Multipotential Differentiation Of Hair Follicle Stem Cells And The Especial Capability Of Differentiating Into Sweat Gland Cells

Stem cells play important roles in organogenesis and development of tissues as well as repair and regeneration of organs. As the significant development of the regenerative medicine, stem cell therapies have been used gradually and effectively in clinic. Up to now, stem cells had become a hot research topic in china as well as all over the world and had been used increasingly to diagnose and cure many refractory diseases.The following tests are mainly about a kind of adult stem cells-hair follicle stem cells(HFSCs),in which investigated the characteristics and applications of HFSCs. The results confirmed the advantages of HFSCs in the cell therapies which indicated an ideal stem cell source for regenerative medicine.Objectives:1.To isolate and culture HFSCs in vitro; to establish the culture and proliferate models in vitro; to determine the possibility for the following tests;2.To determine the multi-differentiation potential of HFSCs by the inducing tests in which the HFSCs would be induced into sebaceous gland cells and epidennal cells;3.To confirm the capability of HFSCs in differentiating into sweat gland cells (SGCs) by the special co-culture system;to determin the function of the induced-sweat gland cells(i-SGCs) by the transplantation tests;4.Using HFSCs as the seed cells to construct tissue engineering skin which would be better than epidermal stem cells(ESCs) in the structures and functions.Methods:1.Isolated and cultured the HFSCs by using two different ways,and made a comparison between the two groups in cell activities and cell proliferation; high proportion of HFSCs were selected by using type collagen IV adhesion method.HFSCs were incubated in the lower compartment of the transwell in company with mitomycin C inactivated fibroblasts as a feeder layer (FL).2.Established two different induction systems in order to induce the HFSCs transfomi into sebaceous gland cells and epidermal cells;after induction, we identified the cell phenotypes by using the cell special molecular markers and Oil Red O staining;3.The designed co-culture microenvironment in the transwell was consisted of two critial factors:heat shock SGCs and the dermis-like mesenchymal tissue, which appeared independently in the two control groups,the dermis-like mesenchymal tissue was consisted of isolated dermal mesenchymal stem cells(d-MSCs)、Matrigel and FBS;then determined the cell activities and proliferative abilities by using trypan-blue exclusion test as well as Edu (5’-ethynyl-2’-deoxyuridine) incorporation;4.Produced third-degree burn wounds on both posterior paws of the BALB/c nude mice by a minitype scald-causing meter. The purified i-SGCs were transplanted into the full-thickness scalded wounds of nude mice,after4weeks,the reconstructed sweat gland(SG)-like structures were identified by immunohistochemical and immunofluorescence analysis;5.Determined the EDA/EDAR genes and proteins in i-SGCs by using western bolt and RT-PCR、real-time fluorescence quantitative PCR analysis;6.Took the ESCs and HFSCs as the seed cells to construct tissue engineering skin,and observed the tissue structures by HE staining, found and confirmed the hair follicle-like structure by CD29、CK19immunohistochemistry staining.Results:1.The cultured HFSCs expressed CD200、CD29and CK15confirmed by immunohistochemistry staining、immunofluorescence staining and flow cytometry analysis;2.After the directed induction, HFSCs finally differentiated into sebaceous gland cells and epidermal cells;3.Cells in control group1were failed in transforming into SGCs, however the other two groups had made outstanding achievements.the experimental group was more effectively than control group2in the differentiation which with a significant difference. The genes expression of i-SGCs in experimental group were higher in EDA and EDAR, meanwhile be lower in CD29and CD200.Besides,the EDA and EDAR expressions in experimental group were stronger than that in normal HFSCs, the difference between them was significant.4. According to Western blotting、RT-PCR results, EDA gene and EDAR gene as well as their proteins in experimental group expressed significantly higher than the other two control groups,and these genes and proteins aslo positive in the control group2but were less evidently, in the control group1and normal HFSCs EDA and EDAR genes were negative.5.After4weeks transplantation,all the scalded wounds were healed compeletly,in the i-SGCs injected group the SG-like structures appeared in our sights by HE staining, most of which located at the dermis nearby the basal layer looked like the sweat gland duct. According to immunohistochemistry and immunofluorescence results,these newborn structures were both positive in anti-human CEA and anti-human CK14. By contrast, in the Nacl injected control group can rarely find any reconstructed SGs, so the expression of CEA and CK14was negative naturally, this is a dear indication that the i-SGCs were helpful to the SG regeneration.6. The tissue engineered skin were composed of dermal and epidermal, the structures of tissue in B group was better than A group.By HE staining we could find the hair follicle-like structures near the dermal.By immunohistochemistry staining the CD29+and CK19+cells were distributed mainly over the basement membrane,furthermore in the B group, both of CD29+and CK19+cells were located in the dermis.Conclusions:1.HFSCs would be isolated and cultured in vitro under our culture system;2.The HFSCs are multipotential and could be induced into sebaceous gland cells and epidermal cells in vitro;3.HFSCs are capable in differentiating into SGCs which promises a potential stem cell reservoir for future use; our special co-culture microenvironment is brilliant for HFSCs differentiating,which may relate to the activation of EDA and EDAR genes; the induced SGCs are functional and could work well in the regeneration of SGs.4.HFSCs did better than ESCs in constructing tissue engineering skin.