Cell numbers were determined by the trypan blue (Gibco) dye exclusion method and they were reported by considering the number HKI272 of expanded cells cultivated in the differentiation stage. (i) In order to assess the degree of megakaryocytic differentiation, CD41 (Mk lineage cells) expression was analyzed by flow cytometry (FACSCalibur, BD) using an anti-CD41 antibody (Biolegend). CD34 and CD33 expression was also determined using appropriate antibodies and isotype controls. (ii) Mk ploidy was determined by double-staining technique with flow cytometry (FACSCalibur, BD) and using CellQuest Pro software (BD) by choosing CD41+ events as a respected gate

[13]. Briefly, the cell cultures incubated 15 min with anti-CD41 antigen (Biolegend) and then fixed by 70% cold ethanol (4 °C). Cells were re-suspended in a staining solution

containing propidium iodide (50 μg/mL; Sigma), sodium citrate (4 mM; Sigma), RNase A (0.1 mg/mL; Sigma), Triton X-100 (0.1%; Sigma) in pH 7.8 1 h before performing the flow cytometry. (i) For scanning electron microscopy imaging, cell population were first fixed in a solution of glutaraldehyde (Sigma) 1.5% (v/v) in PBS (Gibco), then post-fixed in a solution of osmium tetroxide (0.05%; Sigma) cAMP inhibitor in PBS (Gibco); both for 30 min at room temperature. Cells were then dehydrated by gradually increase of ethanol (Sigma) concentration (50%, 75% and 100% in distilled water). Finally, cell populations were coated with gold and observed using scan electron microscope (Hitachi S2400). (ii) In order to observe internal structure of Mks by transmission electron microscopy (TEM), culture-derived cells were fixed in a solution containing 2% paraformaldehyde (Sigma) and 2.5% glutaraldehyde

(Sigma) in 0.1 M sodium cacodylate Florfenicol buffer (Sigma) (pH 7.4) for 1 h at room temperature (22 °C). After rinsing with cacodylate buffer (Sigma), cells were post-fixed with a 1% osmium tetroxide (Sigma) in 0.1 M cacodylate buffer (Sigma) for 1 h at room temperature. Cells were then fixed with uranyl acetate (Sigma) (0.5%) in citrate–acetate acid buffer (pH: 5–6) and dehydrated by graduate increasing ethanol (Sigma) concentration (50%, 75% and 100% in distilled water). Finally, cell populations were embedded in Epon (Sigma), cut and Mks ultrastructure observed with TEM apparatus (Hitachi 8100). Results are presented as a mean ± standard error of mean (SEM). Results were statistically analyzed using two-sided non-paired Student’s t-test by Microsoft Excel and considered significant when p < 0.05. CD34+-enriched cells from UCB were expanded using a previously optimized protocol [12] and differentiated toward Mk lineage using a simple protocol containing only two cytokines (TPO and IL-3). Expanded cells were also differentiated, as a control, using the same protocol but without any supplemented cytokines.