title: [Study on the induction and differentiation of megakaryocyte progenitor cell derived from umbilical cord blood].
abstract: OBJECTIVE
  To build a protocol of separation and induction of megakaryocytes derived from cord blood mononuclear cells.
  
  
  METHODS
  Red blood cells were precipitated by hydroxyethyl starch (HES). Mononuclear cells were obtained by density gradient centrifugation with Ficoll. The inducing efficiencies of megakaryocytes by using of different cytokine cocktails and culture media were analyzed.
  
  
  RESULTS
  The best choice for erythrocyte sedimentation and high efficiency of nucleated cells retrieving were obtained by using of 1.5% HES. The isolated cord blood mononuclear cells were cultured with domestic serum-free medium supplemented with 116t (IL-11, IL-6, TPO), st36(SCF, TPO, IL-3, IL-6), pt36 (PDGF,TPO,IL-3,IL-6) or pst36 for 7 days. St36 group (50 ng/ml SCF, 50 ng/ml TPO, 20 ng/ml IL-3 and 50 ng/ml IL-6) yielded the most CD41/CD61 positive [(6.79±1.97)×10⁴]. The cell viability [(82.85 ± 0.64)%] of st36 group by using of imported serum-free medium was better than [(60.90±6.93)%] that in domestic medium on day 7 after induction, and CD41/CD61 positive cells count [(18.60±1.97)×10⁴] were more than domestic serum-free medium group. Therefore, we chose imported serum-free medium containing st36 to induce cord blood mononuclear cells. After a prolonged culture, the total cell numbers increased accompanied with an elevated percentage of CD41/CD61 positive cells, which reached (54.27 ± 6.31)% on day 14. Wright-Giemsa staining showed that different phase cells, such as megakaryoblast, promegakaryocyte and granular megakaryocyte, occurred after 10 days'culture. Clone forming unit-megakarocytes (CFU-MK) assay showed that the colonies count increased with the prolonged incubation. CFU-MK colonies were [1 236.0±32.9] on day 14, which was higher than that in medium without induction (P<0.01). Platelets from megakaryocytes showed agglutination function after 10 days'culture.
  
  
  CONCLUSIONS
  1.5% HES was the best solution to precipitate erythrocytes. The combination of an imported serum-free medium with IL-3, IL-6, SCF and TPO showed better induction efficiency than domestic medium or other cytokine cocktails. Meanwhile, induced megakaryocytes produced functional platelets.
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Chen
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Xie
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Daqing
authors.initials: D
authors.lastname: Liu
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Lyu
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Yue
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Shi
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Jiafei
authors.initials: J
authors.lastname: Xi
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Xiuyuan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Nan
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Jingxue
authors.initials: J
authors.lastname: Wang
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Junnian
authors.initials: J
authors.lastname: Zhou
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Li
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: He
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Hailei
authors.initials: H
authors.lastname: Yao
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Siting
authors.initials: S
authors.lastname: Li
authors.affiliation: The lab of stem cell and regenerative medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Xuetao
authors.initials: X
authors.lastname: Pei
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002455:Cell Division; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D005312:Fetal Blood / Q000166:cytology*; D006801:Humans; D055016:Megakaryocyte Progenitor Cells / Q000166:cytology*
chemical_list: D016895:Culture Media, Serum-Free