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Huang C, Jiang H, Dong J, Jiang L, Li J, Xu J, Cui T, Wang L, Li X, Feng G, Zhang Y, Li T, Li W, Zhou Q. Functional mouse hepatocytes derived from interspecies chimeric livers effectively mitigate chronic liver fibrosis. Stem Cell Reports 2024; 19:877-889. [PMID: 38729156 PMCID: PMC11390683 DOI: 10.1016/j.stemcr.2024.04.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2024] [Revised: 04/14/2024] [Accepted: 04/15/2024] [Indexed: 05/12/2024] Open
Abstract
Liver disease is a major global health challenge. There is a shortage of liver donors worldwide, and hepatocyte transplantation (HT) may be an effective treatment to overcome this problem. However, the present approaches for generation of hepatocytes are associated with challenges, and interspecies chimera-derived hepatocytes produced by interspecies blastocyst complementation (IBC) may be promising donor hepatocytes because of their more comprehensive hepatic functions. In this study, we isolated mouse hepatocytes from mouse-rat chimeric livers using IBC and found that interspecies chimera-derived hepatocytes exhibited mature hepatic functions in terms of lipid accumulation, glycogen storage, and urea synthesis. Meanwhile, they were more similar to endogenous hepatocytes than hepatocytes derived in vitro. Interspecies chimera-derived hepatocytes could relieve chronic liver fibrosis and reside in the injured liver after transplantation. Our results suggest that interspecies chimera-derived hepatocytes are a potentially reliable source of hepatocytes and can be applied as a therapeutic approach for HT.
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Affiliation(s)
- Cheng Huang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Haiping Jiang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jingxi Dong
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Liyuan Jiang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Jie Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jing Xu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Tongtong Cui
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Leyun Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Xin Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Guihai Feng
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Ying Zhang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Tianda Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China.
| | - Wei Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China.
| | - Qi Zhou
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Organ Regeneration and Reconstruction, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China.
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2
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Yuri S, Murase Y, Isotani A. Generation of rat-derived lung epithelial cells in Fgfr2b-deficient mice retains species-specific development. Development 2024; 151:dev202081. [PMID: 38179792 DOI: 10.1242/dev.202081] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2023] [Accepted: 11/29/2023] [Indexed: 01/06/2024]
Abstract
Regenerative medicine is a tool to compensate for the shortage of lungs for transplantation, but it remains difficult to construct a lung in vitro due to the complex three-dimensional structures and multiple cell types required. A blastocyst complementation method using interspecies chimeric animals has been attracting attention as a way to create complex organs in animals, although successful lung formation using interspecies chimeric animals has not yet been achieved. Here, we applied a reverse-blastocyst complementation method to clarify the conditions required to form lungs in an Fgfr2b-deficient mouse model. We then successfully formed a rat-derived lung in the mouse model by applying a tetraploid-based organ-complementation method. Importantly, rat lung epithelial cells retained their developmental timing even in the mouse body. These findings provide useful insights to overcome the barrier of species-specific developmental timing to generate functional lungs in interspecies chimeras.
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Affiliation(s)
- Shunsuke Yuri
- Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
| | - Yuki Murase
- Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
| | - Ayako Isotani
- Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
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3
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Szpila M, Humięcka M, Bożyk K, Paterczyk B, Suwińska A, Maleszewski M, Tarkowski AK. Attempts to obtain fully xenogeneic fetuses in rat ↔ mouse model†,‡. Biol Reprod 2021; 102:499-510. [PMID: 31511860 DOI: 10.1093/biolre/ioz185] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2019] [Revised: 08/06/2019] [Accepted: 09/06/2019] [Indexed: 11/15/2022] Open
Abstract
The full-term development of the xenogeneic embryo in the uterus of the mother of different species is very restricted and can occur only in certain groups of closely related mammals. In the case of mouse ↔ rat chimeras, the interspecific uterine barrier is less hostile to interspecific chimeric fetuses. In current work, we tested the development of mouse and rat fetuses in uteri of females of the opposite species. We created chimeric mouse ↔ rat blastocysts by injection of mouse embryonic stem cells (ESCs) into eight-cell rat embryos and rat ESCs into eight-cell mouse embryos. Chimeras were transferred to the foster mothers of the opposite species. Despite a huge number of transferred embryos (>1000 in total for both variants), only one live fetus derived solely from the mouse ESCs was isolated at E13.5 from the rat uterus. All other fetuses and newborns were chimeric or were built only from the cells of the recipient embryo. We examined the possible reason for such an outcome and found that the xenogeneic fetuses are eliminated at the perigastrulation stage of development. Thus, we conclude that in the rat ↔ mouse combination even when extraembryonic tissues of the chimeric embryo are composed solely of the cells of the same species as the female to which embryos are transferred, the full-term development of the pure xenogeneic fetus is very unlikely.
