The Study on Difference in Megakaryocyte Expansion between 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 bowel hemorrhage and intracranial hemorrhage. To transfuse ex vivo-expanded Mk cells into patients can reinforce the ability of platelet formation and shorten the time of platelet recovery. So it is one of the effective approaches to protect the danger. To explore the differences in megakaryocyte (MK) expansion between CD34⁺ stem cells derived from 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. CD34⁺ cells were isolated by positive selection using an immunomagnetic separation system. selected CD34⁺ cells were seeded in Iscove’s modified Kulbecco’s medium (IMDM) supplemented with fetal calf serum (FCS) and all kinds of cytokines. After 15–17 days of culture, the cells were measured and determined the content of CD41⁺ cells by flow cytometry, and simultaneously measured the number of megakaryocyte colony-forming unit (CFU-MK).

Results After the definite days of culture, the cytokine combination of TPO+FL+IL-6+IL-3 is most suitable both for PB and CB to obtain high numbers of MK, and better than any other three groups(P<0.05). The fold-increase of CD41⁺ cells from CB on day 14 was 193.23±25.24 fold, and that from PB on day 10 was 131.23±18.26 fold respectively. With the time gone, the number of CD41⁺ cells both decreased.

Conclusion For PB and CB, the cytokine combination of TPO+FL+IL-6+IL-3 is most suitable to obtain high numbers of MK and is the best combination for MK ex vivo expansion. It is suggested that MKs from CB 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. The study explore the differences in MK expansion between CD34⁺ stem cells derived from CB and PB and establish experimental foundation for further application to pre-clinic and clinic.