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Huang T, Radley A, Yanagida A, Ren Z, Carlisle F, Tahajjodi S, Kim D, O'Neill P, Clarke J, Lancaster MA, Heckhausen Z, Zhuo J, de Sousa JPA, Hajkova P, von Meyenn F, Imai H, Nakauchi H, Guo G, Smith A, Masaki H. Inhibition of PRC2 enables self-renewal of blastoid-competent naive pluripotent stem cells from chimpanzee. Cell Stem Cell 2025; 32:627-639.e8. [PMID: 40015279 DOI: 10.1016/j.stem.2025.02.002] [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: 03/11/2024] [Revised: 10/11/2024] [Accepted: 02/04/2025] [Indexed: 03/01/2025]
Abstract
Naive pluripotent stem cells (PSCs) are counterparts of early epiblast in the mammalian embryo. Mouse and human naive PSCs differ in self-renewal requirements and extraembryonic lineage potency. Here, we investigated the generation of chimpanzee naive PSCs. Colonies generated by resetting or reprogramming failed to propagate. We discovered that self-renewal is enabled by inhibition of Polycomb repressive complex 2 (PRC2). Expanded cells show global transcriptome proximity to human naive PSCs and embryo pre-implantation epiblast, with shared expression of a subset of pluripotency transcription factors. Chimpanzee naive PSCs can transition to multilineage competence or can differentiate into trophectoderm and hypoblast, forming tri-lineage blastoids. They thus provide a higher primate comparative model for studying pluripotency and early embryogenesis. Genetic deletions confirm that PRC2 mediates growth arrest. Further, inhibition of PRC2 overcomes a roadblock to feeder-free propagation of human naive PSCs. Therefore, excess deposition of chromatin modification H3K27me3 is an unexpected barrier to naive PSC self-renewal.
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Affiliation(s)
- Tao Huang
- Living Systems Institute, University of Exeter, Exeter EX4 4QD, UK
| | - Arthur Radley
- Living Systems Institute, University of Exeter, Exeter EX4 4QD, UK
| | - Ayaka Yanagida
- Department of Veterinary Anatomy, The University of Tokyo, Tokyo 113-8657, Japan; Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
| | - Zhili Ren
- Living Systems Institute, University of Exeter, Exeter EX4 4QD, UK
| | | | | | - Dongwan Kim
- Stem Cell Therapy Division, Institute of Integrated Research, Institute of Science, Tokyo 113-8510, Japan
| | - Paul O'Neill
- University of Exeter Sequencing Facility, University of Exeter, Exeter EX4 4QD, UK
| | - James Clarke
- Wellcome-MRC Stem Cell Institute, University of Cambridge, Cambridge CB2 0AW, UK
| | - Madeline A Lancaster
- MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK
| | - Zoe Heckhausen
- MRC Laboratory of Medical Sciences (LMS), Du Cane Rd, London W12 0HS, UK; Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, W12 0NN, UK
| | - Jingran Zhuo
- Department of Health Sciences and Technology, ETH Zurich, 8603 Schwerzenbach, Switzerland
| | | | - Petra Hajkova
- MRC Laboratory of Medical Sciences (LMS), Du Cane Rd, London W12 0HS, UK; Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, W12 0NN, UK
| | - Ferdinand von Meyenn
- Department of Health Sciences and Technology, ETH Zurich, 8603 Schwerzenbach, Switzerland
| | - Hiroo Imai
- Department of Cellular and Molecular Biology, Center for the Evolutionary Origins of Human Behavior, Kyoto University, Inuyama, Aichi 484-8506, Japan
| | - Hiromitsu Nakauchi
- Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; Stem Cell Therapy Division, Institute of Integrated Research, Institute of Science, Tokyo 113-8510, Japan; Institute for Stem Cell Biology and Regenerative Medicine, Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Ge Guo
- Living Systems Institute, University of Exeter, Exeter EX4 4QD, UK
| | - Austin Smith
- Living Systems Institute, University of Exeter, Exeter EX4 4QD, UK.
| | - Hideki Masaki
- Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; Stem Cell Therapy Division, Institute of Integrated Research, Institute of Science, Tokyo 113-8510, Japan.
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Marcó S, Muñoz S, Bosch F, Jimenez V. Rat models of musculoskeletal lysosomal storage disorders and their role in pre-clinical evaluation of gene therapy approaches. Mamm Genome 2025:10.1007/s00335-025-10121-3. [PMID: 40100425 DOI: 10.1007/s00335-025-10121-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2024] [Accepted: 03/05/2025] [Indexed: 03/20/2025]
Abstract
Mice have been a cornerstone of biomedical research for decades for studying a wide range of biological processes, disease mechanisms, and the assessment of therapies. Moreover, mice present several practical advantages such as small size, low cost and ease of genetic manipulation. While mice offer numerous benefits, for certain disease areas, rat models provide a closer representation of human disease progression, offering better insights for translational research and therapeutic development. This closer resemblance is particularly important for research focusing on diseases involving the cardiovascular and musculoskeletal system. In rats, the pathophysiology of these diseases mirrors the clinical alterations observed in humans. This review focuses on the key phenotypic differences between mouse and rat models of lysosomal storage disorders that specifically manifest with cardiac, skeletal muscle, and bone and joint involvement (Pompe and Danon diseases, and Maroteaux-Lamy and Morquio A syndromes). Furthermore, we discuss the therapeutic potential of various adeno-associated viral vector-mediated gene therapies that have been evaluated in these rat models, highlighting their contributions to advancing treatment options for these debilitating conditions.
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Affiliation(s)
- Sara Marcó
- Center of Animal Biotechnology and Gene Therapy, Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain
- Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain
| | - Sergio Muñoz
- Center of Animal Biotechnology and Gene Therapy, Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain
- Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain
| | - Fatima Bosch
- Center of Animal Biotechnology and Gene Therapy, Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain
- Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain
| | - Veronica Jimenez
- Center of Animal Biotechnology and Gene Therapy, Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain.
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Guo J, Lin R, Liu J, Liu R, Chen S, Zhang Z, Yang Y, Wang H, Wang L, Yu S, Zhou C, Xiao L, Luo R, Yu J, Zeng L, Zhang X, Li Y, Wu H, Wang T, Li Y, Kumar M, Zhu P, Liu J. Capture primed pluripotency in guinea pig. Stem Cell Reports 2025; 20:102388. [PMID: 39793577 PMCID: PMC11864139 DOI: 10.1016/j.stemcr.2024.102388] [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: 05/15/2024] [Revised: 12/02/2024] [Accepted: 12/06/2024] [Indexed: 01/13/2025] Open
Abstract
Guinea pigs are valuable models for human disease research, yet the lack of established pluripotent stem cell lines has limited their utility. In this study, we isolate and characterize guinea pig epiblast stem cells (gpEpiSCs) from post-implantation embryos. These cells differentiate into the three germ layers, maintain normal karyotypes, and rely on FGF2 and ACTIVIN A signaling for self-renewal and pluripotency. Wingless/Integrated (WNT) signaling inhibition is also essential for their maintenance. GpEpiSCs express key pluripotency markers (OCT4, SOX2, NANOG) and share transcriptional similarities with human and mouse primed stem cells. While many genes are conserved between guinea pig and human primed stem cells, transcriptional analysis also reveals species-specific differences in pluripotency-related pathways. Epigenetic analysis highlights bivalent gene regulation, underscoring their developmental potential. This work demonstrates both the evolutionary conservation and divergence of primed pluripotent stem cells, providing a new tool for biomedical research and enhancing guinea pigs' utility in studying human diseases.
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Affiliation(s)
- Jing Guo
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Runxia Lin
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China; Laboratory of Cell Fate Control, School of Life Sciences, Westlake University, Hangzhou, China
| | - Jinpeng Liu
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Rongrong Liu
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Shuyan Chen
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Zhen Zhang
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Yongzheng Yang
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Haiyun Wang
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Luqin Wang
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Shengyong Yu
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Chunhua Zhou
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Lizhan Xiao
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Rongping Luo
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Jinjin Yu
- Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China; Department of Pediatric Cardiology, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, China
| | - Lihua Zeng
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Xiaoli Zhang
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Yusha Li
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China
| | - Haokaifeng Wu
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China; Centre for Regenerative Medicine and Health, Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, Hong Kong SAR, P.R. China; Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou 510000, China
| | - Tao Wang
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Yi Li
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Manish Kumar
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China.
| | - Ping Zhu
- Guangdong Provincial Key Laboratory of Pathogenesis, Targeted Prevention and Treatment of Heart Disease, Guangzhou Key Laboratory of Cardiac Pathogenesis and Prevention, Guangzhou, China; Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong 510100, China.
| | - Jing Liu
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China, Guangzhou Medical University, Guangzhou 511436, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; Centre for Regenerative Medicine and Health, Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, Hong Kong SAR, P.R. China.
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4
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Bu W, Li Y. Rat Models of Breast Cancer. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2025; 1464:123-148. [PMID: 39821024 DOI: 10.1007/978-3-031-70875-6_8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2025]
Abstract
As the first mammal to be domesticated for research purposes, rats served as the primary animal model for various branches of biomedical research, including breast cancer studies, up until the late 1990s and early 2000s. During this time, genetic engineering of mice, but not rats, became routine, and mice gradually supplanted rats as the preferred rodent model. But recent advances in creating genetically engineered rat models, especially with the assistance of CRISPR/Cas9 technology, have rekindled the significance of rats as a critical model in exploring various facets of breast cancer research. This is particularly pronounced in the study of the formation and progression of the estrogen receptor-positive subtype, which remains challenging to model in mice. In this chapter, we embark on a historical journey through the evolution of rat models in biomedical research and provide an overview of the general and histological characteristics of rat mammary glands. Next, we critically review major rat models for breast cancer research, including those induced by carcinogens, hormones, radiation, germline transgenes, germline knockouts, and intraductal injection of retrovirus/lentivirus to deliver oncogenic drivers into mature mammary glands. We also discuss the advances in building rat models using somatic genome editing powered by CRISPR/Cas9. This chapter concludes with our forward-looking perspective on future applications of advanced rat models in critical areas of breast cancer research that have continued to challenge the mouse model community.
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Affiliation(s)
- Wen Bu
- Lester & Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA
| | - Yi Li
- Lester & Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA.
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Ying Q, Nichols J. Relationship of PSC to embryos: Extending and refining capture of PSC lines from mammalian embryos. Bioessays 2024; 46:e2400077. [PMID: 39400400 PMCID: PMC11589693 DOI: 10.1002/bies.202400077] [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: 04/02/2024] [Accepted: 09/07/2024] [Indexed: 10/15/2024]
Abstract
Pluripotent stem cell lines derived from preimplantation mouse embryos have opened opportunities for the study of early mammalian development and generation of genetically uncompromised material for differentiation into specific cell types. Murine embryonic stem cells are highly versatile and can be engineered and introduced into host embryos, transferred to recipient females, and gestated to investigate gene function at multiple levels as well as developmental mechanisms, including lineage segregation and cell competition. In this review, we summarize the biomedical motivation driving the incremental modification to culture regimes and analyses that have advanced stem cell research to its current state. Ongoing investigation into divergent mechanisms of early developmental processes adopted by other species, such as agriculturally beneficial mammals and birds, will continue to enrich knowledge and inform strategies for future in vitro models.
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Affiliation(s)
- Qi‐Long Ying
- Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of MedicineUniversity of Southern CaliforniaLos AngelesCaliforniaUSA
| | - Jennifer Nichols
- MRC Human Genetics Unit, Institute for Genetics and CancerUniversity of EdinburghEdinburghUK
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Smith A. Propagating pluripotency - The conundrum of self-renewal. Bioessays 2024; 46:e2400108. [PMID: 39180242 PMCID: PMC11589686 DOI: 10.1002/bies.202400108] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2024] [Revised: 07/29/2024] [Accepted: 08/06/2024] [Indexed: 08/26/2024]
Abstract
The discovery of mouse embryonic stem cells in 1981 transformed research in mammalian developmental biology and functional genomics. The subsequent generation of human pluripotent stem cells (PSCs) and the development of molecular reprogramming have opened unheralded avenues for drug discovery and cell replacement therapy. Here, I review the history of PSCs from the perspective that long-term self-renewal is a product of the in vitro signaling environment, rather than an intrinsic feature of embryos. I discuss the relationship between pluripotent states captured in vitro to stages of epiblast in the embryo and suggest key considerations for evaluation of PSCs. A remaining fundamental challenge is to determine whether naïve pluripotency can be propagated from the broad range of mammals by exploiting common principles in gene regulatory architecture.
