Landry et al. 1 first showed the reconstruction of a threedimensional cyto-architecture consisting of differentiated hepatocytes, bile duct-like cells, and deposited extracellular matrix (ECM) by the use of spheroidal aggregate culture of hepatic cells isolated from newborn rats. Thereafter, some attempts have been made to grow a hepatic organoid by the coculture of hepatocytes and fibroblasts. 2,3 As neither fibroblasts derived from the liver nor hepatocytes could proliferate, the size of the cell aggregates was limited and the position of the cells was disorderly, although differentiated functions were well maintained for a long time. To develop a hepatic organoid, the interactions not only between hepatocytes and nonparenchymal cells (NPCs) but also between ECM and hepatocytes must be well coordinated as many differentiated functions of mature hepatocytes (MHs), through which the homeostasis of life is sustained, should be maintained.We have reported that the proliferation of adult rat hepatocytes is observed in serum-free medium supplemented with 10 mmol/L nicotinamide and epidermal growth factor (EGF). [4][5][6][7][8] Other laboratories also reported that supplementation of the medium with nicotinamide and EGF stimulates the growth of the primary rat hepatocytes. 9,10 Most hepatocytes cultured in nicotinamide-supplemented medium can divide, and small hepatocytes (SHs), which are less than half as large as but are morphologically similar to MHs, proliferate so fast that the cells form colonies after 4 to 5 days of culture. They have been shown immunocytochemically and ultrastructurally to possess hepatic characteristics. 6 Recently, it was shown by our 11 and other laboratories 12 that a single SH can clonally proliferate and form a large colony consisting of more than 30 cells at day 10. The cells can grow and survive for more than 5 months. Thus, we previously proposed that rat hepatocytes may be classified into three types of cells with respect to their ability to divide: (1) cells that have a high potential to proliferate and form colonies in primary culture (type I cells; SHs), (2) cells for which the number of possible cell divisions is limited (type II cells), and (3) cells that lose the ability to divide (type III cells). 7,8 In addition, type II and type III cells may possess fully differentiated functions. Therefore, in this report we call both type II and type III cells MHs. Furthermore, we call the cell islands, which consist of SHs themselves and of a mixture of SHs and MHs, colonies.In the present experiment we showed that SHs could differentiate to MHs that interacted with hepatic NPCs and

Colonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum-free modified Dulbecco's modified Eagle's medium containing 10 mmol/L nicotinamide and 10 ng/ml epidermal growth factor. Each colony consisted of cells that had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically these cells were stained with albumin and transferrin. Ultrastructurally these cells had mitochondria, peroxisomes and desmosomes, indicating that they were derived from hepatocytes. When 6 x 10(5) cells were plated on 35-mm dishes, about 5.5 colonies/mm2 were observed. This result suggested that about 1.5% of adult rat hepatocytes has the potential for multiple replications and of forming a focal colony. These cell populations had higher proliferative activities than surrounding hepatocytes. DNA synthetic activity could not be inhibited by 2% dimethyl sulfoxide. Flow cytometric analysis showed that both 2N and 4N nuclei synthesized their DNA until day 4 but that the number of 2N nuclei rapidly increased at day 5. This result correlated with the observation of the appearance of small cell populations indicating that the cells of these focal colonies were predominantly diploid.

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The nucleoids of microbodies of rat liver cells were isolated in a highly homogeneous and pure state, by treating the microbody-rich fraction, prepared from 10% Folyvinylpyrrolidone-0.25 M sucrose homogenate, with Triton X-100. Three treatments with 0.1% detergent were enough to render the nucleoids free from contamination with mitochondria, microsomes, lysosomes, and intact microbodies. Electron microscopically, the nucleoids were found to consist of parallel bundles of highly dense hollow tubules, the outer and inner diameters of which are approximately 150 and 50 A, respectively. Ten tubules are arranged around a longitudinal space 190 X 200 A in width. The nucleoids thus show a honeycomb appearance in the cross-plane and a parallel-packed structure in the longitudinal plane. Biochemically, the nucleoids were found to bear only urate oxidase among probably microbody-enzymes, and they might be the only cytoplasmic particles of rat liver cells in which the enzyme locates. Urate oxidase activity, on a unit protein basis, of the nucleoid preparation is approximately 380 times as high as that of the whole homogenate, and is almost comparable with that of a commercial type I enzyme preparation. No enzymes of mitochondrial, microsomal, and lysosomal origins were detected in the nucleoids. The fine structure of the nucleoids is described in detail, and a probable schematic diagram is presented.