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1
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Moorman A, Benitez EK, Cambulli F, Jiang Q, Mahmoud A, Lumish M, Hartner S, Balkaran S, Bermeo J, Asawa S, Firat C, Saxena A, Wu F, Luthra A, Burdziak C, Xie Y, Sgambati V, Luckett K, Li Y, Yi Z, Masilionis I, Soares K, Pappou E, Yaeger R, Kingham TP, Jarnagin W, Paty PB, Weiser MR, Mazutis L, D'Angelica M, Shia J, Garcia-Aguilar J, Nawy T, Hollmann TJ, Chaligné R, Sanchez-Vega F, Sharma R, Pe'er D, Ganesh K. Progressive plasticity during colorectal cancer metastasis. Nature 2025; 637:947-954. [PMID: 39478232 PMCID: PMC11754107 DOI: 10.1038/s41586-024-08150-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2023] [Accepted: 10/02/2024] [Indexed: 11/06/2024]
Abstract
As cancers progress, they become increasingly aggressive-metastatic tumours are less responsive to first-line therapies than primary tumours, they acquire resistance to successive therapies and eventually cause death1,2. Mutations are largely conserved between primary and metastatic tumours from the same patients, suggesting that non-genetic phenotypic plasticity has a major role in cancer progression and therapy resistance3-5. However, we lack an understanding of metastatic cell states and the mechanisms by which they transition. Here, in a cohort of biospecimen trios from same-patient normal colon, primary and metastatic colorectal cancer, we show that, although primary tumours largely adopt LGR5+ intestinal stem-like states, metastases display progressive plasticity. Cancer cells lose intestinal cell identities and reprogram into a highly conserved fetal progenitor state before undergoing non-canonical differentiation into divergent squamous and neuroendocrine-like states, a process that is exacerbated in metastasis and by chemotherapy and is associated with poor patient survival. Using matched patient-derived organoids, we demonstrate that metastatic cells exhibit greater cell-autonomous multilineage differentiation potential in response to microenvironment cues compared with their intestinal lineage-restricted primary tumour counterparts. We identify PROX1 as a repressor of non-intestinal lineage in the fetal progenitor state, and show that downregulation of PROX1 licenses non-canonical reprogramming.
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Affiliation(s)
- Andrew Moorman
- Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Elizabeth K Benitez
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program, New York, NY, USA
| | - Francesco Cambulli
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- New York Genome Center, New York, NY, USA
| | - Qingwen Jiang
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Ahmed Mahmoud
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Pharmacology Program, Weill Cornell Graduate School, New York, NY, USA
| | - Melissa Lumish
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Case Western Reserve University, Cleveland, OH, USA
| | - Saskia Hartner
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Sasha Balkaran
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Jonathan Bermeo
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Simran Asawa
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Canan Firat
- Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Asha Saxena
- Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Fan Wu
- Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Anisha Luthra
- Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Cassandra Burdziak
- Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Yubin Xie
- Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Tri-Institutional PhD Program in Computational Biology and Medicine, New York, NY, USA
| | - Valeria Sgambati
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Kathleen Luckett
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program, New York, NY, USA
| | - Yanyun Li
- Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Bristol Myers Squibb, Princeton, NJ, USA
| | - Zhifan Yi
- Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Ignas Masilionis
- Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Kevin Soares
- Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Emmanouil Pappou
- Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Rona Yaeger
- Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - T Peter Kingham
- Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - William Jarnagin
- Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Philip B Paty
- Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Martin R Weiser
- Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Linas Mazutis
- Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Michael D'Angelica
- Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Jinru Shia
- Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Julio Garcia-Aguilar
- Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Tal Nawy
- Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Travis J Hollmann
- Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Bristol Myers Squibb, Princeton, NJ, USA
| | - Ronan Chaligné
- Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | | | - Roshan Sharma
- Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Dana Pe'er
- Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
- Howard Hughes Medical Institute, Chevy Chase, MD, USA.
| | - Karuna Ganesh
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
- Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
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2
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Sharma P, Chaudhary NR, Devi S, Negi S, Tandel N, Tyagi RK. Oleuropein mediated autophagy begets antimalarial drug resistance. Front Microbiol 2024; 15:1453998. [PMID: 39228384 PMCID: PMC11369837 DOI: 10.3389/fmicb.2024.1453998] [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: 06/24/2024] [Accepted: 08/02/2024] [Indexed: 09/05/2024] Open
Abstract
Drug resistance in Plasmodium falciparum presents a formidable challenge to the humanity. And, unavailability of an effective vaccine worsens the situation further. Autophagy is one of the mechanisms employed by parasite to evade drug pressure to survive. Autophagy induced by the P. falciparum in response to the oleuropein pressure may answer many questions related to the parasite survival as well as evolving drug tolerance. The survival/autophagy axis could be an important avenue to explore in order to address certain questions related to the evolution of drug resistance. In addition, humanized mouse model of P. falciparum infection could serve as an important preclinical tool to investigate the oleuropein-induced autophagy, potentially helping to dissect the mechanisms underlying the development of antimalarial drug resistance.
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Affiliation(s)
- Prakriti Sharma
- Division of Cell Biology and Immunology, Biomedical Parasitology and Translational-Immunology Lab, CSIR Institute of Microbial Technology (IMTECH), Chandigarh, India
| | - Neil Roy Chaudhary
- Division of Cell Biology and Immunology, Biomedical Parasitology and Translational-Immunology Lab, CSIR Institute of Microbial Technology (IMTECH), Chandigarh, India
| | - Sonia Devi
- Division of Cell Biology and Immunology, Biomedical Parasitology and Translational-Immunology Lab, CSIR Institute of Microbial Technology (IMTECH), Chandigarh, India
- Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India
| | - Sushmita Negi
- Division of Cell Biology and Immunology, Biomedical Parasitology and Translational-Immunology Lab, CSIR Institute of Microbial Technology (IMTECH), Chandigarh, India
- Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India
| | - Nikunj Tandel
- Institute of Science, Nirma University, Ahmedabad, Gujarat, India
| | - Rajeev K. Tyagi
- Division of Cell Biology and Immunology, Biomedical Parasitology and Translational-Immunology Lab, CSIR Institute of Microbial Technology (IMTECH), Chandigarh, India
- Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India
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3
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Moorman AR, Cambuli F, Benitez EK, Jiang Q, Xie Y, Mahmoud A, Lumish M, Hartner S, Balkaran S, Bermeo J, Asawa S, Firat C, Saxena A, Luthra A, Sgambati V, Luckett K, Wu F, Li Y, Yi Z, Masilionis I, Soares K, Pappou E, Yaeger R, Kingham P, Jarnagin W, Paty P, Weiser MR, Mazutis L, D'Angelica M, Shia J, Garcia-Aguilar J, Nawy T, Hollmann TJ, Chaligné R, Sanchez-Vega F, Sharma R, Pe'er D, Ganesh K. Progressive plasticity during colorectal cancer metastasis. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.08.18.553925. [PMID: 37662289 PMCID: PMC10473595 DOI: 10.1101/2023.08.18.553925] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/05/2023]
Abstract
Metastasis is the principal cause of cancer death, yet we lack an understanding of metastatic cell states, their relationship to primary tumor states, and the mechanisms by which they transition. In a cohort of biospecimen trios from same-patient normal colon, primary and metastatic colorectal cancer, we show that while primary tumors largely adopt LGR5 + intestinal stem-like states, metastases display progressive plasticity. Loss of intestinal cell states is accompanied by reprogramming into a highly conserved fetal progenitor state, followed by non-canonical differentiation into divergent squamous and neuroendocrine-like states, which is exacerbated by chemotherapy and associated with poor patient survival. Using matched patient-derived organoids, we demonstrate that metastatic cancer cells exhibit greater cell-autonomous multilineage differentiation potential in response to microenvironment cues than their intestinal lineage-restricted primary tumor counterparts. We identify PROX1 as a stabilizer of intestinal lineage in the fetal progenitor state, whose downregulation licenses non-canonical reprogramming.
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Kutle I, Dittrich A, Wirth D. Mouse Models for Human Herpesviruses. Pathogens 2023; 12:953. [PMID: 37513800 PMCID: PMC10384569 DOI: 10.3390/pathogens12070953] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2023] [Revised: 07/13/2023] [Accepted: 07/15/2023] [Indexed: 07/30/2023] Open
Abstract
More than one hundred herpesviruses have been isolated from different species so far, with nine infecting humans. Infections with herpesviruses are characterized by life-long latency and represent a significant challenge for human health. To investigate the consequences of infections and identify novel treatment options, in vivo models are of particular relevance. The mouse has emerged as an economical small animal model to investigate herpesvirus infections. However, except for herpes simplex viruses (HSV-1, HSV-2), human herpesviruses cannot infect mice. Three natural herpesviruses have been identified in mice: mouse-derived cytomegalovirus (MCMV), mouse herpesvirus 68 (MHV-68), and mouse roseolovirus (MRV). These orthologues are broadly used to investigate herpesvirus infections within the natural host. In the last few decades, immunocompromised mouse models have been developed, allowing the functional engraftment of various human cells and tissues. These xenograft mice represent valuable model systems to investigate human-restricted viruses, making them particularly relevant for herpesvirus research. In this review, we describe the various mouse models used to study human herpesviruses, thereby highlighting their potential and limitations. Emphasis is laid on xenograft mouse models, covering the development and refinement of immune-compromised mice and their application in herpesvirus research.
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Affiliation(s)
- Ivana Kutle
- Research Group Model Systems for Infection, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany
- Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany
| | - Anne Dittrich
- Research Group Model Systems for Infection, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany
- InSCREENeX GmbH, Inhoffenstraße 7, 38124 Braunschweig, Germany
| | - Dagmar Wirth
- Research Group Model Systems for Infection, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany
- Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany
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5
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Karnik I, Her Z, Neo SH, Liu WN, Chen Q. Emerging Preclinical Applications of Humanized Mouse Models in the Discovery and Validation of Novel Immunotherapeutics and Their Mechanisms of Action for Improved Cancer Treatment. Pharmaceutics 2023; 15:1600. [PMID: 37376049 DOI: 10.3390/pharmaceutics15061600] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2023] [Revised: 05/22/2023] [Accepted: 05/24/2023] [Indexed: 06/29/2023] Open
Abstract
Cancer therapeutics have undergone immense research over the past decade. While chemotherapies remain the mainstay treatments for many cancers, the advent of new molecular techniques has opened doors for more targeted modalities towards cancer cells. Although immune checkpoint inhibitors (ICIs) have demonstrated therapeutic efficacy in treating cancer, adverse side effects related to excessive inflammation are often reported. There is a lack of clinically relevant animal models to probe the human immune response towards ICI-based interventions. Humanized mouse models have emerged as valuable tools for pre-clinical research to evaluate the efficacy and safety of immunotherapy. This review focuses on the establishment of humanized mouse models, highlighting the challenges and recent advances in these models for targeted drug discovery and the validation of therapeutic strategies in cancer treatment. Furthermore, the potential of these models in the process of uncovering novel disease mechanisms is discussed.
