EXPOSURE OF PRIMARY RAT HEPATOCYTES IN LONG-TERM DMSO CULTURE TO SELECTED TRANSITION-METALS INDUCES HEPATOCYTE PROLIFERATION AND FORMATION OF DUCT-LIKE STRUCTURES

We previously showed that primary rat hepatocytes plated on a rat-tail collagen coated dish and fed a chemically-defined medium supplemented with 2% dimethylsulfoxide (DMSO) can be maintained in a well-differen tiated, non-replicating state for periods of several months. In this s tudy, we show that the addition of copper, iron, and zinc to the DMSO- containing chemically defined medium induced DNA synthesis and cell re plication during the first two months in culture without loss of hepat ic differentiation. DNA synthesis occurred throughout the hepatocyte p opulation without regard to cellular size. No changes were observed in properties indicative of well-differentiated hepatocytes, including c ellular morphology, ultrastructure, albumin, or cytokeratin-8 expressi on. During the third month in culture, after the hepatocytes had becom e confluent, pseudoduct structures became apparent. Examination of cel ls lining the ducts by immunohistochemistry showed that these cells lo st the ability to express albumin and stained more intensely for cytok eratin 19 and laminin. The ultrastructure of the cells lining the duct s was altered and became more characteristic of bile duct cells. Immun oelectron microscopy revealed that connexin 43, a marker of bile-duct proliferation, was expressed in the duct-like cells, We conclude that under these specific nutritive conditions, primary rat hepatocytes pro liferate and, with time, begin to form duct-like structures with alter ed gene expression and ultrastructural properties.