Limited LT-HSC expansion after induced HSPC depletion. (A) HSC-CreERT/R26^DTA/DTA mice were TAM-induced (3 × 0.3 mg/g BW within 10 days) and analyzed for BM LT-HSC and EC counts at the indicated time points. Mean absolute LT-HSC/EC number per 2 femora of Cre⁻ mice was set to 1 (means ± SD, n = 3-6 per group). (B) WBMCs from HSC-CreERT/R26^DTA/DTA mice isolated 17 weeks after TAM induction (see also supplemental Figure 3A) were transplanted along with B6.CD45.1-competitor WBMCs into irradiated B6.CD45.1/CD45.2 recipients. WBMCs of 3 donors per genotype were each transplanted into 2 recipients. Cre⁺ or Cre⁻ donor contribution to recipient PB neutrophils (NEUT, CD11b⁺Gr-1^hi) and lymphoid cells (LYMPH, FSC^loSSC^loCD11b⁻Gr-1⁻) were analyzed (means ± SD, experiment representative of 2 individual replicates). (C) Different doses (supplemental Table 1) of HSC-CreERT/R26^DTA/DTA (Cre⁺ or Cre⁻) WBMCs were transplanted either directly (left data plot) or 13 weeks (right data plot) after TAM induction alongside with B6.CD45.1-competitor WBMCs into irradiated B6.CD45.1/CD45.2 recipients. Sixteen to 22 weeks after transplantation, recipients showing <0.01% multilineage R26^DTA/DTA donor repopulation in PB were scored as negative. Frequencies of LT-HSCs and statistics (bottom) were calculated using ELDA software. (D) Irradiated congenic B6.CD45.1/CD45.2 recipients were transplanted with 6 × 10⁴ sorted CD117⁺lin⁻ BM donor cells from uninduced HSC-CreERT/R26^DTA/DTA (Cre⁺ and Cre⁻) mice. Ten weeks after transplantation, chimeras were TAM-induced (left) and LT-HSC (LSK CD48⁻ CD150⁺) numbers in 2 femora were analyzed 2 or 11 weeks after TAM induction (right). Mean absolute LT-HSC number of Cre⁻ was set to 1 (means ± SD, n = 2-4 per group). CI, confidence interval; ln, natural logarithm.