Consistent with previous findings (Pronk et al., 2007), 1 or 5 SN-CD150+ cells (designated MEP in Physique 1B) almost exclusively generated Meg/E lineage cells (Physique 1B). 1 or 5 SN-CD150+ cells (designated MEP in Physique 1B) almost exclusively generated Meg/E lineage cells (Physique 1B). By D14, SN Flk-2-CD150-7-integrin+ (SN-7+) cells gave rise only to Meg/E or mast cells. Cultures of DGAT-1 inhibitor 2 5 SN-7+ cells also exhibited a few c-Kit-FcRI+ colonies at D7, but such cells were not detectable at D14. DGAT-1 inhibitor 2 Open in a separate window Figure 1 Evidence that Sca-1-lin-c-Kit+ (SN, Sca-1 negative) cells have already committed to the GM, Meg/E, or mast cell lineage(A) SN cells were sorted into four populations based on surface DGAT-1 inhibitor 2 expression of Flk-2, CD150, CD27, and 7-integrin. (B) Of the four populations that were able to expand analysis reveals that lineage commitment is already initiated in the Sca-1lolin-c-Kit+ (SL) bone marrow fraction(A) SL bone marrow progenitor cells were sorted into four populations based on surface expression of CD27, 7-integrin, Flk-2, and CD150. (B) The four CD27+ fractions were able to expand at both a 5 cell and 1 cell per well density. The SL Mmp27 CD150lo fraction retained the most mixed lineage potential at each plating density, suggesting that this is the most immature population. However, SL Flk-2+ cells were already restricted to the GM lineage, notably without any detectable mast cell potential. The data in B summarize all the experiments we performed. See Table S2 for the total number of wells analyzed and the numbers of individual sorts performed for each progenitor population. For the initial 3-4 sorts, we always analyzed wells plated with single cells and 5 cells; for the remaining 2-3 sorts, we analyzed only wells seeded with single cells. See Table S3 for the results of Chi-square analyses comparing colony output (i.e., proportion of various types of colonies derived from 1 or 5 cell assays at day 7 or 14) for the various SL progenitor cells. CMPs were originally defined as within the Sca-1-lin-c-Kit+ fraction of mouse bone marrow cells (Akashi et al., 2000), but the application of improved antibody labeling and flow cytometric separation technologies, especially using the Sca-1 monoclonal antibody (mAb), has questioned this definition (Pronk et al., 2007; Arinobu et al., 2007). Hypothesizing that some lineage restriction might occur within the Sca-1lolin-c-Kit+ fraction (Sca-1lo cells were formerly contained within the Sca-1- gate; new stains reveal Sca-1lo and Sca-1- subsets), we plated four fractions of Sca-1lolin-c-Kit+ cells, based on expression of CD27, 7-integrin, CD150 and Flk-2 (Figure 2). Among the Sca-1lolin-c-Kit+CD27+ (SL; Sca-1lo) cells analyzed, SL Flk-2-CD150lo cells (SL-CD150lo in Figure 2B) had the greatest mixed lineage potential at D7; there also was substantial mixed lineage potential in SL Flk-2-CD150hi and SL Flk-2-CD150- cells (SL-CD150 hi and SL-CD150- in Figure 2B) but with greater bias towards Meg/E or GM lineages, respectively, suggesting that these could be transitional populations (Figure 2B and Tables S2 and S3). Mast cells were present at D14 in most wells seeded with Flk-2- SL-CD150lo, SL-CD150hi or SL-CD150- cells that were scored on D7 or D14 as having mixed lineage potential, including those derived from 1 or 5 cells (Table S4), and wells plated with 5 cells included some which, at D14, contained only mast cells or only cells with Meg/E-restricted potential (Figure 2B). By contrast, SL-Flk-2+ (SL-GMP in Figure 2B) cells yielded almost exclusively GM colonies, and no mast cells (Table S4), at D7 or D14. These data support the conclusions that: DGAT-1 inhibitor 2 (1) lineage specification is initiated in the SL.