Innovations, challenges, and minimal information for standardization of humanized mice
2020; Springer Nature; Volume: 12; Issue: 7 Linguagem: Inglês
10.15252/emmm.201708662
ISSN1757-4684
AutoresRenata Stripecke, Christian Münz, Jan Jacob Schuringa, Karl‐Dimiter Bissig, Brian W. Soper, Terrence Meeham, Li‐Chin Yao, James P. Di Santo, Michael A. Brehm, Estefanía Rodríguez, Anja K. Wege, Dominique Bonnet, Silvia Guionaud, Kristina E. Howard, Scott G. Kitchen, Florian Klein, Kourosh Saeb‐Parsy, Johannes Sam, Amar Deep Sharma, Andreas Trumpp, Livio Trusolino, Carol Bult, Leonard D. Shultz,
Tópico(s)Cell Image Analysis Techniques
ResumoReview24 June 2020Open Access Innovations, challenges, and minimal information for standardization of humanized mice Renata Stripecke Corresponding Author [email protected] orcid.org/0000-0001-7756-8460 Regenerative Immune Therapies Applied, Hannover Medical School, Hannover, Germany German Center for Infection Research (DZIF), Hannover Region, Germany Search for more papers by this author Christian Münz orcid.org/0000-0001-6419-1940 Viral Immunobiology, Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland Search for more papers by this author Jan Jacob Schuringa orcid.org/0000-0001-8452-8555 Department of Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Karl-Dimiter Bissig Department of Pediatrics, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Brian Soper The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Terrence Meeham Kymab Biotechnology, Cambridge, UK Search for more papers by this author Li-Chin Yao The Jackson Laboratory, Sacramento, CA, USA Search for more papers by this author James P Di Santo Institut Pasteur, INSERM U1223, Paris, France Search for more papers by this author Michael Brehm University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Estefania Rodriguez Virology Dept., Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany Search for more papers by this author Anja Kathrin Wege Department of Gynecology and Obstetrics, University Cancer Center Regensburg, Regensburg, Germany Search for more papers by this author Dominique Bonnet The Francis Crick Institute, London, UK Search for more papers by this author Silvia Guionaud Guionaud Nonclinical Consulting, Canterbury, UK Search for more papers by this author Kristina E Howard U.S. Food & Drug Administration, Silver Spring, MD, USA Search for more papers by this author Scott Kitchen University of California, Los Angeles, Los Angeles, CA, USA Search for more papers by this author Florian Klein University of Cologne, Cologne, Germany Search for more papers by this author Kourosh Saeb-Parsy University of Cambridge, Cambridge, UK Search for more papers by this author Johannes Sam Roche Innovation Center Zurich, Zurich, Switzerland Search for more papers by this author Amar Deep Sharma Regenerative Immune Therapies Applied, Hannover Medical School, Hannover, Germany Search for more papers by this author Andreas Trumpp orcid.org/0000-0002-6212-3466 Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany Search for more papers by this author Livio Trusolino Department of Oncology, University of Torino Medical School, Turin, Italy Candiolo Cancer Institute FPO IRCCS, Candiolo, Italy Search for more papers by this author Carol Bult The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Leonard Shultz The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Renata Stripecke Corresponding Author [email protected] orcid.org/0000-0001-7756-8460 Regenerative Immune Therapies Applied, Hannover Medical School, Hannover, Germany German Center for Infection Research (DZIF), Hannover Region, Germany Search for more papers by this author Christian Münz orcid.org/0000-0001-6419-1940 Viral Immunobiology, Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland Search for more papers by this author Jan Jacob Schuringa orcid.org/0000-0001-8452-8555 Department of Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Karl-Dimiter Bissig Department of Pediatrics, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Brian Soper