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

Abstract

Summary. 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 1 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 apoptosis. Human CB CD34 1 cells were expanded in serum-free conditions with TPO. Multidimensional flow cytometry using simultaneous measurement of apoptosis and immunophenotyping showed that the TPO-induced apoptotic cells appeared in CD611 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 vivo expansion with TPO and then proceeded to apoptosis.

Gene Expression Profile of Megakaryocytes from Human Cord Blood CD34+ Cells Ex Vivo Expanded by Thrombopoietin

“…However, the expression of survivin, an inhibitor of apoptosis, was low in MKs. These results are consistent with our previous report that TPO-induced apoptosis was closely associated with megakaryocytic differentiation [5]. TGF-β1, which was highly expressed in MKs (twofold greater than in non-MKs), plays a pivotal role in the control of differentiation, proliferation, and the state of activation of many different cell types, including immune cells [13].…”

“…The purified CB CD34 + cells were cultured in a serum-free liquid culture system stimulated with TPO for 10 days. Under these conditions, the cells differentiated into CD41 + (GPIIb) cells with characteristic MK morphology [5]. The total RNA prepared from these MK and non-MK fractions was processed with SAGE analysis.…”

“…However, in vitro culture methods, along with the introduction of thrombopoietin (TPO) as an MK growth and developmental factor, provide a sufficient number of MKs, which can be used for biological as well as for structural analyses [3]. Previously, we investigated the process of megakaryocytopoiesis during ex vivo expansion of human cord blood (CB) CD34 + cells using TPO and found that megakaryocytopoiesis was closely associated with apoptosis [4,5]. However, the genes involved in the regulation of megakaryocytopoiesis have not yet been characterized.…”

Role of Apoptotic Processes in Platelet Biogenesis

“…However, the expression of survivin, an inhibitor of apoptosis, was low in megakaryocytic cells. These results are consistent with the previous report that TPO-induced megakaryocytic differentiation and apoptosis are closely associated [95]. TGF-β 1 receptor initiates intracellular signaling through the activation of specific downstream intracellular effectors termed SMAD proteins that become phosphorylated and associate with other proteins of SMAD family.…”

SLE Thrombocytopenia: From Peripheral Platelet Destruction to Central Hemopoietic Defect

“…Anti-TPO positive sera significantly inhibited mature CFU-MK compared to anti-TPO negative SLE sera and were associated with lower circulating TPO concentrations. TPO-induced CFU-MK are small and mature, with low proliferative capacity, suggesting that TPO works mostly late in megakaryocyte progenitor development [71,105]. These findings suggest an antibodyassociated restriction of the TPO-mediated effect on megakaryopoiesis.…”