Application Of R6-feeder In Embryonic Stem Cell Construction

Mouse ES cells are indispensable tools in biomedical research.In order to be more efficient and get better stem cell lines,researchers have been trying to establish embryonic stem cell lines earlier than blastocysts derived using different culture systems.At present,the morula can be pushed from the earliest to the stage of 4-cell,with a high expression of totipotency.However,there is no report about the establishment of ES cell line derived from 2-cell stage embryo.Xist gene has been proved as the key regulator of XCI,and the high expression of Xist gene is positively correlated with X chromosome inactivation.Studies showed that the inhibited Tale-dTF R6 which bound to the Xist first intron region can significantly increasing the induction efficiency from MEF to iPSC,and the efficiency of SCNT and the expression of pluripotent genes Oct4,Sox2,Klf4 and c-Myc in mouse embryonic fibroblasts.It was suggested that R6-feeder could establish an ES cell line with higher totipotency expression and derived from earlier stage than 4-cell.In this study,the R6-MEF cell line was prepared by transfecting MEF with the Xist Tale inhibitory transcription factor R6,and the R6-MEF cell line with high expression of the multipotent gene was screened.The optimum MMC concentration for R6-MEF was found by EDU test and Trypan Blue,and then the R6-feeder was successfully prepared.Using conventional STO-feeder as a control,the E3.5-derived and E1.5-derived stem cell lines were successfully established and their preliminary pluripotency identification was performed.Finally,the extended pluripotency of the E1.5-derived stem cell line was identified.Importantly,it is the first time that the E1.5-derived stem cell line been successfully established,and the pluripotency of cell lines was detected from molecular level,protein level and developmental potential.1.Establishment of an E3.5-derived and E.5-derived embryonic stem cell lines using R6-feederThe R6-MEF cell line was made into an R6-feeder by screening.E3.5-derived and E1.5-derived cell lines were established on R6-feeder,during a series of experimental such as observing the morphological changes,detecting cell growth,karyotype,immunofluorescence,real-time PCR and in vivo teratoma differentiation,found that the E1.5-derived stem cell line established by R6-feeder(2-cell full embryonic cell line is abbreviated as E1.5-derived ES,2-cell single blastomere embryo cell line is abbreviated as E1.5-derived sES)not only expressed the basic characteristics of traditional ESCs(STO-feeder establishment),but also has a slightly superior feature to traditional mouse ESCs in pluripotency identification.2.The pluripotency test of obtain cell lineCompared with the ESCs established by the traditional STO-feeder,the E3.5-derived and E1.5-derived ES cell lines establishedby the R6-feeder show basic characteristics in several aspects: the stem cell line has a typical stem cell morphology;the result of Phosphatase staining was strongly positive;no abnormality in chromosome number of cells;the expression levels of pluripotency genes Oct4,Sox2,Nanog,Klf4 were similar.Compared with ESCs established by traditional STO-feeder,E1.5-derived ES cells showed a higher level proliferation.The expression level of differentiation genes c-Myc,Sox17,Sox17 for E1.5-derived ES cell line was lower than ESCs;and E1.5-derived sES cell line specifically expressed 2-cell genes Tcstv1,Tcstv3.3.The development potential detection of obtain cell lineThe results of teratoma experiments showed that the E1.5-derived sES cells have the ability to differentiate into three germ layer tissues;the technique of preparing chimera proved that the E1.5-derived sES cell line contributes ICM and TE part at the blastocyst stage.More importantly,the E1.5-derived sES cell line contributes to the embryonic ectoderm,extraembryonic ectoderm,anterior placenta cone and primitive endoderm of the E6.5 chimera compared to ESCs and EPSC cell lines.In summary,compared with the ESCs established by the traditional STO-feeder and the 8-cell embryo-derived EPSC cell line,the new R6-feeder of this experiment efficiently established the E3.5-derived cell line,and successfully established E1.5-derived ES and sES cell lines for the first time.In terms of gene expression level and developmental potential,E1.5-derived ES and sES cell lines have a stronger developmental potential,and they are ES cell lines earlier than 4-cell.However,the mechanism of establishing early embryonic stem cells by R6-feeder still needs to be further studied.