IFN-α enhances competitive transplant by enhancing HSC quiescence without affecting stem cell frequency or homing. (A) Limiting dilution assays with IFN-γ–treated, IFN-α–treated, untreated wild-type (WT), or Ifnαr1^−/− E11.5 AGMs; n = 13 for a total of 89 mice. (B) Homing assay for detection of CD45⁺GFP⁺Lin⁺ and CD45⁺GFP⁺Lin⁻ in the BM 18 hours posttransplantation; n = 5 (right). Representative flow cytometry plot showing gating for CD45 and GFP (left); n = 5. (C) Boxplots showing donor chimerism attributable to control or IFN-α treatment during AGM transplantation. (D) Boxplots showing engraftment from competitive transplant with IFN-α–treated, untreated, or Ifnαr1^−/− AGMs against 3 × 10⁵ competitor BM cells. Two-way ANOVA was performed. (E) MFI of MHC class I molecules detected on donor-derived hematopoietic cells 6 hours after IFN-α treatment (top) or in the periphery at 6 weeks posttransplantation (bottom); n = 3-6. (F) Cell-cycle analysis of the donor-derived (CD45.2⁺) LSK HSCs in the BM of transplantations involving IFN-α–treated or untreated AGMs. Samples were analyzed at 4 weeks posttransplant in the primary recipients (n = 3; middle) or 36 weeks posttransplant in the secondary recipients (n = 4-5; bottom) by flow cytometry with Pyronin Y and Hoescht 33342 (top); n = 4-5. (G) Cell-cycle analysis of the donor-derived (CD45.2⁺) LSK HSCs in the BM of transplantations involving IFN-α–treated or untreated AGMs. Samples were analyzed at 4 weeks posttransplant in the primary recipients (n = 3) or 36 weeks posttransplant in the secondary recipients (n = 4-5) by flow cytometry with Pyronin Y and Hoescht 33342. (H) Boxplots showing engraftment from competitive transplant with 5000 Sca1⁺ cells from E14.0 FL from WT, Ifnαr1^−/−, or Ifnγr1^−/− against 2 × 10⁵ competitor BM cells. Two-way ANOVA was performed. Statistical significance: *P < .05; ***P < .001. ns, not significant; SLAM, signaling lymphocyte activation molecules.