); (A.M.); (K.K.); (H.T.)
| | - Hideki Taniguchi
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan; (Y.K.-H.); (A.M.); (K.K.); (H.T.)
- Division of Regenerative Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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Cell therapy for advanced liver diseases: Repair or rebuild. J Hepatol 2021; 74:185-199. [PMID: 32976865 DOI: 10.1016/j.jhep.2020.09.014] [Citation(s) in RCA: 83] [Impact Index Per Article: 20.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/17/2020] [Revised: 08/18/2020] [Accepted: 09/14/2020] [Indexed: 12/15/2022]
Abstract
Advanced liver disease presents a significant worldwide health and economic burden and accounts for 3.5% of global mortality. When liver disease progresses to organ failure the only effective treatment is liver transplantation, which necessitates lifelong immunosuppression and carries associated risks. Furthermore, the shortage of suitable donor organs means patients may die waiting for a suitable transplant organ. Cell therapies have made their way from animal studies to a small number of early clinical trials. Herein, we review the current state of cell therapies for liver disease and the mechanisms underpinning their actions (to repair liver tissue or rebuild functional parenchyma). We also discuss cellular therapies that are on the clinical horizon and challenges that must be overcome before routine clinical use is a possibility.
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Okamoto R, Takayama K, Akita N, Nagamoto Y, Hosokawa D, Iizuka S, Sakurai F, Suemizu H, Ohashi K, Mizuguchi H. Human iPS Cell-based Liver-like Tissue Engineering at Extrahepatic Sites in Mice as a New Cell Therapy for Hemophilia B. Cell Transplant 2019; 27:299-309. [PMID: 29637813 PMCID: PMC5898695 DOI: 10.1177/0963689717751734] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Instead of liver transplantation or liver-directed gene therapy, genetic liver diseases are expected to be treated effectively using liver tissue engineering technology. Hepatocyte-like cells (HLCs) generated from human-induced pluripotent stem (iPS) cells are an attractive unlimited cell source for liver-like tissue engineering. In this study, we attempted to show the effectiveness of human iPS cell–based liver-like tissue engineering at an extrahepatic site for treatment of hemophilia B, also called factor IX (FIX) deficiency. HLCs were transplanted under the kidney capsule where the transplanted cells could be efficiently engrafted. Ten weeks after the transplantation, human albumin (253 μg/mL) and α-1 antitrypsin (1.2 μg/mL) could be detected in the serum of transplanted mice. HLCs were transplanted under the kidney capsule of FIX-deficient mice. The clotting activities in the transplanted mice were approximately 5% of those in wild-type mice. The bleeding time in transplanted mice was shorter than that in the nontransplanted mice. Taken together, these results indicate the success in generating functional liver-like tissues under the kidney capsule by using human iPS cell–derived HLCs. We also demonstrated that the human iPS cell–based liver-like tissue engineering technology would be an effective treatment of genetic liver disease including hemophilia B.
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Affiliation(s)
- Ryota Okamoto
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.,2 Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan
| | - Kazuo Takayama
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.,2 Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan.,3 PRESTO, Japan Science and Technology Agency, Saitama, Japan
| | - Naoki Akita
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.,2 Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan
| | - Yasuhito Nagamoto
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.,2 Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan
| | - Daiki Hosokawa
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.,2 Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan
| | - Shunsuke Iizuka
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
| | - Fuminori Sakurai
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
| | - Hiroshi Suemizu
- 4 Central Institute for Experimental Animals, Kanagawa, Japan
| | - Kazuo Ohashi
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
| | - Hiroyuki Mizuguchi
- 1 Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.,2 Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan.,5 Global Center for Medical Engineering and Informatics, Osaka University, Osaka, Japan
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Barahman M, Asp P, Roy-Chowdhury N, Kinkhabwala M, Roy-Chowdhury J, Kabarriti R, Guha C. Hepatocyte Transplantation: Quo Vadis? Int J Radiat Oncol Biol Phys 2018; 103:922-934. [PMID: 30503786 DOI: 10.1016/j.ijrobp.2018.11.016] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2018] [Revised: 10/10/2018] [Accepted: 11/10/2018] [Indexed: 12/21/2022]
Abstract
Orthotopic liver transplantation (OLT) has been effective in managing end-stage liver disease since the advent of cyclosporine immunosuppression therapy in 1980. The major limitations of OLT are organ supply, monetary cost, and the burden of lifelong immunosuppression. Hepatocyte transplantation, as a substitute for OLT, has been an exciting topic of investigation for several decades. HT is potentially minimally invasive and can serve as a vehicle for delivery of personalized medicine through autologous cell transplant after modification ex vivo. However, 3 major hurdles have prevented large-scale clinical application: (1) availability of transplantable cells; (2) safe and efficient ex vivo gene therapy methods; and (3) engraftment and repopulation efficiency. This review will discuss new sources for transplantable liver cells obtained by lineage reprogramming, clinically acceptable methods of genetic manipulation, and the development of hepatic irradiation-based preparative regimens for enhancing engraftment and repopulation of transplanted hepatocytes. We will also review the results of the first 3 patients with genetic liver disorders who underwent preparative hepatic irradiation before hepatocyte transplantation.
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Affiliation(s)
- Mark Barahman
- Department of Pathology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York
| | - Patrik Asp
- Department of Surgery, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York
| | - Namita Roy-Chowdhury
- Department of Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York
| | - Milan Kinkhabwala
- Department of Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York
| | - Jayanta Roy-Chowdhury
- Department of Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York; Department of Genetics, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York
| | - Rafi Kabarriti
- Department of Radiation Oncology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York
| | - Chandan Guha
- Department of Pathology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York; Department of Radiation Oncology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York; Department of Urology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York.
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Srinivasan RC, Kannisto K, Strom SC, Gramignoli R. Evaluation of different routes of administration and biodistribution of human amnion epithelial cells in mice. Cytotherapy 2018; 21:113-124. [PMID: 30409699 DOI: 10.1016/j.jcyt.2018.10.007] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2018] [Revised: 09/28/2018] [Accepted: 10/07/2018] [Indexed: 01/10/2023]
Abstract
Placenta is a non-controversial and promising source of cells for the treatment of several liver diseases. We previously reported that transplanted human amnion epithelial cells (hAECs) differentiate into hepatocyte-like cells, resulting in correction of mouse models of metabolic liver disease or acute hepatic failure. As part of preclinical safety studies, we investigated the distribution of hAECs using two routes of administration to efficiently deliver hAECs to the liver. Optical imaging is commonly used because it can provide fast, high-throughput, whole-body imaging, thus DiR-labeled hAECs were injected into immunodeficient mice, via the spleen or the tail vein. The cell distribution was monitored using an in vivo imaging system over the next 24 h. After splenic injection, the DiR signal was detected in liver and spleen at 1, 3 and 24 h post-transplant. The distribution was confirmed by analysis of human DNA content at 24 h post-transplant and human-specific cytokeratin 8/18 staining. Tail vein infusion resulted in cell engraftment mainly in the lungs, with minimal detection in the liver. Delivery of cells to the portal vein, via the spleen, resulted in efficient delivery of hAECs to the liver, with minimal, off-target distribution to lungs or other organs.
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Affiliation(s)
- Raghuraman C Srinivasan
- Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden
| | - Kristina Kannisto
- Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden
| | - Stephen C Strom
- Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden
| | - Roberto Gramignoli
- Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden.
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Gupta S, Kim S, Vemuru R, Aragona E, Yerneni P, Burk R, Rha C. Hepatocyte Transplantation: An Alternative System for Evaluating Cell Survival and Immunoisolation. Int J Artif Organs 2018. [DOI: 10.1177/039139889301600310] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
To evaluate systems for barrier immunoisolation of transplanted hepatocytes, we used transgenic mouse hepatocytes that secrete HBsAg. Hepatocytes were rapidly encapsulated in chitosan, a cationic polymer derived by deacetylation of chitin. Chitosan was allowed to electrostatically bond with anionic sodium alginate for creating an outer bipolymer membrane of the capsules. After encapsulation, hepatocyte viability remained unchanged for seven days in vitro with secretion of HBsAg into the culture medium throughout this period. Following intraperitoneal transplantation of encapsulated hepatocytes, HBsAg promptly appeared in blood of recipients. In congeneic recipients, serum HBsAg peaked at two weeks. Hepatocytes were present in recovered chitosan capsules and expressed HBsAg mRNA. In allogeneic recipients, however, serum HBsAg disappeared within one week and recovered chitosan capsules showed lymphomononuclear cells but not hepatocytes. Transplantation of chitosan encapsulatd HbsAg secreting hepatocytes failed to induce an anti-HBs response, suggesting modulation of the host immune response. These results indicate that transplantation systems using genetically modified hepatocytes which secrete gene products in the blood of recipients should facilitate evaluation of hepatocyte encapsulation.
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Affiliation(s)
- S. Gupta
- Departments of Medicine, Obstetrics and Gynecology and the Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY
| | - S.K. Kim
- Biomaterials Science and Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, MA - USA
| | - R.P. Vemuru
- Departments of Medicine, Obstetrics and Gynecology and the Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY
| | - E. Aragona
- Departments of Medicine, Obstetrics and Gynecology and the Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY
| | - P.R. Yerneni
- Departments of Medicine, Obstetrics and Gynecology and the Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY
| | - R.D. Burk
- Pediatrics, Microbiology and Immunology, Obstetrics and Gynecology and the Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY
| | - C.K. Rha
- Biomaterials Science and Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, MA - USA
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7
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Tranchart H, Gaillard M, Diop PS, Goulinet S, Lainas P, Dagher I. Transplantation of genetically modified hepatocytes after liver preconditioning in Watanabe heritable hyperlipidemic rabbit. J Surg Res 2017; 224:23-32. [PMID: 29506845 DOI: 10.1016/j.jss.2017.11.060] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2017] [Revised: 11/06/2017] [Accepted: 11/21/2017] [Indexed: 12/20/2022]
Abstract
BACKGROUND Hepatocyte transplantation is a potentially less invasive alternative to liver transplantation for treating inherited metabolic liver diseases. We developed an autotransplantation protocol of ex vivo genetically modified hepatocytes combining lentiviral transduction and transplantation after liver preconditioning by partial portal vein embolization. We investigated the metabolic efficiency of this approach in Watanabe rabbits, animal model of familial hypercholesterolemia. METHODS Our autotransplantation experimental protocol was used in two groups of rabbits (n = 10), experimental and sham, receiving transduced and control hepatocytes, respectively. Isolated hepatocytes from left liver lobes were transduced using recombinant lentiviruses. Median lobe portal branches were embolized under fluoroscopic control. Functional measurement of low-density lipoprotein (LDL) receptor expression was assessed by LDL internalization assays. Cholesterol level evolution was monitored. Rabbits were killed 20 wk after the procedure. RESULTS Three rabbits of each group died several hours after hepatocyte transplantation; autopsy revealed portal vein thrombosis in two rabbits from each group. The protocol was therefore modified with hepatocytes being transplanted through splenic injection. Lentiviral hepatocyte transduction efficacy was 64.5%. Fluorescence microscopy revealed Dil-LDL internalization of transduced hepatocytes. Seven rabbits in each group were considered for lipid analysis. Four weeks after autotransplantation, median total cholesterol level decreased in the experimental group, without reaching statistical significance (8.9 [8.0-9.8] g/L versus 6.3 [0.5-8.3]; P = 0.171). In the experimental group, enzyme-linked immunosorbent assay detected significant antibody expression against human low-density lipoprotein receptor. CONCLUSIONS Autotransplantation protocol allowed a nonstatistically significant improvement of the lipid profile in Watanabe rabbits. Further experiments involving a larger number of animals are necessary to confirm or refute our findings.
