Endothelial protein C receptor CD201 is a better marker than SCA1 to identify mouse long-term reconstituting hematopoietic stem cells following septic challenge

Abstract Stem cell antigen-1 (SCA1) is widely used to identify mouse hematopoietic stem cells (HSC) and multipotent progenitors (MPP) among lineage-negative KIT + (LK) cells. However, SCA1 is expressed only in a few inbred mouse strains and becomes strongly upregulated on LK cells following in vivo challenge with interferons, lipopolysaccharide (LPS) or pathogens leading to incorrect analysis of HSC function subsets and delineation of HSC, MPP and lineage-restricted progenitor subsets. Endothelial protein C receptor CD201can be used as an alternative marker for mouse and even human HSC. However, whether CD201 expression changes following infectious challenge is unknown. Unlike SCA1, CD201 expression did not change on mouse LK cells in response to LPS in vivo. Long-term competitive transplantations with CD201 + , CD201 − or SCA1 + LK cells showed that most reconstituting HSCs are within the LK CD201 + population after LPS challenge. However long-term competitive repopulation potential of LK SCA1 + cells from LPS-treated mice was much more severely reduced than that of LK CD201 + cells from the same LPS-treated donors suggesting that the LK SCA1 + population in challenged donors becomes contaminated with CD201 − progenitors devoid of long-term repopulation potential. Based on CD201 gating strategy, we re-assessed the effect of LPS on HSC and MPP cycling and mobilization, and their dependency on MY88 and TRIF adaptors. In conclusion, CD201 enables a more accurate analysis of mouse HSC and MPP subsets in all inbred strains in septic conditions or steady-state. Highlights SCA1 expression is markedly enhanced on MPPs and myeloid progenitors in vivo following LPS treatment whereas CD201 expression is not. Over 90% of the long-term competitive repopulation potential resides within LK CD201+ cells after LPS treatment. LK CD201+ cells sorted from LPS-treated mice give 4.4-fold higher multi-lineage chimerism than LK SCA1+ cells. SCA1 upregulation leads to overestimation of LT-HSC and MPP subsets, and underestimation of myeloid progenitors in femoral BM of LPS-treated mice.