The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Terrence Meeham Kymab Biotechnology, Cambridge, UK Search for more papers by this author Li-Chin Yao The Jackson Laboratory, Sacramento, CA, USA Search for more papers by this author James P Di Santo Institut Pasteur, INSERM U1223, Paris, France Search for more papers by this author Michael Brehm University of Massachusetts Medical School, Worcester, MA, USA Search for more papers by this author Estefania Rodriguez Virology Dept., Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany Search for more papers by this author Anja Kathrin Wege Department of Gynecology and Obstetrics, University Cancer Center Regensburg, Regensburg, Germany Search for more papers by this author Dominique Bonnet The Francis Crick Institute, London, UK Search for more papers by this author Silvia Guionaud Guionaud Nonclinical Consulting, Canterbury, UK Search for more papers by this author Kristina E Howard U.S. Food & Drug Administration, Silver Spring, MD, USA Search for more papers by this author Scott Kitchen University of California, Los Angeles, Los Angeles, CA, USA Search for more papers by this author Florian Klein University of Cologne, Cologne, Germany Search for more papers by this author Kourosh Saeb-Parsy University of Cambridge, Cambridge, UK Search for more papers by this author Johannes Sam Roche Innovation Center Zurich, Zurich, Switzerland Search for more papers by this author Amar Deep Sharma Regenerative Immune Therapies Applied, Hannover Medical School, Hannover, Germany Search for more papers by this author Andreas Trumpp orcid.org/0000-0002-6212-3466 Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany Search for more papers by this author Livio Trusolino Department of Oncology, University of Torino Medical School, Turin, Italy Candiolo Cancer Institute FPO IRCCS, Candiolo, Italy Search for more papers by this author Carol Bult The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Leonard Shultz The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Author Information Renata Stripecke *,1,2,‡, Christian Münz3,‡, Jan Jacob Schuringa4,‡, Karl-Dimiter Bissig5,‡, Brian Soper6,‡, Terrence Meeham7,‡, Li-Chin Yao8, James P Di Santo9, Michael Brehm10, Estefania Rodriguez11, Anja Kathrin Wege12, Dominique Bonnet13, Silvia Guionaud14, Kristina E Howard15, Scott Kitchen16, Florian Klein17, Kourosh Saeb-Parsy18, Johannes Sam19, Amar Deep Sharma1, Andreas Trumpp20,21, Livio Trusolino22,23, Carol Bult6 and Leonard Shultz6,‡ 1Regenerative Immune Therapies Applied, Hannover Medical School, Hannover, Germany 2German Center for Infection Research (DZIF), Hannover Region, Germany 3Viral Immunobiology, Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland 4Department of Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 5Department of Pediatrics, Duke University Medical Center, Durham, NC, USA 6The Jackson Laboratory, Bar Harbor, ME, USA 7Kymab Biotechnology, Cambridge, UK 8The Jackson Laboratory, Sacramento, CA, USA 9Institut Pasteur, INSERM U1223, Paris, France 10University of Massachusetts Medical School, Worcester, MA, USA 11Virology Dept., Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany 12Department of Gynecology and Obstetrics, University Cancer Center Regensburg, Regensburg, Germany 13The Francis Crick Institute, London, UK 14Guionaud Nonclinical Consulting, Canterbury, UK 15U.S. Food & Drug Administration, Silver Spring, MD, USA 16University of California, Los Angeles, Los Angeles, CA, USA 17University of Cologne, Cologne, Germany 18University of Cambridge, Cambridge, UK 19Roche Innovation Center Zurich, Zurich, Switzerland 20Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany 21Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany 22Department of Oncology, University of Torino Medical School, Turin, Italy 23Candiolo Cancer Institute FPO IRCCS, Candiolo, Italy ‡These authors contributed equally to this work *Corresponding author. Tel: +49 (511) 532-6999; Fax: +49 (511) 532-6975; E-mail: [email protected] EMBO Mol Med (2020)12:e8662https://doi.org/10.15252/emmm.201708662 