Kazuto Yamazaki

The tumor microenvironment (TME) is deeply involved in cancer progression and treatment resistance. Although humanized mouse models have been developed by transplanting human cells into immunodeficient mice, they fail to fully reconstitute the TME. Blastocyst complementation using Flk-1 (Vegfr2, Kdr) knockout hosts offers a potential solution. However, the generation of interspecies human–mouse chimeras using blastocyst complementation has not yet been successful. As a foundational step, this study aims to demonstrate that donor-derived TME can be constructed using this method in intraspecies chimeric mice. We generated chimeric mice by injecting Azami-Green (AG)-positive C57BL/6 (B6) mouse-derived embryonic stem cells (ESCs) into ICR Flk-1 knockout embryos. We observed that vascular endothelial cells (VECs), hematopoietic cells, and tissue-resident macrophages were derived from the injected AG-positive ESCs. We engrafted B6-derived tumor cells into the chimeras and identified tumor-infiltrating lymphocytes, tumor-associated macrophages, and VECs derived from donor cells. Moreover, tumor-infiltrating CD8⁺ T cells in these chimeric mice showed cytotoxic activity comparable to that in wild-type mice. We anticipate that this intraspecies chimeric mouse model can serve as a valuable tool for basic research. Furthermore, future humanized tumor models generated via blastocyst complementation have the potential to significantly advance anticancer drug development in the preclinical phase.

SUMMARY: The generation of human organs in animals through blastocyst complementation offers a promising solution to the shortage of transplantable organs. While human pluripotent stem cells (hPSCs) can contribute to interspecies chimaeric embryos when injected into preimplantation embryos of mice, pigs, or monkeys, their integration is often limited due to low chimaerism and segregation from host tissues, significantly impeding progress toward exogenic organ generation. Here, we demonstrate that co-overexpression of the anti-apoptotic gene BCL2 and the proto-oncogene MYCL , along with various cell cycle regulators significantly enhances human cell chimaerism by promoting cell proliferation. This strategy facilitates the generation of viable mouse pups containing hPSC-derived tissues without tumorigenesis. scRNA-seq analysis revealed that hPSCs already exit pluripotent by early post-implantation stage yet hPSCs with enhanced proliferation were able to integrate effectively into the cardiomyocytes and vasculature of both embryonic and extraembryonic tissues with gene expression profiles reflecting their structural integration. These findings highlight the critical role of cell cycle regulation in overcoming xenogeneic barriers. Our findings offer new insights into strategies for enhancing interspecies organogenesis and advancing the field of regenerative medicine. Highlight: Enhancing hPSC proliferation increases human cell contribution in interspecies chimaeras Achieving the generation of viable human-mouse chimaeras without tumour formation hPSCs in post-implantation mouse epiblast exit pluripotency but retain the capacity to integrate into mouse embryogenesis hPSCs derivatives integrate into vasculature and cardiac tissues with lineage-matched transcriptional profiles

Blastocyst complementation is an intriguing way of generating humanized animals for organ preparation in regenerative medicine and establishing novel models for drug development. Confirming that complemented organs and cells work normally in chimeric animals is critical to demonstrating the feasibility of blastocyst complementation. Here, we generated thymus-complemented chimeric mice, assessed the efficacy of anti-PD-L1 antibody in tumor-bearing chimeric mice, and then investigated T-cell function. Thymus-complemented chimeric mice were generated by injecting C57BL/6 (B6) embryonic stem cells into Foxn1^nu/nu morulae or blastocysts. Flow cytometry data showed that the chimeric mouse thymic epithelial cells (TECs) were derived from the B6 cells. T cells appeared outside the thymi. Single-cell RNA-sequencing analysis revealed that the TEC gene-expression profile was comparable to that in B6 mice. Splenic T cells of chimeric mice responded very well to anti-CD3 stimulation in vitro; CD4⁺ and CD8⁺ T cells proliferated and produced IFNγ, IL-2, and granzyme B, as in B6 mice. Anti-PD-L1 antibody treatment inhibited MC38 tumor growth in chimeric mice. Moreover, in the chimeras, anti-PD-L1 antibody restored T-cell activation by significantly decreasing PD-1 expression on T cells and increasing IFNγ-producing T cells in the draining lymph nodes and tumors. T cells produced by complemented thymi thus functioned normally in vitro and in vivo. To successfully generate humanized animals by blastocyst complementation, both verification of the function and gene expression profiling of complemented organs/cells in interspecific chimeras will be important in the near future.