Mouse embryo cells cultured in vitro in serum-supplemented media undergo growth crisis, resulting in the loss of genomically normal cells prior to the appearance of established, aneuploid cell lines. Mouse embryo cells established and maintained for multiple passages in the absence of serum did not exhibit growth crisis or gross chromosomal aberration. Cells cultured under these conditions were dependent on epidermal growth factor for survival. Proliferation was reversibly inhibited by serum or platelet-free plasma, suggesting that mouse embryo cultures maintained by conventional procedures are under the influence of inhibitory factors.

We have derived serum-free mouse embryo (SFME) cultures in a basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein (HDL), and fibronectin. These cells are nontumorigenic, lack gross chromosomal aberrations, and exhibit several other unique properties, including dependence on EGF for survival and growth inhibition by serum. We have examined the concentration dependence of the growth stimulatory effects of protein supplements used in the SFME medium formulation and surveyed other supplements that might act as alternative or complementary additions to the culture medium. Insulin could be replaced by insulin-like growth factor I and EGF could be replaced by transforming growth factor alpha in the same concentration range. Transferrin could be replaced by higher concentrations of lactoferrin. Deterioration of cultures in the absence of EGF began within 8 hours of the removal of the growth factor, and could be prevented by the addition of fibroblast growth factor/heparin-binding growth factor. Attachment proteins other than fibronectin were effective on SFME cells, but limited success was obtained when substituting other lipid preparations for HDL. These data introduce a precise system for exploring the unusual characteristics of SFME cells and contribute additional information that may be useful in the extension of these approaches to other cell types and species.

Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, display distinctive properties: (i) SFME cells do not lose proliferative potential or show gross chromosomal aberration upon extended culture, (ii) these cells depend on epidermal growth factor for survival; and (ii) SFME cell proliferation is reversibly inhibited by serum. Treatment of SFME cells with serum or transforming growth factor j3 led to the appearance of glial fibrillary acidic protein, a specific marker for astrocytes. The appearance of glial fibrillary acidic protein in cultures was reversed upon removal of transforming growth factor (3 or serum. Cells with properties similar to SFME cells were also isolated from adult mouse brain. These results suggest a role for transforming growth factor (3 in astrocyte differentiation in developing organisms and in response to injury and identify the cell type that has the unusual properties of SFME cells.Serum-free mouse embryo (SFME) cells initiated and maintained multipassage in a rich basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein, and fibronectin display several distinctive properties (1)(2)(3)(4)(5)(6). Mouse embryo cells derived in conventional serum-supplemented medium undergo a period in which proliferative potential is lost or reduced, and then genetically altered cells with indefinite proliferative potential emerge (2,7,8). SFME cells do not exhibit any loss of proliferative potential or gross chromosomal aberration and maintain a relatively stable karyotype when cultured for >10 times the population doublings achieved with karyotypically normal mouse embryo cells in serum-containing medium. Although the period in which reduced growth rate can be detected may be minimal or absent in cultures in serumcontaining medium maintained in a passaging protocol involving high plating densities, the protocols used to derive SFME cells lead to a well-defined period of limited proliferative potential in cultures maintained in serum-containing medium (2, 7) that is not seen when the cells are cultured in serum-free medium. SFME cells also depend on EGF for survival, and cycloheximide or actinomycin D prevents death from EGF deprivation (5), suggesting that cell death requires the synthesis of RNA and protein, a phenomenon of programmed cell death similar to that reported for neuronal cell death in the absence of nerve growth factor (NGF) (9). In addition, proliferation of SFME cells is reversibly inhibited by serum or platelet-free plasma (1, 4, 6), and thus these cells would not be propagated under conventional culture conditions using serum-supplemented medium. As for most mouse embryo cell lines, the precise nature of the cells giving rise to the continuously growing SFME cultures was unknown. The unusual characteristics of SFME cells led us to attempt to determine the cell type represented by these cells.We found that treatment of SFME cells with serum or We a...

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