[Culture and characterization of and lentiviral vectors mediated glial cell derived neurotrophic factor expression in mesenchymal stem cells from human umbilical cord blood].
OBJECTIVE
To isolate and culture mesenchymal stem cells from umbilical cord blood (UCB-MSCs), study its biological characterization in vitro, transfect UCB-MSCs using lentiviral vectors encoding glial cell derived neurotrophic factor (GDNF) gene, evaluate the biological function change of UCB-MSCs, and detect GDNF expression level in vitro.


METHODS
We isolated monocyte by Ficoll density gradient, separated two kinds of adherent cells through different trypsin digestion time, and detected the cells surface markers by fluorescence activated cell sorting when it was proliferated for P7 passages. At the same time, we sub-cloned GDNF gene into lentiviral vectors and packaged lentiviral supernatant through three plasmids co-transfection method, then transfected the UCB-MSCs using lentiviral vectors encoding GDNF at different multiplicity of infection, and evaluated the change of biological function by observing the ability of proliferation and differentiation, morphology, and the cells surface markers. We detected the GDNF mRNA and protein expression level by using real-time polymerase chain reaction (real-time PCR) and enzyme-link immunosorbent assay (ELISA).


RESULTS
The UCB-MSCs were successfully isolated and cultured in vitro, and induced it to differentiate into fat cells. FACS results showed that the UCB-MSCs expressed CD90, CD73, and CD105 positively, and CD14, CD34, CD45, CD19, HLA-DR, Stro-1, and CD106 negatively. Real-time PCR and ELISA showed that the expressions of GDNF protein and mRNA were correlated with the copy number of transfected cells: high copy number of transfected cells were associated with high GDNF expression. The biological characterization of UCB-MSCs did not obviously change after sub-cloning with GDNF.


CONCLUSIONS
UCB-MSCs was successfully isolated and cultured in vitro. By transfecting UCB-MSCs with GDNF gene-containing lentiviral vectors, the secretion of GDNF protein and mRNA expression level can be controlled by the copy number of transfected cells, and thus make it constantly express GDNF at high level.
Huang
Ai-hua
AH
Cell Therapy Center, Xuanwu Hospital, Capital Medical University, Beijing 100053, China.
Wang
Shu-yan
SY
Liang
Qing-cheng
QC
Wu
Yun
Y
Guan
Yun-qian
YQ
Zhang
Yu
Y
Chen
Zan
Z
D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D005312:Fetal Blood / Q000166:cytology*; D005822:Genetic Vectors; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D014162:Transfection
D051100:Glial Cell Line-Derived Neurotrophic Factor