Early Appearance and Rapid, Selective Expansion of Lymphoid-Deficient Fetal HSCs Associated with Their Preferential Localization in the Marrow

Abstract 1271

Recent tracking of the primitive and mature progeny of more than 500 single cell transplants of highly purified hematopoietic stem cells (HSCs) in cell suspensions isolated from the bone marrow of young adult mice (8–12 weeks old) has shown that this pool is intrinsically heterogeneous. This heterogeneity is manifested as differences both in the durability (beyond 6 months) of the self-renewal activity of the HSCs detected in irradiated recipients as well as in the specific lineage contributions maintained in secondary and tertiary recipients of cells generated within each clone. Interestingly, durable HSC self-renewal activity (serial transplantability) was found to be exclusively associated with a robust and sustained ability to produce myeloid cells, regardless of their ability to produce lymphoid progeny at any stage of lymphoid progenitor development. Thus 2 subtypes of HSCs with durable self-renewal activity could be distinguished depending on whether the sustained myelopoietic activity was accompanied by an equivalent robust lymphoid differentiation activity (β-HSCs), or not (α-HSCs). Preliminary examination of fetal liver HSCs with durable self-renewal activity showed that this compartment is dominated by β-HSCs in contrast to the marrow of young adult mice where ∼30% of this HSC pool are α-HSCs. Since previous studies have documented a switch between 3 and 4 weeks after birth in several key properties of HSCs (indicative of an abrupt change from a fetal to an adult HSC program), we were interested in the question of whether the appearance of α-HSCs might be another aspect of this switch or, alternatively, whether the onset of hematopoiesis in locations other than the fetal liver (e.g., the marrow and spleen) might be a contributing factor. Accordingly, we initiated experiments to examine the proportional representation and absolute number of cells with an E-SLAM phenotype (CD45⁺EPCR⁺CD48⁻CD150⁺) and their functional activity (4-month repopulation of irradiated mice) specific for HSCs in tissues where hematopoiesis is seen during different stages of development between E14.5 and 4 weeks after birth. The number of E-SLAM cells increased ∼15 fold in the fetal liver between E14.5 and E18.5 by which time they were also present in the marrow and spleen but altogether comprising only ∼5% of the fetal liver compartment (assuming the femurs, tibiae and pelvis represent ∼30% of the total marrow). Analysis of the clonal outputs obtained in vivo from single E-SLAM cell transplants from each of these sources showed that α-HSCs are already present as a rare subet of all HSCs in the E14.5 liver and amplify equally with β-HSCs maintaining a ratio of 1:10 α-HSCs to β-HSCs in this organ. Interestingly, despite the small numbers of E-SLAM cells in the E18.5 marrow, these contained readily detectable HSCs in transplant assays and showed a ratio of α-HSCs to β-HSCs of 1:2. This ratio was mirrored in the marrow of adult mice and was also similar to that seen in the marrow and spleen of 3 and 4 week-old mice. These findings show that α-HSCs, by comparison to β-HSCs, have a greater ability to localize in the marrow which appears to then selectively promote the expansion of α-HSCs. These results also show that the initial appearance and early selective expansion of α-HSCs during fetal and early post-natal life is unlikely to be regulated by the same mechanisms that impose an adult program on murine HSCs between 3 and 4 weeks after birth. Rather these results favor a separate, intrinsically determined developmentally controlled mechanism that endows α-HSCs with a higher self-renewal probability, a more rapid turnover, a lower apoptotic activity, or a combination of these differences as compared to β-HSCs. This would explain the initial rapid and marked selective expansion of α-HSCs that occurs during the late fetal and early neonatal period of high HSC proliferation which extends to 3 weeks after birth and is then followed by a much slower rate of selective expansion of α-HSCs during adulthood when the turnover of HSCs is minimal.