Establishment And Charactorization Of Mouse Embryonic Stem Cell Lines

Mouse embryonic stem cells( ESCs) have been widely applied to such research fields as developmental biology, human disease and drug development. The establishing techniques of mouse ESCs lines provide reference standard and theoretical basis for the establishment of human and animals ESCs lines. At present,most of mouse ESCs lines are derived from 129 or hybrid mice, and a small number from C57BL/6J and BALB / c mice due to its low successful efficiency in the establishment of ESCs lines. Kunming mice have been generally used as experimental animals in China, but the isolation and establishment of its ESCs lines was very difficult. In most cases Kunming mouse ESCs which can not fully proliferate in vitro, only be subcultured for a few passage. In this research, different additions were added into culture medium to compare the influences on the isolation of Kunming mouse ESCs, on the basis of optimizing preparation of mouse embryonic fibroblast (MEF) feeder layers. Further, mouse embryos from 129♂×KM♀hybrid F1 mice, were used as the starting material to development the system of isolation and cultivation of mouse ESCs. At last, 1 ESCs strain from normaly in vivo fertilized embryos which was subcultured to 52 passages, and 1 ESCs (pESCs) strain from the parthenogenetic embryos in vitro which was subcultured to 38 passages, were generated so as to laid the foundation for a in-depth study. The study results were as following:1. Optimizatical preparation of mouse embryonic fibroblasts (MEF) feeder layersThe vitality of MEF treated by mitomycin C (10ug/mL final conc.) was detected by MTT method to compare the maintaining time of MEF vitality in different conditions. The result showed that the MEF vitality could keep about 10~12 days stably under this condition when MEFs within 5 passages were used to prepare feeder layer, treating MEFs with mitomycin C (10ug/mL final conc.) for 2 to 3 hours, and inoculating into 96-wells plate (1.2~1.6×104 per well) with 10% NBS culture medium. This was the optimical condition to preparing MEF feeder layer.2. Research of influence factors on the isolation and cultivation of Kunming mouse ESCsA total of 287 ICM outgrowths were generated from the 493 Kunming mouse embryos cultured on inactivated MEF feeder layer. After digestion of ICM outgrowths and ESCs by trypsinization combined with manual dissection, Two strains ESCs which expressed Oct–4 and Nanog protein respectively and also possesed alkaline phosphatase (AKP) activity, were subcultured to 7 passages.①KSR and FBS were respectively added into the ESCs medium to observe the effect on formation of ICM outgrowths and undifferentiated growth of ESCs. The result showed it took 4.58±1.05 d for embryos to attach, and the attachment rate of embryos in KSR medium was significantly lower than that in FBS medium (P <0.05), while the formation rate of ICM outgrowths had no significant difference between KSR medium and FBS medium(P>0.05). The result of ESCs subculture from 1 to 5 passages indicated that KSR could effectively promote the undifferentiated status of Kunming mouse ESCs compared with FBS.②It was observed that it took 5.56±1.15 d for embryos to attach in the ESCs medium supplemented with FBS+ PD98059(50μmol/L), and the attachment rate of embryos and the formation rate of ICM outgrowths were significantly lower than that in KSR or FBS group (P <0.05), and FBS+ PD98059(50μmol/L)could not effectively promote the undifferentiated status of Kunming mice ESCs compared with KSR or FBS according to ESCs subculture from 1 to 5 passages.③The optimization method in dissecting ESCs and ICM outgrowths of Kunming mouse was to use 0.5 g/L typsin-0.2 g/L EDTA combined with manual dissection.3.The isolation and identification of hybrid mouse ESCs①135 mouse embryos derived from 129♂×KM♀F1 hybrid mice, were grown on inactivated MEF feeder layer to emerge 72 ICM outgrowths. A total of 7 cell strains subcultured for above 9 passages were obtained, three of which were passed to 52 passages (named for 060728084), 40 passages (named for 070109137) and 7 passages (named for 070109153) respectively. The cell strains named for 060728084 was identified. The result showed that over 75% cells maintained normal karyotype of XY within 33 passages, and had the enzyme activity (AKP, telomerase), expression pattern of marker antigens (SSEA-1, SSEA-3, SSEA-4, OCT-4 and Nanog ) and genes (Oct-4, Nanog and Gdf-3) of mouse ESCs, and the cell strain could differentiate in vitro and in vivo into various types of cells originated from three germ lines (7 marker antigens and 5 marker genes detected by immunohistoche- mistry and RT-PCR respectively).②It was observed that In the four different media simultaneously supplemented with KSR and FBS according to the different ratios, there were no significant differences on the embryo-attaching rates (P>0.05); in the three media supplemented with 3.75%FBS+ 11.25%KSR, 7.5%FBS+ 7.5%KSR and 15%FBS, the embryo-attaching rates were significantly higher than that in the medium supplemented with 15% KSR (P<0.05); in the media supplemented with 3.75%FBS+ 11.25%KS and 7.5%FBS+ 7.5%KSR, the formation rates of ICM outgrowths were significantly higher than that in the media supplemented with 15% KSR or 15% FBS (P<0.05). However, it would be beneficial to keep the undifferentiated status of ICM outgrowths when adding 1%FBS+14%KSR or 3.75%FBS+11.25%KS compared with adding 7.5%FBS+ 7.5%KSR or 15%FBS.③They could help to promote the undifferentiation growth of ESCs when adding 15%KSR,1%FBS+ 14%KSR or 3.75%FBS+11.25%KSR in the media compared with adding 7.5% KSR+7.5% FBS or 15% FBS. The result of single–cell inoculation showed that the clone-forming efficiency of ESCs was significantly higher in 1%FBS+14%KSR, 3.75%FBS+11.25%KSR and 7.5%FBS+ 7.5%KSR groups than that in 15%KSR or 15%FBS group.4. The isolation and identification of parthenogenesis-derived stem cells (pESCs) of hybrid mice①35 blastocysts derived from the MⅡoocytes of 129×KM hybrid F1 mice, were generated by pharenogenetic activation and in vitro culture. A total of 30 parthenogenetic blastocysts were cultivated on MEF feeder layers to get 11 ICM outgrowths, Eight of which were cocultured with the inactivated MEF feeder layers after typsinization combined with manual dissection. At last, one stable pESCs strain named for P061221132, was obtained from the parthenogenetic mouse embryos, and identified. The result showed that over 70% cells in the cell strain retained normal karyotype of XX within 23 passages, possessed enzyme activity (AKP, telomerase), expression pattern of marker antigens (SSEA-1, SSEA-3, SSEA-4, OCT-4 and Nanog ) and genes (Oct-4, Nanog and Gdf-3) characteristic of mouse ESCs and could differentiated in vivo and in vitro into various types of cells originated from three germ lines by immunohistochemical and RT-PCR detection of embryoid bodies and teratocarcinomas, which basically meet the criteria concerning the establishment of mouse ESCs line, except the chimera experiment.②The result of single-cell inoculation showed that clone-forming rate of the pESCs strain P061221132 was ranged from 33.3% to 47.2% when adding 1%FBS+14%KSR, 3.75%FBS+11.25%KSR or 7.5%KSR+7.5%FBS into ESCs medium. There was no significant difference(P>0.05)on clone-forming rate of cells at different passages in different media. And, 1%FBS+14%KSR,3.75%FBS+ 11.25%KSR in ESCs media could promote to form the typical ESC-colony morphology compared with adding 7.5%KSR+7.5%FBS.