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

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SummaryA procedure for a time series of three-dimensional Förster resonance energy transfer observation of a cell has been established. It employs quantitative deconvolution and threedimensional views of intensity-modulated displays. The development was done with Raichu, a synthetic Ras protein capable of producing Förster resonance energy transfer upon its activation, for which two-dimensional imaging has been established. This method gave much clearer images of Förster resonance energy transfer than does the usual method without deconvolution, even though the signals were relatively weak. The results suggest that this procedure is compatible with weak fluorescent light, which is prone to photobleaching.