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Affiliation(s)
- Hadrien Tranchart
- INSERM U1193, Paul Brousse Hospital, Villejuif, France; Department of Minimally Invasive Surgery, Antoine Béclère Hospital, AP-HP, Paris-Sud University, Clamart, France.
| | - Martin Gaillard
- INSERM U1193, Paul Brousse Hospital, Villejuif, France; Department of Minimally Invasive Surgery, Antoine Béclère Hospital, AP-HP, Paris-Sud University, Clamart, France
| | - Papa Saloum Diop
- INSERM U1193, Paul Brousse Hospital, Villejuif, France; Department of Minimally Invasive Surgery, Antoine Béclère Hospital, AP-HP, Paris-Sud University, Clamart, France
| | | | - Panagiotis Lainas
- INSERM U1193, Paul Brousse Hospital, Villejuif, France; Department of Minimally Invasive Surgery, Antoine Béclère Hospital, AP-HP, Paris-Sud University, Clamart, France
| | - Ibrahim Dagher
- INSERM U1193, Paul Brousse Hospital, Villejuif, France; Department of Minimally Invasive Surgery, Antoine Béclère Hospital, AP-HP, Paris-Sud University, Clamart, France
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Spinelli SV, Rodríguez JV, Quintana AB, Mediavilla MG, Guibert EE. Engraftment and Function of Intrasplenically Transplanted Cold Stored Rat Hepatocytes. Cell Transplant 2017. [DOI: 10.3727/096020198389889] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
Hepatocellular transplant may potentially be efficacious for the treatment of selected liver metabolic disorders and acute hepatic failure. On the other hand, the use of hepatocyte cold preservation techniques in these transplantation protocols would allow to have available cells at the right time and place and, consequently, make an optimal use of scarce human hepatocytes. In our experiments we evaluated the biodistribution and functionality of cold preserved hepatocytes transplanted in the spleen of syngeneic rats. Isolated hepatocytes were labeled with the fluorescent dye 5(6)-carboxyfluorescein diacetate succinimidyl-ester, cold-preserved in modified University of Wisconsin (UW) solution for 48 or 96 h, and then transplanted into the spleen. Recipient animals were euthanized at 0 and 3 h, and at 1, 2, 3, 5, 10, and 14 days after transplantation for tissue analysis. Labeled hepatocytes were clearly identifiable in the recipient tissues up to 14 days later. Fluorescence microscopy also showed no significant differences in biodistribution when either cold stored or freshly isolated hepatocytes were transplanted. In addition, functional activity of transplanted cells was demonstrated by immunohistochemical detection of albumin at levels comparable to those found in normal hepatocytes. Our findings establish that cold preserved hepatocytes appear morphologically and biochemically normal after intrasplenic transplantation. Consequently, it indicates that modified UW solution makes it possible to safety preserve hepatocytes for up to 96 h before transplantation, perhaps providing sufficient time for hepatocyte allocation and potential recipient preparation, if applicable clinically.
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Affiliation(s)
- Silvana V. Spinelli
- Biología Molecular, Dto. Cs. Biológicas, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario
| | - Joaquín V. Rodríguez
- Farmacología, Dto. Cs. Fisiológicas, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario
| | - Alejandra B. Quintana
- Morfología, Dto. Cs. Biológicas, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario
| | - María G. Mediavilla
- Biología Molecular, Dto. Cs. Biológicas, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario
| | - Edgardo E. Guibert
- Biología Molecular, Dto. Cs. Biológicas, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario
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9
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Fujino M, Li XK, Kitazawa Y, Funeshima N, Guo L, Okuyama T, Amano T, Amemiya H, Suzuki S. Selective Repopulation of Mice Liver after Fas-Resistant Hepatocyte Transplantation. Cell Transplant 2017. [DOI: 10.3727/000000001783986701] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Hepatocyte transplantation has been proposed as a potential therapeutic method to treat irreversible liver failure and inherited hepatic disorders, although transplanted cells do not easily reconstruct the liver tissue under intact conditions. This study was aimed at modulating the recipient liver conditions to promote repopulation of the liver after hepatocyte transplantation. Hepatocytes isolated from male MRL-lpr/lpr (lpr) mice with a mutation of Fas antigen were transplanted in a number of 1 × 106 cells in female MRL-+/+ (wildtype mice) by intrasplenic injection. An agonistic anti-Fas antibody (0.15 mg/kg) was administered intravenously 24 h after cell transplantation. We also administrated the antibody at 0.3 mg/kg 1 week after grafting and at 0.6 mg/kg 2 weeks after transplantation. The liver specimens were taken at different time intervals for histological examination. The reconstructed male lpr hepatocytes in the female wild-type mice were determined by a real-time quantitative PCR assay using the primers and probe for the sry gene. The pathologic findings of the recipient livers after treatment with anti-Fas antibody revealed a large number of apoptotic hepatocytes. The grafted lpr hepatocytes were observed to reconstruct as much as 6.9% of the recipient liver in the anti-Fas antibody-treated group 3 months after transplantation. In contrast, we observed the transplanted cells at lower than 0.1% in the nontreated livers. These findings demonstrated that repeated induction of apoptosis in recipient hepatocytes shifts the environment of the liver to a regenerative condition. This method may be useful to promote the reconstruction of transplanted hepatocytes in a recipient liver.
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Affiliation(s)
- Masayuki Fujino
- Department of Experimental Surgery and Bioengineering, Tokyo, Japan
- Department of Zootechnical Science, Tokyo University of Agriculture, Tokyo, Japan
| | - Xiao-Kang Li
- Department of Experimental Surgery and Bioengineering, Tokyo, Japan
| | - Yusuke Kitazawa
- Department of Experimental Surgery and Bioengineering, Tokyo, Japan
| | - Naoko Funeshima
- Department of Experimental Surgery and Bioengineering, Tokyo, Japan
| | - Lei Guo
- Department of Experimental Surgery and Bioengineering, Tokyo, Japan
| | - Torayuki Okuyama
- Genetics, National Children's Medical Research Center, Tokyo, Japan
| | - Takashi Amano
- Department of Zootechnical Science, Tokyo University of Agriculture, Tokyo, Japan
| | - Hiroshi Amemiya
- Department of Experimental Surgery and Bioengineering, Tokyo, Japan
| | - Seiichi Suzuki
- Department of Experimental Surgery and Bioengineering, Tokyo, Japan
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Moscioni AD, Rozga J, Chen S, Naim A, Scott HS, Demetriou AA. Long-Term Correction of Albumin Levels in the Nagase Analbuminemic Rat: Repopulation of the Liver by Transplanted Normal Hepatocytes under a Regeneration Response. Cell Transplant 2017; 5:499-503. [PMID: 8800518 DOI: 10.1177/096368979600500409] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Numerous studies have reported successful transplantation of hepatocytes with demonstration of function. However, none have shown long-term correction of a liver-related metabolic defect. Male Nagase analbuminemic rats, immunosuppressed with cyclosporin-A, were transplanted with normal hepatocytes (2 × 107 cells/rat) isolated from allogeneic male Sprague–Dawley rat donors. Hepatocytes were selectively transplanted via the portal vein tributary into the posterior liver lobes of Nagase analbuminemic rats. Following 2 wk, to allow engraftment, selected transplanted rats (Group I) were reoperated and the portal venous branch supplying the anterior liver lobes was permanently ligated, resulting in their atrophy and induction of regeneration in the residual transplant-bearing lobes. Control rats consisted of: Group II—transplanted with normal hepatocytes without portal branch ligation; Group III—transplanted with analbuminemic hepatocytes with portal branch ligation; and Group IV—nontransplanted analbuminemic rats with portal branch ligation. The experimental period extended to 3 mo posttransplantation. All rats transplanted with normal hepatocytes demonstrated a significant elevation in serum albumin levels (ELISA). Group I rats had dramatic elevations in serum albumin to near normal levels (1.78 ± 0.20 g/dl), and maintained these levels until the end of the experiment Albumin levels in Group II rats reached 0.26 ± 0.07 g/dl (p < 0.001), whereas Group III and IV rats showed no changes in serum albumin levels throughout the experiment Immunohistology of liver tissue obtained from Group I rats, demonstrated large numbers (22.6 ± 7.5%) of albumin-positive hepatocytes populating the recipient liver. This is the first report of near-total and sustained correction of a genetic defect in liver function in an experimental animal model following allogeneic hepatocyte transplantation.
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Affiliation(s)
- A D Moscioni
- Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
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Gupta S, Vasa SR, Rajvanshi P, Zuckier LS, Palestro CJ, Bhargava KK. Analysis of Hepatocyte Distribution and Survival in Vascular Beds with Cells Marked by 99mTC or Endogenous Dipeptidyl Peptidase IV Activity. Cell Transplant 2017; 6:377-86. [PMID: 9258511 DOI: 10.1177/096368979700600404] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Knowledge of the kinetics of cell distribution in vascular beds will help optimize engraftment of transplanted hepatocytes. To noninvasively localize transplanted cells in vivo, we developed conditions for labeling rat hepatocytes with 99mTc–pertechnetate. The incorporated 99mTc was bound to intracellular proteins and did not impair cell viability. When 99mTc hepatocytes were intrasplenically injected into normal rats, cells entered liver sinusoids with time–activity curves demonstrating instantaneous cell translocations. 99mTc activity in removed organs was in liver or spleen, and lungs showed little activity. However, when cells were intrasplenically transplanted into rats with portasystemic collaterals, 99mTc appeared in both liver sinusoids and pulmonary alveolar capillaries. To further localize cells, we transplanted DPPIV+ F344 rat hepatocytes into syngeneic DPPIV – recipients. Histochemical staining for DPPIV activity demonstrated engraftment of intrasplenically transplanted cells in liver parenchyma. In contrast, when 99mTc hepatocytes were injected into a peripheral vein, cells were entrapped in pulmonary capillaries but were subsequently broken down with redistribution of 99mTc activity elsewhere. Intact DPPIV+ hepatocytes were identified in lungs, whereas only cell fragments were present in liver, spleen, or kidneys. These findings indicate that although the pulmonary vascular bed offers advantages of easy accessibility and a relatively large capacity, significant early cell destruction is an important limitation.
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Affiliation(s)
- S Gupta
- Marion Bessin Liver Research Center, Long Island Jewish Hospital, New Hyde Park, NY, USA
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12
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Aoki T, Umehara Y, Ferraresso C, Sugiyama N, Middleton Y, Avital I, Inderbitzin D, Demetriou AA, Rozga J. Intrasplenic Transplantation of Encapsulated Cells: A Novel Approach to Cell Therapy. Cell Transplant 2017. [DOI: 10.3727/000000002783985549] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Cell therapy is likely to succeed clinically if cells survive at the transplantation site and are protected against immune rejection. We hypothesized that this could be achieved with intrasplenic transplantation of encapsulated cells because the cells would have instant access to oxygen and nutrients while being separated from the host immune system. In order to provide proof of the concept, primary rat hepatocytes and human hepatoblastoma-derived HepG2 cells were used as model cells. Rat hepatocytes were encapsulated in 100-kDa hollow fibers and cultured for up to 28 days. Rat spleens were implanted with hollow fibers that were either empty or contained 1 × 107 rat hepatocytes. Human HepG2 cells were encapsulated using alginate/poly-l-lysine (ALP) and also transplanted into the spleen; control rats were transplanted with free HepG2 cells. Blood human albumin levels were measured using Western blotting and spleen sections were immunostained for albumin. Hepatocytes in monolayer cultures remained viable for only 6–10 days, whereas the cells cultured in hollow fibers remained viable and produced albumin throughout the study period. Allogeneic hepatocytes transplanted in hollow fibers remained viable for 4 weeks (end of study). Free HepG2 transplants lost viability and function after 7 days, whereas encapsulated HepG2 cells remained viable and secreted human albumin at all time points studied. ALP capsules, with or without xenogeneic HepG2 cells, produced no local fibrotic response. These data indicate that intrasplenic transplantation of encapsulated cells results in excellent survival and function of the transplanted cells and that the proposed technique has the potential to allow transplantation of allo- and xenogeneic cells (e.g., pancreatic islets) without immunosuppression.
