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A. O’Neill et al.
Pre-MegE cells that give rise to megakaryocytes are affected by the loss of THPO. Analysis of MPL expres- sion among these progenitor populations suggests that, while MPL was detectable in all populations, most cell populations showed baseline levels. Only those popula- tions with megakaryocytic potential showed levels of expression significantly above the baseline (Online Supplementary Figure S2).
Thrombopoietin is required to maintain CD150+ hematopoietic stem cell numbers
To further analyze the hematopoietic compartment the CD34-LSK HSC fraction was subdivided into three HSC populations based on CD41 and CD150 expression (Figure 3A). Previous studies have defined the CD34- CD41-CD150+ LT-HSC population as being enriched for long-term repopulating HSC, while the CD150- HSPC population contains short-term repopulating HSC and lymphoid-biased HSC.13,14 The CD41+ HSPC population has been proposed to be enriched for common myeloid progenitors with the potential to repopulate the bone marrow of lethally irradiated mice.15 This method of defining populations was found to be optimal for distin- guishing those LSK populations that express high levels of MPL from those with reduced MPL expression (Online Supplementary Figure S3) as well as for separating popula- tions based on differentiation potential. Analysis revealed that the two CD150+ LSK populations, LT-HSC and CD41+ HSPC, showed significant loss of cell numbers with LT-HSC numbers reduced from 16.3±4.35 cells per 106 bone marrow mononuclear cells (BMMNC) to 1.8±0.89 in ThpoΔ/Δ Alb-Cre mice and from 19.9±8.21 to 1.4±0.94 in Thpo-/- mice (Figure 3B). CD41+ HSPC also decreased drastically from 9.4±3.92 to 0.7±0.52 in ThpoΔ/Δ Alb-Cre mice and from 10.5±3.68 to 0.2±0.08 in Thpo-/- mice, a reduction of over 90% (Figure 3D). The CD150- HSPC
population, however, showed no significant difference in either Thpofl/flAlb-Cre or Thpo-/- mice, suggesting that this population may have reduced dependence on THPO sig- naling (Figure 3C).
CD150- hematopoietic stem and progenitor cells have reduced MPL expression
As CD48 is expressed by lineage-committed cells we analyzed CD48 expression in the HSPC populations. While a high proportion of LT-HSC and CD41+ HSPC did not express CD48, the majority of CD150- HSPC were CD48+ (Figure 3E and F). To investigate why the two CD150+ populations were highly affected by loss of THPO signaling while CD150- HSPC were not, we looked at the surface expression of MPL of the three pop- ulations. The CD48-CD150- HSPC expressed MPL, but a significantly smaller proportion of these cells expressed MPL compared to the other two HSC populations (Figure 3G). Among the MPL+CD48-CD150- HSPC the MPL mean fluorescence intensity was lower than in MPL+CD48- LT-HSC, suggesting that MPL is being down- regulated as LT-HSC differentiate into CD150- HSPC (Figure 3H). To confirm that MPL expression is reduced we sorted CD48-MPL+ LT-HSC as well as CD48- MPL+CD150- HSPC and CD48-MPL-CD150- HSPC and analyzed MPL mRNA expression (Figure 3I). We observed that even in the MPL+CD150- HSPC, MPL mRNA expression was significantly reduced compared to that in LT-HSC and was further reduced in MPL- CD150- HSPC. This suggests that MPL is downregulated as LT-HSCs differentiate into CD150- HSPC and that CD150-HSPC may therefore be less dependent on THPO/MPL signaling. Analysis of MPL expression in the KO models showed that smaller proportions of LT-HSC and CD150- HSPC were expressing MPL in both Thpo-/- mice and ThpoΔ/Δ Alb-Cre mice, while MFI of the MPL+ cells
ABCD
E
Figure 5. Maintenance of cell number is inde- pendent of quiescence. (A) Quiescence was
) cells within hematopoietic stem and progenitor cell (HSPC) populations of Thpo-/- and ThpoWT/WT control mice. (B) The concentration of CXCL4/PF4 in bone marrow of knockout mice as measured by enzyme-linked immunosorbent assay (n=4). (C-E)
measured as the percentage of Ki67 negative (G
0
F G
at day 7 of intraperi- toneal treatment with CXCL4/PF4 or phosphate- buffered saline (PBS) in Thpo-/- mice and ThpoWT/WT littermate controls (n=3). (F, G) Cell counts of HSPC populations at day 7 of intraperi- toneal treatment with CXCL4/PF4 or PBS in Thpo- /- mice and ThpoWT/WT littermate controls (n=3). BMEF: bone marrow extracellular fluid; BMMNC:
Percentage of cells in G
bone marrow mononuclear cells.
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haematologica | 2021; 106(7)