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mice xenotransplanted with human cells and/or expressing human gene products (also known as "humanized mice") recapitulate the human evolutionary specialization and diversity of genotypic and phenotypic traits. These models can provide a relevant in vivo context for understanding of human-specific physiology and pathologies. Humanized mice have advanced toward mainstream preclinical models and are now at the forefront of biomedical research. Here, we considered innovations and challenges regarding the reconstitution of human immunity and human tissues, modeling of human infections and cancer, and the use of humanized mice for testing drugs or regenerative therapy products. As the number of publications exploring different facets of humanized mouse models has steadily increased in past years, it is becoming evident that standardized reporting is needed in the field. Therefore, an international community-driven resource called "Minimal Information for Standardization of Humanized Mice" (MISHUM) has been created for the purpose of enhancing rigor and reproducibility of studies in the field. Within MISHUM, we propose comprehensive guidelines for reporting critical information generated using humanized mice. Glossary ADCC antibody-dependent cellular cytotoxicity, is an immune defense mechanism whereby effector cells such as NK cells lyses target cells that have been bound by specific antibodies AML acute myeloid leukemia ART anti-retroviral therapy BDBV Bundibugyo ebolavirus BiTE bispecific T-cell engagers is a registered trademark for a class of recombinant bispecific monoclonal antibodies which bind to the CD3 receptor and to a tumor-specific antigen BLT bone marrow-liver-thymus BM bone marrow bNAbs broadly neutralizing antibodies are antibodies capable of neutralizing different types of viral strains BRGF Balb/c Rag2−/− Il2rg−/− Flt3−/− BRGSA2DR2 BRGS mice expressing human HLA-A2 and DR2 transgenes CAR chimeric antigen receptor CB cord blood CCR5 chemokine receptor targeted by R5 tropic HIV strains CD40L CD40 ligand CDX cell line-derived xenograft CRS cytokine release syndrome is a systemic inflammatory response that can be triggered by infections, drugs, and cell therapies DCs dendritic cells DRAG mouse strain expressing a human HLA-DR gene and derived from the NRG strain EBOV Zaire ebolavirus EBV Epstein–Barr virus ES cell embryonic stem cell FAH −/− knock-out for the fumarylacetoacetate hydrolase gene Flt3L Flt3 ligand G-CSF granulocyte colony-stimulating factor GITR glucocorticoid-induced TNFR family-related protein GM-CSF granulocyte–macrophage colony-stimulating factor gp glycoprotein GVHD graft-versus-host disease HAdV2 human adenovirus 2 HBV hepatitis B virus HCMV human cytomegalovirus HCT HSC transplantation is a routine clinical procedure performed with hematopoietic stem cells from the patient (autologous) or from a donor (allogeneic) with the purpose of combating malignancies or correcting defects of the immune system HCV hepatitis C virus Hematopoiesis is the differentiation of different blood cell lineages derived from multipotent hematopoietic stem cells (HSCs) HIS human immune system HIV human immunodeficiency virus HLAs human leukocyte antigens HSCs human hematopoietic stem cells HSPCs hematopoietic/stem/progenitor cells HSPCs hematopoietic/stem/progenitor cells HSVtk herpes simplex virus type 1 thymidine kinase huPBL human peripheral blood lymphocyte IFN interferon IgG immunoglobulin G IgM immunoglobulin M IL-2 interleukin 2 Il2rg interleukin 2 (IL-2) receptor common gamma chain IL-3 interleukin 3 IO Immuno-oncology iPS cell induced pluripotent stem cells LDL low-density lypoprotein Lin− lineage negative lymphomagenesis is the development of malignancies derived from lymphocytes such as B and T cells mAb monoclonal antibodies M-CSF macrophage colony-stimulating factors MDS myelodysplastic syndrome MERS Middle East respiratory syndrome-related coronavirus MHC major histocompatibility complex MISHUM minimal information for standardization of humanized mice MISTRG-6 