Biological characteristics of JAK2 transduced CD34~+ cells from cord blood during ex vivo expansion / 中国病理生理杂志

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.