Culture Of Fetal Murine Epidermal Stem Cells In Vitro And Their Contributions To The Regeneration Of Epidermis And Follicles

Objective To investigate the methods of isolating and culturing the epidermal stem cells (ESCs) and follicular papilla cells (FPCs) in vitro from murine embryo at first. And then, the new skin equivalent was to be prepared by co-culturing ESCs and FP cells in three-dimension system and the contributions of ESCs to the regeneration of new epidermis and follicles were to be investigated after the skin equivalent being transplanted on the athymic nude mouse (BALB/C). Meanwhile, the feasibility of the exogenous gene transfection and tracing study on ESCs were studied.Methods The whiskers hair follicles were isolated from 15-18 days murine embryo by using microsurgery technique and the FPCs were cultured in vitro. The embryo skin was stripped off under microscope and the epidermal cells suspension (including ESCs) was prepared by digesting the skin with dispase and trypsin. ESCs were isolated by adhering to murine collagen type IV for 5-10 minutes in the 6-well-plate and cultured in conditional medium in vitro. The colony forming efficiency (CFE) and cell cycle profile of ESCs were studied, respectively. The expression of intergrin- β 1 and keratin 15 in ESCs were also detected by immunocytochemistry and fluorescent-antibody technique, while the normalkeratinocytes were used as the control group. The new murine skin equivalent was prepared by seeding FPCs in fibrin-gel firstly and then co-culrufing with ESCs sheet. After this skin equivalent was grafted on the full thickness skin wound of athymic nude mouse (BALB/C) for 8-10 weeks, the regeneration 'of new epidermis and follicles was investigated. By using Lipofectamine, the plasmid pIRES2-EGFP was transfected into ESCs and the positive clones were selected by G418. The EGFP expression was observed under fluorescence microscope. Results The primary FPCs spread out from organ after follicular papilla having been cultured for 1-3 days. After being subcultured, the FPCs grew clusterily. The ESCs were able to be isolated primarily and grew well on the collagen type IV in the conditional medium. The ESCs had a high potential ability to form clones and grow into large clones during the primary passage and subcultures, while the keratinocytes could only form the small size clones. The CFE of ESCs was 15.3±2.3% and that of keratinocytes was 6.6±1.1%, showing a significant difference (p<0.05). The cell cycle profile analysis showed that 94.9% of ESCs were in G1 stage and only 3.5% in S stage. Nevertheless, 74.1% of keratinocytes were in Gl stage and 17.5% in S stage. This indicated that ESCs had a lower replicative rate than that of keratinocytes. Both of the intergrin-β 1 and kerain15 expression were positive in most of ESCs, however they were negative or weakly positive in keratinocytes. After the new skin equivalent having been transplanted on animal model for 8-10 weeks, the ESCs gave rise to the regeneration of new epidermis and follicles with FPCs together, while ESCs could only form the new epidermis with the fibroblasts. The pIRES2-EGFP could be transfected into ESCs by Lipofectamine 2000 successfully and the positive could be selected by G418. The EGFP had a strong positive expression in the first few passages, however theexpression decreased during the subculture.Conclusion The ESCs could be isolated successfully from epidermal cellssuspension by adhering to collagen type IV and cultured in conditional medium invitro. The FPCs could be cultured from follicular papilla and grew well and stablyin fibrin gel. The results of the regeneration of new epidermis and follicles basedon animal model indicated that ESCs could give rise to all kinds of epidermal cellsboth in epidermis and follicles, and meanwhile the FPCs could be the main sourceof dermal cells. It was feasible to transfect the exogenous gene into ESCs but itsexpression was not stable. Thus the plasmid pIRES2-EGFP could not be used as amarker for the tracing study of ESCs.