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Affiliation(s)
- Takeshi Aoki
- Liver Support Research Laboratory, Department of Surgery, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048
| | - Yutaka Umehara
- Liver Support Research Laboratory, Department of Surgery, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048
| | - Chiara Ferraresso
- Liver Support Research Laboratory, Department of Surgery, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048
| | - Nozomu Sugiyama
- Liver Support Research Laboratory, Department of Surgery, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048
| | - Yvette Middleton
- Liver Support Research Laboratory, Department of Surgery, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048
| | - Itzhak Avital
- Liver Support Research Laboratory, Department of Surgery, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048
| | - Daniel Inderbitzin
- Liver Support Research Laboratory, Department of Surgery, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048
| | - Achilles A. Demetriou
- Liver Support Research Laboratory, Department of Surgery, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048
| | - Jacek Rozga
- Liver Support Research Laboratory, Department of Surgery, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048
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13
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Markus PM, Koenig S, Krause P, Becker H. Selective Intraportal Transplantation of DiI-marked Isolated Rat Hepatocytes. Cell Transplant 2017; 6:455-62. [PMID: 9331496 DOI: 10.1177/096368979700600504] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Transplantation of isolated hepatocytes is a promising alternative to orthotopic liver transplantation in experimental animal models with acute hepatic failure and hereditary enzyme defects. Conventional light microscopy identification of hepatocytes within recipient livers has been limited due to the inability to distinguish between donor and recipient liver cells. In this study, we labeled hepatocytes intracellularly with the fluorescent dye DiI-18 prior to selective intraportal or intrasplenic transplantation. Syngeneic LEW rat hepatocytes were isolated and 2 × 107 fluorescence-labeled cells were transplanted by intraportal infusion selectively into 2/3 of the recipient liver lobules to avoid lethal portal hypertension. Rats were sacrificed on postop days 1, 3, 5, 10, 20, and 40. Histological examination was performed using light and fluorescence microscopy counterstained by light green dye. The quantity of transplanted hepatocytes residing within the recipient liver was determined by FACS analysis after enzymatic digestion of the recipient liver lobules. Engrafted hepatocytes were identified in the periportal regions of transplanted liver lobules. The stained hepatocytes were retrieved up to 20 days postop using fluorescent microscopy. Using FACS analysis the number of labeled hepatocytes was found to diminish over time following transplantation from 2.1% on postop day 1 to 0.5% on day 10. Labeled hepatocytes transplanted into the spleen were retrieved in clusters up to 20 days postop (the last day of observation). Furthermore, the migration of labeled hepatocytes from spleen to liver parenchyma was observed following intrasplenic transplantation. However, after selective intraportal transplantation, only fluorescent debris was found in splenic and pulmonary tissue upon examination of various organs. This article describes the method of fluorescent labeling of rat hepatocytes and reports the feasibility and limitations of using DiI-18 as a marker.
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Affiliation(s)
- P M Markus
- Department of General Surgery, Georg August University of Göttingen, Germany
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14
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Krishna Vanaja D, Sivakumar B, Jesudasan RA, Singh L, Janardanasarma MK, Habibullah CM. In Vivo Identification, Survival, and Functional Efficacy of Transplanted Hepatocytes in Acute Liver Failure Mice Model by Fish Using Y-Chromosome Probe. Cell Transplant 2017; 7:267-73. [PMID: 9647436 DOI: 10.1177/096368979800700305] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Hepatocyte transplantation has excited much interest in lending temporary metabolic support to a failing liver following acute liver injury. The exact site from which they act and the clinical, biochemical, and histological changes in the recipient body following hepatocyte transplantation is yet to be worked out. The present study is an attempt to delineate location and function of transplanted hepatocytes and also the overall survival of these cells with a fluorescent in situ hybridization (FISH) technique using a Y-chromosome–specific probe in a carbon tetrachloride (CCl4)-induced mice model of fulminant hepatic failure. Fifty-five syngenic adult Swiss female mice of approximately the same age and body weight were divided into three groups. Group-1 (n = 15), which received mineral oil, served as a negative control. Group-II (n = 15) received CCl4 (3 mL/kg) 40% vol/vol in mineral oil, by gavage served as positive control for hepatic failure. Group-III (n = 25) received intrasplenic transplantation of syngenic single cell suspension of hepatocytes in Hanks medium, after 30 h of CCl4 administration. Male Swiss adult mice (n = 15) served as donors of hepatocytes. The overall survival of animals in groups I to III was 100, 0, and 70%, respectively, by 2 wk of the study period. Transplanted hepatocytes were identified by Periodic Acid Schiff (PAS) staining and confirmed with a FISH technique using the Y-chromosome probe. The majority of exogenously transplanted hepatocytes were found in the liver and spleen sections even after 1 wk of hepatocyte transplantation. Transplanted cells were mostly found to be translocated into the sinusoids of the liver. Transplanted hepatocytes were found to be beneficial as a temporary liver support in a failing liver, significantly improving the survival of the animals. In the present study, the FISH technique was used to unequivocally distinguish the transplanted cells from the host, and thus describes a model for studying the distribution and survival of the transplanted cells.
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15
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Markus PM, Krause P, Fayyazi A, Honnicke K, Becker H. Allogeneic Hepatocyte Transplantation Using FK 506. Cell Transplant 2017; 6:77-83. [PMID: 9040958 DOI: 10.1177/096368979700600112] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Hepatocyte transplantation is an intriguing alternative to orthotopic liver transplantation. While engraftment of syngeneic hepatocytes can be achieved with relative ease, engraftment of allogeneic hepatocytes has been far more complicated. We used FK 506 (Tacrolimus), a novel and highly efficient immunosuppressant, which has been reported to augment liver regeneration in rats. Recipients of isolated syngeneic (LEW) and allogeneic (Wistar F.) rat hepatocytes (major histocompatibility barrier) recieved different immunosuppressive regiments with FK 506 or Cyclosporine A (CsA). Mature syngeneic hepatocytes could be retrieved up to post op day 300 with the lowest number of hepatocytes on post op day 20. Following allogeneic transplantation, no mature hepatocytes could be identified after post op day 10, though ductular like structures within the spleen were found in FK 506 but not CsA-treated animals. The epithelial cells of ductular like structures exhibit cytological features of CK-19 positive cells. Our results suggest that under CsA or FK 506 immunosuppression long-term survival of mature allogeneic hepatocytes within the spleen cannot be achieved across a major histocompatibility barrier though FK 506 allows engraftment of allogeneic donor type ductular cells. Copyright © 1997 Elsevier Science Inc.
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Affiliation(s)
- P M Markus
- Department of General Surgery and Pathology, Georg August University of Göttingen, Germany
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16
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Tada K, Roy-Chowdhury N, Prasad V, Kim BH, Manchikalapudi P, Fox IJ, van Duijvendijk P, Bosma PJ, Roy-Chowdhury J. Long-Term Amerlioration of Bilirubin Glucuronidation Defect in Gunn Rats by Transplanting Genetically Modified Immortalized Autologous Hepatocytes. Cell Transplant 2017; 7:607-16. [PMID: 9853589 DOI: 10.1177/096368979800700611] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Ex vivo gene therapy, in which hepatocytes are harvested from mutants, retrovirally transduced with a normal gene and transplanted back into the donor, has been used for correction of inherited metabolic defects of liver. Major drawbacks of this method include limited availability of autologous hepatocytes, inefficient retroviral transduction of primary hepatocytes, and the limited number of hepatocytes that can be transplanted safely. To obviate these problems, we transduced primary hepatocytes derived from inbred bilirubin–UDP–glucuronosyl–transferase (BUGT)-deficient Gunn rats by infection with a recombinant retrovirus expressing temperature-sensitive mutant SV40 large T antigen (tsT). The immortalized cells were then transduced with a second recombinant retrovirus expressing human B-UGT, and a clone expressing high levels of the enzyme was expanded by culturing at permissive temperature (33°C). At 37°C, tsT antigen was degraded and the cells expressed UGT activity toward bilirubin at a level approximately twice that present in normal rat liver homogenates. For seeding the cells into the liver bed, 1 × 107 cells were injected into the spleens of syngeneic Gunn rats five times at 10-day intervals. Excretion of bilirubin glucuronides in bile was demonstrated by HPLC analysis and serum bilirubin levels were reduced by 27 to 52% in 40 days after the first transplantation and remained so throughout the duration of the study (120 days). None of the transplanted Gunn rats or SCID mice transplanted with the immortalized cells developed tumors. © 1998 Elsevier Science Inc.
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Affiliation(s)
- K Tada
- Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10462, USA
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17
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Jauregui HO, Chowdhury NR, Chowdhury JR. Use of Mammalian Liver Cells for Artificial Liver Support. Cell Transplant 2017; 5:353-67. [PMID: 8727004 DOI: 10.1177/096368979600500302] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Advances in orthotopic liver transplantation have improved the survival rate of both acute and chronic liver failure patients to nearly 70%. However, the success of this treatment modality has created an international organ shortage. Many patients die while awaiting transplantation in part due to the minimal capacity to store viable transplantable livers beyond 24 h. Additionally, for many areas of the world, routine use of whole liver transplantation to treat liver disease is impractical due to the demands on both financial and technical resources. Potentially, these issues may be alleviated, at least in part, by the use of liver cell transplantation or cellular-based liver assist devices. The well-documented regenerative capacity of the liver may obviate the need for whole organ transplantation in some instances of acute failure, if the patient may be provided temporary metabolic support. Although other patients ultimately may require transplantation, a longer period of time to find a suitable organ for transplantation may be gained by that supportive therapy. The field of liver cell transplantation may offer solutions to patients with inherited metabolic deficiencies or chronic liver disease. The potential to treat an hepatic disorder by using only a fraction of the whole liver would increase the number of whole organs available for orthotopic liver transplantation. Research in the fields of hepatocyte based intra- and extra-corporeal liver support is providing evidence that these therapeutic modalities may ultimately become routine in the treatment of severe liver disease. A historic overview of that technology along with its current status is discussed.
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Affiliation(s)
- H O Jauregui
- Department of Pathology, Rhode Island Hospital, Providence 02903, USA
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18
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Fanti M, Gramignoli R, Serra M, Cadoni E, Strom SC, Marongiu F. Differentiation of amniotic epithelial cells into various liver cell types and potential therapeutic applications. Placenta 2017; 59:139-145. [PMID: 28411944 DOI: 10.1016/j.placenta.2017.03.020] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/14/2017] [Revised: 03/25/2017] [Accepted: 03/29/2017] [Indexed: 02/07/2023]
Abstract
The aim of Regenerative Medicine is to replace or regenerate human cells, tissues or organs in order to restore normal function. Among all organs, the liver is endowed with remarkable regenerative capacity. Nonetheless, there are conditions in which this ability is impaired, and the use of isolated cells, including stem cells, is being considered as a possible therapeutic tool for the management of chronic hepatic disease. Placenta holds great promise for the field of regenerative medicine. It has long been used for the treatment of skin lesions and in ophthalmology, due to its ability to modulate inflammation and promote healing. More recently, cells isolated from the amniotic membrane are being considered as a possible resource for tissue regeneration, including in the context liver disease. Two cell types can be easily isolated from human amnion: epithelial cells (hAEC) and mesenchymal stromal cells (hAMSC). However only the first cell population has been demonstrated to be a possible source of proficient hepatic cells. This review will summarize current knowledge on the differentiation of hAEC into liver cells and their potential therapeutic application.