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Affiliation(s)
- Austin Smith
- Living Systems InstituteUniversity of ExeterExeterUK
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Neira JA, Conrad JV, Rusteika M, Chu LF. The progress of induced pluripotent stem cells derived from pigs: a mini review of recent advances. Front Cell Dev Biol 2024; 12:1371240. [PMID: 38979033 PMCID: PMC11228285 DOI: 10.3389/fcell.2024.1371240] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2024] [Accepted: 04/10/2024] [Indexed: 07/10/2024] Open
Abstract
Pigs (Sus scrofa) are widely acknowledged as an important large mammalian animal model due to their similarity to human physiology, genetics, and immunology. Leveraging the full potential of this model presents significant opportunities for major advancements in the fields of comparative biology, disease modeling, and regenerative medicine. Thus, the derivation of pluripotent stem cells from this species can offer new tools for disease modeling and serve as a stepping stone to test future autologous or allogeneic cell-based therapies. Over the past few decades, great progress has been made in establishing porcine pluripotent stem cells (pPSCs), including embryonic stem cells (pESCs) derived from pre- and peri-implantation embryos, and porcine induced pluripotent stem cells (piPSCs) using a variety of cellular reprogramming strategies. However, the stabilization of pPSCs was not as straightforward as directly applying the culture conditions developed and optimized for murine or primate PSCs. Therefore, it has historically been challenging to establish stable pPSC lines that could pass stringent pluripotency tests. Here, we review recent advances in the establishment of stable porcine PSCs. We focus on the evolving derivation methods that eventually led to the establishment of pESCs and transgene-free piPSCs, as well as current challenges and opportunities in this rapidly advancing field.
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Affiliation(s)
- Jaime A Neira
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
- Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB, Canada
- Alberta Children's Hospital Research Institute, Calgary, AB, Canada
| | - J Vanessa Conrad
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
- Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB, Canada
- Alberta Children's Hospital Research Institute, Calgary, AB, Canada
| | - Margaret Rusteika
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
- Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB, Canada
- Alberta Children's Hospital Research Institute, Calgary, AB, Canada
- Biomedical Engineering Graduate Program, University of Calgary, Calgary, AB, Canada
| | - Li-Fang Chu
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
- Reproductive Biology and Regenerative Medicine Research Group, University of Calgary, Calgary, AB, Canada
- Alberta Children's Hospital Research Institute, Calgary, AB, Canada
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8
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Lim JJY, Murata Y, Yuri S, Kitamuro K, Kawai T, Isotani A. Generating an organ-deficient animal model using a multi-targeted CRISPR-Cas9 system. Sci Rep 2024; 14:10636. [PMID: 38724644 PMCID: PMC11082136 DOI: 10.1038/s41598-024-61167-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2023] [Accepted: 05/02/2024] [Indexed: 05/12/2024] Open
Abstract
Gene-knockout animal models with organ-deficient phenotypes used for blastocyst complementation are generally not viable. Animals need to be maintained as heterozygous mutants, and homozygous mutant embryos yield only one-fourth of all embryos. In this study, we generated organ-deficient embryos using the CRISPR-Cas9-sgRNAms system that induces cell death with a single-guide RNA (sgRNAms) targeting multiple sites in the genome. The Cas9-sgRNAms system interrupted cell proliferation and induced cell ablation in vitro. The mouse model had Cas9 driven by the Foxn1 promoter with a ubiquitous expression cassette of sgRNAms at the Rosa26 locus (Foxn1Cas9; Rosa26_ms). It showed an athymic phenotype similar to that of nude mice but was not hairless. Eventually, a rat cell-derived thymus in an interspecies chimera was generated by blastocyst complementation of Foxn1Cas9; Rosa26_ms mouse embryos with rat embryonic stem cells. Theoretically, a half of the total embryos has the Cas9-sgRNAms system because Rosa26_ms could be maintained as homozygous.
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Affiliation(s)
- Jonathan Jun-Yong Lim
- Laboratory of Organ Developmental Engineering, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0912, Japan
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore
| | - Yamato Murata
- Laboratory of Organ Developmental Engineering, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0912, Japan
| | - Shunsuke Yuri
- Laboratory of Organ Developmental Engineering, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0912, Japan
| | - Kohei Kitamuro
- Laboratory of Organ Developmental Engineering, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0912, Japan
| | - Taro Kawai
- Laboratory of Molecular Immunobiology, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0912, Japan
- Life Science Collaboration Center (LiSCo), Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0192, Japan
| | - Ayako Isotani
- Laboratory of Organ Developmental Engineering, Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0912, Japan.
- Life Science Collaboration Center (LiSCo), Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0192, Japan.
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9
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Throesch BT, Bin Imtiaz MK, Muñoz-Castañeda R, Sakurai M, Hartzell AL, James KN, Rodriguez AR, Martin G, Lippi G, Kupriyanov S, Wu Z, Osten P, Izpisua Belmonte JC, Wu J, Baldwin KK. Functional sensory circuits built from neurons of two species. Cell 2024; 187:2143-2157.e15. [PMID: 38670072 PMCID: PMC11293795 DOI: 10.1016/j.cell.2024.03.042] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2023] [Revised: 01/18/2024] [Accepted: 03/26/2024] [Indexed: 04/28/2024]
Abstract
A central question for regenerative neuroscience is whether synthetic neural circuits, such as those built from two species, can function in an intact brain. Here, we apply blastocyst complementation to selectively build and test interspecies neural circuits. Despite approximately 10-20 million years of evolution, and prominent species differences in brain size, rat pluripotent stem cells injected into mouse blastocysts develop and persist throughout the mouse brain. Unexpectedly, the mouse niche reprograms the birth dates of rat neurons in the cortex and hippocampus, supporting rat-mouse synaptic activity. When mouse olfactory neurons are genetically silenced or killed, rat neurons restore information flow to odor processing circuits. Moreover, they rescue the primal behavior of food seeking, although less well than mouse neurons. By revealing that a mouse can sense the world using neurons from another species, we establish neural blastocyst complementation as a powerful tool to identify conserved mechanisms of brain development, plasticity, and repair.
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Affiliation(s)
- Benjamin T Throesch
- Department of Neuroscience, The Scripps Research Institute, La Jolla, San Diego, CA, USA; Neuroscience Graduate Program, University of California, San Diego, La Jolla, San Diego, CA, USA
| | - Muhammad Khadeesh Bin Imtiaz
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | | | - Masahiro Sakurai
- Salk Institute for Biological Studies, La Jolla, San Diego, CA, USA; Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Andrea L Hartzell
- Department of Neuroscience, The Scripps Research Institute, La Jolla, San Diego, CA, USA
| | - Kiely N James
- Department of Neuroscience, The Scripps Research Institute, La Jolla, San Diego, CA, USA; Neuroscience Graduate Program, University of California, San Diego, La Jolla, San Diego, CA, USA
| | - Alberto R Rodriguez
- Mouse Genetics Core, The Scripps Research Institute, La Jolla, San Diego, CA, USA
| | - Greg Martin
- Mouse Genetics Core, The Scripps Research Institute, La Jolla, San Diego, CA, USA
| | - Giordano Lippi
- Department of Neuroscience, The Scripps Research Institute, La Jolla, San Diego, CA, USA
| | - Sergey Kupriyanov
- Mouse Genetics Core, The Scripps Research Institute, La Jolla, San Diego, CA, USA
| | - Zhuhao Wu
- Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Pavel Osten
- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
| | - Juan Carlos Izpisua Belmonte
- Salk Institute for Biological Studies, La Jolla, San Diego, CA, USA; Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, San Diego, CA, USA; Altos Labs, San Diego, CA, USA
| | - Jun Wu
- Salk Institute for Biological Studies, La Jolla, San Diego, CA, USA; Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA; Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA; Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA.
| | - Kristin K Baldwin
- Department of Neuroscience, The Scripps Research Institute, La Jolla, San Diego, CA, USA; Neuroscience Graduate Program, University of California, San Diego, La Jolla, San Diego, CA, USA; Department of Genetics and Development, Columbia Stem Cell Initiative, Columbia University Medical Center, New York, NY, USA.
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10
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Du P, Wu J. Hallmarks of totipotent and pluripotent stem cell states. Cell Stem Cell 2024; 31:312-333. [PMID: 38382531 PMCID: PMC10939785 DOI: 10.1016/j.stem.2024.01.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2023] [Revised: 01/18/2024] [Accepted: 01/26/2024] [Indexed: 02/23/2024]
Abstract
Though totipotency and pluripotency are transient during early embryogenesis, they establish the foundation for the development of all mammals. Studying these in vivo has been challenging due to limited access and ethical constraints, particularly in humans. Recent progress has led to diverse culture adaptations of epiblast cells in vitro in the form of totipotent and pluripotent stem cells, which not only deepen our understanding of embryonic development but also serve as invaluable resources for animal reproduction and regenerative medicine. This review delves into the hallmarks of totipotent and pluripotent stem cells, shedding light on their key molecular and functional features.
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Affiliation(s)
- Peng Du
- MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing 100871, China; Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China.
| | - Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA.
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11
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Ji J, Cao J, Chen P, Huang R, Ye SD. Inhibition of protein kinase C increases Prdm14 level to promote self-renewal of embryonic stem cells through reducing Suv39h-induced H3K9 methylation. J Biol Chem 2024; 300:105714. [PMID: 38309502 PMCID: PMC10909794 DOI: 10.1016/j.jbc.2024.105714] [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: 07/28/2023] [Revised: 12/19/2023] [Accepted: 01/28/2024] [Indexed: 02/05/2024] Open
Abstract
Inhibition of protein kinase C (PKC) efficiently promoted the self-renewal of embryonic stem cells (ESCs). However, information about the function of PKC inhibition remains lacking. Here, RNA-sequencing showed that the addition of Go6983 significantly inhibited the expression of de novo methyltransferases (Dnmt3a and Dnmt3b) and their regulator Dnmt3l, resulting in global hypomethylation of DNA in mouse ESCs. Mechanistically, PR domain-containing 14 (Prdm14), a site-specific transcriptional activator, partially contributed to Go6983-mediated repression of Dnmt3 genes. Administration of Go6983 increased Prdm14 expression mainly through the inhibition of PKCδ. High constitutive expression of Prdm14 phenocopied the ability of Go6983 to maintain` mouse ESC stemness in the absence of self-renewal-promoting cytokines. In contrast, the knockdown of Prdm14 eliminated the response to PKC inhibition and substantially impaired the Go6983-induced resistance of mouse ESCs to differentiation. Furthermore, liquid chromatography-mass spectrometry profiling and Western blotting revealed low levels of Suv39h1 and Suv39h2 in Go6983-treated mouse ESCs. Suv39h enzymes are histone methyltransferases that recognize dimethylated and trimethylated histone H3K9 specifically and usually function as transcriptional repressors. Consistently, the inhibition of Suv39h1 by RNA interference or the addition of the selective inhibitor chaetocin increased Prdm14 expression. Moreover, chromatin immunoprecipitation assay showed that Go6983 treatment led to decreased enrichment of dimethylation and trimethylation of H3K9 at the Prdm14 promoter but increased RNA polymerase Ⅱ binding affinity. Together, our results provide novel insights into the pivotal association between PKC inhibition-mediated self-renewal and epigenetic changes, which will help us better understand the regulatory network of stem cell pluripotency.
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Affiliation(s)
- Junxiang Ji
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui, PR China
| | - Jianjian Cao
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui, PR China
| | - Peng Chen
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui, PR China
| | - Ru Huang
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui, PR China
| | - Shou-Dong Ye
- Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, Anhui, PR China.
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12
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Hu S, Li Z, Liu H, Cao W, Meng Y, Liu C, He S, Lin Q, Shang M, Lin F, Yi N, Wang H, Sachinidis A, Ying Q, Li L, Peng L. Kcnh2 deletion is associated with rat embryonic development defects via destruction of KCNH2‑integrin β1 complex. Int J Mol Med 2024; 53:14. [PMID: 38063256 PMCID: PMC10760793 DOI: 10.3892/ijmm.2023.5338] [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: 05/18/2023] [Accepted: 10/06/2023] [Indexed: 12/18/2023] Open
Abstract
The Kv11.1 potassium channel encoded by the Kcnh2 gene is crucial in conducting the rapid delayed rectifier K+ current in cardiomyocytes. Homozygous mutation in Kcnh2 is embryonically lethal in humans and mice. However, the molecular signaling pathway of intrauterine fetal loss is unclear. The present study generated a Kcnh2 knockout rat based on edited rat embryonic stem cells (rESCs). Kcnh2 knockout was embryonic lethal on day 11.5 of development due to a heart configuration defect. Experiments with human embryonic heart single cells (6.5‑7 weeks post‑conception) suggested that potassium voltage‑gated channel subfamily H member 2 (KCNH2) plays a crucial role in the development of compact cardiomyocytes. By contrast, apoptosis was found to be triggered in the homozygous embryos, which could be attributed to the failure of KCNH2 to form a complex with integrin β1 that was essential for preventing the process of apoptosis via inhibition of forkhead box O3A. Destruction of the KCNH2/integrin β1 complex reduced the phosphorylation level of AKT and deactivated the glycogen synthase kinase 3 β (GSK‑3β)/β‑catenin pathway, which caused early developmental abnormalities in rats. The present work reveals a basic mechanism by which KCNH2 maintains intact embryonic heart development.