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Affiliation(s)
- Isha Karnik
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore 138673, Singapore
- Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
| | - Zhisheng Her
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore 138673, Singapore
| | - Shu Hui Neo
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore 138673, Singapore
| | - Wai Nam Liu
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore 138673, Singapore
| | - Qingfeng Chen
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore 138673, Singapore
- Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
- Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos, Singapore 138648, Singapore
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Chen J, Liao S, Xiao Z, Pan Q, Wang X, Shen K, Wang S, Yang L, Guo F, Liu HF, Pan Q. The development and improvement of immunodeficient mice and humanized immune system mouse models. Front Immunol 2022; 13:1007579. [PMID: 36341323 PMCID: PMC9626807 DOI: 10.3389/fimmu.2022.1007579] [Citation(s) in RCA: 61] [Impact Index Per Article: 20.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Accepted: 10/07/2022] [Indexed: 12/02/2022] Open
Abstract
Animal models play an indispensable role in the study of human diseases. However, animal models of different diseases do not fully mimic the complex internal environment of humans. Immunodeficient mice are deficient in certain genes and do not express these or show reduced expression in some of their cells, facilitating the establishment of humanized mice and simulation of the human environment in vivo. Here, we summarize the developments in immunodeficient mice, from the initial nude mice lacking T lymphocytes to NOD/SCID rgnull mice lacking T, B, and NK cell populations. We describe existing humanized immune system mouse models based on immunodeficient mice in which human cells or tissues have been transplanted to establish a human immune system, including humanized-peripheral blood mononuclear cells (Hu-PBMCs), humanized hematopoietic stem cells (Hu-HSCs), and humanized bone marrow, liver, thymus (Hu-BLT) mouse models. The different methods for their development involve varying levels of complexity and humanization. Humanized mice are widely used in the study of various diseases to provide a transitional stage for clinical research. However, several challenges persist, including improving the efficiency of reconstructing the human B cell immune response, extending lifespan, improving the survival rate of mice to extend the observation period, and improving the development of standardized commercialized models and as well as their use. Overall, there are many opportunities and challenges in the development of humanized immune system mouse models which can provide novel strategies for understanding the mechanisms and treatments of human disease.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | | | - Qingjun Pan
- *Correspondence: Hua-feng Liu, ; Qingjun Pan,
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7
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Patient-derived tumor models are attractive tools to repurpose drugs for ovarian cancer treatment: Pre-clinical updates. Oncotarget 2022; 13:553-575. [PMID: 35359749 PMCID: PMC8959092 DOI: 10.18632/oncotarget.28220] [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: 01/12/2022] [Accepted: 03/08/2022] [Indexed: 11/29/2022] Open
Abstract
Despite advances in understanding of ovarian cancer biology, the progress in translation of research findings into new therapies is still slow. It is associated in part with limitations of commonly used cancer models such as cell lines and genetically engineered mouse models that lack proper representation of diversity and complexity of actual human tumors. In addition, the development of de novo anticancer drugs is a lengthy and expensive process. A promising alternative to new drug development is repurposing existing FDA-approved drugs without primary oncological purpose. These approved agents have known pharmacokinetics, pharmacodynamics, and toxicology and could be approved as anticancer drugs quicker and at lower cost. To successfully translate repurposed drugs to clinical application, an intermediate step of pre-clinical animal studies is required. To address challenges associated with reliability of tumor models for pre-clinical studies, there has been an increase in development of patient-derived xenografts (PDXs), which retain key characteristics of the original patient’s tumor, including histologic, biologic, and genetic features. The expansion and utilization of clinically and molecularly annotated PDX models derived from different ovarian cancer subtypes could substantially aid development of new therapies or rapid approval of repurposed drugs to improve treatment options for ovarian cancer patients.
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Kim YY, Kim JS, Che JH, Ku SY, Kang BC, Yun JW. Comparison of Genetically Engineered Immunodeficient Animal Models for Nonclinical Testing of Stem Cell Therapies. Pharmaceutics 2021; 13:130. [PMID: 33498509 PMCID: PMC7909568 DOI: 10.3390/pharmaceutics13020130] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 01/12/2021] [Accepted: 01/18/2021] [Indexed: 12/23/2022] Open
Abstract
For the recovery or replacement of dysfunctional cells and tissue-the goal of stem cell research-successful engraftment of transplanted cells and tissues are essential events. The event is largely dependent on the immune rejection of the recipient; therefore, the immunogenic evaluation of candidate cells or tissues in immunodeficient animals is important. Understanding the immunodeficient system can provide insights into the generation and use of immunodeficient animal models, presenting a unique system to explore the capabilities of the innate immune system. In this review, we summarize various immunodeficient animal model systems with different target genes as valuable tools for biomedical research. There have been numerous immunodeficient models developed by different gene defects, resulting in many different features in phenotype. More important, mice, rats, and other large animals exhibit very different immunological and physiological features in tissue and organs, including genetic background and a representation of human disease conditions. Therefore, the findings from this review may guide researchers to select the most appropriate immunodeficient strain, target gene, and animal species based on the research type, mutant gene effects, and similarity to human immunological features for stem cell research.
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Affiliation(s)
- Yoon-Young Kim
- Department of Obstetrics and Gynecology, Seoul National University Hospital, Seoul 03080, Korea; (Y.-Y.K.); (S.-Y.K.)
| | - Jin-Soo Kim
- Department of Medical and Biological Sciences, The Catholic University of Korea, Bucheon 14662, Korea;
| | - Jeong-Hwan Che
- Biomedical Center for Animal Resource and Development, Seoul National University College of Medicine, Seoul 03080, Korea;
| | - Seung-Yup Ku
- Department of Obstetrics and Gynecology, Seoul National University Hospital, Seoul 03080, Korea; (Y.-Y.K.); (S.-Y.K.)
| | - Byeong-Cheol Kang
- Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul 03080, Korea
| | - Jun-Won Yun
- Department of Medical and Biological Sciences, The Catholic University of Korea, Bucheon 14662, Korea;
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A human SIRPA knock-in xenograft mouse model to study human hematopoietic and cancer stem cells. Blood 2020; 135:1661-1672. [PMID: 32206775 DOI: 10.1182/blood.2019002194] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2019] [Accepted: 02/20/2020] [Indexed: 12/15/2022] Open
Abstract
In human-to-mouse xenogeneic transplantation, polymorphisms of signal-regulatory protein α (SIRPA) that decide their binding affinity for human CD47 are critical for engraftment efficiency of human cells. In this study, we generated a new C57BL/6.Rag2nullIl2rgnull (BRG) mouse line with Sirpahuman/human (BRGShuman) mice, in which mouse Sirpa was replaced by human SIRPA encompassing all 8 exons. Macrophages from C57BL/6 mice harboring Sirpahuman/human had a significantly stronger affinity for human CD47 than those harboring SirpaNOD/NOD and did not show detectable phagocytosis against human hematopoietic stem cells. In turn, Sirpahuman/human macrophages had a moderate affinity for mouse CD47, and BRGShuman mice did not exhibit the blood cytopenia that was seen in Sirpa-/- mice. In human to mouse xenograft experiments, BRGShuman mice showed significantly greater engraftment and maintenance of human hematopoiesis with a high level of myeloid reconstitution, as well as improved reconstitution in peripheral tissues, compared with BRG mice harboring SirpaNOD/NOD (BRGSNOD). BRGShuman mice also showed significantly enhanced engraftment and growth of acute myeloid leukemia and subcutaneously transplanted human colon cancer cells compared with BRGSNOD mice. BRGShuman mice should be a useful basic line for establishing a more authentic xenotransplantation model to study normal and malignant human stem cells.
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10
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Bürtin F, Mullins CS, Linnebacher M. Mouse models of colorectal cancer: Past, present and future perspectives. World J Gastroenterol 2020; 26:1394-1426. [PMID: 32308343 PMCID: PMC7152519 DOI: 10.3748/wjg.v26.i13.1394] [Citation(s) in RCA: 103] [Impact Index Per Article: 20.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/18/2019] [Revised: 03/05/2020] [Accepted: 03/10/2020] [Indexed: 02/06/2023] Open
Abstract
Colorectal cancer (CRC) is the third most common diagnosed malignancy among both sexes in the United States as well as in the European Union. While the incidence and mortality rates in western, high developed countries are declining, reflecting the success of screening programs and improved treatment regimen, a rise of the overall global CRC burden can be observed due to lifestyle changes paralleling an increasing human development index. Despite a growing insight into the biology of CRC and many therapeutic improvements in the recent decades, preclinical in vivo models are still indispensable for the development of new treatment approaches. Since the development of carcinogen-induced rodent models for CRC more than 80 years ago, a plethora of animal models has been established to study colon cancer biology. Despite tenuous invasiveness and metastatic behavior, these models are useful for chemoprevention studies and to evaluate colitis-related carcinogenesis. Genetically engineered mouse models (GEMM) mirror the pathogenesis of sporadic as well as inherited CRC depending on the specific molecular pathways activated or inhibited. Although the vast majority of CRC GEMM lack invasiveness, metastasis and tumor heterogeneity, they still have proven useful for examination of the tumor microenvironment as well as systemic immune responses; thus, supporting development of new therapeutic avenues. Induction of metastatic disease by orthotopic injection of CRC cell lines is possible, but the so generated models lack genetic diversity and the number of suited cell lines is very limited. Patient-derived xenografts, in contrast, maintain the pathological and molecular characteristics of the individual patient's CRC after subcutaneous implantation into immunodeficient mice and are therefore most reliable for preclinical drug development - even in comparison to GEMM or cell line-based analyses. However, subcutaneous patient-derived xenograft models are less suitable for studying most aspects of the tumor microenvironment and anti-tumoral immune responses. The authors review the distinct mouse models of CRC with an emphasis on their clinical relevance and shed light on the latest developments in the field of preclinical CRC models.
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Affiliation(s)
- Florian Bürtin
- Department of General, Visceral, Vascular and Transplantation Surgery, University Medical Center Rostock, University of Rostock, Rostock 18057, Germany
| | - Christina S Mullins
- Department of Thoracic Surgery, University Medical Center Rostock, University of Rostock, Rostock 18057, Germany
| | - Michael Linnebacher
- Molecular Oncology and Immunotherapy, Department of General, Visceral, Vascular and Transplantation Surgery, University Medical Center Rostock, Rostock 18057, Germany
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11
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Ganesh K, Basnet H, Kaygusuz Y, Laughney AM, He L, Sharma R, O'Rourke KP, Reuter VP, Huang YH, Turkekul M, Emrah E, Masilionis I, Manova-Todorova K, Weiser MR, Saltz LB, Garcia-Aguilar J, Koche R, Lowe SW, Pe'er D, Shia J, Massagué J. L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer. ACTA ACUST UNITED AC 2020; 1:28-45. [PMID: 32656539 DOI: 10.1038/s43018-019-0006-x] [Citation(s) in RCA: 159] [Impact Index Per Article: 31.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Metastasis-initiating cells with stem-like properties drive cancer lethality, yet their origins and relationship to primary-tumor-initiating stem cells are not known. We show that L1CAM+ cells in human colorectal cancer (CRC) have metastasis-initiating capacity, and we define their relationship to tissue regeneration. L1CAM is not expressed in the homeostatic intestinal epithelium, but is induced and required for epithelial regeneration following colitis and in CRC organoid growth. By using human tissues and mouse models, we show that L1CAM is dispensable for adenoma initiation but required for orthotopic carcinoma propagation, liver metastatic colonization and chemoresistance. L1CAMhigh cells partially overlap with LGR5high stem-like cells in human CRC organoids. Disruption of intercellular epithelial contacts causes E-cadherin-REST transcriptional derepression of L1CAM, switching chemoresistant CRC progenitors from an L1CAMlow to an L1CAMhigh state. Thus, L1CAM dependency emerges in regenerative intestinal cells when epithelial integrity is lost, a phenotype of wound healing deployed in metastasis-initiating cells.
