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CD27, CD201, FLT3, CD48, and CD150 identify HSC in mice
(Figure 5K) showed a 32-fold enrichment (P=1.86 x 10-5) in CRU frequency in the CD48- CD150+ gate (1 in 179 cells) compared to the NOT GATE cells (1 in 5,786 cells) con- firming that the FLT3 CD48- CD150+ phenotype comple- ments the CD27+ CD201+ phenotype for further enrich- ment in functional LT-HSC. It is also important to note that the 1/5,786 CRU frequency found in the NOT GATE was due to a single recipient of the highest donor cell dose which had a very low level of engraftment (less than 3%) compared to recipients of CD48- CD150+ cells (Online Supplementary Table S4). Therefore the CRU frequency in the NOT GATE could be overestimated. Nevertheless, by multiplying the CRU frequency obtained from each gate by the number of cells in each gate, we found that 70% of the CRU contained within LK CD27+ CD201+ FLT3- cells were within the CD48- CD150+ subset (Online Supplementary Table S5).
SCA1 expression in unaltered in bone marrow endothelial and mesenchymal cells in NSG mice
As NSG mice and NOD-scid mice blood cells have low SCA1 expression in hematopoietic stem and progenitor cells (HSPC) (Figure 1I), we compared SCA1 expression in BM endothelial cells and MSC from C57BL/6 mice and NSG mice (Figure 6). Endosteal cells were collected from collagenase-treated femurs, magnetically enriched in non- hematopoietic cells, and stained against CD45, Lin, CD31, CD51, SCA1, and PDGFRα antibodies (gating strategy in Figure 6A-D). CD45- Ter119- CD31+ BM endothelial cells (Figure 6E, F) expressed equivalent levels of SCA1 in C57BL/6 and NSG mice (Figure 6E, F, I). Likewise, BM MSC, defined as CD45- Ter119- CD31- CD51+ cells (Figure 6G, H), expressed similar levels of SCA1 in the PDGFRα+ subset which defines the PαS cells30 (Figure 6J).
Finally, we found that plastic-adherent BM MSC derived from NSG mice also expressed high levels of SCA1 (Figure 6K).
Discussion
Considering that all the LT-HSC reconstituting activity resides within the Lin- CD27+ CD201+ population,13 we sought to determine the expression profile of these cells for FLT3, CD48 and CD150 antigens, which are classically used to identified LT-HSC and various subsets of multipo- tent progenitors.4,5,26 We found that in all three strains, irre- spective of SCA1 expression levels, only a small subset of LK CD27+ CD201+ cells was also FLT3- CD48- CD150+, a phenotype that defines LT-HSC when used in combina- tion with SCA1 positivity.26 Conversely, only a minority of LK FLT3- CD48- CD150+ cells were double-positive for CD27 and CD201. Using a stringent serial dilution long- term transplantation assay, we demonstrated that CRU were 32-fold enriched in the small FLT3- CD48- CD150+ subset of the LK CD27+ CD201+ population from NSG mice despite negative to low levels of SCA1 expression. This demonstrates that CD27 and CD201 positivity is complementary to the FLT3- CD48- CD150+ phenotype to identify functional LT-HSC and can be used to replace SCA1. This is particularly advantageous in strains that express low levels of SCA1 in hematopoietic cells such as NOD-scid and NSG strains, or because of treatments that increase or decrease SCA1 expression, such as irradiation and lipopolysaccharide administration.13,27 We also noted a
lower CRU frequency compared to the reported 50% CRU frequency in LK CD48-CD150+ cells sorted from C57BL/6 mice.6 Competitive assays in lethally irradiated recipient mice with congenic whole BM cells as a source of competing HSC were used in these studies6 whereas in the present study, we sublethally irradiated our recipient mice (2.5 Gy) without exogenous competing HSC. This irradiation dose depresses circulating granulocytes and monocytes for only 8 days without transplantation (data not shown) and therefore spares an unknown number of host HSC. Consequently, this sublethal irradiation of the hosts creates a competitive assay between the residual female host HSC and the transplanted male HSC. This could in part explain the relatively low frequency of recon- stituting cells that we measured in LK CD27+ CD201+ FLT3- CD48- CD150+ cells from NSG mice. An additional factor to consider regarding this relatively low frequency of reconstituting cells in the LK CD27+ CD201+ FLT3- CD48- CD150+ fraction from NSG mice is the known engraftment defect of HSC caused by the scid mutation, which would consequently reduce the reconstitution potential of the sorted cell populations.31,32
Our flow cytometry data revealed that the expression of CD48 was unusually higher in LK CD27+ CD201+ FLT3- cells from the NOD-scid and NSG mice compared to C57BL/6 mice. We found that the expression of CD244, the physiological ligand of CD48, was dramatically reduced in myeloid cells and lymphocytes from NOD-scid and NSG BM and spleen. Although NOD-scid and NSG mice have very low frequencies of T and NK cells that would express CD244, expression of CD244 in all myeloid lineages was also markedly reduced in the BM and spleen of NOD-scid and NSG mice. It is, therefore, tempting to speculate that CD48 upregulation in NOD- scid-derived strains is caused by the low expression of its ligand CD244. However, this potential mechanism will need to be confirmed in C57BL/6 mice with CD244 gene deficiency.
Interestingly, NOD-scid mice still contain a NK-cell pop- ulation33 but we did not detect higher numbers of NK1.1+ cells compared to the numbers in NSG mice. As we did not perform functional assays, we cannot conclude from our experiments that NSG mice had less functional NK cells compared to the NOD-scid mice. The literature indi- cates that CD3+ and primitive B cells are present in these mice but do not develop into mature functional lympho- cytes.33 The scid mutation is known to eliminate B and T cells at the education stage of development during VDJ recombination.34 This means that the BM will develop immature B and T precursors, which migrate into the cir- culation but cannot fully mature into functional lympho- cytes. With age, NOD-scid mice are known to have some ‘leakiness’ and develop functional B and T cells while NSG do not.33 Previous studies on these mice have focused on the spleen and/or blood,20,33 which are locations of mature B and T cells, and did not examine the BM in which these cells develop initially. As we used 8-week old mice, this small percentage of CD3ε+ and B220+ cells may represent immature lymphoid cells. The use of markers of more mature B cells, such as CD19 and surface IgM (sIgM), could have confirmed the absence of mature CD19+ sIgM+ B cells in these mice.33,35
Beside hematopoietic cells, SCA1 is expressed by vari- ous cell types such as mesenchymal and endothelial cells and is considered a progenitor/stem cell marker in many
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