Induction Of Dermal Fibroblasts Into Dermal Papilla Cell-like Cells In Hydrogel Microcapsules For Hair Follicle Regeneration

Current therapeutic options for hair loss include medication and autologous hair follicle transplantation,none of which can promote the formation of new hair follicles.Dermal papilla cells(DPCs),essential for hair follicle morphogenesis and regulation of hair cycling,are considered as one of the target cell types for hair follicle regeneration.However,hDPCs tend to lose their hair-inducing ability in vitro.Therefore,how to obtain sufficient DPCs with the hair-inducing ability has become a bottleneck problem in hair regeneration.The main purpose of this study was to investigate whether the three-dimensional microenvironment of sodium alginate can participate in the transdifferentiation of fibroblasts to dermal papilla cell-like cells and the related mechanism.In order to enhance the aggregation of human dermal fibroblasts(hDFs)and facilitate the formation of cell spherois,we encapsulated hDFs into alginate-poly-L-lysine-alginate(APA)microspheres by electrostatic spray.The proliferation and the expression of DPC-specific genes were detaected by CCK-8 analysis and RT-PCR,respectively.Alkaline phosphatase staining and quantification and flow cytometry method were used to detect the protein expression levels of human ALP and ?-SMA,respectively.It was found that APA microspheres have good biocompatibility over the period of cultivation,and the transcripitional and protein levels of DPC-specific genes were increased following the APA culture time.hDPC-like cells induced in APA microspheres and newborn mouse epidermal cells were subcutaneously injected into the dorsal skin of nude mice in order to verify the ability of hDPC-like cells in inducing hair formation.The source of cells and function of newly formed hair follicles were determined by H&E,immunofluorescence and immunohistochemical staining.It was found that melanin-like and hair follicle-like structures similar to the positive control post injection(4 weeks).Human mitochondrial staining confirmed that the new hair follicle structure was derived from the hDPC-like cells,while the staining of DPC marker genes demonstrated that the formed hair follicle-like structures were functionally active.hDPCs,hDFs,and hDP-like cells were sequenced at the transcriptome level.RNA-Seq analyses suggested the activation of the Wnt signaling pathway in the hDF-DPC conversion.The inhibition and activation of Wnt signaling in APA treatment confirmed the important role of the pathway in the transdifferentiation of hDFs to hDPC-like cells.Additionally,we showed that APA treatment induced the conversion of mDFs into mDPC-like cells.The mDPC-like cells were endowed with not only the similarities to primary mDPCs on the molecular level,but also the ability to induce the formation of hair follicle-like structures and pigmentation of hairs when co-implanted with newborn mouse epidermal cells into nude mice,demonstrating the universality of APA cultivation in inducing DF-DPC conversion.These results collecitively proved that APA treatment induced the conversion of hDFs or mDFs into hDPC-like or mDPC-like cells,respectively.The resultant DPC-like cells were aggregative and functionally active in inducing hair follicle regeneration.In the conversion process,Wnt signaling pathway played an important regulatory role.The derivation of hDPC-like cells through APA treatment would provide a novel,sufficient,and the most importantly safe source of DPCs for hair follicle regeneration.