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CD27, CD201, FLT3, CD48, and CD150 identify HSC in mice
vated cell sorting. LK CD27+CD201+FLT3-CD48-CD150+ and LK CD27+CD201+FLT3-CD48±CD150- (NOT GATE) were sorted on a FACS Aria Fusion sorter (BD Bioscience). Sorted cells were washed, counted and defined cell doses were resuspended in saline with 2% heat-inactivated FBS containing 100,000 irradiated (15 Gy) BM carrier cells. Grafts were then injected retro-orbitally into female recipients 24 h after 2.5 Gy total-body γ irradiation (137Cs, Gammacell 40 Exactor, Best Theratronics, Ontario, Canada).
Engraftment was monitored with regular bleeds. At 18 weeks after transplantation, BM, spleen, and blood were harvested. Chimerism by donor male cells was determined by Y-chromo- some polymerase chain reaction analysis based on previous proto- cols,6,25 as outlined in the Online Supplementary Methods. Engraftment was considered positive when female recipients had >1% male DNA in the blood
Results
Comparison of SCA1, CD48 and CD150 expression in LK CD27+ CD201+ cells in C57BL/6, NOD-scid and NSG mice
BM cells from C57BL/6, NOD-scid, and NSG mice were stained with a cocktail of antibodies combining the tradi- tional markers (Lin, c-KIT, SCA1, FLT3, CD48, CD150)26 together with more recently proposed markers CD27 and CD201.13 After gating live cells, LK cells were examined for CD27 and CD201 expression (see gating strategy in Online Supplementary Figure S1). LK cells had a similar profile for CD27 and CD201 expression as previously reported for C57BL/6 and NOD strains13 (Figure 1A-C). The LK CD27+ CD201+ population labeled 0.019% ± 0.007% (mean ± standard deviation) of live BM nucleated cells in C57BL/6 mice, 0.100% ± 0.012% in NOD-scid mice and 0.041% ± 0.014% in NSG mice (Figure 1D). When calculated as cells per femur, NOD-scid mice had significantly more LK CD27+ CD201+ cells than had C57BL/6 and NSG mice (Online Supplementary Table S2 and Figure 1E). When the LK CD27+ CD201+ were back-gated for SCA1 and c-KIT expression, SCA1 staining was lower in NOD-scid and NSG mice than in C57BL/6 mice, which were predomi- nantly SCA1+ (Figure 1F-I). This resulted in a large propor- tion of the phenotypic HSC defined by the LK CD27+ CD201+ phenotype13 in NOD-scid and NSG mice falling in the SCA1- gate compared to the proportion from C57BL/6 mice (Figure 1J). Consequently, any calculation of pheno- typic HSC numbers using the classic LSK phenotype may underestimate the actual number of HSC in NOD-scid and NSG mice when calculated as cells per femur (Figure 1K).
Next, we investigated whether CD27 and CD201 stain- ing was compatible or complementary with FLT3-, CD48- and CD150+ staining to phenotypically identify LT-HSC. Live LK CD27+ CD201+ cells were gated for FLT3-, CD150, and CD48 expression analyzed for each mouse strain (Figure 2A-C). A similar CD150+ and CD48- LT-HSC pro- file was observed in the three strains. The frequency of LK CD27+ CD201+ FLT3- CD48- CD150+ cells among live BM nucleated cells was similar in C57BL/6 and NOD-scid mice but reduced in NSG mice (Online Supplementary Table S2 and Figure 2D). When calculated as cells per femur, C57BL/6 and NOD-scid mice had similar levels of pheno- typic LT-HSC per femur, whereas NSG had a significantly lower number of phenotypic LT-HSC cells per femur (Online Supplementary Table S2 and Figure 2E).
As it has been proposed that in the absence of SCA1 staining, the LK FLT3- CD48- CD150+ phenotype is suffi- cient to quantify mouse HSC,27 we further examined the expression of CD27 and CD201 in this population. In C57BL/6 mice, only 17.6% of LK FLT3- CD48- CD150+ cells were positive for both CD27 and CD201 (Online Supplementary Figure S2). As it has been previously report- ed that all HSC reconstitution activity is within the Lin- CD27+ CD201+ population,13 this suggests that it is neces- sary to add CD27 and CD201 stains in order to further enrich HSC within the Lin- CD117+ FLT3- CD48- CD150+ population. Likewise, in NOD-scid and NSG mice, only 28.6-32.9% of LK FLT3- CD48- CD150+ cells were positive for both CD27 and CD201.
High expression of CD48 in NOD-scid and NSG mice
In this analysis of CD48 and CD150 HSC detection (Figure 2A-C), we noticed that CD48 was more highly expressed in NOD-scid and NSG mice than in C57BL/6 mice as revealed by CD48 expression overlays of LK CD27+ CD201+ FLT3- cells from the different mouse strains (Figure 2F). In addition, the CD48 mean fluores- cence intensity for the whole LK CD27+ CD201+ FLT3- population was significantly reduced in C57BL/6 mice
compared to that in the other mouse strains (Figure 2G). The ligand for CD48 is CD24428 and is expressed by NK cells, some T cells, and monocytes.29 As the NOD-scid and NSG mice are devoid of functionally mature B and T cells, and NSG lack NK cells (Online Supplementary Figures S3 and S4) we speculated that CD48 upregulation in NSG and NOD-scid mice may be due to low expression of the ligand CD244. To assess this, we performed a lineage and CD244 stain on BM and spleen cells (Figures 3 and 4) to measure CD244 expression on each cell subset defined in Online Supplementary Figures S3 and S4. In C57BL/6 mice, subsets of CD244+ cells were observed on all BM lineages (Figure 3C, F) but predominantly on NK cells (Online Supplementary Table S3 and Figure 3F). Within the C57BL/6 spleen (Figure 4), CD244 was highly expressed on a subset of NK cells as well as on monocytes, macrophages, and neutrophil/myeloid progenitors. In NOD-scid and NSG mice, the frequency of CD244+ was less than 1% of all lin-
eages examined (Figures 3 and 4).
We detected some lymphocyte-type cells that were
B220+ in BM and spleen in both NOD-scid and NSG mouse BM (Online Supplementary Figures S3 and S4). In addition, NSG mice had rare NK1.1+ cells whereas both NSG and NOD-scid had few CD3ε+ cells.
Long-term hematopoietic stem cells are enriched in the Lin- KIT+ CD27+ CD201+ FLT3- CD48- CD150+ subset in NSG mice
Finally, we tested whether the combination of CD27 and CD201 with FLT3, CD48, and CD150 markers could identify functional LT-HSC in NSG mice by serial dilution transplantation assay into non-lethally irradiated syngene- ic recipients (Figure 5). As it has been previously shown that the whole competitive repopulation unit (CRU) activ- ity is contained within the Lin- CD27+ CD201+ fraction of the BM in NOD mice,13 we further characterized the func- tional properties of these cells stained additionally with FLT3, CD48, and CD150 antibodies. We sorted two sub- sets of the LK CD27+ CD201+ population from the BM of male NSG mice, namely (i) LK CD27+ CD201+ FLT3- CD48- CD150+ cells (CD48-CD150+ gate) and (ii) LK
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