Hematopoietic stem/progenitor cells originating from extra-embryonic arterial vessels are the major contributors to mouse foetal lympho-myelopoiesis

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: In mammals, embryonic hematopoiesis takes place in discrete but overlapping waves. Hematopoietic Stem Cells (HSC) generation is preceded by the appearance of HSC-independent progenitors, both emerging from a specialized transient population of endothelial cells termed hemogenic endothelium (HE). Recently, several reports showed that HSC contribution to fetal hematopoiesis is limited and that, in contrast, HSC-independent progenitor play key roles in fetal and postnatal life. Understanding the dynamics of fetal hematopoiesis has a number of important implications. Indeed, gaining a better knowledge of the cellular and molecular processes underlying the production of hematopoietic cells in the embryo would aid establishing new methodologies for the in vitro generation of different hematopoietic cells from pluripotent stem cells. Moreover, given the prenatal origin of many pediatric blood cancers, a better understanding of fetal hematopoiesis could lead to the identification of potential cell(s) of origin and relevant therapeutic vulnerabilities. Aims: To precisely define the origin, identity and extent of contribution of the distinct waves of embryonic hematopoietic stem and progenitor cells. Methods: To this aim, we took advantage of a genetic fate-mapping strategy in mouse that allows in vivo labeling and tracking of distinct subsets of HE. We combined this strategy with whole-mount embryo imaging, single-cell RNA sequencing and a range of functional assays. Results: Time-course labeling of HE revealed that the major lympho-myeloid contribution towards the end of gestation was derived from progenitors appearing between E8.5 and E9.5, a time window in which dorsal aorta definitive-type HSCs have not yet emerged. This contribution was transient and fetal-restricted, as it exhibited a sharp decline during postnatal life. Remarkably, we were able to localize the emergence of fetal-restricted hematopoietic stem/progenitor cells (HSPCs) to Kit+ hematopoietic clusters emerging from HE in the vitelline and umbilical (extraembryonic) arteries. Lineage tracing using a different, myeloid-specific, transgenic mouse line established that these clusters contained cells other than erythro-myeloid progenitors (EMPs). Moreover, single-cell RNA sequencing showed that fetal-restricted HSPCs express a transcriptional signature characteristic of HSCs. Accordingly, ex vivo co-cultures showed that B- and T-lymphoid potential were enriched in these progenitors. Finally, transplantation assays demonstrated that fetal-restricted HSPCs are endowed with in vivo multi-lineage repopulation potential. Summary/Conclusion: In summary, here we identify a wave of fetal-restricted HSPCs that physiologically contribute the majority of lymphoid and myeloid cells other than macrophages during fetal development, and we demonstrate that its emergence is segregated in space and time from that of EMPs and adult definitive-type HSCs. Keywords: Hematopoietic stem and progenitor cells, Hematopoiesis, Development, Mouse model