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Affiliation(s)
- Sangyu Hu
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Zhigang Li
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Huan Liu
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Wenze Cao
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Yilei Meng
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Chang Liu
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Siyu He
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Qin Lin
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Mengyue Shang
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Fang Lin
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Na Yi
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Hanrui Wang
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
| | - Agapios Sachinidis
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Center for Physiology, Working Group Sachinidis, Center for Molecular Medicine, D-50931 Cologne, Germany
| | - Qilong Ying
- Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Li Li
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Research Units of Origin and Regulation of Heart Rhythm, Chinese Academy of Medical Sciences, Beijing 100730, P.R. China
- Department of Medical Genetics Tongji University School of Medicine, Shanghai 200331, P.R. China
| | - Luying Peng
- State Key Laboratory of Cardiology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Institute of Medical Genetics, Tongji University, Shanghai 200331, P.R. China
- Heart Health Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China
- Research Units of Origin and Regulation of Heart Rhythm, Chinese Academy of Medical Sciences, Beijing 100730, P.R. China
- Department of Medical Genetics Tongji University School of Medicine, Shanghai 200331, P.R. China
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13
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Wei Y, Zhang E, Yu L, Ci B, Sakurai M, Guo L, Zhang X, Lin S, Takii S, Liu L, Liu J, Schmitz DA, Su T, Zhang J, Shen Q, Ding Y, Zhan L, Sun HX, Zheng C, Xu L, Okamura D, Ji W, Tan T, Wu J. Dissecting embryonic and extraembryonic lineage crosstalk with stem cell co-culture. Cell 2023; 186:5859-5875.e24. [PMID: 38052213 PMCID: PMC10916932 DOI: 10.1016/j.cell.2023.11.008] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2022] [Revised: 09/01/2023] [Accepted: 11/02/2023] [Indexed: 12/07/2023]
Abstract
Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-β, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs), and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts. This approach facilitates the co-culture of embryonic and extraembryonic stem cells, revealing a growth inhibition effect exerted by extraembryonic endoderm cells on pluripotent cells, partially through extracellular matrix signaling. Additionally, our cross-species analysis identified both shared and unique transcription factors and pathways regulating FTW-XENs. The embryonic and extraembryonic stem cell co-culture strategy offers promising avenues for developing more faithful embryo models and devising more developmentally pertinent differentiation protocols.
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Affiliation(s)
- Yulei Wei
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
| | - E Zhang
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
| | - Leqian Yu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Baiquan Ci
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
| | - Masahiro Sakurai
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Lei Guo
- Quantitative Biomedical Research Center, Peter O'Donnell Jr. School of Public Health, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Xin Zhang
- State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Sirui Lin
- State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - Shino Takii
- Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University, Nakamachi, Nara 631-8505, Japan
| | - Lizhong Liu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Jian Liu
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
| | - Daniel A Schmitz
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Ting Su
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
| | - Junmei Zhang
- State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing 100193, China; State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Qiaoyan Shen
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Yi Ding
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Linfeng Zhan
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
| | | | - Canbin Zheng
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Lin Xu
- Quantitative Biomedical Research Center, Peter O'Donnell Jr. School of Public Health, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Daiji Okamura
- Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University, Nakamachi, Nara 631-8505, Japan
| | - Weizhi Ji
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China.
| | - Tao Tan
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China.
| | - Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
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14
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Kanegi R, Hatoya S, Kimura K, Yodoe K, Nishimura T, Sugiura K, Kawate N, Inaba T. Generation, characterization, and differentiation of induced pluripotent stem-like cells in the domestic cat. J Reprod Dev 2023; 69:317-327. [PMID: 37880086 PMCID: PMC10721851 DOI: 10.1262/jrd.2022-038] [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: 03/23/2022] [Accepted: 09/28/2023] [Indexed: 10/27/2023] Open
Abstract
Induced pluripotent stem (iPS) cells are generated from somatic cells and can differentiate into various cell types. Therefore, these cells are expected to be a powerful tool for modeling diseases and transplantation therapy. Generation of domestic cat iPS cells depending on leukemia inhibitory factor has been reported; however, this strategy may not be optimized. Considering that domestic cats are excellent models for studying spontaneous diseases, iPS cell generation is crucial. In this study, we aimed to derive iPS cells from cat embryonic fibroblasts retrovirally transfected with mouse Oct3/4, Klf4, Sox2, and c-Myc. After transfection, embryonic fibroblasts were reseeded onto inactivated SNL 76/7 and cultured in a medium supplemented with basic fibroblast growth factor. Flat, compact, primary colonies resembling human iPS colonies were observed. Additionally, primary colonies were more frequently observed in the KnockOut Serum Replacement medium than in the fetal bovine serum (FBS) medium. However, enhanced maintenance and proliferation of iPS-like cells occurred in the FBS medium. These iPS-like cells expressed embryonic stem cell markers, had normal karyotypes, proliferated beyond 45 passages, and differentiated into all three germ layers in vitro. Notably, expression of exogenous Oct3/4, Klf4, and Sox2 was silenced in these cells. However, the iPS-like cells failed to form teratomas. In conclusion, this is the first study to establish and characterize cat iPS-like cells, which can differentiate into different cell types depending on the basic fibroblast growth factor.
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Affiliation(s)
- Ryoji Kanegi
- Department of Advanced Pathobiology, Graduate School of Veterinary Sciences, Osaka Metropolitan University, Osaka 598-8531, Japan
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531, Japan
| | - Shingo Hatoya
- Department of Advanced Pathobiology, Graduate School of Veterinary Sciences, Osaka Metropolitan University, Osaka 598-8531, Japan
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531, Japan
| | - Kazuto Kimura
- Department of Advanced Pathobiology, Graduate School of Veterinary Sciences, Osaka Metropolitan University, Osaka 598-8531, Japan
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531, Japan
| | - Kyohei Yodoe
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531, Japan
| | - Toshiya Nishimura
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531, Japan
| | - Kikuya Sugiura
- Department of Advanced Pathobiology, Graduate School of Veterinary Sciences, Osaka Metropolitan University, Osaka 598-8531, Japan
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531, Japan
| | - Noritoshi Kawate
- Department of Advanced Pathobiology, Graduate School of Veterinary Sciences, Osaka Metropolitan University, Osaka 598-8531, Japan
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531, Japan
| | - Toshio Inaba
- Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531, Japan
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15
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Cao J, Liu Z. Generation of chimeric monkeys using embryonic stem cells. Zool Res 2023; 44:1154-1155. [PMID: 37990421 PMCID: PMC10802105 DOI: 10.24272/j.issn.2095-8137.2023.357] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2023] [Accepted: 11/21/2023] [Indexed: 11/23/2023] Open
Affiliation(s)
- Jing Cao
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Zhen Liu
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China. E-mail:
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16
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Anwised P, Moorawong R, Samruan W, Somredngan S, Srisutush J, Laowtammathron C, Aksoy I, Parnpai R, Savatier P. An expedition in the jungle of pluripotent stem cells of non-human primates. Stem Cell Reports 2023; 18:2016-2037. [PMID: 37863046 PMCID: PMC10679654 DOI: 10.1016/j.stemcr.2023.09.013] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2023] [Revised: 09/20/2023] [Accepted: 09/21/2023] [Indexed: 10/22/2023] Open
Abstract
For nearly three decades, more than 80 embryonic stem cell lines and more than 100 induced pluripotent stem cell lines have been derived from New World monkeys, Old World monkeys, and great apes. In this comprehensive review, we examine these cell lines originating from marmoset, cynomolgus macaque, rhesus macaque, pig-tailed macaque, Japanese macaque, African green monkey, baboon, chimpanzee, bonobo, gorilla, and orangutan. We outline the methodologies implemented for their establishment, the culture protocols for their long-term maintenance, and their basic molecular characterization. Further, we spotlight any cell lines that express fluorescent reporters. Additionally, we compare these cell lines with human pluripotent stem cell lines, and we discuss cell lines reprogrammed into a pluripotent naive state, detailing the processes used to attain this. Last, we present the findings from the application of these cell lines in two emerging fields: intra- and interspecies embryonic chimeras and blastoids.
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Affiliation(s)
- Preeyanan Anwised
- University Lyon, University Lyon 1, INSERM, Stem Cell and Brain Research Institute U1208, 69500 Bron, France; Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
| | - Ratree Moorawong
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
| | - Worawalan Samruan
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
| | - Sirilak Somredngan
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
| | - Jittanun Srisutush
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
| | - Chuti Laowtammathron
- Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
| | - Irene Aksoy
- University Lyon, University Lyon 1, INSERM, Stem Cell and Brain Research Institute U1208, 69500 Bron, France.
| | - Rangsun Parnpai
- Embryo Technology and Stem Cell Research Center, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
| | - Pierre Savatier
- University Lyon, University Lyon 1, INSERM, Stem Cell and Brain Research Institute U1208, 69500 Bron, France.
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17
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Cao J, Li W, Li J, Mazid MA, Li C, Jiang Y, Jia W, Wu L, Liao Z, Sun S, Song W, Fu J, Wang Y, Lu Y, Xu Y, Nie Y, Bian X, Gao C, Zhang X, Zhang L, Shang S, Li Y, Fu L, Liu H, Lai J, Wang Y, Yuan Y, Jin X, Li Y, Liu C, Lai Y, Shi X, Maxwell PH, Xu X, Liu L, Poo M, Wang X, Sun Q, Esteban MA, Liu Z. Live birth of chimeric monkey with high contribution from embryonic stem cells. Cell 2023; 186:4996-5014.e24. [PMID: 37949056 DOI: 10.1016/j.cell.2023.10.005] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2022] [Revised: 07/18/2023] [Accepted: 10/03/2023] [Indexed: 11/12/2023]
Abstract
A formal demonstration that mammalian pluripotent stem cells possess preimplantation embryonic cell-like (naive) pluripotency is the generation of chimeric animals through early embryo complementation with homologous cells. Whereas such naive pluripotency has been well demonstrated in rodents, poor chimerism has been achieved in other species including non-human primates due to the inability of the donor cells to match the developmental state of the host embryos. Here, we have systematically tested various culture conditions for establishing monkey naive embryonic stem cells and optimized the procedures for chimeric embryo culture. This approach generated an aborted fetus and a live chimeric monkey with high donor cell contribution. A stringent characterization pipeline demonstrated that donor cells efficiently (up to 90%) incorporated into various tissues (including the gonads and placenta) of the chimeric monkeys. Our results have major implications for the study of primate naive pluripotency and genetic engineering of non-human primates.
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Affiliation(s)
- Jing Cao
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China; Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
| | - Wenjuan Li
- Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Jie Li
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Md Abdul Mazid
- Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Chunyang Li
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yu Jiang
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun 130062, China
| | - Wenqi Jia
- Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Liang Wu
- Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Zhaodi Liao
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shiyu Sun
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Weixiang Song
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Jiqiang Fu
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yan Wang
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yong Lu
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yuting Xu
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yanhong Nie
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Xinyan Bian
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Changshan Gao
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Xiaotong Zhang
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Liansheng Zhang
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Shenshen Shang
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yunpan Li
- Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Lixin Fu
- Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Hao Liu
- Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Junjian Lai
- Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
| | - Yang Wang
- BGI-Research, Hangzhou 310030, China
| | - Yue Yuan
- BGI-Research, Hangzhou 310030, China
| | - Xin Jin
- BGI-Research, Shenzhen 518083, China; School of Medicine, South China University of Technology, Guangzhou, China
| | - Yan Li
- BGI-Research, Shenzhen 518083, China
| | | | - Yiwei Lai
- BGI-Research, Hangzhou 310030, China
| | | | - Patrick H Maxwell
- School of Clinical Medicine, University of Cambridge, Cambridge CB2 0ST, United Kingdom
| | - Xun Xu
- BGI-Research, Hangzhou 310030, China; BGI-Research, Shenzhen 518083, China; Guangdong Provincial Key Laboratory of Genome Read and Write, Shenzhen 518120, China
| | | | - Muming Poo
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China; Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, Shanghai, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xiaolong Wang
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
| | - Qiang Sun
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China; Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, Shanghai, China; University of Chinese Academy of Sciences, Beijing 100049, China.
| | - Miguel A Esteban
- BGI-Research, Hangzhou 310030, China; Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, CAS Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun 130062, China.
| | - Zhen Liu
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China; Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, Shanghai, China; University of Chinese Academy of Sciences, Beijing 100049, China.
