The Effect Of Low-passage Dermal Papilla Cell Derived Exosomes On Hair Regeneration And Hair Cycling And Its Its Mechanism Research

Background and objectiveThe hair follicle(HF)is a complex mini-organ of mammals,which is composed of epithelial and mesenchymal components that interact with each other.Dermal papilla stem cells(DPCs)serve as a signaling center in hair follicles and regulate hair formation and cycling.Increasing evidences indicate that DPCs exert their regulatory function of HF growth mainly through their unique paracrine properties,opening up a way to exosome therapies.Exosomes are nanovesicles that can originate from various cell types and carry different kinds of regulatory proteins,mRNAs,and microRNAs(miRNAs).They play a significant role in regulating cell-to-cell communication,ultimately affecting physiological and pathological conditions.Recently,several studies suggested that exosomes derived from stem cells exhibited notably beneficial functions similar to their mother cells,In the field of dermatology,keratinocyte-derived exosomes were confirmed to modulate melanocyte pigmentation,promote wound healing,and inhibit scar formation.In addition,the use of exosomes for therapeutic treatment offers specific advantages compared with stem cell therapy.Therefore,in the study we isolated exosomes from both human and mice DPs and demonstrated their therapeutic effect in vitro and in vivo.We explored the influence of DP-Exos on injured hair regeneration and hair cycling.Finally,we further study the target genes and related pathways of DP-Exo by using exosome small RNA sequencing and communication analysis,and explore its mechanism..Methods1.The influence of DP-Exos on cell viability of human hair matrix cells.Media were collected from low-passage mice and human DPC cultures(P1-P3)and stored at-20 ? and exosomes were isolated using ultracentrifugation.To investigate the influence of DP-Exos on hair matrix cells,we added DP-Exos with different concentrations to cultured hair matrnx cells.Hair matrix cells proliferation determined by CCK-8 assay and microscopy.The migration ability was measured by transwell migration assay and wound healing assay.2.The effects of DP-Exos on hair follicle stem cells(HFSCs)DP-Exos with different concentrations were added to the culture medium of HFSCs.CCK-8 assay was applied to investigate cell viability of HFSCs.The migration ability was measured by transwell migration assay.3.The effects of DP-Exos on hair follicle regeneration after partly injury.To investigate the role of dermal papilla derived exosomes(DP-Exos)on hair follicle regeneration,lower third amputated vibrissa follicles were transplanted to nude mice.Macroscopic and histological staining were preformed to assess the regeneration process on amputated follicles treated with DPCs,DP-Exos or PBS.Hair follicle regeneration was analysis by photograph and H&E staining4.The influence of DP-Exos on hair cycle.To investigate the influence of DP-Exos on hair cycle,mice dorsal hair in telogen were subcutaneously injected with DP-Exos after depilation.Histological assessment were proceed to analysis skin thickness and bulb diameter,as well as hair cycling phase of hair follicles in each group.5.The underlying mechanism of hair promoting effect of DP-Exos.Libraries for mRNA and miRNA sequencing were respectively generated using the NEBNext Ultra RNA Library Prep Kit for Illumine and the NEBNext Multiplex Small RNA Library Prep Kit for Illumine.We predicted the potential target genes of the top 30 miRNAs using miRWalk 2.0.Using DAVID 6.8,GO analysis was performed to help elucidate the concrete biological process of specific genes,and KEGG pathways enrichment was employed to identify the critical signal pathways of the PTG.The PTGs were mapped to the STRING database to screen PPI,and networks were visualized by Cytoscape.The most significant modules were identified with the plug-in MCODE of Cytoscape with a cutoff MCODE score of>5(30).Hub genes were identified with the plug-in CytoHubba of Cytoscape with top 10 nodes ranked by Maximal Clique Centrality.Immunofluorescence staining was utilize to detect the change of HFSCs marker and cell proliferation after exosome treatment.RT-PCR,Western-blot and immunofluorescence staining were applied to identify the target gene expression change in the middle and late process of treatment.Results1.The influence of DP-Exos on cell viability of human hair matrix cells.The result of CCK-8 and Ki67 immunofluorescence staining indicated that human DPC-Exo could significantly promote cell proliferation of hair matrix cells,DP-Exos with 10?g/ml concentration had strongest effect.The result of transwell assay and wound healing assay revealed that DP-Exos promoted hair matrix cell migration.10?g/mDP-Exosl exhibited best effect.2.The effects of DP-Exos on hair follicle stem cells(HFSCs)The result of CCK-8,Ki67 immunofluorescence staining and transwell assay indicated that human DPC-Exo could significantly promote HFSCs proliferation and migration.3.The effects of DP-Exos on hair follicle regeneration after partly injury.By day 30,some transplanted amputated vibrissa follicles were observed.Regenerated HFs in PBS-treated group had relatively smaller dermal papilla,some were not successfully regenerated which were small dermal condensations with no visible hair fibers,hair bulb structures.The hair regeneration rate was relatively low compared to DPCs and DP-Exo treated groups.However,no significant difference among DPCs and DP-Exo treated groups.30 days after transplantation,Dil signals were detected in dermal sheath and dermal papilla,both in DPCs and DP-Exos treatment group.These signals were red dots inside DPCs without specific cell structure encompassing Dapi-labeled nucleus.4.The influence of DP-Exos on hair cycle.The shaved area of mice in the DP-Exos treatment groups darkened and displayed more than 70%hair regrowth by 14 days after treatment initiation,and almost full hair regrowth by day 21.The control group showed about 6%hair regrowth at day 14,and only 35%hair regrowth was observed at the final time point.Mice follicle in DP-Exo group exhibited typical anagen appearance with thicker skin and bigger hair bulb.Within 14 days after the beginning of DP-Exo treatment,more than 80%of the hair follicles grew out of the skin,and the hair follicles regenerated completely on the 21st day.The hair regeneration rate was 77%in minoxidil group and 35%in PBS group respectively after 21 days.At 14 days,DP-Exo was 3.8 times higher than Minodil group and 14 times higher than control group.After 21 days of treatment,the skin thickness of DP-Exo group was most acutely increased,compared with minoxidil group and PBS group.5.The underlying mechanism of hair promoting effect of DP-Exos.The exosomal miRNA-predicted target genes were enriched in 66 gene ontology(GO)terms and 41 Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways.Using Cytoscape,hub gene Wnt5a,BMP2,and BMP4 were obtained from the PPI network.Multiple miRNAs might serve as novel biomarkers of exosome-regulated hair follicle regeneration were identified.Finally,immunofluorescence staining,RT-PCR and western blot validated that DP-Exos down-regulated Wnt5a,BMP2,and BMP4 in regenerative vibrissa follicles as well as in dorsal skin in the middle and late stage of treatment.Immunofluorescence staining revealed cellular marker for proliferation Ki67,stem cell marker K15 and CD34 were over-expressed after being treated with DP-Exos.Conclusion1.Low-passage DP-Exos promote hair matrix cell proliferation and migration.2.Low-passage DP-Exos promote HFSCs proliferation and migration.3.DP-Exos facilitate amputated hair follicle regeneration.4.DP-Exos accelerated hair follicle telogen-to-anagen transition.5.DP-Exos influence hair signaling pathway by miRNA,which downregulate of inhibitory molecules such as Wnt5a,BMP2 and BMP4.