Distinct patterns of apoptosis in association with modulation of CD44 induced by thrombopoietin and granulocyte-colony stimulating factor during ex vivo expansion of human cord blood CD34(+) cells

The insufficient number of haemopoietic stem cells (HSCs) In cord blood (CB ) is the major potential limitation to widespread use of CB for marrow repl acement. Cytokine-mediated ex vivo expansion has been proposed as a means o f increasing the number of CB HSCs for transplantation. However, the biolog y of CB HSCs during cytokine-mediated ex vivo expansion; such as apoptosis or expression of adhesion molecules, has not yet been elucidated. We have i nvestigated the patterns of apoptosis and CD44 expression on human CB CD34( +) cells during ex, vivo expansion. CD34(+) cells isolated from human CB we re cultured in a stroma-free liquid culture system with thrombopoietin (TPO ), flt3-ligand (FL), stem cell factor (SCF), and/or granulocyte-colony stim ulating factor (G-CSF). During the culture, for up to 5 weeks, apoptosis wa s measured by staining with 7-amino-actinomycin D (7-AAD) along with concur rent immunophenotyping of CD34 and CD44 with three-colour flow cytometry. L n the cultures with TPO, an apoptotic fraction with down-regulated CD44 app eared from the fourth day up to the second week. G-CSF also induced apoptos is but in a different manner; the apoptotic fraction without down-regulatio n of CD44 appeared unremittingly for up to 5 weeks. FL did not induce apopt osis or down-regulation of CD44. These findings show that apoptosis is inde ed involved in the regulation of CB CD34(+) cells in ex vivo expansion and the patterns of apoptosis are dependent on the type of cytokines used. The distinct patterns of apoptosis suggest different mechanisms of TPO and G-CS F in inducing apoptosis, which still remains to be elucidated.