The Effects Of HADSCs-Exos On Osteogenic Differentiation Of Adipose-derived Stem Cells

Objective: To isolate and identify the exosomes(hADSC-Exos)secreted by human adipose-derived stem cells(hADSCs)from different osteogenic differentiation stages.To explore the appropriate extraction time of hADSC-Exos to directly induce the osteogenesis of native hADSCs and prove that they can enter into the hADSCs.To identify the appropriate concentation of hADSC-Exos to enhance the proliferation,migration and osteogenesis of native hADSCs.Methods:Primary hADSCs were isolated and cultured by enzyme digestion.The different time span of hADSC-Exos(Exo0,Exo14,Exo28)were isolated by ultracentrifugation and they were identified by TEM.Western Blot,dynamic light scattering.Alizarin red staining was used to evalute the mineralization of the native hADSCs after seeding with different time-span of hADSC-Exos;PKH67 labeled Exo14 to detect the internalization ability by hADSCs;Different Western Blot was used to detect the effect of different concentrations of Exo14 on the expression of osteoblast-associated protein in hADSCs.Results: The hADSCs obtained from the experiment met the criteria for identification of mesenchymal stem cells.TEM and DLS showed that the obtained exosomes had typical morphological characteristics.Western blot showed that CD63 and CD9 were positive expression in hADSC-Exos.Alizarin red stain showed that the content of mineralized nodules in Exo14 group was significantly higher than that in Exo28 group after 21 days of culture.PKH67-labeled exosomes can be taken up into cells by hADSCs and aggregated around the nucleus.Western Blot showed that after co-culturing for 3 days,bone-related protein ALP and RUNX2 was positive in Exos groups.The results showed that the 15?g/mlExo14 group promoted the expression of RUNX2 and the osteogenic induction group(positive control group).There was no significant difference in the expression of protein between the 10?g/mlExo14 group(0.583±0.045)and 20?g/mlExo14(0.499±0.010).Conclusion:Exosomes secreted by hADSCs for 1~14 days can be efficiently taken upby the native hADSCs and had a role to induce osteogenic differentiation of hADSCs.The low concentration Exo14 can directly enhance the proliferation and migration of native hADSCs.The optimal concentration of Exo14 to promote osteogenic differentiation was 15 ?g/ml.