| BackgroundSkin,as the largest organ in the human body,is the first barrier against external damage and plays a key role in maintaining the homeostasis of the body’s internal environment.It is also the organ most susceptible to burns and scalds.In large and deep skin injuries,such as burning wounds,the patient’s skin completely loses its ability to repair itself,and the extensive exposure of the wound is easy to cause complications such as infection,hypermetabolism and internal environment disorders,which could be further aggravated to induce sepsis,and the patient even dies due to multiple organ failure.For many years,the culture of skin cells in vitro has been the cornerstone of dermatological research.Amniotic epithelial stem cells(AESCs)have been identified as a potential alternative to keratinocytes(KCs)among tissue engineered skin alternatives used to treat skin injuries.However,their clinical use is limited because the similarities and differences between AESCs and KCs remain unclear.In addition,skin replacement systems often lack the full functionality of natural skin.Notably,skin appendages(such as hair follicles and sweat glands)remain a challenge to generate or maintain in cell cultures and rebuild in damaged skin.Emerging skin organoids hold promise for the construction of three-dimensional skin tissues containing skin appendages in vitro.MethodsAmniotic epithelial stem cells,keratinocytes,transcriptomic analysis,and functional assessment were used to understand the commonalities and differences between AESCs and KCs.The differences and similarities between amniotic epithelial stem cells and keratinocytes were compared from the aspects of gene level,protein level,transcriptome sequencing and melanin particle transfer in co-culture with melanocytes.The efficacy of amniotic epithelial stem cells in three-dimensional skin construction was also evaluated.The possibility of constructing artificial skin with amniotic epithelial stem cells was explored by the experiment of hair follicle reconstruction.Then direct reprogramming was used to induce the fate convergence of amniotic epithelium towards keratinocytes.In addition,pluripotent stem cells were used to induce skin organoids and detect early related molecular markers.Results1.RNA sequencing has shown that AESCs are involved in several cuticle-related biological processes shared by KCs,including transcriptome analysis that reveals similarities in molecular characteristics between AESCs and KCs.Especially in the biological processes related to epidermis(epidermal development,epithelial cell morphogenesis,regulation of epidermal development,cell connective tissue,molting cycle and hair cycle),and the similarity to early immature KCs are higher than that of adult KCs.2.It is observed that AESCs are heterogeneous,and some have mixed mesenchymal and epithelial characteristics that differ from KCs.3.The ability of AESCs to interact with melanocytes and phagocytic melanocyte bodies and reconstruct colored 3D skin equivalents when co-cultured with human melanocytes and fibroblasts.AESCs can also interact with melanocytes and phagocytose transferred melanosomes in 2D and 3D co-culture systems.4.Overexpression of TP63 and activation of NOTCH signal can promote the dryness of AESCs and improve their differentiation characteristics,respectively,bridging the gap between AESCs and KCs.These changes cause the fate of AESCs to converge with that of KCs.5.Skin organoids could be successfully induced by the three-dimensional induction system.Skin ectodermal markers PAX6 and SALL3 are highly expressed,and early skin markers TP63 and KRT5 are also highly expressed.ConclusionsHuman amniotic epithelial cells are similar but heterogeneous to keratinocytes,and improved reprogramming strategies(e.g.,using small molecules)may facilitate further modulation of human AESCs for skin regeneration and three-dimensional skin construction;The early structure of skin organoids showed preliminary appearance. |
