Research On Overexpression Of Proxl Induces The Differentiation Of Human Adipose-derived Stem Cells Into Lymphatic Endothelial-like Cells In Vitro

Objective Homeobox transcription factor Proxl is a decisive gene for the maintenance of lymphatic endothelial cells(LECs)migration,proliferation,and lymphatic vessel differentiation and maturation.Recent studies have demonstrated that using human LECs for cell transplantation therapy may improve secondary lymphedema.The aim of this study was to investigate the inducing effect of Proxl on the differentiation of human adipose-derived stem cells(hADSCs)towards LECsMethods Lentiviral vectors encoding Proxl gene were constructed,and h ADSCs were obtained from adipose tissue harvested from healthy adult donors.Proxl was overexpressed in hADSCs by using the transfection of lentiviral vectors.After Prox1 was overexpressed in hADSCs,the proliferation and viability of transfected cell were measured by CCK8 assay and trypan blue staining.LECs and endothelial cells(ECs)specific phenotypes were evaluated by flow cytometry analysis,RT-PCR,western blot analysis and immunofluorescent staining.Tube formation assay of transfected cells was used for functional evaluationResults Overexpression of Proxl in hADSCs had no influence on the cell proliferation and viability measured by CCK8 assay and trypan blue staining.The morphologic transition of hADSCs from a fibroblast-like appearance to a typical EC-specific cobblestone-like appearance could be clearly observed at day 14.Flow cytometry analysis found the expression of LECs specific markers Podoplanin and VEGFR3,together with the ECs marker CD31 were significantly increased,while the expression of CD29,CD44 and CD90 were decreased obviously at day 14 and 28 Besides,RT-PCR indicated that Proxl-overexpressed cells expressed significantly increased mRNA levels of Podoplanin,LYVE1,VEGFR3 at days 7,14,21 and 28.Western blot and immunofluorescence staining results further confirmed that overexpression of Proxl in hADSCs significantly increased the protein levels of Podoplanin,LYVE1 and VEGFR3,as well as that of the EC-specific markers such as VWF and CD144,at day 14.Tube formation assay found that Proxl-overexpressed cells in matrigel acquired vascular-like structures.Conclusion These findings suggested that overexpression of Proxl in hADSCs was able to induce the differentiation of hADSCs into lymphatic endothelial-like cells,suggesting an attractive therapeutic potential of ADSC for treating limb lymphedema and lymphatic system related diseases.