Dissect the mechanisms underlying interspecies pluripotent stem cell competition

PROJECT SUMMARY/ABSTRACT Shortage of organs for transplantation is one of the largest unmet medical needs. Researchers are currently working on a variety of ways to increase the number of organs available. Recently, the prospect of producing human organs in animals via interspecies blastocyst complementation has raised important attention and may constitute an innovative approach to overcome the worldwide shortage of donor organs. Interspecies blastocyst complementation works by injecting donor pluripotent stem cells (PSCs) from one species into the organogenesis-disabled blastocyst of a different species. The growing host embryo can provide an emptied developmental organ niche for donor cells to occupy. Despite the potential, however, recent work revealed that although donor PSCs can initially engraft to inner cell mass (ICM) of blastocysts from distantly related host species, their chimeric contribution to early post-implantation stages is low. These results suggest the existence of embryonic xenogeneic barriers between evolutionarily distant species. Unlike chimera formation within the same species, a multitude of factors can differ significantly between species, which preclude robust chimerism. Cell competition, a conserved fitness-sensing process during which fitter cells eliminate neighboring less-fit but viable cells, has been proposed as a surveillance mechanism to ensure normal development and tissue homeostasis, and has also been suggested to act as a barrier to interspecies chimerism. During chimera formation, xenogeneic donor PSCs may be treated as unfit or aberrant cells and targeted for elimination. Most recently, we developed an interspecies PSC co-culture strategy and discovered a previously unknown mode of cell competition between species. Interspecies competition between PSCs occurred in primed but not naive pluripotent cells, and between evolutionarily distant species. By comparative transcriptome analysis, we found that genes related to the NF-κB and p53 signaling pathways, among others, were upregulated in less-fit ‘loser’ human cells. Genetic inactivation of TP53, a core component (P65, also known as RELA) and an upstream regulator (MYD88) of the NF-κB complex in human PSCs could overcome their competitive elimination by co- cultured mouse PSCs, thereby improving the survival and chimerism of human cells in early mouse embryos. Based on extensive preliminary results, in this proposal, we will further dissect the mechanisms underlying interspecies primed PSC competition in both loser and winner cells. And, by suppressing interspecies PSC competition, we aim to improve chimerism and provide the proof-of-concept of interspecies blastocyst complementation from donor PSCs of rhesus monkey, our close kin, in distantly related mouse host.