Ex vivo large-scale generation of human platelets from cord blood CD34+ cells.
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 10(6) CD34(+) cells) was 1.26 x 10(11) - 1.68 x 10(11) 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.
Matsunaga
Takuya
T
Fourth Department of Internal Medicine, Sapporo Medical University, School of Medicine, S1W17, Sapporo, Japan.
Tanaka
Ikuta
I
Kobune
Masayoshi
M
Kawano
Yutaka
Y
Tanaka
Maki
M
Kuribayashi
Kageaki
K
Iyama
Satoshi
S
Sato
Tsutomu
T
Sato
Yasushi
Y
Takimoto
Rishu
R
Takayama
Tetsuji
T
Kato
Junji
J
Ninomiya
Takafumi
T
Hamada
Hirofumi
H
Niitsu
Yoshiro
Y
D018952:Antigens, CD34 / Q000276:immunology*; D001792:Blood Platelets / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage / Q000187:drug effects; D016207:Cytokines / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D005312:Fetal Blood / Q000166:cytology*; D006680:HLA Antigens / Q000378:metabolism; D006801:Humans; D008533:Megakaryocytes / Q000187:drug effects / Q000648:ultrastructure; D040201:Platelet Membrane Glycoprotein IIb / Q000276:immunology; D011003:Ploidies; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects; D019098:Telomerase / Q000378:metabolism
D018952:Antigens, CD34; D016207:Cytokines; D006680:HLA Antigens; D040201:Platelet Membrane Glycoprotein IIb; D019098:Telomerase