Cocultivation of umbilical cord blood CD34+ cells with retro-transduced hMSCs leads to effective amplification of long-term culture-initiating cells.
OBJECTIVE
To establish a novel coculture system for ex vivo expansion of umbilical cord blood(UCB) hematopoietic progenitors using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human marrow-derived mesenchymal stem cells (tfhMSCs) as feeder.


METHODS
UCB CD34+ cells were isolated and cultured using four culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient (SCID) mouse-repopulating cell (SRC) assay was performed to confirm ability of the cultured cells to reconstitute long-term hematopoiesis.


RESULTS
There were no significant differences in the number of total nucleated cells among different culture systems in serum-containing medium during 21-d culture. However, on d 14, the outputs of CD34+ cells, CFU-C and CFU-GEMM in tfhMSCs coculture system were significantly enhanced. LTC-IC assay demonstrated that the tfhMSCs coculture system had the most powerful activity. The severe-combined immunodeficient (SCID) mouse repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. Furthermore, PCR analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of NOD/SCID mice.


CONCLUSIONS
The TPO/FL-transduced hMSCs, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro and the tfhMSCs coculture system may be a suitable system for ex vivo manipulation of primitive progenitor cells under contact culture conditions.
College of Life Sciences, Zhejiang University, 232 Wen San Road, Hangzhou 310012, Zhejiang Province, China.
Chun-Gang
CG
Xie
Jin-Fu
JF
Wang
Ying
Y
Xiang
Li-Yan
LY
Qiu
Bing-Bing
BB
Jia
Li-Juan
LJ
Wang
Guo-Zhong
GZ
Wang
Guo-Ping
GP
Huang
D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism*; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology*; D006801:Humans; D016130:Immunophenotyping; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D013926:Thrombopoietin / Q000235:genetics / Q000378:metabolism
D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D016207:Cytokines; D008565:Membrane Proteins; C084881:flt3 ligand protein; D013926:Thrombopoietin