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Affiliation(s)
- Marcin Szpila
- Department of Embryology, Institute of Zoology, Faculty of Biology, University of Warsaw, Warsaw, Poland
| | - Monika Humięcka
- Department of Embryology, Institute of Zoology, Faculty of Biology, University of Warsaw, Warsaw, Poland
| | - Katarzyna Bożyk
- Department of Embryology, Institute of Zoology, Faculty of Biology, University of Warsaw, Warsaw, Poland
| | - Bohdan Paterczyk
- Laboratory of Electron and Confocal Microscopy, Faculty of Biology, University of Warsaw, Warsaw, Poland
| | - Aneta Suwińska
- Department of Embryology, Institute of Zoology, Faculty of Biology, University of Warsaw, Warsaw, Poland
| | - Marek Maleszewski
- Department of Embryology, Institute of Zoology, Faculty of Biology, University of Warsaw, Warsaw, Poland
| | - Andrzej K Tarkowski
- Department of Embryology, Institute of Zoology, Faculty of Biology, University of Warsaw, Warsaw, Poland
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Stepien BK, Vaid S, Naumann R, Holtz A, Huttner WB. Generation of interspecies mouse-rat chimeric embryos by embryonic stem (ES) cell microinjection. STAR Protoc 2021; 2:100494. [PMID: 34189467 PMCID: PMC8219896 DOI: 10.1016/j.xpro.2021.100494] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Interspecies chimerism is a useful tool to study interactions between cells of different genetic makeup in order to elucidate the mechanisms underlying non-cell-autonomous processes, including evolutionary events. However, generating interspecies chimeras with high efficiency and chimerism level remains challenging. Here, we describe a protocol for generating chimeras between mouse and rat. Donor embryonic stem cells of one species are microinjected into early embryos of the other species (recipient), which are implanted into host foster mothers of the recipient species. For complete details on the use and execution of this protocol, please refer to Stepien et al. (2020).
Isolation of ES cells from mouse blastocysts and culture of rodent ES cells Microinjection of mouse ES cells into early rat embryos and vice versa Transfer of chimeric embryos into pseudo-pregnant foster mothers Generation of mouse-rat chimeric brains
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Affiliation(s)
- Barbara K Stepien
- Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.,Institute of Anatomy, Medical Faculty Carl-Gustav-Carus, Technische Universität Dresden, 01307 Dresden, Germany
| | - Samir Vaid
- Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
| | - Ronald Naumann
- Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
| | - Anja Holtz
- Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
| | - Wieland B Huttner
- Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
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Kitahara A, Ran Q, Oda K, Yasue A, Abe M, Ye X, Sasaoka T, Tsuchida M, Sakimura K, Ajioka Y, Saijo Y, Zhou Q. Generation of Lungs by Blastocyst Complementation in Apneumic Fgf10-Deficient Mice. Cell Rep 2021; 31:107626. [PMID: 32402288 DOI: 10.1016/j.celrep.2020.107626] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2020] [Revised: 04/09/2020] [Accepted: 04/18/2020] [Indexed: 01/13/2023] Open
Abstract
The shortage of donor lungs hinders lung transplantation, the only definitive option for patients with end-stage lung disease. Blastocyst complementation enables the generation of transplantable organs from pluripotent stem cells (PSCs) in animal models. Pancreases and kidneys have been generated from PSCs by blastocyst complementation in rodent models. Here, we report the generation of lungs using mouse embryonic stem cells (ESCs) in apneumic Fgf10 Ex1mut/Ex3mutmice by blastocyst complementation. Complementation with ESCs enables Fgf10-deficient mice to survive to adulthood without abnormalities. Both the generated lung alveolar parenchyma and the interstitial portions, including vascular endothelial cells, vascular and parabronchial smooth muscle cells, and connective tissue, largely originate from the injected ESCs. These data suggest that Fgf10 Ex1mut/Ex3mutblastocysts provide an organ niche for lung generation and that blastocyst complementation could be a viable approach for generating whole lungs.
