Humanized mouse

A humanized mouse is a genetically modified mouse that has functioning human genes, cells, tissues and/or organs.^[1] Humanized mice are commonly used as small animal models in biological and medical research for human therapeutics.^[2]

A humanized mouse or a humanized mouse model is one that has been xenotransplanted with human cells and/or engineered to express human gene products, so as to be utilized for gaining relevant insights in the in vivo context for understanding of human-specific physiology and pathologies.^[3] A lot of knowledge about several human biological processes has been obtained from studying animal models like rodents and non-human primates. In particular, small animals such as mice are advantageous in such studies owing to their small size, brief reproductive cycle, easy handling and due to the genomic and physiological similarities with humans; moreover, these animals can also be genetically modified easily. Nevertheless, there are several incongruencies of these animal systems with those of humans, especially with regard to the components of the immune system. To overcome these limitations and to realize the full potential of animal models to enable researchers to get a clear picture of the nature and pathogenesis of immune responses mounted against human-specific pathogens, humanized mouse models have been developed. Such mouse models have also become an integral aspect of preclinical biomedical research.^[4]

History

The discovery of the athymic mouse, commonly known as the nude mouse, and that of the SCID mouse were major events that paved the way for humanized mice models. The first such mouse model was derived by backcrossing C57BL/Ka and BALB/c mice, featuring a loss of function mutation in the PRKDC gene. The PRKDC gene product is necessary for resolving breaks in DNA strands during the development of T cells and B cells. A mutation in the Foxn1 gene on chromosome 11 resulted in impaired thymus development, leading to a deficiency in mature T lymphocytes. Dysfunctional PRKDC gene leads to impaired development of T and B lymphocytes which gives rise to severe combined immunodeficiency (SCID). In spite of the efforts in developing this mouse model, poor engraftment of human hematopoietic stem cells (HSCs) was a major limitation that called for further advancement in the development humanized mouse models.^[5] Nude mice were the earliest immunodeficient mouse model. These mice primarily produced IgM and had minimal or no IgA. As a result, they did not exhibit a rejection response to allogeneic tissue. Commonly utilized strains included BALB/c-nu, Swiss-nu, NC-nu, and NIH-nu, which were extensively employed in the research of immune diseases and tumors. However, due to the retention of B cells and NK cells, they were unable to fully support engraftment of human immune cells, thus making them unsuitable as an ideal humanized mouse model.

The next big step in the development of humanized mice models came with transfer of the scid mutation to a non-obese diabetic mouse. This resulted in the creation of the NOD-scid mice which lacked T cells, B cells, and NK cells. This mouse model permitted for a slightly higher level of human cell reconstitution. Nevertheless, a major breakthrough in this field came with the introduction of the mutant IL-2 receptor (IL2rg) gene in the NOD-scid model. This accounted for the creation of the NOD-scid-γcnull mice (NCG, NSG or NOG) models which were found to have defective signaling of interleukins IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. Researchers evolved this NSG model by knocking out the RAG1 and RAG2 genes (recombination activation genes), resulting into the RAG^null version of the NSG model that was devoid of major cells of the immune system including the natural killer cells, B lymphocytes and T lymphocytes, macrophages and dendritic cells, causing the greatest immunodeficiency in mice models so far. The limitation with this model was that it lacked the human leukocyte antigen. In accordance to this limitation, the human T cells when engrafted in the mice, failed to recognize human antigen-presenting cells, which consequated in defective immunoglobulin class switching and improper organization of the secondary lymphoid tissue.^[6]

To circumvent this limitation, the next development came with the introduction of transgenes encoding for HLA I and HLA II in the NSG RAG^null model that enabled buildout of human T-lymphocyte repertoires as well as the respective immune responses.^[7] Mice with such human genes are technically human-animal hybrids.

Types

Engrafting an immunodeficient mouse with functional human cells can be achieved by intravenous injections of human cells and tissue into the mouse, and/or creating a genetically modified mouse from human genes. These models have been instrumental in studying human diseases, immune responses, and therapeutic interventions. This section highlights the various humanized mice models developed using the different methods.

