Ex Vivo Large Scale Generation of Human Platelets from Cord Blood CD34+ Cells.

To obtain a large quantity of platelets (PLTs) from cord blood stem cells (CBSC) in vitro, we employed three-phase culture system. We first expanded CBSC on a monolayer of human telomerase catalytic subunit gene-transduced human stromal cells (hTERT stroma) in serum-free medium supplemented with stem cell factor (SCF), Flt-3/Flk-2 ligand (FL) and thrombopoietin (TPO) for 14 days (1st phase), and then cultured them to differentiate into megakaryocytes for another 14 days with refreshing medium which contain interleukin-11 (IL-11) in addition to original cytokine cocktail (2nd phase). Subsequently, we transferred the cells to a liquid culture medium containing SCF, FL, TPO and IL-11, and cultured them for 5 days (3rd phase) to recover PLTs in the culture medium. The quantity of PLTs recovered from one CB unit (5 x 10⁶ CD34+ cells) was calculated to be 10.5 units (2 x 10¹¹ PLTs). These CB-derived PLTs exhibited quite similar feature as those from peripheral blood in morphology as revealed by electron micrograph and in functions as revealed by aggregation assay and by FACS detecting expression of P-selectin and activated glycoprotein IIb-IIIa antigens upon fibrinogen/ADP stimulation. Thus our three-phase culture system was considered to be useful for large scale generation of PLTs from CB for clinical usage.