Construction Of Recombinant Lentivirus Encoding MiR-302-367Cluster And Study Of Its Expression In Human Umilical Cord Mesenchymal Stem Cells

Objective： To study the the feasibility of reprogramming humanumilical cord mesenchymal stem cells into induced pluripotent stem cells bylentivirus carrying miR-302-367cluster, and to lay the foundation for thenext research.Methods:1)Using tissue adherence technique to separate and culturehUCMSCs,Observing cells growth under the inverted microscope,analysingcell cycle and surface markers by flow cytometry;2)The miR-302-367cluster gene sequence was inserted into lentiviral expression vectorpLenO-DCE-Puro encoding green fluorescent protein(GFP)，and then therecombinant vector pLenO-DCE-Puro-miR-302-367was verified by doubleenzyme digestion and sequencing identification.The viral supematant washarvested from293T cells which were co-transfected with correctrecombinant plasmid and packaging plasmids, concentrated and tested thetiter of virus;3)Firstly,Using lentivirus expressing GFP to infect hUCMSCs，GFP expression efficiency in293T cell was observed under the Fluorescence microscope and flow cytometry to determine the best MOIvalue.Then, hUCMSCs were transfected with lentiviral supernatants fromthe lentivirus encoding for miR-302-367cluster, cultured on mouse feedercells and observed the changes of cellular morphology.Results:1)Successfully cultured hUCMSCs and displayed fusiformshape as fibroblasts in vitro.Cell cycle analysed that the majority of cellswere in G0/G1phases(70.15%),only the minority in S phase(5.85%).Flowcytometry revealed that the cells highly expressed CD29,CD105andCD90,while lowly expressed CD34and CD45.2)The lentiviral expressionvector pLenO-DCE-Puro-miR-302-367had been successfullyconstructed,confirmed to be correct clone by enzyme digestion and DNAsequencing，producted to lentivirus.One titer of recombinant lentivirus was2×10~8TU/ml, the other was1×10~9TU/ml.3)The optimal MOI was20.Themember of miR-302-367cluster highly expressed in transfection group,butboth in negative and non-transfection group were not detected at all. Wealso observed obvious changes of cellular morphology in transfectiongroup,which gradually changed from fusiform shape into EpithelialCell-like or ellipse,and even clone groups with ES cell-like morphology，but cells in the other groups was still spindle shape.Conclusion:1)The mesenchymal stem cells were successfully isolatedfrom human umbilical cord.2)Successfully constructed recombinantlentiviral expression vector pLenO-DCE-Puro-miR-302-367and packed lentivirus with high titer.3)The cellular morphology changed, but needed tobe further identified the induction condition.