Isolation of HSC in these cases has been enabled by the Hoechst dye exclusion approach, but this method is more technically challenging than antibody staining and thus alternative antibody staining approaches are needed to facilitate studies in which Kit and Sca-1 are insufficient10. One model system where an alternative HSC stain is needed is the non-obese diabetic (NOD) mouse, which is the predominant mouse model of spontaneous autoimmune diabetes. of long-term KCY antibody HSC in NOD mice, as well as in other strains including SJL, FVB, AKR, BALB/c, C3H and CBA. We also find that HSC appear to maintain expression of CD201 and CD27 after hematopoietic injury, when Kit expression is downregulated. These results suggest a widely applicable yet simple alternative for HSC isolation in settings where Kit and Sca-1 expression are insufficient. Introduction Hematopoietic stem cells (HSC) are defined by their ability to durably give rise to all lineages of the blood and immune system; they are essential for bone marrow (BM) transplantation, and they are usually isolated based on their expression of unique combinations of cell surface proteins. Studies of HSC in wild type C57BL/6 (B6) mice have predominantly used Kit and Sca-1 (also called Ly-6A/E) as well as the absence of markers of lineage committed cells (lin?), to identify HSC, termed KLS (or LSK) staining1-4. Although additional markers including CD34, CD150 and CD48 can be used to further enrich HSC, they are commonly used in combination with the KLS stain4-6. However, Sca-1 is not robustly expressed in all mouse strains, hindering the application of this stain to diverse model systems7. Mice of the Ly6.1 haplotype, including BALB/c, C3H and CBA strains, express very low levels of Sca-17,8. In addition, both Kit and Sca-1 expression levels are dynamically regulated in response to hematopoietic injury9. Isolation of HSC in these cases has been enabled by the Hoechst dye exclusion approach, but this method is more technically challenging than antibody staining and thus alternative antibody staining approaches are needed to facilitate studies in which Kit and Sca-1 are insufficient10. One model system where an alternative HSC stain is needed is the non-obese diabetic (NOD) mouse, which is the predominant mouse model of spontaneous autoimmune diabetes. Several studies have reported on the ability of HSC transplantation to prevent, halt or reverse progression of diabetes in NOD mice11,12. Although Sca-1 is used as an identifying marker for HSC in some of these transplantation studies, NOD HSC fail to express high levels of Sca-1 (despite the fact that NOD have the Ly6.2 haplotype), suggesting that these studies may Jasmonic acid have been impacted by transplantation of progenitor populations that were poorly enriched for HSC7,13. We investigated the use of alternative markers that could identify HSC in NOD mice. CD201, a type I transmembrane receptor, is expressed at high levels on murine HSC14. Although CD201 is a highly specific marker for HSC, it is still used in combination with Sca-1 and SLAM-family markers CD150 and CD48 to identify a more enriched HSC population14-16. Jasmonic acid CD27 is another marker that is expressed on HSC and downstream progenitors17,18. Although it has been proposed that the CD27 positive subset of hematopoietic progenitors does not contain long-term HSC, other studies suggest that most CD34? long-term HSC express CD27 at moderately high levels17,19. We show here that CD27 and CD201 identify HSC independently of Sca-1 in NOD mice. This identification method was applicable in several other strains, including C57B/6, SJL, FVB/N, AKR, BALB/c, C3H/He and CBA. In addition, these markers identify HSC and progenitors in mice that have downregulated Kit as a result of hematopoietic injury. CD27 and CD201 therefore enable identification and isolation of highly enriched hematopoietic stem and progenitor Jasmonic acid cells in models where Sca-1 and Kit are unable to identify a distinct progenitor population. Methods Mice C57BL/6J (stock no. 000664), NOD/ShiLtJ (001976), SJL/J (000686), FVB/NJ (001800), AKR/J (000648), BALB/cJ (000651), C3H/HeJ (000659) and CBA/J (000656) mice were purchased from Jackson Laboratories. NOD-mRaspberry (mRasp) transgenic mice were provided by Dr. Jason Gaglia. NOD, NOD-mRaspberry transgenic, B6-GFP transgenic and Rag?/? transgenic mice were bred at the Joslin Diabetes Center Animal Facility. Ages of donor and recipient mice ranged from 4 -12 weeks at time of initial treatment and sacrifice. All strains were maintained at the Joslin Diabetes Center Animal Facility and fed with standard mouse chow and water. All animal procedures were approved by the Joslin IACUC. Isolation and staining of.