Abstract Body: Ischemic heart disease is the leading cause of death globally, largely driven by inability of the adult heart to restore tissue structure and function after injury. However, the neonatal mouse heart is able to regenerate up to one week after birth, circumventing scarring and functional impairment. Neonatal heart regeneration is a spatially and temporally regulated process, involving coordinated communication of different cardiac cell types. Our previous work identified a regenerative cardiomyocyte population (CM4) that proliferates after myocardial infarction (MI), but the intercellular communication that induces their regenerative response is not fully understood. To study the cellular environment of CM4 cells during neonatal heart regeneration, we profiled spatial transcriptomes of regenerative P1 hearts at 3 days and 7 days after MI and compared them to nonregenerative P8 hearts. We identified unique spatial regions with distinct gene expression signatures, that we refer to as “molecular niches”, in the ischemic and border zones (IZ, BZ) of the P1 heart post-MI compared to P8 hearts. Cell type deconvolution of molecular niches showed a distinct temporal change of fibroblast and macrophage abundance in the IZ of P1 hearts compared to P8 hearts and also showed the localization of CM4 cells to the BZ of P1 hearts at 7 days post-MI. To identify intercellular communication that contributes to the differential injury response in P1 and P8 hearts, we analyzed ligand-receptor pairs that were expressed at higher levels in the IZ and BZ of P1 compared to P8 hearts. Among the nearly 50 signaling pairs identified, OSM and SLIT signaling have been previously implicated in heart regeneration. Notably, many of these signaling factors, including cytokines, chemokines and growth factors, have receptors expressed in multiple cardiac cell types. Thus, ongoing studies are systematically distilling the effect of each secreted factor on individual cell types in the heart using a cardiac cell co-culture and multiplexed single-cell RNA sequencing approach. In summary, our study provides insight into the spatial organization and tissue microenvironment of neonatal heart regeneration and identifies a number of candidate signaling pathways that potentially mediate critical intercellular communication during neonatal heart regeneration.