Chapter 40 - Isolation, Characterization and Maintenance of Primate Embryonic Stem Cells

Abstract

This chapter focuses on techniques for the derivation, characterization, maintenance, and differentiation of primate embryonic stem (ES) cells. Both human and nonhuman primate ES cell lines were derived from embryos at the blastocyst stage. All primate ES cell lines were shown to be capable of continuous culture at an undifferentiated state, but expression of high levels of OCT 4 had been demonstrated only for human ES cell (hES) cells. The derivation of hES cell lines is a relatively simple procedure, with success rates of up to 30% and 50% for mice and humans, respectively. The isolation can be conducted using three methods: immunosurgical isolation, mechanical isolation, or the use of an intact embryo. Human embryonic stem cell lines are derived from the ICM, which may not represent a homogenous cell population. To eliminate the possibility that the pluripotency of the lines reflects a collection of several distinct committed multipotential cell types in the culture, parental lines have to be single-cell cloned. The main aim of the first derivation of hES single-cell clones was to establish the parental lines' pluripotency, but as with mouse ES (mES) cells, single-cell cloning has additional advantages. Although primate and mES cells share most of the features of ES cells, there are some differences between them. On the single-cell level, there is no difference: both mouse and primate ES cells demonstrate typical spaces between the cells, a high nucleus-to-cytoplasm ratio, and the presence of one or more nucleoli.