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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 replacement. Cytokine‐mediated ex vivo expansion has been proposed as a means of increasing the number of CB HSCs for transplantation. However, the biology of CB HSCs during cytokine‐mediated ex vivo expansion, such as apoptosis or expression of adhesion molecules, has not yet been elucidated. We have investigated the patterns of apoptosis and CD44 expression on human CB CD34+ cells during ex vivo expansion. CD34+ cells isolated from human CB were cultured in a stroma‐free liquid culture system with thrombopoietin (TPO), flt3‐ligand (FL), stem cell factor (SCF), and/or granulocyte‐colony stimulating factor (G‐CSF). During the culture, for up to 5 weeks, apoptosis was measured by staining with 7‐amino‐actinomycin D (7‐AAD) along with concurrent immunophenotyping of CD34 and CD44 with three‐colour flow cytometry. In the cultures with TPO, an apoptotic fraction with down‐regulated CD44 appeared from the fourth day up to the second week. G‐CSF also induced apoptosis but in a different manner; the apoptotic fraction without down‐regulation of CD44 appeared unremittingly for up to 5 weeks. FL did not induce apoptosis or down‐regulation of CD44. These findings show that apoptosis is indeed 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‐CSF in inducing apoptosis, which still remains to be elucidated.