Study Of Wnt Signaling Pathway-mediated Regulation In The Differentiation Of Hair Follicle Stem Cells

Background & Objective Hair follicle is one of most important appendages in skin. The discovery and successful culture of hair follicle stem cells (HFSCs) in vitro provided a newly idea and method for preparing a new kind of tissue-engineered skin used in clinical cutaneous deficiency. Nowadays people have acknowledged that the progenitor cells of HFSCs were situated in the outer root sheath of upper hair follicle named the bulge region, where located neared the attachment point of arrector pilli muscle. A great deal of studies showed that HFSCs were defined as multipotential stem cells, with pluripotent differentiation potential and the capability for self-renewal, could not only differentiate into the main phenotype of follicular keratinocytes, but also differentiate into epidermic cells, nerve cells, osteoblasts and so on. The HFSCs involved in the formation of hair follicles ,sebaceous gland and the interfollicular epidermis,which played an important role in the hair follicle growth cycle and morphology formation process. At the resting state, the cells showed slow periodic. When HFSCs under external stimuli, they demonstrated a strong capacity of proliferation and colony formation .In previous, although many studies about HFSCs had been done,but there were still have a great deal of works need to be done because their exact characteristics were still unknown. In our study, we used a bi-enzymatic digestion method to get integrity of the hair follicle structure first, then harvested the ORS(outer root sheath) cells by microdissection method on the basis of anatomic structure. The successful culture of HFSCs laid the foundation for further study of cells physiological function, differentiation and biological characteristics.Recent studies had showed that Wnt/β-catenin signaling pathway played important roles in the HFSCs growth and development. As a downstream signal molecule of Wnt signaling pathway,β-catenin controlled essential steps in many aspects, such as embryogenesis ,neoplasia and required for the initiation of hair follicle development. Binding of wnt proteins to their receptors could activate the signaling pathway and cause stabilization of intracellularβ-catenin. Accumulatedβ-catenin may transfer into the nucleus and co-activated with T cell specific factor (Tcf)/lymphoid enhancer binding factor l (LEF-l) transcription factor, which served as transcriptional factors to activate various target genes such as cyelinD1. Besides endogenous autogene factors, there were still some other factors played synergetic effect atβ-catenin level such as cell growth factor, chemical material and other foreign signals. As regulative factor,β-catenin was essential for fate decisions of skin stem cells and required for the initiation of hair follicle development. None the less, the concrete relationships between Wnt/β-catenin signaling pathway and other signal molecule factors had not been fully elucidated. In order to make it clear about the roles ofβ-catenin on the proliferation and differentiation of hair follicle stem cells,this research was designed from fo1low aspects:Method1. HFSCs were cultured in vitro and the biological feature was observed;And the characteristic of cells growth at different seeding density were detected by Hoechst assay. Then filtered out the best subculture density in vitro. HFSCs and their differentiated cells were identified by Using immunofluorescence staining.2. HFSCs were cultured with lithium chloride (LiCl) and keratinocyte growth factor (KGF) in different concentration gradient(0mmol / L, 0.5mmol / L, 1.5mmol / L, 10mmol / L, 25mmol / L LiCl and 0ug/ L, 10ug/ L, 25ug/ L, 50ug/ L, 100ug/ L KGF). The effect of different revulsion on cell proliferation was explored by Hoechst assay to determine the optimal concentration. The ability of HFSCs differentiate into epidermal cells or papilla cells also detected by immunofluorescence. The expression ofβ-catenin, APC, Axin, GSK-3βand LEF-1 mRNA at different time point were investigated by real-time quantitative polymerase chain reaction, respectively. Ultimately, we could explore the role of Wnt/β-catenin signaling pathway and the relationships with other correlated signal molecules at the HFSCs differentiation processing.3. The experimental study of poly lactic-co-glycolic acid (PLGA) nanocapsules in tissue-engineered skin construction: The KGF-loaded PLGA nanocapsules were prepared by double emulsion-solvent evaporation and freeze drying methods as cytokine carrier material. Then cross-linked nanocapsules to the acellular dermal (ADM) serve as scaffold materials. Seeded HFSCs on the ADM in vitro and cocultured the formation of cells graft to construct the tissue-engineered skin. At last we evaluated the graft by DIO staining, cells activity staining and SEM.Results1.HFSCs were isolated from Human scalp and were purified and characterized after being cultured in vitro. After continuous subculture,HFSCs retained strong reproductive activity and multipotency. The best cells inoculation density was about 1×10~5ml.2. With LiCl concentration increasing, the capacity of proliferation was inferior than the control group,while the KGF group had a good proliferation correspondingly. In our study, we could find that the morphology and cell differentiation level of HFSCs change obviously in mediums containing LiCl, especially the concentration exceed 10mmol/L. At the same time, the effect of KGF on cell differentiation was not obvious. The differentiate potency of HFSCs induced by LiCl and KGF were observed in vitro may be associated with the Wnt pathway,and theβ-catenin and its target gene on the process of oriented differentiation played a key role.3.HFSCs grew well on the surface of PLGA-ADM material observed by SEM. With the culture time went on, secretion of extracellular matrix became rich and gradually increased. DIO staining showed HFSCs uniformly distributed on the surface of dermal substitute. Cell growth curve showed that HFSCs grew well on the material and cell masses were found.Conclusions:1. HFSCs were cultured successfully in vitro and the best cells inoculation density was about 1×10~5ml. 2. The effect of Wnt/β-catenin signaling on HFSCs proliferation and oriented differentiation may induced by LiCl and KGF.3. This research intends to provide a theoretical ground for constructing new type of tissue-engineered skin with hair follicle.