Isolation and culture of bovine embryonic stem-like cells using whole zona-free embryo culture.

Abstract

Zona membranes of bovine embryos were dissolved using 5 mg/ml pronase to collect zona-free embryos. Whole embryo culture was applied for bovine embryonic stem cell establishment. Bovine embryonic stem cells were isolated and cultured from bovine morula stage embryos and blastocyst stage embryos. Nine morula stage embryos were cultured on mouse embryonic fibroblast inactivated by 10 µg/ml mitomycin C. However, there was no attachment or proliferation of morula blastomere on the feeder cell layer. Whole blastocyst stage embryo culture were also used to establish embryonic stem cells. One blastocyst stage embryo attached to feeder cell layer and proliferated to embryonic stem-like cell colony in three weeks. The primary bovine embryonic-like colony were collected and passaged to passage one. Six embryonic-like colonies were formed on feeder layer in passage one. The colonies’ diameter of passage one is from 50µm to 100 µm. Four colonies were degenerated. Colony 4 and 6 proliferated continuously in one week. Colony 4 was picked up and passaged to passage 2. Five embryonic-like colonies were formed on the feeder layer in passage two. The colonies’ diameter of passage two was from 70 µm to 140 µm. However, all embryonic-like colonies of passage two degenerated in one week. These colonies of passage one and two had embryonic-like morphology, and were surrounded by clear boundaries. This characteristic is very important to distinguish embryonic-like colonies from feeder cell cluster. Colony 6 of passage one was treated to Trysin-EDTA and culture in petri dish with embryonic stem cell medium without leukemia inhibitory factor. Some embryoid bodies were formed in two weeks. This result supported the pluripotent property of these embryonic stem-like cells.