Endothelial Protein C Receptor CD201 Enables Identification of Transplantable Mouse Hematopoietic Stem Cells in Inflammatory Conditions Unlike SCA1

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 not expressed by HSCs and MPPs from Ly6A.1 haplotype inbred strains such as BALB/c, C3H, CBA and nonobese diabetic (NOD), precluding proper HSC isolation and characterization in these mice. Furthermore, SCA1 is strongly induced on lineage-committed progenitor cells even in Ly6A.1 haplotype strains following treatment with type 1 and type 2 interferons, lipopolysaccharide (LPS) or after bacterial infection or sepsis which further compromises the ability SCA1 to correctly distinguish HSC/MPP from lineage-committed progenitors in inflammatory conditions. The endothelial protein C receptor CD201 has been suggested as an alternative marker for mouse and human HSC. However, whether CD201 expression changes during inflammation has not been investigated. Here we show in a mouse model of acute sepsis that LPS dramatically upregulates SCA1 expression in LK cells with 3-fold increase in mean fluorescence intensity and frequency. In sharp contrast, CD201 expression remains invariant on mouse bone LK cells in response to in vivo challenge with LPS, suggesting that CD201 is more suitable than SCA1 to identify HSC and MPP during inflammation. We then performed long-term competitive transplantations assays to assess the engraftment potential of LK201⁺ versus LK201^- versus LK SCA1⁺ (LKS⁺) cells. Following LPS treatment, HSC from LK201⁺ engrafted 4.4-fold better than LKS⁺ and 75-fold better than LK201^- cells suggesting that during inflammation, true HSCs are within the LK201⁺ population. Based upon CD201⁺ gating, LPS increased HSC and MPP cell proliferation but reduced their numbers in the BM. Also based on CD201⁺ gating, LPS suppressed myeloid and erythroid progenitors, decreased expression of HSC retention factors in the BM, and increased HSC mobilization. Finally using knock-out mice, LPS upregulated SCA1 expression on HSC and MPP via both MYD88 and TRIF signaling. Likewise, LPS increased HSPC mobilization via both TRIF and MYD88 pathway. However, LPS induced suppression of myeloid and erythroid progenitors required MYD88 but not TRIF signaling. In conclusion, CD201 enables a more accurate analysis of mouse HSC and MPP in all inbred strains under inflammatory conditions unlike SCA1. Activation of TLR4-TRIF and TLR4-MYD88 pathways play a distinct role on BM myeloid and erythroid progenitors and HSPC mobilization.

No relevant conflicts of interest to declare.