Modeling Complexity: Multi-Cytokine and Multi-Organ Humanization

Preclinical Disease Models That Deliver Better Predictive Power

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.

MISTRG Mice

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].

Cytokine Knock-ins and Hematopoiesis

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].

Applications in Myelodysplastic Syndrome and Cancer

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].

NSG-SGM3 and Specialized Cytokine Models

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].

IL-3/GM-CSF Knock-ins for Pulmonary Immunity

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].

Dual Humanization Platforms

Next-generation approaches extend cytokine-driven humanization into multi-organ systems:

Immune + Liver (FAH KO): Allows concurrent human hepatocyte and immune system engraftment, supporting studies of hepatitis virus infections and drug-induced liver injury.
Immune + Lung (BLT-L): Supports parallel human immune and lung tissue reconstitution, enabling research into chronic pulmonary inflammation and viral pathogenesis.

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].

Conclusion

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].

References

Rongvaux A, et al. Nat Biotechnol. 2014. https://pubmed.ncbi.nlm.nih.gov/24633240/
Demonstrates the creation and utility of MITRG/MISTRG mice with human cytokine knock-ins, enabling robust development of human myeloid and NK cells—core for multi-cytokine modeling.
Song Y, et al. Nat Commun. 2019. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341122/
Validates that MISTRG mice can faithfully reproduce patient-derived myelodysplastic syndromes, confirming this model’s translational power in hematopoietic disease research.
Stocks H, et al. Trends Immunol. 2025. https://pubmed.ncbi.nlm.nih.gov/40781011/
Reviews recent technical advances and clinical applications of NSG-SGM3 and MISTRG mice, especially improvements in human myeloid cell engraftment for preclinical disease modeling.
Munteanu R, Gulei D, Moldovan CS, et al. Humanized mouse models in MDS. Cell Death Dis. 2025. https://pubmed.ncbi.nlm.nih.gov/40675972/
Details new-generation humanized mouse platforms for MDS—including enhanced cytokine knock-in approaches—highlighting their superior disease fidelity and relevance for drug testing.
Wunderlich M, et al. Blood. 2018. https://pubmed.ncbi.nlm.nih.gov/30237223/
Shows how NSG-SGM3 mice enable robust engraftment and propagation of patient-derived AML, which is essential for expanding leukemia therapeutic research.
Li F, et al. J Exp Med. 2020. https://pubmed.ncbi.nlm.nih.gov/32715931/
Proves that IL-3/GM-CSF double knock-in mice support functional development of human alveolar macrophages, facilitating high-fidelity modeling of pulmonary immunity.

Modeling Complexity: Multi-Cytokine and Multi-Organ Humanization

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