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TRANSLATIONAL AND CLINICAL RESEARCH

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

^aFourth Department of Internal Medicine,
^bFirst Department of Anatomy, and
^cDepartment of Molecular Medicine, Sapporo Medical University, School of Medicine, Sapporo, Japan

Key Words. Platelet • Megakaryocyte • Cord blood • CD34⁺ cell • Stromal cell

Correspondence: Yoshiro Niitsu, M.D., Ph.D., Fourth Department of Internal Medicine, Sapporo Medical University, S1W17, Sapporo, Japan. Telephone: 81-11-611-2111; Fax: 81-11-612-7987; e-mail: niitsu{at}sapmed.ac.jp

Received May 23, 2006; accepted for publication August 10, 2006.
First published online in STEM CELLS EXPRESS   September 7, 2006.



In the present investigation, we generated platelets (PLTs) from cord blood (CB) CD34⁺ cells using a three-phase culture system. We first cultured 500 CB CD34⁺ cells on telomerase 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. We then transferred the cells to hTERT stroma and cultured for another 14 days with fresh medium containing interleukin-11 (IL-11) in addition to the original cytokine cocktail. Subsequently, we cultured the cells in a liquid culture medium containing SCF, FL, TPO, and IL-11 for another 5 days to recover PLT fractions from the supernatant, which were then gel-filtered to purify the PLTs. The calculated yield of PLTs from 1.0 unit of CB (5 x 10⁶ CD34⁺ cells) was 1.26 x 10¹¹–1.68 x 10¹¹ PLTs. These numbers of PLTs are equivalent to 2.5–3.4 units of random donor-derived PLTs or 2/5–6/10 of single-apheresis PLTs. The CB-derived PLTs exhibited features quite similar to those from peripheral blood in morphology, as revealed by electron micrographs, and in function, as revealed by fibrinogen/ADP aggregation, with the appearance of P-selectin and activated glycoprotein IIb-IIIa antigens. Thus, this culture system may be applicable for large-scale generation of PLTs for future clinical use.

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