Hu-PBL-scid model

The human peripheral blood lymphocyte-severe combined immunodeficiency mouse model has been employed in a diverse array of research, encompassing investigations into Epstein-Barr virus (EBV)-associated lymphoproliferative disease, toxoplasmosis, human immunodeficiency virus (HIV) infection, and autoimmune diseases.^[8] These studies have highlighted the effectiveness of the hu-PBL-SCID mouse model in examining various facets of human diseases, including pathogenesis, immune responses, and therapeutic interventions. Furthermore, the model has been utilized to explore genetic and molecular factors linked to neuropsychiatric disorders such as schizophrenia, offering valuable insights into the pathophysiology and potential therapeutic targets for these conditions.^[9] This model is developed by intravenously injecting human PBMCs into immunodeficient mice. The peripheral blood mononuclear cells to be engrafted into the model are obtained from consented adult donors. The advantages associated with this method are that it is comparatively an easy technique, the model takes relatively less time to get established and that the model exhibits functional memory T cells.^[10] It is particularly very effective for modelling graft vs. host disease.^[7] The model lacks engraftment of B lymphocytes and myeloid cells. Other limitations with this model are that it is suitable for use only in short-term experiments (<3 months) and the possibility that the model itself might develop graft vs. host disease.^[7]

Hu-SRC-scid model

The humanized severe combined immunodeficiency (SCID) mouse model, also known as the hu-SRC-scid model, has been extensively utilized in various research areas, including immunology, infectious diseases, cancer, and drug development. This model has been instrumental in studying the human immune response to xenogeneic and allogeneic decellularized biomaterials, providing valuable insights into the biocompatibility and gene expression regulation of these materials.^[11] Hu-SRC-scid mice are developed by engrafting CD34+ human hematopoietic stem cells into immunodeficient mice. The cells are obtained from human fetal liver, bone marrow or from blood derived from the umbilical cord,^[12] and engrafted via intravenous injection. The advantages of this model are that it offers multilineage development of hematopoietic cells, generation of a naïve immune system, and if engraftment is carried out by intrahepatic injection of newborn mice within 72 hours of birth, it can lead to enhanced human cell reconstitution. Nevertheless, limitations associated with the model are that it takes a minimum of 10 weeks for cell differentiation to occur, it harbors low levels of human RBCs, polymorphonuclear leukocytes, and megakaryocytes.^[7]

BLT (bone marrow/liver/thymus) model

The BLT model is constituted with human HSCs, bone marrow, liver, and thymus. The engraftment is carried out by implantation of liver and thymus under the kidney capsule and by transplantation of HSCs obtained from fetal liver. The BLT model has a complete and totally functional human immune system with HLA-restricted T lymphocytes. The model also comprises a mucosal system that is similar to that of humans. Moreover, among all models the BLT model has the highest level of human cell reconstitution.^[13]

However, since it requires surgical implantation, this model is the most difficult and time-consuming to develop. Other drawbacks associated with the model are that it portrays weak immune responses to xenobiotics, sub-optimal class switching and may develop GvHD.^[7]

Transplanted human organoids

Bio- and electrical engineers have shown that human cerebral organoids transplanted into mice functionally integrate with their visual cortex.^[14]^[15] Such models may raise similar ethical issues as organoid-based humanization of other animals.

Mouse-human hybrid

A mouse-human hybrid is a genetically modified mouse whose genome has both mouse and human genes, thus being a murine form of a human-animal hybrid. For example, genetically modified mice may be born with human leukocyte antigen genes in order to provide a more realistic environment when introducing human white blood cells into them in order to study immune system responses.^[7] One such application is the identification of hepatitis C virus (HCV) peptides that bind to HLA, and that can be recognized by the human immune system, thereby potentially being targets for future vaccines against HCV.^[16]

Established models for human diseases

Several mechanisms underlying human maladies are not fully understood. Utilization of humanized mice models in this context allows researchers to determine and unravel important factors that bring about the development of several human diseases and disorders falling under the categories of infectious disease, cancer, autoimmunity, and GvHD.

Infectious diseases

Among the human-specific infectious pathogens studied on humanized mice models, the human immunodeficiency virus has been successfully studied.^[7] Besides this, humanized models for studying Ebola virus,^[17] Hepatitis B,^[18] Hepatitis C,^[19] Kaposi's sarcoma-associated herpesvirus,^[20] Leishmania major,^[21] malaria,^[22] and tuberculosis^[23] have been reported by various studies.

NOD/scid mice models for dengue virus^[24] and varicella-zoster virus,^[25] and a Rag2^null𝛾c^null model for studying influenza virus^[26] have also been developed.

