Study of liver differentiation in vitro.

Abstract

A clonal rat fetal liver cell line that expresses the functions of differentiated liver cells under controllable conditions was established. Normal fetal liver cells were transformed by a temperature-sensitive A (tsA) mutant (tsA209) of SV-40. At the permissive temperature (33.degree. C), the tsA209-transformed liver cell line (RLA209-15) can be cultured indefinitely and cloned readily. The RLA209-15 cells were temperature-sensitive for maintenance of the transformed phenotype. These transformed liver cells selectively lost 4 characteristics of the transformed phenotype at the restrictive temperature (40.degree. C): generation time of the cells increased, the saturation density decreased, the efficiency of growth on nontransformed cell layers decreased and the ability to clone in soft agar was lost. The transformation can be reversed simply by a shift in temperature. RLA209-15 fetal liver cells synthesized .alpha.-fetoprotein, albumin and transferrin. At 33.degree. C, the levels of these liver proteins were relatively low. At 40.degree. C, the transformed phenotype was lost and the levels of .alpha.-fetoprotein, albumin and transferrin were greatly increased. At the restrictive temperature, maximal induction of the synthesis of .alpha.-fetoprotein, albumin and transferrin was achieved 3-4 days after the upward shift in temperature. The synthesis of .alpha.-fetoprotein then decreased; the synthesis of albumin and transferrin was maintained. A 2nd phase of albumin and transferrin synthesis was observed in all cultures after 6 days or more at 40.degree. C. .alpha.-Fetoprotein, albumin and transferrin secreted by RLA209-15 cells were immunologically indistinguishable from authentic .alpha.-fetoprotein, albumin and transferrin, respectively. RLA209-15 cells, like primary cultures of hepatocytes and a SV-40 tsA255-transformed fetal liver cell line (RLA255-4) previously reported, responded to glucagon with markedly elevated levels of cAMP. Thus, it appears that glucagon receptors characteristic of hepatocytes are retained in the SV-40 tsA-transformed fetal liver cells.