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The bulk of the hematopoietic stem cell population is dispensable for murine steady-state and stress hematopoiesis

Kristina B. Schoedel, Mina N. F. Morcos, Thomas Zerjatke, Ingo Roeder, Tatyana Grinenko, David Voehringer, Joachim R. Göthert, Claudia Waskow, Axel Roers and Alexander Gerbaulet

Data supplements

Article Figures & Data

Figures

  • Figure 1

    Inducible HSPC depletion. (A) Mouse model for inducible HSPC depletion; a conditional Rosa26-DTA knock-in allele (R26DTA) was crossed into HSC-CreERT mice. In the latter, the TAM-inducible Cre/estrogen receptor fusion protein (CreERT) is expressed in LT-HSCs and to a lower extent in progenitors. Induction of Cre-mediated recombination by TAM triggers expression of DTA in HSPCs and, thereby, their apoptotic cell death. Scl6E5-CreERT-3′enhancer transgenic mice were designated HSC-CreERT throughout the manuscript. (B) HSC-CreERT/R26DTA/DTA mice were induced by oral gavage of 2 × 0.1 mg/g BW TAM. (C) Flow cytometric analysis of BM HSPCs on day 7 after initiation of TAM. One Cre control (Cre) and 1 HSC-CreERT+R26DTA/DTA (Cre+) animal representative of 4 mice per group are shown. Mean frequencies ± SD of the gated population within WBMCs are shown. (D) Total numbers of HSCs and progenitors recovered from 2 femora and 2 tibiae of the animals described in panel B and shown in panel C. Mean numbers of controls (displayed ± SD) were set to 1 (dotted line). See supplemental Figure 1A for gating strategies and Figure 1E and supplemental Figure 2A-B for alternative TAM-induction protocols. (E) BM, spleen, and PB of HSC-CreERT/R26DTA/DTA were analyzed 8 days after TAM administration (n = 5, normalization and display of data as in panel D). (F) WBMCs from TAM-induced (5 × 0.3 mg/g BW) HSC-CreERT/R26DTA/DTA were mixed with equal numbers of B6.CD45.1 competitor WBMCs and transplanted into irradiated B6.CD45.1/CD45.2 recipients (n = 5 per genotype). Recipients were monitored for contribution of the R26DTA/DTA donor (Cre+ or Cre) to PB neutrophils (NEUT, CD11b+Gr-1hi) and lymphoid cells (LYMPH, FSCloSSCloCD11bGr-1) as well as to BM LSK cells (18 weeks after transfer). Means ± SD are shown. Experiment representative of 2 individual replicates. CLP, common lymphocyte progenitor; FSC, forward scatter; GMP, granulocyte/macrophage progenitor; MEP, megakaryocyte/erythrocyte progenitor; ns, not significant; SD, standard deviation; SSC, side scatter.

  • Figure 2

    Limited LT-HSC expansion after induced HSPC depletion. (A) HSC-CreERT/R26DTA/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/R26DTA/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-1hi) and lymphoid cells (LYMPH, FSCloSSCloCD11bGr-1) were analyzed (means ± SD, experiment representative of 2 individual replicates). (C) Different doses (supplemental Table 1) of HSC-CreERT/R26DTA/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 R26DTA/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/R26DTA/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.

  • Figure 3

    Effects of HSPC depletion on hematopoietic niche cells. (A) HSC-CreERT+R26EYFP/EYFP animals (n = 7-9 per cell population, black bars indicate means) were TAM-treated (4 × 0.2 mg/g BW plus TAM chow) for 4 weeks and BM niche cell subsets were analyzed for EYFP expression. BM LT-HSCs from the same animals served as a positive control for TAM induction. Identification of CXCL12-abundant reticular (CAR) cells, mesenchymal stem/progenitor cells (MSC), osteoblasts (OB), and ECs is shown in supplemental Figure 1C. (B) HSC-CreERT/R26DTA/DTA animals (n = 7-12 per group) were TAM-treated (as described in panel A) and selected BM niche cell subsets were quantified by flow cytometry. Absolute cell numbers from 2 femora and 2 tibiae were determined. Mean population size of Cre controls was set to 1. Means ± SD are shown. (C) HSC-CreERT+R26DTA/DTA (Cre+) and Cre control recipients (Cre) were lethally irradiated and transplanted with 5 × 106 WBMCs from B6.ubiEYFP donors which were wt except for ubiquitous EYFP expression (left). Ten weeks after transfer, chimeras were TAM-induced for 4 weeks (4 × 0.2 mg/g BW plus TAM chow) and absolute numbers of LT-HSCs and ECs per 2 femora and 2 tibiae were determined (right); n = 5 per genotype, black bar mean. (D) HSC-CreERT/R26DTA/DTA recipients (n = 3 to 5 per genotype) were TAM-induced for 4 weeks (4 × 0.2 mg/g BW and TAM chow; see also supplemental Figure 2A,C) and transplanted with 2.5 × 107 WBMCs from B6.ubiRFP donors which were wt except for ubiquitous RFP expression. PB neutrophil (NEUT, CD11b+Gr-1hi) and lymphoid cell (LYMPH, FSCloSSCloCD11bGr-1) chimerism (middle) were monitored thereafter and BM donor chimerism (right) was analyzed 23 weeks after transfer. Experiment is representative of 2 individual replicates.

  • 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 LSK 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 CD48CD150+CD34CD135 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.

  • Figure 5

    Low HSC numbers are sufficient to meet acutely increased demand for mature blood cells. (A) HSC-CreERT/R26DTA/DTA mice (n = 4-7 per group) were TAM-treated (2 × 0.2 mg/g BW) and 12 weeks later injected with PHZ or saline. PB hematocrit (HCT) was measured directly before PHZ injection and 3 and 6 days afterward (middle). One week after PHZ injection, BM populations from 2 femora were quantified by flow cytometry (right). Mean ± SD, absolute counts from 2 femora were normalized to the mean of saline-treated Cre controls (dotted line). (B) HSC-CreERT/R26DTA/DTA animals were TAM-treated (2 × 0.1 mg/g BW) and 19 weeks later i.p. injected with antiplatelet serum. Platelet (PLT) counts in PB were measured at the indicated time points before and after platelet depletion. HSC and progenitor populations in the BM were analyzed 6 days after PLT depletion (PLT-depl). Mean ± SD, absolute counts from 2 femora, normalized to the mean of Cre controls (dotted line). (C) HSC-CreERT/R26DTA/DTA mice (n = 4-5 per group) were TAM-induced and 10 weeks later injected with G-CSF or saline. PB was analyzed 1 day after the last injection for immunophenotypic LT-HSCs (middle). Two weeks after G-CSF treatment BM populations from 2 femora were quantified by flow cytometry (right, normalization and display as in panel A). (D) HSC-CreERT+R26DTA/DTA (Cre+, n = 3-5) and control mice (Cre, n = 4-9) were TAM-induced (2 × 0.2 mg/g BW) and 13 weeks later injected IV with 5-FU or saline. PB WBC counts of 5-FU–treated mice were determined at the indicated time points (middle data plot, means ± SD, Cre+, red; Cre, dotted black line). BM populations of HSC-CreERT+R26DTA/DTA and Cre control mice were measured 13 days after 5-FU injection. Means ± SD are shown; absolute counts from 2 femora were normalized to the mean of 5-FU–treated Cre controls. For data of saline-treated controls, refer to supplemental Figure 5E. Ctrl, control; MkP, megakaryocyte progenitor; Pre MegE (see Pronk et al32 for description); Pro Ery, proerythroblast.