Age-induced alterations in hematopoiesis, including reduction in functional B and T lymphocytes and expansion of myeloid cells, are associated with numerous hematopoietic pathologies (Wahlestedt et al., 2015). These cellular changes are associated with and can be driven by age-dependent decline in hematopoietic stem cell (HSC) function (Morriso n et al., 1996) and biased HSC fate toward myeloerythroid lineages at the expense of lymphoid (Rossi et al., 2005; Beerman et al., 2010; Dykstra et al., 2011). The hierarchical structure of hematopoiesis defines the production of multipotent progenitors (MPPs) from HSCs (Christensen and Weissman, 2001), which serve as effector cells to tailor output of myeloid and lymphoid lineages. Recently, a major role for the MPP compartment in long-term blood production during steady-state hematopoiesis has been uncovered by in vivo lineage-tracing studies (Sun et al., 2014; Busch et al., 2015), highlighting the importance of further study of this compartment and its contribution to hematopoietic aging and pathology. Within the heterogeneous MPP compartment, the brightest∼ 25% of Flk2-expressing cells represent lymphoid-primed MPPs (LMPPs; Adolfsson et al., 2005). Additionally, differential expression of CD150, CD48, and Flk2 defines myeloid-biased MPP2 and MPP3 and lymphoid-primed MPP4 (Wilson et al., 2008; Cabezas-Wallscheid et al., 2014; Pietras et al., 2015). It remains undetermined as to whether the process of aging dynamically alters the composition and functional output of the MPP compartment. To identify age-dependent cellular and molecular changes in the MPP compartment, we systematically examined MPP composition with aging and combined single-cell transcriptome and functional studies of MPP4/LMPP. We found that aging induces increased cycling, loss of lymphoid priming, and differentiation potential of MPP4/LMPP cells. In vivo transplantation of aged LMPPs into a young BM microenvironment demonstrates cell-autonomous defects in lymphoid production and skewing toward myeloid cell production. Together, this suggests that early alterations in the MPP compartment may be the effectors of lymphoid cell loss in aging hematopoiesis. results and dIscussIon aging-induced loss of lMPPs

We began by examining alterations in BM frequency of longterm HSCs (LT-HSC), short-term HSCs (ST-HSCs), MPP2, MPP3, MPP4, and LMPPs with age using defined markers (Fig. 1 A; Adolfsson et al., 2005; Wilson et al., 2008; Piet-ras et al., 2015). Analysis of C57BL/6J female mice between 2 and 28 months old (mo) revealed a significant increase in BM frequency of LT-HSCs and ST-HSCs as early as 8 mo (Fig. 1 B), consistent with known phenotypic HSC expansion with aging (Rossi et al., 2005). Increased frequency of MPP2 was observed at 28 mo, consistent with reported molecular and functional megakaryocyte/erythroid bias of aged HSCs (Grover et al., 2016; Rundberg Nilsson et al., 2016).