Xenophagocytosis blockade enhances interspecies chimerism 2258754

Abstract Introduction Organ shortage remains a major challenge in transplantation medicine. Interspecies blastocyst complementation seeks to generate human organs in livestock by creating an empty organ niche for donor pluripotent cells. Yet donor chimerism often falls at early embryonic stages. We tested whether an innate immunologic barrier emerging with embryonic macrophages causes this drop and whether overcoming it improves donor chimerism. Methods We generated rat to mouse and human to mouse chimeras by injecting donor pluripotent stem cells into blastocyst stage embryos. Donor contribution was quantified by imaging, droplet digital PCR, and flow cytometry, and host myeloid states by index sorted single cell RNA sequencing. Mechanism tests included macrophage depletion or impairment (PU.1 or Csf1r knockout), receptor genetics (Axl knockout), and tuning eat me versus do not eat me cues on donors (BCL2, mouse CD47). Results Donor chimerism declined as macrophages appeared at E9.5 to E11.5. Host macrophages contained donor EGFP puncta while targeted donor cells lacked cleaved caspase 3, consistent with phagoptosis; we term this xenogeneic clearance xenophagocytosis. Depleting host macrophages increased rat and human donor contribution. Xenogeneic donor cells displayed elevated surface phosphatidylserine sensed by Axl on host macrophages. Knocking out Axl in the host increased donor chimerism in mouse embryos. Donor mouse CD47 overexpression increased chimerism. Conclusion An Axl dependent xenophagocytosis checkpoint restricts interspecies chimerism by enabling macrophages to remove viable xenogeneic cells with high surface phosphatidylserine. Targeting this checkpoint improves donor persistence and provides routes to increase the efficiency of blastocyst complementation for human organ generation. Funding Source NIH (R01DK121851) the Ludwig Foundation the Leducq Foundation the Centers for Clinical Application Research on Specific Disease/Organ of the Research Center Network for Realization of Regenerative Medicine, funded by the Japan Agency for Medical Research and Development (AMED_JP23bm1123041) and the Japan Society of the Promotion of Science Topic Categories Transplantation Immunology (TRAN)