In Vitro Proliferation, Differentiation And Transduction With BFGF Gene Of Human Embryonic Mesenchymal Stem Cell

Object: By exploring the proliferation conditions of human embryonic bone marrow mesenchymal stem cell (MSC), inducing MSC to have a dopaminergic neurons phenotype as well as introducing bFGF gene into MSC with retrovirus vector, we are trying to develop the potential protocols for treatment of Parkinson's disease with MSC.Method: The effect of different cell density, different concentrations of fetal bovine serum (FBS), EGF and PDGF-BB on hMSCs proliferation was assessed by MTT assay. Different passages of MSC were then induced by treatment of RA, bFGF, FGF-8 and BDNF, immunofluorescence and Western blot were further used to detect the cells after induction. Recombinant virus plasmid pLXSN-bFGF was constructed and transfected to PA317 package cells, the cells were screened by G418 treatment, the integration of bFGF gene in G418-resistant package cells was identified by PCR, the retrovirus was then prepared and used to transfect MSC.Results: The best expansion conditions for MSC in vitro was 1 l04/ml cell density, 10%FBS, 10ng/ml EGF and 10ng/ml PDGF-BB. After the induction of RA, MSC differentiated into neurons without dopaminergic neuron phenotype. With the treatment of bFGF, FGF-8 and BDNF, most MSC differentiated into neurons and about 20% cells also adopted a dopaminergic neuron phenotype. There is no significant difference in the efficiency of differentiation between different passages of MSC. After the transfection of pLXSN-bFGF plasmid, G418-resistant package cell was selected and the PCR result was positive. After the transfection of pLXSN-bFGF retrovirus and G418 selection, little MSC survived and little colony was found.Conclusion: The best expansion conditions for MSC was well established and MSC was successfully induced to adopt a dopaminergic neuron phenotype in vitro. This will provide a powerful therapeutic tool for Parkinson's disease. pLXSN-bFGF retrovirus vector was successfully constructed and the retrovirus packaging cell line was well established. This will facilitate our further research in using bFGF as a gene therapy strategy.