DIFFERENTIATION AND PROLIFERATION OF PRIMARY RAT HEPATOCYTES CULTUREDAS SPHEROIDS

We studied spheroid (multicellular aggregate) formation by hepatocytes and the expression of liver-specific functions such as albumin secret ion when hepatocytes were cultured with various extracellular matrices . Hepatocytes cultured on Primaria(R) and poly-D-lysine coated dishes, and in the presence of a polymer, Eudragit, formed spheroids, and the y also exhibited higher liver-specific functions and poor growth compa red to monolayer cultures. The results indicated that the cell morphol ogical change and cell-cell interaction caused by the spheroid formati on were key factors promoting the expression of the liver-specific fun ctions. To elucidate the mechanism underlying the poor growth in spher oids, we examined the HGF signaling pathway. Phosphorylation and down- regulation of the NGF receptor (c-Met proto-oncogene product) were obs erved for the cells from both monolayer and spheroid cultures, but Ras activation was partly blocked in spheroids, Furthermore, we found tha t CDK inhibitors, p21 and p27, were highly expressed in spheroids. The se results suggested that the reduced Ras signaling and high expressio n of the CDK inhibitors might cause the lower growth in spheroids. We then examined the relationship between liver-enriched transcription fa ctors (C/EBP alpha and beta) and liver-specific functions. The results revealed that the high expression of C/EBP alpha was maintained durin g cultures when hepatocytes formed spheroids. Antisense oligonucleotid es of C/EBP alpha repressed albumin secretion and the expression of p2 1, suggesting that the transcription factor, C/EBP alpha, may play a c rucial role in the growth and differentiation of hepatocytes in sphero ids.