Studies of c-Mpl Function Distinguish the Survival and Proliferation of Hematopoietic Stem Cells (HSC) from the Expansion of Differentiating Clones.

HSC are defined by their quiescence, long survival, ability to self-renew, and ability to give rise to a multi-lineage clone of differentiating blood cells. To dissect the signals regulating these events, we studied c-Mpl function in mice. In contrast to previous studies, we quantitated HSC by transplanting marrow cells into tpo−/− recipients, so that the c-mpl−/− cells could compete fairly. In parabiosis studies linking WT and c-mpl−/− mice × 4–6 wk, 0.3–0.9% of WT HSC engrafted in the c-mpl−/− partner mouse’s marrow. This is similar to HSC trafficking between WT mouse pairs. Next, we transplanted various ratios of c-mpl−/− and WT marrow cells into irradiated tpo−/− or WT recipients. As expected, when phenotypically-distinct WT marrow cells were transplanted into tpo−/− mice, the ratio of reconstituting cells was identical to infused cells; and when c-mpl−/− and WT cells were transplanted into WT mice at a 1:1 ratio, the contribution of c-mpl−/− cells was low (4.6±2.0%). Even at a ratio of 8:1, c-mpl−/− cells contributed minimally (12.4±2.1%) at 3 m. Most importantly, however, when c-mpl−/− and WT marrow was transplanted into tpo−/− mice (at ratios of 4:1 and 8:1), c-mpl−/− HSC were efficient competitors, resulting in 71.2±7.1 and 82.1±6.6% engraftment (expected values 80% and 88%), implying that the frequency of HSC in c-mpl−/− marrow is relatively normal. Interestingly, WT cells had a significant advantage when c-mpl−/− to WT ratios were 2:1 and 1:1 (31.1±3.9 and 24.8±12.1% engraftment vs. expected values 66.7 and 50%). Secondary transplantation studies demonstrated this resulted from the small numbers of Tpo-producing mature cells present in donor marrow and attest to the exquisite sensitivity of multilineage WT progenitors to Tpo. Lastly, we performed non-myeloabalative transplantations using AMD3100 (a specific CRCX4 antagonist) to mobilize endogenous HSC, open niches, and allow the competitive engraftment of infused (competitor) HSC. Previously, we showed that transplanting 40×10⁶ WT marrow cells into WT mice results in 1.0±0.2% donor engraftment without AMD3100 and 4.6±1.1% donor engraftment 2 h after AMD3100 (5mg/kg sq), the percent expected should the mobilized and infused HSC compete equally (Blood 107:3764,2006). We confirmed that c-mpl−/− mice mobilize progenitors equivalently to WT mice (CFU-GM/ml blood increased 6.4± 3.0 and 4.6± 1.0 fold, respectively) then transplanted c-mpl−/− mice with WT marrow. 3 m later, the percent of WT HSC was 1.7±0.5 and 5.4±1.6%, values identical to the studies of WT recipients. Thus HSC number, not just frequency, appears normal in c-mpl−/− mice. As 22–26% of granulocytes and marrow CFU-GM had a WT phenotype, WT clones had a major growth advantage in the Tpo-rich (c-mpl−/−) environment. Since a similar competitive advantage should exist for WT clones in children with congenital amegakaryocytic thrombocytopenia, a clinical benefit might derive from the transplantation and engraftment of small numbers of gene-corrected HSC. Our studies suggest that c-Mpl does not promote HSC survival or self-renewal (and thus may not control HSC number, as had been previously argued), but rather facilitates the early expansion of differentiating clones. These experiments document the limitations of traditional competitive reconstitution assays for quantitating HSC number, provide alternative experimental strategies to distinguish the actions of HSC from earliest progenitor cells in vivo, and demonstrate that a selective growth advantage at a level distal to HSC can result in a profound effect on multilineage hematopoiesis.