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

Thrombopoietin (TPO), the primary regulator of megakaryocytopoiesis, plays important roles in early haematopoiesis. Previously, we have demonstrated t hat TPO induces a characteristic pattern of apoptosis during ex vivo expans ion of cord blood (CB) CD34(+) cells. In this study, we have demonstrated t hat the TPO-induced apoptotic cells belong to the megakaryocytic (MK) linea ge and that initially expanding MK progenitors declined along with the appe arance of TPO-induced appptosis. Human CB CD34(+) cells were expanded in se rum-free conditions with TPO. Multi-dimensional flow cytometry using simult aneous measurement of apoptosis and immunophenotyping showed that the TPO-i nduced apoptotic cells appeared in CD61(+) fractions. Immunocytochemical an alysis of the fluorescent activated cell-sorted fractions showed that the a poptosis-associated CD44(low) fraction expressed CD61. Clonogenic assay rev ealed 7.4 +/- 0.50-fold increase of total megakaryocyte colony-forming unit s (CFU-MKs) during the initial 9 d, Thereafter, the number of CFU-MKs decre ased in parallel with the increase of apoptosis. When the MK colonies were subdivided according to size, the proportion of large colonies progressivel y decreased, while that of medium and small colonies increased. In particul ar 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.