Neonatal Recipients Offer Permissive Hematopoietic Microenvironment for Engraftment of Embryonic Murine Hematopoietic Stem Cells

Abstract 2344

The first hematopoietic stem cells (HSC) that give rise to robust, long-term, multi-lineage reconstitution in irradiated adult recipients arise in the murine embryo at embryonic day 11.5 (E11.5). However, long-term multi-lineage engraftment in neonatal recipients has been observed from E9.0 yolk sac, suggesting that the neonatal hematopoietic microenvironment is more permissive for engraftment of embryonic HSCs. To resolve the apparent discrepancy between the numbers of candidate HSCs detected by direct visualization in the early embryo, relative to the numbers that can be measured by limiting dilution, we sought to characterize engraftment of neonatal recipients versus adult recipients with hematopoietic populations dissected from the aorta-gonad-mesonephros (AGM) region of the early embryo, the first putative site of intraembryonic origin of definitive HSCs. We dissected whole AGM from E11.5 embryos and injected cell dilutions from 2 embryo equivalents (ee) to 0.25 ee into the facial vein of day 1–2 neonatal recipients that had received sublethal conditioning with 350 rad irradiation. In neonatal recipients we detected robust, long-term, multi-lineage hematopoietic engraftment from as little as 0.25 ee. From less than 1 ee of whole AGM, the engraftment chimerism ranged from 5–20%. With 2 ee, chimerism was as high as 70%. Most animals showed balanced donor derived myeloid and lymphoid contribution by 10 weeks post-transplant. Interestingly, some animals had predominantly myeloid reconstitution for as long as 18 weeks, suggesting the presence of a novel long-term, myeloid-restricted, embryonic HSC. We also explored the neonatal engraftment potential of VE-cadherin⁺ CD45⁺ and VE-cadherin⁺ CD45⁻ populations. As expected from the literature, only the VE-cadherin⁺ CD45⁺ population engrafted the neonatal recipients. Our data indicate that the neonate harbors a more permissive hematopoietic microenvironment that enables more robust engraftment of early embryonic hematopoietic populations, thereby allowing us to identify potentially novel classes of embryonic hematopoietic progenitors. We are currently exploring the neonatal engraftment potential of E9.5 and E10.5 embryonic populations, additional FACS-purified populations, and hematopoietic populations derived from pluripotent stem cells in vitro.