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18
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Iwatsuki K, Oikawa M, Kobayashi H, Penfold CA, Sanbo M, Yamamoto T, Hochi S, Kurimoto K, Hirabayashi M, Kobayashi T. Rat post-implantation epiblast-derived pluripotent stem cells produce functional germ cells. CELL REPORTS METHODS 2023; 3:100542. [PMID: 37671016 PMCID: PMC10475792 DOI: 10.1016/j.crmeth.2023.100542] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/11/2023] [Revised: 05/10/2023] [Accepted: 07/03/2023] [Indexed: 09/07/2023]
Abstract
In mammals, pluripotent cells transit through a continuum of distinct molecular and functional states en route to initiating lineage specification. Capturing pluripotent stem cells (PSCs) mirroring in vivo pluripotent states provides accessible in vitro models to study the pluripotency program and mechanisms underlying lineage restriction. Here, we develop optimal culture conditions to derive and propagate post-implantation epiblast-derived PSCs (EpiSCs) in rats, a valuable model for biomedical research. We show that rat EpiSCs (rEpiSCs) can be reset toward the naive pluripotent state with exogenous Klf4, albeit not with the other five candidate genes (Nanog, Klf2, Esrrb, Tfcp2l1, and Tbx3) effective in mice. Finally, we demonstrate that rat EpiSCs retain competency to produce authentic primordial germ cell-like cells that undergo functional gametogenesis leading to the birth of viable offspring. Our findings in the rat model uncover principles underpinning pluripotency and germline competency across species.
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Affiliation(s)
- Kenyu Iwatsuki
- Division of Mammalian Embryology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan
- Graduate School of Medicine, Science and Technology, Shinshu University, Nagano 386-8567, Japan
| | - Mami Oikawa
- Division of Mammalian Embryology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan
- Laboratory of Regenerative Medicine, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan
| | - Hisato Kobayashi
- Department of Embryology, Nara Medical University, Nara 634-0813, Japan
| | - Christopher A. Penfold
- Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Site, Cambridge CB2 3EG, UK
- Centre for Trophoblast Research, University of Cambridge, Downing Site, Cambridge CB2 3EG, UK
- Wellcome Trust – Cancer Research UK Gurdon Institute, Henry Wellcome Building of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK
| | - Makoto Sanbo
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Aichi 444-8787, Japan
| | - Takuya Yamamoto
- Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan
- Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto 606-8501, Japan
- Medical-risk Avoidance Based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project, Kyoto 606-8501, Japan
| | - Shinichi Hochi
- Graduate School of Medicine, Science and Technology, Shinshu University, Nagano 386-8567, Japan
- Faculty of Textile Science and Technology, Shinshu University, Nagano 386-8567, Japan
| | - Kazuki Kurimoto
- Department of Embryology, Nara Medical University, Nara 634-0813, Japan
| | - Masumi Hirabayashi
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Aichi 444-8787, Japan
- The Graduate University of Advanced Studies, Aichi 444-8787, Japan
| | - Toshihiro Kobayashi
- Division of Mammalian Embryology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Aichi 444-8787, Japan
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19
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Zhong L, Gordillo M, Wang X, Qin Y, Huang Y, Soshnev A, Kumar R, Nanjangud G, James D, David Allis C, Evans T, Carey B, Wen D. Dual role of lipids for genome stability and pluripotency facilitates full potency of mouse embryonic stem cells. Protein Cell 2023; 14:591-602. [PMID: 37029701 PMCID: PMC10392030 DOI: 10.1093/procel/pwad008] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2022] [Accepted: 01/09/2023] [Indexed: 02/18/2023] Open
Abstract
While Mek1/2 and Gsk3β inhibition ("2i") supports the maintenance of murine embryonic stem cells (ESCs) in a homogenous naïve state, prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential. Additionally, 2i fails to support derivation and culture of fully potent female ESCs. Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin (AlbuMAX) undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture. Mechanistically, lipids in AlbuMAX impact intracellular metabolism including nucleotide biosynthesis, lipid biogenesis, and TCA cycle intermediates, with enhanced expression of DNMT3s that prevent DNA hypomethylation. Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation, which also alleviates X chromosome loss in female ESCs. Importantly, both male and female "all-ESC" mice can be generated from de novo derived ESCs using AlbuMAX-based media. Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.
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Affiliation(s)
- Liangwen Zhong
- Department of Reproductive Medicine, Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medicine, New York, NY 10065, USA
| | - Miriam Gordillo
- Department of Surgery, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA
| | - Xingyi Wang
- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
| | - Yiren Qin
- Department of Reproductive Medicine, Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medicine, New York, NY 10065, USA
| | - Yuanyuan Huang
- Department of Reproductive Medicine, Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medicine, New York, NY 10065, USA
| | - Alexey Soshnev
- Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University, New York, NY 10065, USA
- Department of Neuroscience, Developmental and Regenerative Biology, University of Texas at San Antonio, One UTSA Circle, San Antonio, TX 78249, USA
| | - Ritu Kumar
- Department of Surgery, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA
- Gladstone Institutes, 1650 Owens St, San Francisco, CA 94158, USA
| | - Gouri Nanjangud
- Molecular Cytogenetics Core. Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Daylon James
- Department of Reproductive Medicine, Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medicine, New York, NY 10065, USA
| | - C David Allis
- Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University, New York, NY 10065, USA
| | - Todd Evans
- Department of Surgery, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA
| | - Bryce Carey
- Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University, New York, NY 10065, USA
| | - Duancheng Wen
- Department of Reproductive Medicine, Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medicine, New York, NY 10065, USA
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20
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Matsumura T, Katagiri K, Yao T, Ishikawa-Yamauchi Y, Nagata S, Hashimoto K, Sato T, Kimura H, Shinohara T, Sanbo M, Hirabayashi M, Ogawa T. Generation of rat offspring using spermatids produced through in vitro spermatogenesis. Sci Rep 2023; 13:12105. [PMID: 37495678 PMCID: PMC10372019 DOI: 10.1038/s41598-023-39304-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2023] [Accepted: 07/23/2023] [Indexed: 07/28/2023] Open
Abstract
An in vitro spermatogenesis method using mouse testicular tissue to produce fertile sperm was established more than a decade ago. Although this culture method has generally not been effective in other animal species, we recently succeeded in improving the culture condition to induce spermatogenesis of rats up to the round spermatid stage. In the present study, we introduced acrosin-EGFP transgenic rats in order to clearly monitor the production of haploid cells during spermatogenesis in vitro. In addition, a metabolomic analysis of the culture media during cultivation revealed the metabolic dynamics of the testis tissue. By modifying the culture media based on these results, we were able to induce rat spermatogenesis repeatedly up to haploid cell production, including the formation of elongating spermatids, which was confirmed histologically and immunohistochemically. Finally, we performed a microinsemination experiment with in vitro produced spermatids, which resulted in the production of healthy and fertile offspring. This is the first demonstration of the in vitro production of functional haploid cells that yielded offspring in animals other than mice. These results are expected to provide a basis for the development of an in vitro spermatogenesis system applicable to many other mammals.
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Affiliation(s)
- Takafumi Matsumura
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
| | - Kumiko Katagiri
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
| | - Tatsuma Yao
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
- Research and Development Center, Fuso Pharmaceutical Industries, Ltd., 2-3-30 Morinomiya, Joto-ku, Osaka, 536-8523, Japan
| | - Yu Ishikawa-Yamauchi
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
| | - Shino Nagata
- Laboratory of Biopharmaceutical and Regenerative Sciences, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama, Kanagawa, 230-0045, Japan
| | - Kiyoshi Hashimoto
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
- Department of Urology, Yokohama City University School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
| | - Takuya Sato
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan
| | - Hiroshi Kimura
- Micro/Nano Technology Center, Tokai University, Hiratsuka, Kanagawa, 259-1292, Japan
| | - Takashi Shinohara
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan
| | - Makoto Sanbo
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8787, Japan
| | - Masumi Hirabayashi
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8787, Japan.
| | - Takehiko Ogawa
- Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, 236-0004, Japan.
- Department of Urology, Yokohama City University School of Medicine, Yokohama, Kanagawa, 236-0004, Japan.
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21
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Wei Y, Zhang E, Yu L, Ci B, Guo L, Sakurai M, Takii S, Liu J, Schmitz DA, Ding Y, Zhan L, Zheng C, Sun HX, Xu L, Okamura D, Ji W, Tan T, Wu J. Dissecting embryonic and extra-embryonic lineage crosstalk with stem cell co-culture. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.03.07.531525. [PMID: 36945498 PMCID: PMC10028955 DOI: 10.1101/2023.03.07.531525] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/11/2023]
Abstract
Faithful embryogenesis requires precise coordination between embryonic and extraembryonic tissues. Although stem cells from embryonic and extraembryonic origins have been generated for several mammalian species(Bogliotti et al., 2018; Choi et al., 2019; Cui et al., 2019; Evans and Kaufman, 1981; Kunath et al., 2005; Li et al., 2008; Martin, 1981; Okae et al., 2018; Tanaka et al., 1998; Thomson et al., 1998; Vandevoort et al., 2007; Vilarino et al., 2020; Yu et al., 2021b; Zhong et al., 2018), they are grown in different culture conditions with diverse media composition, which makes it difficult to study cross-lineage communication. Here, by using the same culture condition that activates FGF, TGF-β and WNT signaling pathways, we derived stable embryonic stem cells (ESCs), extraembryonic endoderm stem cells (XENs) and trophoblast stem cells (TSCs) from all three founding tissues of mouse and cynomolgus monkey blastocysts. This allowed us to establish embryonic and extraembryonic stem cell co-cultures to dissect lineage crosstalk during early mammalian development. Co-cultures of ESCs and XENs uncovered a conserved and previously unrecognized growth inhibition of pluripotent cells by extraembryonic endoderm cells, which is in part mediated through extracellular matrix signaling. Our study unveils a more universal state of stem cell self-renewal stabilized by activation, as opposed to inhibition, of developmental signaling pathways. The embryonic and extraembryonic stem cell co-culture strategy developed here will open new avenues for creating more faithful embryo models and developing more developmentally relevant differentiation protocols.
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Affiliation(s)
- Yulei Wei
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
| | - E Zhang
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
| | - Leqian Yu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
- The State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Baiquan Ci
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
| | - Lei Guo
- Quantitative Biomedical Research Center, Department of Population and Data Sciences, Peter O’Donnell Jr. School of Public Health, University of Texas Southwestern Medical Center, Dallas, TX 75390
| | - Masahiro Sakurai
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Shino Takii
- Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University, Nakamachi, Nara 631-8505, Japan
| | - Jian Liu
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
| | - Daniel A. Schmitz
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Yi Ding
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Linfeng Zhan
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
| | - Canbin Zheng
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | | | - Lin Xu
- Quantitative Biomedical Research Center, Department of Population and Data Sciences, Peter O’Donnell Jr. School of Public Health, University of Texas Southwestern Medical Center, Dallas, TX 75390
| | - Daiji Okamura
- Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University, Nakamachi, Nara 631-8505, Japan
| | - Weizhi Ji
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
| | - Tao Tan
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
| | - Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
- Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA
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22
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Sedivy LJ, Brandt G, Martin AL, Abroe HM, Phiel CJ. Mouse Embryonic Stem Cell Pluripotency Factors Regulate RNA Methylation. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.02.23.529801. [PMID: 36865332 PMCID: PMC9980107 DOI: 10.1101/2023.02.23.529801] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/03/2023]
Abstract
The pluripotency of embryonic stem cells (ESCs) is actively promoted by a diverse set of factors, including leukemia inhibitory factor (LIF), glycogen synthase kinase-3 (Gsk-3) and mitogen-activated protein kinase kinase (MEK) inhibitors, ascorbic acid, and α-ketoglutarate. Strikingly, several of these factors intersect with the post-transcriptional methylation of RNA (m 6 A), which has also been shown to play a role in ESC pluripotency. Therefore, we explored the possibility that these factors converge on this biochemical pathway to promote the retention of ESC pluripotency. Mouse ESCs were treated with various combinations of small molecules, and the relative levels of m 6 A RNA were measured, as well as the expression of genes marking naïve and primed ESCs. The most surprising result was the discovery that replacing glucose with high levels of fructose pushed ESCs to a more naïve state and reduced m 6 A RNA abundance. Our results suggest a correlation between molecules previously shown to promote the retention of ESC pluripotency and m 6 A RNA levels, strengthening a molecular connection between reduced m 6 A RNA and the pluripotent state, and provides a foundation for future mechanistic studies on the role of m 6 A and ESC pluripotency.
