Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis

Abstract

Umbilical cord blood (UCB) transplantation has emerged as a promising therapy, but it is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag's effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, and CD41+ cells in bone marrow compartment without effects on mouse bone marrow cells in the NOD/SCID mice xenotransplant model. Consequently, eltrombopag increased peripheral human platelets and white blood cells. We further examined effects in the STAT and AKT signaling pathways in serum-free cultures. Eltrombopag expanded human CD34+ CD38-, CD34+, and CD41+ cells. Both eltrombopag and recombinant human TPO (rhTPO) induced phosphorylation of STAT5 of CD34+ CD41-, CD34- CD41+, and CD34- CD41- cells. rhTPO preferentially induced pSTAT3, pAKT, and more pSTAT5 in CD34- C41+ cells, while eltrombopag had no effects on pSTAT3. In conclusion, eltrombopag enhanced expansion of HSCs/HPCs of human UCB in vivo and in vitro, and promoted multi-lineage hematopoiesis through the expansion of bone marrow HSCs/HPCs of human UCB in vivo. Eltrombopag differed somewhat from rhTPO in the signal transduction pathways by favoring earlier HSC/HPC populations.

Figure 1

Eltrombopag treatment increased circulating human platelets and human WBCs in peripheral blood of NOD/SCID mice after transplantation. The mice were transplanted with human UCB CD34+ cells. Mice were gavaged with 50mg/kg eltrombopag or vehicle daily for 28 days. (A) Human platelets. (B) Mouse platelets. (C) Human WBCs and (D) Mouse WBCs. Data are expressed as mean ± standard error of the mean (n=8–12). *p<0.05, **p<0.01 between eltrombopag and vehicle two-tail Mann-Whitney U Test.

Figure 2

Eltrombopag promoted rapid establishment of human engraftment in bone marrow of NOD/SCID mice after transplantation. The mice were transplanted with human UCB CD34+ cells. Mice were gavaged with 50mg/kg eltrombopag or vehicle daily for 28 days. (A) Human CD45+ bone marrow MNCs. (B) Mouse CD45+ bone marrow MNCs. (C) Human CD34+ bone marrow MNCs. (D) Human CD41+ bone marrow MNCs. Data are expressed as mean ± standard error of the mean (n=7–12). *p<0.05, **p<0.01 between eltrombopag and vehicle two-tail Mann-Whitney U Test.

Figure 3

Expansion of human UCB CD34+ cells in the culture. Human UCB CD34+ cells (> 90% purity) were cultured in the presence of 25ng/ml SCF (S), 50ng/ml FL (F) and 10 or 50ng/ml rhTPO (T10 or T50) or 0.5, 3, 6μg/ml (equivalent to 1.13, 6.78 and 13.6μM, respectively) eltrombopag (E0.5, E3 or E6) for 7 days. The cells were counted and analyzed by flow cytometry. (A) The population distribution after the 7-day culture by flow cytometry. (B) The numbers of total viable cell, CD34+, CD34+CD38− and CD41+ cells are presented in bar graph, while their percentages are shown as squares. 1×10⁵ CD34+ human UCB cells were seeded in the beginning of the culture. Data are showed as mean ± standard error of the mean, n=3~5. Compared with the control culture (SF), *p<0.05, **p<0.01 by two-sided paired t-test.

Figure 4

Phosphorylation of STAT3, STAT5 after treatment with rhTPO or eltrombopag. Human UCB CD34+ cells were expanded in serum-free media with 50 ng/ml FL, 25 ng/ml SCF, and 10 ng/ml rhTPO for 7 days. Then the cells were rendered quiescent in serum-free media without cytokines for 7~8 hours. The cells were exposed to 100 ng/ml rhTPO or 3μg/ml eltrombopag for 10 to 60 min at 37°C. These cells were then fixed, permeabilized, stained for surface markers and intracellular antigens, and analyzed by flow cytometry. (A) Cell population gating. (B) Phosphorylation of STAT3, STAT5 and AKT in CD34+CD41−, CD34−CD41+ and CD34−CD41− cells induced by rhTPO or eltrombopag. Data are shown as mean ± standard error of the mean, n=4. Compared with the untreated control, *p<0.05, **p<0.01 by two-sided paired t-test. (C) Almost all cells expressed TPO receptors (TPO-R) after 7-day culture. The close curve shows the cells stained with anti-TPO-R (anti-c-MPL) antibody, while the open curve represents the cells stained with isotype antibody for non-specific and background binding.