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Affiliation(s)
- Maura Fanti
- University of Cagliari, Department of Biomedical Sciences, Unit of Experimental Medicine, Cagliari, Italy
| | - Roberto Gramignoli
- Karolinska Institutet, Department of Laboratory Medicine, Division of Pathology, Stockholm, Sweden
| | - Monica Serra
- University of Cagliari, Department of Biomedical Sciences, Unit of Experimental Medicine, Cagliari, Italy
| | - Erika Cadoni
- University of Cagliari, Department of Biomedical Sciences, Unit of Experimental Medicine, Cagliari, Italy
| | - Stephen C Strom
- Karolinska Institutet, Department of Laboratory Medicine, Division of Pathology, Stockholm, Sweden
| | - Fabio Marongiu
- University of Cagliari, Department of Biomedical Sciences, Unit of Experimental Medicine, Cagliari, Italy.
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19
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Cellular Mechanisms of Liver Regeneration and Cell-Based Therapies of Liver Diseases. BIOMED RESEARCH INTERNATIONAL 2017; 2017:8910821. [PMID: 28210629 PMCID: PMC5292184 DOI: 10.1155/2017/8910821] [Citation(s) in RCA: 76] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/13/2016] [Revised: 11/29/2016] [Accepted: 12/27/2016] [Indexed: 12/14/2022]
Abstract
The emerging field of regenerative medicine offers innovative methods of cell therapy and tissue/organ engineering as a novel approach to liver disease treatment. The ultimate scientific foundation of both cell therapy of liver diseases and liver tissue and organ engineering is delivered by the in-depth studies of the cellular and molecular mechanisms of liver regeneration. The cellular mechanisms of the homeostatic and injury-induced liver regeneration are unique. Restoration of the mass of liver parenchyma is achieved by compensatory hypertrophy and hyperplasia of the differentiated parenchymal cells, hepatocytes, while expansion and differentiation of the resident stem/progenitor cells play a minor or negligible role. Participation of blood-borne cells of the bone marrow origin in liver parenchyma regeneration has been proven but does not exceed 1-2% of newly formed hepatocytes. Liver regeneration is activated spontaneously after injury and can be further stimulated by cell therapy with hepatocytes, hematopoietic stem cells, or mesenchymal stem cells. Further studies aimed at improving the outcomes of cell therapy of liver diseases are underway. In case of liver failure, transplantation of engineered liver can become the best option in the foreseeable future. Engineering of a transplantable liver or its major part is an enormous challenge, but rapid progress in induced pluripotency, tissue engineering, and bioprinting research shows that it may be doable.
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20
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Yovchev MI, Locker J, Oertel M. Biliary fibrosis drives liver repopulation and phenotype transition of transplanted hepatocytes. J Hepatol 2016; 64:1348-57. [PMID: 26855174 PMCID: PMC5137249 DOI: 10.1016/j.jhep.2016.01.036] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/05/2015] [Revised: 01/12/2016] [Accepted: 01/29/2016] [Indexed: 01/11/2023]
Abstract
BACKGROUND & AIMS Current research focuses on developing alternative strategies to restore decreased liver mass prior to the onset of end-stage liver disease. Cell engraftment/repopulation requires regeneration in normal liver, but we have shown that severe liver injury stimulates repopulation without partial hepatectomy (PH). We have now investigated whether a less severe injury, secondary biliary fibrosis, would drive engraftment/repopulation of ectopically transplanted mature hepatocytes. METHODS Ductular proliferation and progressive fibrosis in dipeptidyl-peptidase IV (DPPIV)(-) F344 rats was induced by common bile duct ligation (BDL). Purified DPPIV(+)/green fluorescent protein (GFP)(+) hepatocytes were infused without PH into the spleen of BDL rats and compared to rats without BDL. RESULTS Within one week, transplanted hepatocytes were detected in hepatic portal areas and at the periphery of expanding portal regions. DPPIV(+)/GFP(+) repopulating cell clusters of different sizes were observed in BDL rats but not untreated normal recipients. Surprisingly, some engrafted hepatocytes formed CK-19/claudin-7 expressing epithelial cells resembling cholangiocytes within repopulating clusters. In addition, substantial numbers of hepatocytes engrafted at the intrasplenic injection site assembled into multicellular groups. These also showed biliary "transdifferentiation" in the majority of intrasplenic injection sites of rats that received BDL but not in untreated recipients. PCR array analysis showed upregulation of osteopontin (SPP1). Cell culture studies demonstrated increased Itgβ4, HNF1β, HNF6, Sox-9, and CK-19 mRNA expression in hepatocytes incubated with osteopontin, suggesting that this secreted protein promotes dedifferentiation of hepatocytes. CONCLUSIONS Our studies show that biliary fibrosis stimulates liver repopulation by ectopically transplanted hepatocytes and also stimulates hepatocyte transition towards a biliary epithelial phenotype.
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Affiliation(s)
- Mladen I Yovchev
- Dept. of Pathology (Division of Experimental Pathology), University of Pittsburgh, Pittsburgh, PA, United States
| | - Joseph Locker
- Dept. of Pathology (Division of Experimental Pathology), University of Pittsburgh, Pittsburgh, PA, United States
| | - Michael Oertel
- Dept. of Pathology (Division of Experimental Pathology), University of Pittsburgh, Pittsburgh, PA, United States; McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, United States.
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21
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Nagamoto Y, Takayama K, Ohashi K, Okamoto R, Sakurai F, Tachibana M, Kawabata K, Mizuguchi H. Transplantation of a human iPSC-derived hepatocyte sheet increases survival in mice with acute liver failure. J Hepatol 2016; 64:1068-1075. [PMID: 26778754 DOI: 10.1016/j.jhep.2016.01.004] [Citation(s) in RCA: 110] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/16/2014] [Revised: 01/05/2016] [Accepted: 01/06/2016] [Indexed: 12/17/2022]
Abstract
BACKGROUND & AIMS Hepatocyte transplantation is one of the most attractive approaches for the treatment of patients with liver failure. Because human induced pluripotent stem cell-derived hepatocyte-like cells (iPS-HLCs) can be produced on a large scale and generated from a patient with liver failure, they are expected to be used for hepatocyte transplantation. However, when using conventional transplantation methods, i.e., intrasplenic or portal venous infusion, it is difficult to control the engraftment efficiency and avoid unexpected engraftment in other organs because the transplanted cells are delivered into blood circulation before their liver engraftment. METHODS In this study, to resolve these issues, we attempted to employ a cell sheet engineering technology for experimental hepatocyte transplantation. The human iPS-HLC sheets were attached onto the liver surfaces of mice with liver injury. RESULTS This method reduced unexpected engraftment in organs other than the liver compared to that by intrasplenic transplantation. Human albumin levels in the mice with human iPS-HLC sheets were significantly higher than those in the intrasplenically-transplanted mice, suggesting the high potential for cell engraftment of the sheet transplantation procedure. In addition, human iPS-HLC sheet transplantation successfully ameliorated lethal acute liver injury induced by the infusion of carbon tetrachloride (CCl4). Moreover, we found that the hepatocyte growth factor secreted from the human iPS-HLC sheet played an important role in rescuing of mice from acute hepatic failure. CONCLUSIONS Human iPS-HLC sheet transplantation would be a useful and reliable therapeutic approach for a patient with severe liver diseases.
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Affiliation(s)
- Yasuhito Nagamoto
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; Laboratory of Hepatocyte Regulation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
| | - Kazuo Takayama
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; Laboratory of Hepatocyte Regulation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; The Keihanshin Consortium for Fostering the Next Generation of Global Leaders in Research (K-CONNEX), Kyoto University, Kyoto 606-8302, Japan
| | - Kazuo Ohashi
- Laboratory of Drug Development and Science, Osaka University, Osaka 567-0085, Japan; iPS Cell-Based Projects on Cell Transplantation and Cell Dynamics, Osaka University, Osaka 567-0085, Japan
| | - Ryota Okamoto
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; Laboratory of Hepatocyte Regulation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
| | - Fuminori Sakurai
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; Laboratory of Regulatory Sciences for Oligonucleotide Therapeutics, Osaka University, Osaka 567-0085, Japan
| | - Masashi Tachibana
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
| | - Kenji Kawabata
- Laboratory of Stem Cell Regulation, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan
| | - Hiroyuki Mizuguchi
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; Laboratory of Hepatocyte Regulation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; iPS Cell-Based Research Project on Hepatic Toxicity and Metabolism, Clinical Drug Development Unit, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; Global Center for Medical Engineering and Informatics, Osaka University, Osaka 565-0871, Japan.
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22
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Yarygin KN, Lupatov AY, Kholodenko IV. Cell-based therapies of liver diseases: age-related challenges. Clin Interv Aging 2015; 10:1909-24. [PMID: 26664104 PMCID: PMC4671765 DOI: 10.2147/cia.s97926] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The scope of this review is to revise recent advances of the cell-based therapies of liver diseases with an emphasis on cell donor's and patient's age. Regenerative medicine with cell-based technologies as its integral part is focused on the structural and functional restoration of tissues impaired by sickness or aging. Unlike drug-based medicine directed primarily at alleviation of symptoms, regenerative medicine offers a more holistic approach to disease and senescence management aimed to achieve restoration of homeostasis. Hepatocyte transplantation and organ engineering are very probable forthcoming options of liver disease treatment in people of different ages and vigorous research and technological innovations in this area are in progress. Accordingly, availability of sufficient amounts of functional human hepatocytes is crucial. Direct isolation of autologous hepatocytes from liver biopsy is problematic due to related discomfort and difficulties with further expansion of cells, particularly those derived from aging people. Allogeneic primary human hepatocytes meeting quality standards are also in short supply. Alternatively, autologous hepatocytes can be produced by reprogramming of differentiated cells through the stage of induced pluripotent stem cells. In addition, fibroblasts and mesenchymal stromal cells can be directly induced to undergo advanced stage hepatogenic differentiation. Reprogramming of cells derived from elderly people is accompanied by the reversal of age-associated changes at the cellular level manifesting itself by telomere elongation and the U-turn of DNA methylation. Cell reprogramming can provide high quality rejuvenated hepatocytes for cell therapy and liver tissue engineering. Further technological advancements and establishment of national and global registries of induced pluripotent stem cell lines homozygous for HLA haplotypes can allow industry-style production of livers for immunosuppression-free transplantation.
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Affiliation(s)
| | - Alexei Y Lupatov
- Laboratory of Cell Biology, Institute of Biomedical Chemistry, Moscow, Russia
| | - Irina V Kholodenko
- Laboratory of Cell Biology, Institute of Biomedical Chemistry, Moscow, Russia
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23
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Landis CS, Zhou H, Liu L, Hetherington HP, Guha C. Liver regeneration and energetic changes in rats following hepatic radiation therapy and hepatocyte transplantation by ³¹P MRSI. Liver Int 2015; 35:1145-51. [PMID: 25775097 PMCID: PMC4363097 DOI: 10.1111/liv.12507] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/25/2013] [Accepted: 02/14/2014] [Indexed: 12/11/2022]
Abstract
BACKGROUND & AIMS Radiation-induced liver damage (RILD) is a poorly understood and potentially devastating complication of hepatic radiation therapy (RT) for liver cancers. Previous work has demonstrated that hepatocyte transplantation (HT) can ameliorate RILD in rats. We hypothesized that RT inhibits generation of cellular ATP and suppresses hepatic regeneration. METHODS To study the metabolic changes that occur in RILD with and without HT, (31)P MRSI data were acquired in rats treated with partial hepatectomy (PH) alone, PH with hepatic irradiation (PHRT) or PHRT with HT (PHRT+HT). RESULTS Both [γ -ATP] and ATP/Pi (31)P MRSI signal ratio initially decreased and subsequently returned to baseline levels within 2 weeks after PH, which is consistent with other published data. Persistently reduced [γ-ATP] and ATP/Pi (31)P MRSI signal ratio were observed in rats up to 20 weeks after PHRT. However, progressive increases in [γ -ATP] were observed over time in the group of rats receiving PHRT+HT. Normal [γ -ATP] was observed 20 weeks after PHRT+HT (vs. PH alone), although, ATP/Pi levels did not return to normal after PHRT +HT. Ex vivo histological studies were performed to confirm liver repopulation with transplanted hepatocytes and the amelioration of pathologic changes of RILD. CONCLUSIONS These findings suggest that (31)P MRSI can be used to monitor the progress of RILD and its amelioration using transplanted hepatocytes to simultaneously restore metabolic function while replacing host hepatocytes damaged by RT.
