Differentiation and growth of rat egg-cylinders cultured in vitro in a serum-free and protein-free medium.

Modified organ cultures of rat egg-cylinders were grown for 2 weeks in Eagle's minimal essential medium (MEM) without serum. Differentiation of epidermis and cartilage in the cultures deprived of serum was comparable to that in fully serum-supplemented medium, whereas other differentiated tissues were rare or absent. The purpose of the experiment was to determine whether terminal tissue differentiation is modified by various added factors. The factors used affected the growth and/or differentiation of explants as follows: bovine serum albumin and human transferrin had a positive permissive influence on the appearance of neuroblasts; human transferrin alone stimulated the formation of lentoids, a relatively rare tissue. Retinoic acid inhibited cartilage formation and stimulated the differentiation of cylindrical epithelium; neural growth factor inhibited the growth of explants; and 5-azacytidine impeded the survival of explants. One can conclude that these factors influenced the growth and differentiation of the early rat embryos cultured in a chemically defined medium.

Retinoic acid can change normal differentiation of rat egg-cylinders cultured in vitro.

The modified organ culture of rat egg cylinders provides favorable conditions for 2 weeks for the differentiation of main tissue types. To study the effect of retinoids on early rodent differentiation, retinoic acid (RA) was added in various concentrations to serum-supplemented or serum-free medium. Explant survival decreased when RA was added to serum-free medium. Although the cartilage was well differentiated even in cultures deprived of serum, RA inhibited chondrogenesis in all cultures without or with serum. The frequency of columnar epithelium was higher and its folds more often present when RA was added to the medium. Keratinization of squamous epithelium depended on the RA concentration added to the medium, and was almost absent when the concentration was high. Other tissues often present in serum-supplemented medium (such as neuroblasts and myotubes) were not affected by RA, a result that differs from those obtained in other experimental systems.

Neural growth factor inhibits the growth of rat egg-cylinders cultured in vitro.

Rat egg-cylinders at the primitive streak stage were grown in modified organ culture for 2 weeks using a chemically defined medium. Differentiation of the epidermis and cartilage was comparable to that in fully serum-supplemented medium, whereas neuroblasts were very scarce. In explants treated either with bovine serum albumin or transferrin, neuroblasts were observed, whereas the addition of NGF did not improve neuroblast differentiation. On the contrary, NGF impaired growth and tissue differentiation when compared with explants grown in serum-free medium.

Transferrin enhances lentoid differentiation in rat egg cylinders cultivated in a chemically defined medium.

Rat egg cylinders at the primitive streak stage were grown in modified organ culture for 2 weeks using a chemically-defined medium. The purpose of the experiment was to determine whether the terminal tissue differentiation is modified by human transferrin. The control sets were grown in medium with or without rat serum. In explants treated with transferrin, groups of atypical cells of the ocular lens (lentoids) appeared more frequently than in both control sets; however neuroblasts were observed as often as in the serum-supplemented medium. Bovine serum albumin (BSA) stimulated the differentiation of neuroblasts but did not promote lentoid formation. We conclude that human transferrin does stimulate the differentiation of lentoids in rat embryonic explants, but the mechanism of its action remains unknown.

Partial differentiation of rat egg cylinders in serum-free and protein-free medium.

Modified organ cultures of rat egg cylinders were grown for 2 weeks in Eagle's MEM without serum or with serum added at different times. Explant survival was decreased only in cultures grown for the entire 2 weeks in serum-free medium, whereas the explant growth was impeded in all but the cultures grown for 2 weeks in 50% MEM plus 50% serum. Differentiation of epidermis and cartilage in the cultures deprived of serum for the entire 2-week period was comparable to that in fully serum-supplemented medium, whereas other differentiated tissues were rare or absent. In explants cultivated without serum for only the first week, neuroblasts were scarce.

Cyclic nucleotides affect growth and differentiation of cultured rat embryonic shields.

The modified organ culture of rat embryonic shields provides favorable conditions during 2 weeks for the differentiation of main tissue types. Since the terminal differentiation in explants is inferior to that obtained in the homografts of the same shields under the kidney capsule, we tried to improve the culture medium by adding some known regulatory molecules: db-cAMP, db-cGMP, ATP, AMP, and butyric acid. These agents were added to the liquid medium in the concentration of 1 mM. In the first part of the study the explants were fixed and weighed after 8 or 14 days in vitro culture, and histological sections were examined. When the explants were treated with db-cAMP during the second week of culture, the skeletal muscle appeared more frequently in the treated series than in controls, and the weight of the treated explants was sometimes increased when compared with the control series. The db-cGMP had no effect on differentiation, but stimulated the growth of the explants when applied during the first week of culture. On the contrary, the db-cAMP when added during the first week, severely impeded the growth of explants. Other agents seem to be ineffective. In the second part, the content of cAMP and cGMP was measured in normal explants. The radioimmunoassay showed the same content of cAMP and cGMP during the entire culture period. In the third part of our study the incorporation of tritiated uridine and tritiated thymidine was measured during the second week of culture after the addition of db-cAMP. During the first days of treatment with db-cAMP the uptake of tritiated uridine and thymidine was inhibited, whereas on the seventh day the uptake was similar to that of the control. We can conclude that both cyclic nucleotides have a visible effect on growth whereas only cAMP has a positive impact on the differentiation of myotubes in cultured rat embryonic shields.

Effect of various sera on cultured rodent embryonic shields.

The modified organ culture of rodent embryonic shields, developed in our laboratory, has been shown to provide favorable conditions for the differentiation of the main tissues in teratoma-like explants. The purpose of the experiment was to discover whether tissue differentiation is modified by different sorts of sera used in the liquid medium. Rat explants showed an increase of the incidence of cartilage when cultured in at least 20% homologous serum in comparison with human and fetal bovine sera. Mouse explants did not grow in mouse serum, whereas rat serum stimulated the development of endoderm-like epithelium, and fetal bovine serum brought about more neural tissue.

Effect of dibutyryl cAMP and theophylline on cultured rat embryonic shields.

Effects of N6, O2 dibutyryl adenosine 3',5'-cyclic monophosphate (db-cAMP) and theophylline (Th) on cultured rat embryonic shields were studied. After addition of db-cAMP to the culture medium, an increase of the weight of explants and of the incidence of the skeletal muscle was observed. Theophylline seems to be ineffective.

Origin of embryo-derived yolk sac carcinomas.

The origin of yolk sac carcinoma obtained from rat embryos transplanted to extrauterine sites was traced to the extraembryonic portion of 9-day egg-cylinders. Under appropriate conditions cells of the extraembryonic portion of the egg cylinder differentiate into cells of parietal yolk sac epithelium, continue to proliferate and form retransplantable malignant tumors. Serum concentrations of alpha-fetoprotein were elevated in rats bearing yolk sac carcinomas and in some animals bearing teratomas admixed with yolk sac carcinoma. Possible factors that regulate the survival and proliferation of yolk sac epithelium in extrauterine sites are briefly discussed.