Blocking macrophage 'eat-me' signals lets foreign stem cells thrive in chimeric embryos

Organ shortages drive interest in growing patient-matched human organs inside livestock embryos, a strategy called interspecies blastocyst complementation. Its efficiency is low because donor cells from another species are lost during development. In rat-to-mouse chimeras, donor contribution fell sharply between embryonic days 9.5 and 11.5 (about 22% to 3%), a window when only primitive macrophages, not adaptive immune cells, are present.

The authors used rat and mouse pluripotent stem cells and embryos. Flow cytometry, imaging flow cytometry and immunostaining showed host macrophages containing donor-derived GFP. This was seen in both rat-to-mouse and mouse-to-rat chimeras, and phagocytic activity was higher against cross-species than same-species cells. Most engulfed donor cells were negative for cleaved caspase-3, so they were alive rather than dying. This is consistent with phagoptosis, the elimination of viable cells by phagocytes.

Genetic loss of host macrophages (PU.1 or Csf1r knockout) markedly raised rat donor chimerism at E18.5, for example blood from 9% to 33%, heart 14% to 38% and lung 18% to 45%, with peaks up to 77% in lung. It did not raise mouse-to-mouse chimerism, so the barrier is specific to xenogeneic cells. Rat cells became cardiomyocytes and pericytes in heart but mostly endothelial and mesenchymal cells in lung, with few lung epithelial cells.

Single-cell RNA-seq showed that macrophages that had engulfed rat cells upregulated Axl. Axl knockout increased chimerism and reduced phagocytosis, while Mertk knockout alone did not. Xenogeneic donor cells showed more surface phosphatidylserine, the Axl ligand. Overexpressing mouse CD47 in rat cells raised chimerism, while Bcl2 overexpression (anti-apoptosis) did not. Overexpressing the flippase ATP11C lowered phosphatidylserine exposure and phagocytosis and increased chimerism. TMEM16F knockout had no effect, and CDC50A overexpression unexpectedly nearly eliminated donor cells, possibly through impaired proliferation. The abstract also reports improved human donor chimerism in mouse embryos and better rat pancreas complementation.

These results identify an innate immune barrier to interspecies chimerism with several ways to relieve it. Caveats: the full text provided is truncated, so calcium-signaling, human-cell and pancreas data are not detailed here. Macrophage ablation is not a simple clinical strategy, and rat-to-mouse is a much closer pairing than human-to-pig. Lineage skewing suggests other barriers remain.