Wnt Signaling Pathway Mediated Gene Regulate Hair Follicle Stem Cells Committed Differentiation

Background and ObjectiveIt has become a hot spot in medical researches that hair follicle stem cells(HFSCs) are selected to be seed cells to construct tissue engineered skin to repair skin missing cau-sed by burns, traumas and chronic ulcers. The hair follicle is an important subsidiary organ of the skin, and is consist of hair shaft, internal root sheath and external root sheath. T-he hair follicle bulge is in the external root sheath near the epidermal side, at the arrector muscle attaching below sebaceous glands. The cells at this place cultured in vitro turn o-ut to becharacteristics of stem cells, which are abilities of self-duplicating, slow cyclicity and multi-directional differentiation. These cells are proved to be HFSCs. HFSCs have the ability of multi-directional differentiation, which participate epidermal renewal, sebaceous glands maintaining and hair follicle formation. The differentiation of HFSCs is co-instructed by multi-factors, including elements and genes in cells and microenvironment, even signaling pathways. With the effortsof many researchers, Wnt signaling pathway is proved to have critical effectson proliferation and differentiation in HFSCs.Wnt/β-catenin signaling pathway exists in nearly all cells. Wnt protein is released after cell stimulation, activates a series of molecular signaling substances in downstream and f-inally changes gene expression, which is a molecular cascade pathway in inducing cell differentiation and proliferation, so as to embryogenesis and tumorigenesis. In HFSCs, β-catenin is accumulated in cytoplasm after activation of Wnt/β-catenin pathway, then moves into nucleus to integrate intranuclear transcription factor Tcf3/Lefl, activates target genes c-myc and cyclin D1to promote HSFCs differentiation. In recent years researches, external interference factor LiCl suppressed degeneration of β-catenin in Wnt signaling pathway, accumulated in cytoplasm, activated downstream pathway, and finally affected phenotype of HFSCs differentiation. However, the molecular mechanisms are still unclear and restricted in cytoplasm. This study uses LiCl to activateWnt signaling pathway to investigate the effects of Wnt/β-catenin signaling pathway on HFSCs differentiation to hair follicle or epidermic cell and the interrelations with other signaling pathway, especially changes of molecular level in nucleus, and the application of drugs controlled release system on tissue-engineered skin.Methods1. Hair follicle eminence was obtained by modified methods and two-stepenzymatic digestion from scalp tissue, and then cut into mud. HFSCs were harvested by type IV collagen different speed adherence method, which were identified by immunofluorescent staining method, and then inoculated into96-wellculture broad with different density:2.5×10~3/ml,5×10~3/ml,1×10~4/ml,1.5×10~4/ml and2.5×10~4/ml, to observe cells proliferation in different time to screen thebest subculturing density of HFSCs by curves of cells growth portrayed by MTT method.2. HFSCs were induced to differentiate with0,0.1,1,5,10,20,40and100mM/l LiCl to observe the HFSCs proliferation to define toxicity of LiCl; HFSCs were cultured with0,5and10mM/l LiCl to compare the proliferation between the three groups using MTT method, and5d later, real-time PCR wasadministrated to determine the mRNA level of biomacromolecules in Wnt signaling pathway, including β-catenin, GSK-3β, Tcf3, c-myc and cyclin D1, to investigate the effects of different concentration of LiCl on Wnt/β-catenin pathwayand interaction between them during HFSCs differentiation; HFSCs were cultured with0,0.1,1,10,50,100,200and400μg/ml to detect toxicity of LiCland to find the best concentration of LiCl to promote HFSCs differentiation.3. LiCl-PLGA nanometer microsphere preparation: Poly (lactic-co-glycolic acid)(PLGA) was selected as microcapsule dressings to load certain concentration LiCl by multiple emulsion-solvent volatilization method. Human fibroblastswere cultured in complete medium. Variant acellular dermal matrix (ADM) wasselected as frame. Amplifiable HFSCs were inoculated at epidermis side of ADM as seed cells, and fibroblasts were inoculated at dermis side of ADM to c onstruct tissue-engineered skin. Using5mM/l LiCl to interfere constructed tissue-engineered skin,5d later, observing seed cells proliferation and differentiationin ADM with HE staining, immunofluorescent staining and immunohistostaining to establish a good foundation for animal experiments.Results1.HFSCs, harvested from human scalp tissue, still had strong abilities of proliferation and multi-directional differentiation after high passage in vitro. Whendensity was1x10~4ml, the cells proliferation and extension were best and the cells growth curve was classical S type.2. As the concentration of LiCl became higher, the proliferation of cells became weaker. The shape of HFSCs changed a lot in K-SFM conditioned medium with LiCl. Each groups had significant difference. When LiCl>10mmol/L,differentiation proportion was large. When LiCl>200μg/ml, cells growth were dramatically suppressed, which showed toxicity of LiCl while inducing Wnt signaling pathway in HFSCs. When LiCl=5mM/l, the transcriptions of β-catenin, GSK3β, Tcf3, c-myc and cyclin D1were up-regulated, while10mM/l, the transcriptions were down-regulated except cyclin D1, which demonstrated that LiCl could activate Wnt/β-catenin signaling pathway during HFSCs differentiation.3. Observed by scanning electron microscope, cells on PLGA-ADM grewwell and secretory extracellular matrix was abundant. As time going on, cells turned to be fused and secretory extracellular matrix became more. HE stainingshowed that, HFSCs grew well at epidermis side of ADM with monolayer row; in LiCl interference group, hair follicle-like dimple was formed at epidermisside and fibroblasts were stick to dermis side of ADM, secreted extracellularmatrix to form a continuous cell layer to encase ADM.Conclusions1. Human HFSCs were harvested conveniently by our modified culture methodwith strong abilities of proliferation and passage.1×10~4/ml was the best density topromote HFSCs proliferation.2. LiCl could significantly promote HFSCs proliferation with a borderline concentration of toxicity.200μg/ml was the best concentration to promote HFSCs proliferation, while high over that, cells growth were suppressed obviously.These might be concerned with activation of Wnt/β-catenin signaling pathway and up-regulated transcription of factors in downstream. Tcf3was effective onmaintain undifferentiation of cells, while transcription of cyclin D1played a critical role in HFSCs differentiation, which was positive correlated with LiCl.3. Nanometer microcapsule loaded LiCl could be prepared quickly by multiple emulsion-solvent volatilization method. PLGA had good compatibility withcells so that human HFSCs could adhere and grow well. Induced by LiCl, seed cells with molecular changes constructed hair follicle-like dimple, which made possibilities of constructing tissue engineered-surrogate with cutaneous appendages.