Effects of mitogens and co‐mitogens on the formation of small‐cell colonies in primary cultures of rat hepatocytes

Abstract

Colonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum-free Dulbecco's modified Eagle's medium containing 10 mM nicotinamide and 10 ng/ml of epidermal growth factor (EGF), acidic and basic fibroblast growth factors (FGF), hepatocyte growth factor (HGF), or transforming growth factor-alpha (TGF-alpha). Every colony consisted of cells that each had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically the cells induced by any mitogen were stained with albumin, transferrin, cytokeratin-8 and -18. But these cells expressed neither cytokeratin-7 nor -19. When 6 x 10(5) cells were plated on 35-mm dishes, about 15 colonies per 1,000 attached cells were observed in the cultures treated with EGF, HGF, and TGF-alpha. Although FGFs could also induce colonies, their number was less than half of the number induced by EGF. Furthermore, the numbers of colonies induced by the combinations of EGF+HGF, EGF+TGF-alpha, and HGF+TGF-alpha were not different from those of the colonies induced by each mitogen alone. To examine the ability of co-mitogenic factors to induce small-cell colonies, angiotensin-II, insulin-like growth factor-I, norepinephrine, tumor necrosis factor, and vasopressin were used. In the cells cultured without EGF, these co-mitogens neither stimulated DNA synthesis nor induced colonies. On the other hand, in cells cultured with both EGF and each co-mitogen, although the DNA synthesis of the hepatocytes was enhanced, the number of colonies detected was not significantly different from the number which EGF alone could induce. These results showed that the small-cell colonies in primary cultures of rat hepatocytes were inducible by EGF, HGF, TGF-alpha, or FGFs and that the co-mitogens did not influence the formation of the small-cell colonies.

Immunohistochemical study of hepatic oval cells in human chronic viral hepatitis

“…We detected π-GST as a fetal GST in the majority of oval cells, supporting the view that oval cells d i s p l a y c h a r a c t e r i s t i c s w h i c h r e s e m b l e fetalhepatocytes or liver stem cells [29,39] . CK19n positive staining suggests that oval cells have characteristics of biliary epithelium [40] . The combined use of these antibodies and the knowledge of morphology allows us to reliably identify oval cells.…”

Selective proliferation of rat hepatocyte progenitor cells in serum-free culture

“…We found that fewer than 2.5 Â 10 3 cells cm À2 resulted in a smaller number of SH colonies than we expected (ii) Total number of SH colonies is dependent on rats. Cells from younger rats make many colonies per dish 19 Step 19B(viii) No pellet Poor mixture Mix the cell suspension again and repeat Step 8. Good mixing of cells and Percoll solution is important to obtain high viability and purity of mature hepatocytes (MHs)…”

Reconstruction of Hepatic Organoid by Rat Small Hepatocytes and Hepatic Nonparenchymal Cells

“…25,57,59,62 CK7,8,18, and 19 are expressed in bile duct cells but CK7 and 19 are rarely in hepatocytes. All SHs showed positive staining for CK8 and 18 in the whole culture period.…”

“…[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.…”

“…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.…”