Study Of Serum-free Expansion Of Human Skin Hair F Ollicle Stem Cells

BackgroundSkin epithelial cells have been used as seed cells in artificial skin and wound repair for many years.However,the seed cells are usually single differentiation potential that can not reconstruct any appendages to recover fully functional skin.Studies have shown that hair follicle stem cells are ideal seed cells for wound repair and artificial skin,because hair follicle stem cells can repair the epidermis and differentiate into hair,sebaceous glands,and sweat glands.However,the proportion of hair follicle stem cells in human normal skin tissue is rare,for that a hair follicle can only extract 200-400 hair follicle stem cells.And we usually need no less than multimillion of cells in treatment;therefore,the application of hair follicle stem cells is limited by the inadequate numbers.However,hair follicle stem cells are still difficult to expand in vitro,especially serum-free and feeder-free.Therefore,how to extract HFSCs accurately with limited samples and establish a stable technological process for cell collection and purification is great significance.Small molecule inhibitors typically yield highly penetrant effects across whole cell populations.Y-27632 is a selective ROCK1 inhibitor.In human embryonic stem(hES)cells,Y-27632 treatment at 10 μM markedly diminishes dissociation-induced apoptosis even in serum-free suspension(SFEB)culture,increases cloning efficiency,facilitates subcloning after gene transfer,and enables SFEB-cultured hES cells to survive.A83-01 blocks phosphorylation of Smad2 and inhibits TGF-β-induced epithelial-to-mesenchymal transition,and a cocktail of small molecules,Y-27632,A83-01,and CHIR-99021,can convert rat and mouse mature hepatocytes in vitro into proliferative bipotent cells.Furthermore,a cocktail of A83-01,DMH-1,CHIR-99021,Y-27632 can amplify certain epithelial cells in serum-free medium in vitro.These results indicate that small molecule inhibitors in cell culture can remedy deficiencies in serum-free culture,open up excellent opportunities to researchers studying multiple aspects of stem cells culture.Using A83-01,DMH-1,CHIR-99021,Y-27632 in hair follicle stem cells in vitro serum-free amplification has not been reported before.In our study,the technological proliferation for human HFSCs was employed,and the small molecule inhibitors A83-01,DMH-1,CHIR-99021 and Y-27632 were used to affect the proliferation of human HFSCs serum-free in vitro.Our study demonstrated that HFSCs could be effectively enriched by microsurgery,enzymatic digestion and differential attachment and culture in serum-free medium CNT-Prime Epithelial Culture Medium with Y-27632.Our study also showed a cocktail of A83-01,Y-27632 or A83-01,Y-27632,DMH-1 can increase the expansion efficiency for HFSCs.Objective1.To extract HFSCs accurately with limited samples and establish a stable technological process for cell collection and purification.2.To explore the condition for culturing serum-free HFSCs and optimize the conditions for expansion and maintaining pluripotency of HFSCs.Methods1.Cell Isolation Hair follicles were plucked from the scalp after Dispase digestion,the sebaceous glands were removed and the bulk of the hair shafts cut off.Incubate the isolated follicle fragments in 0.05% Trypsin-Versene,collect the hair follicle outer root sheath cells after spinning down the cell suspensions.Results are tested by tissue immunofluorescence.2.Enrichment human hair follicle stems cell and primary culture Human hair follicle outer root sheath cells were cultivated on Coating Matrix coated flasks,the un-adhering cells were removed 15 minutes later.Then add enough CNT-Prime Epithelial Culture Medium with Y-27632 to the flask for culturing the remaining cells.3.Observed the Small molecule inhibitors effect on Human HFSCs culture in vitro HFSCs were randomly divided into six groups: control group,the A83-01 group,the DMH-1 group,the A83-01+DMH-1 group,the CHIR-99021 group and the A83-01+DMH-1+CHIR-99021 group.Cells viability was examined by CCK-8 kit.Flow analysis and immunofluorescence were used to observe the long-term effect of different treatment.Result1.Cut off the bulks and remove the sebaceous glands can increase hair follicle stem cell purification effect,for our immunofluorescence results indicated that HFSCs located in the middle of hair shafts.2.The validity of differential attachment for enrichment of HFSCs using Coating Matrix were proved by flow analysis that the proportion of CD49f+/CD200+ cells were dramatically increased(p< 0.01).3.The primary stem cells were cultured serum-free in vitro for 16 ± 3 days,and an average number of 4.5 ± 0.7 × 104 K15 positive cells were collected from each hair follicle.4.A83-01 or A83-01+DMH-1 in CNT-Prime epithelial culture medium improved the expansion of HFSCs cells,and shortened the expansion time(P <0.05).5.Successfully establish an experimental process for isolation,enrichment and culture of HFSCs in the condition of serum-free,feeder-free and definite medium.ConclusionOur studies establish an effective and stable procedure for isolation,enrichment of HFSCs by microdissection and differential attachment,and culturing human HFSCs serum-free in vitro,and also demonstrate that A83-01 could improve the efficiency and pluripotency maintenance of human HFSCs.