Efficient Non-integrating And Growth Factors-free Culture System For Induction And Maintenance Of Human Pluripotent Stem Cells

Research Background:iPSCs successfully avoids the problems including scarce sources of embryonic stem cells,immune rejection,as well as ethical controversy.Therefore,iPSC have shown multiple application values in disease modeling,drug screening and cell therapy.However,there are still unclear in the study of somatic reprogramming:1)low reprogramming efficiency,2)the mechanism of reprogramming,3)expensive culture system for iPSC,4)unstable Stem cell culture medium(E8)with growth factor bFGF.These have strongly limited the clinical application of iPSC.Small molecule compounds have been reported to have the ability of promoting somatic cell reprogramming efficiency by replacing reprogramming factors and accurate targeting,and have been playing an important role of overcoming research disadvantages in the iPSC field.Given the discussed above,we focus on screening small molecule combination that can promote somatic cell reprogramming,establishing growth factor-free iPSC preparation systems and culture systems,and exploring the mechanism of somatic cell reprogramming.Research result:1.In the non-integrated free plasmid(OSK,L-MYC,LIN28,shP53)-mediated human cell reprogramming,our study found that dual-specificity tyrosine phosphorylation-regulated kinase(DYRK)inhibitor ID-8 can dramatically enchance the reprogramming efficiency of human fibroblasts by about 13 times and human astrocytes by approximately 17 times.2.We found that using ID-8 and TGF? signaling pathway activator(Kartogenin)can replace the growth factors bFGF and TGF? to maintain the growth of the TRA-1-60+clones,and finally can establish stable iPSC lines.The iPSC lines show high expression of pluripotency-related marker genes similar to ESC.3.Our results have demonstrated that Kartogenin facilitates cell differentiation in long-term culture of stem cells.By further optimizing the growth factor-free pluripotent stem cell culture system,the combination of compounds ID-8,CHIR99021 and SB203580/SB431542 have demonstreted the pluripotency more than three passages.4.According to the recent literatures,the glycolytic pathway has promoted iPSC formation.We discovered that metabolism-related genes were significantly up-regulated by RNA-seq.PDK4 and KCNJ11 correated glycolytic pathway was significantly up-regulated,indicting that ID-8 might potentially accelerate somatic cell reprogramming by promoting glycolysis.In this study,we successfully established a safe and efficient reprogramming system and a growth factor-free high-quality iPSCs preparation and culture system.In addition,reducing the cost of gerenaration iPSC lines and pluripotent stem cells expansion increase a great potential for clinical application of iPSCs.