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CD27, CD201, FLT3, CD48, and CD150 identify HSC in mice
expressed low levels and BALB/c-derived MSC were neg- ative.41 As SCA1 has been recently described as an activa- tion marker facilitating cell cycling8,42 and mesenchymal progenitor cell self-renewal in vivo,43 culturing these cells in vitro could activate SCA1 expression and may explain this discordance. In our experiments, we found that low SCA1 expression is restricted to HSPC in NSG mice. Both freshly isolated BM endothelial cells (Figure 5G) and primitive mesenchymal progenitor cells (Figure 5H) from NSG mice expressed high levels of SCA1 similar to C57BL/6 mice as previously reported.44,45 The high expres- sion level of SCA1 on mesenchymal cells from NOD-scid and NSG mice is consistent with the absence of the osteoporotic phenotype that is observed in SCA1 knock- out mice.43 Our results therefore suggest that lower SCA1 expression may be limited to hematopoietic cells in NSG mice and may be a result of the original source of the scid mutation that was derived from the BALB/c background, a LY6.1 haplotype mouse or from the NOD back- ground.20,46
In conclusion, co-staining for CD27 and CD201 can be used in place of SCA1 to identify HSC in NOD-scid and NSG mice in circumstances that SCA1 expression is weak. However, when the Lin- CD27+ and CD201+ phe- notype is combined with the FLT3- CD48- CD150+ pheno- type, HSC with long-term engraftment potential are fur- ther enriched in NOD-scid and NSG mice. Compared to recent studies that focused only on Lin- CD27+ CD201+ cells,32,47 we show that within this population the small subset that is FLT3-, CD48- and CD150+ is enriched in LT- HSC activity in NSG mice in a rigorous serial dilution long-term competitive transplantation assay. This allevi- ates the need to stain for SCA1, which is expressed at very low levels in these mice. In addition, we identified a non-reported upregulation of CD48 in NOD-scid and NSG mice possibly due to the low expression of its ligand CD244. Finally, the low SCA1 expression in NSG mice seems limited to the hematopoietic compartment as SCA1 expression remains high in primary BM endothelial and mesenchymal cells. Overall, our new strategy may provide a more accurate method to quantify murine HSC
within xenograft models using NOD-scid-derived strains. For instance, in previous work48,49 humanized scaffolds seeded with human MSC were transplanted into NOD- scid mice and once humanized ectopic bone organoid had been established, were injected with human BM or cord blood CD34+ cells. The relative quantification of the seed- ing of humanised ectopic bone scaffolds by human vs. murine HSC was difficult due to low SCA1 expression by NOD-scid and NSG HSC. Likewise, in a common xeno- transplanted model of NSG mice engrafted with human cold blood CD34+ HSC, we were able to demonstrate that hypoxia-inducible factor prolyl hydroxylase inhibitor can rescue a human HSPC mobilization defect in NSG mice but we were unable to show a similar effect on murine HSC due to their low SCA1 expression.50 Therefore, this new staining strategy identifying Lin- KIT+ CD27+ CD201+ FLT3- CD48- CD150+ cells as mouse HSC in NOD-scid-derived strains will enable a more accurate measurement of the relative colonization or distribution of mouse bones or ectopic bone organoids by endoge- nous mouse HSC vs. xenotransplanted human HSC.
Acknowledgments
The authors acknowledge the Translational Research Institute (TRI) for providing an excellent research environment and core facilities that enabled this research. We particularly thank the Flow Cytometry and the Biological Resources Core Facilities. BN was supported by an Australian Government Research Training Program Scholarship during her PhD studies. JPL is funded by Research Fellowship APP1136130 from the National Health and Medical Research Council of Australia (NHMRC). MRD is funded by a Career Development Fellowship APP1130013 and Project Grant APP1108043 from the NHMRC. EDW is sup- ported by funding from the Movember Foundation and the Prostate Cancer Foundation of Australia through a Movember Revolutionary Team Award. The APCRC-Q is supported by funding from the Australian Government Department of Health. The TRI is supported by Therapeutic Innovation Australia (TIA). TIA is supported by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program.
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