Isolation And In Vitro Culture Of Mouse Embryonic Stem Cells Of KM Species

Objective The target of this expriement is to investigate how to isolate totipotentmouse embryonic stem cell (ES cells) from blastocysts of Kunming species mouseand to analysis some biological characteristics of ES cells.Methods The blastocysts of 3.5d from Kunming species after over-ovulationwere collected and cultured on mouse embryonic fibroblast (MEF) feeder layerprepaerd from 13.5d-old embryo.The culture medium is DMEM with 4500mg/lglucose , supplemented with 15% fetal bovine serum (FBS),2mmol/l L-glutamine,0.1mmol/l β -mercaptoethanol , 1000IU/ml mouse leukemia inhibitory factor(mLIF),penicillin and streptomycin. Inner cell mass (ICM) which has prolifered for2~3 days was mechanically isolated with self-made glass neddles. ICM was removedand transferred into liquid drops of 0.25%Trypsin-0.04%EDTA.After incubation for3~5minutes, the disaggregated clumps of ICM were implanted into newly-preparedfeeder layer and were cultured continously. The changes of cell colonies wereobserved and recorded. ES-cells like colonies were contionuly disaggregated andcultured. AKP staining to testify those cell colonies was carried out. Blastocysts thatwere flushed out from mouse uterus at the same time were divided evenly into 7culture dish containing the same feeder layer. (5 blastocysts in one dish).The changesof AKP staining of ICM were testified every day.Results ES cells colonies were obtained. ES cells differentiated intocardiomyocytes which could contrast spontaneously and rhythmically in vitro. AfterAKP staining, ICM of early stage and ES cells colonies exhibited obvious AKPpositive. ICM which prolifered for 2~5days expressed strong AKP activity, but ICMwhich prolifered for 7 days began to differentiate and results of AKP staining werenegative. 4Conclusions ①ICM which prolifered less than 5 days can be used to isolate EScells.But ICM which prolifered for more than 7 days begin to differentiate and cann,tbe used to isolate ES cells. ②ES cells colonies which are totipotent can be isolatedfrom ICM of blastocyst of Kunming species mouse. ③ES cells can be induced todifferentiate into cardiomyocytes that can beat spontaneously in certain condition invitro.