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Affiliation(s)
- Charles S. Landis
- Department of Radiation Oncology, Division of Gastroenterology and Hepatology, Department of Medicine, University of Washington, Seattle, WA
| | - Hongchao Zhou
- Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York
| | - Laibin Liu
- Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York
| | | | - Chandan Guha
- Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York
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24
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Forbes SJ, Gupta S, Dhawan A. Cell therapy for liver disease: From liver transplantation to cell factory. J Hepatol 2015; 62:S157-69. [PMID: 25920085 DOI: 10.1016/j.jhep.2015.02.040] [Citation(s) in RCA: 218] [Impact Index Per Article: 21.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/29/2014] [Revised: 02/20/2015] [Accepted: 02/27/2015] [Indexed: 02/08/2023]
Abstract
Work over several decades has laid solid foundations for the advancement of liver cell therapy. To date liver cell therapy in people has taken the form of hepatocyte transplantation for metabolic disorders with a hepatic basis, and for acute or chronic liver failure. Although clinical trials using various types of autologous cells have been implemented to promote liver regeneration or reduce liver fibrosis, clear evidence of therapeutic benefits have so far been lacking. Cell types that have shown efficacy in preclinical models include hepatocytes, liver sinusoidal endothelial cells, mesenchymal stem cells, endothelial progenitor cells, and macrophages. However, positive results in animal models have not always translated through to successful clinical therapies and more realistic preclinical models need to be developed. Studies defining the optimal repopulation by transplanted cells, including routes of cell transplantation, superior engraftment and proliferation of transplanted cells, as well as optimal immunosuppression regimens are required. Tissue engineering approaches to transplant cells in extrahepatic locations have also been proposed. The derivation of hepatocytes from pluripotent or reprogrammed cells raises hope that donor organ and cell shortages could be overcome in the future. Critical hurdles to be overcome include the production of hepatocytes from pluripotent cells with equal functional capacity to primary hepatocytes and long-term phenotypic stability in vivo.
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Affiliation(s)
- Stuart J Forbes
- MRC Centre for Regenerative Medicine, Scottish Centre for Regenerative Medicine, 5 Little France Drive, Edinburgh EH16 4UU, United Kingdom.
| | - Sanjeev Gupta
- Departments of Medicine and Pathology, Albert Einstein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Avenue, Ullmann Building, Room 625, Bronx, NY 10461, United States
| | - Anil Dhawan
- Paediatric Liver GI and Nutrition Center and NIHR/Wellcome Cell Therapy Unit, King's College Hospital at King's College, London SE59RS, United Kingdom
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Xiang D, Liu CC, Wang MJ, Li JX, Chen F, Yao H, Yu B, Lu L, Borjigin U, Chen YX, Zhong L, Wangensteen KJ, He ZY, Wang X, Hu YP. Non-viral FoxM1 gene delivery to hepatocytes enhances liver repopulation. Cell Death Dis 2014; 5:e1252. [PMID: 24853430 PMCID: PMC4047909 DOI: 10.1038/cddis.2014.230] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2014] [Revised: 04/03/2014] [Accepted: 04/14/2014] [Indexed: 01/08/2023]
Abstract
Hepatocyte transplantation as a substitute strategy of orthotopic liver transplantation is being studied for treating end-stage liver diseases. Several technical hurdles must be overcome in order to achieve the therapeutic liver repopulation, such as the problem of insufficient expansion of the transplanted hepatocytes in recipient livers. In this study, we analyzed the application of FoxM1, a cell-cycle regulator, to enhance the proliferation capacity of hepatocytes. The non-viral sleeping beauty (SB) transposon vector carrying FoxM1 gene was constructed for delivering FoxM1 into the hepatocytes. The proliferation capacities of hepatocytes with FoxM1 expression were examined both in vivo and in vitro. Results indicated that the hepatocytes with FoxM1 expression had a higher proliferation rate than wild-type (WT) hepatocytes in vitro. In comparison with WT hepatocytes, the hepatocytes with FoxM1 expression had an enhanced level of liver repopulation in the recipient livers at both sub-acute injury (fumaryl acetoacetate hydrolase (Fah)–/– mice model) and acute injury (2/3 partial hepatectomy mice model). Importantly, there was no increased risk of tumorigenicity with FoxM1 expression in recipients even after serial transplantation. In conclusion, expression of FoxM1 in hepatocytes enhanced the capacity of liver repopulation without inducing tumorigenesis. FoxM1 gene delivered by non-viral SB vector into hepatocytes may be a viable approach to promote therapeutic repopulation after hepatocyte transplantation.
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Affiliation(s)
- D Xiang
- 1] Department of Cell Biology, Second Military Medical University, Shanghai, China [2] Center for Stem Cell and Medicine, The Graduate School, Second Military Medical University, Shanghai, China
| | - C-C Liu
- 1] Department of Cell Biology, Second Military Medical University, Shanghai, China [2] Center for Stem Cell and Medicine, The Graduate School, Second Military Medical University, Shanghai, China
| | - M-J Wang
- 1] Department of Cell Biology, Second Military Medical University, Shanghai, China [2] Center for Stem Cell and Medicine, The Graduate School, Second Military Medical University, Shanghai, China
| | - J-X Li
- 1] Department of Cell Biology, Second Military Medical University, Shanghai, China [2] Center for Stem Cell and Medicine, The Graduate School, Second Military Medical University, Shanghai, China
| | - F Chen
- 1] Department of Cell Biology, Second Military Medical University, Shanghai, China [2] Center for Stem Cell and Medicine, The Graduate School, Second Military Medical University, Shanghai, China
| | - H Yao
- 1] Department of Cell Biology, Second Military Medical University, Shanghai, China [2] Center for Stem Cell and Medicine, The Graduate School, Second Military Medical University, Shanghai, China
| | - B Yu
- 1] Department of Cell Biology, Second Military Medical University, Shanghai, China [2] Center for Stem Cell and Medicine, The Graduate School, Second Military Medical University, Shanghai, China
| | - L Lu
- 1] Department of Cell Biology, Second Military Medical University, Shanghai, China [2] Center for Stem Cell and Medicine, The Graduate School, Second Military Medical University, Shanghai, China
| | - U Borjigin
- The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Huhhot, China
| | - Y-X Chen
- 1] Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA [2] Department of Medicine, University of Minnesota, Minneapolis, MN, USA
| | - L Zhong
- 1] School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China [2] Shenzhen Center for ADR Monitoring, Shenzhen, China
| | - K J Wangensteen
- Division of Gastroenterology, Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Z-Y He
- 1] Department of Cell Biology, Second Military Medical University, Shanghai, China [2] Center for Stem Cell and Medicine, The Graduate School, Second Military Medical University, Shanghai, China
| | - X Wang
- 1] The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Huhhot, China [2] Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA [3] Hepatoscience Incorporation, Palo Alto, CA, USA
| | - Y-P Hu
- 1] Department of Cell Biology, Second Military Medical University, Shanghai, China [2] Center for Stem Cell and Medicine, The Graduate School, Second Military Medical University, Shanghai, China
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The generation of hepatocytes from mesenchymal stem cells and engraftment into the liver. Curr Opin Organ Transplant 2013; 16:69-75. [PMID: 21150616 DOI: 10.1097/mot.0b013e3283424f5b] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
PURPOSE OF REVIEW Liver transplantation is the ultimate therapeutic option for the treatment of end-stage liver diseases, which, however, is restricted by the shortage of donor organs. Instead hepatocyte transplantation seemed to be a way out, but again marginal donor livers for the isolation of primary human hepatocytes are scarce. The hepatocyte differentiation capacity of mesenchymal stem cells might open a new cell resource to generate hepatocyte-like cells for therapeutical use. RECENT FINDINGS Apart from their potency of hepatocyte differentiation mesenchymal stem cells display pleiotropic biological features including modulation of immunogenicity, anti-inflammatory and anti-apoptotic as well as pro-proliferative impact at the site of tissue or organ lesions. They are mobilized from the bone marrow and migrate to the liver along chemoattractive gradients thus contributing to the humoral and cellular response in tissue repair. The cause of different liver diseases is varying depending on, for example, viral, toxic, nutritional, neoplastic challenges. As known from animal studies mesenchymal stem cells seem to have a beneficial impact on liver regeneration and tissue repair under a variety of liver disease conditions. SUMMARY Their versatile biological features render mesenchymal stem cells an alternate cell resource for the treatment of liver diseases. It is important to know the mechanisms of integration of transplanted cells into the recipient tissue and to understand the communication between donor cells and the host tissue on the molecular level in order to support efficacy of cell transplantation and thus optimize the therapeutical outcome.
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Shao X, Qian Y, Xu C, Hong B, Xu W, Shen L, Jin C, Wu Z, Tong X, Yao H. The protective effect of intrasplenic transplantation of Ad-IL-18BP/IL-4 gene-modified fetal hepatocytes on ConA-induced hepatitis in mice. PLoS One 2013; 8:e58836. [PMID: 23516562 PMCID: PMC3596329 DOI: 10.1371/journal.pone.0058836] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2012] [Accepted: 02/07/2013] [Indexed: 12/19/2022] Open
Abstract
BACKGROUND Concanavalin A (ConA)-induced hepatitis is an experimental murine model mirroring the pathology of human autoimmune hepatitis. AIM To investigate the effects of intrasplenically transplanted fetal hepatocytes (BNL.CL2) transfected with recombinant adenovirus vector expressing the IL-18 binding protein (IL-18BP) and IL-4 fusion protein on ConA-induced hepatitis in mice. METHODS Ad-IL-18BP/IL-4 was used to infect BNL.CL2 cells. IL-4 and IL-18BP fusion protein expression were detected by ELISA and Western blotting. BNL.CL2 cells infected with Ad-IL-18BP/IL-4 were intrasplenically transplanted into mice. After 10 days, mice were injected with ConA (15 mg/kg), and sacrificed 18 hours later. Liver injury was assessed by serum transaminase and liver histology. TNF-α, IL-18, IL-4, IL-10, IL-12p70 and monocyte-chemoattracting protein (MCP)-1 levels in serum and liver homogenates were detected by ELISA. Signaling molecules in liver homogenates were analyzed by Western blotting. RESULTS Ad-IL-18BP/IL-4 effectively expressed the IL-18BP/IL-4 fusion protein for more than 14 days in BNL.CL12 cells. Treatment of mice with Ad-IL-18BP/IL-4-BNL.CL2 before ConA injection significantly reduced the elevated plasma levels of transaminases compared with ConA control groups. TNF-α, IL-18, IL-12p70 and MCP-1 levels in serum and liver homogenates from mice transplanted with Ad-IL-18BP/IL-4-BNL.CL2 were lower and IL-4 and IL-10 levels were higher than control groups. Phosphorylation levels of NF-κB p65, AKT, p38 and JNK1/2 in liver homogenates were markedly suppressed by Ad-IL-18BP/IL-4. CONCLUSIONS Ad-IL-18BP/IL-4 was effectively transfected into mouse BNL.CL2 cells. Intrasplenic transplantation of Ad-IL-18BP/IL-4-BNL.CL12 cells alleviated the severity of inflammation in ConA-induced experimental hepatitis and provides a useful basis for the targeted gene therapy of liver disease.