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Affiliation(s)
- Karuna Ganesh
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.,Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA.,Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Harihar Basnet
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.,These authors contributed equally: Harihar Basnet, Yasemin Kaygusuz, Ashley M. Laughney
| | - Yasemin Kaygusuz
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.,Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA.,These authors contributed equally: Harihar Basnet, Yasemin Kaygusuz, Ashley M. Laughney
| | - Ashley M Laughney
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.,Present address: Institute for Computational Biomedicine, Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA.,These authors contributed equally: Harihar Basnet, Yasemin Kaygusuz, Ashley M. Laughney
| | - Lan He
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Roshan Sharma
- Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.,Department of Applied Physics and Applied Math, Columbia University, New York, NY, USA.,Present address: New York Genome Center, New York, NY, USA
| | - Kevin P O'Rourke
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.,Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program, New York, NY, USA
| | - Vincent P Reuter
- Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Yun-Han Huang
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.,Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA.,Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program, New York, NY, USA
| | - Mesruh Turkekul
- Molecular Cytology Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Ekrem Emrah
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.,Present address: Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL, USA
| | - Ignas Masilionis
- Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Katia Manova-Todorova
- Molecular Cytology Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Martin R Weiser
- Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Leonard B Saltz
- Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Julio Garcia-Aguilar
- Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Richard Koche
- Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Scott W Lowe
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Dana Pe'er
- Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Jinru Shia
- Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Joan Massagué
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
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12
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Barve A, Casson L, Krem M, Wunderlich M, Mulloy JC, Beverly LJ. Comparative utility of NRG and NRGS mice for the study of normal hematopoiesis, leukemogenesis, and therapeutic response. Exp Hematol 2018; 67:18-31. [PMID: 30125602 DOI: 10.1016/j.exphem.2018.08.004] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2018] [Revised: 07/25/2018] [Accepted: 08/10/2018] [Indexed: 12/25/2022]
Abstract
Cell-line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) in immune-deficient mice have revolutionized our understanding of normal and malignant human hematopoiesis. Transgenic approaches further improved in vivo hematological research, allowing the development of human-cytokine-producing mice, which show superior human cell engraftment. The most popular mouse strains used in research, the NOG (NOD.Cg-Prkdcscid Il2rγtm1Sug/Jic) and the NSG (NOD/SCID-IL2Rγ-/-, NOD.Cg-PrkdcscidIl2rγtm1Wjl/SzJ) mouse, and their human-cytokine-producing (interleukin-3, granulocyte-macrophage colony-stimulating factor, and stem cell factor) counterparts (huNOG and NSGS), rely partly on a mutation in the DNA repair protein PRKDC, causing a severe combined immune deficiency (SCID) phenotype and rendering the mice less tolerant to DNA-damaging therapeutics, thereby limiting their usefulness in the investigation of novel acute myeloid leukemia (AML) therapeutics. NRG (NOD/RAG1/2-/-IL2Rγ-/-) mice show equivalent immune ablation through a defective recombination activation gene (RAG), leaving DNA damage repair intact, and human-cytokine-producing NRGS (NRG-SGM3) mice were generated, improving myeloid engraftment. Our findings indicate that unconditioned NRG and NRGS mice can harbor established AML CDXs and can tolerate aggressive induction chemotherapy at higher doses than NSG mice without overt toxicity. However, unconditioned NRGS mice developed less clinically relevant disease, with CDXs forming solid tumors throughout the body, whereas unconditioned NRG mice were incapable of efficiently supporting PDX or human hematopoietic stem cell engraftment. These findings emphasize the contextually dependent utility of each of these powerful new strains in the study of normal and malignant human hematopoiesis. Therefore, the choice of mouse strain cannot be random, but must be based on the experimental outcomes and questions to be addressed.
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Affiliation(s)
- Aditya Barve
- Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY, USA
| | - Lavona Casson
- James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA
| | - Maxwell Krem
- James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA; Department of Medicine, University of Louisville, Louisville, KY, USA
| | - Mark Wunderlich
- Department of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
| | - James C Mulloy
- Department of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
| | - Levi J Beverly
- Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY, USA; James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA; Department of Medicine, University of Louisville, Louisville, KY, USA.
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13
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14
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Baiu DC, Marsh IR, Boruch AE, Shahi A, Bhattacharya S, Jeffery JJ, Zhao Q, Hall LT, Weichert JP, Bednarz BP, Otto M. Targeted Molecular Radiotherapy of Pediatric Solid Tumors Using a Radioiodinated Alkyl-Phospholipid Ether Analog. J Nucl Med 2017; 59:244-250. [PMID: 28747518 DOI: 10.2967/jnumed.117.193748] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2017] [Accepted: 06/06/2017] [Indexed: 01/10/2023] Open
Abstract
External-beam radiotherapy plays a critical role in the treatment of most pediatric solid tumors. Particularly in children, achieving an optimal therapeutic index to avoid damage to normal tissue is extremely important. Consequently, in metastatic disease, the utility of external-beam radiotherapy is limited. Molecular radiotherapy with tumor-targeted radionuclides may overcome some of these challenges, but to date there exists no single cancer-selective agent capable of treating various pediatric malignancies independently of their histopathologic origin. We tested the therapeutic potential of the clinical-grade alkyl-phospholipid ether analog CLR1404, 18-(p-iodophenyl)octadecyl phosphocholine, as a scaffold for tumor-targeted radiotherapy of pediatric malignancies. Methods: Uptake of CLR1404 by pediatric solid tumor cells was tested in vitro by flow cytometry and in vivo by PET/CT imaging and dosimetry. The therapeutic potential of 131I-CLR1404 was evaluated in xenograft models. Results: In vitro, fluorescent CLR1404-BODIPY showed significant selective uptake in a variety of pediatric cancer lines compared with normal controls. In vivo tumor-targeted uptake in mouse xenograft models using 124I-CLR1404 was confirmed by imaging. Single-dose intravenous injection of 131I-CLR1404 significantly delayed tumor growth in all rodent pediatric xenograft models and extended animal survival while demonstrating a favorable side effect profile. Conclusion:131I-CLR1404 has the potential to become a tumor-targeted radiotherapeutic drug with broad applicability in pediatric oncology. Because 131I-CLR1404 has entered clinical trials in adults, our data warrant the development of pediatric clinical trials for this particularly vulnerable patient population.
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Affiliation(s)
- Dana C Baiu
- Department of Pediatrics, Carbone Cancer Center, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin
| | - Ian R Marsh
- Department of Medical Physics, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin
| | - Alexander E Boruch
- Department of Pediatrics, Carbone Cancer Center, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin
| | - Ankita Shahi
- Department of Pediatrics, Carbone Cancer Center, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin
| | - Saswati Bhattacharya
- Department of Pediatrics, Carbone Cancer Center, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin
| | - Justin J Jeffery
- Department of Radiology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin; and
| | - Qianqian Zhao
- Department of Biostatistics and Medical Informatics, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin
| | - Lance T Hall
- Department of Radiology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin; and
| | - Jamey P Weichert
- Department of Medical Physics, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin.,Department of Radiology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin; and
| | - Bryan P Bednarz
- Department of Medical Physics, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin
| | - Mario Otto
- Department of Pediatrics, Carbone Cancer Center, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin
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15
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Ibeh BO, Furuta Y, Habu JB, Ogbadu L. Humanized mouse as an appropriate model for accelerated global HIV research and vaccine development: current trend. Immunopharmacol Immunotoxicol 2016; 38:395-407. [PMID: 27604679 DOI: 10.1080/08923973.2016.1233980] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Humanized mouse models currently have seen improved development and have received wide applications. Its usefulness is observed in cell and tissue transplant involving basic and applied human disease research. In this article, the development of a new generation of humanized mice was discussed as well as their relevant application in HIV disease. Furthermore, current techniques employed to overcome the initial limitations of mouse model were reviewed. Highly immunodeficient mice which support cell and tissue differentiation and do not reject xenografts are indispensable for generating additional appropriate models useful in disease study, this phenomenom deserves emphases, scientific highlight and a definitive research focus. Since the early 2000s, a series of immunodeficient mice appropriate for generating humanized mice has been successively developed by introducing the IL-2Rγnull gene (e.g. NOD/SCID/γcnull and Rag2nullγcnull mice) through various genomic approaches. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γcnull and Rag2nullγcnull mouse backgrounds. The application of these techniques serves as a quick and appropriate mechanistic model for basic and therapeutic investigations of known and emerging infections.
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Affiliation(s)
- Bartholomew Okechukwu Ibeh
- a Immunovirology and Vaccine Development Laboratory, Medical Biotechnology Department , National Biotechnology Development Agency , Abuja , Nigeria
| | - Yasuhide Furuta
- b RIKEN CDB CLST (Center for Life Science Technologies) , Kobe , Japan
| | - Josiah Bitrus Habu
- c Bioresources Development Center Odi, Bayelsa , National Biotechnology Development Agency , Abuja , Nigeria
| | - Lucy Ogbadu
- d National Biotechnology Development Agency , Abuja , Nigeria
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16
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Yurino A, Takenaka K, Yamauchi T, Nunomura T, Uehara Y, Jinnouchi F, Miyawaki K, Kikushige Y, Kato K, Miyamoto T, Iwasaki H, Kunisaki Y, Akashi K. Enhanced Reconstitution of Human Erythropoiesis and Thrombopoiesis in an Immunodeficient Mouse Model with Kit(Wv) Mutations. Stem Cell Reports 2016; 7:425-438. [PMID: 27499200 PMCID: PMC5031955 DOI: 10.1016/j.stemcr.2016.07.002] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2016] [Revised: 07/04/2016] [Accepted: 07/04/2016] [Indexed: 01/18/2023] Open
Abstract
In human-to-mouse xenograft models, reconstitution of human hematopoiesis is usually B-lymphoid dominant. Here we show that the introduction of homozygous Kit(Wv) mutations into C57BL/6.Rag2(null)Il2rg(null) mice with NOD-Sirpa (BRGS) strongly promoted human multi-lineage reconstitution. After xenotransplantation of human CD34(+)CD38(-) cord blood cells, these newly generated C57BL/6.Rag2(null)Il2rg(null)NOD-Sirpa Kit(Wv/Wv) (BRGSK(Wv/Wv)) mice showed significantly higher levels of human cell chimerism and long-term multi-lineage reconstitution compared with BRGS mice. Strikingly, this mouse displayed a robust reconstitution of human erythropoiesis and thrombopoiesis with terminal maturation in the bone marrow. Furthermore, depletion of host macrophages by clodronate administration resulted in the presence of human erythrocytes and platelets in the circulation. Thus, attenuation of mouse KIT signaling greatly enhances the multi-lineage differentiation of human hematopoietic stem and progenitor cells (HSPCs) in mouse bone marrow, presumably by outcompeting mouse HSPCs to occupy suitable microenvironments. The BRGSK(Wv/Wv) mouse model is a useful tool to study human multi-lineage hematopoiesis.
