Megakaryocytic Differentiation of Human Cord Blood CD34+Cells During ex vivo Expansion / 대한혈액학회지

ABSTRACT

BACKGROUND: Thrombopoietin (TPO) has been currently used for ex vivo expansion of hematopoietic progenitor cells. Previously, we have reported that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of cord blood (CB) CD34+cells. In the present study, we have investigated on the relationship between the TPO-induced apoptosis and megakaryocytic differentiation. METHODS: CD34+cells, purified from human CBs, were expanded in serum-free conditions stimulated with TPO. Multidimensional flow cytometry and TUNEL assay as well as electron microscopy were applied for analysis of apoptosis. Asociation of megakaryocytic differentiation and apoptosis was studied by FACS-sorting and immunocytochemistry. Clonogenic potential was studied by CFU-MK assay. RESULTS: The TPO-induced apoptotic cells appeared in CD61+fractions. Immunocytochemical analysis of the FACS-sorted fractions showed that the apoptosis-associated CD44low fraction expressed CD61. Clonogenic assay revealed 7.4+-0.50-fold increase of total CFU-MKs during the initial 9 days. Thereafter, the number of CFU-MKs decreased, which was 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 day 6, small colonies became predominant. CONCLUSION: These results suggested that the MK progenitors matured as they were expanded during ex vivo expansion with TPO, and then proceeded to apoptosis.