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Affiliation(s)
- Xueting Shao
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Yun Qian
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Chenhuai Xu
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Bo Hong
- Department of Pathology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Wanhong Xu
- Hangzhou High Throughput Drug Screening Center, ACEA Bio, Hangzhou, Zhejiang, China
| | - Ling Shen
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Changzhong Jin
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Zhigang Wu
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Xiangmin Tong
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Hangping Yao
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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Wan Z, Zhang XG, Liu ZW, Lv Y. Therapeutic liver repopulation for metabolic liver diseases: Advances from bench to bedside. Hepatol Res 2013; 43:122-130. [PMID: 22971121 DOI: 10.1111/j.1872-034x.2012.01081.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/23/2012] [Revised: 07/20/2012] [Accepted: 07/23/2012] [Indexed: 12/14/2022]
Abstract
Metabolic liver diseases are characterized by inherited defects in hepatic enzymes or other proteins with metabolic functions. Therapeutic liver repopulation (TLR), an approach of massive liver replacement by transplanted normal hepatocytes, could be used to provide the missing metabolic function elegantly. However, partial and transient correction of the underlying metabolic defects due to very few integrated donor cell mass remains the major obstacle for the effective and widespread use of this approach. Little engraftment and proliferation insufficiency lead to the poor outcome. This article reviews the advances in the mechanisms of initial engraftment and selective proliferation and suggests some effective treatment strategies, from pharmacological preconditioning to stem cell transplantation, to optimize liver repopulation with liver cell transplantation. Enhancing cell viability and plating efficiency, increasing sinusoidal spaces, regulation of sinusoidal endothelial cell barrier and controlling inflammatory reaction may promote initial cell engraftment. Liver-directed irradiation, reversible portal vein embolization and fetal liver stem/progenitor cell transplantation induce preferential proliferation of donor cells substantially without severe side-effects. Furthermore, it seems better to use combined approaches to achieve a high level of liver repopulation for the management of metabolic liver diseases.
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Affiliation(s)
- Zhen Wan
- Hepatobiliary Surgery; Institute of Advanced Surgical Techniques and Tissue Engineering Research, Xi'an Jiaotong University, Xi'an, China
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29
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Abstract
The liver has an enormous potential to restore the parenchymal tissue loss due to injury. This is accomplished by the proliferation of either the hepatocytes or liver progenitor cells in cases where massive damage prohibits hepatocytes from entering the proliferative response. Under debate is still whether hepatic stem cells are involved in liver tissue maintenance and regeneration or even whether they exist at all. The definition of an adult tissue-resident stem cell comprises basic functional stem cell criteria like the potential of self-renewal, multipotent, i.e. at least bipotent differentiation capacity and serial transplantability featuring the ability of functional tissue repopulation. The relationship between a progenitor and its progeny should exemplify the lineage commitment from the putative stem cell to the differentiated cell. This is mainly assessed by lineage tracing and immunohistochemical identification of markers specific to progenitors and their descendants. Flow cytometry approaches revealed that the liver stem cell population in animals is likely to be heterogeneous giving rise to progeny with different molecular signatures, depending on the stimulus to activate the putative stem cell compartment. The stem cell criteria are met by a variety of cells identified in the fetal and adult liver both under normal and injury conditions. It is the purpose of this review to verify hepatic stem cell candidates in the light of the stem cell definition criteria mentioned. Also from this point of view adult stem cells from non-hepatic tissues such as bone marrow, umbilical cord blood or adipose tissue, have the potential to differentiate into cells featuring functional hepatocyte characteristics. This has great impact because it opens the possibility of generating hepatocyte-like cells from adult stem cells in a sufficient amount and quality for their therapeutical application to treat end-stage liver diseases by stem cell-based hepatocytes in place of whole organ transplantation.
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Affiliation(s)
- Bruno Christ
- Translational Centre for Regenerative Medicine-TRM, University of Leipzig, Philipp-Rosenthal-Straße 55, D-04103 Leipzig, Germany.
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Yu YD, Kim KH, Lee SG, Choi SY, Kim YC, Byun KS, Cha IH, Park KY, Cho CH, Choi DH. Hepatic differentiation from human embryonic stem cells using stromal cells. J Surg Res 2011; 170:e253-61. [PMID: 21816427 DOI: 10.1016/j.jss.2011.06.032] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2011] [Revised: 06/13/2011] [Accepted: 06/15/2011] [Indexed: 10/18/2022]
Abstract
OBJECTIVE The derivation of hepatocytes from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report the generation of hepatocyte-like cells derived from hES cells. METHODS Hepatic endoderm cells were generated by adding activin A for 5 d- to 1-d-old embryoid bodies formed from hES cells. The hepatic endoderm cells were cocultured with mitomycin treated 3T3-J2 feeder cells. RESULTS After co-culture with mitomycin treated 3T3-J2 feeder cells, these hepatic endodermal cells yielded hepatocyte-like cell colonies, which possessed the proliferation potential to be cultured for an extended period of more than 30 d. With extensive expansion, they co-expressed the hepatic marker AFP and albumin, indicating that they were hepatocyte-like cells. CONCLUSIONS We report the generation of proliferative hepatocyte-like cells from hES cells. These hES cell derived hepatic cells can effectively be used as in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.
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Affiliation(s)
- Young-Dong Yu
- Division of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
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Török E, Lutgehetmann M, Bierwolf J, Melbeck S, Düllmann J, Nashan B, Ma PX, Pollok JM. Primary human hepatocytes on biodegradable poly(l-lactic acid) matrices: a promising model for improving transplantation efficiency with tissue engineering. Liver Transpl 2011; 17:104-14. [PMID: 21280182 DOI: 10.1002/lt.22200] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Liver transplantation is an established treatment for acute and chronic liver disease. However, because of the shortage of donor organs, it does not fulfill the needs of all patients. Hepatocyte transplantation is promising as an alternative method for the treatment of end-stage liver disease and as bridging therapy until liver transplantation. Our group has been working on the optimization of matrix-based hepatocyte transplantation. In order to increase cell survival after transplantation, freshly isolated human hepatocytes were seeded onto biodegradable poly(l-lactic acid) (PLLA) polymer scaffolds and were cultured in a flow bioreactor. PLLA discs were seeded with human hepatocytes and exposed to a recirculated medium flow for 6 days. Human hepatocytes formed spheroidal aggregates with a liver-like morphology and active metabolic function. Phase contrast microscopy showed increasing numbers of spheroids of increasing diameter during the culture period. Hematoxylin and eosin histology showed viable and intact hepatocytes inside the spheroids. Immunohistochemistry confirmed sustained hepatocyte function and a preserved hepatocyte-specific cytoskeleton. Albumin, alpha-1-antitrypsin, and urea assays showed continued production during the culture period. Northern blot analysis demonstrated increasing albumin signals. Scanning electron micrographs showed hepatocyte spheroids with relatively smooth undulating surfaces and numerous microvilli. Transmission electron micrographs revealed intact hepatocytes and junctional complexes with coated pits and vesicles inside the spheroids. Therefore, we conclude that primary human hepatocytes, precultured in a flow bioreactor on a PLLA scaffold, reorganize to form morphologically intact liver neotissue, and this might offer an optimized method for hepatocyte transplantation because of the expected reduction of the initial cell loss, the high regenerative potential in vivo, and the preformed functional integrity.
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Affiliation(s)
- Eva Török
- Departments of Hepatobiliary and Transplant Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
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Inagaki M, Furukawa H, Satake Y, Okada Y, Chiba S, Nishikawa Y, Ogawa K. Replacement of liver parenchyma in analbuminemic rats with allogenic hepatocytes is facilitated by intrabone marrow-bone marrow transplantation. Cell Transplant 2010; 20:1479-89. [PMID: 21176395 DOI: 10.3727/096368910x547453] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Although hepatocyte transplantation (HCTx) is expected to become a useful therapy for human liver diseases, allogenic hepatocytes still tend to be rejected within a short period due to host immunosurveillance. In the present study, we investigated the effect of prior bone marrow transplantation (BMTx) for the engraftment of allogenic hepatocytes using the analbuminemic rat transplantation model. The hepatocytes of Lewis (LEW) rats were not accepted in the liver of retrorsine (RS)/partial hepatectomy (PH)-treated analbuminemic F344 (F344-alb) rats, which express the disparate major histocompatibility complex (MHC) against that of LEW rats. Prior BMTx with the LEW bone marrow cells (BMCs) after sublethal irradiation achieved acceptance and repopulation of LEW hepatocytes in the liver of the RS/PH-treated F344-alb rats, associated with elevation of serum albumin. The replacement of hepatic parenchyma with albumin positive (Alb(+)) donor hepatocytes and elevation of serum albumin levels were dependent on the bone marrow reconstitution by donor BMCs, which was more efficiently achieved by intrabone marrow (IBM)-BMTx than by intravenous (IV)-BMTx. Our results demonstrate that efficient bone marrow reconstitution by IBM-BMTx enables the replacement of the hepatic parenchyma with allogenic hepatocytes in RS/PH-treated analbuminemic rats without immunosuppressants.
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Affiliation(s)
- Mitsuhiro Inagaki
- Department of Surgery, Asahikawa Medical University, Asahikawa, Japan.
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Roll GR, Willenbring H. Transplanted nonviable human hepatocytes produce appreciable serum albumin levels in mice. Stem Cell Res 2010; 5:267-70. [PMID: 20829142 DOI: 10.1016/j.scr.2010.07.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/27/2010] [Revised: 07/24/2010] [Accepted: 07/28/2010] [Indexed: 10/19/2022] Open
Abstract
In animal models of liver cell therapy serum human albumin levels are universally measured as a marker for the engraftment and function of transplanted human hepatocytes, or hepatocyte-like cells derived from human stem cells. However, even the most efficient cell transplantation protocols encounter a significant amount of graft cell death. If albumin released from dying cells could be detected for prolonged periods of time in the serum of the recipient, it may misleadingly suggest engraftment and function of the transplanted cells. While a half-life of approximately 20 days of human albumin in humans is established, the duration of its detectability in mice is unknown. Here we show that human albumin is readily detectable in the serum of mice injected with nonviable human hepatocytes. Human albumin levels peak 24h after injection of hepatocyte debris, and remain detectable at significant levels for at least 8 days. Our finding suggests that long-term, or in situ, analyses are needed to prove functional engraftment of human primary or stem cell-derived hepatocytes.
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Affiliation(s)
- Garrett R Roll
- Department of Surgery, Division of Transplantation, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA.
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Wu YM, Kao CY, Huang YJ, Yu IS, Lee HS, Lai HS, Lee PH, Lin CN, Lin SW. Genetic modification of donor hepatocytes improves therapeutic efficacy for hemophilia B in mice. Cell Transplant 2010; 19:1169-80. [PMID: 20412633 DOI: 10.3727/096368910x503398] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Hepatocyte transplantation (Tx) holds promise for curing genetic liver diseases. However, a limited number of donor hepatocytes can be transplanted into the host liver. Recipient preconditioning and donor cell engineering are under investigation to improve cell engraftment. In theory, genetically engineered cells secreting therapeutic proteins with superior function could compensate for poor engraftment efficiency. We have generated a bioengineered human coagulation factor IX (FIX) with augmented specific activity (named FIX-Triple). The aim of this study was to evaluate therapeutic efficacy of cell therapy using hemophilia B (HB) as a disease model by transplanting FIX-Triple-secreting hepatocytes. The donor hepatocytes were isolated from FIX-Triple knock-in (KI) or FIX-WT (wild-type) KI mice and transplanted intrasplenically into FIX knock-out (KO) mice. FIX-Triple KI recipients exhibited fourfold higher plasma FIX clotting activity than FIX-WT KI recipients. By repeated Txs, the clotting activity of FIX-Triple KI recipients even increased to more than 10% of normal mouse plasma. The engraftment and FIX production efficiencies of transplanted cells were equivalent between the FIX-WT KI and FIX-Triple KI donors. A hemostatic function assay showed that FIX-Triple KI recipients with repeated Txs had more enhanced clot kinetics and a greater maximum rate of thrombus generation than those with a single Tx. Moreover, FIX inhibitors in these recipients rarely developed. In conclusion, hepatocyte Tx with genetically engineered donor cells is an effective therapeutic strategy for HB.
