Establishment of Transgenic Cell Strains Expressing Recombinant Retroviral Vector of hLIF Gene and Their Roles in Proliferation of Umbilical Cord Blood CD34~+ HSPC in vitro

Objective To establish the transgenic cell strains expressing recombinant retroviral vector of human leukemia inhibitory factor (hLIF) gene which was supposed to be used as feeder layer cells for the proliferation of umbilical cord blood CD34+ hematopoietic stem/progenitor cell (HSPC) in vitro. Methods The recombinant retroviral vector of hLIF gene was transfected into human embryo kidney fibroblasts cells 293T ,and the objective gene was detected by RT-PCR and ELISA .The purity of umbilical cord blood CD34+ HSPC separated by magnetic-activated cell sorting (MACS) was detected by the flow cytometry. After culturing with feeder layer cells for 7 days, the rate of proliferation was detected by flow cytometry. Results The green fluorescence was observed by fluorescence microscope in the transgenic cell strains,and the objective gene was confirmed by RT-PCR and ELISA.The purity of umbilical cord blood CD34+ HSPC separated by magnetic-activated cell sorting (MACS) could reach to (95.6±2.58)%. After culturing with feeder layer cells for 7 days , the CD34+ cells were 8.74 times of that in the group containing hLIF than that in group without hLIF, the rate of adhension molecules’ expression on the surface of CD34+ cells was also higher in the group containing hLIF than that without hLIF. Conclusion Recombinant retroviral vector of hLIF gene as feeder layer cells could provide a better microenvironment for the growth and proliferation of umbilical cord blood CD34+ HSPC in vitro which contributes to maintain its multipotency and undifferentiation state.