Derivation And Culture Of Mouse And Porcine Embryonic Stem Cells

Embryonic stem cells can be maintained indefinitely in culture and yet maintain the competence to produce all the cells within a fetus. The well-accepted criteria for totipotency of embryonic stem cells inculde 1) differentiating into three germ layers in vitro; 2) teratoma formation ability; 3) germline transmission and/or tetraploid-complementation.. Only mouse, rat and chicken embryonic stem cell lines could satisfy all the standards since Kaufman MH and Evans MJ derived the first mouse embryonic stem cell line in 1981. The pigs are widely used as drug experimental models and disease models because of the physiologically similarity with humans. Besides, pigs are the main donors for organ transplantation. Although the derivation of porcine embryonic stem cell are highly needed, no convincing embryonic stem cell lines have been produced in this species to date.We derived several mouse embryonic stem cell lines and tested the pluripotency of one line. Then we studied the early developmental process of porcine embryo. On the basis of the mouse embryonic stem cell derivation technology and the knowledge to porcine embryo, we tried to establish a genuine porcine embryonic stem cell line. The following are brief experimental designs and results:Firstly we derived several mouse embryonic stem cell lines with the method of combining KOSR embryonic stem cell culture medium and FBS embryonic stem cell culture medium during the process of derivation. It turned out that the efficiency of our method could be as high as 71.4%. We chose one of the derived lines to test the pluripotency. The experimental data showed that this line could satisfy the standards of mouse embryonic stem cell line.Then we collected porcine embryos at early ages to observe morphology and measure diameter. We found that for the early stage porcine embryos, there were no positive correlation between the age and the diameter. We also found it was common that the embryos from the same batch were not at the same developmental stage. So we thought it was not scientific to judge porcine early embryos only by age.Last, we tried to derive porcine embryonic stem cell line with both in-vivo and in-vitro embryos. We compared the effect of three kinds of culture medium: KOSR embryonic stem cell culture medium, FBS embryonic stem cell culture medium and N2/B27 embryonic stem cell culture medium. The data showed that KOSR culture medium was more suitable for in-vivo embryos. With the optional culture system, we got colonies looked like human embryonic stem cell colonies and passaged them three times.