External Hair Follicle Mesenchymal Stem Cells Promote Hair Growth Through Homing

Background and objectiveStem cell therapy has received increasing attention in the treatment of androgenetic alopecia(AGA).Theoretically,its mechanism for treating the hair loss may be to improve the internal microenvironment of the target tissue by homing or to improve the peripheral microenvironment by paracrine.Since homing is more targeted to improve the internal microenvironment,homing has a wider application space in disease treatment.In recent years,a large number of studies have explored the effectiveness of cell therapy for AGA,such as the use of bone marrow mesenchymal stem cells,hair follicle stem cells,adipose stem cells and hair follicle dermal sheath cup cells.Studies have shown that the above cells can promote hair growth to a certain extent,but only through paracrine function,the efficacy is limited.No research has been reported on the treatment of AGA by homing of stem cells.Studies have found that injection of SKPs can homing into the DP and DS of the recipient’s hair follicles,increasing the diameter of the DP and thickening the hair shaft.Dermal papilla cells(DPCs)and dermal sheath cells(DSCs)belong to the type of mesenchymal stem cells,which also contain SKPs,suggesting that DPCs and DSCs may have homing properties.Therefore,this study explores the effects of DPCs and DSCs on hair follicle growth through animal experiments,explores the homing phenomenon of DPCs and DSCs and their effects on hair follicles,and clearly determines the factors that affect homing efficiency.It lays the foundation for the application of DPCs and DSCs in stem cell therapy for AGA.Methods1.Exogenous DPCs and DSCs injection promote hair follicle growthDPCs and DSCs of EGFP mouse tentacle hair follicles were isolated and cultured,and the characteristics of P3 DPCs and DSCs were detected by immunofluorescence.Cells were collected and injected into the dermis of mice after epilation,and samples were taken on days 8,14,and 21,respectively,to observe the hair follicle cycle and hair growth,and to detect hair follicle diameter and expression of related markers.2.Homing of exogenous DPCs and DSCs and its effect on recipient hair folliclesDPCs and DSCs cells were injected subcutaneously into NOD/SCID mice.Samples were taken 2 weeks and 6 months after injection to observe the location of the injected cells in the hair follicles of recipient mice.The hair follicles with cell homing,hair follicles without cell homing and the control group were compared in diameter,anagen period,cell proliferation,apoptosis and β-catenin expression in the hair follicles.The survival and location of the injected cells were detected 6 months after injection.3.Factors affecting the homing efficiency of exogenous DPCs and DSCsThe effects of plucking and shaving,growth phase and resting phase,injection of 1 × 105/ml,1 × 106/ml,and 2 × 106/ml on the homing efficiency of the recipient mice were investigated.Explore the best conditions to improve cell homing efficiency.Transcriptome sequencing was used to analyze the differences in gene expression between high and low generation cells in order to explore the mechanism of differences in homing of different generations of cells.Results1.Exogenous DPCs and DSCs injection promote hair follicle growthThe DPCs and DSCs cultured from the primary to the P3 generation grew well.Immunofluorescence showed that the DPCs and DSCs cultured to the P3 generation still expressed their own markers(ALP,α-SMA,NCAM)and the stem cell marker SOX2.Injecting DPCs and DSCs into the dermis of NOD/SCID mice after plucking showed that the hair follicle growth process accelerated on the 8th day.Two weeks later,it was found that the hair shaft growth of the injected cell group was faster than that of the control group.The diameter of the hair follicles became thicker,and some cells homed into the DP and DS of the hair follicles.2.Homing of exogenous DPCs and DSCs and its effect on recipient hair folliclesIn terms of the hair follicle cycle,compared with non-homing hair follicles,the anagen period of homing hair follicles was significantly longer,and the expression ofβ-catenin and hair matrix ki-67 was significantly higher than that of non-homing hair follicles and control hair follicles.In terms of hair follicle diameter,the diameter of hair follicles with cells homing was significantly larger than that of non-homing and control hair follicles.After 6 months of injection,the homing cells were still alive and expressed DPCs and DSCs-related markers.3.Factors affecting the homing efficiency of exogenous DPCs and DSCsThe number of hair follicles with integrated exogenous cells and the number of exogenous cells integrated into a single hair follicle in the hair plucking group were greater than those in the hair shaving group.Hair plucking during growth and resting periods had no effect on cell homing efficiency.The homing efficiency is the highest when the cell concentration is 2 × 106,;among P3,P6,P9 generation of DPCs and DSCs,the homing efficiency of the P3 generation cells is the highest;KEGG pathway analysis confirms that the differential genes of the high and low generation cells are concentrated in "micro-environmental signal transduction","gene replication and repair",and "cell growth and death" related signal pathways.Meanwhile,stem-related genes of higher generation cells are significantly down-regulated.Conclusions1.DPCs and DSCs have a homing behavior after injection into the dermis of mice.The homing cells have normal functions consistent with the recipient mouse hair follicle cells.2.DPCs and DSCs increase hair follicle diameter,prolong anagen period,and promote hair follicle growth by homing.3.The homing efficiency of the cells was the highest under the condition that the mice were plucked,the cell concentration was 2 × 106,and the cells are P3 generation.4.DPCs and DSCs cell therapy have great potential for clinical treatment of AGA.