The Study Of Hair Follicle Stem Cells' Markers And Purification

Background:Nowadays, hair restoration surgery technique, although its curative effect affirmation, hold out for long time, it got a large number of bald-headed sufferer's approve.But no matter which repair technique of adoption, all with the sufferer own to amplely provide hair area from the body for premise. For those sufferer whose bald rea area is too big or the even hair complete shattered, these repair of the technique often can't use. Even these techniques are used, under the effect of the shortage of providing area and the transplant rate unsteady, the hair density after operation can't attain satisfaction. Besides none surgical operation can creat new hair and can't increase the amount of hair. Many bald-headed sufferer's hair is losing every day, hair restoration surgery technique can't make it stop, either, can't estimate it develop circumstance. So, the shortage of the resources of hair area is obvious and become the present hair repair surgery more difficult problem for solve. And also to be a hurry request for the development of the hair follicle engineering technology.Hair follicle is the most complicated skin organization, crossing epidermis and dermis.It is structure is very different than other organization, and has a special immunity.Hair follicle is the result of the interaction between epidermis and dermis. In the embryo, the skin begins as a single layer of epidermal stem cells.Soon after, as mesenchymal cells populate the skin to form the underlying collagenous dermis, morphogenesis of the hair follicle begins. Hair follicle stem cells (HFSCs) can indeed influence hair growth. Therefore, there is most important to establish the HFSCs' development model.And it will help to the study of the hair follicle morphogenesis and hair follicle switch from the vellus to the terminal state.On the other hand, we have to face the purification of the stem cell during the studying the stem cell. The previous belief that stem cells reside in the bulbar region of hair follicles.But the old method is hard to manipulate, cells are hard to adhere and grow slowly, can't get the pure stem cells,it is also easy to be contaminated , this is a laboring task.Objective:1. To confirm the fixed position of the HFSCs.Determinate and improve the method of how to isolate and culture HFBCs.2. To confirm the HFSCs surface marks and attempts seeks the method of how to purifies HFSCs3. To establish the method of reconstructing hair follicles by mixes the HFSCswhich after purification and dermal papilla cells (DPCs).Methods:1. The microdissection of mouse hair follicle and human hair follicle. Separates the mouse and person's complete hair-follicle by microdissection.Put them under the inverted microscope to observe the difference of their shape and structure.2. Mouse HFBCs' separation and cultivation.Micromanipulation:takes Wistar big mouse on 7th day age skin under the aseptic condition. After two rinses in D-Hanks' contains penicillin and streptomycin. And the skin fragments were incubated in 0.25% dispase for 2h at 4℃. Those in anagen phase, were carefully selected under the dissecting microscope. After two rinses, then the bulge region was amputated from upper follicle by making two transversal cuts respectively at the site of the enlargement spots of bulge area with a fine needle.And immerse them in a supplemented mixture medium of Dulbecco's modified Eagle's medium and Ham's F12 medium (DMEM/F12) containing 10% fetal bovine serum as described. Enzyme digestion: put the bulge region which was got by micromanipulation in to 0.25% dispase mixed with 0.1% collagen enzyme for 20min at 37℃.Then blow the organization into cells.Collect the cells to the supplemented mixture medium of DMEM/F12 containing 10% fetal bovine serum to stop digesting.Then immersed in a DMEM/F12.The culture was incubated at 37℃and 5% CO₂ in air, and the medium changed twice a week.3. Human HFBCs' separation and cultivation.Plastic surgery specimens of human scalp skin were obtained from the nape of the neck. After rinsing with 70% alcohol, the tissues were trimmed into small pieces, and the skin fragments were incubated in 0.25% dispase for 2h at 37℃. The hair follicles were drawed out form the side of dermis carefully. Those in anagen phase, were carefully selected under the dissecting microscope. After two rinses, then the bulge region was amputated from upper follicle by making two transversal cuts respectively at the site of the enlargement spots of bulge area with a fine needle. All surgical procedures were operated under a sterile environment. After additional two rinses, them were transferred into a new the follicles were transferred into a 25mL bottle, immersed in a supplemented mixture medium of DMEM/F12 containing 10% fetal bovine serum as described. The culture was incubated at 37℃and 5% CO₂ in air, and the medium changed twice a week.4. Human HFBCs' examination and purification. Takes few HFBCs to put in the culture dish, After pasteing the wall in 48 hours. Then them were rinsed 3 times by PBS (each time 5min). Precooling acetone fixed 10min. Immunohistochemical staining. Examined the K19's expression. Float the HFBCs again.Adjust the density for 1×10⁸/mL, according to the density of 1.0μg/mL, immersed in FITC-mouse-anti-person CD200 30min.Separate the HFSCs by magnetic cell sorting.5. Human HFSCs' examination. Float the purify cell again with 0.4% trypan bule.Then the viability of these purified HFSCs was detected under light microscope. The purification rate was analyzed by flow cytometry. The preand post-purification cells were compared by immunofluorescence staining.6. DPCs of human scalp hair follicles were isolated by digesting-filtrating method, and cultured in supplemented mixture medium of DMEM/F12 containing 10% fetal bovine serum as described.7. The mixture of HFSCs and DPCs were implanted onto the back skin of nude mouse by hypodermal injection.The histologic examination of the skin was performed after 21 days.Results:1. Both mouse and human's bulge area were detected in the contiguous part of bulge area, that provides the insertion point for arrector pili muscle and marks the bottom of the permanent portion of hair follicles. In mouse pelage hair follicles, the bulge can be detected as discrete protuberances of the outer root sheath. Although the bulge can also be detected as a clearly defined structure in embryonic hair follicles.2. Compared with micromanipulation and enzyme digestion. In the former method, the adhering rate was high.It can't waste too much cells.3. Cells proliferated rapidly in vitro by micromanipulation.Floated the cells again,the HFBCs were mixed with the other cells.4. After purification, the viability of cells was not significantly impaired. And the cells proliferated rapidly.5. Flow cytometry and immunofluorescence staining examination, the CD200⁺ cell rate was 8.31% before cell sorting purification while that was 82.31% after cell sorting purification.6. The earliest HFSCs was observed within 2 days after inoculation.Cells proliferated rapidly after 3 days.Then kept rapid growth until day 7.The cells began to decelerate after day 10.7. New formed hair follicles can be seen under microscopy after 21 days.Conclusion:Highly purified and viable human hair foll icle stem cells could be obtained by micromanipulation and magnetic cell sorting assay. This method can isolate HFSCs of human scalp hair follicless on a large scale rapidly and efficiently.The mixture of HFSCs and DPCs can form new hair follicles under particular conditions.