The Study Of A High-efficient Generation Of Keloid Patient-derived Induced Pluripotent Stem Cells By MRNA Reprogramming System

Objective: Keloid become troublesome for dermatologists by its high recurrence rate and low cure rate,we need to develop new diagnostic and therapeutic techniques for it.At present,the research on induced pluripotent stem cells(iPSCs)is very popular.By reprogramming fibroblasts from the keloid patient's lesions into iPSCs in vitro,we sought new ideas for stem cell therapy for keloid patients.Method: First,we isolated fibroblasts from keloid lesions and examined the expression of disease-associated genes from both diseased fibroblasts and tissues.Then,we produced corresponding iPSCs by transfecting patients' fibroblasts with non-modified mRNA cocktails,expressing OCT4,SOX2,K LF4,c MYC,NANOG,and LIN28 reprogramming factors.Finally,the function of these patient-derived iPSCs were identified by alkaline phosphatase staining,cellular immunofluorescence,pluripotency gene detection,the silence of fibroblastic gene,embryoid body formation in vitro,and teratoma formation in severely immunodeficient mice.Results: Fibroblastic genes were expressed in the fibroblast derived from diseased individua ls.The expression of TGF-?1,COLIA2,FGF7 and MMP14 in keloid tissue was higher than that in normal tissue(P>0.05);The expression of cTGF,TGF-?1 and COLIA2 was found higher in fibroblasts derived from keloid lesions than in normal fibroblasts,with statistical significance(P>0.05).After four times' mRNA transfection,fibroblasts appeared epithelial-like changes,and gradually emerged as dense and well-defined Embryonic Stem cell-like clones.iPSCs from keloid patients showed positive expression of pluripotency markers,while their endogenous pluripotency genes were highly expressed,fibroblastic genes are silenced and can be continuously cultued in vitro.In addition,keloid patient-derived iPSCs can form embryoid bodies in vitro,and the ability of forming teratomas in vivo to differentiate into endoderm,mesoderm,and ectodermal three germ layers has also been demonstrated.Conclusion: Fibroblasts derived from the lesions of patients with keloid can be successfully reprogrammed to iPSCs in vitro.We use an efficient method by mRNA transfection,which has a high reproducibility rate and can provide the basis for the production of individualized genetically modified artificial skin.This achievement not only prevent the immune rejection of xenogeneic skin grafts but also the trauma after autologous skin grafts.In addition,these cells can provide cell models for the study of the pathogenesis of keloids and the application of stem cells for personalized medicine.