Preliminary Analysis Of Side Population Phenotype In Human Epidermal Cells

Background:Skin, the largest organ of human body, is also one of continuously renewing tissue. It has been widely believed that this self-renewal is maintained by epidermal stem cells located in the basal layer and the follicle bugle of epidermis. Epidermal stem sells are tumor initiating cells and gene therapy targets, and they play an important role in injury recovering and homeostasis. So epidermal stem sells have becoming the hot spot in stem cells research domain. The fundamental problem is how to isolate, purify, culture and identify epidermal stem sells from skin tissues. Lately, a new method for enriching stem cells is applying by more and more investigators. The cells isolated by this method are called side population cells, which can efflux DNA binding fluorescent dye, Hoechst33342, and they demonstrate low fluorescence after be excited by the UV light. The SP cells have been discovered from various tissues of human, such as, live, lung, brain, nerve, small intestine and trachea. In addition, they are shown to have stem cell characteristics and enrich the stem cell population.Objective:To identify whether there are SP cells in human epidermal cells and to observe the expression of stem cells marker ABCG2,α₆ integrin andβ₁ integrin on SP cells.Methods:1,Epidermal cell suspension obtaining and cells culture: Epidermal cell suspension was prepared from epidermal sheets harvested from 86 cases 16—25 years old circumcision patients (without urinary infection) by enzymatic treatment. Then epidermal cells were cultured with K-SFM in flasks incubated with IV type human placental collagen. 2,Cell dying and SP analysis: The 2 to 4 generation epidermal cells were obtainedand stained with Hoechst33342 dye, control group was incubated with verapamilin addition. After incubation, PI was added, and cells were analyzed on a flowcytometer.3,ABCG2,α6integrin andβ1integrin expression analysis: Harvested SP cells andanalyzed the expression of ABCG2,α6integrin andβ1integrin on them by a flowcytometer and RT-PCR.4,Statistic analysis: Data were presented as mean values±SD and analyzed byMann-Whitney U test of SPSS software. P values less than 0.05 were consideredto be significant.Results:1,Partial were adhesive to the vessel walls after 24 hours of seeding. Cellmonolayers were observed as slabstones 5 days later, with polygonal appearance,transparent cytoplasm and large nucleus. Epidermal cells reached confluence 10days later.2,SP cells had been detected in human epidermal cells which accounted for0.2%～0.3% of total epidermal cells, decreased(<0.01%) by treated with verapamil.3,Only a small part of cells expressed ABCG2,α₆integrin andβ₁integrin of SP cellsdetected by both flow cytometer and RT-PCR.4,According to statistic analyze, no significant difference was found in the positiverates between SP cells and total human epidermal cells (P>0.05).Conclusion:1,SP cells had been detected in human epidermal cells which accounted for 0.2%～0.3% of total epidermal cells.2,Compared with the total human epidermal cells, the expression of ABCG2,α6integrin andβ1integrin on SP cells was not upregulated.3,SP cells sorted by FACS combined with Hoechst33342 were not epidermal stemcells. This method can not enrich epidermal stem cells from human totalepidermal cells. Future research will be required to make the method fit for sortingepidermal stem cells.