Studies On Isolation And Cloning Of Pluripotent Stem Cells From ICR Mouse Embryos

Pluripotent stem cells have been derived from two embryonic sources. Embryonic stem (ES) cells were derived from the Inner Cell Mass (ICM) of preimplantation embryos, and embryonic germ (EG) cells from primordial germ cells (PGC). Both ES and EG cells were cultured to determine the factors affecting on isolation, cloning and passage of pluripotent stem cells from ICR mouse embryos. The purpose of this study was to establish ES or EG cells lines in the future for further research. The results attained were as follows: 1.Derivation of Embryonic stem cells from ICR mouse preimplantation embryos.Mouse blastocysts and morulas were cultured in Rat Heart Conditioned Medium (RH-CM).As far as the following indexes attaching ratio, ICM growth ratio and ES clone formation ratio are concerned, the choice of blastocysts is much better than morulas (P<0.05), therefore mouse blastocysts were more suitable than morulas to get ES cells. Interval between two passages (38 h) of expriment groups was significant higher than control group (P<0.05). It was showed that RH-CM not only could maintain ES cells in the undifferentiated state, inhibiting the spontaneous differentiation effectively, but also promote adherence and proliferation of mouse ES cells. The effect of the three culture systems on formation of ES colonies at primary passage was not remarkable but significant in. ES colonies formation after passage and the highest times of passage. The combination of RH-CM with mitotically inactive MEF feeder layer, was the best culture system.The concentration of FBS (fetal bovine serum) and NCS (newborn calf serum) was influential on the culture process of MEF (mouse embryonic fibroblast) and ES cells. The result indicated: the basic medium supplemented 15%~20% NBS or FCS was much easier to culture MEF and ES cells in vitro. Medium which is serum-free or in the low concentration serum inhibit the proliferation of cells. A certain degree of increase serum concentration can promote the growth rate as well as prolong the life. The feeder layer cellswere provided by MEF, MUE (mouse uterus epithelium) and RMC (rat myocardial cells). During the course of the primary culture and passage of ES cells, co-culture of mouse ES cells and MEF was the best. In this research, cell digestive juice with 0.125% trypsin+0.02%EDTA was relative gentle to cells. 2.isolation and cloning of mouse embryonic germ cells of ICR speciesPrimordial germ cells (PGC) were isolation from 8.5-15.5 dpc (days post coitum) embryos. EG cell lines with the characteristic of murine ES cell line were established and continuously cultured to 6th passage. To a certain extent, the paper showed that there was not sure species-dependent relation between the in vitro culture and the establishment of the lines. In the experiment, in case of the proliferative capability of EG cells, the composition of 0.25% trypsin+0.04EDTA was optimal. The growth behavior of ICR murine EG cells from PGC was observed. With the increase of passage, the total of colonies decreased, but the number of classical nest-like or island-like EG cell colonies relatively increased. The growth of EG cells was dependent on the feeder layer. PGC and EG cells derived from PGC characterized by a typical colony appearance with very scarce individual cell. EG cells of the 2th and 4th passage were AKP (alkaline phosphate) positive. When cultured on degenerated feeder layers or in suspension, EG cells formed embryoid bodies (EBs) in vitro. The EBs maintained for a total of 2-3 days with a colony or individual appearance could be further differentiated into neural lineages, epithelium and blood vessel-like structure.