|Dr Wei-Hua WangÂ’s research team collected 7-9 day embryos from Chinese miniature pigs by flushing the uteri of sows. The recovered embryos were divided into two groups. For the first group, the zona pellucida was removed from the embryo by enzyme digestion and the intact embryos were cultured on a feeder layer of mouse embryonic fibroblasts (MEF) with an ES cell medium. The ES medium contained antibiotics, human fibroblast growth factor-basic (bFGF) and human leukemia inhibitory factor (LIF). The growth and development of colonies from the embryos was examined daily. For the second group of embryos, zona pellucida-free blastocysts were treated in a microdrop of a trypsin/EDTA solution for several minutes. The embryos were observed under a microscope during treatment and when the trophoblasts began to disperse, the blastocysts were transferred to another microdrop of culture medium. Inner cell masses (ICMs) were isolated from the blastocysts using two fine needles and a pulled mouth micropipette. Isolated ICMs were seeded onto MEF feeder cell layers and cultured in 96-well plates with ES medium.
After 2-5 days of culture, ES-like colonies from each culture group were picked off the feeder cells, disaggregated and reseeded onto new feeder layers. From the intact embryo cultures, five ES-colonies (28%) were detected. A significantly higher number, 68%, of ES-like colonies were formed from cultures based on ICMs. Most of the colonies examined showed alkaline phosphatase activity, a marker for ES cells. All of the colonies isolated from intact embryos and most of the colonies from ICMs only survived 3-5 passages in culture before differentiating or degenerating. Two ICM-derived colonies continued for 6-9 passages but did not form new colonies after 10 passages. ES-like cells were seen to differentiate into several types of cells and form embryoid bodies, suggesting that they were pluripotent.
The authors of the paper suggest that this simple enzymatic method of isolating the ICM from blastocysts is easier than using immunochemical means as reported in previous studies. When compared with previous isolation methods, the enzymatic method was found to produce greater numbers of healthy ICM cells and give better subsequent ES cell culture.
The paper describing the isolation of the embryonic stem cells is entitled `Isolation and culture of embryonic stem cells from porcine blastocysts` and is by Ming Li, Dong Zhang, Yi Hou, Lihong Jiao, Xing Zheng and Wei-Hua Wang. It was published online in July 2003 and will be printed in the August 2003 edition of the Molecular Reproduction and Development journal.
Source: AgBiotechNet, the online service for agricultural biotechnology
The paper was published online in a recent issue of Molecular Reproduction and Development.
Contact: Dr Wei-Hua Wang, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Science, Beijing 100080, China
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