Small molecules facilitate single factor?mediated sweat gland cell reprogramming

Background:Large skin defects severely disrupt the overall skin structure and can irreversibly damage sweat glands(SGs), thus impairing the skin’s physiological function. This study aims to develop a stepwise reprogramming strategy to convert fibroblasts into SG lineages, which may provide a promising method to obtain desirable cell types for the functional repair and regeneration of damaged skin.Methods:The expression of the SG markers cytokeratin 5 (CK5), cytokeratin 10 (CK10), cytokeratin 18 (CK18), carcinoembryonicantigen(CEA),aquaporin5(AQP5)and α-smoothmuscleactin(α-SMA)wasassessedwithquantitative P CR (qPCR), immunofluorescence and flow cytometry. Calcium activity analysis was conducted to test the function of induced SG-like cells (iSGCs). Mouse xenograft models were also used to evaluate the in vivo regeneration of iSGCs.BALB/c nude mice were randomly divided into normal group, SGM treatment group and iSGC transplantation group.Immunocytochemical analyses and starch-iodine sweat tests were used to confirm the in vivo regeneration of iSGCs.Results:Ectodermal dysplasia antigen (EDA) overexpression drove human dermal fibroblast (HDF) conversion into i SGCs in SG culture medium (SGM). qPCR indicated significantly increased mRNA levels of the SG markers CK5, CK18and CEA in iSGCs, and flow cytometry data demonstrated (4.18±0.04)%of iSGCs were CK5 positive and (4.36±0.25)%of iSGCs were CK18 positive. The addition of chemical cocktails greatly accelerated the SG fate program. qPCR results revealedsignificantlyincreasedmRNAexpressionofCK5,CK18andCEAiniSGCs,aswellasactivationoftheduct marker CK10 and luminal functional marker AQP5. Flow cytometry indicated, after the treatment of chemical cocktails,(23.05±2.49)%of iSGCs expressed CK5+and (55.79±3.18)%of iSGCs expressed CK18+, respectively. Calcium activity analysis indicated that the reactivity of iSGCs to acetylcholine was close to that of primary SG cells [(60.79±7.71)%vs.(70.59±0.34)%, ns]. In vivo transplantation experiments showed approximately (5.2±1.1)%of the mice were sweat test positive, and the histological analysis results indicated that regenerated SG structures were present in iSGCs-treated mice.Conclusions:WedevelopedaSGreprogrammingstrategytogeneratefunctionaliSGCsfromHDFsbyusingthe single factor EDA in combination with SGM and small molecules. The generation of iSGCs has important implications for future in situ skin regeneration with SG restoration.