Introduction: spatial origin of murine hematopoietic stem cells.

Hematopoiesis is the developmentally regulated and tissue-specific process of blood cell production. The predominant anatomic site of hematopoiesis changes several times during murine and human ontogeny. Blood and endothelial cells become morphologically identifiable on embryonic day 7.5 (E7.5) in the developing murine yolk sac blood islands.1 Upon initiation of blood flow through the systemic circulation, blood island–derived progenitor cells are carried throughout the yolk sac and embryo.2,3 The liver becomes colonized with hematopoietic progenitor and stem cells at the 28 somite pair (sp) stage of murine development.4,5 By E12 the liver is the predominant site of hematopoiesis. Hematopoietic stem cells (HSCs) in the rodent fetal liver subsequently migrate to the bone marrow and contribute to lifelong hematopoiesis.6 For nearly 100 years, hematologists have been challenged by the question of how hematopoiesis is sequentially initiated in different organs during mammalian development. At the turn of the century, morphologic evidence that all blood cells appeared to develop from a common precursor cell was accumulating. The “mother” stem cell was identifiable as a mononuclear cell with a large nucleus, prominent nucleoli and deeply basophilic staining cytoplasm.7-9 This cell was present in every tissue displaying hematopoietic activity, leading Maximow8 and Danchakoff7 to hypothesize that the stem cell must arise independently in each hematopoietic tissue at a specific time or under specific circumstances. Seventy-five years later, the fetal liver has clearly been established as the site of development for HSCs that seed the bone marrow compartment;6,10 however, the temporal and spatial origin of the HSCs that seed the fetal liver remains controversial.11,12 More than 30 years ago, Moore and Owen13 hypothesized that the sequential emergence of hematopoietic organs throughout ontogeny required an inflow of circulating HSCs from the bloodstream and that HSCs are first formed in the yolk sac. The results of transplantation experiments in which yolk sac cells were injected into irradiated chick embryos14 served as the basis for this hypothesis. Follow-up studies in the murine system confirmed that the yolk sac was critical to the establishment of hepatic hematopoiesis. Removal of the fetal liver before the 28 sp stage (E9.5) and grafting of the tissue beneath the kidney capsule of an adult recipient resulted in survival of the fetal hepatic tissue, but no hematopoietic elements were present. But administration of hematopoietic cells into the circulation of the recipient mice bearing the fetal tissue grafts resulted in multilineage engraftment in the implanted fetal liver tissue.4,5 These data suggested that hematopoiesis did not arise from precursors endogenous to the fetal liver, but the liver tissue did promote growth and differentiation of circulating precursors that lodged therein. Likewise, E7 embryos dissected free from yolk sac membranes grew and developed normally in vitro for 2 days, but no blood cells or hematopoietic colonyforming cells were present in any portion of the embryo, including the liver.15 These studies supported the theory of organ seeding by blood-borne stem cells and the yolk sac as the source of HSCs.13 In contrast, some studies in both avian and murine systems have provided data that refutes the hypothesis of the yolk sac as the site of origin of HSCs. Using chick-quail and chick-chick chimeric models, a number of investigators failed to observe yolk sac cell contributions to adult myleopoiesis or lymphopoiesis (reviewed in Dieterlen-Lievre and Le Douarin16 and Dieterlen-Lievre et al17). The results of these avian studies contrasted with those of Moore and Owen,14 but the experimental methods differed in age of donor embryonic tissue, model (unirradiated chimeric host versus irradiated recipient), and period of analysis after transplantation or chimeric grafting. Efforts to assay directly for the presence of HSCs in the murine yolk sac via transplantation of these cells into recipient animals have yielded results that support or refute the Moore-Owen hypothesis largely dependent upon the age of the recipient. Weissman et al18 reported that in utero transplantation of E8 yolk sac cells into the yolk sacs of congenic nonablated recipients contributed to lymphomyelopoiesis in surviving mice that grew into adulthood. In utero transplantation of E9 yolk sac cells into the placenta of stem-cell deficient congenic fetal mice resulted in long-term reconstitution (more than 5 months) of the erythroid lineage in some surviving animals.19 Thus HSCs that engraft in embryonic or fetal mice and then contribute to adult hematopoiesis can be observed to be present in the E8-9 murine yolk sac. In contrast, the first HSCs that directly engraft in lethally irradiated adult mice are present at E10 in the aorto-gonad-mesonephros (AGM) region within the embryo.20 Adult-repopulating HSCs cannot be identified in the yolk sac until E11, at which time HSCs are already present in the AGM region, the liver, and vitelline and umbilical vessels.20-23 These results are generally interpreted to infer that the yolk sac is not a site where fetal-liver-seeding (and likewise, adult-marrow-repopulating) HSCs develop. The inability to demonstrate engraftment of yolk sac cells isolated earlier than E11 in lethally irradiated adult mice may be due to a failure of the yolk sac HSCs to home and engraft in an adult microenvironment. Based on the observation that the liver of the newborn mouse continues as an active hematopoietic organ for 1 to 2 weeks following birth,24-26 sublethally myeloablated newborn mice were transplanted with E9 or E10 yolk sac cells. In multiple experiments, yolk sac cells were noted to give rise to all lineages of blood cells long-term (more than 11 months) and that marrow from the primary E9 and E10 yolk sac–engrafted recipients also reconstituted Band Tlymphocyte, granulocyte, and erythroid lineages in secondary lethally irradiated adult recipient mice.27-29 These results suggested that yolk sac HSCs capable of repopulating lymphoid and myeloid lineages in adult animals were present prior to E11 but were not identifiable upon direct transplantation into adult recipients. What then is the relationship between yolk sac HSCs that engraft in newborn recipients and contribute to long-lived multilineage repopulation and HSCs from the AGM region, the liver, and vitelline and umbilical cord vessels that directly repopulate all lineages of a lethally irradiated adult mouse?