Exosomes From Human Adipose-derived Mesenchymal Stem Cells Promote Epidermal Stem Cell Proliferation Through Upregulating ?-catenin,c-Myc And Cyclins Expression

Epidermal stem cells(EpSCs)are not only implicated in the treatment of large skin defects but also ideal seed cells for the fabrication of tissue engineering skin.However,the source of EpSCs is limited,and EpSCs proliferate slowly in vitro.At present,there are few studies on the regulation of EpSC proliferation by endogenous factors.Identification of endogenous factors that can promote the proliferation of EpSCs will be helpful for expanding EpSCs in vitro.Adipose derived mesenchymal stem cells are rich in sources.It has been reported that the exosomes secreted by adipose mesenchymal stem cells(ADSC-Exos)could promote skin wound healing by promoting proliferation and migration of keratinocytes and fibroblasts.However,whether ADSC-Exos could promote EpSC proliferation is unclear.ObjectiveThis study aimed to investigate the effect ofA DSC-Exos on the proliferation of human EpSCs and explore the underlying mechanisms.Methods1.Human skin tissues were digested with neutral protease and trypsin to obtain epidermal cells.EpSCs were isolated by rapid adherent method.Epidermal stem cell markers(?1 integrin,CK19,CD71 and a6 integrin)were detected by immunofluorescence staining and flow cytometry.Human adipose mesenchymal stem cells were isolated from subcutaneous fat tissues by type I collagenase digestion.The marrkers of adipose mesenchymal stem cells(CD73,CD90)were detected by flow cytometry.The ability of adipose mesenchymal stem cells to undergo osteogenic and adipogenic differentiation was determined.2.The ADSC-Exos were isolated from the supernatant of cultured adipose mesenchymal stem cells by ExoQuick-TC reagent The protein concentration of ADSC-Exos was determined by the BCA method.The morphology of ADSC-Exos was observed under transmission electron microscope.The size of ADSC-Exos was analyzed by nano-particle size analyzer.The protein markers of exosomes(CD63,Alix)were detected by Western blotting.3.The effect of ADSC-Exos on the proliferation of EpSCs was examined by MTT and BrdU incorporation assays,as well as immunofluorescence staining of Ki67.The effect ofA DSC-Exos on cell cycle of EpSCs was detected by flow cytometry.4.H&E staining,immunohistochemical staining of cell proliferation marker PCNA and epidermal stem cell markers ?1 integrin,?6 integrin were performed to observe the structure of cultured human skin tissues and the proliferation of EpSCs..5.The expression of ?-catenin,c-Myc and cyclin was detected by RT-PCR and Western blotting.Results1.94.36%of the EpSCs isolated from human skin tissues expressed high level of epidermal stem cell marker ?6 integrin and low level of CD71.These cells also highly expressed ?1 integrin and CK19,the other two markers of EpSCs.These results indicate that the isolated and cultured EpSCs have high purity.The adipose mesenchymal stem cells isolated from human subcutaneous fat tissues expressed high level of adipose mesenchymal stem cell markers CD73 and CD90,low level of vascular endothelial marker CD34 and leukocyte marker CD45.Adipogenic and osteogenic differentiation of adipose mesenchymal stem cells showed lipid droplets and calcium deposition,respectively.These results indicate that the isolated and cultured adipose mesenchymal stem cells have high purity and multi-directional differentiation potential.2.The ADSC-Exos showed a round bilayer structure under transmission electron microscope.The size of ADSC-Exos was 50-150 nm.ADSC-Exos expressed exosome markers Alix and CD63,but not GAPDH.These results indicate that the purity of ADSC-Exos is high.3.ADSC-Exos at 5-20 ?g/ml promoted EpSC proliferation in a concentration-dependent manner.ADSC-Exos significantly increase the ratio of Ki67 positive cells in cultured EpSCs.Treatment of EpSCs with ADSC-Exos significantly reduced the number of cells in G1 phase and increased the number of cells in S phase.These results indicate that ADSC-Exos promote the proliferation of EpSCs through accelerating G1 to S phase transition.4.Human skin tissues were treated with ADSC-Exos by intradermal injection of ADSC-Exos and cultured for 2 and 5 days respectively.ADSC-Exos treatment significantly increased the thickness of epidermis,markedly increased the number of PCNA,?6 integrin and ?1 integrin positive cells in the epidermal basal layer.These results demonstrated that ADSC-Exos could promote cutaneous EpSC proliferation.5.Mechanistic studies showed that ?-catenin inhibitor XAV-939 and c-Myc inhibitor 10058-F4 partially inhibited the pro-proliferation effect of ADSC-Exos on EpSCs.RT-PCR results showed that the ADSC-Exos promoted the expression of ?-catenin,c-Myc,Cyclins D1,E1 and A2 at both mRNA and protein level.XAV-939 significantly inhibited ADSC-Exos-induced expression of ?-catenin,c-Myc,Cyclins E1,A2 and D1,10058-F4 significantly inhibited the upregulation of c-Myc and Cyclins E1,A2,D1 by ADSC-Exos.These results indicate that the pro-promoting effect of ADSC-Exos on EpSCs proliferation is mediated partly by stimulating ?-catenin,c-Myc and Cyclins E1,A2,D1 expression.ConclusionADSC-Exos promote EpSCs proliferation partly through upregulating the expression of ?-catenin,c-Myc and cyclins.