Sorting And Functional Study Of Dermal Papilla Cells

Hair follicles are tiny organs and skin appendage that play an important role in our radiation protection,personal image-building,thermoregulation,tactile perception,skin physical protection,sebum secretion,and many other functions.Dermal papilla(DP)is located at the bottom of the hair follicle,which regulates hair growth,determines hair types,and participates in dermal regeneration during skin injury repair.DP cells can induce hair follicle regeneration when injected into the skin,and can reconstitute hair when combined with hair follicle stem cells.In addition,DP cell itself possesses stem cell characteristics.Compared with fibroblasts,DP cells are easier to be induced to differentiate into other types of functional cells in vitro,such as neurons,smooth muscle cells,and adipocytes.Therefore,DP cells have the potential to treat diseases associated with cell defects.Due to the abundance of hair follicles,easy access,and no ethical dilemma,DP cells have become a research focus in the field of stem cells.Isolation of a large number of DP cells is not only the key to hair regeneration to solve the problem of hair loss,but also will help to the development of new stem cell products and cell therapy based on DP cells.At present,there are very few literature reports on the technology of large-scale collection of DP cells,and there are many limitations in these methods.The most conventional method of obtaining a small amount of DP cells from single hair follicle through physical microdissection is very inefficient,and it is only applicable to large hair follicles;the method of labeling and sorting DP cells by genetic tool mouse cannot be universally applied to other organisms,especially humans;the method of labeling and sorting DP cells with PROM1 antibody is not specific,and the expression of PROM1 decreases sharply with the increase of mouse age,which makes the method unsuitable for sorting DP cells of adult animals.Therefore,our project is to establish a large-scale method for isolation of DP cells.This mothed must be highly efficient,specific and universal.First,we found candidate proteins specifically expressed on the DP cell membrane by analyzing the gene expression profile of DP cells from public database,and verified these proteins with tissue immunofluorescence technology.Finally,we found two antibodies against LEPR and SCARA5 which can specifically label hair follicle DP cells.After identifying the antibodies that label the DP cells,we used flow cytometry to sort the DP cells.For the dorsal skin of newborn mice,the yield of DP cells is about 1.16% by sorting with LEPR antibody.The new method significantly increased the number of DP cells obtained by physical dissection from a few hundred to several hundred thousand using the same amount of time.In view of the small proportion of DP cells in the whole hair follicle,the number of hundreds of thousands of DP cells is still difficult to meet the requirements of application.Therefore,we have also established an in vitro culture and expansion system for DP cells.To verify the reliability of this method,newly sorted or cultured DP cells were further tested.The sorted cells were subjected to flow cytometry and fluorescence detection again.It was found that the positive cells all fell into the signal-positive gate and all of cells could be seen with the fluorescence signal under the microscope.In addition,positive cells have DP-specific alkaline phosphatase activity.When the cultured cells were re-labeled with antibodies described above,the fluorescent signal could still be detected.Further,we performed functional verification on the sorted and cultured DP cells.Using the chamber grafting technique,we mixed the cultured DP cells with the epidermal cells of newborn mice,and successfully reconstituted the hair on nude mice.We injected cultured DP cells subcutaneously into the mice in the quiescent phase.Compared with the control group,LEPR positive cells accelerated the transition of hair from telogen to anagen in the treated skin.We tested with q PCR and found that the expression levels of RSPO1,RSPO2,and EDN3 in Lepr-positive DP were significantly higher than those in Lepr-negative cells,suggesting that these factors highly expressed in DP may promote hair reconstitution and growth.In conclusion,we have successfully established a technique for sorting DP cells.This technical solution can specifically collect a large number of DP cells,and is suitable for a variety of mammals.The establishment of this technique has laid a solid foundation for the future use of DP cells in the clinical treatment of hair loss and skin injury repair.