Apoptosis and megakaryocytic differentiation during ex vivo expansion of human cord blood CD34(+) cells using thrombopoietin

Abstract

Thrombopoietin (TPO), the primary regulator of megakaryocytopoiesis, plays important roles in early haematopoiesis. Previously, we have demonstrated that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of cord blood (CB) CD34(+) cells. In this study, we have demonstrated that the TPO-induced apoptotic cells belong to the megakaryocytic (MK) lineage and that initially expanding MK progenitors declined along with the appearance of TPO-induced appptosis. Human CB CD34(+) cells were expanded in serum-free conditions with TPO. Multi-dimensional flow cytometry using simultaneous measurement of apoptosis and immunophenotyping showed that the TPO-induced apoptotic cells appeared in CD61(+) fractions. Immunocytochemical analysis of the fluorescent activated cell-sorted fractions showed that the apoptosis-associated CD44(low) fraction expressed CD61. Clonogenic assay revealed 7.4 +/- 0.50-fold increase of total megakaryocyte colony-forming units (CFU-MKs) during the initial 9 d, Thereafter, the number of CFU-MKs decreased in parallel with the increase of apoptosis. When the MK colonies were subdivided according to size, the proportion of large colonies progressively decreased, while that of medium and small colonies increased. In particular from d 6 small colonies became predominant. These results suggested that the MK progenitors matured as they expanded during ex viva expansion with TPO and then proceeded to apoptosis.