mouse strain expressing macrophage colony-stimulating factors (M-CSF), IL-3, IL-6, GM-CSF, and thrombopoietin (TPO) MSCs mesenchymal stromal cells Myelo-ablated mice are mice treated with irradiation or chemotherapy in order to decrease the bone marrow activity in order to improve the engraftment of transplanted stem cells Myelodysplasia is an abnormal accumulation of immature blood cells in the bone marrow Myelofibrosis is the replacement of the bone marrow with scar tissue due to proliferation of immature blood cells NASH non-alcoholic steatohepatitis NIH National Institutes of Health NK natural killer NOD Non-obese diabetic NOG NOD.Cg-PrkdcscidIl2rgtm1Sug/Jic NRGF NOD-Rag1−/− Il2rg−/− Flk2−/− NRG NOD-Rag1tm1Mom Il2rgtm1Wjl/SzJ NSG NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ PBMCs peripheral blood mononuclear cells PD-1 programmed death receptor 1 PD-L1 PD-1 ligand 1 PDX-MI PDX Model Minimal Information standard PDX patient-derived xenograft PIRF POR−/−/Il2rg−/−/Rag2−/−/FAH−/− Rag1 recombination activating gene 1 Rag2 recombination activating gene 2 RESTV Reston ebolavirus RSV Respiratory syncytial virus SC-beta stem cell-derived human beta cells SCF stem cell factor scid mice CB17-Prkdcscid severely compromised immunodeficient SCID-hu SCID-humanized SHIV simian and human immunodeficiency virus Sirpa signal regulatory protein alpha SUDV Sudan ebolavirus T1D type 1 diabetes TAFV Tai Forest ebolavirus TCB T-cell bispecific antibodies TCRs T-cell receptors Tim-3 T-cell immunoglobulin and mucin domain-containing protein 3 TK-NOG NOG mice expressing transgenic herpes simplex virus type 1 thymidine kinase (HSVtk) under the albumin promoter (NOD.Cg-PrkdcscidIl2rgtm1SugTg(Alb-Tk)7-2/ShiJic) TPO Thrombopoietin Tregs regulatory T cells TSLP thymic-stromal-cell-derived lymphopoietin uPA urokinase-type plasminogen activator expressed under the albumin promoter Studies of human stem cell engraftment, hematopoiesis, and immunity Studies using immunocompetent mice have provided critical insights into the development and regulation of hematopoiesis and immunity. However, such studies do not always reflect responses in humans because of multiple species-specific differences. Therefore, mice developing components of the human immune system (HIS) mice were created. These models have provided tools for the understanding of human hematopoiesis and immunity in vivo and to test new therapies or vaccines without incurring risks to patients. The simplest engraftment method is the adoptive administration of human peripheral blood mononuclear cells (PBMCs) into severely immunodeficient mice (Fig 1A, Table 1). Since the adoptive human T cells react forcefully against the xenogeneic major histocompatibility complex (MHC) class I and II expressed by mouse tissues, this so-called "huPBL" model faces the hardship of fulminant xenograft graft-versus-host disease (GVHD) occurring 2–4 weeks after PBMC transfer. These models have limited applicability to follow specific antigenic responses, but can be used to test human immunosuppressive agents. Improvement of the huPBL model has been described with novel mouse strains lacking mouse MHC class I and II, resulting in lower occurrences of GVHD (Yaguchi et al, 2018; Brehm et al, 2019). Figure 1. Development and applications of humanized mouse modelsSchematic representation of the human materials (in blue), immunodeficient mouse strain characteristics and handling (black), and analyses performed (red) for different types of humanized mouse models: (A) human immunity; (B) human metabolism; (C) human infections; (D) human malignancies; (E) human immuno-oncology. Abbreviated items are spelled out in the glossary. Download figure Download PowerPoint Table 1. Checklist as a guideline for reporting the "Minimal Information for Standardization of Humanized Mice" (MISHUM) MISHUM Section 1: human donor •. *Ethical approval and informed consent •. *(Gestational) age •. Sex •. Ethnic origin •. Human leukocyte antigens (HLA-A, B, C, DR) •. Known latent viral infections (EBV, HCMV, HIV, HCV, LCMV, HBV) •. Exome