Stem Cell Factor Inhibits Premature Senescence of Megakaryocytes during ex vivo Expansion of Human Cord Blood CD34+ Cells Using Thrombopoietin / 대한소아혈액종양학회지

ABSTRACT

PURPOSE: Thrombopoietin (TPO) has been currently used for ex vivo expansion of hematopoietic stem cells. Previously, we have reported that TPO induces apoptosis during ex vivo expansion of cord blood CD34 cells. In this study, we have investigated the stem cell factor (SCF) to determine whether it affects the TPO-induced apoptosis. METHPDS CD34+ cells, purified from four separate human cord bloods by magnetic bead selection, were expanded in Iscoves modified Dulbeccos medium with several cytokines. Apoptosis has been confirmed by several ways; 7-amino-actinomycin D (7-AAD) or annexin V staining, and morphologic analysis with light and electron microscopic examinations. And cell maturation was also analysed with DNA staining. RESULTS: Apoptosis was reached peak (67.8+/-5.24% of total cells) at day 9 in the cultures with TPO alone. Morphological examination of Giemsa-stained cytospin preparations revealed many immature cells with cytoplasmic blebbing and eccentric nuclei. However, multilobed nuclei were rarely observed while bilobed nuclei were frequently observed. These results suggest that premature senescence occurs in the megakaryocytic cells before full polyploidization. SCF was affective for the TPO-induced apoptosis; the peak apoptotic fraction was significantly decreased to 37.2+/-3.31% at day 9. In the cultures with SCF, the fractions of CD41+ cells were also decreased in parallel with decrease in apoptosis. Notwithstanding reduction of differentiation into megakaryocytic lineage, polyploidization(> 6N) in CD41 cell faction was significantly increased in the cultures with SCF at day 9 and day 11, respectively (P<0.05). CONCLUSION: These results suggest that SCF inhibits premature senescence of the differentiating cells of megakaryocytic lineage during ex vivo expansion using TPO.