Establishing Of The Optimization Of Mouse STO Cell Lines Reprogram To Pluripotent Stem Cells

Induced pluripotent stem cells(iPSCs) is coming from the terminal differentiation cellsby importing some exogenous genes which maintain the pluripotency of embryonic stemcells(ESCs).These pluripotent stem cells have mutiplex potential of differentiation and cancontinuously update ability.At present,iPSCs are potential resource which can be used toreplace of the stem cells to treat human diseases and have beening achieved exciting results inmany fields,such as molecular biology,cellular biology,developmental biology, new drugscreening and the clinical cell alternative treatment.But there are some shortcomings in theefficiency and security of iPSCs. Researchers have been looking for efficient and safe way tomake up for the defect of the iPSCs.Research of the induced pluripotent stem cells by inducing the STO cell line. Lentivirusvector FUW-OSKM which contain Oct4,Sox2,Klf4,c-Myc genes were transfected to293FTcells by calcium phosphate method.Then,using the virus infected STO cells after lentiviruswas packaged and the cells were cultured in the ESC media.In order to improving theefficiency of induction,we added valproic acid (VPA, histone acetylation inhibitors) duringthe induced process.The clones were isolated on the fifteenth day infection and cultured onthe none feed layer culture dishs. To identifying the biology characteristic of STO-iPSCs,wedid kinds of detections including morphological observation, alkaline phosphatase dyeing,immunofluorescence stain, western blot detection, real-time quantitative PCR and retinoicacid directional differentiation to nerve cells, etc. Moreover,we compared the morphologicaldifferences between the STO-iPSCs cultured on gelatin coated dishes and matrigel coateddishes after freezing and thawing.Through the biological characteristics analysis,we found the STO-iPSCs have the typicalmorphological characteristics of embryonic stem cells, a positive result by alkalinephosphatase staining,expressed embryonic stem cells marker Nanog and Oct4by theimmunocytochemistry and high quantity of Nanog,Oct4mRNAs by qPCR,especially candifferentiate to nerve cells in vitro.Besides we found that matrigel coated dishes are moresuitable to STO-iPSCs growth.In conclusion,we obtained STO-iPSCs and stablished the STO-iPSCs training system without raising layer.We use cell lines as reprogramming research donor cells for the first time, and inducedmouse embryos fibroblast STO cell lines into STO-iPSCs successfully. Because the cell linescan be cultured-passaged and freezed-thawed long-term in vitro. If we use cell lines as adonor cells in the research of iPSCs signaling pathways and drug testing, it will reduce thecomplex steps of preparing the donor cells, which not only enriches the donor cell types, butalso makes for the generation of iPSCs and helps to experimental research progress.