3041 - Robust Autonomous Generation of Definitive Hematopoietic Stem Cells From Pre-Circulation Murine Embryos

Abstract

The embryonic anatomical site of origin of the definitive Hematopoietic Stem and Progenitor Cells (HSPCs) is still under debate in the field. An intra-embryonic origin (IE) is well accepted. In contrast, a yolk sac (YS) contribution to adult hematopoiesis remains controversial. The same developmental origin (e.g. endothelial cells with a mesodermal ancestor) makes it difficult to identify specific markers to discern between an IE- vs. YS-origin using a lineage trace approach. This together with the highly migratory nature of blood cells and the inability of cells isolated from pre-circulatory embryos to robustly engraft in transplantation assays, even after ex vivo culture, has precluded scientists from properly answering these questions. We have established ex vivo culture conditions to allow for the derivation of robust multi-lineage engraftment activity from pre-circulation murine embryos. CD45.2 + embryos at the 2–7 somite pairs (s.p.) stage were dissected from their YS, cultured as explants in our optimized conditions and transplanted into lethally irradiated CD45.1 + /CD45.2 + recipients. Here, we observed multilineage reconstitution of the peripheral blood (PB) for >16 weeks post-transplant in 16/18 recipients. This engraftment activity was serially transplantable. In primary recipients, PB chimerism was >50% in 8/18 total recipients. This robust PB chimerism allowed us to rigorously interrogate the corresponding CD45.2 + YS (cultured under the same conditions). In these transplants, we observed PB engraftment in 0/18 recipients of cells isolated from cultured YS. These data suggest that either YS does not autonomously generate HSPCs or that it can not support their maturation ex vivo. Additionally, we found that the engraftment ability from the IE is confined to an emerging CD31+CD45+c-Kit+CD41- population. Importantly, this population also develops in the YS explants, but lacks engraftment ability. In sum, our work supports a model in which the embryo, not the YS, is the major source of lifelong hematopoiesis.