Endothelial protein C receptor CD201 is a better marker than stem cell antigen-1 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 (HSCs) and multipotent progenitors (MPPs) among lineage-negative KIT⁺ (LK) cells. However, SCA1 is expressed only in a few inbred mouse strains and becomes strongly upregulated in LK cells following in vivo challenge with interferons, lipopolysaccharide (LPS), or pathogens, leading to incorrect analysis of HSC functional subsets and delineation of HSC, MPP, and lineage-restricted progenitor phenotypes. Endothelial protein C receptor CD201 can 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, the 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 the CD201 gating strategy, we reassessed 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.