Cancers

On the basis of the type of human cells/tissues that have been used for engraftment, humanized mouse models for cancer can be classified as patient-derived xenografts or cell line-derived xenografts.^[27] PDX models are considered to retain the parental malignancy characteristics at a greater extent and hence these are regarded as the more powerful tool for evaluating the effect of anticancer drugs in pre-clinical studies.^[27]^[28] Humanized mouse models for studying cancers of various organs have been designed. A mouse model for the study of breast cancer has been generated by the intrahepatic engraftment of SK-BR-3 cells in NSG mice.^[29] Similarly, NSG mice intravenously engrafted with patient-derived AML cells,^[30] and those engrafted (via subcutaneous, intravenous or intra-pancreatic injections) with patient-derived pancreatic cancer tumors^[31] have also been developed for the study of leukemia and pancreatic cancer respectively. Several other humanized rodent models for the study of cancer and cancer immunotherapy have also been reported.^[32]

Autoimmune diseases

Problems posed by the differences in the human and rodent immune systems have been overcome using a few strategies, so as to enable researchers to study autoimmune disorders using humanized models. As a result, the use of humanized mouse models has extended to various areas of immunology and disease research. For instance, humanized mice have been utilized to study human-tropic pathogens, liver cancer models, and the comparison of mouse models to human diseases NSG mice engrafted with PBMCs and administered with myelin antigens in Freund's adjuvant, and antigen-pulsed autologous dendritic cells have been used to study multiple sclerosis.^[33] Similarly, NSG mice engrafted with hematopoietic stem cells and administered with pristane have been used for studying lupus erythematosus.^[34] Furthermore, NOG mice engrafted with PBMCs has been used to study mechanisms of allografts rejection in vivo.^[35] The development of humanized mouse models has significantly advanced the study of autoimmune disorders and various areas of immunology and disease research. These models have provided a platform for investigating human diseases, immune responses, and therapeutic interventions, bridging the gap between human and rodent immune systems and offering valuable insights into disease pathogenesis and potential therapeutic strategies.

