Generation of tetraploid organs in mice.

Tetraploidy occurs infrequently in mammals but remains widespread in amphibians. Blastocyst complementation using xenogeneic transplantation of tetraploid embryonic stem cells (4 N-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. 4 N-ESCs (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 Hhex- and Pdx1-deficient blastocysts and Nkx2.5 lineage-ablated blastocysts, which were engineered to lack relevant organ-forming lineages. Tetraploid pups exhibited significantly reduced body mass and organ mass (liver and heart) relative to diploid controls ( P<0.05). Fluorescence-activated cell sorting demonstrated a significant 4 N-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 4 N-ESCs showed preferential integration into tetraploid liver and heart with a substantial contribution to pancreatic regeneration. Collectively, these findings support the feasibility of 4 N-ESC-based blastocyst complementation for human organ regeneration and establish a framework for developing strategies to alleviate organ shortages in clinical settings.