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In part 2 of this series on humanized mouse models, we examined how immune system engineering, through precise HLA class I and II knock-ins, transformed the fidelity of human immune responses in vivo. By replacing murine MHC genes with human alleles, these models can achieve tissue-appropriate expression and support robust, epitope-specific responses, from CTL activation to full antibody maturation. Innovations such as monochain HLA class I knock-ins, double HLA allele models, and class II replacements like HLA-DQ2.5 extend their utility to cancer immunotherapy, vaccine development, and autoimmune disease research. Collectively, these advances pushed humanized mice beyond earlier limitations, enabling preclinical studies that now capture the complexity of human immunity with unprecedented accuracy. In this part 3, we will delve into how Multi-cytokine and dual-organ humanized mouse platforms such as MISTRG, NSG-SGM3, and immune–liver or immune–lung chimeras are transforming preclinical research by enabling physiologically faithful modeling of human hematopoiesis, immunity, and multi-organ disease.
The MISTRG platform (Macrophage colony-stimulating factor, Interleukin-3, Stem cell factor, Thrombopoietin, Rag2-/-, IL2Rγ-/-) introduced a breakthrough in multi-cytokine support for human hematopoietic and immune development. By replacing murine loci with human M-CSF, IL-3/GM-CSF, SCF, and TPO, these mice provide physiological cytokine expression that enables robust engraftment and differentiation of human HSCs into macrophages, dendritic cells, granulocytes, and NK cells [1].
The physiological knock-in strategy overcomes the pitfalls of ectopic or transgenic cytokine overexpression, ensuring correct spatiotemporal signaling to support multilineage hematopoiesis. These improvements strengthen human myeloid and NK cell development, addressing limitations of earlier NSG hosts [1-3].
MISTRG mice have been validated as superior hosts for modeling myelodysplastic syndromes (MDS). Song et al. showed that MISTRG faithfully reproduced patient-derived MDS with preservation of dysplastic morphology, lineage diversity (including erythroid and megakaryocytic), and clonal architecture [2-3]. Newer enhanced cytokine knock-in variants, such as MISTRG6kitW41, further overcome fidelity gaps in MDS modeling, providing improved translational tools for drug development [4].
The robust immune reconstitution in MISTRG also benefits immuno-oncology studies, where tumor–immune cell interactions can be faithfully modeled [1-3].
The NSG-SGM3 strain carries human SCF, GM-CSF, and IL-3, which together create a highly supportive niche for hematopoiesis and acute myeloid leukemia (AML) xenografts. This platform enables engraftment of patient-derived AML samples otherwise non-engraftable in conventional NSG, thereby expanding translational leukemia research [5]. A broader perspective on the strengths and limitations of NSG-SGM3 in modeling myeloid biology has been reviewed in detail [3].
Human IL-3/GM-CSF double knock-in mice enable the development of functional human alveolar macrophages upon CD34+ HSPC engraftment. These macrophages populate the lung niche and mediate human-specific responses to pathogens, enabling in vivo study of respiratory immunity and inflammation [6].
Next-generation approaches extend cytokine-driven humanization into multi-organ systems:
Though newer dual-humanization systems are still maturing, reviews highlight their importance in expanding the translational scope from isolated immune niches to interconnected human physiology [3].
The transition from single cytokine supplementation to multi-cytokine, dual-organ humanization reflects a paradigm shift toward in vivo systems capable of recapitulating human hematopoiesis, immunity, and organ-specific pathophysiology. MISTRG, NSG-SGM3, and enhanced variants such as MISTRG6kitW41 now provide unprecedented fidelity for hematopoietic disease, cancer, pulmonary immunity, and drug safety studies [1-6].