Isolation And Culture Of Mouse And Sheep Embryonic Stem-Like Cells

Stable ES cells(ESCs) lines have been considered as perfect cells for making bio-reactor, but there is no report about having established sheep ES cells lines so far. KB mouse is a mostly used trail animal in our country. While most of the established mouse ES cells lines were from129,C57BL/6J and BALB/C mouse and very few reports have showed the establishment of KB mouse ES cells lines. So it is very important to establish the ES cells lines of sheep and mouse for stem cell research in our country. In this study, we isolate and culture ESCs from sheep and mouse embryos of different development stages, and to compare some factors influencing the efficiency of establishing those stem cell lines by adopting the methods of mouse and human. The results obtained were as follows:1. We obtained 120 embryos from Kunbai mouse and 98 ICMs grew out ICMs were plated on mitomycin-inactivated feeders and cultured in a humidified environment of 5%CO2 in the air with temperature at 37℃. 68 ES-like colonies were gained from 98 ICM and 2 of them had maintained undifferentiating for 18 passages. The effects of adding KSR or FBS to basic medium to the isolation and cloning of mouse ES cells was eliminated, the result showed that KSR could improve the ratio of the stably passaging of ES cells, and ES-like colonies gained from medium supplanted with KSR had maintained undifferentiating for18 passages.2. The result shows that blastocysts are the most suitable material for the isolation and cloning of sheep ES-like cells. Although the plantation ratio of blastocysts (80.0%) was a little lower than that of hatching embryos (87.5%) (p>0.05), but ICM growing rate and passages rate of blastocysts were significantly better than that of morula and hatching embryos.3. The effects of different feeder cells on the isolation and cloning of sheep ES-like cells was Eliminated. Embryos were cultured on MEF, SEF that were inactivated by mitomycin. There were no distinct differences on the ratio of hatched embryos and growth of ICM. Some differences existed on the forming of ES colonies and passaging, the result indicated that MEF is the best feeders for cloning of sheep ES-like cells.4. Using MEF as feeder layers. To eliminate the influence of KSR and mouse ES cells conditioned medium (ESCCM) on self-renewal of sheep ES-like cells, KSR was used to replace serum, and then supplanted with ESCCM. Sheep ES-like cells could remain undifferentiated for 5 passages when cultured in the KSR medium.The result indicates that KSR culture system was more suitable for cloning of sheep ES-like cells compared to FBS culture system. Finally we applied basic medium with 15%KSR and supplanted with 40% ESCCM as a new culture system to isolate sheep ES-like cells, finally, two ES-like cell line still maintained undifferentiating for 8 passages.5. ES cells gained from both sheep and KB embryos were identified by AKP activity, expression of OCT-4 or Nanog and differentiation in vitro for there pluripotenty. Two ES cell lines of ES cells isolated from sheep and KB respectively were dertermined by karyotype analysis. The result showed that both of the mouse and sheep ES cells isolated in these experiment have pluripotentiality and normal diploid.