[Ex vivo expansion of megakaryocyte progenitor cells for CD133(+) cells derived from human umbilical cord blood].
To study the expansion potentiality of megakaryocyte progenitor cells (MPCs) derived from human umbilical cord blood CD133(+) (UCB-CD133(+)) cells and determine the optimal harvest time. UCB-CD133(+) cells were purified from mononuclear cells (MNCs) by magnetic activated cell sorting (MACS) and seeded in serum-free liquid culture medium supplemented with thrombopoietin (TPO), interleukin-3 (IL-3), and stem cell factor (SCF) to expand MPCs. At day 0, 6, 10 and 14 of culture, the total cell number was counted and the dynamic changes of CD133, CD34, and CD41 antigen expression during ex vivo expansion were analyzed by flow cytometry (FCM). At different expansion times, the CD133(+) cells were collected and cultured in collagen semisolid medium to carry out CFU-MK colony culture. The incidence of CFU-MK was calculated and the morphology of MPCs and CFU-MK were detected by immunohistochemistry and Wright-Giemsa staining. The results showed that UCB-CD133(+) cells optimally expanded at day 7 with expansion multiple of 8.2 +/- 2.2 in serum-free liquid culture systems and the total cell number was expanded by 116-fold at day 14. At 10 days, each UCB-CD133(+) cell can form 2.5 +/- 1.0, 2.6 +/- 0.5 and 20.3 +/- 5.9 cells of CD133(+)CD41(+), CD34(+)CD41(+) and CD41(+) respectively, from which the number of CD133(+)CD41(+) and CD34(+)CD41(+) cells reach the highest. UCB-CD133(+) cells both before and after expansion could form CFU-MK, the total number of CFU-MK reached the peak from cells of 10 days expansion of UCB-CD133(+) cells and the expansion multiple of CFU-MK was 59.5 +/- 11.8. Immunohistochemical results indicated that the expanded megakaryocytic cells were immature and no sign of platelet formation. It is concluded that the human UCB-CD133(+) cells have a high ability of MPC expansion, 10 days of culture can be result in optimal expansion effect.
Key Laboratory of Cell Engineering of Guizhou Province, The First Affiliated Hospital of Zunyi Medical College, Zunyi 563003, Guizhou Province, China.
Li
L
Wang
Dai-Xiong
DX
Chen
Ning
N
Fang
Zu-Lin
ZL
Liu
Tao
T
Zhang
Wei-Hong
WH
Wan
Ying
Y
Qi
Jin-Wei
JW
Liu
D000071916:AC133 Antigen; D015703:Antigens, CD / Q000097:blood*; D002455:Cell Division; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D005312:Fetal Blood / Q000166:cytology*; D006023:Glycoproteins / Q000097:blood*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology*; D010455:Peptides / Q000097:blood*; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology
D000071916:AC133 Antigen; D015703:Antigens, CD; D016895:Culture Media, Serum-Free; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides; D019089:Stem Cell Factor; D013926:Thrombopoietin