Abstract
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.
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