Effects of eltrombopag on human cord blood hematopoietic stem cell/primitive progenitor cell expansion.

Abstract

6632 Background: Umbilical cord blood transplantation has emerged as a promising therapy but is low in stem cells. Agents enhancing expansion of CB hematopoietic stem cell (HSC)/hematopoietic progenitor cell (HPC) are highly desirable. Eltrombopag (epag) is a non-peptide, c-MPL agonist that activates c-MPL of human and chimps. We investigate effects of epag on expansion of HSC/HPC of human CB. Methods: (1) In vitro: CB CD34+ cells were treated with rhTPO or epag in presence of rhSCF and rhFL for 7 days. After staining with anti-CD41, anti-CD34 and anti-CD38, cells were analyzed by Flow. (2) Intracellular signaling: serum-starved cells expanded from CB CD34+ were treated with rhTPO or epag. Phosphorylation of STAT3, STAT5 and AKT in c-MPL signal pathway was analyzed by Flow after staining with anti-phospho-STAT3, anti-phospho-STAT5, anti-phospho-AKT anti-CD41, anti-CD34, and anti-TPO-receptor. (3) In vivo: Sublethally irradiated NOD/SCID mice were transplanted with CB CD34+ and gavaged daily with epag or water (28 d) and sacrificed between 1-9 wks. Blood count and bone marrow (BM) extracts were analyzed by Flow after staining with anti-human-CD41a-PE, anti-mouse-CD61-FITC, anti-human-CD45-FITC, anti-mouse CD45-PE, anti-human-CD34-FITC and anti-human-CD41a-PE antibodies. Results: (1) Epag increased CD34+ by 26%, CD34+CD38- by 42%, and CD41+ by 400% in culture, compared with130%,160% and 900%, respectively by rhTPO. The % CD34+, CD34+CD38- and CD41+ were similar comparing rhTPO with epag. (2) Both rhTPO and epag induced phosphorylation of STAT5 in CD34+CD41-, CD34-CD41+ and CD34-CD41- cells. rhTPO-induced pSTAT5 was cell-type dependent, while epag-induced pSTAT5 was not. rhTPO-induced the phosphorylation of STAT3 and AKT in CD34-CD41+ cells, while pSTAT3 and pAKT were not detectable in epag treated CD34-CD41+ cells. (3) Epag led to an increase of circulating human platelets and WBCs without affecting murine platelets or WBCs in NOD/SCID. Epag augmented expansion of human CD45+, CD34+ and CD41+ cells in BM without effects on mouse BM cells. Conclusions: Eltrombopag expanded HSC/HPC of CB in vitro, promoted human platelet and WBC proliferation/differentiation in NOD/SCID mice, and human HSC/HPC in mouse BM.