In the stem cell transplantation setting, reconstitution of bone marrow and the subsequent recovery of peripheral blood cells are dependent on the expansion and differentiation of the HSCs and HPCs of donor cells. In this report, we investigated eltrombopag effect on human UCB CD34+ cells in the NOD/SCID mice xenotransplants after receiving human UCB CD34+ cells. We found that eltrombopag treatment accelerated expansion of human CD34+ progenitors, human CD45+ and CD41+ cells in the bone marrow, and promoted the production of circulating human platelets and WBCs without any influence on mouse hematopoietic cells ( and ). By week 9, which was 5 weeks after the cessation of eltrombopag feeding, the differences between the eltrombopag-treated mice and the control mice persisted. The multiple lineage and long-term effect of eltrombopag strongly suggested that it acted on “earlier hematopoiesis” at the HSC/HPC levels in addition to its anticipated effect on promoting megakaryocpoiesis, which is a more committed later stage.
To further examine eltrombopag’s effect on early hematopoiesis through HSCs/HPCs, we tested the ability of eltrombopag on the expansion of human UCB CD34+ cells in the presence of SCF and FL in the serum-free media (). Eltrombopag significantly expanded CD34+CD38− HSCs/HPCs, CD34+ immature progenitors and CD41+ cells. In comparison with the effects of 3μg/ml eltrombopag, 10ng/ml rhTPO did not affect the percentage of CD34+CD38−, CD34+, and CD41+ cells but did markedly increase the expansion, supporting the fact that eltrombopag is as effective as TPO in regulating the balance of proliferation and differentiation among CD34+CD38−, CD34+, and CD41+ cells. However, eltrombopag was not as potent as TPO in promoting proliferation. Our finding was consistent with a recent report showing that eltrombopag was able to expand CD34+CD38− cells in the presence of SCF in the serum-free culture (Nishino et al., 2009
), suggesting that it at least partly retained the function of TPO on early hematopoiesis.
The differential findings between eltrombopag and TPO suggested the possibility that separate intracellular signaling pathways might have taken place in mediating proliferation vs. differentiation of HSC/HPCs mediated by c-Mpl.
To look for the potential mechanisms behind the functional difference between TPO and eltrombopag, we conducted a cytometry-based method to analyze the phosphorylation of STAT3, STAT5 and AKT in the signaling pathway of TPO receptor (c-Mpl)
, in three different cell types: CD34+CD41− HSC/ HPC, CD34−CD41+ megakaryocytes, and CD34−CD41− cells. We found that the level of phosphorylation of STAT5 induced by eltrombopag was similar in all three cell-populations and were comparable to the level stimulated by TPO in CD34+CD41− and CD34−CD41− cells, but TPO stimulated higher levels of STAT5 phosphorylation in CD34−CD41+ cells. This was the cell population that was more committed to thrombopoiesis than the other two cell type populations. We also observed that TPO activated STAT3 and AKT in CD34−CD41+ cell, while eltrombopag did not cause the phosphorylation of STAT3 and AKT pathways in CD34−CD41+ cells. Because eltrombopag promoted CD41+ differentiation as effectively as TPO in our in vivo model, our finding suggested that STAT3 and AKT signaling pathway might not be important for the differentiation of CD34+ cells to CD41+ cells. It is known that the binding of TPO to c-Mpl
activates MAPK p42/p44, AKT, and STAT proteins in normal human CD34+ cells, megakaryocytes, and platelets (Majka et al., 2000
; Majka et al., 2002
), while eltrombopag activates MAPK p42/p44 and STAT5 in N2C-TPO cells, and STAT5 in human megakaryocytes (Erickson-Miller et al., 2009
). In human platelets, eltrombopag induces STAT1, STAT3, and STAT5 activation, but spares AKT phosphorylation (Erhardt et al., 2009
). In bone marrow cells from patients with acute myeloid leukemia and myelodysplastic syndrome, eltrombopag activates STAT5 but not STAT3 (Will et al., 2009
). Combining our findings with the published reports support the observation that the activation of the downstream signaling pathway by eltrombopag differs from TPO, despite the fact both exert their functions through the TPO-R (c-Mpl)
To our knowledge, our manuscript is the first of its kind in finding that the thrombopoietic agent, ie, eltrombopag, has the potential to expand UCB CD34+ “in vivo”. The implication is significant in that despite much work has been done on “ex vivo” UCB CD34+ cell expansion, the current success rate of UCB stem cell transplantation remains poor when compared with PBSC transplantation or bone marrow stem cell transplantation. If eltrombopag can improve UCB transplant directly in vivo, which will need to be tested in future clinical trials, it potentially can either negate the need of “ex vivo” UCB stem cell expansion procedures, or may work synergistically with “ex vivo” expansion of UCB CD34+ cells to enhance UCB transplant success rate.