In vitro induction of terminal differentiation of neonatal rat hepatocytes by direct contact with adult rat hepatocytes [corrected].

Immunocytochemical staining of tryptophan 2, 3-dioxygenase (TO, EC 1.13.11.11), which is a typical marker of terminal differentiation of rat hepatocytes, showed that TO was expressed as early as the first day after birth by a few hepatocytes, and that the number of hepatocytes expressing TO gradually increased during early neonatal development. In contrast, immature hepatocytes grew actively in culture even without growth factors and serum, showing a labelling index with [3H]thymidine of over 80% just after birth, but their ability to show autonomous growth decreased rapidly during postnatal development. Double staining of hepatocytes from neonatal rats indicated that hepatocytes showing DNA synthesis were distinct from those expressing TO, suggesting that immature cells proliferate, but do not express TO, and then become fully differentiated expressing TO, but not proliferating any more. On the contrary, albumin was fully expressed in most hepatocytes of 0-day-old rats, in which the hepatocytes were still growing. When immature hepatocytes without TO from 0-day-old rats were cultured on a feeder layer of adult rat hepatocytes for 3.5 days, their expression of TO increased rapidly to almost the adult level (over 70% of the cells became TO-positive). Conversely, this feeder layer strongly inhibited autonomous growth of the neonatal rat hepatocytes. Other feeder layers, such as non-parenchymal liver cells of adult rats, Reuber hepatoma, and Swiss 3T3 fibroblasts, had no effect on the reciprocal changes of terminal differentiation and autonomous growth of immature rat hepatocytes. A feeder layer of dead adult rat hepatocytes, obtained by treating the cells with cytosine arabinoside for 24 h or drying them at 37 degrees C, had the same ability as a feeder layer of living cells to induce cytodifferentiation of immature cells. When the dead feeder layer was treated with 1 N HCl or digested with 0.1% trypsin, its ability to induce differentiation was almost completely lost. These results suggest that a cell surface component of adult rat hepatocytes, probably an acid-labile protein, controls terminal differentiation and growth of immature hepatocytes.