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Affiliation(s)
- Akihiko Kitahara
- Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan; Division of Thoracic and Cardiovascular Surgery, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan
| | - Qingsong Ran
- Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan
| | - Kanako Oda
- Department of Comparative and Experimental Medicine, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8585, Japan
| | - Akihiro Yasue
- Department of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan
| | - Manabu Abe
- Department of Cellular Neurobiology, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8585, Japan
| | - Xulu Ye
- Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan
| | - Toshikuni Sasaoka
- Department of Comparative and Experimental Medicine, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8585, Japan
| | - Masanori Tsuchida
- Division of Thoracic and Cardiovascular Surgery, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan
| | - Kenji Sakimura
- Department of Animal Model Development, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8585, Japan
| | - Yoichi Ajioka
- Division of Molecular and Diagnostic Pathology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan
| | - Yasuo Saijo
- Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan.
| | - Qiliang Zhou
- Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori Chuo-ku, Niigata 951-8510, Japan.
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6
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Nagaya M, Hasegawa K, Uchikura A, Nakano K, Watanabe M, Umeyama K, Matsunari H, Osafune K, Kobayashi E, Nakauchi H, Nagashima H. Feasibility of large experimental animal models in testing novel therapeutic strategies for diabetes. World J Diabetes 2021; 12:306-330. [PMID: 33889282 PMCID: PMC8040081 DOI: 10.4239/wjd.v12.i4.306] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/09/2021] [Revised: 01/30/2021] [Accepted: 03/11/2021] [Indexed: 02/06/2023] Open
Abstract
Diabetes is among the top 10 causes of death in adults and caused approximately four million deaths worldwide in 2017. The incidence and prevalence of diabetes is predicted to increase. To alleviate this potentially severe situation, safer and more effective therapeutics are urgently required. Mice have long been the mainstay as preclinical models for basic research on diabetes, although they are not ideally suited for translating basic knowledge into clinical applications. To validate and optimize novel therapeutics for safe application in humans, an appropriate large animal model is needed. Large animals, especially pigs, are well suited for biomedical research and share many similarities with humans, including body size, anatomical features, physiology, and pathophysiology. Moreover, pigs already play an important role in translational studies, including clinical trials for xenotransplantation. Progress in genetic engineering over the past few decades has facilitated the development of transgenic animals, including porcine models of diabetes. This article discusses features that attest to the attractiveness of genetically modified porcine models of diabetes for testing novel treatment strategies using recent technical advances.
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Affiliation(s)
- Masaki Nagaya
- Meiji University International Institute for Bio-Resource Research, Meiji University, Kawasaki 214-8571, Kanagawa, Japan
- Department of Immunology, St. Marianna University School of Medicine, Kawasaki 261-8511, Kanagawa, Japan
| | - Koki Hasegawa
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Kanagawa, Japan
| | - Ayuko Uchikura
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Kanagawa, Japan
| | - Kazuaki Nakano
- Meiji University International Institute for Bio-Resource Research, Meiji University, Kawasaki 214-8571, Kanagawa, Japan
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Kanagawa, Japan
- Research and Development, PorMedTec Co. Ltd, Kawasaki 214-0034, Kanagawa, Japan
| | - Masahito Watanabe
- Meiji University International Institute for Bio-Resource Research, Meiji University, Kawasaki 214-8571, Kanagawa, Japan
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Kanagawa, Japan
- Research and Development, PorMedTec Co. Ltd, Kawasaki 214-0034, Kanagawa, Japan
| | - Kazuhiro Umeyama
- Meiji University International Institute for Bio-Resource Research, Meiji University, Kawasaki 214-8571, Kanagawa, Japan
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Kanagawa, Japan
- Research and Development, PorMedTec Co. Ltd, Kawasaki 214-0034, Kanagawa, Japan
| | - Hitomi Matsunari
- Meiji University International Institute for Bio-Resource Research, Meiji University, Kawasaki 214-8571, Kanagawa, Japan
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Kanagawa, Japan
| | - Kenji Osafune
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Kyoto, Japan
| | - Eiji Kobayashi
- Department of Organ Fabrication, Keio University School of Medicine, Shinjuku 160-8582, Tokyo, Japan
| | - Hiromitsu Nakauchi
- Institute for Stem Cell Biology and Regenerative Medicine, Department of Genetics, Stanford University School of Medicine, Stanford University, Stanford, CA 94305, United States
- Division of Stem Cell Therapy, Institute of Medical Science, The University of Tokyo, Minato 108-8639, Tokyo, Japan
| | - Hiroshi Nagashima
