Setd1a-deficient HSCs survive in situ but cannot contribute to hematopoiesis after transplantation. (A) Experimental outline for competitive transplantations using R.26-CreER^T2+;Setd1a^F/F or Setd1a^+/+ Lin⁻ BM donor cells together with wild-type Lin⁻ BM cells (1:1). TAM was induced 3 months after transplantation; the mice were bled regularly and analyzed 2.5 months later. Data for primary transplantation are representative of 4 independent experiments. A total of 10⁷ BM cells from primary recipients were transplanted into secondary recipients; blood chimerism was determined over 16 weeks, and BM chimerism analyzed after 16 weeks. Data for secondary transplantations are representative of 2 independent experiments. (B) Test or control donor cell chimerism in blood neutrophils (PMNs), CD11b⁺ Gr1^−/lo monocytes and eosinophils, and T and B lymphocytes before and after TAM treatment of BM chimeras. (C) Dot plots show the expression of Kit and Sca-1 on Lin⁻ donor BM cells that were further resolved for the expression of CD34 and CD135 from R.26-CreER^T2+;Setd1a^F/F or control BM chimeras that have received TAM 3 months after transplantation and were analyzed 2.5 months later. (D) Cell number of R.26-CreER^T2+;Setd1a^F/F or control donor LT-HSCs (LT), ST-HSCs (ST), MPPs, CMPs, MEPs, and GMPs from 4 independent experiments. (E) Frequency of KSL in Lin⁻ donor cells as shown in panel C. Data from 4 independent experiments are shown. (F) Single-cell PCRs specific for Setd1a^F, Setd1a⁺, or Setd1a^del alleles in donor-derived KSL Slam LT-HSCs from BM chimeras. A total of 165 cells from 2 independent mice were analyzed (39 and 126 cells), and representative PCR results from 16 of them are shown here. (G) R.26-CreER^T2+;Setd1a^F/F (red lines) or control (blue lines) donor cell contributions to blood neutrophils, myeloid, T, and B cells in secondary recipient mice. At the time point of transplantation, LT-HSC numbers were comparable between R.26-CreER^T2+;Setd1a^F/F and wild-type control BM chimeric primary recipients (Figure 2D). (H) R.26-CreER^T2+;Setd1a^F/F (red circles) or control (blue circles) LT-HSCs, ST-HSCs, MPPs, CMPs, MEPs, and GMPs in the BM of secondary recipients. Data are pooled from 2 independent experiments. (I) R.26-CreER^T2+;Setd1a^F/F (red lines) or control (blue lines) mixed BM chimeras were injected with 5-FU 10 weeks after TAM treatment, and PMN blood chimerism was determined 2 weeks later. (J) LT-HSC numbers of donor R.26-CreER^T2+;Setd1a^F/F (red circles) or R.26-CreER^T2+;Setd1a^+/+ (blue circles) in 5-FU-treated BM chimeras as described in panel I. w/o, without; wt, wild-type.