| Hair follicle(HF)morphogenesis and regeneration depend on intensive but wellorchestrated interactions between epithelial and mesenchymal components.Embryonic hair follicle induction and formation are regulated by mesenchymal-epithelial interactions between specialized dermal cells and epidermal stem cells that switch to a hair fate.During postnatal hair growth,communication between mesenchymal dermal papilla and surrounding epithelial matrix cells coordinates hair shaft production.Adult hair follicle regeneration in the hair cycle is thought to be controlled by activating signals originating from the mesenchymal compartment and acting on hair follicle stem cells.Many signaling pathways are implicated in hair follicle formation and growth,the precise nature,timing,and intersection of these inductive and regulatory signals remains complicated.The dermal papilla plays key roles in epithelial-mesenchymal interactions that enable HF development and regeneration.Dermal papilla cells(DPCs)are important part of the mesenchymal compartment and are used to reconstitute HFs.Hair follicle regeneration requires abundant DPCs.However,it is also well known that primary DPCs isolated from hair follicles change their characteristics and lose the ability for hair follicles induction.Successfully induced hair regeneration involves retaining the hair induction capacity of DPCs.In this study,we isolated and cultured DPCs,the DPCs showed dedifferentiation and the characteristics of DPCs changed during subculture,including the morphology,specific staining,and the expression of Sox2,Versican,and ALP.We used HaCaT condition medium containing small molecular compounds including PDGF-AA,SB431542,CHIR99021,to culture DPCs,and observed the change of genes and proteins expression in DPCs.Finally,we conform that HaCaT condition medium comprising small molecule compounds has a potent ability to maintain the characteristics of dermal papilla cells.Additionally,we used the three-dimensional(3D)hanging drop culture method for further observation of sphere forming ability and the change of characteristics in DPCs of higher generation under the influence of HaCaT condition medium and specific small molecular compounds.Results:1.Cultured DPCs showed dedifferentiation and the ALP staining decreased,the expression of Sox2,Versican and ALP decreased gradually.The expression of ?-catenin and Lef-1 in the Wnt pathway was significantly decreased and the level of phospho-Smad2/Smad3 in the TGF-? / Smad pathway significantly upregulated with passaging.2.HaCaT cells were cultured with 1%FBS,the condition medium of 50% volume percentage and 24h-collected HaCaT supernatant is best for DPCs,which showed the most upregulated expression of Sox2 and Versican.3.HaCaT condition medium and small molecule compounds including PDGF-AA and Wnt signal pathway activator CHIR99021 and TGF-? /Smad signal pathway inhibitor SB431542 were used to culture P7 DPCs.Compared with ordinary medium,the optimized medium could promote DPCs with high gene expression of Sox2,ALP and Versican,and could upregulate the proteins of Sox2?ALP and ?-catenin.4.Compared with the ordinary medium,the application of the hanging drop culture method with the optimized medium could increase the sphere diameter and area,and promoted the expression of Sox2 and ALP proteins in immunofluorescence assay.Conclusions:1.Cultured DPCs show dedifferentiation and change their characteristics and downregulate the expression of Sox2,ALP and Versican.2.HaCaT condition medium and small molecule compounds including PDGF-AA?SB431542?CHIR99021 promote the specific genes and proteins expression of DPCs in high generation.3.The application of the hanging drop culture method with the optimized medium can be utilize to form hair papilla-like cell aggregate and prevent DPCs from dedifferentiation and maintain the characteristics of dermal papilla cells. |
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