Isolation And Characterization Of Exosomes Secreted By Human Urine-derived Stem Cells

Objective: Isolation and characterization of exosomes secreted by human urine-derived stem cells(h USCs-Exo)Methods: A modified urine collection device was used to collect fresh urine from healthy male adults.Human urine-derived stem cells(h USCs)were isolated from urine by ‘One centrifuge method 'and then cultured and passaged.Using the CCK-8 method to draw the proliferation curve,identify of its surface markers by flow cytometry,then induce the h USCsto differentiate into osteoblasts and adipocytes,and it was identified by alizarin red,Von Kossa and oil red O staining,respectively.Ultra high speed centrifugation was used to isolate h USCs-Exo from h USCs conditioned medium.Transmission electron microscopy was used to analyze its morphology,NTA method was used to analyze its size and concentration,and protein Western blotting(WB)was used to detect the expression of its specific protein CD9,C D63 and HSP70.Results: The h USCs began to adherent grow after 3-5 days of culturing.They resemble "slabstone" in the primary passage,then resemble "rice" in the first and second passage,in the third passage,they gradually resemble“spindle shape”or "shuttle shape".The h USCs showed a typical "S" growth curve by the CCK-8 method.Flow cytometry showed that the positive expression of h USCs in mesenchymal stem cell specific surface markers CD44,CD73,CD90 and CD105,andnegative expression of CD19,CD34,CD45,CD11 b and HLA-DR,proved that the h USCs accordwith the characteristic of mesenchymal stem cells;and after osteogenic and adipogenic induction medium culturingin vitro,h USCs successfully differentiated into osteoblasts and adipocytes.After Ultra high speed centrifugation,h USCs-Exo was extractedfrom conditioned medium of h USCs,observed by transmission electron microscope showed round or oval membranous vesicles,resembling "saucer",mainly concentrated in the size of 64-141 nm,the protein expression of CD9,CD63 and HSP70.Conclusion: The h USCs were successfully isolated and cultured and the h USCs-Exowas successfully isolated and characterized.