Effects And Mechanisms Study Of Bone Marrow-Derived Mesenchymal Stem Cells On Hair Follicle Cycle Transition

Background and objective:Hair loss(HL)affects patients'appearance,has been shown to have profound negative impact on psychosocial status,which adversely affects their quality of life(QOL).With the increasing expectation of HL treatment,more and more researchers are committed to screening drugs or treatments that effectively promote hair growth and regeneration and and investigating the underlying mechanisms.Bone marrow-derived mesenchymal stem cells(BMSCs)are a type of multipotent adult stem cell,can be easily isolated,cultured and expand,and demonstrate low immunogenicity,as well as the ability to differentiate into a variety of cell lineages in vitro.Based on the properties mentioned above,BMSCs are ideal seed cells,with great potential applications in tissue engineering and regenerative medicine.However,the molecular mechanism underlying BMSCs and conditioned medium(MSCs-CM)-induced hair follicle regeneration remains to be fully elucidated.Tandem mass tags(TMT)-based quantitative proteomic analysis is a highly specific method for explore protein dynamics and their complex regulatory mechanisms.Compared to the performance of two-dimensional electrophoresis,LC-MS/MS yields a comprehensive analysis of low-abundance proteins with high accuracy.Parallel reaction monitoring(PRM)is a mass spectrometry-based targeted verification proteomics technology,which can verify the quantitative proteomics results.Therefore,we turn attention towards exploring the effects of BMSCs and MSCs-CM on hair follicle regrowth in vibrissal follicle organ co-culture model and intradermal injection model.We further search for the regulatory proteins related to hair follicle regeneration,which play a guiding role in the treatment of hair-related diseases.Material and Methods:1.BMSCs were isolated from the femurs and tibia of 7-week-old C57BL/6N female mice using the whole bone marrow adherent method.After culturing,the surface antigens of BMSCs were detected by flow cytometry.To assess their multi-differentiative potential,BMSCs were induced to differentiate toward osteogenic and adipogenic lineages.A CCK-8 kit was used to gauge BMSC proliferation.2.Vibrissal follicles were isolated from 7-week-old C57BL/6N female mice and arranged into four groups:a control group,a CM-control group,an MSCs-CM co-culture group,and a BMSCs co-culture group.Control vibrissal follicle organ was maintained in William's E mouse vibrissal follicle organ medium.vibrissal follicles in CM-control group were maintained in William's E mouse vibrissal follicle organ medium and mouse BMSC complete medium.MSC-CM co-culture group vibrissal follicles were maintained in William's E mouse vibrissal follicle organ medium and cell-free MSC-CM.In BMSC co-culture group,BMSC were cultivated on plates and vibrissal follicle was plated on top of transwell chambers with William's E mouse vibrissal follicle organ medium and mouse BMSC complete medium.The length of each follicle was measured at baseline and 7 days after culture initiation under the dissection microscope and the relative increase length was calculated.Moreover,the levels of VEGF,PDGF-AA,PDGF-BB,IGF-1,b FGF,and EGF were quantified in the supernatants of co-cultures using magnetic Luminex assay.3.Forty-five 7-week-old C57BL/6N female mice were subsequently randomized into three treatment groups(n=15/group):a control group,an MSCs-CM group,and a BMSCs group.Control mice were administered media containing no cells,while mice in the MSCs-CM group were administered cell-free MSCs-CM,and mice in the BMSCs group were administered 1x10~6 BMSCs in fresh CM.A 250?l total injection volume was used for all mice,with 16 separate intradermal injections being made on the dorsal skin of treated mice.One injection was made every other day for two weeks in total.The dorsal skin of control,MSCs-CM treated and BMSCs treated mice was photographed on days 0,7,10,and 15 after the initial injection and the percentage of hair regrowth area was counted.The impact of these injections on hair cycle transition and expression of Krt15 and Ki67,Sox9 and Ki67 was then evaluated via hematoxylin and eosin(H&E)staining and immunofluorescent(IF)staining.The expression of Wnt/?-catenin signaling pathway related proteins,such as Fzd4,Lrp5,?