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Affiliation(s)
- Yao-Ming Wu
- Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
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Abstract
Hepatocyte transplantation has therapeutic potential for multiple hepatic and extrahepatic disorders with genetic or acquired basis. To demonstrate whether cell populations of interest will be effective for clinical applications, it is first necessary to characterize their properties in animal systems. Demonstrating the potential of cells to engraft and proliferate is a critical part of this characterization. Similarly, for stem/progenitor cells, demonstrating the capacity to differentiate along appropriate lineages and generate mature cells that can engraft and proliferate is essential. In various animal models, preconditioning of recipients prior to cell transplantation has been necessary to improve engraftment of cells, to stimulate proliferation of engrafted cells, and to induce extensive repopulation of the host liver by transplanted cells. Although this is an area of active investigation, effective preconditioning protocols should alter the hepatic microenvironment, such that transplanted cells can obtain selective advantages for engrafting and proliferating in the liver. Use of such experimental systems in animals will help generate further strategies for liver repopulation and thereby advance clinical applications of liver cell therapy.
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Affiliation(s)
- Yao-Ming Wu
- Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan
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36
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Yin Z, Ellis ECS, Nowak G. Isolation of mouse hepatocytes for transplantation: a comparison between antegrade and retrograde liver perfusion. Cell Transplant 2008; 16:859-65. [PMID: 18088005 DOI: 10.3727/000000007783465235] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
We compared antegrade with retrograde liver perfusion when isolating mouse hepatocytes for hepatocyte transplantation. Male mouse hepatocytes were isolated by different perfusion methods and transplanted into the spleen of congeneic female mice. Retrograde perfusion yielded a larger number of cells (4.90 x 10(7)) than antegrade (4.09 x 10(7), p < 0.05), but hepatocytes obtained by antegrade perfusion gave higher engraftment efficiency (p < 0.05). More of the transplanted hepatocytes could be recovered from recipient liver with antegrade perfusion than with retrograde perfusion (p < 0.05). Our results indicate that hepatocytes isolated by antegrade perfusion gave a higher engraftment efficiency.
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Affiliation(s)
- Zhaohui Yin
- Division of Transplantation Surgery, Department for Clinical Science, Intervention and Technology CLINTEC, Karolinska University Hospital, Stockholm, Sweden
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37
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Katchman H, Tal O, Eventov-Friedman S, Shezen E, Aronovich A, Tchorsh D, Cohen S, Shtabsky A, Hecht G, Dekel B, Freud E, Reisner Y. Embryonic porcine liver as a source for transplantation: advantage of intact liver implants over isolated hepatoblasts in overcoming homeostatic inhibition by the quiescent host liver. Stem Cells 2008; 26:1347-55. [PMID: 18339772 DOI: 10.1634/stemcells.2007-0631] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Cell therapy as an alternative to orthotopic liver transplantation represents a major challenge, since negligible proliferation of isolated hepatocytes occurs after transplantation because of the stringent homeostatic control displayed by the host liver. Thus, different modalities of liver injury as part of the pretransplant conditioning are a prerequisite for this approach. The major objective of the present study was to test whether xenotransplantation of pig fetal liver fragments, in which potential cell-cell and cell-stroma interactions are spared, might afford more robust growth and proliferation compared with isolated pig fetal hepatoblasts. After transplantation into SCID mice, fetal liver tissue fragments exhibited marked growth and proliferation, in the setting of a quiescent host liver, compared with isolated fetal hepatoblasts harvested at the same gestational age (embryonic day 28). The proliferative advantage of fetal pig liver fragments was clearly demonstrated by immunohistochemical and morphometric assays and was observed not only after implantation into the liver but also into extrahepatic sites, such as the spleen and the subrenal capsule. The presence of all types of nonparenchymal liver cells that is crucial for normal liver development and regeneration was demonstrated in the implants. Preservation of the three-dimensional structure in pig fetal liver fragments enables autonomous proliferation of transplanted hepatic cells in the setting of a quiescent host liver, without any requirement for liver injury in the pretransplant conditioning. The marked proliferation and functional maturation exhibited by the pig fetal liver fragments suggests that it could afford a preferable source for transplantation.
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Affiliation(s)
- Helena Katchman
- Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
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Ju S, Teng GJ, Lu H, Zhang Y, Zhang A, Chen F, Ni Y. In vivo MR tracking of mesenchymal stem cells in rat liver after intrasplenic transplantation. Radiology 2007; 245:206-15. [PMID: 17717324 DOI: 10.1148/radiol.2443061290] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
PURPOSE To prospectively track in vivo in rats intrasplenically transplanted stem cells labeled with superparamagnetic particles by using magnetic resonance (MR) imaging. MATERIALS AND METHODS The study was approved by the institutional Committee on Animal Research. Liver damage in 12 rats was induced with subcutaneous injection of carbon tetrachloride (CCl4). Intrasplenic transplantation of 6x10(6) rodent bone mesenchymal stem cells (BMSCs) with (n=6) and without (n=6) superparamagnetic particle Fe2O3-poly-L-lysine (PLL) labeling was performed via direct puncture. Cell labeling efficiency was assessed in vitro by using Prussian blue stain and an atomic absorption spectrometer. MR examinations were performed immediately before and 3 hours and 3, 7, and 14 days after transplantation. Liver-to-muscle contrast-to-noise ratios (CNRs) on T2*-weighted MR images obtained before and after injection were measured and correlated with histomorphologic studies. Statistical analyses were performed by using repeated-measures analysis of variance. RESULTS Rat BMSCs could be effectively labeled with approximately 100% efficiency. Migration of transplanted labeled cells to the liver was successfully documented with in vivo MR imaging. CNRs on T2*-weighted images decreased significantly in the liver 3 hours after injection of BMSCs (P<.05) and returned gradually to the level achieved without labeled cell injection in 14 days. Histologic analyses confirmed the presence of BMSCs in the liver. The labeled cells primarily localized in the sinusoids of periportal areas and the foci of CCl4-induced liver damage. Quantitative analysis of Prussian blue-stained cells indicated gradual decrease of dye pigments from 3 hours to 3, 7, and 14 days after injection. No free iron particles were found in the interstitium or within hepatic microvessels. CONCLUSION The rat BMSCs could be efficiently labeled with Fe2O3-PLL and the relocation of the labeled cells to rat livers after intrasplenic transplantation could be depicted at in vivo MR imaging.
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Affiliation(s)
- Shenghong Ju
- Laboratory of Molecular Imaging, Department of Radiology, Zhongda Hospital, Laboratory of Molecular and Biomolecular Electronics, and School of Basic Medical Science, Southeast University, 87 Ding Jia Qiao Road, Nanjing 210009, China
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39
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Sgodda M, Aurich H, Kleist S, Aurich I, König S, Dollinger MM, Fleig WE, Christ B. Hepatocyte differentiation of mesenchymal stem cells from rat peritoneal adipose tissue in vitro and in vivo. Exp Cell Res 2007; 313:2875-86. [PMID: 17574236 DOI: 10.1016/j.yexcr.2007.05.020] [Citation(s) in RCA: 140] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2007] [Revised: 04/13/2007] [Accepted: 05/15/2007] [Indexed: 02/08/2023]
Abstract
Mesenchymal tissues harbour stromal cells capable of multilineage differentiation. Here, we demonstrate the isolation of mesenchymal stem cells (MSC) from rat peritoneal adipose tissue capable of osteogenic and adipogenic differentiation. Under in vitro conditions favouring hepatocyte differentiation, these MSC gained characteristic functions of hepatocytes such as the capacity to synthesize urea or store glycogen. Hepatocyte-specific transcripts of dipeptidylpeptidase type IV (CD26), albumin, cytochrome P450 type 1A1 (CYP1A1) and connexin CX32 (CX32) were detected only in differentiated but not undifferentiated cells. Transient transgenic expression of luciferase could be stimulated by cAMP when driven by the hepatocyte-specific promoter of the cytosolic phosphoenolpyruvate carboxykinase (PCK1) gene. Finally, stem cell-derived hepatocytes from wild type (CD26+/+) rats were transplanted into the livers of CD26-deficient animals after lentiviral transduction with the GFP gene under the control of the ubiquitin promoter. GFP-positive cells engrafted in the host liver predominantly in the periportal region of the liver lobule. They continued to express CD26, a prominent feature of differentiated hepatocytes, indicating their topologically and functionally proper integration into the host liver parenchyma. Thus, MSCs from rat peritoneal adipose tissue exhibit the potential to differentiate into hepatocyte-like cells in vitro and in vivo.
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Affiliation(s)
- Malte Sgodda
- First Department of Medicine, Martin-Luther University of Halle-Wittenberg, Heinrich-Damerow-Strasse 1, Halle/Saale, Germany
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40
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Min SH, Lee DC, Lim MJ, Park HS, Kim DM, Cho CW, Yoon DY, Yeom YI. A composite gene delivery system consisting of polyethylenimine and an amphipathic peptide KALA. J Gene Med 2007; 8:1425-34. [PMID: 17009340 DOI: 10.1002/jgm.973] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
BACKGROUND Animal viruses such as enveloped virus carry multi-functional proteins in the virion that can mediate more than two distinct steps of a gene delivery process during the transfer of viral genome into host cells. We tested if the aspects of the viral gene delivery mechanism could be mimicked by forming composite formulae from multi-functional synthetic gene carriers having complementary action modes. METHODS Polyethylenimine (PEI) was chosen as the component responsible for endosome escape and DNA condensation and KALA for cellular entry and DNA condensation. Compact DNA-carrier particles consisting of the core part where DNA chains were tightly condensed by PEI and the outer layer lined with KALA were formulated, characterized and compared with monolithic cationic formulae in terms of gene delivery efficiency and mechanism. RESULTS High-level gene expression was observed when C2C12 cells were transfected with DNA that was first partially condensed with PEI and, then, fully with KALA. In these formulae KALA mediated enhanced cellular entry of DNA by facilitating endocytic vesicle formation, while PEI provided an effective endosomolytic capacity. An optimal PEI/KALA formula showed transfection efficiencies better than or comparable to the commercial cationic liposome in various cell types in culture and in vivo. CONCLUSIONS Gene delivery by combining the membrane-active property of KALA with the endosomolytic activity of PEI can be more efficient than that by either of the properties alone. It appears that, in these formulae, the predominant role of KALA is to facilitate cellular entry of DNA by providing a fusogenic capability, rather than an endosomolytic activity.
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Affiliation(s)
- Sang-Hyun Min
- Laboratory of Human Genomics, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong, Yusong-gu, Taejon 305-333, Korea
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41
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Pasciu D, Montisci S, Greco M, Doratiotto S, Pitzalis S, Pani P, Laconi S, Laconi E. Aging is associated with increased clonogenic potential in rat liver in vivo. Aging Cell 2006; 5:373-7. [PMID: 16911563 DOI: 10.1111/j.1474-9726.2006.00230.x] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Cancer increases with age and often arises from the selective clonal growth of altered cells. Thus, any environment favoring clonal growth per se poses a higher risk for cancer development. Using a genetically tagged animal model, we investigated whether aging is associated with increased clonogenic potential. Groups of 4-, 12-, 18-, and 24-month-old Fischer 344 rats were infused (via the portal vein) with 2x10(6) hepatocytes isolated from a normal syngenic 2-month-old donor. Animals deficient in dipeptidyl-peptidase type IV (DPP-IV-) enzyme were used as recipients, allowing for the histochemical detection of injected DPP-IV+ cells. Groups of animals were sacrificed at various times thereafter. No growth of DPP-IV+ transplanted hepatocytes was present after either 2 or 6 months in the liver of rats transplanted at young age, as expected. In striking contrast, significant expansion of donor-derived cells was seen in animals transplanted at the age of 18 months: clusters comprising 7-10 DPP-IV+ hepatocytes/cross-section were present after 2 months and were markedly enlarged after 6 months (mean of 88+/-35 cells/cluster/cross-section). These results indicate that the microenvironment of the aged liver supports the clonal expansion of transplanted normal hepatocytes. Such clonogenic environments can foster the selective growth of pre-existing altered cells, thereby increasing the overall risk for cancer development associated with aging.