sequence if available MISHUM Section 2: human cells or tissues •. *Cell lines (mycoplasma tested or other tests) •. *Cell lines or primary cells/tissue available through academic collections and material transfer agreement/publicly available through commercial repositories •. *huPBL: Whole blood, PBMC •. *HSC: obtained from fetal liver, cord blood, G-CSF mobilized adult donors, bone marrow •. Hepatocytes (±non parenchymal cells) •. Primary patient tumors (isolation or collection method) •. *Density fractionation (e.g., by Ficoll) •. *Surface markers for positive cell isolation (magnetic beads or sorting) •. *Surface markers for cell depletions (magnetic beads or sorting) •. *Single donor or pooled •. *Fresh or cryopreserved •. *Dose as viable cell numbers •. * Dose of tissue by weight •. *Genetic modifications •. *Genetic reprogramming (e.g., iPSC) •. *Ex vivo expansion •. *Ex vivo activation •. *Use of scaffolds for 3D culture •. *Organoids •. Known if latently infected with pathogens MISHUM Section 3: mouse recipient •. *Institutional approval and approval number •. *Strain/source/publicly available or material transfer agreement/stock number •. *Human transgenes/knock-in •. *Knock-out of mouse genes •. *Sex •. *Age (weeks) •. Health reports •. Microbiota MISHUM Section 4: mouse handling •. *Anesthesia (local, general, type and dose) •. *Preconditioning (radiation dose/schedule for pharmacologic myeloablation or liver cell death) •. *Route of injections (intravenous, intra-peritoneal, intra-femoral, intra-liver, intra-splenic) •. *Surgical implantation (under kidney capsule, intradermal, in mammary fat pad) •. *Collection of blood (intravenous, facial vein, cardiac puncture) •. *Administration of recombinant cytokines (vendor, units per weight, route) •. *Administration of vectors (type, dose, route) •. Non-invasive optical imaging methods (fluorescence, bioluminescence substrate, dose, imaging time, region of interest) MISHUM Section 5: human hematopoiesis and immunity •. *Relative human HSC engraftment and chimerism (% huCD45+ cells in mouse blood at weeks 10, 15, 20 after HCT showing gating strategies) •. Absolute human HSC engraftment and chimerism (absolute numbers of huCD45+ cells and muCD45+ cells in mouse blood at weeks 10, 15, 20 after HCT showing gating and quantification strategies) •. *Kinetics of human lymphocyte development (% huCD45+, huCD3+, huCD4+, huCD8+ huCD19+ cells in mouse blood at weeks 10, 15, 20 after HCT showing gating strategies) •. *Human cytokines or chemokines detectable in plasma at terminal analyses (ELISA, bead array methods with appropriate human control samples) •. *Human immunoglobulins detectable in plasma at terminal analyses (ELISA, bead array methods with appropriate human control samples) •. Kinetics of human myeloid development (% huCD45+, huCD33+, huCD11c+, huCD11b+, huCD14+ cells in mouse blood at weeks 6, 10, 15, 20 after HCT showing gating strategies) •. Kinetics of human NK development (% huCD45+, huNKp46+, hu56+, huCD16+ cells in mouse blood at weeks 6, 10, 15, 20 after HCT showing gating strategies) •. Kinetics of human B cell development (% huCD45+, huCD19+, huCD27+, huIgM+, huIgG+, huIgA+, cells in mouse blood at weeks 10, 15, 20 after HCT showing gating strategies) •. Terminal analyses of human hematopoietic cells in lymphatic tissues (spleen, bone marrow, thymus, peripheral lymph nodes, mesenteric lymph nodes showing total number of cells recovered by tissue). •. Terminal analyses of human hematopoietic cells in organs (liver, lungs, brain, etc.