Optimization Of MRNA-induced Human Ipscs Reprogramming Process And Promotion Of Naive Pluripotency Trnasformation

IPSCs(Induced pluripotent stem cells)play an important role in the establishment of disease models,the application of regenerative medicine,and the drugs discovery,but the bio-safety of iPSCs had plagued the development of iPSCs application.With the improvement of induction strategy,mRNA-induced method had become the safest and effective method s:ince no any exogenous genes intergrate into host genome,however the current mRNA-induced reprogramming process was time consuming and relatively expensive.In order to solve the problems of the above mRNA induction system,we attended to optimize and establish a more time and money saving and easier operating induction system.At the same time,under the existing culture system,we also investigated the effects of the feeder layer,the small molecule compounds and the change of the cellular metabolic mode on human iPSC naive pluripotency transformation and laid the foundation for establishing the stable state naive hiPSCs cell lines under our laboratory platform.(1)The optimization of mRNA-induced human iPSCs reprogramming system Nanog mRNA was added to the original induction system(Oct4,Sox2,Klf4,c-Myc mRNA).The results showed that the number of mRNA induction was reduced from 12(days)to 5(days)and the cost of whole reprogramming was reduced by nearly 50%.During the early reprogramming,we used the Pluriton Medium supplements to replace NuFF(human newborn foreskin fibroblasts)-Conditioned Pluriton Medium which could simplify the entire reprogramming operating process and make the operating system easier and more stable as well as effectively.Using the optimized mRNA reprogramming platform,we have already obtained three hiPSCs cell lines which can stably pass more than 10 generations.(2)The preliminary identification of hiPSCs biological characteristics The results showed that hiPSCs had activity of alkaline phosphatase(AP),all the 6,7,8,9 generation hiPSCs expressed OCT4,SOX2,NANOG,C-MYC,DAPP2,DAPP4,DAPP5,GDF3,REX1,TRET,kLF4,and FGF pluripotency genes.The results of immunofluorescence assay also showed that the protein of OCT4,SOX2,NANOG as well as the surface specific antigens SSEA-3,SSEA-4,Tra-160 and Tra-181 were detected in the hiPSCs.(3)The transformation of naive pluripotency on hiPSCs The results showed that feeding layer could significantly improve the related epigenetic modified genes H3K4me3 and H3K9ac,and surface-specific antigen SSEA-4 and pluripotency genes OCT4,SOX2,NANOG,thus maintained pluripotency and conversed primed to Naive stage.2i/LIF could significantly increase the expression of H3K4me3,H3K9ac,surface specific antigen SSEA-4 and Naive marker genes NANOG,KLF2,SALL4 by regulating the WNT and MAPK signaling pathways,thereby promoted pluripotency and conversed primed to Naive stage.2i/LIF medium supplied with PS48 could strengthen the cell anaerobic glycolysis to change the energy metabolism of hiPSCs,significantly increase the expression of H3K4me3,H3K9ac,surface-specific antigen SSEA-4 and Naive marker genes REX1,NANOG,KLF2,SALL4,STELLA pluripotency gene expression compared to 2i/LIF medium,which promoted the maintenance of pluripotency and Naive transformation.The above results showed that we had established a simple and time-saving as well as cost-saving optimized mRNA reprogramming technology platform,which also promoted human iPSCs Naive transformation by supplied with feeder layer,2i/LIF and PS48.