| Burn is a common traumatic disease in the clinic,which is caused by factors of heat,chemical reagents,electricity,damage symptoms of tissue and organ.Severe burns cannot only cause organ failure and series of complications,but also may endanger the lives of patients.The key to regenerate and repair the skin is the presence of epidermal stem cells on the epidermis of the skin.But large-areas and deep burns are tending to destroy the epidermal stem cells of the skin,lead to difficulties in repairing the wound.Traditional treatment programs include autologous,allogeneic or xenogeneic skin grafting,but the results are not always satisfactory.Skin wound repair requires skin substitute built by tissue engineering,epidermal stem cells are seed cells commonly used in skin substitute.However,epidermal stem cells have low efficiency of extraction and purification,cannot be obtained in large quantities.Therefore,how to enlarge the epidermal stem cells in vitro is the main point of skin tissue engineering research.In 2006,Japanese scientists obtained by the method of involved the four transcriptional factors(c-Myc,Klf4,Sox2 and Oct-3/4)associated with the diversity of embryonic stem cell into mouse fibroblasts cells,which is called induced pluripotent stem cells(iPSCs).Because iPSCs avoid the ethical and legal problems caused by embryonic stem cells,and readily available,it has become an important cell source of tissue engineering.Over the past decade,iPSCs have turned out to be able to differentiate into epidermal stem cells through the induction of cytokines.In the existing methods of inducing epidermal stem cell differentiation,there is amniotic membrane-induced,altered culture environments,frequent replacement of culture medium,etc.The limitations of these culture methods are uncontrollable and fussy operation.In this paper,we focused on the use of the defined ingredients in the medium of epidermal stem cell to improve the efficiency of differentiation and the proliferation of epidermal stem cell.In this paper,I will use inverted microscope(IM),immunocytochemistry(ICC)to discuss the process of preliminary study of human iPSCs(human iPSCs,hiPSCs)differentiate into epidermal stem cells.In this study,iPSCs were cultured with mTeSR 1 complete medium to avoid contact with animal immunoglobulin.After cell passage,iPSCs were divided into the experimental group and control group.The experimental group was co-cultured with RA and BMP4.The control group was induced by amnion extract(AETRA).Two different groups replaced the medium every day,using defined keratinocyte serum free medium(DK-SFM)as a differentiation medium supplemented with RA and BMP4.The cells we obtained were identified by morphological observation and immunocytochemistry.At the same time,a normal epidermal stem cells group was established to observe the difference between differentiated epidermal stem cells and normal human epidermal stem cells.The results showed that the number of epidermal stem cells in the experimental group was significantly more than that in the control group after 6 days of induced differentiation.Immunofluorescence technique was used to detect the expression of CK19 and α6 integrin in epidermal stem cells after 6,8,10 and 12 days The expression of CK19 and α6 integrin in the experimental group were significantly higher than those in the control group.It was found that the efficiency of epidermal stem cell differentiation was higher in the experimental group than in the control group.Compared with normal epidermal stem cells,there was no significant difference in morphology and characteristics between the experimental group and the normal epidermal stem cells.In contrast,amniotic membrane co-culture of epidermal stem cell is inconsistent in morphology and a small number of cells appear differentiation,the expression of CK19 and α6 integrin is also in low level.In conclusion,Epidermal stem cell differentiation medium(ESCDM),which supplemented with RA and BMP4,shortened the time required for the differentiation of hiPSCs into epidermal stem cells,increased the efficiency of epidermal stem cell differentiation and the rate of proliferation and maintained the shape and characteristics of normal epidermal stem cells. |