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Affiliation(s)
- Daniela Pasciu
- Department of Biomedical Sciences and Biotechnology, Section of Experimental Pathology, University of Cagliari, Cagliari, Italy
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Okita K. [Liver regeneration with stem cell transplantation and its clinical application]. NIHON NAIKA GAKKAI ZASSHI. THE JOURNAL OF THE JAPANESE SOCIETY OF INTERNAL MEDICINE 2006; 95:1659-66. [PMID: 17037298 DOI: 10.2169/naika.95.1659] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/12/2023]
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Zender L, Xue W, Cordón-Cardo C, Hannon GJ, Lucito R, Powers S, Flemming P, Spector MS, Lowe SW. Generation and analysis of genetically defined liver carcinomas derived from bipotential liver progenitors. COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY 2006; 70:251-61. [PMID: 16869761 PMCID: PMC4595853 DOI: 10.1101/sqb.2005.70.059] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Hepatocellular carcinoma is a chemoresistant cancer and a leading cause of cancer mortality; however, the molecular mechanisms responsible for the aggressive nature of this disease are poorly understood. In this study, we developed a new liver cancer mouse model that is based on the ex vivo genetic manipulation of embryonic liver progenitor cells (hepatoblasts). After retroviral gene transfer of oncogenes or short hairpin RNAs targeting tumor suppressor genes, genetically altered liver progenitor cells are seeded into the liver of otherwise normal recipient mice. We show that histopathology of the engineered liver carcinomas reveals features of the human disease. Furthermore, representational oligonucleotide microarray analysis (ROMA) of murine liver tumors initiated by two defined genetic hits revealed spontaneously acquired genetic alterations that are characteristic for human hepatocellular carcinoma. This model provides a powerful platform for applications like cancer gene discovery or high-throughput preclinical drug testing.
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Affiliation(s)
- L Zender
- Cold Spring Harbor Laboratory, New York 11724, USA
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Strom SC, Bruzzone P, Cai H, Ellis E, Lehmann T, Mitamura K, Miki T. Hepatocyte transplantation: clinical experience and potential for future use. Cell Transplant 2006; 15 Suppl 1:S105-10. [PMID: 16826802 DOI: 10.3727/000000006783982395] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. There are three main areas where the transplantation of isolated hepatocytes has been proposed and used for clinical therapy. Cell transplantation has been used: 1) for temporary metabolic support of patients in end-stage liver failure awaiting whole organ transplantation, 2) as a method to support liver function and facilitate regeneration of the native liver in cases of fulminant hepatic failure, and 3) in a manner similar to gene therapy, as a "cellular therapy" for patients with genetic defects in vital liver functions. We will briefly review the basic research that leads to clinical hepatocyte transplantation, the published clinical experience with this experimental technique, and some possible future uses of hepatocyte transplantation.
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Affiliation(s)
- Stephen C Strom
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
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Stéphenne X, Najimi M, Sibille C, Nassogne MC, Smets F, Sokal EM. Sustained engraftment and tissue enzyme activity after liver cell transplantation for argininosuccinate lyase deficiency. Gastroenterology 2006; 130:1317-23. [PMID: 16618422 DOI: 10.1053/j.gastro.2006.01.008] [Citation(s) in RCA: 156] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/07/2005] [Accepted: 12/21/2005] [Indexed: 12/17/2022]
Abstract
BACKGROUND & AIMS Donor cell engraftment with expression of enzyme activity is the goal of liver cell transplantation for inborn errors of liver metabolism with a view to achieving sustained metabolic control. METHODS Sequential hepatic cell transplantations using male and female cells were performed in a 3.5-year-old girl with argininosuccinate lyase deficiency over a period of 5 months. Beside clinical, psychomotor, and metabolic follow-up, engraftment was analyzed in repeated liver biopsies (2.5, 5, 8, and 12 months after first infusion) by fluorescence in situ hybridization for the Y-chromosome and by measurement of tissue enzyme activity. RESULTS Metabolic control was achieved together with psychomotor catch-up, changing the clinical phenotype from a severe neonatal one to a moderate late-onset type. The child was no longer hospitalized and was able to attend normal school. Sustained engraftment of male donor liver cells was shown in repeated biopsies, reaching 19% at 8 months and 12.5% at the 12-month follow-up. XXYY tetraploid donor cells were mainly detected during the infusion period (2.5- and 5-month biopsies), whereas in the follow-up 8-month and 1-year biopsies, diploid donor cell subpopulations had become dominant. Moreover, argininosuccinate lyase activity, originally absent, became measurable in 2 different biopsy samples at 8 months, reaching 3% of control activity, indicating in situ metabolic effect and supporting the clinical evolution to a moderate form of the disease. CONCLUSIONS Liver cell transplantation can achieve donor cell engraftment in humans in a significant proportion, leading to sustained metabolic and clinical control with psychomotor catch-up.
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Affiliation(s)
- Xavier Stéphenne
- Laboratoire d'hépatologie Pédiatrique et Transplantation Cellulaire, Département GYPE, Service de Pédiatrie, Université Catholique de Louvain & Cliniques St Luc, Brussels, Belgium
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Gandillet A, Vidal I, Alexandre E, Audet M, Chenard-Neu MP, Stutzmann J, Heyd B, Jaeck D, Richert L. Experimental models of acute and chronic liver failure in nude mice to study hepatocyte transplantation. Cell Transplant 2005; 14:277-90. [PMID: 16052909 DOI: 10.3727/000000005783983061] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Although hepatocyte transplantation is a promising therapy for acute liver failure in human, there is still a lack of animal models suffering from hepatic injury in which the benefits of hepatocyte transplantation could be evaluated solely, without the bias caused by immunosuppression. As a consequence, the aim of the study was first to develop reproducible models of partial hepatectomy and of thioacetamide (TA)- or Jo2-induced acute liver failure in nude mice. Chronic liver disease was also investigated by repeated injections of sublethal doses of thioacetamide. Survival rates, routine histologic observations, alanin aminotransferase sera content, Ki67, and caspase 3 immunodetection were investigated both after 40% partial hepatectomy and after toxic-induced damages. Liver injuries were more severe and/or precocious in nude mice than in Balb/c mice for a given treatment with a maximum of acute injury obtained 24 h after single toxic injection, and were found to be transitory and reversible within 10 days. Toxics induced apoptosis followed by necrosis, confirming recent published data. Onset of fibrosis leading to reproducible chronic cirrhosis in nude mice correlated with increasing number of Ki67-positive cells, indicating that high levels of cell proliferation occurred. Chronic cirrhosis progressively reversed to fibrosis when the treatment ceased. Preliminary results demonstrated that engrafted xenogeneic hepatocytes could be detected in the host liver by anti-MHC class I immunohistochemistry. Fractions enriched in 2n or 4n hepatocytes by cell sorting using a flow cytometer were equivalent to the unpurified fraction in terms of engraftment in control nude mice or in nude mice subjected to PH. However, in mice suffering from liver injury 24 h after Jo2 or TA treatment, the engraftment of 2n hepatocytes was about twice that of an unpurified hepatocyte population or of a population enriched in 4n hepatocytes.
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Affiliation(s)
- Amaud Gandillet
- Laboratoire de Chirurgie Expérimentale, Fondation Transplantation, 67200 Strasbourg, France
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Guha C, Yamanouchi K, Jiang J, Wang X, Roy Chowdhury N, Santana A, Shapiro LJ, Salido E, Roy-Chowdhury J. Feasibility of hepatocyte transplantation-based therapies for primary hyperoxalurias. Am J Nephrol 2005; 25:161-70. [PMID: 15849463 DOI: 10.1159/000085408] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2005] [Accepted: 03/11/2005] [Indexed: 11/19/2022]
Abstract
Primary hyperoxalurias (PHs) are diseases caused by overproduction of oxalate by hepatocytes. Most patients with PHs develop nephrocalcinosis and renal failure. Combined liver-kidney transplantation is often used as a definitive treatment of PHs, but because of a large body oxalate load at the time of transplantation, the procedure is not always successful. Because all hepatocytes overproduce oxalate, partial liver replacement procedures, such as auxiliary transplantation of a liver lobe or hepatocyte transplantation are not expected to be useful in this disorder. In this paper we describe novel techniques, based on preparative hepatic irradiation and stimulation of hepatocyte mitosis, through loss of liver mass or administration of hepatic growth factor, which permit transplanted wild-type hepatocytes to massively repopulate the liver, replacing up to 90% of the hepatocytes in recipient mouse livers. Application of this procedure in a recently developed Agxt-gene-deleted mouse model of PH1 resulted in marked amelioration of hyperoxaluria. We propose that further refinement of the different components of this procedure may permit early cell-based therapies of PHs, thereby preventing renal failure and its complications.
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Affiliation(s)
- Chandan Guha
- Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York, NY, USA
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Gupta S, Inada M, Joseph B, Kumaran V, Benten D. Emerging insights into liver-directed cell therapy for genetic and acquired disorders. Transpl Immunol 2005; 12:289-302. [PMID: 15157922 DOI: 10.1016/j.trim.2003.12.014] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Treatment of acute or chronic liver diseases by cell transplantation is an attractive prospect because organ shortages greatly restrict liver transplantation. Moreover, a variety of genetic deficiency states affecting extrahepatic organs are amenable to liver-directed cell therapy. While the initial clinical experience with liver cell transplantation has been encouraging, further advances in several areas are necessary to improve these results. Insights into how engraftment and proliferation of transplanted cells may be modulated to obtain therapeutically effective masses of transplanted cells will be important in this pursuit. Studies of cell therapy in animal models of specific diseases have provided insights into the development of clinically relevant strategies for various disorders. Also, identification of suitable cell types, including stem/progenitor cells that could be expanded and manipulated in cell culture conditions, has begun to provide important new information for cell therapy. Similarly, advances in cryopreservation of cells and prevention of allograft rejection offer ways to accomplish cell therapy in an effective manner. Taken together, these advances indicate that liver-directed cell therapy will be well positioned in the near future to play significant roles in transplantation medicine.
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Affiliation(s)
- Sanjeev Gupta
- Department of Medicine, Marion Bessin Liver Research Center, Cancer Research Center and General Clinical Research Center, Albert Einstein College of Medicine Ullmann Building, Rm 625, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
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Abstract
Liver gene therapy is being developed as an alternative to orthotopic liver transplantation, which is the only effective therapy for many liver diseases. The liver has unique features that make it attractive for in vivo and ex vivo gene transfer. In vivo approach is far less invasive than ex vivo approach, although in most cases, host immune response directed against the transgene product and/or vector particles severely impairs the efficiency of gene transfer, and precludes long-term transgene expression after in vivo gene delivery. Ex vivo approach allows for an elective targeting of the hepatocytes, avoiding that the transgene be expressed in professional antigen-presenting, but is faced with the low in vitro proliferative ability of hepatocytes, and to the low in vivo liver repopulating ability of transplanted cells. In some specific situations where immune response was controlled or transplanted cells had a strong growth advantage over host hepatocytes, gene transfer resulted in long-term and complete correction of a liver genetic defect. In this review, we will outline the liver diseases that may benefit from gene therapy, the vector technology under investigation, the advances and the problems to be overcome.
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Affiliation(s)
- T H Nguyen
- Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland
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