- Meiji University International Institute for Bio-Resource Research, Meiji University, Kawasaki 214-8571, Kanagawa, Japan
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Kanagawa, Japan
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7
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Babochkina TI, Gerlinskaya LA, Moshkin MP. Generation of donor organs in chimeric animals via blastocyst complementation. Vavilovskii Zhurnal Genet Selektsii 2020; 24:913-921. [PMID: 35088005 PMCID: PMC8763716 DOI: 10.18699/vj20.690] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2020] [Revised: 10/21/2020] [Accepted: 11/17/2020] [Indexed: 11/25/2022] Open
Abstract
The lack of organs for transplantation is an important problem in medicine today. The growth of organs
in chimeric animals may be the solution of this. The proposed technology is the interspecific blastocyst complementation method in combination with genomic editing for obtaining “free niches” and pluripotent stem cell
production methods. The CRISPR/Cas9 method allows the so-called “free niches” to be obtained for blastocyst
complementation. The technologies of producing induced pluripotent stem cells give us the opportunity to obtain human donor cells capable of populating a “free niche”. Taken together, these technologies allow interspecific
blastocyst complementation between humans and other animals, which makes it possible in the future to grow
human organs for transplantations inside chimeric animals. However, in practice, in order to achieve successful
interspecific blastocyst complementation, it is necessary to solve a number of problems: to improve methods for
producing “chimeric competent” cells, to overcome specific interspecific barriers, to select compatible cell developmental stages for injection and the corresponding developmental stage of the host embryo, to prevent apoptosis of donor cells and to achieve effective proliferation of the human donor cells in the host animal. Also, it is
very important to analyze the ethical aspects related to developing technologies of chimeric organisms with the
participation of human cells. Today, many researchers are trying to solve these problems and also to establish new
approaches in the creation of interspecific chimeric organisms in order to grow human organs for transplantation.
In the present review we described the historical stages of the development of the blastocyst complementation
method, examined in detail the technologies that underlie modern blastocyst complementation, and analyzed
current progress that gives us the possibility to grow human organs in chimeric animals. We also considered the
barriers and issues preventing the successful implementation of interspecific blastocyst complementation in practice, and discussed the further development of this method
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Affiliation(s)
- T I Babochkina
- Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
| | - L A Gerlinskaya
- Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
| | - M P Moshkin
- Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
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Miura K, Matoba S, Hirose M, Ogura A. Generation of chimeric mice with spermatozoa fully derived from embryonic stem cells using a triple-target CRISPR method for Nanos3†. Biol Reprod 2020; 104:223-233. [PMID: 32965494 PMCID: PMC7786261 DOI: 10.1093/biolre/ioaa176] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2020] [Revised: 09/02/2020] [Accepted: 09/22/2020] [Indexed: 12/27/2022] Open
Abstract
Conditional knockout (cKO) mice have contributed greatly to understanding the tissue- or stage-specific functions of genes in vivo. However, the current cKO method requires considerable time and effort because of the need to generate two gene-modified mouse strains (Cre transgenic and loxP knockin) for crossing. Here, we examined whether we could analyze the germ cell-related functions of embryonic lethal genes in F0 chimeric mice by restricting the origin of germ cells to mutant embryonic stem cells (ESCs). We confirmed that the full ESC origin of spermatozoa in fertile chimeric mice was achieved by the CRISPR/Cas9 system using three guide RNAs targeting Nanos3, which induced germ cell depletion in the host blastocyst-derived tissues. Among these fertile chimeric mice, those from male ESCs with a Dnmt3b mutation, which normally causes embryo death, also produced F1 mice derived exclusively from the mutant ESCs. Thus, our new chimeric strategy readily revealed that Dnmt3b is dispensable for male germ cell development, in agreement with a previous cKO study. Our new approach enables us to analyze the germ cell functions of embryonic lethal genes in the F0 generation without using the current cKO method.
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Affiliation(s)
- Kento Miura
- RIKEN BioResource Research Center, Ibaraki, Japan.,Department of Disease Model, Research Institute of Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
| | - Shogo Matoba
- RIKEN BioResource Research Center, Ibaraki, Japan.,Cooperative Division of Veterinary Sciences, Tokyo University of Agriculture and Technology, Tokyo, Japan
| | | | - Atsuo Ogura
- RIKEN BioResource Research Center, Ibaraki, Japan.,RIKEN Cluster for Pioneering Research, Saitama, Japan.,Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan
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