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23
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Menzorov AG. Pluripotent Stem Cells of Order Carnivora: Technical Perspective. Int J Mol Sci 2023; 24:ijms24043905. [PMID: 36835318 PMCID: PMC9963171 DOI: 10.3390/ijms24043905] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2022] [Revised: 02/08/2023] [Accepted: 02/12/2023] [Indexed: 02/17/2023] Open
Abstract
Human and mouse induced pluripotent stem cells (PSCs) are widely used for studying early embryonic development and for modeling of human diseases. Derivation and studying of PSCs from model organisms beyond commonly used mice and rats may provide new insights into the modeling and treating human diseases. The order Carnivora representatives possess unique features and are already used for modeling human-related traits. This review focuses on the technical aspects of derivation of the Carnivora species PSCs as well as their characterization. Current data on dog, feline, ferret, and American mink PSCs are summarized.
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Affiliation(s)
- Aleksei G. Menzorov
- Sector of Cell Collections, Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia;
- Natural Sciences Department, Novosibirsk State University, 630090 Novosibirsk, Russia
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24
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Baral I, Shirude MB, Jothi DL, Mukherjee A, Dutta D. Characterization of a Distinct State in the Continuum of Pluripotency Facilitated by Inhibition of PKCζ in Mouse Embryonic Stem Cells. Stem Cell Rev Rep 2023; 19:1098-1115. [PMID: 36781773 DOI: 10.1007/s12015-023-10513-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/26/2023] [Indexed: 02/15/2023]
Abstract
Inhibition of PKC (PKCi) signaling maintains pluripotency of embryonic stem cells (ESCs) across different mammalian species. However, the position of PKCi maintained ESCs in the pluripotency continuum is largely unknown. Here we demonstrate that mouse ESCs when cultured continuously, with PKCi, for 75 days are retained in naïve state of pluripotency. Gene expression analysis and proteomics studies demonstrated enhanced naïve character of PKCi maintained ESCs in comparison to classical serum/LIF (S/L) supported ESCs. Molecular analysis revealed that activation of PKCζ isoform associate with primed state of pluripotency, present in epiblast-like stem cells generated in vitro while inhibition of PKCζ phosphorylation associated with naïve state of pluripotency in vitro and in vivo. Phosphoproteomics and chromatin modification enzyme array based studies showed loss in DNA methyl transferase 3B (DNMT3B) and its phosphorylation level upon functional inhibition of PKCζ as one of the crucial components of this regulatory pathway. Unlike ground state of pluripotency maintained by MEK/GSK3 inhibitor in addition to LIF (2i/LIF), loss in DNMT3B is a reversible phenomenon in PKCi maintained ESCs. Absence of phosphorylation of c-MYC, RAF1, SPRY4 while presence of ERF, DUSP6, CIC and YAP1 phosphorylation underlined the phosphoproteomics signature of PKCi mediated maintenance of naïve pluripotency. States of pluripotency represent the developmental continuum and the existence of PKCi mediated mouse ESCs in a distinct state in the continuum of pluripotency (DiSCo) might contribute to the establishment of stages of murine embryonic development that were non-permissible till date.
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Affiliation(s)
- Ishita Baral
- Regenerative Biology Program, Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, 695014, Kerala, India.,Manipal Academy of Higher Education, Karnataka State, Manipal, 576104, India
| | - Mayur Balkrishna Shirude
- Regenerative Biology Program, Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, 695014, Kerala, India.,Manipal Academy of Higher Education, Karnataka State, Manipal, 576104, India
| | - Dhana Lakshmi Jothi
- Regenerative Biology Program, Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, 695014, Kerala, India
| | - Ananda Mukherjee
- Cancer Biology Program, Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, 695014, Kerala, India
| | - Debasree Dutta
- Regenerative Biology Program, Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, 695014, Kerala, India.
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25
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Yoshimatsu S, Nakajima M, Sonn I, Natsume R, Sakimura K, Nakatsukasa E, Sasaoka T, Nakamura M, Serizawa T, Sato T, Sasaki E, Deng H, Okano H. Attempts for deriving extended pluripotent stem cells from common marmoset embryonic stem cells. Genes Cells 2023; 28:156-169. [PMID: 36530170 DOI: 10.1111/gtc.13000] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2021] [Revised: 12/13/2022] [Accepted: 12/13/2022] [Indexed: 12/23/2022]
Abstract
Extended pluripotent stem cells (EPSCs) derived from mice and humans showed an enhanced potential for chimeric formation. By exploiting transcriptomic approaches, we assessed the differences in gene expression profile between extended EPSCs derived from mice and humans, and those newly derived from the common marmoset (marmoset; Callithrix jacchus). Although the marmoset EPSC-like cells displayed a unique colony morphology distinct from murine and human EPSCs, they displayed a pluripotent state akin to embryonic stem cells (ESCs), as confirmed by gene expression and immunocytochemical analyses of pluripotency markers and three-germ-layer differentiation assay. Importantly, the marmoset EPSC-like cells showed interspecies chimeric contribution to mouse embryos, such as E6.5 blastocysts in vitro and E6.5 epiblasts in vivo in mouse development. Also, we discovered that the perturbation of gene expression of the marmoset EPSC-like cells from the original ESCs resembled that of human EPSCs. Taken together, our multiple analyses evaluated the efficacy of the method for the derivation of marmoset EPSCs.
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Affiliation(s)
- Sho Yoshimatsu
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan.,Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science, Saitama, Japan
| | - Mayutaka Nakajima
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Iki Sonn
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Rie Natsume
- Department of Animal Model Development, Brain Research Institute, Niigata University, Niigata, Japan
| | - Kenji Sakimura
- Department of Animal Model Development, Brain Research Institute, Niigata University, Niigata, Japan
| | - Ena Nakatsukasa
- Department of Animal Model Development, Brain Research Institute, Niigata University, Niigata, Japan
| | - Toshikuni Sasaoka
- Department of Animal Model Development, Brain Research Institute, Niigata University, Niigata, Japan
| | - Mari Nakamura
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Takashi Serizawa
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Tsukika Sato
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Erika Sasaki
- Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science, Saitama, Japan.,Department of Marmoset Biology and Medicine, Central Institute for Experimental Animals, Kanagawa, Japan
| | - Hongkui Deng
- Stem Cell Research Center, Peking University, Beijing, China
| | - Hideyuki Okano
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan.,Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science, Saitama, Japan
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26
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Lee J, Wang J, Ally R, Trzaska S, Hickey J, Mujica A, Miloscio L, Mastaitis J, Morse B, Smith J, Atanasio A, Chiao E, Chen H, Latuszek A, Hu Y, Valenzuela D, Romano C, Zambrowicz B, Auerbach W. Production of large, defined genome modifications in rats by targeting rat embryonic stem cells. Stem Cell Reports 2023; 18:394-409. [PMID: 36525967 PMCID: PMC9860120 DOI: 10.1016/j.stemcr.2022.11.012] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Revised: 11/14/2022] [Accepted: 11/15/2022] [Indexed: 12/23/2022] Open
Abstract
Rats were more frequently used than mice to model human disease before mouse embryonic stem cells (mESCs) revolutionized genetic engineering in mice. Rat ESCs (rESCs) were first reported over 10 years ago, yet they are not as frequently used as mESCs. CRISPR-based gene editing in zygotes is widely used in rats but is limited by the difficulty of inserting or replacing DNA sequences larger than about 10 kb. We report here the generation of germline-competent rESC lines from several rat strains. These rESC lines maintain their potential for germline transmission after serial targeting with bacterial artificial chromosome (BAC)-based targeting vectors, and CRISPR-Cas9 cutting can increase targeting efficiency. Using these methods, we have successfully replaced entire rat genes spanning up to 101 kb with the human ortholog.
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Affiliation(s)
- Jeffrey Lee
- Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA.
| | | | - Roxanne Ally
- Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA
| | - Sean Trzaska
- Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA
| | | | - Alejo Mujica
- Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA
| | | | | | - Brian Morse
- Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA
| | - Janell Smith
- Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA
| | | | - Eric Chiao
- Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA
| | - Henry Chen
- Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA
| | | | - Ying Hu
- Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA
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27
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Men H, Davis DJ, Bryda EC. Gene Targeting in Rat Embryonic Stem Cells. Methods Mol Biol 2023; 2631:341-353. [PMID: 36995676 DOI: 10.1007/978-1-0716-2990-1_15] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/31/2023]
Abstract
Rat germline-competent embryonic stem (ES) cell lines have been available since 2008, and rat models with targeted mutations have been successfully generated using ES cell-based genome targeting technology. This chapter will focus on the procedures of gene targeting in rat ES cells.
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Affiliation(s)
- Hongsheng Men
- Rat Resource and Research Center, University of Missouri, Columbia, MO, USA.
| | - Daniel J Davis
- Animal Modeling Core, University of Missouri, Columbia, MO, USA
| | - Elizabeth C Bryda
- Rat Resource and Research Center, University of Missouri, Columbia, MO, USA
- Animal Modeling Core, University of Missouri, Columbia, MO, USA
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28
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Bryda EC, Men H, Stone BJ. Rat Embryonic Stem Cell Transgenesis. Methods Mol Biol 2023; 2631:355-370. [PMID: 36995677 DOI: 10.1007/978-1-0716-2990-1_16] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/31/2023]
Abstract
The availability of reliable germline competent rat embryonic stem cell (ESC) lines that can be genetically manipulated provides an important tool for generating new rat models. Here we describe the process for culturing rat ESCs, microinjecting the ESCs into rat blastocysts, and transferring the embryos to surrogate dams by either surgical or non-surgical embryo transfer techniques to produce chimeric animals with the potential to pass on the genetic modification to their offspring.
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Affiliation(s)
- Elizabeth C Bryda
- University of Missouri, Rat Resource and Research Center, Columbia, MO, USA.
| | - Hongsheng Men
- University of Missouri, Rat Resource and Research Center, Columbia, MO, USA
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29
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The Role of Genetically Modified Human Feeder Cells in Maintaining the Integrity of Primary Cultured Human Deciduous Dental Pulp Cells. J Clin Med 2022; 11:jcm11206087. [PMID: 36294410 PMCID: PMC9605397 DOI: 10.3390/jcm11206087] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2022] [Revised: 09/30/2022] [Accepted: 10/07/2022] [Indexed: 11/06/2022] Open
Abstract
Tissue-specific stem cells exist in tissues and organs, such as skin and bone marrow. However, their pluripotency is limited compared to embryonic stem cells. Culturing primary cells on plastic tissue culture dishes can result in the loss of multipotency, because of the inability of tissue-specific stem cells to survive in feeder-less dishes. Recent findings suggest that culturing primary cells in medium containing feeder cells, particularly genetically modified feeder cells expressing growth factors, may be beneficial for their survival and proliferation. Therefore, the aim of this study was to elucidate the role of genetically modified human feeder cells expressing growth factors in maintaining the integrity of primary cultured human deciduous dental pulp cells. Feeder cells expressing leukemia inhibitory factor, bone morphogenetic protein 4, and basic fibroblast growth factor were successfully engineered, as evidenced by PCR. Co-culturing with mitomycin-C-treated feeder cells enhanced the proliferation of newly isolated human deciduous dental pulp cells, promoted their differentiation into adipocytes and neurons, and maintained their stemness properties. Our findings suggest that genetically modified human feeder cells may be used to maintain the integrity of primary cultured human deciduous dental pulp cells.
