Abstract
CD150 is a member of the signaling lymphocyte activation molecule (SLAM) family of receptors which can be used as a positive selection marker for hematopoietic stem cells (HSCs) from mouse bone marrow (BM) and fetal liver (Kiel et al., Cell 2005; Yilmaz et al., Blood 2006; Kim et al., Blood 2006). In the current study, we combined CD150 with a set of lineage-specific markers to enrich HSCs, comparing normal C57BL/6 (B6) mice with B6-Trp53-deficient (Trp53 null) mice which were previously shown to have elevated HSC activity. Using an anti-mouse CD150 antibody (Clone TC 15-12F 12.2, BioLegend), we defined a population of Lin−CD41−CD48−CD150+(SLAM) cells that is 0.0078 ± 0.0010% and 0.0135 ± 0.0010% of total BM cells from B6 and Trp53 null mice. The size of the SLAM cell fraction was strongly correlated (R2= 0.7116, P<0.0013) to the population of well defined Lin−Sca1+CD117+ (LSK) cells (at 0.0165 ± 0.0036% and 0.0276 ± 0.0036% respectively) for the same B6 and Trp53 null mice we have tested (results consistent with previous findings indicating that the HSC pool is larger in Trp53 null mice than in B6 mice). To test HSC function in vivo, we sorted SLAM cells and Lin−CD41−CD48−CD150+ (SLAM−) cells from B6 and Trp53 null donors and transplanted them into lethally irradiated (11 Gys) B6 recipients. In a day 12 colony forming unit-spleen (CFU-S) assay, both SLAM and SLAM−cells formed colonies but the SLAM cells contained 12–15 fold higher day 12 CFU-S than did SLAM−cells. In a competitive repopulation assay, when donor SLAM and SLAM−cells were mixed with fresh B6-CD45.1 BM cells and engrafted into lethally-irradiated B6 recipients for six months, the density of repopulating units (RUs) was 22.48 ± 12.05 and 11.06 ± 8.78 per thousand SLAM cells from B6 and Trp53 null donors, dramatically higher than the RU density of 0.43 ± 0.09 and 0.55 ± 0.43 per thousand SLAM−cells from the same B6 and Trp53 null donors. When BM cells from the competitive repopulation recipients were serially-transplanted into secondary recipients, the SLAM donor cell contribution increased while the SLAM−donor cell contribution decreased with time when measured at two, six and fourteen weeks in the secondary recipients, indicating that SLAM cells were the primitive HSCs that sustained hematopoiesis long-term. No recipient that received Try53 null SLAM or SLAM−cells died, significantly different from our previous observation that whole BM cells from Trp53 null donors yielded high donor reconstitution in a competitive repopulation assay but 56% recipient died within five months after reconstitution (TeKippe et al., Exp Hematol 2003). SLAM markers may have enriched HSCs by excluding any Try53 null BM cell responsible for early recipient death. Analysis of intracellular reactive oxygen species (ROS) revealed higher proportions of ROS−cells in the CD150+ than in the CD150− BM cell fraction in both B6 and Trp53 null mice, consistent with the hypothesis that HSCs are metabolically inactive and therefore contain lower level of ROS. While SLAM− cells may still contain some residual HSC activity, results from the present study are in strong support of utilization of SLAM receptor CD150 as a positive selection marker for HSCs. The Lin− CD41−CD48−CD150+SLAM combination provides a simple and effective method for HSC enrichment.
Isolation of insertion elements from Gram-positiveBrevibacterium, CorynebacteriumandRhodococcusstrains using theBacillus subtilis sacBgene as a positive selection marker