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First published online September 7, 2006
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2006-0309v1
24/12/2877    most recent
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Submitted on May 23, 2006
Accepted on August 10, 2006

Translational and Clinical Research

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

Takuya Matsunaga 1, Ikuta Tanaka 1, Masayoshi Kobune 1, Yutaka Kawano 1, Maki Tanaka 1, Kageaki Kuribayashi 1, Satoshi Iyama 1, Tsutomu Sato 1, Yasushi Sato 1, Rishu Takimoto 1, Tetsuji Takayama 1, Junji Kato 1, Takafumi Ninomiya 2, Hirofumi Hamada 3, Yoshiro Niitsu 1*

1 Fourth Department of Internal Medicine, Sapporo Medical University, School of Medicine, Sapporo, Japan
2 First Department of Anatomy, Sapporo Medical University, Sapporo, Japan
3 Department of Molecular Medicine, Sapporo Medical University, Sapporo, Japan

* To whom correspondence should be addressed. E-mail: niitsu{at}sapmed.ac.jp.


  Abstract

In the present investigation, we generated platelets (PLT) from cord blood (CB) CD34+ cells employing 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 PLT. The calculated yield of PLT from 1.0 unit CB (5x106 CD34+ cells) was 1.26 x1011 - 1.68 x1011 PLT. These numbers of PLT are equivalent to 2.5 - 3.4 units of random donor-derived PLT or 2/5 - 6/10 of a single apheresis PLT. The CB-derived PLT 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 PLT for future clinical usage.

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




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