References

^ Chuprin J, Buettner H, Seedhom MO, Greiner DL, Keck JG, Ishikawa F, et al. (March 2023). "Humanized mouse models for immuno-oncology research". Nature Reviews. Clinical Oncology. 20 (3): 192–206. doi:10.1038/s41571-022-00721-2. PMC 10593256. PMID 36635480.
^ Brehm MA, Wiles MV, Greiner DL, Shultz LD (August 2014). "Generation of improved humanized mouse models for human infectious diseases". Journal of Immunological Methods. 410: 3–17. doi:10.1016/j.jim.2014.02.011. PMC 4155027. PMID 24607601.
^ Stripecke R, Münz C, Schuringa JJ, Bissig KD, Soper B, Meeham T, et al. (July 2020). "Innovations, challenges, and minimal information for standardization of humanized mice". EMBO Molecular Medicine. 12 (7): e8662. doi:10.15252/emmm.201708662. PMC 7338801. PMID 32578942.
^ Walsh NC, Kenney LL, Jangalwe S, Aryee KE, Greiner DL, Brehm MA, Shultz LD (January 2017). "Humanized Mouse Models of Clinical Disease". Annual Review of Pathology. 12 (1): 187–215. doi:10.1146/annurev-pathol-052016-100332. PMC 5280554. PMID 27959627.
^ Ito R, Takahashi T, Katano I, Ito M (May 2012). "Current advances in humanized mouse models". Cellular & Molecular Immunology. 9 (3): 208–14. doi:10.1038/cmi.2012.2. PMC 4012844. PMID 22327211.
^ Bosma GC, Custer RP, Bosma MJ (February 1983). "A severe combined immunodeficiency mutation in the mouse". Nature. 301 (5900): 527–30. Bibcode:1983Natur.301..527B. doi:10.1038/301527a0. PMID 6823332. S2CID 4267981.
^ ^a ^b ^c ^d ^e ^f ^g Yong KS, Her Z, Chen Q (August 2018). "Humanized Mice as Unique Tools for Human-Specific Studies". Archivum Immunologiae et Therapiae Experimentalis. 66 (4): 245–266. doi:10.1007/s00005-018-0506-x. PMC 6061174. PMID 29411049.
^ Ahmed EH, Baiocchi RA (2016-03-31). "Murine Models of Epstein-Barr Virus-Associated Lymphomagenesis". ILAR Journal. 57 (1): 55–62. doi:10.1093/ilar/ilv074. PMC 6302257. PMID 27034395.
^ Nomura J, Takumi T (2012). "Animal models of psychiatric disorders that reflect human copy number variation". Neural Plasticity. 2012: 589524. doi:10.1155/2012/589524. PMC 3414062. PMID 22900207.
^ Tary-Lehmann M, Saxon A, Lehmann PV (November 1995). "The human immune system in hu-PBL-SCID mice". Immunology Today. 16 (11): 529–533. doi:10.1016/0167-5699(95)80046-8. PMID 7495490.
^ Wang RM, Johnson TD, He J, Rong Z, Wong M, Nigam V, et al. (June 2017). "Humanized mouse model for assessing the human immune response to xenogeneic and allogeneic decellularized biomaterials". Biomaterials. 129: 98–110. doi:10.1016/j.biomaterials.2017.03.016. PMC 5434867. PMID 28334641.
^ Pearson T, Greiner DL, Shultz LD (May 2008). "Creation of "humanized" mice to study human immunity". Current Protocols in Immunology. Chapter 15 (1): 15.21.1–15.21.21. doi:10.1002/0471142735.im1521s81. PMC 3023233. PMID 18491294.
^ Karpel ME, Boutwell CL, Allen TM (August 2015). "BLT humanized mice as a small animal model of HIV infection". Current Opinion in Virology. Animal models for viral diseases / Oncolytic viruses. 13: 75–80. doi:10.1016/j.coviro.2015.05.002. PMC 4550544. PMID 26083316.
^ Firtina N (3 January 2023). "In a first, human brain organoids placed in the mouse cortex react to visual stimuli". Interesting Engineering. Retrieved 17 January 2023.
^ Wilson MN, Thunemann M, Liu X, Lu Y, Puppo F, Adams JW, et al. (December 2022). "Multimodal monitoring of human cortical organoids implanted in mice reveal functional connection with visual cortex". Nature Communications. 13 (1): 7945. Bibcode:2022NatCo..13.7945W. doi:10.1038/s41467-022-35536-3. PMC 9792589. PMID 36572698.
^ "Mouse strain C57BL/6-Mcph1^{Tg(HLA-A2.1)1Enge}". The Jackson Laboratory. Retrieved 2023-01-06.
^ Lüdtke A, Oestereich L, Ruibal P, Wurr S, Pallasch E, Bockholt S, et al. (April 2015). "Ebola virus disease in mice with transplanted human hematopoietic stem cells". Journal of Virology. 89 (8): 4700–4. doi:10.1128/JVI.03546-14. PMC 4442348. PMID 25673711.
^ Bility MT, Cheng L, Zhang Z, Luan Y, Li F, Chi L, et al. (March 2014). "Hepatitis B virus infection and immunopathogenesis in a humanized mouse model: induction of human-specific liver fibrosis and M2-like macrophages". PLOS Pathogens. 10 (3): e1004032. doi:10.1371/journal.ppat.1004032. PMC 3961374. PMID 24651854.
^ Bility MT, Zhang L, Washburn ML, Curtis TA, Kovalev GI, Su L (September 2012). "Generation of a humanized mouse model with both human immune system and liver cells to model hepatitis C virus infection and liver immunopathogenesis". Nature Protocols. 7 (9): 1608–17. doi:10.1038/nprot.2012.083. PMC 3979325. PMID 22899330.
