Isolation And Culture Endothelial Progenitor Cells From Human Adipose Tissue And Analysis Its Biological Characteristics

Background:Endothelial progenitor cells(EPCs)are precursor cells of endothelial cells,which can migrate to ischemic tissue,differentiate into mature endothelial cells and play an important role in vascular endothelial regeneration.According to the recent researches on endothelial progenitor cells,it has wide application prospect in clinical diagnosis,prognosis judgement and treatment of ischemic diseases.Some studies have shown that EPCs can be obtained from bone marrow,peripheral blood and umbilical cord blood,and embryo,heart,skeletal muscle and blood vessels.However,EPCs from these sources present some limitations.Thus,there is a need to identify alternative sources of EPCs.Fortunately,recent studies show that adipose tissues contain a variety of cells,including EPCs.Adipose tissue is plentiful in the human body and can be easily harvested through a minimally invasive method.And autologous transplantation of adipose-derived cells is appropriate.Therefore,adipose tissues are ideal cell sources for EPCs,but currently there is no effective method of isolating and culturing adipose-derived EPCs at home or abroad.Objectives:In this study,we aimed to establish an effective,economical and feasible method for simultaneous isolation and culture of EPCs from human adipose tissue and to explore its biological characteristics.Methods:Cells from the stromal vascular fraction(SVF)were isolated from human adipose tissues by enzymatic digestion.Immunophenotype of SVF was examined by flow cytometry to analyze cellular components.And then EPCs and ASCs were then separated with their different respond to trypsin.Observe the morphology of the cultured cells,draw the cell growth curve and calculate population doublings(PD)and doubling times(DT)to test the proliferation ability of the cultured cells.EPCs were characterized based on the expression of endothelial cell markers(CD31,CD34,CD 133,VEGFR2,CD45)by flow cytometry.Additionally,biological functions of EPCs were examined by measuring FITC-UEA-1 absorption,Dil-ac-low-density lipoprotein(LDL)uptake.Finally,we used vascular network formation in Matrigel to explore the angiogenic ability of vascular endothelial cells.Results:We successfully isolated microvascular EPCs and ASCs from human adipose tissue.EPCs were confirmed by the constitutive expression of a range of vascular endothelial markers(CD31,CD34,CD 133,and VEGFR2).Importantly,cultures were negative for the hematopoietic marker CD45,and after expansion by in vitro culture,EPCs exhibited typical cobblestone morphology.And internalization of Dil-Ac-LDL and FITC-UEA-1,which were detected as red and green fluorescence,respectively.Furthermore,it formed tube-like structures in a Matrigel assay.Meanwhile,ASCs were identified based on a series of mesenchymal markers(CD29,CD73,CD90,and CD 105)and cells presented with long spindle-shaped fibrocyte-like adherent growth after expansion by in vitro culture.Conclusion:We successfully isolate and culture EPCs from adipose tissue based on their differential responses to trysinization.An effective,economical and feasible method for isolation and culture of EPCs from human adipose tissue was established,which provide abundant seeding cells for various ischemic diseases and plays crucial roles in regenerative medicine and have broad applications in the therapeutic angiogenesis.