-Catenin,Tcf1 and Lef1 were detected by western blot.4.We conducted a TMT-based quantitative proteomic analysis of control mice and mice treated with BMSCs or MSCs-CM to identify differentially expressed proteins(DEPs)associated with hair follicle regeneration and hair cycle transition.PRM was utilized as a means of verifying our proteomic analysis results.Results:1.Isolation and culture of mouse BMSCs:The cells that we isolated and cultured were adherent cells and exhibited uniform fibroblast-like spindle-shaped morphology on the third passage.BMSCs exhibited a high expression of CD44,CD90 and CD105 surface antigens and had multiple potentials to differentiate into osteoblasts and adipocytes.Based upon CCK8 proliferation assay,we elected to utilize third-passaged BMSC collected on the fifth day of culture for the next intradermal injection experiments.2.Isolation and co-culture of mouse vibrissal follicle organ:After 7 days co-culture,there was hair growth in all the experimental groups with significantly longer vibrissal follicles being observed in the BMSCs co-culture and MSCs-CM co-culture groups relative to the control and CM-control groups(P<0.01).The level of VEGF,PDGF-AA and IGF-1 in the BMSCs co-culture and MSCs-CM co-culture groups were significantly higher than in the control group and CM-control groups,BMSCs co-culture group relative to the MSCs-CM co-culture group(P<0.05).However,there were no statistically significant differences observed with regard to b FGF,PDGF-BB and EGF levels in the above-mentioned groups.3.The effect of BMSCs and MSCs-CM inducing hair follicle cycle transition in mice:After intradermal injection of BMSCs and MSCs-CM,Gross observation revealed that that the mice in MSCs-CM group and BMSCs group entered anagen earlier than the control group,and the hair growth rate was faster and BMSC treatment significantly enhanced hair growth relative to control treatment on days 7,10,and 15 post-treatment(p<0.05),BMSC treatment significantly enhanced elative to MSCs-CM treatment on days 7 and 15 post-treatment(p<0.05).H&E staining confirmed the consistency with the gross observation at histopathological level.Quantitative analyses confirmed that hair follicle length increased over time after injection,with significantly longer hair follicles being observed in the BMSCs and MSCs-CM treatment groups relative to the control group(p<0.05),BMSCs treatment group relative to the MSCs-CM treatment group on days 7 and 15 post-treatment(p<0.05).IF staining revealed that there were significantly more Krt15+proliferating cells in the bulge region and Ki67+proliferating cells in the bulb area and infundibulum of hair follicles in the BMSCs and MSCs-CM groups on days 7,10,and 15 of treatment relative to the control group.We also detected a significant increase in Sox9 expression in the bulge and outer root sheath(ORS)region and Ki67 expression in the bulb area and infundibulum of follicles from BMSCs-and MSCs-CM-treated mice.Western blot showed that Fzd4,Lrp5,?-Catenin,Tcf1 and Lef1expression were markedly upregulated in BMSCs and MSCs-CM groups as compared with that in the control group.4.TMT-based quantitative proteomic analysis of full-thickness dorsal skin after BMSCs and MSCs-CM intradermal injections:In total,5,046 quantifiable proteins were identified.Those proteins with a fold change?1.30 or?1/1.3 and P<0.05 when comparing the BMSC or MSC-CM groups to the control group were identified as DEPs.We identified 1,060 DEPs when comparing the BMSCs and control group samples(638upregulated,422 downregulated),770 were identified when comparing the MSC-CM and control group samples(511 upregulated,259 downregulated),while 100 were identified when comparing the BMSCs and MSC-CM group samples(45 upregulated,55downregulated).Based on functional classifications and enrichment analyses,these DEPs are involved in genetic material replication,transcription and translation,signal transduction,cell cycle control and metabolic regulation.We selected nine upregulated DEPs(Krt25,Cpm,Ctps1,Flg,Ncapd2,Tyrp1,Dct,Tgm3,Stmn1)and five downregulated DEPs(Mb,Hspb7,Cox7a1,Scara5,Fbp2)for subsequent PRM validation.Our quantitative PRM results confirmed that these candidate DEPs exhibited trends comparable to those observed upon TMT analysis.Those proteins were related to the hair shaft scaffolding,melanin biosynthesis,immune cell recruitment and regulation of cell proliferation.Conclusion:1.The whole bone marrow adherence method can isolate and cultivate BMSCs with high purity,self-renewal and multi-directional differentiation abilities,and specific surface antigens expression.2.BMSCs can promote the growth of vibrissal follicle organ through paracrine growth factors such as VEGF,PDGF-AA and IGF-1.3.Intradermal injection of BMSCs and MSCs-CM promotes hair follicle telogen-to-anagen transition by activating hair follicle stem cells proliferation and Wnt/?-catenin pathway initiation.4.BMSCs and MSCs-CM-induced DEPs related to hair follicle regeneration showed similarity in functional classification and enrichment analysis.Krt25?Tyrp1?Dct?Cpm?Ctps1?Flg?Stmn1 and Tgm3 were optimized upregulated and Mb,Hspb7,Cox7a1,Scara5 and Fbp2 were optimized downregulated.Stmn1,Ncapd2,Krt25 and Ctps1 are the crucial proteins of hair regeneration.