Characterization of liquid cultures of human CD34⁺ cells stimulated by TPO.

<p>CD34⁺ cells (1×10⁴/ml) purified by immunomagnetic positive selection were cultured in IMDM containing 5% human serum and TPO (25 ng/ml added at days one and seven of the cell culture). At the indicated culture times, (A) total cell count was determined with a hemocytometer and (B) cell size was analyzed by flow cytometry. (C) CD41 and CD34 expression were evaluated by labeling cells with specific mAbs or corresponding matched isotypes and establishing the percentage of positive cells by flow cytometry analysis. (D) Platelet (Plt) count was evaluated by flow cytometry and cellular apoptosis was determined by detecting nuclear morphological changes of cells stained with acridine orange and ethidium bromide by fluorescence microscopy. (E) Culture morphology was assessed by phase contrast microscopy [original magnification 450×, except day 12 inset (1200×)]. The values expressed in panels A–D represent the mean ± SEM of five independent experiments. Panel E shows a representative experiment of five similar replicates.