Biological characteristics of JAK2 transduced CD34+ cells from cord blood during ex vivo expansion |
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Authors: | ZHAO Sheng-ming GU Xi-chun CHANG Nai-bai XU Xiao-dong PEI Lei |
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Institution: | Department of Hematology,Beijing Hospital,Beijing 100730,China.E-mail: mgz2001@hotmail.com |
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Abstract: | AIM: To explore the feasibility and biological characterization of long-term regulated expansion of JAK2 transduced human CD34+ cord blood cells in vitro.METHODS: A retrovirus (RV) vector which contains JAK2 catalytic domain and two binding sites for a chemical inducer,dimerization (AP20187),was cloned (designated MGI-F2JAK2).CD34+cells were enriched from cord blood with a MiniMACS system.The purified CD34+ cells were transfected with supernatant from the retrovirus packaging cell line that expressed JAK2.Following transduction,cells were expanded into four groups: AP20187 alone,FL alone,TPO,alone,AP20187+FL+TPO,respectively.The expanded cells were monitored by GFP expression,immunophenotyping,progenitor colony assay,karyotype analysis as well as tumorigenesis in nude mice.RESULTS: The purity of selected CD34+ cells was over 91% and gene transfer rate was 49.32%±6.21%.Only the group of AP20187 +FL+ TPO was obtained a significant sustained outgrowth of the transduced CD34+ cord blood cells.The percentage of GFP+ cells consistently produced a rise to the 90% peak level by the end of 8th week of culture.Flow cytometry analysis showed that the phenotype of the expanded cells was CD33+,CD61+ and Gly-A+ partial positive;CD38+ and HLA-DR+ strong positive,while CD2,CD7 and CD19 were almost negative.Colony assays performed in methycelluos,which can give rise to BFU-E,CFU-GM and CFU-Mix,the CFU-GM was predominantly in all colonies.The tumor was not observed in nude mice and the karyotype analysis was normal from expanded cells.CONCLUSION: The results demonstrate that AP20187-mediated activation of JAK2 signaling is capable of stimulating expansion JAK2 transduced CB CD34+ cells in combination with FL and TPO.This system may have applications for studies in signaling transduction,hematopoiesis,and for gene and cell therapy. |
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Keywords: | Tyrosine kinase JAK2 Gene therapy CD34+ cells Expansion |
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