Abstract Abstract 215 The role of osteoblasts (OB) in maintaining hematopoietic stem cells (HSC) in their niche is well elucidated, but the exact definition, both phenotypically and hierarchically of OB responsible for these functions is not clearly known. We previously demonstrated (Chitteti et al. Blood 115(16):3239–48, 2010) that OB identified by their expression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) or CD166, represent a class of OB capable of mediating high levels of hematopoiesis enhancing activity (HEA). We also demonstrated that OB maturational status influences HSC function whereby immature OB with high Runx2 expression promote hematopoietic expansion. Here, we show that CD166 expression tracks the maturational status of OB and is directly correlated with Runx2 expression and high HEA. Fractionation of 2d calvariae-derived OB with lineage markers (CD45, CD31, and Ter119), Sca1, osteopontin (OPN), CD166, CD44, and CD90 revealed that Lin-Sca1-OPN+CD166+ cells (CD166+) and their subpopulations fractionated with CD44 and CD90 expressed high levels of Runx2 and low levels of osteocalcin (OC) demonstrating the relatively immature status of these cells. Conversely, the majority of the Lin-Sca1-OPN+CD166- cells (CD166-) expressed high levels of OC suggesting that CD166- OB are more mature. We then used a co-culture system previously described by our group to assess the potential of different groups of OB to mediate HEA and sustain the expansion of clonogenic cells in culture. In vitro hematopoietic potential of bone marrow-derived Lineage-Sca1+ckit+ (LSK) cells co-cultured for 7 days with fresh OB or OB pre-cultured for 0, 1, 2, or 3 weeks (followed by an additional 1 week of co-culture with LSK cells) declined precipitously with increasing culture duration concomitant with loss of CD166 expression. Percentage of cells expressing CD166 dropped from 63.5% at week 1 to 2.5% at week 4 (measured as total culture duration). During the same time period, the number of total colony forming units per culture dropped from 34,300 ± 4,000 (at week 1) to 1,800 ± 800 (at week 4) reflecting also the drop in the plating efficiency of cultured hematopoietic cells (22.9% ± 1.6% at week 1 versus 5.5% ± 0.5% at week 4). To assess the ability of OB to sustain stem cell function in vitro, we transplanted the progeny of LSK cells maintained in co-culture with OB for 7 days. Levels of chimerism 4mo post-transplantation in primary recipients of LSK progeny harvested from co-cultures containing Lin-Sca1-OPN+CD166+CD90+CD44+ OB were not significantly different from those in recipients of fresh LSK cells 71.8% ± 7.4% vs 86.1% ± 5.2%, respectively. Interestingly, when cells from primary recipients were transplanted into secondary recipients (4mo post primary transplantation), chimerism in mice receiving LSK progeny harvested from co-cultures containing Lin-Sca1-OPN+CD166+CD90+CD44+ OB increased to 95.8% ± 1.2% suggesting that these cells maintained the full potential of their self-renewal capacity. Chimerism levels in secondary recipients of LSK progeny harvested from co-cultures containing other fractions of OB were significantly lower than those observed for cells co-cultured with Lin-Sca1-OPN+CD166+CD90+CD44+ OB. These data suggest that robust HEA activity is best mediated by immature CD166+ OB with high Runx2 and low OC expression. Furthermore, these studies begin to define the hierarchical organization of osteoblastic cells and provide a more refined phenotypic definition of OB that can mediate HEA and maintain stem cell function. Disclosures: No relevant conflicts of interest to declare.