…). •. Phenotypic characterization of T cells (naïve, central memory, terminal effector, terminal effector memory) •. Antigen-specific characterization of T cells (ELISpot, intracellular staining of IFN-γ or TNF-α, tetramer analyses) •. Antigen-specific characterization of antibodies produced by B cells (ELISA, dot-plot, antigen binding by flow cytometry) •. Analyses of antibody functionality against infections (neutralization) •. Immune composition by CyTOF •. Gene expression analyses (microarrays, RNAseq) MISHUM Section 6: regeneration of human tissues •. Liver engraftment of hepatoblast, hepatocytes and stem cell-derived cells (ES or iPSC protocols), lung, gut, endocrine pancreas, kidney or other tissue •. Validation of chimerism in the murine blood (ELISA human albumin other secreted proteins) •. Functional validation: exogenous test drugs with known and different human metabolism, viral titers or antigens of human hepatotropic viruses (HBV, HCV, etc.) •. Validation of chimerism postmortem by immunostaining (human nuclei or other human-specific antibodies) •. Onset of autoimmunity or diabetes. MISHUM Section 7: human infections •. *Scientific and informal nomenclature for clinical or laboratory pathogen isolates •. *Availability through academic collections with material transfer agreement or publicly available through commercial repositories •. Biosafety level containment: BSL-2, BSL-3, BSL-3**, BSL-4 •. *Gene modification or reporter gene •. *Route of infection: intravenous, intra-peritoneal, intranasal, intrarectal, intra-splenic •. *Determination of titer and dose of challenge •. *Analyses of infection dissemination by PCR (primers, methods) •. *Analyses of infection dissemination by histology (antibodies, methods) •. Analyses of pathogenesis (load in different tissues, survival, weight loss, liver enzymes, virus-induced tumor formation) •. Analyses of infected cells (FACS, FISH, IF, PrimeFlow, single-cell sequencing) •. Non-invasive optical imaging methods (fluorescence, bioluminescence substrate, dose, imaging time, region of interest) MISHUM Section 8: human oncology and immuno-oncology •. *Donor (age, sex, HLA type) •. Primary human tumor or passaged as xenograft •. Isolation or selection method of tumor tissue •. *Tumor information (HLA expression level, exome sequencing, mutations) •. *Cancer identity and metastasis in vivo by histopathological analyses •. *Autologous or allogeneic to HSCs used in HIS mice •. Characteristics after growth (infiltration and activation of human lymphocytes) •. Immune modulation of tumor growth MISHUM Section 9: preclinical testing of human drugs and vaccines •. *Chemical or commercial name •. *Vendor or collaboration agreements •. *Dose, route, schedule •. Pharmacokinetics and pharmacodynamics •. Antibody characteristics for passive vaccination •. Characteristics of attenuated viruses, of antigen carrying receptor targeting antibodies, of virus-like particles, and of recombinant viral vaccine candidates •. Human drug metabolism in the liver: degree of humanization upon testing, next-generation strains with human drug metabolism (PIRF or other). •. Detection of AST/ALT (liver damage), cytokine release symptom (cytokine storm) MISHUM Section 10: testing of human cell therapies •. *Production in laboratory scale, GMP-like or GMP •. *Dose of viable cells •. *Route, schedule •. Pharmacokinetics and pharmacodynamics •. Readouts as described above MISHUM Section 11: statistical and correlative analyses •. Commercially available statistical software (e.g., t-test, ANOVA, etc.…) •. Specialized tests used by professional biostatisticians •. Heat-map analyses •. Principal component analyses •. Neural network analyses •. Isogenic control groups or different donors Asterisks indicate information that should be required in publications. A more complex approach covered here in detail is the hematopoietic stem cell transplantation (HCT) of preconditioned immunodeficient mice with human hematopoietic stem cells (HSCs). Despite the full mismatch between the human leukocyte antigens (HLA) expressed on the human hematopoietic cells and the mouse MHC expressed on tissues, HCT leads to "fully" humanized HIS models (Fig 1A, Table 1). Human HSCs can differentiate into multiple human hematopoietic lineages, giving rise to mature leukocytes, including several lineages of the human immune system. Robust engraftment with human hematopoietic and lymphoid cells was pioneered back in 1988 with the description of the CB17-Prkdcscid severely compromised immunodeficient (scid) strain engrafted with human fetal liver hematopoietic cells and autologous