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Affiliation(s)
- Ayano Yurino
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan
| | - Katsuto Takenaka
- Center for Cellular and Molecular Medicine, Kyushu University Hospital, Fukuoka 812-8582, Japan
| | - Takuji Yamauchi
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan
| | - Takuya Nunomura
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan
| | - Yasufumi Uehara
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan
| | - Fumiaki Jinnouchi
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan
| | - Kohta Miyawaki
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan
| | - Yoshikane Kikushige
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan
| | - Koji Kato
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan
| | - Toshihiro Miyamoto
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan
| | - Hiromi Iwasaki
- Center for Cellular and Molecular Medicine, Kyushu University Hospital, Fukuoka 812-8582, Japan
| | - Yuya Kunisaki
- Center for Cellular and Molecular Medicine, Kyushu University Hospital, Fukuoka 812-8582, Japan
| | - Koichi Akashi
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan; Center for Cellular and Molecular Medicine, Kyushu University Hospital, Fukuoka 812-8582, Japan.
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17
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Humanized NOD-SCID IL2rg–/– mice as a preclinical model for cancer research and its potential use for individualized cancer therapies. Cancer Lett 2014; 344:13-19. [PMID: 24513265 DOI: 10.1016/j.canlet.2013.10.015] [Citation(s) in RCA: 76] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2013] [Revised: 10/16/2013] [Accepted: 10/21/2013] [Indexed: 11/24/2022]
Abstract
Humanized mouse models have been developed and utilized in cancer research for decades. Newly developed combined immunodeficient NOD-SCID-IL2rg–/– mice are more permissive for human cells and tissue engraftment. In this review, we discuss the use of NOD-SCID-IL2rg(–/–) mice as a preclinical tool in cancer research and its potential use for individualized cancer therapies.
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18
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Zhou J, Shen B, Zhang W, Wang J, Yang J, Chen L, Zhang N, Zhu K, Xu J, Hu B, Leng Q, Huang X. One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering. Int J Biochem Cell Biol 2014; 46:49-55. [PMID: 24269190 DOI: 10.1016/j.biocel.2013.10.010] [Citation(s) in RCA: 96] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2013] [Revised: 10/20/2013] [Accepted: 10/22/2013] [Indexed: 01/26/2023]
Abstract
Taking advantage of the multiplexable genome engineering feature of the CRISPR/Cas9 system, we sought to generate different kinds of immunodeficient mouse strains by embryo co-microinjection of Cas9 mRNA and multiple sgRNAs targeting mouse B2m, Il2rg, Prf1, Prkdc, and Rag1. We successfully achieved multiple gene modifications, fragment deletion, double knockout of genes localizing on the same chromosome, and got different kinds of immunodeficient mouse models with different heritable genetic modifications at once, providing a one-step strategy for generating different immunodeficient mice which represents significant time-, labor-, and money-saving advantages over traditional approaches. Meanwhile, we improved the technology by optimizing the concentration of Cas9 and sgRNAs and designing two adjacent sgRNAs targeting one exon for each gene, which greatly increased the targeting efficiency and bi-allelic mutations.
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Affiliation(s)
- Jiankui Zhou
- MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing 210061, China
| | - Bin Shen
- MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing 210061, China
| | - Wensheng Zhang
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
| | - Jianying Wang
- MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing 210061, China
| | - Jing Yang
- MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing 210061, China
| | - Li Chen
- MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing 210061, China
| | - Na Zhang
- Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 225 South Chongqing Road, Shanghai, China
| | - Kai Zhu
- Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 225 South Chongqing Road, Shanghai, China
| | - Juan Xu
- MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing 210061, China
| | - Bian Hu
- MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing 210061, China
| | - Qibin Leng
- Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 225 South Chongqing Road, Shanghai, China.
| | - Xingxu Huang
- MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing 210061, China.
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19
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Iwamoto C, Takenaka K, Urata S, Yamauchi T, Shima T, Kuriyama T, Daitoku S, Saito Y, Miyamoto T, Iwasaki H, Kitabayashi I, Itoh K, Kishimoto J, Kohda D, Matozaki T, Akashi K. The BALB/c-specific polymorphic SIRPA enhances its affinity for human CD47, inhibiting phagocytosis against human cells to promote xenogeneic engraftment. Exp Hematol 2013; 42:163-171.e1. [PMID: 24269920 DOI: 10.1016/j.exphem.2013.11.005] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2013] [Revised: 11/05/2013] [Accepted: 11/07/2013] [Indexed: 02/04/2023]
Abstract
It has been shown that in xenotransplantation of human cells into immunodeficient mice, the mouse strain background is critical. For example, the nonobese diabetic (NOD) strain is most efficient, the BALB/c is moderate, and the C57BL/6 is inefficient for human cell engraftment. We have shown that the NOD-specific polymorphism of the signal regulatory protein-alpha (Sirpa) allows NOD SIRPA to bind human CD47, and the resultant "don't eat me" signaling by this binding prevents host macrophages to engulf human grafts, thereby inhibiting rejection. Here we tested whether the efficient xenotransplantation capability of the BALB/c strain is also mediated by the SIRPA-CD47 self-recognition system. BALB/c SIRPA was capable of binding to human CD47 at an intermediate level between those of C57BL/6 SIRPA and NOD SIRPA. Consistent with its binding activity, BALB/c-derived macrophages exhibited a moderate inhibitory effect on human long-term culture-initiating cells in in vitro cultures, and showed moderate phagocytic activity against human hematopoietic stem cells. The increased affinity of BALB/c SIRPA for human CD47 was mounted at least through the BALB/c-specific L29V SNP within the IgV domain. Thus, the mouse strain effect on xenogeneic engraftment might be ascribed mainly to the binding affinity of strain-specific polymorphic SIRPA with human CD47. This information should be useful for developing a novel immunodeficient strain with superior efficiency for xenogeneic transplantation of human cells.
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Affiliation(s)
- Chika Iwamoto
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
| | - Katsuto Takenaka
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
| | - Shingo Urata
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
| | - Takuji Yamauchi
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
| | - Takahiro Shima
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
| | - Takuro Kuriyama
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
| | - Shinya Daitoku
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
| | - Yasuyuki Saito
- Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan
| | - Toshihiro Miyamoto
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
| | - Hiromi Iwasaki
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
| | - Issay Kitabayashi
- Division of Hematological Malignancy, National Cancer Center Research Institute, Tokyo, Japan
| | - Katsuhiko Itoh
- Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, Kyoto, Japan
| | - Junji Kishimoto
- Digital Medicine Initiative, Kyushu University, Fukuoka, Japan
| | - Daisuke Kohda
- Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
| | - Takashi Matozaki
- Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan; Division of Molecular and Cellular Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan
| | - Koichi Akashi
- Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.
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20
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Casazza K, Hanks LJ, Clines GA, Tse HM, Eberhardt AW. Diabetes-related impairment in bone strength is established early in the life course. World J Diabetes 2013; 4:145-150. [PMID: 23961325 PMCID: PMC3746087 DOI: 10.4239/wjd.v4.i4.145] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/06/2013] [Revised: 04/24/2013] [Accepted: 06/19/2013] [Indexed: 02/05/2023] Open
Abstract
AIM: To evaluate properties of bone quantity/quality using young non-obese Type 1 (T1D)-diabetic (NOD) prone and syngenic non-diabetic (NOD.scid) mice.
METHODS: Quantitative bone assessment of tibia was conducted using dual-energy X-ray absorptiometry (DXA) for the evaluation of body mass, bone mineral content, body fat mass and lean mass. Qualitative assessment was accomplished by three-point breakage for assessment of force to failure and micro-computed tomography for evaluation of trabecular and cortical properties of bone. In addition, fasting blood was evaluated prior to sacrifice at week eleven and fifteen to evaluate and compare glucose homeostasis between the strains of mice.
RESULTS: Our findings support a perturbation in the relationship between bone quantity, quality, and subsequently, the association between structure and strength. There were no differences in DXA-assessed body composition (body fat, % fat mass and lean mass) and bone composition (bone mineral content and bone mineral density) between strains. However, relative to NOD.scid, NOD mice had lower trabecular bone volume, relative trabecular bone volume, trabecular number and trabecular total material density (P < 0.05). Conversely, NOD mice had greater cortical total mean volume (P < 0.05). General linear models analysis adjusted for body weight revealed a significant contribution of T1D to bone health as early as 5 wk.
CONCLUSION: It is well-established that diabetes is a significant risk factor for increased fractures, although the underlying mechanisms are not fully understood. Investigation of bone parameters encompassing strength and structure early in the life course will facilitate the elucidation of the pathogenesis of impaired bone integrity.
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21
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Brehm MA, Powers AC, Shultz LD, Greiner DL. Advancing animal models of human type 1 diabetes by engraftment of functional human tissues in immunodeficient mice. Cold Spring Harb Perspect Med 2013; 2:a007757. [PMID: 22553498 DOI: 10.1101/cshperspect.a007757] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
Despite decades of studying rodent models of type 1 diabetes (T1D), no therapy capable of preventing or curing T1D has successfully been translated from rodents to humans. This inability to translate otherwise promising therapies to clinical settings likely resides, to a major degree, from significant species-specific differences between rodent and human immune systems as well as species-related variances in islets in terms of their cellular composition, function, and gene expression. Indeed, taken collectively, these differences underscore the need to define interactions between the human immune system with human β cells. Immunodeficient mice engrafted with human immune systems and human β cells represent an interesting and promising opportunity to study these components in vivo. To meet this need, years of effort have been extended to develop mice depleted of undesirable components while at the same time, allowing the introduction of constituents necessary to recapitulate physiological settings as near as possible to human T1D. With this, these so-called "humanized mice" are currently being used as a preclinical bridge to facilitate identification and translation of novel discoveries to clinical settings.
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Affiliation(s)
- Michael A Brehm
- University of Massachusetts Medical School, Program in Molecular Medicine, Worcester, Massachusetts, USA
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Rongvaux A, Takizawa H, Strowig T, Willinger T, Eynon EE, Flavell RA, Manz MG. Human hemato-lymphoid system mice: current use and future potential for medicine. Annu Rev Immunol 2013; 31:635-674. [PMID: 23330956 DOI: 10.1146/annurev-immunol-032712-095921] [Citation(s) in RCA: 266] [Impact Index Per Article: 22.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
To directly study complex human hemato-lymphoid system physiology and respective system-associated diseases in vivo, human-to-mouse xenotransplantation models for human blood and blood-forming cells and organs have been developed over the past three decades. We here review the fundamental requirements and the remarkable progress made over the past few years in improving these systems, the current major achievements reached by use of these models, and the future challenges to more closely model and study human health and disease and to achieve predictive preclinical testing of both prevention measures and potential new therapies.
