Abstract 4267: Eltrombopag, a non-peptide thrombopoietin receptor agonist, enhances the ex vivo expansion of human hematopoietic stem cells
Authors: Jeong Jee-Yeong , Vanasse K. Gary 
Published: February 2011

Abstract
Hematopoietic stem cells (HSCs) are characterized by their capacity for self-renewal and pluripotency, allowing them to replenish all mature, differentiated cells of the hematopoietic lineage. HSCs are not immortal but have a limited replicative potential and finite lifespan, properties which may be further negatively influenced by increased age or malignant disorders of the bone marrow. To overcome this problem, various combinations of cytokines, including recombinant human thrombopoietin (rhTPO), have been developed to support in vitro culture as well as ex vivo expansion of HSCs. Although rhTPO appears to enhance the mobilization of hematopoietic progenitors, its use in humans has been limited due to the generation of neutralizing antibodies. Eltrombopag is an orally-active, non-peptide small molecule TPO receptor agonist that has been shown to promote megakaryocyte proliferation and differentiation in a manner similar to that seen with rhTPO. We hypothesized that eltrombopag may enhance the ex vivo expansion of human HSCs to a degree equivalent to that of rhTPO. To test this hypothesis, human bone marrow derived CD34+ cells isolated from 5 healthy individuals were incubated in serum-free expansion medium containing SCF (50 ng/ml), IL-3 (10 ng/ml), IL-6 (10 ng/ml), LDL (40 μg/ml) and various concentrations of eltrombopag (0 - 100 μM) or rhTPO (0 - 100 ng/ml) and CD34+ cell numbers determined using FACS analysis over the course of a 14-day ex vivo culture. Both eltrombopag and rhTPO exhibited dose-dependent increases in CD34+ cell numbers, with 10 μM eltrombopag and 100 ng/ml rhTPO providing the most robust CD34+ cell expansion. Under these conditions, both 10 µM eltrombopag and 100 ng/ml rhTPO resulted in a five-fold greater CD34+ cell expansion compared with control (p &lt; 0.05), an effect seen most prominently at day 11 of liquid culture. To confirm that expanded CD34+ cells maintained their pluripotency, hematopoietic colony-forming assays were performed in methylcellulose cultures containing SCF (50 ng/ml), IL-3 (10 ng/ml), G-CSF (10 ng/ml) and erythropoietin (1 U/ml). CD34+ cells expanded with eltrombopag or rhTPO increased the total number of granulocyte, monocyte, and erythroid colony forming units (CFUs) by 81% and 95%, respectively, compared to control (p &lt; 0.01). The difference in total CFU formation observed between eltrombopag- and rhTPO-treated CD34+ cells was not statistically significant (p = 0.59). Taken together, these results serve as proof-of-principle showing eltrombopag to be an effective alternative to rhTPO for promoting the ex vivo expansion of human HSCs. Furthermore, these pre-clinical studies support the design of future clinical trials examining the use of eltrombopag in combination with G-CSF for the enhanced mobilization of human CD34+ hematopoietic progenitors in patients undergoing peripheral blood stem cell transplantation.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4267.