The Effect Of HLIF Transgenic Feeder Layer Cells On Proliferation And Differentiation Of Umbilical Cord Blood CD34~+ HSPC

Objective:To establish retroviral vector of human leukemia inhibitory factor (hLIF) gene and Ad-hLIF transgenic feeder cells, respectively, which were used for the expansion of umbilical cord blood CD34+ HSPC in vitro and study the SCID mice model of HSPC transplantation.Methods:Recombinant retroviral vector of hLIF gene constructed in our laboratory was transfected into human embryo kidney fibroblasts cell line 293T by Lipofectamine reagent, and the transgenic cells stains expressed hLIF was obtained in the medium which contained Zeocin and confirmed by RT-PCR and ELISA on mRNA and protein level. Ad-hLIF constructed in our laboratory was transfected into human embryo lung fibroblasts cell line WI-38 and confirmed by RT-PCR and ELISA on mRNA and protein level.The transgenic feeder cells were used for the expansion of umbilical cord blood CD34+ HSPC separated by magnetic-activated cell sorting (MACS), and the cell proliferation was detected by flow cytometry. The expanded cells stained with CFDA SE were injected to the sublethally irradiated SCID mice, Humanize gene Alu was detected by RT-PCR and fluorescence microscope.Results:The retroviral vector of hLIF gene transgenic cells were observed green fluorescence and the expression of hLIF gene was confirmed by RT-PCR and ELISA. The green fluorescence was also observed in the 95% transgenic cells infected with 50MOI Ad-hLIF for 48h, hLIF gene expression was confirmed by RT-PCR and ELISA. The purity of umbilical cord blood CD34+ HSPC separated by MACS could reach to 95.6±2.58%, after culturing with retroviral vector of hLIF gene transgenic feeder layer cells for 7 days, the total cells number was expanded to12.6±1.32 fold, the expressions of adhesion molecules CXCR4 and CD54 were 55.0%,59.3%; Ad-hLIF transgenic feeder cells and various cytokines system expanded total cells to 18.45±1.24 fold, CXCR4 and CD54 expressions were 57.2%,59.8%, co-culturing for 28d continuely, total cells number was expanded to 356.95±0.87 fold, and maximal expansion of CD34+ cells was observed during 0~14 days of culture, then down-expansion gradually. The expanded cells stained with CFDA SE were transplanted in SCID mice for four weeks, fluorescently-labeled humanize cells were still observed and the humanized gene Alu was confirmed by RT-PCR.Conclusion:Retroviral vector of human leukemia inhibitory factor (hLIF) gene and Ad-hLIF transgenic feeder cells were established, respectively, which effectively proliferated and differentiated umbilical cord blood CD34+ HSPC in vitro, the expanded cells could engraft the bone marrow of sublethally irradiated SCID mice and reconstitute hematopoiesis.