1. Granulocyte colony-stimulating factor mobilizes dormant hematopoietic stem cells without proliferation in mice
by Jeffrey M. Bernitz, Michael G. Daniel, Yesai S. Fstkchyan, and Kateri Moore
Blood
Volume 129(14):
April 6, 2017
©2017 by American Society of Hematology

2. G-CSF does not induce proliferation of dormant HSCs
G-CSF does not induce proliferation of dormant HSCs. (A) Experimental schematic.
G-CSF does not induce proliferation of dormant HSCs. (A) Experimental schematic. Mice were placed on dox for 12 to 16 weeks to turn off expression of H2BGFP. Four days before analysis, mice were serially treated with G-CSF, and then BM, PB, and spleen were harvested to assay GFP label dilution, CFU, and HSC (LSKCD48−CD150+) quantification. (B) Histogram of HSC GFP label dilution. (C) Quantification of LR-HSC in panel B. (D) Absolute number of LR-HSCs in the BM. (E) CFU analysis from PB. (F) HSC quantification in the spleen. (G) Updated experimental schematic. Mice were placed on dox for 7 weeks, treated with G-CSF, and then chased for an additional 8 weeks to allow homeostasis to reestablish and all HSCs to return to the bone marrow before analysis. (H) Histogram of HSC GFP label dilution. (I) Quantification of LR-HSC in panel H. (J) Absolute number of LR-HSCs in the BM. (K) CFU analysis from PB immediately after the final G-CSF treatment. (L) HSC quantification in spleen. Data are represented as mean ± SEM of 6 and 7 mice for control and G-CSF treatments, respectively. *P < .05, **P < .01, ***P < .001 by Welch’s t test.
Jeffrey M. Bernitz et al. Blood 2017;129:
©2017 by American Society of Hematology

3. G-CSF preferentially mobilizes dormant HSCs. (A) Experimental schematic.
G-CSF preferentially mobilizes dormant HSCs. (A) Experimental schematic. Mice were placed on dox for 12 weeks and were treated with G-CSF either 4 days or 2 weeks before analysis. BM, spleen, and PB HSPCs were analyzed for GFP label distribution. (B) Histograms of GFP distribution across HSC (BM) and HSPC (spleen and PB) compartments without, 3 hours after, and 2 weeks after G-CSF treatment. (C) Quantification of HSPCs found across GFP gates. Data are represented as mean ± SEM of 6, 7, and 4 mice for control, 3-hour, and 2-week treatments, respectively. Statistical significance was determined by comparing control with 3-hour and 2-week data. *P < .05, **P < .01, ***P < .001 by Welch’s t test.
Jeffrey M. Bernitz et al. Blood 2017;129:
©2017 by American Society of Hematology

4. Mobilized HSPC transplantation defects are divisional history independent.
Mobilized HSPC transplantation defects are divisional history independent. (A) Schematic of BM/PB chimeric HSPC transplantations. LSKCD48− cells were sorted from the bone marrow of 34/H2B mice (CD45.2+) chased with dox for 12 weeks, based on label dilution. These cells were mixed together to mimic the label dilution ratios of LSKCD48− cells found in mobilized PB 3 hours after G-CSF treatment (Figure 2C). This BM HSPC mix was then further mixed at equal ratios with LSKCD48− cells sorted from mobilized PB of UBC-GFP mice (CD45.2+). This BM/PB HSPC mix was then competitively transplanted into CD45.1+ recipient mice. (B) Plot displaying GFP intensities of PB derived from UBC-GFP and 34/H2B mice. (C-D) PB- and BM-derived HSPC contribution to PB in primary (C) and secondary (D) transplantations. (E) Gating strategy for the BM HSPC compartment. (F-G) PB and BM derived contributions to the regeneration the BM HSPC compartments. In secondary transplant, regeneration in the HPC-2 and HSC compartments was exclusively confined to BM-derived cells, which prevented calculation of statistical significance between the BM- and PB-derived groups; not determined (n.d.). Data are represented as mean ± SEM of 8 and 9 mice for primary and secondary transplants, respectively. *P < .05, **P < .01, ***P < .001 by paired t test.
Jeffrey M. Bernitz et al. Blood 2017;129:
©2017 by American Society of Hematology