thymic tissues (McCune et al, 1988). This SCID-humanized (SCID-hu) system showed initially only a transient presence of human T cells and human immunoglobulin G (IgG) in the circulation. The critical relevance of the strain background for engraftment success of human cells was later appreciated when it was observed that non-obese diabetic (NOD)-scid mice had a much higher capacity to support human HSC engraftment. This was elucidated to be due to the expression of a human-like signal regulatory protein alpha (Sirpa) allele in the NOD strain, popularly known as the "don't eat me signal", bypassing phagocytosis of human cells by mouse macrophages (Takenaka et al, 2007; Shultz et al, 2012). Targeting the interleukin 2 (IL-2) receptor common gamma chain (Il2rg) resulted in the absence of mouse natural killer (NK) cell activity as well as ablation of T and B lymphocyte lineages. In addition, the development of mice lacking the expression of recombination activating gene 1 (Rag1)−/− and Rag2−/− provided radioresistant mouse models lacking mature host T cells as well as B cells (Shultz et al, 2007). Currently, there are approximately 50 diverse humanized mouse models available from biorepositories. Most of these models are homozygous for the scid, Il2rg, Rag1, or Rag2 mutations and express the NOD or human Sirpa allele. The NOD-scid IL2rg(−/−) (NSG), the NOD-Rag1−/− IL2rg−/− (NRG), and the NOD/Shi-scid IL2rg(−/−) (NOG) are broadly used strains for xenografting a large variety of human cells, but several other strains are prospering (for recent reviews, see Shultz et al, 2019; Allen et al, 2019). It is important to be thoughtful also about the nature of the human HSCs. Although humans and mice differ greatly in their biological characteristics, human HSCs can essentially engraft in myelo-ablated or irradiated mice and reside in the mouse bone marrow (BM) niche. This HCT approach opened several doors for the understanding of the basic properties for long-term durable repopulation of human HSCs. As sources of human HSCs, cord blood (CB) or fetal liver are mostly used, as they have high frequencies of HSCs. Generally, a range of 1 × 104–105 isolated HSCs is administered per mouse in order to enable efficient human hematopoietic engraftment and long-term reconstitution. Several laboratories have opted to use fetal tissues due to the higher abundance in the numbers of HSCs, which can be explored to generate larger cohorts of humanized mice (n = 30–40) compared with cord blood (n = 10–20). Some groups have tried to overcome this limitation by pooling HSCs from several donors, but upon development of immune systems that are not HLA-matched, once the T cells develop, allograft reactions among donors can complicate the analyses of the immune responses. Additionally, it is important to take into consideration that HSCs in fetal and neonatal tissues may be intrinsically different regarding the stage of the hematopoietic development. Further, it is important to consider ethical constraints and difficulties in procurement of human fetal tissues. In fact, the US National Institutes of Health (NIH) is currently supporting investigators to seek and develop humanized mouse models that do not rely on human fetal tissues (Allen et al, 2019). Human HSC cell surface markers have been used to allow their identification, purification, and analyses, in order to define the HSC populations with highest engraftment and/or repopulation capacity. Xenotransplantation of human CD34+ HSCs into preconditioned immunodeficient mice is the most broadly used procedure to generate HIS mice, and this approach is corroborated by the clinical evidence that transplantation with human-enriched CD34+ hematopoietic/stem/progenitor cells (HSPCs) is a salvage procedure when the HLA is not optimally matched between patients and donors. Remarkably, a defined CD93hi sub-fraction within the lineage negative (Lin−) CD34− CD38− cell present in CB has high repopulating capacity in NOD-scid mice (Danet et al, 2002). CD49f is an adhes
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