IFN-α treatment promotes long-term engraftment of AGM HSCs. (A) Flow cytometry for IFN receptors Ifnγr1 (top) and Ifnαr1 (bottom), which are present in CD45⁺ cells of the AGM; n = 3. (B) Example of phospho-Stat1 activation in CD45⁺ E11.5 AGM cells treated with IFN-α (bottom) with isotype control (top). (C) Phospho-Stat1⁺ cells in the different cell compartments in the E11.5 AGM in response to IFN-α. The column indicates the absolute number of positive cells per embryo; n = 3. (D) Phospho-Stat3⁺ response in the different cell compartments in the E11.5 AGM in response to IFN-α. The column indicates the absolute number of positive cells per embryo; n = 3. (E) Phospho-Stat5⁺ response in the different cell compartments in the E11.5 AGM in response to IFN-α. The column indicates the absolute number of positive cells per embryo; n = 3. (F) Immunoblot for phospho-Stat1 in response to dose-titrations of IFN-α in adult splenocytes. (G) Example of donor chimerism analysis for multilineage engraftment. (H) Boxplots showing the effect of IFN-α on long-term hematopoietic engraftment of E11.5 AGM HSCs in the peripheral blood; 2 e.e. were transplanted with 2 × 10⁵ helper splenocytes. Two-way analysis of variance (ANOVA) was performed. (I) Boxplots showing the effect of IFN-α on long-term hematopoietic engraftment of AGM HSCs in the BM at 21 weeks posttransplantation. Wilcoxon rank-sum test was performed; n = 4-6. (J) Quantification of lineage contributions of B, T, and myeloid cells at 21 weeks posttransplantation in the peripheral blood; n = 4-6. (K) Boxplots showing engraftment from secondary transplantation of 2 × 10⁶ BM cells from IFN-α–treated and control AGMs in (H) with 3 × 10⁵ competitor BM cells. Two-way ANOVA was performed. Statistical significance: *P < .05; **P < .01; ***P < .001. n.s., not significant.