Differentiated Epidermal Cells Regain The Ability To Regenerate A Skin Equivalent

Background and ObjectivesScar outcome will inevitably appear in patients with severe and large area burns treatedwith conventional surgery. Epidermal stem cells are important for tissue engineering, butseverely burned patients remained extremely limited normal skin for stem cell therapy. Thenumber of not sufficient epidermal stem cells in normal skin only accounts for1%-10%ofskin base layer cells. This greatly limits the clinical application of epidermal stem cell tissueengineering technology. Is there any other method that can completely solve the shortage ofepidermal stem cells, produce them enough for transplant, and even realize "perfect woundhealing" eventually?Cell dedifferentiation, a popular phenomenon founded recently, is a good way to produceabundant epidermal stem cells. Mature terminally differentiating epidermal cells can reverseto their precursor cells by dedifferentiation, that to say, Epidermal cells can reverse from "old"differentiated state to the not fully differentiated "young" state, even "naive" state with thecharacteristics of epidermal stem cells. These dedifferentiated yong epidermal cells can beused for the severe wound mentioned above. How can we actively induce differentiatedmature cells into lower differentiated immature state with a regenerative ability or directlyinto dedifferentiated epidermal stem cells with a strong regenerative ability?The answer is obviously YES. It was reported that epidermal cell dedifferentiation hadbe induced by some specific factors. Therefore, it is important to establish a reliable andstable cell differentiation way to solve the stem cell shortage. Dedifferentiation isalternatively a moral and ethical way for disease therapy, with no genetic incompatibility ortissue rejection risk.Several signaling pathways have been implicated in dedifferentiation of mature cellssuch as the Wnt/β-catenin, p38, extracellular signal-regulated kinase (ERK) and Janus kinase(JAK)/signal transducer and activator of transcription (STAT) signaling pathways. However,the pathway that induces differentiated epidermal cells to reverse into an immature state with a regenerative ability has not been reported. There is increasing evidence that Wnt signaling isnecessary for normal skin development, and a key effector in the Wnt signaling pathway isβ-catenin that is involved in maintenance of the progenitor cell population in skin. Studieshave also shown that elevated β-catenin levels lead to proliferation of epidermal stem cellsand induce trans-differentiation into hair follicle stem cells.Based on our early epidermal stem cell research work, we continue to explore and try toestablish a stable skin stem cell culture system in the first part of the present study, which isimportant to further cell differentiation research. In the following part, the effect of β-cateninactivation on the characteristics of differentiated epidermal cells was investigated. First,β-catenin expression was induced by applying LiCl and a highly specific GSK-3β inhibitor.Then, the effects of β-catenin activation on morphological, phenotypic and growthcharacteristics of the dedifferentiated cells were observed. Finally, the ability ofdedifferentiated cells to regenerate a skin equivalent in vitro was investigated.MethodsDifferentiated human epidermal cells were isolated from epidermal sheets by differentialadhesion. Human foreskin specimens were digested at4℃with2mg/ml protease for10-12hand then the epidermis was isolated from the dermis. The isolated epidermis sheets were cutinto pieces, digested with0.25%trypsinase for20min at37℃and made into single-cellsuspensions. Then observe characteristics of epidermal stem cells under light and electronicmicroscopy, and immunohistochemical expression of epidermal stem cell series surfacemarkers.For the second part of the experiment, differentiated human epidermal cells, isolatedfrom epidermal sheets by differential adhesion, were plated into6-well plates and divided intothree groups. LiCl and a highly specific GSK-3β inhibitor were added to the desired wells,and sodium chloride was added to the control without inhibitors according to the experimentalconditions. First, β-catenin expression was induced by applying LiCl and a highly specificGSK-3β inhibitor. Then, the effects of β-catenin activation on morphological, phenotypic andgrowth characteristics of the dedifferentiated cells were observed. Finally, the ability ofdedifferentiated cells to regenerate a skin equivalent in vitro was investigated. ResultsThe isolated epidermal cells exhibited the characteristics of epidermal stem cells, smallround pearl shape with a large nuclear-cytoplasmic ratio and gradually changing into squareslab shape. In addition, less organelles were observed under electronic microscope. Twoweeks later the original cultured cells covered the bottle bottom. Cells cultured in growthpromoting factor DMEM/F12stick to the bottom24h later and covered the bottom in3-4d.The original generation epidermal stem cells immunohistochemically expressed epidermalstem cell series surface markers except for CK10.Elevated expression of β-catenin in the nuclei of epidermal cells cultured with LiCl orGSK-3β inhibitor. β-catenin expression tested by Western Blot and measured by densitometricanalysis. There are striking differences in morphology between differentiated epidermal cellsand epidermal stem cells. The former are large flat-shaped cells with a smallnuclear-cytoplasmic ratio and the latter are small round shaped cells with a largenuclear-cytoplasmic ratio. These results demonstrated that the induced cells had morphologiccharacteristics of epidermal stem cells. The expression of CK10, CK19and β1integrin incultured epidermal cells in the presence of LiCl or GSK-3β inhibitor was studiedimmunohistochemically. LiCl or GSK-3β inhibitor significantly decreased the number andproportion of CK10positive cells. In contrast, CK19and β1integrin expression was enhancedby the addition of LiCl or GSK-3β inhibitor to the culture medium. Real-time PCR analysisrevealed that the induced cells were>4-6-fold enriched for both Oct4and Nanog comparedwith control. Cells in the control group grew dispersedly, but the cells treated with LiCl orGSK-3β inhibitor obviously formed colonies and the colonies gradually enlarged. However,there was no obvious difference in clonogenic capacity between LiCl or GSK-3βinhibitor-treated cells. In the induced groups, more cells were in the S and G2/M phase of thecell cycle compared with the control group. In the LiCl or GSK-3β inhibitor groups more cellswere in the proliferative phase. LiCl or GSK-3β inhibitor-induced cells have long-termgrowth potential. After11d, we found that differentiated epidermal cells could not form anepidermal monolayer, the induced epidermal cells and epidermal stem cells were capable ofregenerating a stratified epidermal layer on the dermal equivalent. ConclusionsEpidermal stem cells can be effectively isolated by differential adhesion after early stageelectively digesting connections between dermis and epidermis. Low carbohydrate DMEM/F12(10%FBS) is a high-efficient medium for epidermal stem cells culture to keep theircharacteristics.Differentiated epidermal cells regain the characteristics of epidermal stem cells byincreasing β-catenin expression. LiCl and GSK-3β inhibitor treatments resulted in anaccumulation of β-catenin in the nuclei of differentiated epidermal cells. The dedifferentiatedcells exhibited morphological, phenotypic and growth features of epidermal stem cells. Inaddition, dedifferentiated cells regenerated a skin equivalent similar to that of epidermal stemcells. These data suggest that abundant epidermal stem cells can be generated with an efficientdedifferentiation process by manipulating the expression of β-catenin. The Wnt/β-cateninsignaling pathway plays an important role in skin wound repair and regeneration...