Abstract Body: Introduction: During development, various cells intricately collaborate to form embryonic and fetal hearts. We can harness this naturally occurring generative mechanism to advance regenerative medicine for cardiac repair. Studies reported that C-X3-C Motif Chemokine Receptor 1 (CX3CR1)⁺ cells play vital roles in cardiac development and disease. However, the origin and the fate of CX3CR1⁺ cells during cardiogenesis remain unclear. Specifically, the existence of CX3CR1⁺ cells before E8.5 and their commitment other than to macrophages have not been investigated.

Methods: We investigated the origin and fate of CX3CR1⁺ cells using various knock-in reporter mice with CX3CR1 drivers for spatiotemporal genetic lineage tracing of CX3CR1⁺ cells. In addition, we developed a method to generate CX3CR1⁺ cells from differentiating mouse embryonic stem cells (mESCs). mESC-derived CX3CR1⁺ cells were characterized in vitro, in the fetal mouse heart ex vivo, and in the adult heart in vivo. Their heterogeneity and trajectory were analyzed via single cell RNA sequencing (scRNA-seq).

Results: Genetic fate mapping of CX3CR1⁺ cells identified epiblasts at E6.5, the parietal endoderm at E7.0, yolk sac cells at E8.0, and cardiomyocytes at E9.5. Temporal genetic labeling (Cx3cr1-CreERT2;R26-tdTomato) showed that CX3CR1⁺ cells at E6.5 can contribute to cardiomyocytes (CMs) and endothelial cells (ECs) during prenatal development via both de novo differentiation and fusion with pre-existing CMs or ECs, respectively. In the adult heart, cells that have ever expressed Cx3cr1 comprised 13.5 ± 0.2% of CMs and 30.9 ± 1.3% of ECs. In addition, CX3CR1⁺ cells, which were differentiated from mESCs generated CMs, ECs and macrophages in vitro, in the fetal mouse heart ex vivo, and in the adult heart in vivo. Single cell RNA sequencing showed that Cx3cr1⁺ cells represent an intermediate cell population differentiating into the mesoderm from pluripotent stem cells.

Conclusions: Our data demonstrate that CX3CR1 serves as a marker for a unique subset of progenitors that contribute to the formation of CMs and ECs as well as macrophages, providing new insights into the versatile role of CX3CR1⁺ cells in cardiogenesis.