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Affiliation(s)
- Anthony Rongvaux
- Department of Immunobiology, Yale University, New Haven, Connecticut 06520
| | - Hitoshi Takizawa
- Division of Hematology, University Hospital Zürich, CH-8091 Zürich, Switzerland
| | - Till Strowig
- Department of Immunobiology, Yale University, New Haven, Connecticut 06520
| | - Tim Willinger
- Department of Immunobiology, Yale University, New Haven, Connecticut 06520
| | - Elizabeth E Eynon
- Department of Immunobiology, Yale University, New Haven, Connecticut 06520
| | - Richard A Flavell
- Department of Immunobiology, Yale University, New Haven, Connecticut 06520.,Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520;
| | - Markus G Manz
- Division of Hematology, University Hospital Zürich, CH-8091 Zürich, Switzerland
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Polymorphic Sirpa is the genetic determinant for NOD-based mouse lines to achieve efficient human cell engraftment. Blood 2013; 121:1316-25. [PMID: 23293079 DOI: 10.1182/blood-2012-06-440354] [Citation(s) in RCA: 101] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Current mouse lines efficient for human cell xenotransplantation are backcrossed into NOD mice to introduce its multiple immunodeficient phenotypes. Our positional genetic study has located the NOD-specific polymorphic Sirpa as a molecule responsible for its high xenograft efficiency: it recognizes human CD47 and the resultant signaling may cause NOD macrophages not to engulf human grafts. In the present study, we established C57BL/6.Rag2(nullIl2rgnull) mice harboring NOD-Sirpa (BRGS). BRGS mice engrafted human hematopoiesis with an efficiency that was equal to or even better than that of the NOD.Rag1(nullIl2rgnull) strain, one of the best xenograft models. Consequently, BRGS mice are free from other NOD-related abnormalities; for example, they have normalized C5 function that enables the evaluation of complement-dependent cytotoxicity of antibodies against human grafts in the humanized mouse model. Our data show that efficient human cell engraftment found in NOD-based models is mounted solely by their polymorphic Sirpa. The simplified BRGS line should be very useful in future studies of human stem cell biology.
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Abstract
Humanized mouse models that have received human cells or tissue transplants are extremely useful in basic and applied human disease research. Highly immunodeficient mice, which do not reject xenografts and support cell and tissue differentiation and growth, are indispensable for generating additional appropriate models. Since the early 2000s, a series of immunodeficient mice appropriate for generating humanized mice has been successively developed by introducing the IL-2Rγ(null) gene (e.g., NOD/SCID/γc(null) and Rag2(null)γc(null) mice). These strains show not only a high rate of human cell engraftment, but also generate well-differentiated multilineage human hematopoietic cells after human hematopoietic stem cell (HSC) transplantation. These humanized mice facilitate the analysis of human hematology and immunology in vivo. However, human hematopoietic cells developed from HSCs are not always phenotypically and functionally identical to those in humans. More recently, a new series of immunodeficient mice compensates for these disadvantages. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γc(null) and Rag2(null)γc(null) mice. In this review, we describe the current knowledge of human hematopoietic cells developed in these mice. Various human disease mouse models using these humanized mice are summarized.
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Membrane-bound human SCF/KL promotes in vivo human hematopoietic engraftment and myeloid differentiation. Blood 2012; 119:2768-77. [PMID: 22279057 DOI: 10.1182/blood-2011-05-353201] [Citation(s) in RCA: 81] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
In recent years, advances in the humanized mouse system have led to significantly increased levels of human hematopoietic stem cell (HSC) engraftment. The remaining limitations in human HSC engraftment and function include lymphoid-skewed differentiation and inefficient myeloid development in the recipients. Limited human HSC function may partially be attributed to the inability of the host mouse microenvironment to provide sufficient support to human hematopoiesis. To address this problem, we created membrane-bound human stem cell factor (SCF)/KIT ligand (KL)-expressing NOD/SCID/IL2rgKO (hSCF Tg NSG) mice. hSCF Tg NSG recipients of human HSCs showed higher levels of both human CD45(+) cell engraftment and human CD45(+)CD33(+) myeloid development compared with NSG recipients. Expression of hSCF/hKL accelerated the differentiation of the human granulocyte lineage cells in the recipient bone marrow. Human mast cells were identified in bone marrow, spleen, and gastrointestinal tissues of the hSCF Tg NSG recipients. This novel in vivo humanized mouse model demonstrates the essential role of membrane-bound hSCF in human myeloid development. Moreover, the hSCF Tg NSG humanized recipients may facilitate investigation of in vivo differentiation, migration, function, and pathology of human mast cells.
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Ablamunits V, Klebanov S, Giese SY, Herold KC. Functional human to mouse adipose tissue xenotransplantation. J Endocrinol 2012; 212:41-7. [PMID: 22007021 DOI: 10.1530/joe-11-0201] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
White adipose tissue (WAT) produces a number of metabolically important factors and, therefore, some inborn errors of metabolism may potentially be corrected by transplantation of normal allogeneic WAT. To explore the ability of human WAT (HuWAT) to compensate for a missing factor and to induce allogeneic immune response, we created leptin-deficient, immunodeficient mice and transplanted them with either 2·5 or 5 ml HuWAT. Recipient mice showed stable levels of human leptin in circulation, reduced body mass gain, and amelioration of hepatic steatosis. Food consumption and plasma insulin levels were reduced only in recipients of 5 ml WAT. Transfer of 2×10(7) human mononuclear cells to reject WAT as an allograft was ineffective and resulted only in some reduction of circulating leptin and a limited damage to the WAT grafts followed by the loss of human leukocytes.
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Affiliation(s)
- Vitaly Ablamunits
- Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA.
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27
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Improving human hemato-lymphoid-system mice by cytokine knock-in gene replacement. Trends Immunol 2011; 32:321-7. [DOI: 10.1016/j.it.2011.04.005] [Citation(s) in RCA: 102] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2011] [Revised: 04/14/2011] [Accepted: 04/15/2011] [Indexed: 11/17/2022]
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Frecha C, Fusil F, Cosset FL, Verhoeyen E. In vivo gene delivery into hCD34+ cells in a humanized mouse model. Methods Mol Biol 2011; 737:367-90. [PMID: 21590405 DOI: 10.1007/978-1-61779-095-9_15] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
In vivo targeted gene delivery to hematopoietic stem cells (HSCs) would mean a big step forward in the field of gene therapy. This would imply that the risk of cell differentiation and loss of homing/-engraftment is reduced, as there is no need for purification of the target cell. In vivo gene delivery also bypasses the issue that no precise markers that permit the isolation of a primitive hHSC exist up to now. Indeed, in vivo gene transfer could target all HSCs in their stem-cell niche, including those cells that are "missed" by the purification criteria. Moreover, for the majority of diseases, there is a requirement of a minimal number of gene-corrected cells to be reinfused to allow an efficient long-term engraftment. This requisite might become a limiting factor when treating children with inherited disorders, due to the low number of bone marrow (BM) CD34(+) HSCs that can actually be isolated. These problems could be overcome by using efficient in vivo HSC-specific lentiviral vectors (LVs). Additionally, vectors for in vivo HSC transduction must be specific for the target cell, to avoid vector spreading while enhancing transduction efficiency. Of importance, a major barrier in LV transduction of HSCs is that 75% of HSCs are residing in the G0 phase of the cell cycle and are not very permissive for classical VSV-G-LV transduction. Therefore, we engineered "early-activating-cytokine (SCF or/and TPO)" displaying LVs that allowed a slight and transient stimulation of hCD34(+) cells resulting in efficient lentiviral gene transfer while preserving the "stemness" of the targeted HSCs. The selective transduction of HSCs by these vectors was demonstrated by their capacity to promote selective transduction of CD34(+) cells in in vitro-derived, long-term culture-initiating cell colonies and long-term NOD/SCID repopulating cells. A second generation of these "early-acting-cytokine"-displaying lentiviral vectors has now been developed that is fit for targeted in vivo gene delivery to hCD34(+) cells. In the method presented here, we describe the in vivo gene delivery into hCD34(+) cells by intramarrow injection of these new vectors into humanized BALB/c Rag2( null )/IL2rgc ( null ) (BALB/c RAGA) mice.
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Affiliation(s)
- Cecilia Frecha
- Human Virology Department, INSERM U758, Ecole Normale Supérieure de Lyon, and Université de Lyon 1, Lyon, France
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Brehm MA, Bortell R, Diiorio P, Leif J, Laning J, Cuthbert A, Yang C, Herlihy M, Burzenski L, Gott B, Foreman O, Powers AC, Greiner DL, Shultz LD. Human immune system development and rejection of human islet allografts in spontaneously diabetic NOD-Rag1null IL2rgammanull Ins2Akita mice. Diabetes 2010; 59:2265-70. [PMID: 20570944 PMCID: PMC2927949 DOI: 10.2337/db10-0323] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
OBJECTIVE To create an immunodeficient mouse model that spontaneously develops hyperglycemia to serve as a diabetic host for human islets and stem cell-derived beta-cells in the absence or presence of a functional human immune system. RESEARCH DESIGN AND METHODS We backcrossed the Ins2(Akita) mutation onto the NOD-Rag1(null) IL2rgamma(null) strain and determined 1) the spontaneous development of hyperglycemia, 2) the ability of human islets, mouse islets, and dissociated mouse islet cells to restore euglycemia, 3) the generation of a human immune system following engraftment of human hematopoietic stem cells, and 4) the ability of the humanized mice to reject human islet allografts. RESULTS We confirmed the defects in innate and adaptive immunity and the spontaneous development of hyperglycemia conferred by the IL2rgamma(null), Rag1(null), and Ins2(Akita) genes in NOD-Rag1(null) IL2rgamma(null) Ins2(Akita) (NRG-Akita) mice. Mouse and human islets restored NRG-Akita mice to normoglycemia. Insulin-positive cells in dissociated mouse islets, required to restore euglycemia in chemically diabetic NOD-scid IL2rgamma(null) and spontaneously diabetic NRG-Akita mice, were quantified following transplantation via the intrapancreatic and subrenal routes. Engraftment of human hematopoietic stem cells in newborn NRG-Akita and NRG mice resulted in equivalent human immune system development in a normoglycemic or chronically hyperglycemic environment, with >50% of engrafted NRG-Akita mice capable of rejecting human islet allografts. CONCLUSIONS NRG-Akita mice provide a model system for validation of the function of human islets and human adult stem cell, embryonic stem cell, or induced pluripotent stem cell-derived beta-cells in the absence or presence of an alloreactive human immune system.
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Affiliation(s)
- Michael A Brehm
- Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
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Reconstitution of huPBL-NSG mice with donor-matched dendritic cells enables antigen-specific T-cell activation. J Neuroimmune Pharmacol 2010; 6:148-57. [PMID: 20532647 PMCID: PMC3028099 DOI: 10.1007/s11481-010-9223-x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2010] [Accepted: 05/18/2010] [Indexed: 11/18/2022]
Abstract
Humanized mouse models provide a unique opportunity to study human immune cells in vivo, but traditional models have been limited to the evaluation of non-specific T-cell interactions due to the absence of antigen-presenting cells. In this study, immunodeficient NOD/SCID/IL2r-γnull (NSG) mice were engrafted with human peripheral blood lymphocytes alone or in combination with donor-matched monocyte-derived dendritic cells (DC) to determine whether antigen-specific T-cell activation could be reconstituted. Over a period of 3 weeks, transferred peripheral blood lymphocytes reconstituted the spleen and peripheral blood of recipient mice with predominantly human CD45-positive lymphocytes. Animals exhibited a relatively normal CD4/CD8 ratio (average 1.63:1) as well as reconstitution of CD3/CD56 (averaging 17.8%) and CD20 subsets (averaging 4.0%). Animals reconstituted with donor-matched CD11c+ DC also demonstrated a CD11c+ population within their spleen, representing 0.27% to 0.43% of the recovered human cells with concurrent expression of HLA-DR, CD40, and CD86. When immunized with adenovirus, either as free replication-incompetent vector (AdV) or as vector-transduced DC (DC/AdV), there was activation and expansion of AdV-specific T-cells, an increase in Th1 cytokines in serum, and skewing of T-cells toward an effector/memory phenotype. T-cells recovered from animals challenged with AdV in vivo proliferated and secreted a Th1-profile of cytokines in response to DC/AdV challenge in vitro. Our results suggest that engrafting NSG mice with a combination of lymphocytes and donor-matched DC can reconstitute antigen responsiveness and allow the in vivo assessment of human immune response to viruses, vaccines, and other immune challenges.