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30
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Endoh M, Niwa H. Stepwise pluripotency transitions in mouse stem cells. EMBO Rep 2022; 23:e55010. [PMID: 35903955 PMCID: PMC9442314 DOI: 10.15252/embr.202255010] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2022] [Revised: 05/13/2022] [Accepted: 07/01/2022] [Indexed: 07/31/2023] Open
Abstract
Pluripotent cells in mouse embryos, which first emerge in the inner cell mass of the blastocyst, undergo gradual transition marked by changes in gene expression, developmental potential, polarity, and morphology as they develop from the pre-implantation until post-implantation gastrula stage. Recent studies of cultured mouse pluripotent stem cells (PSCs) have clarified the presence of intermediate pluripotent stages between the naïve pluripotent state represented by embryonic stem cells (ESCs-equivalent to the pre-implantation epiblast) and the primed pluripotent state represented by epiblast stem cells (EpiSCs-equivalent to the late post-implantation gastrula epiblast). In this review, we discuss these recent findings in light of our knowledge on peri-implantation mouse development and consider the implications of these new PSCs to understand their temporal sequence and the feasibility of using them as model system for pluripotency.
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Affiliation(s)
- Mitsuhiro Endoh
- Department of Pluripotent Stem Cell Biology, Institute of Molecular Embryology and Genetics (IMEG)Kumamoto UniversityKumamotoJapan
| | - Hitoshi Niwa
- Department of Pluripotent Stem Cell Biology, Institute of Molecular Embryology and Genetics (IMEG)Kumamoto UniversityKumamotoJapan
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31
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Carbonaro M, Lee J, Pefanis E, Desclaux M, Wang K, Pennington A, Huang H, Mujica A, Rojas J, Ally R, Kennedy D, Brown M, Rogulin V, Moller-Tank S, Sabin L, Zambrowicz B, Thurston G, Li Z. Efficient engraftment and viral transduction of human hepatocytes in an FRG rat liver humanization model. Sci Rep 2022; 12:14079. [PMID: 35982097 PMCID: PMC9388686 DOI: 10.1038/s41598-022-18119-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2022] [Accepted: 08/05/2022] [Indexed: 11/09/2022] Open
Abstract
Humanized liver rodent models, in which the host liver parenchyma is repopulated by human hepatocytes, have been increasingly used for drug development and disease research. Unlike the leading humanized liver mouse model in which Fumarylacetoacetate Hydrolase (Fah), Recombination Activating Gene (Rag)-2 and Interleukin-2 Receptor Gamma (Il2rg) genes were inactivated simultaneously, generation of similar recipient rats has been challenging. Here, using Velocigene and 1-cell-embryo-targeting technologies, we generated a rat model deficient in Fah, Rag1/2 and Il2rg genes, similar to humanized liver mice. These rats were efficiently engrafted with Fah-expressing hepatocytes from rat, mouse and human. Humanized liver rats expressed human albumin and complement proteins in serum and showed a normal liver zonation pattern. Further, approaches were developed for gene delivery through viral transduction of human hepatocytes either in vivo, or in vitro prior to engraftment, providing a novel platform to study liver disease and hepatocyte-targeted therapies.
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Affiliation(s)
| | - Jeffrey Lee
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA
| | | | | | - Kehui Wang
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA
| | | | - Hui Huang
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA
| | - Alejo Mujica
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA
| | - Jose Rojas
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA
| | - Roxanne Ally
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA
| | | | | | | | | | - Leah Sabin
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA
| | | | | | - Zhe Li
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.
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32
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Zvick J, Tarnowska-Sengül M, Ghosh A, Bundschuh N, Gjonlleshaj P, Hinte LC, Trautmann CL, Noé F, Qabrati X, Domenig SA, Kim I, Hennek T, von Meyenn F, Bar-Nur O. Exclusive generation of rat spermatozoa in sterile mice utilizing blastocyst complementation with pluripotent stem cells. Stem Cell Reports 2022; 17:1942-1958. [PMID: 35931077 PMCID: PMC9481912 DOI: 10.1016/j.stemcr.2022.07.005] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2022] [Revised: 07/06/2022] [Accepted: 07/07/2022] [Indexed: 11/17/2022] Open
Abstract
Blastocyst complementation denotes a technique that aims to generate organs, tissues, or cell types in animal chimeras via injection of pluripotent stem cells (PSCs) into genetically compromised blastocyst-stage embryos. Here, we report on successful complementation of the male germline in adult chimeras following injection of mouse or rat PSCs into mouse blastocysts carrying a mutation in Tsc22d3, an essential gene for spermatozoa production. Injection of mouse PSCs into Tsc22d3-Knockout (KO) blastocysts gave rise to intraspecies chimeras exclusively embodying PSC-derived functional spermatozoa. In addition, injection of rat embryonic stem cells (rESCs) into Tsc22d3-KO embryos produced interspecies mouse-rat chimeras solely harboring rat spermatids and spermatozoa capable of fertilizing oocytes. Furthermore, using single-cell RNA sequencing, we deconstructed rat spermatogenesis occurring in a mouse-rat chimera testis. Collectively, this study details a method for exclusive xenogeneic germ cell production in vivo, with implications that may extend to rat transgenesis, or endangered animal species conservation efforts.
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Affiliation(s)
- Joel Zvick
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Monika Tarnowska-Sengül
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Adhideb Ghosh
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland; Functional Genomics Center Zurich, ETH Zurich and University of Zurich, Zurich 8057, Switzerland
| | - Nicola Bundschuh
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Pjeter Gjonlleshaj
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Laura C Hinte
- Laboratory of Nutrition and Metabolic Epigenetics, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Christine L Trautmann
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Falko Noé
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland; Functional Genomics Center Zurich, ETH Zurich and University of Zurich, Zurich 8057, Switzerland
| | - Xhem Qabrati
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Seraina A Domenig
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Inseon Kim
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Thomas Hennek
- ETH Phenomics Center, ETH Zurich, Zurich 8049, Switzerland
| | - Ferdinand von Meyenn
- Laboratory of Nutrition and Metabolic Epigenetics, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland
| | - Ori Bar-Nur
- Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland.
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33
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Zhang J, Zhi M, Gao D, Zhu Q, Gao J, Zhu G, Cao S, Han J. Research progress and application prospects of stable porcine pluripotent stem cells. Biol Reprod 2022; 107:226-236. [PMID: 35678320 DOI: 10.1093/biolre/ioac119] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2021] [Revised: 05/26/2022] [Accepted: 05/30/2022] [Indexed: 11/14/2022] Open
Abstract
Pluripotent stem cells (PSCs) harbor the capacity of unlimited self-renewal and multi-lineage differentiation potential which are crucial for basic research and biomedical science. Establishment of PSCs with defined features were previously reported from mice and humans, while generation of stable large animal PSCs has experienced a relatively long trial stage and only recently has made breakthroughs. Pigs are regarded as ideal animal models for their similarities in physiology and anatomy to humans. Generation of porcine PSCs would provide cell resources for basic research, genetic engineering, animal breeding and cultured meat. In this review, we summarize the progress on the derivation of porcine PSCs and reprogrammed cells and elucidate the mechanisms of pluripotency changes during pig embryo development. This will be beneficial for understanding the divergence and conservation between different species involved in embryo development and the pluripotent regulated signaling pathways. Finally, we also discuss the promising future applications of stable porcine PSCs.
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Affiliation(s)
- Jinying Zhang
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Minglei Zhi
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Dengfeng Gao
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Qianqian Zhu
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Jie Gao
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Gaoxiang Zhu
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
| | - Suying Cao
- Animal Science and Technology College, Beijing University of Agriculture, Beijing, China
| | - Jianyong Han
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
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34
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Souto EP, Dobrolecki LE, Villanueva H, Sikora AG, Lewis MT. In Vivo Modeling of Human Breast Cancer Using Cell Line and Patient-Derived Xenografts. J Mammary Gland Biol Neoplasia 2022; 27:211-230. [PMID: 35697909 PMCID: PMC9433358 DOI: 10.1007/s10911-022-09520-y] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/04/2022] [Accepted: 05/19/2022] [Indexed: 11/24/2022] Open
Abstract
Historically, human breast cancer has been modeled largely in vitro using long-established cell lines primarily in two-dimensional culture, but also in three-dimensional cultures of varying cellular and molecular complexities. A subset of cell line models has also been used in vivo as cell line-derived xenografts (CDX). While outstanding for conducting detailed molecular analysis of regulatory mechanisms that may function in vivo, results of drug response studies using long-established cell lines have largely failed to translate clinically. In an attempt to address this shortcoming, many laboratories have succeeded in developing clinically annotated patient-derived xenograft (PDX) models of human cancers, including breast, in a variety of host systems. While immunocompromised mice are the predominant host, the immunocompromised rat and pig, zebrafish, as well as the chicken egg chorioallantoic membrane (CAM) have also emerged as potential host platforms to help address perceived shortcomings of immunocompromised mice. With any modeling platform, the two main issues to be resolved are criteria for "credentialing" the models as valid models to represent human cancer, and utility with respect to the ability to generate clinically relevant translational research data. Such data are beginning to emerge, particularly with the activities of PDX consortia such as the NCI PDXNet Program, EuroPDX, and the International Breast Cancer Consortium, as well as a host of pharmaceutical companies and contract research organizations (CRO). This review focuses primarily on these important aspects of PDX-related research, with a focus on breast cancer.
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Affiliation(s)
- Eric P Souto
- Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, 77030, USA
| | - Lacey E Dobrolecki
- Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, 77030, USA
| | - Hugo Villanueva
- Otolaryngology-Head and Neck Surgery, Baylor College of Medicine, Houston, TX, 77030, USA
| | - Andrew G Sikora
- Department of Head and Neck Surgery, Division of Surgery, University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA
| | - Michael T Lewis
- Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, 77030, USA.
- Departments of Molecular and Cellular Biology and Radiology, Baylor College of Medicine, Houston, TX, 77030, USA.
- Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, 77030, USA.
- Baylor College of Medicine, One Baylor Plaza, BCM-600; Room N1210, Houston, TX, 77030, USA.
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35
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Liu C, Li W. Advances in haploid embryonic stem cell research. Biol Reprod 2022; 107:250-260. [PMID: 35639627 DOI: 10.1093/biolre/ioac110] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2022] [Revised: 05/12/2022] [Accepted: 05/25/2022] [Indexed: 11/14/2022] Open
Abstract
Haploid embryonic stem cells are embryonic stem cells of a special type. Their nuclei contain one complete set of genetic material, and they are capable of self-renewal and differentiation. The emergence of haploid embryonic stem cells has aided research in functional genomics, genetic imprinting, parthenogenesis, genetic screening, and somatic cell nuclear transfer. This article reviews current issues in haploid stem cell research based on reports published in recent years and assesses the potential applications of these cells in somatic cell nuclear transfer, genome imprinting, and parthenogenesis.
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Affiliation(s)
- Chao Liu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.,Institute for Stem Cell and Regenerative Medicine, 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.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing 100101, China.,Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China.,University of Chinese Academy of Sciences, Beijing 100049, China
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36
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Abstract
Wnt signaling pathways have been extensively studied in the context of several diseases, including cancer, coronary artery disease, and age-related disorders. β-Catenin plays a central role in the most studied Wnt pathways, the Wnt/β-catenin signaling pathway, commonly referred to as the canonical Wnt signaling pathway. β-catenin is a substrate of glycogen synthase kinase 3β (GSK-3β), and the phosphorylated β-catenin by GSK-3β can be degraded by the proteasome through ubiquitination. Thus, GSK-3β inhibitors have become a widely used chemical biology tool to study the canonical Wnt signaling pathway. Among the varied GSK-3β inhibitors, a compound known as CHIR-99021 is one of the most widely used. Although these inhibitors contribute greatly to our understanding of the canonical Wnt pathway, certain pitfalls associated with such an approach may have been overlooked. In many published studies, micromolar concentrations of CHIR-99021 are used to activate the canonical Wnt pathway. Although CHIR-99021 is a specific GSK-3β inhibitor, it specifically inhibits the kinase at the nanomolar level. Therefore, caution is required when micromolar levels of CHIR-99021 are used for the purpose of activating the canonical Wnt signaling pathway.
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37
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Oikawa M, Kobayashi H, Sanbo M, Mizuno N, Iwatsuki K, Takashima T, Yamauchi K, Yoshida F, Yamamoto T, Shinohara T, Nakauchi H, Kurimoto K, Hirabayashi M, Kobayashi T. Functional primordial germ cell-like cells from pluripotent stem cells in rats. Science 2022; 376:176-179. [PMID: 35389778 DOI: 10.1126/science.abl4412] [Citation(s) in RCA: 30] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The in vitro generation of germ cells from pluripotent stem cells (PSCs) can have a substantial effect on future reproductive medicine and animal breeding. A decade ago, in vitro gametogenesis was established in the mouse. However, induction of primordial germ cell-like cells (PGCLCs) to produce gametes has not been achieved in any other species. Here, we demonstrate the induction of functional PGCLCs from rat PSCs. We show that epiblast-like cells in floating aggregates form rat PGCLCs. The gonadal somatic cells support maturation and epigenetic reprogramming of the PGCLCs. When rat PGCLCs are transplanted into the seminiferous tubules of germline-less rats, functional spermatids-that is, those capable of siring viable offspring-are generated. Insights from our rat model will elucidate conserved and divergent mechanisms essential for the broad applicability of in vitro gametogenesis.
