BACKGROUND: Human umbilical cord blood (CB) is an alternative source of hematopoietic stem cells (HSCs) for allogeneic transplantation. Cytokine-mediated ex vivo expansion has been proposed as a means of increasing the number of HSCs. We investigated the ability to generate megakaryocytic (MK) progenitors during ex vivo expansion of CB CD34(+) cells using thrombopoietin (TPO), flt3-ligand (FL), and granulocyte-colony stimulating factor (G-CSF).
METHODS: CB CD34(+) cells were cultured for four weeks
in a stroma-free liquid culture system using TPO, FL, and/or G-CSF. Expression of CD34, CD61(GPIII), CD41(GPIIb/IIIa), and CD42b (GPIb) were investigated by flow cytometry, along with simultaneous measurement of apoptosis by 7-aminoactinomycin D staining method. Morphologic differentiation was studied by cytospin preparation with Wright stain, PAS stain and the dual esterase stain.
RESULTS: Total viable cells increased continuously until the 4th week. The megakaryocytic cells with CD61, CD41,
and CD42b expression appeared in the culture with TPO from the 4th day to the 2nd week of culture. Apoptotic cells were generated from CD34(-)CD61(+), CD34(-)CD41(+), and CD34(-)CD42b(+) fractions during this period. Morphologically, the majority of the cells were mono-nuclear cells with increased cytoplasm, except a few binuclear cells even in the presence of TPO. Some immature cells with cytoplasmic bleb appeared in the cultures with TPO, which were positive for nonspecific esterase and were also positive for the PAS stain in block pattern.
CONCLUSION: TPO is able to induce megakaryocytic differentiation without any antagonistic effect of FL and G-CSF during the ex vivo expansion of cord blood CD34(+) cells. MK progenitors expanded ex vivo may
be an appropriate cell population as treatment prolonged thrombocytopenia. The efficacy of this procedure will be tested prospectively in a clinical trial.

Keywords: Cord blood, CD34(+) cell, ex vivo expansion, Megakaryocytic differentiation