Establishment Of Serum-free Rat Hair Follicle Stem Cell Culture System

Background The skin can block the intrusion of foreign bodies,pathogenic microorganisms and other assaults from the external environment,which is an important protective barrier for the body.After skin damages,different types of stem cells proliferate and differentiate to replace and supplement normal dead cells,maintaining homeostasis.Hair follicle stem cells(Hair Follicle Stem Cells)are adult stem cells that are self-renewing and pluripotent,can maintain hair shafts growth and hair follicles regeneration under normal physiological conditions.After wound,HFSCs would proliferate and differentiate to form hair follicles,epidermis,sebaceous glands,to maintain the integrity and functionality of the skin under the joint action of multiple signaling pathways.In addition,HFSCs can directly inhibit wound scars formation by regenerating hair follicles during wound healing.HFSCs have a huge application prospect in clinical treatment.At present,in vitro culture of hair follicle stem cells usually requires the use of medium supplemented with fetal bovine serum(FBS)to maintain the biological characteristics of the cells.Our previous studies have found a way to obtain large amounts of HFSCs from a rat tentorial hair follicle,but the serum still needs to be added to the culture.However,cells cultured using serum-added media for clinical treatment also face biosafety issues,ethical issues and many uncertain risks.The Knock Out TM Serum Replacement(KSR)is a defined serum-free formulation that avoids the use of serum for culture.KSR has been used in the culture of clinical embryonic stem cells to maintain the biological characteristics of embryonic stem cells.Objective To establish an in vitro serum-free culture system of rat hair follicle stem cells by using KSR instead of FBS in culture medium.Methods Knock Out serum substitutes determined by chemical composition were used to replace the fetal bovine serum in the existing hair follicle stem cell growth medium and the morphological characteristics and growth of hair follicle stem cells cultured in the two media were compared.Through hair follicle reconstruction experiments on the back skin of nude mice serum-free cultured hair follicle stem cells were mixed with neonatal rat dermal cells in proportion and transplanted to fresh wounds on the back of nude mice to determine whether the stem cells cultured in the serum-free medium could be maintain "stemness" of hair follicle stem cells in vitro and reconstruct functional skin including skin appendages such as hair follicles.The expression of highly expressed proteins(?6-integrin,keratin K14,and P63)in HFSC was detected in stem cells cultured in serum-free medium using immunofluorescence.Results HFSCs grew normally in this serum-free medium.The serum-free cultured hair follicle stem cells were transplanted to the back of nude mice and were capable of reconstructing hair follicle-containing functional skin.Serum-free cultured HFSCs also express ?6-integrin,keratin K14,and P63.Conclusion This experiment initially established an in vitro serum-free culture system for rat hair follicle stem cells which could maintain the biological characteristics of stem cells and provide theoretical basis and technical support for clinical research and application of hair follicle stem cells.