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Thrombopoietin maintains megakaryopoietic HSPC
was not affected (Online Supplementary Figure S4). This suggests specific loss of MPL+ cells in the absence of THPO. Despite this, MPL expression in CD41+ HSPC was unaffected by loss of THPO, suggesting that this population of HSPC is more homogeneous than the other two HSPC populations and that MPL expression is intrinsic to CD41+ HSPC.
CD150- hematopoietic stem and progenitor cells have reduced dependence on thrombopoietin and low megakaryopoietic potential
To examine whether the CD150- HSPC are dependent on THPO signaling for proliferation, single MPL+CD48- CD150- HSPC and MPL-CD48-CD150- HSPC were cul- tured with THPO and SCF for 7 days. Proliferation of CD48-MPL+ LT-HSC and CD41+ HSPC was also analyzed (Figure 4A). A high proportion of the LT-HSC and CD41+ HSPC gave rise to colonies at day 10 in the presence of THPO, but in the absence of THPO few colonies were observed. This indicates that the two CD150+ popula- tions are dependent on THPO for proliferation in vitro. Both MPL+CD150- HSPC and MPL-CD150- HSPC pro- duced few colonies in the presence of THPO and while the MPL+CD150- HSPC produced slightly fewer colonies in the absence of THPO, the colony number among MPL- CD150- HSPC was not significantly different. This sug- gests that the CD150- HSPC population has reduced dependence on THPO for proliferation in vitro, with the
MPL-CD150- HSPC population showing limited depend- ence on THPO/MPL signaling. Downregulation of MPL expression has previously been linked to lymphoid line- age commitment,16 suggesting that the CD150- HSPC would have reduced myeloid differentiation potential compared to the other LSK populations. To test this we utilized an adapted method of a previously described in vitro culture assay.17 The CD48- fractions of each popula- tion were cultured with SCF, THPO, EPO, IL3 and IL6 to assess their differentiation potentials (Figure 4B). At day 10 fewer than 50% of single cells from the two CD150- HSPC populations produced colonies, suggesting that the myeloid differentiation cytokines in the media were insufficient to support the proliferation of many of the cells in these populations. Both MPL+CD48-CD150- HSPC and MPL-CD48-CD150- HSPC populations produced sig- nificantly fewer Mk colonies than LT-HSC and CD41+ HSPC. Previous studies have shown that MPL is down- regulated as HSC become lymphoid-biased. Taken together this suggests that megakaryopoietic potential is lost as the CD150- HSPC become lymphoid-primed. Over 50% of CD48-MPL+ LT-HSC gave rise to colonies contain- ing megakaryocytes, while approximately 90% of CD48- MPL+CD41+ HSPC give rise to colonies with megakary- ocytes. Subsequent mRNA expression analysis revealed that while LT-HSC and CD41+ HSPC both highly expressed the megakaryocyte marker, von Willebrand factor (Vwf), the two CD150- HSPC populations showed
Figure 6. Proposed model of maintenance of megakaryopoietic hematopoietic stem and progenitor cell number and quiescence. Thrombopoietin (THPO) from liver maintains megakaryopoietic cell numbers in the bone marrow. This preserves homeostatic levels of megakaryocyte-derived CXCL4, which induces quiescence in hematopoietic stem and progenitor cell (HSPC) populations. CD150- HSPC rely on CXCL4 for quiescence but are not dependent on THPO for maintenance of cell num- bers. LT-HSC: long-term hematopoietic stem cell; MkP: megakaryocyte progenitor; MK: megakaryocyte.
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