5. Proliferation in response to G-CSF is limited to CD41 expressing cells within the HSC compartment.
Proliferation in response to G-CSF is limited to CD41 expressing cells within the HSC compartment. (A) Representative plots displaying cell cycle status of the HSC compartment in response to G-CSF. (B) Quantification of G0 HSCs in panel A. (C) Distribution of CD41 expression on HSCs across GFP gates after a 12-week chase during homeostasis (no G-CSF). (D) Quantification of CD41− and CD41+ HSC GFP fluorescence intensities during homeostasis. (E) Quantification of total, CD41+, and CD41− HSCs in G0 with and without G-CSF treatment. (F) Representative plots of CD41 expression on HSCs with and without G-CSF treatment. (G) Quantification of CD41Hi cells within the HSC compartment. (H) Absolute numbers of CD41Hi HSCs in the BM. (I) Label dilution experimental set-up. (J) GFP histograms of CD41−, CD41+, and CD41Hi HSCs after G-CSF treatment. (K) Representation of CD41−, CD41+, and CD41Hi HSCs within each GFP gate after G-CSF treatment. (L) GFP fluorescence intensities after G-CSF treatment. (M) Cell cycle analysis of CD41−, CD41+, and CD41Hi HSCs after G-CSF treatment. Data are represented as mean ± SEM. n = 14 and 9 mice for control and G-CSF treatments, respectively (B, E, G, and H); n = 10 mice (C and D); n = 7 mice (K and L); n = 9 mice (M). *P < .05, **P < .01, ***P < .001 by Welch’s t test (B,E,G-H), paired t test (D), or 1-way repeated measures ANOVA with Tukey’s multiple comparison test (L-M).
Jeffrey M. Bernitz et al. Blood 2017;129:
©2017 by American Society of Hematology

6. Competitive transplantation of CD41−/+/Hi HSCs after G-CSF treatment.
Competitive transplantation of CD41−/+/HiHSCs after G-CSF treatment. (A) Experimental schematic. UBC-GFP mice were treated with G-CSF for 4 days. Three hours after the final treatment, BM was harvested and the CD41−/+/Hi cells from within the HSC compartment were sorted and competitively transplanted. (B) Five-lineage repopulation of mice transplanted with 20 CD41−/+/Hi cells. Horizontal line represents threshold for repopulation. (C) Chart summarizing data from B. (D) Five-lineage repopulation of mice transplanted with 100 CD41+/Hi cells. (E) Chart summarizing data from D. (F) Analysis of the BM HSPC compartment 16 weeks after transplantation with 20 CD41−/+ cells. Only mice that demonstrated PB reconstitution were analyzed. (G) Analysis of the BM HSPC compartment 16 weeks after transplantation with 100 CD41+/Hi cells. All mice were analyzed, regardless of PB reconstitution.
Jeffrey M. Bernitz et al. Blood 2017;129:
©2017 by American Society of Hematology

7. CD41Hi HSCs rapidly generate megakaryocytes in culture without proliferation.
CD41HiHSCs rapidly generate megakaryocytes in culture without proliferation. Single CD41−/+/Hi HSCs from UBC-GFP mice treated with G-CSF (Figure 5A) were sorted into wells of a 96-well plate and cultured in the presence of Tpo, Epo, IL-6, IL-3, and SCF. Cells were observed daily, and cell number, cell size, and megakaryocyte emergence was quantified. (A) Proliferation of single CD41−/+/Hi cells over 14 days in culture. (B) Representative images of sorted CD41−/+/Hi over time. (C) CD41−/+/Hi cell fate assayed at day 14. (D) Quantification of colony diameter at day 14. “Colony size” of CD41Hi cells was most often the diameter of remaining single cells. (E) Quantification of the largest cell diameter visible in culture over time. The dashed line at 40 μm represents the threshold above which a cell was considered “large.” (F) Size of single CD41−/+/Hi cells before culture. (G) Quantification of megakaryocyte emergence over time. (H) Hematoxylin and eosin staining of a megakaryocyte from a CD41− cell colony. Megakaryocyte is large and contains a multilobule nucleus. (I) Live imaging of representative single CD41Hi cell on day 5 of culture. Cell stains positive for megakaryocyte marker CD41 and has a multilobule nucleus. Data are representative of 2 to 3 independent experiments. ***P < .001 by 1-way ANOVA with Tukey’s multiple comparisons test.
Jeffrey M. Bernitz et al. Blood 2017;129:
©2017 by American Society of Hematology

8. Model of BM proliferative response to G-CSF treatment.
Model of BM proliferative response to G-CSF treatment. In response to G-CSF, dormant HSCs are mobilized to the circulation without proliferation. These cells will accumulate in the spleen and return to the BM with time. The remaining cells in the BM after G-CSF treatment can be organized based on CD41 expression, which positively correlates with divisional history. Proliferative response to G-CSF is exclusively associated with CD41 expressing cells, and the CD41Hi population rapidly matures into megakaryocytes. Label dilution is illustrated by the various shades of green in the nuclei of the cells. Circular arrows represent cell proliferation.
Jeffrey M. Bernitz et al. Blood 2017;129:
©2017 by American Society of Hematology