Figure 4
Persistently low LT-HSC numbers do not impair steady-state hematopoiesis. (A) HSC and precursor populations in the BM of TAM-treated HSC-CreERT/R26DTA/DTA mice were analyzed at the indicated time points (as in Figure 2A). Absolute size of populations per 2 femora of Cre+ animals (n = 4-6 per time point) were normalized to the respective mean of Cre− controls (n = 3-5 per time point, not shown), which was set to 1 (dotted line). Means ± SD of Cre+ are shown. (B) Numbers (count per microliter) of WBC and red blood cells (RBC), platelets (PLT), and neutrophils (NEUT) in PB of HSPC-depleted HSC-CreERT+R26DTA/DTA and Cre− controls were determined at different time points after TAM treatment (as in Figure 2A). Cre− control means were set to 1 (means ± SD, n = 3-13 per group). (C) Frequencies (means ± SD are shown) of LT-HSCs, ST-HSCs, MPPs, and LS−K cells in G0, G1, and S/G2/M phase were determined by Ki67 expression and DNA content analysis (see supplemental Figure 1D) 1, 4, and 30 weeks after TAM. Cell-cycle analysis of LT-HSCs isolated from Cre+ mice 1 week after depletion was not possible due to very low abundance of these cells. Only significant results are indicated. (D) Correlation of LT-HSCs in G0 (%) and HSC-depletion efficiency (cell counts normalized to Cre− controls, mean set to 1) from Cre+ individuals 4 (black) and 30 (blue) weeks after TAM induction. (E) HSC-CreERT/R26DTA/rtTA/Col1A1tetO-H2B-RFP/wt mice were TAM-treated (2 × 0.2 mg/g BW) and H2B-RFP expression was induced by Dox treatment. Cre+ (n = 8) and Cre− animals (n = 7) were chased for 6 weeks and H2B-RFP label retention in BM LT-HSCs was analyzed. Frequencies (mean ± SD) of LT-HSCs retaining high (RFPhi) or low (RFPlo) level or complete dilution (RFP−) of H2B-RFP are shown. Median H2B-RFP fluorescence intensities (MFI) of RFP+ LT-HSCs from individual mice are shown (see supplemental Figure 4E for RFP gating of LT-HSCs). (F) One hundred LSK CD48−CD150+CD34−CD135− cells were sorted from individual HSPC-depleted mice (Cre+, n = 8) or controls (Cre−, n = 6) 17 weeks after TAM induction and transplanted together with 3 × 105 B6.CD45.1 competitor WBMCs into lethally irradiated recipients (a single recipient mouse for each donor). Neutrophil, B-cell, and T-cell donor chimerism was measured in PB. Donor chimerism of LSK cells in recipient BM was analyzed 23 weeks after transfer. Means ± SD are shown.

Persistently low LT-HSC numbers do not impair steady-state hematopoiesis. (A) HSC and precursor populations in the BM of TAM-treated HSC-CreERT/R26DTA/DTA mice were analyzed at the indicated time points (as in Figure 2A). Absolute size of populations per 2 femora of Cre+ animals (n = 4-6 per time point) were normalized to the respective mean of Cre− controls (n = 3-5 per time point, not shown), which was set to 1 (dotted line). Means ± SD of Cre+ are shown. (B) Numbers (count per microliter) of WBC and red blood cells (RBC), platelets (PLT), and neutrophils (NEUT) in PB of HSPC-depleted HSC-CreERT+R26DTA/DTA and Cre− controls were determined at different time points after TAM treatment (as in Figure 2A). Cre− control means were set to 1 (means ± SD, n = 3-13 per group). (C) Frequencies (means ± SD are shown) of LT-HSCs, ST-HSCs, MPPs, and LS−K cells in G0, G1, and S/G2/M phase were determined by Ki67 expression and DNA content analysis (see supplemental Figure 1D) 1, 4, and 30 weeks after TAM. Cell-cycle analysis of LT-HSCs isolated from Cre+ mice 1 week after depletion was not possible due to very low abundance of these cells. Only significant results are indicated. (D) Correlation of LT-HSCs in G0 (%) and HSC-depletion efficiency (cell counts normalized to Cre− controls, mean set to 1) from Cre+ individuals 4 (black) and 30 (blue) weeks after TAM induction. (E) HSC-CreERT/R26DTA/rtTA/Col1A1tetO-H2B-RFP/wt mice were TAM-treated (2 × 0.2 mg/g BW) and H2B-RFP expression was induced by Dox treatment. Cre+ (n = 8) and Cre− animals (n = 7) were chased for 6 weeks and H2B-RFP label retention in BM LT-HSCs was analyzed. Frequencies (mean ± SD) of LT-HSCs retaining high (RFPhi) or low (RFPlo) level or complete dilution (RFP−) of H2B-RFP are shown. Median H2B-RFP fluorescence intensities (MFI) of RFP+ LT-HSCs from individual mice are shown (see supplemental Figure 4E for RFP gating of LT-HSCs). (F) One hundred LSK CD48−CD150+CD34−CD135− cells were sorted from individual HSPC-depleted mice (Cre+, n = 8) or controls (Cre−, n = 6) 17 weeks after TAM induction and transplanted together with 3 × 105 B6.CD45.1 competitor WBMCs into lethally irradiated recipients (a single recipient mouse for each donor). Neutrophil, B-cell, and T-cell donor chimerism was measured in PB. Donor chimerism of LSK cells in recipient BM was analyzed 23 weeks after transfer. Means ± SD are shown.

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