Generation of tetraploid organs in miceOA

Tetraploidy occurs infrequently in mammals but remains widespread in amphibians.Blastocyst complementation using xenogeneic transplantation of tetraploid embryonic stem cells(4N-ESCs)represents a promising approach to mitigate organ shortages,yet robust generation of fully reconstituted organs in mammalian hosts remains elusive.In this study,CRISPR/Cas9,the Cre-LoxP system,and blastocyst complementation were combined to generate tetraploid mouse liver,heart,and pancreatic tissues.4NESCs(tdTomato-labeled)were established and shown to maintain stable pluripotency and tetraploidy,as confirmed by karyotyping and immunofluorescence analyses.Subsequently,these cells were microinjected into Hhexand Pdx1-deficient blastocysts and Nkx2.5 lineage-ablated blastocysts,which were engineered to lack relevant organforming lineages.Tetraploid pups exhibited significantly reduced body mass and organ mass(liver and heart)relative to diploid controls(P<0.05).Fluorescenceactivated cell sorting demonstrated a significant 4N-ESC(tdTomato-labeled)contribution within tetraploid organs(4N population)at E18.5,with tdTomato-positive fractions reaching 84.3%of hepatic cells,67.8%of cardiac cells,and 73.4%of pancreatic cells.Single-cell transcriptome sequencing further revealed that tetraploidy markedly altered developmental trajectories and differentiation programs in liver and heart tissues,and 4N-ESCs showed preferential integration into tetraploid liver and heart with a substantial contribution to pancreatic regeneration.Collectively,these findings support the feasibility of 4NESC-based blastocyst complementation for human organ regeneration and establish a framework for developing strategies to alleviate organ shortages in clinical settings.