Introduction

Primordial germ cells (PGCs) in the embryo give rise to functional gametes in the adult animal. Considering their importance in the continuation of the species, it is no wonder that there is much interest in understanding the biology of these highly specialized cells. Much has been learned from the analysis of mouse mutants that are defective in germ-cell proliferation and survival. However, given the relative inaccessibility of PGCs in the embryo, the ability to culture these cells in vitro has led to a greater understanding of the mechanism by which growth factors control their proliferation, migration, and differentiation in vivo. This chapter will outline methods to obtain PGCs from various embryonic stages, culture them in vitro, stain them for endogenous alkaline phosphatase activity, and finally generate embryonic germ (EG) cell lines.

Successful in vitro culture systems for PGCs include the use of a mitotically inactivated feeder layer of somatic cells. However, even under these conditions, the number of surviving PGCs in culture decreases dramatically after 5 d. The addition of various growth factors to the culture medium can greatly increase the survival and proliferation of PGCs in vitro and, in some cases, lead to the generation of cell lines resembling blastocyst-derived embryonic stem cells (ES cells). These growth factors include stem cell factor (SCF), leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF). SCF, which is also known as mast cell growth factor, kit ligand, and Steel factor is encoded at the Steel locus. Embryos mutant for SCF have PGCs, but they fail to divide and resulting Steel mutant mice are sterile. SCF is a transmembrane

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

protein that can be alternatively spliced to produce a soluble form. When PGCs are cultured on feeder cells that express the membrane-bound form of SCF, their survival and proliferation are greatly enhanced (1-3). Another factor made by the feeders is LIF, also known as differentiation inhibiting activity (DIA). It has been shown that by using LIF alone, it is possible to establish and maintain pluripotent blastocyst-derived ES cell lines (4-6) and soluble LIF stimulates PGC proliferation in the presence of other factors (2,3). The receptor for LIF is made up of two subunits, one that binds LIF directly and the other, which is a signal transducer called gp130. The gp130 subunit is shared by other cytokines, such as oncostatin M (OSM) and ciliary neurotropic factor (CNTF). OSM and CNTF can substitute for LIF in PGC culture, and neutralizing antibodies against gp130 block PGC survival in culture (7,8), suggesting a role for this receptor in PGC survival and proliferation in vivo. Tumor necrosis factor-a (TNF-a) selectively stimulates the proliferation of early PGCs (9), interleukin-4 (IL-4) is a survival factor (10), and retinoic acid is a growth activator of PGCs (11). Also agents, such as dibutyryl cAMP and forskolin, that raise the intercellular concentrations of cAMP also stimulate the proliferation of PGCs in vitro (12). The in vivo functional significance of these growth factors is not yet known.

Long-term proliferation of PGCs was demonstrated by the addition of bFGF to a mixture of SCF and LIF in the in vitro culture system outlined in this chapter (13-16). It has been determined that these growth factors act directly on the PGCs and are not merely an indirect effect on the feeder layers to produce even more growth factors (7). EG cell lines derived from PGCs have many characteristics of ES cells. They can form embryoid bodies in vitro, produce teratomas in nude mice, and even contribute to the germ line of chimeric mice (13-17). However, EG cells are not identical to ES cells with regard to their genomic imprinting. These cell lines may be used in the future to study genomic imprinting, the potency of PGCs at various stages during embryogenesis, and finally as a route to generate pluripotent stem cells from species other than mice.

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