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Brehm MA, Cuthbert A, Yang C, Miller DM, DiIorio P, Laning J, Burzenski L, Gott B, Foreman O, Kavirayani A, Herlihy M, Rossini AA, Shultz LD, Greiner DL. Parameters for establishing humanized mouse models to study human immunity: analysis of human hematopoietic stem cell engraftment in three immunodeficient strains of mice bearing the IL2rgamma(null) mutation. Clin Immunol 2010; 135:84-98. [PMID: 20096637 DOI: 10.1016/j.clim.2009.12.008] [Citation(s) in RCA: 215] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2009] [Revised: 12/14/2009] [Accepted: 12/21/2009] [Indexed: 01/09/2023]
Abstract
"Humanized" mouse models created by engraftment of immunodeficient mice with human hematolymphoid cells or tissues are an emerging technology with broad appeal across multiple biomedical disciplines. However, investigators wishing to utilize humanized mice with engrafted functional human immune systems are faced with a myriad of variables to consider. In this study, we analyze HSC engraftment methodologies using three immunodeficient mouse strains harboring the IL2rgamma(null) mutation; NOD-scid IL2rgamma(null), NOD-Rag1(null) IL2rgamma(null), and BALB/c-Rag1(null) IL2rgamma(null) mice. Strategies compared engraftment of human HSC derived from umbilical cord blood following intravenous injection into adult mice and intracardiac and intrahepatic injection into newborn mice. We observed that newborn recipients exhibited enhanced engraftment as compared to adult recipients. Irrespective of the protocol or age of recipient, both immunodeficient NOD strains support enhanced hematopoietic cell engraftment as compared to the BALB/c strain. Our data define key parameters for establishing humanized mouse models to study human immunity.
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Affiliation(s)
- Michael A Brehm
- Diabetes Division, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
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King MA, Covassin L, Brehm MA, Racki W, Pearson T, Leif J, Laning J, Fodor W, Foreman O, Burzenski L, Chase TH, Gott B, Rossini AA, Bortell R, Shultz LD, Greiner DL. Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex. Clin Exp Immunol 2009; 157:104-18. [PMID: 19659776 DOI: 10.1111/j.1365-2249.2009.03933.x] [Citation(s) in RCA: 316] [Impact Index Per Article: 19.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
Immunodeficient non-obese diabetic (NOD)-severe combined immune-deficient (scid) mice bearing a targeted mutation in the gene encoding the interleukin (IL)-2 receptor gamma chain gene (IL2rgamma(null)) engraft readily with human peripheral blood mononuclear cells (PBMC). Here, we report a robust model of xenogeneic graft-versus-host-like disease (GVHD) based on intravenous injection of human PBMC into 2 Gy conditioned NOD-scid IL2rgamma(null) mice. These mice develop xenogeneic GVHD consistently (100%) following injection of as few as 5 x 10(6) PBMC, regardless of the PBMC donor used. As in human disease, the development of xenogeneic GVHD is highly dependent on expression of host major histocompatibility complex class I and class II molecules and is associated with severely depressed haematopoiesis. Interrupting the tumour necrosis factor-alpha signalling cascade with etanercept, a therapeutic drug in clinical trials for the treatment of human GVHD, delays the onset and progression of disease. This model now provides the opportunity to investigate in vivo mechanisms of xenogeneic GVHD as well as to assess the efficacy of therapeutic agents rapidly.
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Affiliation(s)
- M A King
- Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
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Schmidt MR, Appel MC, Giassi LJ, Greiner DL, Shultz LD, Woodland RT. Human BLyS facilitates engraftment of human PBL derived B cells in immunodeficient mice. PLoS One 2008; 3:e3192. [PMID: 18784835 PMCID: PMC2527131 DOI: 10.1371/journal.pone.0003192] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2008] [Accepted: 08/20/2008] [Indexed: 01/07/2023] Open
Abstract
The production of fully immunologically competent humanized mice engrafted with peripheral lymphocyte populations provides a model for in vivo testing of new vaccines, the durability of immunological memory and cancer therapies. This approach is limited, however, by the failure to efficiently engraft human B lymphocytes in immunodeficient mice. We hypothesized that this deficiency was due to the failure of the murine microenvironment to support human B cell survival. We report that while the human B lymphocyte survival factor, B lymphocyte stimulator (BLyS/BAFF) enhances the survival of human B cells ex vivo, murine BLyS has no such protective effect. Although human B cells bound both human and murine BLyS, nuclear accumulation of NF-κB p52, an indication of the induction of a protective anti-apoptotic response, following stimulation with human BLyS was more robust than that induced with murine BLyS suggesting a fundamental disparity in BLyS receptor signaling. Efficient engraftment of both human B and T lymphocytes in NOD rag1−/− Prf1−/− immunodeficient mice treated with recombinant human BLyS is observed after adoptive transfer of human PBL relative to PBS treated controls. Human BLyS treated recipients had on average 40-fold higher levels of serum Ig than controls and mounted a de novo antibody response to the thymus-independent antigens in pneumovax vaccine. The data indicate that production of fully immunologically competent humanized mice from PBL can be markedly facilitated by providing human BLyS.
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Affiliation(s)
- Madelyn R Schmidt
- Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
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Pearson T, Shultz LD, Miller D, King M, Laning J, Fodor W, Cuthbert A, Burzenski L, Gott B, Lyons B, Foreman O, Rossini AA, Greiner DL. Non-obese diabetic-recombination activating gene-1 (NOD-Rag1 null) interleukin (IL)-2 receptor common gamma chain (IL2r gamma null) null mice: a radioresistant model for human lymphohaematopoietic engraftment. Clin Exp Immunol 2008; 154:270-84. [PMID: 18785974 DOI: 10.1111/j.1365-2249.2008.03753.x] [Citation(s) in RCA: 171] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Immunodeficient hosts engrafted with human lymphohaematopoietic cells hold great promise as a preclinical bridge for understanding human haematopoiesis and immunity. We now describe a new immunodeficient radioresistant non-obese diabetic mice (NOD) stock based on targeted mutations in the recombination activating gene-1 (Rag1(null)) and interleukin (IL)-2 receptor common gamma chain (IL2rgamma(null)), and compare its ability to support lymphohaematopoietic cell engraftment with that achieved in radiosensitive NOD.CB17-Prkdc(scid) (NOD-Prkdc(scid)) IL2rgamma(null) mice. We observed that immunodeficient NOD-Rag1(null) IL2rgamma(null) mice tolerated much higher levels of irradiation conditioning than did NOD-Prkdc(scid) IL2rgamma(null) mice. High levels of human cord blood stem cell engraftment were observed in both stocks of irradiation-conditioned adult mice, leading to multi-lineage haematopoietic cell populations and a complete repertoire of human immune cells, including human T cells. Human peripheral blood mononuclear cells also engrafted at high levels in unconditioned adult mice of each stock. These data document that Rag1(null) and scid stocks of immunodeficient NOD mice harbouring the IL2rgamma(null) mutation support similar levels of human lymphohaematopoietic cell engraftment. NOD-Rag1(null) IL2rgamma(null) mice will be an important new model for human lymphohaematopoietic cell engraftment studies that require radioresistant hosts.
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Affiliation(s)
- T Pearson
- Diabetes Division, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
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Pearson T, Shultz LD, Lief J, Burzenski L, Gott B, Chase T, Foreman O, Rossini AA, Bottino R, Trucco M, Greiner DL. A new immunodeficient hyperglycaemic mouse model based on the Ins2Akita mutation for analyses of human islet and beta stem and progenitor cell function. Diabetologia 2008; 51:1449-56. [PMID: 18563383 PMCID: PMC2719841 DOI: 10.1007/s00125-008-1057-1] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2008] [Accepted: 04/24/2008] [Indexed: 11/25/2022]
Abstract
AIMS/HYPOTHESIS To develop and validate a new immunodeficient mouse strain that spontaneously develops a non-autoimmune hyperglycaemia to serve as a diabetic host for human islets and human beta stem and progenitor cells without the need for induction of hyperglycaemia by toxic chemicals with their associated side effects. METHODS We generated and characterised a new strain of immunodeficient spontaneously hyperglycaemic mice, the NOD-Rag1null Prf1null Ins2Akita strain and compared this strain with the NOD-scid Il2rgammanull (also known as Il2rg) immunodeficient strain rendered hyperglycaemic by administration of a single dose of streptozotocin. Hyperglycaemic mice were transplanted with human islets ranging from 1,000 to 4,000 islet equivalents (IEQ) and were monitored for normalisation of blood glucose levels. RESULTS NOD-Rag1null Prf1null Ins2Akita mice developed spontaneous hyperglycaemia, similar to Ins2Akita-harbouring strains of immunocompetent mice. Histological examination of islets in the host pancreas validated the spontaneous loss of beta cell mass in the absence of mononuclear cell infiltration. Human islets transplanted into spontaneously diabetic NOD-Rag1null Prf1null Ins2Akita and chemically diabetic NOD-scid Il2rgammanull mice resulted in a return to euglycaemia that occurred with transplantation of similar beta cell masses. CONCLUSIONS/INTERPRETATION The NOD-Rag1null Prf1null Ins2Akita mouse is the first immunodeficient, spontaneously hyperglycaemic mouse strain described that is based on the Ins2Akita mutation. This strain is suitable as hosts for human islet and human beta stem and progenitor cell transplantation in the absence of the need for pharmacological induction of diabetes. This strain of mice also has low levels of innate immunity and can be engrafted with a human immune system for the study of human islet allograft rejection.
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Affiliation(s)
- T. Pearson
- Diabetes Division, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA
| | | | - J. Lief
- Diabetes Division, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA
| | | | - B. Gott
- The Jackson Laboratory, Bar Harbor, ME, USA
| | - T. Chase
- The Jackson Laboratory, Bar Harbor, ME, USA
| | - O. Foreman
- The Jackson Laboratory, Bar Harbor, ME, USA
| | - A. A. Rossini
- Diabetes Division, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA
| | - R. Bottino
- Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA, USA
| | - M. Trucco
- Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA, USA
| | - D. L. Greiner
- Diabetes Division, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA
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Giassi LJ, Pearson T, Shultz LD, Laning J, Biber K, Kraus M, Woda BA, Schmidt MR, Woodland RT, Rossini AA, Greiner DL. Expanded CD34+ human umbilical cord blood cells generate multiple lymphohematopoietic lineages in NOD-scid IL2rgamma(null) mice. Exp Biol Med (Maywood) 2008; 233:997-1012. [PMID: 18653783 PMCID: PMC2757278 DOI: 10.3181/0802-rm-70] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Umbilical cord blood (UCB) is increasingly being used for human hematopoietic stem cell (HSC) transplantation in children but often requires pooling multiple cords to obtain sufficient numbers for transplantation in adults. To overcome this limitation, we have used an ex vivo two-week culture system to expand the number of hematopoietic CD34(+) cells in cord blood. To assess the in vivo function of these expanded CD34(+) cells, cultured human UCB containing 1 x 10(6) CD34(+) cells were transplanted into conditioned NOD-scid IL2rgamma(null) mice. The expanded CD34(+) cells displayed short- and long-term repopulating cell activity. The cultured human cells differentiated into myeloid, B-lymphoid, and erythroid lineages, but not T lymphocytes. Administration of human recombinant TNFalpha to recipient mice immediately prior to transplantation promoted human thymocyte and T-cell development. These T cells proliferated vigorously in response to TCR cross-linking by anti-CD3 antibody. Engrafted TNFalpha-treated mice generated antibodies in response to T-dependent and T-independent immunization, which was enhanced when mice were co-treated with the B cell cytokine BLyS. Ex vivo expanded CD34(+) human UCB cells have the capacity to generate multiple hematopoietic lineages and a functional human immune system upon transplantation into TNFalpha-treated NOD-scid IL2rgamma(null) mice.
