OBJECTIVE OF THE EXPERIMENT The aim of the experiments is (1) to establish mouse embryonic stem (ES) cell lines and (2) to study the differentiation of ES cells into cardiac and neuronal cells.
DEGREE OF DIFFICULTY Determination of alkaline phosphatase activity: easy. Immunofluorescence staining: moderate. All experiments concerning the establishment of ES cell lines and in vitro differentiation: difficult.
INTRODUCTION
Embryonic stem (ES) cells have been established as undifferentiated cell lines from the inner cell mass (ICM) of mouse and human blastocysts (Evans and Kaufman, 1981; Thomson et al., 1998). These pluripotent ES cells are characterized by a high proliferative capacity and the ability to develop into terminally differentiated cells of all three primary germ layers, the endodermal, ectodermal and mesodermal lineage (Figure 25.1). Mouse and human ES cells can differentiate in vitro to give rise to various somatic cell types (reviewed in Schuldiner et al., 2000; Wobus, 2001). In vivo, after transfer into blastocysts or by aggregation with blastomeres, mouse ES cells participate in the development of the embryo including the germ line (Bradley et al., 1984), whereas human ES cells after transplantation into nude mice give rise to teratomas (Thomson et al., 1998).
Besides ES cells, two other types of pluripotent embryonic cell types have been established as permanent lines: embryonic carcinoma (EC) cells derived from teratocarcinomas and embryonic germ (EG) cells isolated from primordial germ cells of early embryos (Stewart et al., 1994; Shamblott et al., 1998; see Figure 25.1).