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Affiliation(s)
- Mami Oikawa
- Division of Mammalian Embryology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.,Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan
| | - Hisato Kobayashi
- Department of Embryology, Nara Medical University, Kashihara, Nara 634-0813, Japan
| | - Makoto Sanbo
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan
| | - Naoaki Mizuno
- Division of Stem Cell Therapy, Distinguished Professor Unit, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan
| | - Kenyu Iwatsuki
- Division of Mammalian Embryology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.,Graduate School of Medicine, Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan
| | - Tomoya Takashima
- Department of Embryology, Nara Medical University, Kashihara, Nara 634-0813, Japan.,Department of Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan
| | - Keiko Yamauchi
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan
| | - Fumika Yoshida
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan
| | - Takuya Yamamoto
- Center for iPS Cell Research and Application, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.,Institute for the Advanced Study of Human Biology, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.,Medical-risk Avoidance Based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project, Sakyo-ku, Kyoto 606-8507, Japan
| | - Takashi Shinohara
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
| | - Hiromitsu Nakauchi
- Division of Stem Cell Therapy, Distinguished Professor Unit, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.,Institute for Stem Cell Biology and Regenerative Medicine, Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Kazuki Kurimoto
- Department of Embryology, Nara Medical University, Kashihara, Nara 634-0813, Japan
| | - Masumi Hirabayashi
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan.,The Graduate University of Advanced Studies, Okazaki, Aichi 444-8787, Japan
| | - Toshihiro Kobayashi
- Division of Mammalian Embryology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.,Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan
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38
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Xu M, Zhao Y, Zhang W, Geng M, Liu Q, Gao Q, Shuai L. Genome-scale screening in a rat haploid system identifies Thop1 as a modulator of pluripotency exit. Cell Prolif 2022; 55:e13209. [PMID: 35274380 PMCID: PMC9055895 DOI: 10.1111/cpr.13209] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2022] [Revised: 02/06/2022] [Accepted: 02/09/2022] [Indexed: 11/29/2022] Open
Abstract
OBJECTIVES The rats are crucial animal models for the basic medical researches. Rat embryonic stem cells (ESCs), which are widely studied, can self-renew and exhibit pluripotency in long-term culture, but the mechanism underlying how they exit pluripotency remains obscure. To investigate the key modulators on pluripotency exiting in rat ESCs, we perform genome-wide screening using a unique rat haploid system. MATERIALS AND METHODS Rat haploid ESCs (haESCs) enable advances in the discovery of unknown functional genes owing to their homozygous and pluripotent characteristics. REX1 is a sensitive marker for the naïve pluripotency that is often utilized to monitor pluripotency exit, thus rat haESCs carrying a Rex1-GFP reporter are used for genetic screening. Genome-wide mutations are introduced into the genomes of rat Rex1-GFP haESCs via piggyBac transposon, and differentiation-retarded mutants are obtained after random differentiation selection. The exact mutations are elucidated by high-throughput sequencing and bioinformatic analysis. The role of candidate mutation is validated in rat ESCs by knockout and overexpression experiments, and the phosphorylation of ERK1/2 (p-ERK1/2) is determined by western blotting. RESULTS High-throughput sequencing analysis reveals numerous insertions related to various pathways affecting random differentiation. Thereafter, deletion of Thop1 (one candidate gene in the screened list) arrests the differentiation of rat ESCs by inhibiting the p-ERK1/2, whereas overexpression of Thop1 promotes rat ESCs to exit from pluripotency. CONCLUSIONS Our findings provide an ideal tool to study functional genomics in rats: a homozygous haploid system carrying a pluripotency reporter that facilitates robust discovery of the mechanisms involved in the self-renewal or pluripotency of rat ESCs.
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Affiliation(s)
- Mei Xu
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China
| | - Yiding Zhao
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China
| | - Wenhao Zhang
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China.,Chongqing Key Laboratory of Human Embryo Engineering, Chongqing Health Center for Women and Children, Chongqing, China
| | - Mengyang Geng
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China
| | - Qian Liu
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China
| | - Qian Gao
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China
| | - Ling Shuai
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China.,Tianjin Central Hospital of Gynecology Obstetrics, Tianjin Key Laboratory of Human Development and Reproductive Regulation, Tianjin, China.,National Clinical Research Center for Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China.,Frontiers Science Center for Cell Responses, Nankai University, Tianjin, China
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39
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Sato M, Nakamura S, Inada E, Takabayashi S. Recent Advances in the Production of Genome-Edited Rats. Int J Mol Sci 2022; 23:ijms23052548. [PMID: 35269691 PMCID: PMC8910656 DOI: 10.3390/ijms23052548] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Revised: 02/17/2022] [Accepted: 02/21/2022] [Indexed: 12/14/2022] Open
Abstract
The rat is an important animal model for understanding gene function and developing human disease models. Knocking out a gene function in rats was difficult until recently, when a series of genome editing (GE) technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the type II bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Cas9 (CRISPR/Cas9) systems were successfully applied for gene modification (as exemplified by gene-specific knockout and knock-in) in the endogenous target genes of various organisms including rats. Owing to its simple application for gene modification and its ease of use, the CRISPR/Cas9 system is now commonly used worldwide. The most important aspect of this process is the selection of the method used to deliver GE components to rat embryos. In earlier stages, the microinjection (MI) of GE components into the cytoplasm and/or nuclei of a zygote was frequently employed. However, this method is associated with the use of an expensive manipulator system, the skills required to operate it, and the egg transfer (ET) of MI-treated embryos to recipient females for further development. In vitro electroporation (EP) of zygotes is next recognized as a simple and rapid method to introduce GE components to produce GE animals. Furthermore, in vitro transduction of rat embryos with adeno-associated viruses is potentially effective for obtaining GE rats. However, these two approaches also require ET. The use of gene-engineered embryonic stem cells or spermatogonial stem cells appears to be of interest to obtain GE rats; however, the procedure itself is difficult and laborious. Genome-editing via oviductal nucleic acids delivery (GONAD) (or improved GONAD (i-GONAD)) is a novel method allowing for the in situ production of GE zygotes existing within the oviductal lumen. This can be performed by the simple intraoviductal injection of GE components and subsequent in vivo EP toward the injected oviducts and does not require ET. In this review, we describe the development of various approaches for producing GE rats together with an assessment of their technical advantages and limitations, and present new GE-related technologies and current achievements using those rats in relation to human diseases.
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Affiliation(s)
- Masahiro Sato
- Department of Genome Medicine, National Center for Child Health and Development, Tokyo 157-8535, Japan
- Correspondence: (M.S.); (S.T.); Tel.: +81-3-3416-0181 (M.S.); +81-53-435-2001 (S.T.)
| | - Shingo Nakamura
- Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama 359-8513, Japan;
| | - Emi Inada
- Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan;
| | - Shuji Takabayashi
- Laboratory Animal Facilities & Services, Preeminent Medical Photonics Education & Research Center, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan
- Correspondence: (M.S.); (S.T.); Tel.: +81-3-3416-0181 (M.S.); +81-53-435-2001 (S.T.)
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40
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Asano K, Takahashi Y, Ueno M, Fukuda T, Otani M, Kitamoto S, Tomigahara Y. Lack of human relevance for rat developmental toxicity of flumioxazin is revealed by comparative heme synthesis assay using embryonic erythroid cells derived from human and rat pluripotent stem cells. J Toxicol Sci 2022; 47:125-138. [DOI: 10.2131/jts.47.125] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Affiliation(s)
- Koji Asano
- Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd
| | | | - Manako Ueno
- Bioscience Research Laboratory, Sumitomo Chemical Co., Ltd
| | - Takako Fukuda
- Bioscience Research Laboratory, Sumitomo Chemical Co., Ltd
| | - Mitsuhiro Otani
- Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd
| | - Sachiko Kitamoto
- Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd
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41
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Porcine OCT4 reporter system as a tool for monitoring pluripotency states. JOURNAL OF ANIMAL REPRODUCTION AND BIOTECHNOLOGY 2021. [DOI: 10.12750/jarb.36.4.175] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
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42
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Yeh CY, Huang WH, Chen HC, Meir YJJ. Capturing Pluripotency and Beyond. Cells 2021; 10:cells10123558. [PMID: 34944066 PMCID: PMC8700150 DOI: 10.3390/cells10123558] [Citation(s) in RCA: 6] [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] [Received: 09/16/2021] [Revised: 12/13/2021] [Accepted: 12/14/2021] [Indexed: 11/16/2022] Open
Abstract
During the development of a multicellular organism, the specification of different cell lineages originates in a small group of pluripotent cells, the epiblasts, formed in the preimplantation embryo. The pluripotent epiblast is protected from premature differentiation until exposure to inductive cues in strictly controlled spatially and temporally organized patterns guiding fetus formation. Epiblasts cultured in vitro are embryonic stem cells (ESCs), which recapitulate the self-renewal and lineage specification properties of their endogenous counterparts. The characteristics of totipotency, although less understood than pluripotency, are becoming clearer. Recent studies have shown that a minor ESC subpopulation exhibits expanded developmental potential beyond pluripotency, displaying a characteristic reminiscent of two-cell embryo blastomeres (2CLCs). In addition, reprogramming both mouse and human ESCs in defined media can produce expanded/extended pluripotent stem cells (EPSCs) similar to but different from 2CLCs. Further, the molecular roadmaps driving the transition of various potency states have been clarified. These recent key findings will allow us to understand eutherian mammalian development by comparing the underlying differences between potency network components during development. Using the mouse as a paradigm and recent progress in human PSCs, we review the epiblast's identity acquisition during embryogenesis and their ESC counterparts regarding their pluripotent fates and beyond.
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Affiliation(s)
- Chih-Yu Yeh
- Department of Medicine, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan; (C.-Y.Y.); (W.-H.H.)
| | - Wei-Han Huang
- Department of Medicine, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan; (C.-Y.Y.); (W.-H.H.)
| | - Hung-Chi Chen
- Department of Medicine, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan; (C.-Y.Y.); (W.-H.H.)
- Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Linkou 333, Taiwan
- Correspondence: (H.-C.C.); (Y.-J.J.M.)
| | - Yaa-Jyuhn James Meir
- Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Linkou 333, Taiwan
- Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan
- Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan
- Correspondence: (H.-C.C.); (Y.-J.J.M.)
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43
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Kinoshita M, Kobayashi T, Planells B, Klisch D, Spindlow D, Masaki H, Bornelöv S, Stirparo GG, Matsunari H, Uchikura A, Lamas-Toranzo I, Nichols J, Nakauchi H, Nagashima H, Alberio R, Smith A. Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species. Development 2021; 148:273644. [PMID: 34874452 PMCID: PMC8714072 DOI: 10.1242/dev.199901] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2021] [Accepted: 10/26/2021] [Indexed: 12/12/2022]
Abstract
Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated ‘The people behind the papers’ interview. Summary: We report the derivation of similar, stable and continuously expandable pluripotent stem cells related to embryonic disc epiblast from embryos of pig, sheep and cow.
