Pre-aggregation Of Scalp Dermal Progenitor And Epidermal Stem Cells Activates The WNT Pathway And Promotes Hair Follicle Formation

BackgroundBillions of dollars are invested annually by pharmaceutical companies in search of new options for treating hair loss conditions;nevertheless,the challenge remains.One of the major limitations to hair follicle research is the lack of effective and efficient drug screening systems using human cells.Organoids,three-dimensional structures derived from stem cells have characteristics similar to actual organs,providing new opportunities for studying organ development,tissue regeneration,and disease pathogenesis.Although multiple epithelial organoids,such as mammary glands,salivary glands,and colon and liver ducts,have been successfully generated recently,there are still challenges to generate some other types of organoids such as teeth and hair follicles in vitro.In this study,epidermal stem cells and scalp dermal progenitor cells were suspended and co-cultured to form aggregates in order to achieve efficient culture of hair follicle organoids,and to explore their effects on hair regeneration potential and study the role of the WNT signaling pathway during the aggregation.Objectives1.To explore an effective experimental method for constructing hair follicle organoids by using pre-aggregating epidermal and dermal cells.2.To conduct histopathological and genetic evaluation on successfully constructed human dermal-epidermal aggregations.3.To study the role of the WNT signaling pathway during the aggregation4.To investigate whether transplantation of pre-aggregating epidermal and dermal cells can enhance hair formation in vivo.Methods1.According to the "one-step digestion method" established by our group,the epidermal stem cells were isolated from the neonatal foreskin tissue,and dermal progenitor cells were isolated from either fetal or adult scalp tissues.The biological characteristics of epidermal stem cell was confirmed by colony formation and flow cytometry analysis.The dermal progenitor cells derived from adult and fetal scalp tissues were proved to be multipotential by analysis of their ablility to differentiate into different tissue-type of cells.2.Dissociated scalp dermal progenitor and epidermal stem cells were mixed and suspended in supplemented DMEM/F12(3:1)culture medium,and then the mixture of cells was plated into ultra-low attachment Costar(?)6-well plates for aggregating to form hair organoids.The morphology of organoids during culture was monitored by optical microscopy.3.Frozen sections made from dermal-epidermal aggregation were stained with hematoxylin and eosin.Immunofluorescence staining was also conducted to analyze the structure of organoids.4.qRT-PCR was performed to analyze the expression of genes known to be involved in hair follicle development.5.WNT3a recombinant protein and WNT inhibitor IWP-2 were added to the culture medium to further test the importance of the WNT pathway in the formation of aggregates.6.Grafting assays were performed to test the hair regeneration ability of dermal-epidermal aggregations in vivo.Results1.Epidermal progenitor cells were successful isolated and cultured able to form holoclones,a defining characteristic of epidermal stem cells.Immunofluorescence results show that dermal progenitor cells derived from adult and fetal scalp tissues express osteogenic,adipogenic,myogenic,and neurogenic marker proteins after differentiation induction.2.Dermal-epidermal cells interacted to form two major types of aggregates in vitro,and Type I aggregates expressed hair follicle markers.3.qRT-PCR analysis indicated that dermal-epidermal aggregation enhances the WNT pathway.4.Adding WNT3a recombinant protein and WNT pathway inhibitor to the culture medium can promote and inhibit the formation of Type I aggregates respectively,which indicate that WNT pathway activation is essential to Type I aggregates formation during the dermal-epidermal aggregation process.5.Pre-aggregation of fetal scalp derived dermal progenitor and epidermal stem cells enhances hair follicle formation in vivo.6.In the process of 3D co-culture with epidermal stem cells,the efficiency of dermal progenitor cells derived from fetal scalp to form Type Ⅰ aggregates(hair follicle organoids)is significantly higher than that of dermal progenitor cells derived from adult scalp,indicating that the formation of Type I aggregates can reflect the ability of cells to regenerate hair in vivo.ConclusionsIn summary,our system supports the rapid formation of a large number of hair follicle organoids(Type Ⅰ aggregates).This system provides a platform for testing the trichogenic potential of hair stem cells,for assessing inductive hair stem cells and for screening compounds that support hair follicle regeneration.The present study also clearly showed that it is still a big challenge to efficiently regenerate hair follicles both in vivo and in vitro using dermal progenitor cells derived from adult scalp tissue.Due to ethical considerations,the use of cells derived from fetal tissues is not recommended,especially for clinical applications.Therefore,future work will be directed at developing trichogenic cells from induced pluripotent stem cells(iPSCs)and to reprogram adult cells into fetal-like cells to replace the fetal cells in our system.