GROWTH AND MATURATION OF SMALL HEPATOCYTES

Proliferation of adult rat hepatocytes is observed in serum-free Dulbe cco's modified Eagle's medium (DMEM) supplemented with 10 mmol/L nicot inamide and 10 ng/mL epidermal growth factor (EGF). The proliferating cells are mainly mononucleate and form small cell colonies surrounded by mature hepatocytes. Although these cells in focal colonies have a l ess-differentiated appearance, immunocytochemically and ultrastructura lly they possess hepatic characteristics. The size of small hepatocyte s is one-third to half that of mature hepatocytes. Therefore, we call the cells forming a colony, small hepatocytes. The small hepatocytes c an be subcultured for several passages. Furthermore, the cells are ric h in the supernatant following 50 g centrifugation for 1 min after col lagenase liver perfusion. When the cells are cultured in DMEM suppleme nted with 10% foetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L as corbic acid 2-phosphate, 10 ng/mL EGF and 1% dimethyl sulphoxide, each small hepatocyte can clonally proliferate for more than 3 months. A s mall hepatocyte divides to form a colony and the number of cells reach es more than 100 within 20 days. With time in culture, cells with a la rge cytoplasm appear within a colony. They have many mitochondria and large peroxisomes with crystalline nucleoids and are typical, mature h epatocytes. Immunoreactivity to connexin 32 and well-developed bile ca naliculus structures are often observed in the cell-cell borders. Thus , we suggest that small hepatocytes may be considered to be 'committed progenitor cells' that can further differentiate into mature hepatocy tes.