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Affiliation(s)
- Lisa J. Giassi
- Division of Diabetes, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605
| | - Todd Pearson
- Division of Diabetes, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605
| | | | | | | | - Morey Kraus
- Viacell, Inc., Cambridge, Massachusetts 02142
| | - Bruce A. Woda
- Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
| | - Madelyn R. Schmidt
- Department of Molecular Genetics and Microbiology University of Massachusetts Medical School, Worcester, Massachusetts 01655
| | - Robert T. Woodland
- Department of Molecular Genetics and Microbiology University of Massachusetts Medical School, Worcester, Massachusetts 01655
| | - Aldo A. Rossini
- Division of Diabetes, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605
| | - Dale L. Greiner
- Division of Diabetes, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605
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Zhang B, Duan Z, Zhao Y. Mouse models with human immunity and their application in biomedical research. J Cell Mol Med 2008; 13:1043-58. [PMID: 18419795 PMCID: PMC4496103 DOI: 10.1111/j.1582-4934.2008.00347.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
Biomedical research in human beings is largely restricted to in vitro studies that lack complexity of a living organism. To overcome this limitation, humanized mouse models are developed based on immunodeficient characteristics of severe combined immunodeficiency (SCID) or recombination activating gene (Rag)(null) mice, which can accept xenografts. Peripheral constitution of human immunity in SCID or Rag(null) mice has been achieved by transplantation of mature human immune cells, foetal human thymus, bone marrow, liver tissues, lymph nodes or a combination of these, although efficiency needs to be improved. These mouse models with constituted human immunity (defined as humanized mice in the present text) have been widely used to investigate the basic principles of human immunobiology as well as complex pathomechanisms and potential therapies of human diseases. Here, elements of an ideal humanized mouse model are highlighted including genetic and non-genetic modification of recipient mice, transplantation strategies and proposals to improve engraftments. The applications of the humanized mice to study the development and response of human immune cells, human autoimmune diseases, virus infections, transplantation biology and tumour biology are reviewed as well.
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Affiliation(s)
- Baojun Zhang
- Institute of Zoology, Chinese Academy of Sciences, Beijing, China
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Abstract
There is a growing need for effective animal models to carry out experimental studies on human hematopoietic and immune systems without putting individuals at risk. Progress in development of small animal models for the in vivo investigation of human hematopoiesis and immunity has seen three major breakthroughs over the last three decades. First, CB 17-Prkdc(scid) (abbreviated CB 17-scid) mice were discovered in 1983, and engraftment of these mice with human fetal tissues (SCID-Hu model) and peripheral blood mononuclear cells (Hu-PBL-SCID model) was reported in 1988. Second, NOD-scid mice were developed and their enhanced ability to engraft with human hematolymphoid tissues as compared with CB17-scid mice was reported in 1995. NOD-scid mice have been the "gold standard" for studies of human hematolymphoid engraftment in small animal models over the last 10 years. Third, immunodeficient mice bearing a targeted mutation in the IL-2 receptor common gamma chain (IL2rgamma(null)) were developed independently by four groups between 2002 and 2005, and a major increase in the engraftment and function of human hematolymphoid cells as compared with NOD-scid mice has been reported. These new strains of immunodeficient IL2rgamma(null) mice are now being used for studies in human hematopoiesis, innate and adaptive immunity, autoimmunity, infectious diseases, cancer biology, and regenerative medicine. In this chapter, we discuss the current state of development of these strains of mice, the remaining deficiencies, and how approaches used to increase the engraftment and function of human hematolymphoid cells in CB 17-scid mice and in previous models based on NOD-scid mice may enhance human hematolymphoid engraftment and function in NOD-scid IL2rgamma(null) mice.
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Ito M, Kobayashi K, Nakahata T. NOD/Shi-scid IL2rgamma(null) (NOG) mice more appropriate for humanized mouse models. Curr Top Microbiol Immunol 2008; 324:53-76. [PMID: 18481452 DOI: 10.1007/978-3-540-75647-7_3] [Citation(s) in RCA: 75] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
"Humanized mice," in which various kinds of human cells and tissues can be engrafted and retain the same functions as in humans, are extremely useful because human diseases can be studied directly. Using the newly combined immunodeficient NOD-scid IL2rgamma(null) mice and Rag2(null) IL2rgamma(null) humanized mice, it has became possible to expand applications because various hematopoietic cells can be differentiated by human hematopoietic stem cell transplantation, and the human immune system can be reconstituted to some degree. This work has attracted attention worldwide, but the development and use of immunodeficient mice in Japan are not very well known or understood. This review describes the history and characteristics of the NOD/Shi-scid IL2rgamma(null) (NOG) and BALB/cA-Rag2(null) IL2rgamma(null) mice that were established in Japan, including our unpublished data from researchers who are currently using these mice. In addition, we also describe the potential development of new immunodeficient mice that can be used as humanized mice in the future.
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Affiliation(s)
- M Ito
- Laboratory of Immunology, Central Institute for Experimental Animals, 1430 Nogawa, Miyamae, Kawasaki 216-0001, Japan.
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King M, Pearson T, Shultz LD, Leif J, Bottino R, Trucco M, Atkinson MA, Wasserfall C, Herold KC, Woodland RT, Schmidt MR, Woda BA, Thompson MJ, Rossini AA, Greiner DL. A new Hu-PBL model for the study of human islet alloreactivity based on NOD-scid mice bearing a targeted mutation in the IL-2 receptor gamma chain gene. Clin Immunol 2007; 126:303-14. [PMID: 18096436 DOI: 10.1016/j.clim.2007.11.001] [Citation(s) in RCA: 143] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2007] [Revised: 11/07/2007] [Accepted: 11/12/2007] [Indexed: 11/25/2022]
Abstract
Immunodeficient NOD-scid mice bearing a targeted mutation in the IL2 receptor common gamma chain (Il2rgamma(null)) readily engraft with human stem cells. Here we analyzed human peripheral blood mononuclear cells (PBMC) for their ability to engraft NOD-scid Il2rgamma(null) mice and established engraftment kinetics, optimal cell dose, and the influence of injection route. Even at low PBMC input, NOD-scid Il2rgamma(null) mice reproducibly support high human PBMC engraftment that plateaus within 3-4 weeks. In contrast to previous stocks of immunodeficient mice, we observed low intra- and inter-donor variability of engraftment. NOD-scid Il2rgamma(null) mice rendered hyperglycemic by streptozotocin treatment return to normoglycemia following transplantation with human islets. Interestingly, these human islet grafts are rejected following injection of HLA-mismatched human PBMC as evidenced by return to hyperglycemia and loss of human C-peptide. These data suggest that humanized NOD-scid Il2rgamma(null) mice may represent an important surrogate for investigating in vivo mechanisms of human islet allograft rejection.
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Affiliation(s)
- Marie King
- Department of Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01655, USA
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Thomsen M, Galvani S, Canivet C, Kamar N, Böhler T. Reconstitution of immunodeficient SCID/beige mice with human cells: applications in preclinical studies. Toxicology 2007; 246:18-23. [PMID: 18055093 DOI: 10.1016/j.tox.2007.10.017] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2007] [Revised: 10/16/2007] [Accepted: 10/20/2007] [Indexed: 10/22/2022]
Abstract
Experimental studies of the in vivo behaviour of human cells and tissues have become possible with the development of immunodeficient mice strains. Such mice accept readily allogeneic or xenogeneic grafts, including grafts of human cells or tissues, without rejection. In this review we describe different immunodeficient mouse strains that have been used for reconstitution by human immune cells. We subsequently go through the experience that we and others have had with reconstitution, and mention the adverse effects, in particular xenogeneic graft versus host reactions. The use of haematopoietic stem cells avoids such reactions but the immunological reconstitution may take several months. We then report the use of immunodeficient mice for the study of chronic vascular rejection of human mesenteric arteries due to cellular or humoral alloreaction. We have shown that SCID/beige mice grafted with a human artery at the place of the aorta developed a thickening of the intima of the human artery after 5-6 weeks, when they were reconstituted with spleen cells from another human donor. The thickening is mainly due to a proliferation of smooth muscle cells. The same type of lesion developed if they received injection of antibodies towards HLA class I antigens. The arteries of the mouse did not develop any lesion. The arterial lesions closely resembled those seen after clinical organ transplantation. Mice that received spleen cells from the same human donor developed little or no lesions. An important aspect of this experimental transplantation model is the possibility to test drugs that may be used in clinical transplantation. In recent experiments we have shown that novel immunosuppressive drugs can inhibit the hyperproliferation of smooth muscle cells in vitro. Preclinical testing in reconstituted SCID/beige mice grafted with human arteries will permit the evaluation of the potential use of these drugs to prevent chronic vascular rejection. The model also allows pharmacodynamic studies that give information on the biological impact of different drugs that may be used in experimental or clinical transplantation.
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Affiliation(s)
- Mogens Thomsen
- Institute of Molecular Medicine of Rangueil (I2MR), Centre Hospitalier Universitaire de Rangueil, BP 84225, 31432 Toulouse Cedex 4, France.
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Camacho RE, Wnek R, Fischer P, Shah K, Zaller DM, Woods A, La Monica N, Aurisicchio L, Fitzgerald-Bocarsly P, Koo GC. Characterization of the NOD/scid-[Tg]DR1 mouse expressing HLA-DRB1*01 transgene: a model of SCID-hu mouse for vaccine development. Exp Hematol 2007; 35:1219-30. [PMID: 17662890 DOI: 10.1016/j.exphem.2007.05.002] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2007] [Revised: 03/29/2007] [Accepted: 05/07/2007] [Indexed: 11/24/2022]
Abstract
OBJECTIVE We previously showed enhanced engraftment of human T cells in the transgenic NonObese Diabetic/severe combined immunodeficient (NOD/scid)-DR1 mice, compared to NOD/scid mice. We now characterize their immunobiology, innate immunity, and intrahepatic neonatal engraftment of cord blood mononuclear cells (CBMNC), and test immune responses of these chimeric mice to an experimental cancer vaccine. METHODS Fluorescence in situ hybridization analysis, blood biochemistry, hematology, and fluorescein-activated cell sorting analyses of cellular subsets were performed on NOD/scid-DR1 mice, in comparison to parental NOD/scid mice. Innate immunity and lifespan were examined. Histology of engrafted tissues and short-term intrahepatic engraftment of CBMNC were performed. Intracellular interferon-gamma (IFN-gamma) production was assessed in mice immunized with cancer vaccine. RESULTS The DR1 transgene was located on chromosome 5 and no significant changes were observed in blood chemistry, peripheral blood counts, lymphoid subsets, natural killer cell and lipopolysaccharide response, and antigen presentation in the NOD/scid-DR1 mice, compared to NOD/scid mice. Interestingly, NOD/scid-DR1 mice had a significantly longer lifespan (approximately 14 months) than NOD/scid mice (approximately 8.5 months). Engraftment with human cord blood cells resulted in slight changes in the architecture/structure of spleens. No correlation was found between DR1 genotype of the donor CBMNC and extent of engraftment of human T cells. Enhanced engraftment of human cells was observed with intrahepatic injections of CBMNC in neonatal NOD/scid DR1 mice. Intracellular IFN-gamma was detected in human cells, when chimeric mice were immunized with a cancer vaccine. CONCLUSION NOD/scid-DR1 mice were similar in most of the physiological parameters as the NOD/scid mice, with the exception of longer lifespan. Intrahepatic engraftment of neonatal mice is the preferred protocol of xenotransplantation in this model and the engrafted human cells can respond to a cancer vaccine.