^ Wang LX, Kang G, Kumar P, Lu W, Li Y, Zhou Y, et al. (February 2014). "Humanized-BLT mouse model of Kaposi's sarcoma-associated herpesvirus infection". Proceedings of the National Academy of Sciences of the United States of America. 111 (8): 3146–51. Bibcode:2014PNAS..111.3146W. doi:10.1073/pnas.1318175111. PMC 3939909. PMID 24516154.
^ Wege AK, Florian C, Ernst W, Zimara N, Schleicher U, Hanses F, et al. (2012-07-24). "Leishmania major infection in humanized mice induces systemic infection and provokes a nonprotective human immune response". PLOS Neglected Tropical Diseases. 6 (7): e1741. doi:10.1371/journal.pntd.0001741. PMC 3404120. PMID 22848771. S2CID 7657105.
^ Amaladoss A, Chen Q, Liu M, Dummler SK, Dao M, Suresh S, et al. (2015-06-22). "De Novo Generated Human Red Blood Cells in Humanized Mice Support Plasmodium falciparum Infection". PLOS ONE. 10 (6): e0129825. Bibcode:2015PLoSO..1029825A. doi:10.1371/journal.pone.0129825. PMC 4476714. PMID 26098918. S2CID 543860.
^ Calderon VE, Valbuena G, Goez Y, Judy BM, Huante MB, Sutjita P, et al. (2013-05-17). "A humanized mouse model of tuberculosis". PLOS ONE. 8 (5): e63331. Bibcode:2013PLoSO...863331C. doi:10.1371/journal.pone.0063331. PMC 3656943. PMID 23691024. S2CID 17215038.
^ Frias-Staheli N, Dorner M, Marukian S, Billerbeck E, Labitt RN, Rice CM, Ploss A (February 2014). "Utility of humanized BLT mice for analysis of dengue virus infection and antiviral drug testing". Journal of Virology. 88 (4): 2205–18. doi:10.1128/JVI.03085-13. PMC 3911540. PMID 24335303.
^ Moffat JF, Stein MD, Kaneshima H, Arvin AM (September 1995). "Tropism of varicella-zoster virus for human CD4+ and CD8+ T lymphocytes and epidermal cells in SCID-hu mice". Journal of Virology. 69 (9): 5236–42. doi:10.1128/jvi.69.9.5236-5242.1995. PMC 189355. PMID 7636965.
^ Zheng J, Wu WL, Liu Y, Xiang Z, Liu M, Chan KH, et al. (2015-08-18). "The Therapeutic Effect of Pamidronate on Lethal Avian Influenza A H7N9 Virus Infected Humanized Mice". PLOS ONE. 10 (8): e0135999. Bibcode:2015PLoSO..1035999Z. doi:10.1371/journal.pone.0135999. PMC 4540487. PMID 26285203. S2CID 10461525.
^ ^a ^b Tian H, Lyu Y, Yang YG, Hu Z (2020). "Humanized Rodent Models for Cancer Research". Frontiers in Oncology. 10: 1696. doi:10.3389/fonc.2020.01696. ISSN 2234-943X. PMC 7518015. PMID 33042811. S2CID 221589508.
^ Hausser HJ, Brenner RE (July 2005). "Phenotypic instability of Saos-2 cells in long-term culture". Biochemical and Biophysical Research Communications. 333 (1): 216–22. doi:10.1016/j.bbrc.2005.05.097. PMID 15939397.
^ Wege AK, Schmidt M, Ueberham E, Ponnath M, Ortmann O, Brockhoff G, Lehmann J (2014-05-08). "Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice: a novel approach to generate tumor cell specific human antibodies". mAbs. 6 (4): 968–77. doi:10.4161/mabs.29111. PMC 4171030. PMID 24870377. S2CID 34234807.
^ Her Z, Yong KS, Paramasivam K, Tan WW, Chan XY, Tan SY, et al. (October 2017). "An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia". Journal of Hematology & Oncology. 10 (1): 162. doi:10.1186/s13045-017-0532-x. PMC 5639594. PMID 28985760.
^ Her Z, Yong KS, Paramasivam K, Tan WW, Chan XY, Tan SY, et al. (October 2017). "An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia". Journal of Hematology & Oncology. 10 (1): 162. doi:10.1186/s13045-017-0532-x. PMC 5639594. PMID 28985760. S2CID 25506701.
^ Chen Q, Wang J, Liu WN, Zhao Y (July 2019). "Cancer Immunotherapies and Humanized Mouse Drug Testing Platforms". Translational Oncology. 12 (7): 987–995. doi:10.1016/j.tranon.2019.04.020. PMC 6529825. PMID 31121491.
^ Zayoud M, El Malki K, Frauenknecht K, Trinschek B, Kloos L, Karram K, et al. (September 2013). "Subclinical CNS inflammation as response to a myelin antigen in humanized mice". Journal of Neuroimmune Pharmacology. 8 (4): 1037–47. doi:10.1007/s11481-013-9466-4. PMID 23640521. S2CID 503830.
^ Gunawan M, Her Z, Liu M, Tan SY, Chan XY, Tan WW, et al. (November 2017). "A Novel Human Systemic Lupus Erythematosus Model in Humanised Mice". Scientific Reports. 7 (1): 16642. Bibcode:2017NatSR...716642G. doi:10.1038/s41598-017-16999-7. PMC 5709358. PMID 29192160. S2CID 5604139.
^ King M, Pearson T, Shultz LD, Leif J, Bottino R, Trucco M, et al. (March 2008). "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". Clinical Immunology. 126 (3): 303–314. doi:10.1016/j.clim.2007.11.001. PMID 18096436.