On the other hand, HSCs (thought as Lin?KIT+SCA-1+FLK2? Compact disc150+Compact disc34?) got a bimodal appearance of Compact disc11A (Statistics 1A and 1B). (HSCs) possess arguably end up being the most well-characterized tissue-specific adult stem cell. HSCs reside atop the hematopoietic hierarchy and present rise to useful effector cells through a succession of significantly dedicated Rabbit Polyclonal to RHG17 downstream progenitor cell levels (Seita and Weissman, 2010). Our knowledge of the molecular basis for lineage perseverance and self-renewal provides depended critically on our capability to recognize and isolate HSCs and their downstream progeny with high purity. HSCs are quiescent primarily, but their instant downstream progeny, multipotent progenitors (MPPs), are transit-amplifying cells and quickly proliferate and differentiate to replenish the blood circulation. Thus, reliably separating HSCs from MPPs is paramount to characterizing their specific differentiation and self-renewal potentials, and considerable interest continues TUG-891 to be paid to markers that may better different these?populations, such as SCA-1, KIT, Compact disc34, and Compact disc150 (Kiel et?al., 2005). Analyses of purified HSCs transplanted into lethally irradiated mice at low amounts (1 to 50 cells per mouse) possess revealed useful heterogeneity within phenotypic HSCs (Beerman et?al., 2010; Benz et?al., 2012; Lu et?al., 2011). Beerman et?al. confirmed that higher degrees of Compact disc150 (SLAMF1) proclaimed HSCs that are skewed toward myeloid cell fates, in comparison to Compact disc150int HSCs, which screen a more well balanced lineage result (Beerman et?al., 2010). Various 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 population 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, em Itgal /em ) 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 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 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 (Figure?1A). All mature lymphocytes 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 (Figure?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 population, depending on the strain and TUG-891 age of the mouse (Figure?1B). Open in a separate window Figure?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 negative control, 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 TUG-891 shown here for additional resolution. (C) CD11A? (?) and CD11A+ (+) HSC subfractions (50 cells/mouse) were transplanted into five lethally irradiated congenic recipients along with.