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Abstract
Psoriasis is a T-cell-mediated chronic inflammatory skin disease believed to be of autoimmune nature that can be triggered or worsened by streptococcal throat infections. In addition to conventional chronic inflammatory changes, psoriasis is characterized by complex and striking alterations in epidermal growth and differentiation. Psoriasis is generally not observed in animals other than man, and this lack of a suitable animal model has greatly hindered research into the pathogenesis of psoriasis. Multiple transgenic, knockout, and reconstituted models of psoriasis have been developed over the past two decades. Despite their limitations, these models have demonstrated that keratinocyte hyperplasia, vascular hyperplasia, and cell-mediated immunity in the skin are closely interrelated. Xenograft models, in which involved and uninvolved psoriatic skin are transplanted onto immunodeficient mice, are the only models that come close to incorporating the complete genetic, immunologic, and phenotypic changes of the disease. They have shown conclusively that psoriasis is a T-cell-mediated disease, and have been used to elucidate novel pathogenic pathways. In this review, we describe various animal models, detail the immunologic and intracellular pathways that mediate these phenotypes and assess the utility of these models to better understand this disease.
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Affiliation(s)
- Johann E Gudjonsson
- Department of Dermatology, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
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King M, Pearson T, Shultz LD, Leif J, Bottino R, Trucco M, Atkinson M, Wasserfall C, Herold K, Mordes JP, Rossini AA, Greiner DL. Development of new-generation HU-PBMC-NOD/SCID mice to study human islet alloreactivity. Ann N Y Acad Sci 2007; 1103:90-3. [PMID: 17376822 DOI: 10.1196/annals.1394.011] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
The use of "humanized" mice represents an appealing translational model for studies of the pathogenesis of immune-mediated diseases and for the evaluation of potential therapeutics. The utility of humanized mice depends on their ability to model the human immune system with high fidelity, and, in this respect, previous models have fallen short. The recently developed NOD-scid Il2rgamma(null) mouse, however, exhibits greatly enhanced ability to support the engraftment of human peripheral blood mononuclear cells. Herein, we describe the challenges of recapitulating human immunity in humanized mice and features of NOD-scid Il2rgamma(null) mice that help overcome them.
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Affiliation(s)
- Marie King
- Diabetes Division, University of Massachusetts Medical School, 373 Plantation Street, Suite 218, Worcester, MA 01605, USA
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Abstract
The culmination of decades of research on humanized mice is leading to advances in our understanding of human haematopoiesis, innate and adaptive immunity, autoimmunity, infectious diseases, cancer biology and regenerative medicine. In this Review, we discuss the development of these new generations of humanized mice, how they will facilitate translational research in several biomedical disciplines and approaches to overcome the remaining limitations of these models.
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Affiliation(s)
- Leonard D Shultz
- The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, USA.
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Shultz LD, Pearson T, King M, Giassi L, Carney L, Gott B, Lyons B, Rossini AA, Greiner DL. Humanized NOD/LtSz-scid IL2 receptor common gamma chain knockout mice in diabetes research. Ann N Y Acad Sci 2007; 1103:77-89. [PMID: 17332083 DOI: 10.1196/annals.1394.002] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
There are many rodent models of autoimmune diabetes that have been used to study the pathogenesis of human type 1 diabetes (T1D), including the non-obese diabetic (NOD) mouse, the biobreeding (BB) rat, and the transgenic mouse models. However, mice and rats are not humans, and these rodent models do not completely recapitulate the autoimmune pathogenesis of the human disease. In addition, many of the reagents, tools, and therapeutics proposed for use in humans may be species specific and cannot be investigated in rodents. Researchers have used nonhuman primates to more closely mimic the human immune system and, to study species-specific therapeutics, but these studies are associated with additional ethical and economic constraints and, to date, no model of autoimmune diabetes in this species has been described. New animal models are needed that will permit the in vivo investigation of human immune systems and analyses of the pathogenesis of human T1D without putting individuals at risk. To fill this need, we are developing humanized mouse models for the in vivo study of T1D. These models are based on our newly generated stock of NOD-scid IL2rgamma(null) mice, which engraft at higher levels with human hematolymphoid cells and exhibit enhanced function of the engrafted human immune systems compared with previous humanized mouse models. Overall, development of these new generations of humanized mice should facilitate in vivo studies of the human immune system as well as permit the investigation of the pathogenesis and effector phases of human T1D.
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Affiliation(s)
- Leonard D Shultz
- Division of Diabetes, Department of Medicine, 373 Plantation Street, Biotech 2, Suite 218, Worcester, MA 01605, USA
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Tian X, Woll PS, Morris JK, Linehan JL, Kaufman DS. Hematopoietic engraftment of human embryonic stem cell-derived cells is regulated by recipient innate immunity. Stem Cells 2006; 24:1370-80. [PMID: 16456127 DOI: 10.1634/stemcells.2005-0340] [Citation(s) in RCA: 116] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Human embryonic stem cells (hESCs) provide an important means to characterize early stages of hematopoietic development. However, the in vivo potential of hESC-derived hematopoietic cells has not been well defined. We demonstrate that hESC-derived cells are capable of long-term hematopoietic engraftment when transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Human CD45(+) and CD34(+) cells are identified in the mouse bone marrow (BM) more than 3 months after injection of hESCs that were allowed to differentiate on S17 stromal cells for 7-24 days. Secondary engraftment studies further confirm long-term repopulating cells derived from hESCs. We also evaluated two mechanisms that may inhibit engraftment: host immunity and requirement for homing to BM. Treatment with anti-ASGM1 antiserum that primarily acts by depletion of natural killer cells in transplanted mice leads to improved engraftment, likely due to low levels of HLA class I expressed on hESCs and CD34(+) cells derived from hESCs. Intra-BM injection also provided stable engraftment, with hematopoietic cells identified in both the injected and contra-lateral femur. Importantly, no teratomas are evident in animals injected with differentiated hESCs. These results demonstrate that SCID-repopulating cells, a close surrogate for hematopoietic stem cells, can be derived from hESCs. Moreover, both adaptive and innate immune effector cells may be barriers to engraftment of these cells.
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Affiliation(s)
- Xinghui Tian
- Stem Cell Institute, Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455, USA
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Abstract
Animal models have been instrumental in increasing the understanding of human physiology, particularly immunity. However, these animal models have been limited by practical considerations and genetic diversity. The creation of humanized mice that carry partial or complete human physiological systems may help overcome these obstacles. The National Institute of Allergy and Infectious Diseases convened a workshop on humanized mouse models for immunity in Bethesda, MD, on June 13–14, 2005, during which researchers discussed the benefits and limitations of existing animal models and offered insights into the development of future humanized mouse models.
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Affiliation(s)
- Francesca Macchiarini
- Division of Allergy, Immunology, and Transplantation, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
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Minamiguchi H, Wingard JR, Laver JH, Mainali ES, Shultz LD, Ogawa M. An assay for human hematopoietic stem cells based on transplantation into nonobese diabetic recombination activating gene-null perforin-null mice. Biol Blood Marrow Transplant 2005; 11:487-94. [PMID: 15983548 DOI: 10.1016/j.bbmt.2005.04.003] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Abstract Nonobese diabetic recombination activating gene-null perforin-null (NOD- Rag1 null Prf1 null ) mice, which totally lack mature T and B cells and natural killer cell cytotoxic function, survive longer and are easier to breed than NOD-severe combined immunodeficiency ( scid ) or NOD- scid /beta 2 -microglobulin null mice. We have tested the use of NOD- Rag1 null Prf1 null mice as recipients in a long-term xenograft assay for human hematopoietic stem cells (HSCs) by adopting Yoder and colleagues' method of conditioned newborn mice, with minor modifications. Pregnant NOD- Rag1 null Prf1 null dams were treated with busulfan 22.5 mg/kg. On the day of delivery, the busulfan-exposed pups underwent transplantation with 4 to 5 million T cell--depleted human cord blood mononuclear cells via the facial vein. At 2 months after transplantation, all 11 transplanted mice showed human hematopoietic engraftment in the peripheral blood. At 6 months after transplantation, human cells were detected in 5 mice, which showed higher than 0.9% human cell engraftment at 2 months. The mean percentage of human CD45 + cells in the bone marrow of engrafted mice was 43.9% +/- 36.5% (range, 2.0%-79.9%). Next, we tested the usefulness of conditioned newborn NOD- Rag1 null Prf1 null mice for applications to characterize the dye efflux capability and phenotypic features of human HSCs. Given that cord blood HSCs have the ability to efflux rhodamine 123 (Rho), we attempted transplantations of sorted cells that retained a low level (Rho low ) or high level (Rho high ) of Rho. Six-month engraftment was found only with the Rho low cells, which contained high percentages of CD34 + CD38 - cells and side population cells with Hoechst 33324 efflux activity. These observations suggest that Rho low cells are highly enriched for primitive hematopoietic cells. Accordingly, conditioned newborn NOD- Rag1 null Prf1 null mice provide a desirable model for an assay of long-term transplantable human HSCs.
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Affiliation(s)
- Hitoshi Minamiguchi
- Department of Veterans Affairs Medical Center, Medical University of South Carolina, Charleston, South Carolina 29401-5799, USA
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50
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Thomsen M, Yacoub-Youssef H, Marcheix B. Reconstitution of a human immune system in immunodeficient mice: models of human alloreaction in vivo. ACTA ACUST UNITED AC 2005; 66:73-82. [PMID: 16029426 DOI: 10.1111/j.1399-0039.2005.00409.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Rodents have been widely used for studies in transplantation immunology because of their short reproduction period and the relative ease of generating inbred mutant or transgenic strains. However, although many biological mechanisms are similar between rodents and humans, several features clearly distinguish the immune system in these species. Consequently, it is rarely possible to extrapolate observations from rodent models directly into clinical practice. In vitro studies with human cells are useful for elucidation of basic mechanisms, but in order to study complex biological phenomena, in vivo studies are indispensable. In later years, a number of interesting models have been described where immunodeficient mice have been reconstituted with human cells, so-called humanized mice, in order to study human immune responses in vivo. This has opened a new field of experimental immunology that has been applied to areas such as cancer, autoimmunity, allergy, infections, and transplantation biology. In this review, we shall concentrate on the use of severe combined immunodeficient mice reconstituted with human immune or stem cells for studies of human alloreaction in vivo.
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Affiliation(s)
- M Thomsen
- INSERM U466, CHU Rangueil, Toulouse, France.
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