We then harvested BM from CFP+ mice and stained them with CD11A Stomach (with 100?ng/106 cells), or left them untreated
We then harvested BM from CFP+ mice and stained them with CD11A Stomach (with 100?ng/106 cells), or left them untreated. atop the hematopoietic hierarchy and give rise to functional effector cells through a succession of increasingly committed downstream progenitor cell stages (Seita and Weissman, 2010). Our understanding of the molecular basis for lineage determination and self-renewal has depended critically on our ability to identify and isolate HSCs and their downstream progeny with high purity. SB-277011 dihydrochloride HSCs are primarily quiescent, but their immediate downstream progeny, multipotent progenitors (MPPs), are transit-amplifying cells and rapidly proliferate and differentiate to replenish the blood supply. Thus, reliably separating HSCs from MPPs is key to characterizing their distinct self-renewal and differentiation potentials, and considerable attention has been paid to markers that can better individual these?populations, which include SCA-1, KIT, CD34, and CD150 (Kiel et?al., 2005). Analyses of purified HSCs transplanted into lethally irradiated mice at low numbers (1 to 50 cells per mouse) have revealed functional heterogeneity within phenotypic HSCs (Beerman et?al., 2010; Benz et?al., 2012; Lu et?al., 2011). Beerman et?al. exhibited that higher levels of CD150 (SLAMF1) marked HSCs that are skewed toward myeloid cell fates, compared to CD150int HSCs, which display a more balanced lineage output (Beerman et?al., 2010). Other groups have shown heterogeneity of HSCs using a variety of markers such as cytokine receptors, other Slam family members, and adhesion molecules (Arai et?al., 2004; Kiel et?al., 2005; Wagers et?al., 2002). Thus, even with the existing panel of markers, the HSC populace is likely heterogeneous. Based on our own gene expression analyses of HSCs and downstream progenitors (Seita and Weissman, 2010), we identified integrin alpha L (CD11A, Itgal) as?a?possible marker to better purify HSCs. CD11A heterodimerizes with CD18 (integrin beta-2) to form the adhesion molecule LFA-1 (lymphocyte function-associated anigten-1) (Cornwell et?al., 1993). LFA-1 is usually expressed on all leukocytes and plays important roles in many immunological processes, including transendothelial migration toward sites of inflammation (Van Epps et?al., 1989), lymphocyte costimulation and effector-target cell interactions (Davis et?al., 1999), and formation of the T?cell immunological synapse (Grakoui et?al., 1999). In this study, we show that CD11A has bimodal expression on phenotypic HSCs (Lin?KIT+SCA-1+FLK2?CD150+CD34?). Our data show that the CD11A? fraction of HSCs contains all functional HSC activity, with the CD11A+ fraction composed of more differentiated cells that lack long-term self-renewal activity. Results and Discussion Bimodal Expression of SB-277011 dihydrochloride CD11A on Phenotypic HSCs in Mice Based on a screen of a microarray database spanning over?35 mouse hematopoietic populations (Seita and Weissman, 2010), we discovered that HSCs express much lower levels of CD11A than downstream progenitors (Figures S1A and S1B available online). We examined mouse whole bone marrow (BM) with anti-CD11A antibodies (Abs) to measure CD11A surface expression by flow cytometry (Physique?1A). All mature lymphocytes SB-277011 dihydrochloride were positive for CD11A on their cell surface (data not shown), and almost all hematopoietic progenitor populations expressed high levels of CD11A, including MPPs and both myeloid (CMP, GMP) and lymphoid (CLP, BLP) committed progenitors (Physique?1A, see Supplemental Experimental Procedures for definitions and surface marker phenotypes). Only the megakaryocyte/erythrocyte progenitor (MEP) expressed low levels of CD11A. In contrast, HSCs (defined as Lin?KIT+SCA-1+FLK2? CD150+CD34?) had a bimodal expression of CD11A (Figures 1A and 1B). The CD11A? fraction accounts for anywhere from 30%C70% of the phenotypic HSC populace, depending on the strain and age of the mouse (Physique?1B). Open in a separate window Physique?1 Bimodal Expression of CD11A on Phenotypic HSCs (A) BM populations were analyzed for cell-surface expression of CD11A. Fluorescence minus one (FMO) was used as the unfavorable control, Mouse monoclonal to SORL1 gated on Lin? cells. (B) Gating scheme of murine HSCs. Phenotypic HSCs are gated on live cells (PI?), Lin? (CD3?, CD19?, NK1.1?, GR1?, TER119?), IL-7R?, FcRlo, KIT+, SCA-1+, FLK2?, CD150+, and CD34?. The markers IL-7R and FcR are typically not necessary to identify HSCs but are shown here for additional resolution. (C) CD11A? (?) and CD11A+.