[Difference in ex vivo expansion of megakaryocytes derived from umbilical cord blood and peripheral blood].
OBJECTIVE
Currently, thrombocytopenia is typically observed in patients undergoing hematopioetic stem cell transplantation (HSCT), high-dose chemotherapy or irradiation. Severe thrombocytopenia can cause intestinal and intracranial hemorrhage. To transfuse ex vivo-expanded megakaryocytes (MK) into patients can reinforce the ability of platelet formation and shorten the time of platelet recovery. Therefore it is one of the effective approaches to reduce the danger. The purpose of the present study was to explore the differences in MK expansion between CD(34)(+) stem cells derived from umbilical cord blood (CB) and peripheral blood (PB) and to establish the most optimal culture system.


METHODS
Mononuclear cells were isolated by density gradient centrifugation over Ficoll-Hypaque gradient solution. CD(34)(+) cells were isolated by positive selection using an immunomagnetic separation system and the selected CD(34)(+) cells were seeded in Iscove's modified Dulbecco's medium (IMDM) supplemented with fetal calf serum (FCS) and certain kinds of cytokines. After 15 - 17 days of culture, the cells were counted and the content of CD(41)(+) cells was determined by using flow cytometry, and the number of megakaryocyte colony-forming unit (CFU-MK) was simultaneously measured.


RESULTS
After the defined days of culture, the cytokine combination of thrombopoietin (TPO) + fetal liver tyrosine kinase ligand (FL) + IL-6 + IL-3 showed to be the most suitable for both PB and CB to obtain high numbers of MK, and to be better than any of the other three groups (P < 0.05). The CD(41)(+) cells from CB were expanded by193 +/- 25 fold on day 14, and those from PB were expanded by 131 +/- 18 fold on day 10. The number of CD(41)(+) cells from both CB and PB decreased.


CONCLUSIONS
For PB and CB, the cytokine combination of TPO + FL + IL-6 + IL-3 is most suitable for obtaining large number of MK and is the best combination for ex vivo MK expansion. MKs from CB seemed to have higher proliferation potential than that from PB, and the optimal culture duration of MK from PB is shorter than that of MK from CB.
Fang
Jian-pei
JP
Department of Pediatrics, The Second Affiliated Hospital of Sun Yat-sen University, Guangzhou 510120, China.
Xia
Ting
T
Chen
Guo-hua
GH
Wu
Yan-feng
YF
Xu
Hong-gui
HG
Wei
Jing
J
Huang
Shao-liang
SL
D018952:Antigens, CD34; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D002469:Cell Separation; D003470:Culture Media; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology*
D018952:Antigens, CD34; D003470:Culture Media