In vitro culture and differentiation of normal mouse blastocysts

Abstract

WHEN preimplantation mouse embryos are transplanted to extrauterine sites, up to 20% develop almost normally to the egg cylinder stage (equivalent to about 6–7 d in utero). They then become disorganised and give rise to tumours, which are usually mature teratomas containing a wide range of differentiated tissues, but less frequently are progressively growing teratocarcinomas containing nests of undifferentiated pluripotent embryonal cells^1,2. There are many reasons why it would be interesting to reproduce such normal and abnormal development efficiently in vitro. Although a number of techniques have been used to culture preimplantation embryos, none of them has proved entirely satisfactory^3–5; in the best conditions only 5–20% of 3.5–4-d blastocysts develop in vitro to form differentiated, foetal tissues. Most only give rise to extraembryonic endoderm and its associated mesoderm; these cell types proliferate extensively in vitro, and consequently most of the continuous lines derived from blastocyst cultures have probably originated from extraembryonic tissues⁶. In this paper we describe a simple and reproducible method for culturing mouse blastocysts in vitro so that around 70% give rise to colonies which continue to grow for up to 4 weeks and produce a variety of differentiated tissues, including skin, nerve, beating muscle, cartilage and fibroblasts. The method relies on the technique of immunosurgery⁷ for killing and removing first the trophectoderm and then the hypoblast (primary endoderm) layers of the embryo, to yield clumps of isolated epiblast (embryonic ectoderm) (Fig. 1).