Direct Reprogramming Of HUC-MSCs Into Eccrine Sweat Gland-like Cells By Overexpressing EDARADD:An Experimental Study

Objective: To construct human umbilical cord mesenchymal stem cell lines stably overexpressing ectodermal dysplasia receptor-associated death structural domain(EDARADD-hUCMSCs).Their reprogramming into eccrine sweat-like cells was explored by inducing differentiation through conditioned sweat cell culture medium.Methods: 一、Isolation and culture identification of hUC-MSCs:(1)Tissue apposition method to isolate hUC-MSCs.(2)Flow cytometry to identify hUC-MSCs.(3)Lipogenic,osteogenic and chondrogenic three-way differentiation assay to confirm hUC-MSCs.(4)MTS assay to compare the proliferation ability of hUC-MSCs in different generations.二、preparation of stable strain EDARADD-hUCMSCs:(1)1/2 small volume transfection method for hUC-MSCs with overexpression of EDARADD lentiviral vector(2)Puromycin(puromycin)screening of stable strain EDARADD-hUCMSCs.(3)MTS method to compare the proliferation ability of stable strain EDARADD-hUCMSCs and hUC-MSCs proliferation ability.(4)RT-q PCR was performed to detect the transcript levels of EDARADD in stable strain EDARADD-hUCMSCs and hUC-MSCs;Western blot was performed to detect the expression of EDARADD protein in stable strain EDARADD-hUCMSCs and hUC-MSCs.三、Induced differentiation of stable EDARADD-hUCMSCs into eccrine sweat-like cells in conditioned sweat cell medium:(1)Enzymatic digestion to isolate human eccrine sweat tissue masses,and tissue apposition to obtain human eccrine sweat cells.(2)Induced differentiation of stable EDARADD-hUCMSCs into eccrine sweat-like cells by conditioned sweat cell medium.(3)Morphological and statistical analysis of the ratio of the longest axis to the shortest axis of the induced cells,stable EDARADD-hUCMSCs,eccrine sweat cells and hUC-MSCs fitted ellipse,cell cross-sectional area and cell fitted circle approximation ratio.(4)Comparison of the MTS method proliferation ability of induced cells,stable transfer strain EDARADD-hUCMSCs,and hUC-MSCs.(5)RT-q PCR to detect the transcription of m RNA of the focal gene of induced cells,hUC-MSCs CK18,CK19,α-SMA eccrine sweat cells;Western bolt to detect the expression of CK18,CK19,α-SMA protein in induced cells,hUC-MSCs,eccrine sweat cells.Results: 一、(1)hUC-MSCs were successfully isolated and identified from human umbilical cord Huatong gum tissue.(2)Cell proliferation assay showed that the third generation(P3)hUC-MSCs had the best proliferation ability.二、(1)The number of green fluorescent protein(GFP)-positive cells and fluorescence intensity analysis showed that the stable transfer strain EDARADD-hUCMSCs were successfully prepared.(2)The proliferation ability of stable transfer strain EDARADD-hUCMSCs was significantly decreased.(3)The transcript levels of EDARADD and its protein translation were significantly higher in stable strain EDARADD-hUCMSCs compared with hUC-MSCs,indicating that the target gene had successfully incorporated into the genome of hUC-MSCs.三、(1)Human eccrine sweat cells were successfully isolated.(2)The morphological analysis showed no significant difference between the induced cells and human eccrine sweat cells.(3)The proliferation ability of induced cells was significantly decreased compared with that of stable transfected EDARADD-hUCMSCs and hUC-MSCs,suggesting that induced cells differentiated into mature cells.(4)CK18 transcript level was significantly higher in induced cells compared with hUC-MSCs;CK18 and CK19 proteins were expressed in induced cells,but not in hUC-MSCs;CK5,CK19 and hedgehog factor(Shh)proteins were expressed in human eccrine sweat cells,but not in hUC-MSCs,suggesting that induced cells may be directed to differentiate into the secretory part of eccrine sweat tissue cells.Conclusion: In this study,EDARADD,a key gene in the development of eccrine sweat gland,was efficiently integrated into hUC-MSCs and stably expressed.The stable transgenic EDARADD-hUCMSCs were induced to differentiate into eccrine sweat-like cells under conditioned sweat cell culture medium.This study provides high quality seed cells for the cultivation of eccrine sweat-like organs in vitro,and provides new ideas and methods to promote wound healing in patients with large burns and regenerative treatment of eccrine sweat glands.