Effect And Preliminary Mechanism Of Conditioned Medium Derived From Mesenchymal Stem Cells Cultured In Gelatin Sponge On Hair Follicles Cycle Transition

Objective1.To investigate the effect of conditioned medium(CM)derived from mesenchymal stem cells(MSCs)cultured in gelatin sponge scaffolds on hair follicles(HFs)cycle transition.2.To investigate the mechanism of CM derived from MSCs cultured in gelatin sponge scaffolds on HFs cycle transition.Methods1.The umbilical cord of healthy pregnant women in the obstetrics department of our hospital was obtained,primary MSCs were obtained by tissue adherent culture,digestion and subculture,and third-generation MSCs were amplified,which were identified by flow cytometry,immunofluorescence staining and trilineage differentiation.MSCs were grown in gelatin sponges for3 D culture.Add 50 μg/m L of ascorbic acid,perform continuous culture,change it every two days,and detect its viability by live/dead staining.CM on days 3,7,and 10 were collected and concentrated by lyophilization for subsequent experiments.2.Eighteen 7-week-old male C57BL/6 mice were divided into 3 groups,PBS group,CM group,minoxidil group,PBS group and CM group were injected subcutaneously 300 μL,minoxidil was applied daily for two weeks,and the color change and hair growth of the back skin were recorded by taking pictures.On day 14,mice were sacrificed and dorsal skin samples were collected,some samples frozen at-80°C for WB analysis,and the remainder fixed with 4% PFA for histological analysis.The number of dorsal skin HFs was observed by HE staining,the ALP activity of the dermal papilla was observed by ALP staining,and the positive number of K15 and PCNA was observed by immunofluorescence staining.3.Human scalp dermal papilla cells were cultured and subcultured to the third generation.The effects of CM on cell migration,proliferation and ALP gene expression were observed by cell scratch experiment,CCK-8 proliferation experiment,and q RT-PCR experiment.4.The expression of β-catenin and LEF-1,the key proteins of wnt/β-catenin pathway,was detected by WB to study the mechanism of action of CM.Results1.Primary MSCs were successfully cultured by tissue adherent culture,and high expression of CD73,CD105,CD44,CD29,CD90,low expression of CD45,HLA-DR,CD34,CD31,CD14,CD19 were detected by flow cytometry,and immunofluorescence was detected by cells with high expression of CD73,CD44,CD105,CD90,Oct4,c-MYC,low expression of CD29,nanog,CD31,HLA-DR,CD45.Trilineage differentiation suggested that cells can be induced to differentiate into adipocyte,osteocytes,and chondrocytes in vitro.Cells grew well in gelatin sponge,and live/dead staining indicated that cells maintain high viability throughout the culture process.CM for days 3,7,and 10 of culture was collected and lyophilized and concentrated for subsequent experiments.2.After subcutaneous injection of CM,general observation showed that the dorsal skin of mice in the CM treatment group achieved a faster transition from telogen phase to anagen phase,grayscale analysis results showed that the CM treatment group showed darker skin color(P<0.05)than the PBS group at the 11 th and 14 th days,HE staining results showed that the CM group had more HFs(P<0.001),ALP staining showed a larger staining range of dermal papilla area in the CM treatment group(P<0.001),immunofluorescence staining showed that the CM treatment group showed more K15,PCNA positive number(P<0.001).In summary,CM facilitated the transition from telogen to anagen.3.CM promoted the migration,proliferation and expression of the ALP gene in the dermal papilla cells.4.WB results showed that the expressions of β-catenin and LEF-1 were up-regulated in the CM treatment group.ConclusionsCM derived from MSCs cultured in gelatin sponge scaffolds promotes the transition from telogen to anagen phase of HFs,which is probably achieved by activating the Wnt/β-catenin pathway,and derivatives of MSCs may be a promising option for the treatment of alopecia.