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Affiliation(s)
- Masaki Kinoshita
- Wellcome-MRC Cambridge Stem Cell Institute, Jeffery Cheah Biomedical Centre, University of Cambridge, Cambridge CB2 0AW, UK
| | - Toshihiro Kobayashi
- Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan.,Division of Mammalian Embryology, Centre for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan
| | - Benjamin Planells
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham LE12 5RD, UK
| | - Doris Klisch
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham LE12 5RD, UK
| | - Daniel Spindlow
- Wellcome-MRC Cambridge Stem Cell Institute, Jeffery Cheah Biomedical Centre, University of Cambridge, Cambridge CB2 0AW, UK.,Living Systems Institute, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
| | - Hideki Masaki
- Division of Stem Cell Therapy, Distinguished Professor Unit, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan
| | - Susanne Bornelöv
- Wellcome-MRC Cambridge Stem Cell Institute, Jeffery Cheah Biomedical Centre, University of Cambridge, Cambridge CB2 0AW, UK
| | - Giuliano Giuseppe Stirparo
- Wellcome-MRC Cambridge Stem Cell Institute, Jeffery Cheah Biomedical Centre, University of Cambridge, Cambridge CB2 0AW, UK.,Living Systems Institute, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
| | - Hitomi Matsunari
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama, Kawasaki 214-8571, Japan
| | - Ayuko Uchikura
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama, Kawasaki 214-8571, Japan
| | - Ismael Lamas-Toranzo
- Wellcome-MRC Cambridge Stem Cell Institute, Jeffery Cheah Biomedical Centre, University of Cambridge, Cambridge CB2 0AW, UK.,School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham LE12 5RD, UK
| | - Jennifer Nichols
- Wellcome-MRC Cambridge Stem Cell Institute, Jeffery Cheah Biomedical Centre, University of Cambridge, Cambridge CB2 0AW, UK.,Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 1GA, UK
| | - Hiromitsu Nakauchi
- Division of Stem Cell Therapy, Distinguished Professor Unit, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.,Institute for Stem Cell Biology and Regenerative Medicine, Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305USA
| | - Hiroshi Nagashima
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama, Kawasaki 214-8571, Japan
| | - Ramiro Alberio
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham LE12 5RD, UK
| | - Austin Smith
- Wellcome-MRC Cambridge Stem Cell Institute, Jeffery Cheah Biomedical Centre, University of Cambridge, Cambridge CB2 0AW, UK.,Living Systems Institute, University of Exeter, Stocker Road, Exeter EX4 4QD, UK
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44
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Inhibition of ubiquitin-specific protease 13-mediated degradation of Raf1 kinase by Spautin-1 has opposing effects in naïve and primed pluripotent stem cells. J Biol Chem 2021; 297:101332. [PMID: 34688658 PMCID: PMC8577099 DOI: 10.1016/j.jbc.2021.101332] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2021] [Revised: 10/10/2021] [Accepted: 10/19/2021] [Indexed: 11/20/2022] Open
Abstract
Embryonic stem cells (ESCs) are progenitor cells that retain the ability to differentiate into various cell types and are necessary for tissue repair. Improving cell culture conditions to maintain the pluripotency of ESCs in vitro is an urgent problem in the field of regenerative medicine. Here, we reveal that Spautin-1, a specific small-molecule inhibitor of ubiquitin-specific protease (USP) family members USP10 and USP13, promotes the maintenance of self-renewal and pluripotency of mouse ESCs in vitro. Functional studies reveal that only knockdown of USP13, but not USP10, is capable of mimicking the function of Spautin-1. Mechanistically, we demonstrate that USP13 physically interacts with, deubiquitinates, and stabilizes serine/threonine kinase Raf1 and thereby sustains Raf1 protein at the posttranslational level to activate the FGF/MEK/ERK prodifferentiation signaling pathway in naïve mouse ESCs. In contrast, in primed mouse epiblast stem cells and human induced pluripotent stem cells, the addition of Spautin-1 had an inhibitory effect on Raf1 levels, but USP13 overexpression promoted self-renewal. The addition of an MEK inhibitor impaired the effect of USP13 upregulation in these cells. These findings provide new insights into the regulatory network of naïve and primed pluripotency.
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45
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Abstract
[Figure: see text].
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Affiliation(s)
- Mitinori Saitou
- Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.,Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.,Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
| | - Katsuhiko Hayashi
- Department of Developmental Stem Cell Biology, Faculty of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.,Department of Germline Genetics, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
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46
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Gerri C, Menchero S, Mahadevaiah SK, Turner JMA, Niakan KK. Human Embryogenesis: A Comparative Perspective. Annu Rev Cell Dev Biol 2021; 36:411-440. [PMID: 33021826 DOI: 10.1146/annurev-cellbio-022020-024900] [Citation(s) in RCA: 39] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Understanding human embryology has historically relied on comparative approaches using mammalian model organisms. With the advent of low-input methods to investigate genetic and epigenetic mechanisms and efficient techniques to assess gene function, we can now study the human embryo directly. These advances have transformed the investigation of early embryogenesis in nonrodent species, thereby providing a broader understanding of conserved and divergent mechanisms. Here, we present an overview of the major events in human preimplantation development and place them in the context of mammalian evolution by comparing these events in other eutherian and metatherian species. We describe the advances of studies on postimplantation development and discuss stem cell models that mimic postimplantation embryos. A comparative perspective highlights the importance of analyzing different organisms with molecular characterization and functional studies to reveal the principles of early development. This growing field has a fundamental impact in regenerative medicine and raises important ethical considerations.
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Affiliation(s)
- Claudia Gerri
- Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom;
| | - Sergio Menchero
- Sex Chromosome Biology Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom;
| | - Shantha K Mahadevaiah
- Sex Chromosome Biology Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom;
| | - James M A Turner
- Sex Chromosome Biology Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom;
| | - Kathy K Niakan
- Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom;
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47
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Sherstyuk VV, Zakian SM. Generation of Transgenic Rat Embryonic Stem Cells Using the CRISPR/Cpf1 System for Inducible Gene Knockout. BIOCHEMISTRY (MOSCOW) 2021; 86:843-851. [PMID: 34284709 DOI: 10.1134/s0006297921070051] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Rat embryonic stem cells (ESCs) play an important role in the studies of genes involved in maintaining of pluripotent state and early development of this model organism. To study functions of the essential genes, as well as the processes of cell differentiation, the method of induced knockout is widely used. The CreERT2/loxP system allows obtaining an inducible knockout in cells expressing tamoxifen-inducible Cre recombinase (CreERT2) and containing loxP sites flanking the target gene by adding 4-hydroxy tamoxifen to the culture medium. However, the rat ESC lines expressing CreERT2 are absent. In this work, we tested three CRISPR/Cas systems for introduction of double-strand breaks into the Rosa26 locus in the rat ESCs and inserted tamoxifen-dependent Cre recombinase into this locus using the CRISPR/Cpf1 system. It was shown that the obtained transgenic rat ESC lines retained the characteristics of pluripotent cells. Tamoxifen-inducible Cre recombinase activity was analyzed using a reporter vector.
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Affiliation(s)
- Vladimir V Sherstyuk
- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.
| | - Suren M Zakian
- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia
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48
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Liu C, Cui Z, Yan Y, Wu NL, Li L, Ying Q, Peng L. An optimized proliferation system of embryonic stem cells for generating the rat model with large fragment modification. Biochem Biophys Res Commun 2021; 571:8-13. [PMID: 34298338 DOI: 10.1016/j.bbrc.2021.07.053] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2021] [Accepted: 07/15/2021] [Indexed: 11/25/2022]
Abstract
Rats have long been an ideal model for disease research in the field of biomedicine, but the bottleneck of in vitro culture of rat embryonic stem (ES) cells hindered the wide application as genetic disease models. Here, we optimized a special medium which we named 5N-medium for rat embryonic stem cells, which improved the in vitro cells with better morphology and higher pluripotency. We then established a drug selection schedule harboring a prior selection of 12 h that achieved a higher positive selection ratio. These treatments induced at least 50% increase of homologous recombination efficiency compared with conventional 2i culture condition. Moreover, the ratio of euploid ES clones also increased by 50% with a higher germline transmission rate. Finally, we successfully knocked in a 175 kb human Bacterial Artificial Chromosome (BAC) fragment to rat ES genome through recombinase mediated cassette exchange (RMCE). Hence, we provide a promising system for generating sophisticated rat models which could be benefit for biomedical researches.
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Affiliation(s)
- Chang Liu
- Key Laboratory of Arrhythmias, Ministry of Education of China, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Department of Medical Genetics, Tongji University School of Medicine, Shanghai, 200092, China
| | - Zhonglin Cui
- Division of Hepatobiliopancreatic Surgery, Department of General Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, 510515, China; Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA
| | - Youzhen Yan
- USC/Norris Cancer Center Transgenic/Knockout Rodent Core Facility, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA
| | - Nancy L Wu
- USC/Norris Cancer Center Transgenic/Knockout Rodent Core Facility, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA
| | - Li Li
- Key Laboratory of Arrhythmias, Ministry of Education of China, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Department of Medical Genetics, Tongji University School of Medicine, Shanghai, 200092, China; Research Units of Origin and Regulation of Heart Rhythm, Chinese Academy of Medical Sciences, Beijing, 100730, China.
| | - Qilong Ying
- Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA; USC/Norris Cancer Center Transgenic/Knockout Rodent Core Facility, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033, USA.
| | - Luying Peng
- Key Laboratory of Arrhythmias, Ministry of Education of China, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Department of Medical Genetics, Tongji University School of Medicine, Shanghai, 200092, China; Research Units of Origin and Regulation of Heart Rhythm, Chinese Academy of Medical Sciences, Beijing, 100730, China.
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Mao Y, Wang L, Zhong B, Yang N, Li Z, Cui T, Feng G, Li W, Zhang Y, Zhou Q. Continuous expression of reprogramming factors induces and maintains mouse pluripotency without specific growth factors and signaling inhibitors. Cell Prolif 2021; 54:e13090. [PMID: 34197016 PMCID: PMC8349648 DOI: 10.1111/cpr.13090] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2021] [Revised: 05/06/2021] [Accepted: 06/09/2021] [Indexed: 12/11/2022] Open
Abstract
Objectives Derivation and maintenance of pluripotent stem cells (PSCs) generally require optimized and complex culture media, which hinders the derivation of PSCs from various species. Expression of Oct4, Sox2, Klf4, and c‐Myc (OSKM) can reprogram somatic cells into induced PSCs (iPSCs), even for species possessing no optimal culture condition. Herein, we explored whether expression of OSKM could induce and maintain pluripotency without PSC‐specific growth factors and signaling inhibitors. Methods The culture medium of Tet‐On‐OSKM/Oct4‐GFP mouse embryonic stem cells (ESCs) was switched from N2B27 with MEK inhibitor, GSK3β inhibitor, and leukemia inhibitory factor (LIF) (2iL) to N2B27 with doxycycline. Tet‐On‐OSKM mouse embryonic fibroblast (MEF) cells were reprogrammed in N2B27 with doxycycline. Cell proliferation was traced. Pluripotency was assessed by expression of ESC marker genes, teratoma, and chimera formation. RNA‐Seq was conducted to analyze gene expression. Results Via continuous expression of OSKM, mouse ESCs (OSKM‐ESCs) and the resulting iPSCs (OSKM‐iPSCs) reprogrammed from MEF cells propagated stably, expressed pluripotency marker genes, and formed three germ layers in teratomas. Transcriptional landscapes of OSKM‐iPSCs resembled those of ESCs cultured in 2iL and were more similar to those of ESCs cultured in serum/LIF. Furthermore, OSKM‐iPSCs contributed to germline transmission. Conclusions Expression of OSKM could induce and maintain mouse pluripotency without specific culturing factors. Importantly, OSKM‐iPSCs could produce gene‐modified animals through germline transmission, with potential applications in other species.
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Affiliation(s)
- Yihuan Mao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Libin Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China.,Beijing Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China
| | - Bei Zhong
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.,College of Life Science, Northeast Agricultural University of China, Harbin, China
| | - Ning Yang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Zhikun Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China.,Beijing Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China
| | - Tongtong Cui
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China.,Beijing Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China
| | - Guihai Feng
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China.,Beijing Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China
| | - Wei Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China.,University of Chinese Academy of Sciences, Beijing, China.,Beijing Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China
| | - Ying Zhang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China.,Beijing Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China
| | - Qi Zhou
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.,Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China.,University of Chinese Academy of Sciences, Beijing, China.,Beijing Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China
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Zheng C, Ballard EB, Wu J. The road to generating transplantable organs: from blastocyst complementation to interspecies chimeras. Development 2021; 148:dev195792. [PMID: 34132325 PMCID: PMC10656466 DOI: 10.1242/dev.195792] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Growing human organs in animals sounds like something from the realm of science fiction, but it may one day become a reality through a technique known as interspecies blastocyst complementation. This technique, which was originally developed to study gene function in development, involves injecting donor pluripotent stem cells into an organogenesis-disabled host embryo, allowing the donor cells to compensate for missing organs or tissues. Although interspecies blastocyst complementation has been achieved between closely related species, such as mice and rats, the situation becomes much more difficult for species that are far apart on the evolutionary tree. This is presumably because of layers of xenogeneic barriers that are a result of divergent evolution. In this Review, we discuss the current status of blastocyst complementation approaches and, in light of recent progress, elaborate on the keys to success for interspecies blastocyst complementation and organ generation.
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Affiliation(s)
- Canbin Zheng
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
- Department of Microsurgery, Orthopaedic Trauma and Hand Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China
| | - Emily B. Ballard
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
- Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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