Interspecies blastocyst complementation generates functional rat cell-derived forebrain tissues in mice

Interspecies organogenesis via blastocyst complementation provides a unique platform to study development in an evolutionarily context and holds potential to overcome world-wide organ shortages1. By using this technique, rat pancreas, thymus, heart, and eye tissues have been generated in mice2-4. To date, however, xeno-generation of brain tissues has not been achieved through blastocyst complementation. Here, we developed an optimized one-step blastocyst complementation strategy based on C-CRISPR5, which facilitated rapid screening of candidate genes to support blastocyst complementation. Among the seven WNT pathway-related genes selected for targeting, only Dkk1 or Hesx1 deficiency supported forebrain complementation by blastocyst injection of mouse embryonic stem cells (mESCs). Further, injecting rat ESCs (rESCs) into mouse blastocysts deficient for Hesx1 but not Dkk1 supported the development of adult chimeric forebrains comprised a large proportion of rat cells that were structurally and functionally similar to the mouse forebrains. Our analysis revealed that the rESC-derived forebrains developed along the spatial-temporal trajectory with the mouse forebrains rather than rat forebrains, but gene expression profiles of rESC-derived nerve cells surprisingly maintained the characteristics of the rat cells. We noted that the chimeric rate gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late prenatal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRIPSR based IBC strategy developed here holds great potential to broaden the study and application of interspecies organogenesis.