Virchows Archiv B Cell Pathol (1992) 62:329-335

VirchowsArch&B CellPathology

IncludingMolecularPathology

9 Springer-Verlag1992

Characteristics of small cell colonies developing in primary cultures of adult rat hepatocytes Toshihiro Mitaka 1'3, Gerald L. Sattler 2, Henry C. Pitot 2, and Yohichi Mochizuki 1 1 Department of Pathology,Cancer Research Institute, Sapporo Medical CollegeChuo-Ku, S-1, W-17, Sapporo 060, Japan 2 McArdle Laboratoryfor Cancer Research, Universityof Wisconsin, 1400 UniversityAvenue, Madison,WI 53706, USA Received November 21, 1991 / AcceptedJuly 9, 1992

Summary. Phenotypes of the cells developing into small colonies after days of primary culture of adult rat hepatocytes in serum-flee modified Dulbecco Modified Eagles' medium containing 10 mM nicotinamide and 10 ng/ml epidermal growth factor were analyzed immunocytochemically, cytochemically and ultrastructurally. Albumin, cytokeratin 8 and 18 were seen by immunocytochemical techniques in the cells of the small colonies at Day 6. Transferrin, ~l-antitrypsin, ceruloplasmin, and haptoglobin, proteins secreted by mature hepatocytes, were faintly stained in these cells as was ~-fetoprotein. These proteins were secreted into the culture medium as evidenced by immunoblot analysis. 7-Glutamyltransferase, alkaline phosphatase and glucose 6-phosphatase were not present in the cells of the small colonies as well as the surrounding hepatocytes at Day 6 of culture. In addition, ultrastructural examinations of the cells in the small colonies indicated that these cells not only had many characteristic mitochondria and desmosomes, but also a few small peroxisomes. Such cells, even after 20 days in culture were proliferating, as evidenced by the intranuclear presence of the proliferating cell nuclear antigen. The potential relation of these cells to hepatocytes which may serve as the principal reserve for replicating hepatocytes is discussed.

to stimulate an initial rise in DNA synthesis (Richman et al. 1976; Koch et al. 1980; Hasegawa et al. 1981 ; Tomita et al. 1981; Enat R et al. 1984; McGowan 1986; Sawada et al. 1986; Bucher 1987; Luetteke and Michalopoulos 1987). Eckl et al. (1987) reported that low Ca z+ concentration in the medium enhanced a second round of replication of hepatocytes in vitro, but the proportion of cells which entered the second mitosis was less than 1% of the initial wave 72 h after plating. Recently we showed that adult rat hepatocytes cultured in media supplemented with nicotinamide (Mitaka et al. 1991 a) or in modified L-15 medium (Mitaka et al. 1991b) could replicate several times, when stimulated by EGF. In those culture conditions, focal populations of small cells which formed distinct colonies appeared after hepatic parenchymal cells divided two to three times. The existence of putative liver stem cells has been argued (Kinosita 1937; Price et al. 1952; Sell 1990), but no one has clearly proven their existence. A study of the characteristics of the cells which appear in focal colonies in primary culture of adult rat hepatocytes may be important in determining whether these cells represent or are related to putative hepatic stem cells. In this study, we have investigated some cytological and ultrastructural characteristics of these small cells appearing as focal colonies in primary cultures of rat hepatocytes.

Key words: Hepatocytes - Primary culture - Colony Proliferation - Cytochemistry Materials and methods Introduction A primary aim in developing techniques for the primary culture of hepatocytes is in increasing and maintaining their proliferative capacity. However, there are relatively few reports in which multiple cell divisions can be induced in primary cultures of adult rat hepatocytes although many factors and conditions have been found Correspondence to: T. Mitaka

Materials. Collagenasewas purchased from Yakuruto Co., Tokyo, Japan. L-15 medium was obtained from GIBCO Lab., Grand Island, NY, USA. ModifiedDMEM (100 mg/L L-ornithineinstead of L-arginine, 2 g/L galactose instead of glucose, addition of 30 mg/L L-proline) was obtained from Kyokuto Pharm. Co., Tokyo, Japan. Galaetose, nicotinamide and DMSO were obtained from KatayamaChem. Co., Osaka, Japan. L-Proline,insulin,dexamethasone, BrdU and heavymetals were purchased from SigmaChemical Co. St. Louis, MO, USA. BSA was purchased from Seikagaku Co., Tokyo, Japan. Penicillin G and streptomycinwere obtained from BanyuPham. Co., Tokyoand Meiji Seika Co., Tokyo,respectively. Percoll was purchased from Pharmacia, Uppsala, Sweden. Tf was obtained from Wako Pure Chem., Osaka, Japan. HEPES

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Fig. 1 A-H. Cytochemical studies in primary cultured rat hepatocytes. A Double immunocytochemistry for AFP and BrdU of the small cells in a focal colony. BrdU (40 ~tM) was added to the medium between 96 h to 120 h after plating. The cells were fixed with cold absolute ethanol at 120 h. Darkened nuclei are those stained with BrdU. Cells with darkened cytoplasm are those stained with AFP. Small mononuclear cells in the focal colonies are stained with both BrdU and AFP ( • 340). B Double immunocytochemistry for Tf and BrdU of the small cells in focal colony. BrdU (40 I.tM) was added to the medium between 96 h to 144 h after plating. The cells were fixed at 144 h. Darkened nuclei are those stained with BrdU. Cells with darkened cytoplasm are those stained with Tf. Small mononuclear ceils in the focal colonies are stained with both BrdU and Tf (Arrow heads, x 118). C Immunocytochemistry for ~I-AT of the cells in focal colonies at day 6. Arrow heads

surround the colony (x 118). D Immunocytochemistry for CP of the cells in focal colonies at day 6. Arrow heads surround the colony ( x 235). E Immunocytochemistry for Hg of the cells in focal colonies at day 6. Arrow heads surround the colony ( x 118). F Enzymecytochemistry for GGT in the cells at day 5. Some cells (arrow heads) are faintly stained with GGT but the cells in colonies are not specifically stained (x 85). G Immunocytochemistry for CK-8 in the cells at day 5. All parenchymal cells are stained with CK-8 but a non-parenchymal cell shown by an arrow is not similarly stained (x 118). H Immunocytochemistry for vimentin in the cells at day 6. Most cells are stained with anti-vimentin antibodies but the ceils in some focal colonies shown by arrow heads are not stained. Arrows show the positively stained cells in the focal colonies ( z 85)

was obtained from Dojindo, Kumamoto, Japan. Culture dishes were purchased from Corning Glass Works, Corning, NY, USA EGF was obtained from Collaborative Res. Inc., Lexington, MA, USA. Vectastain ABC Alkaline phosphatase Kit and Elite kit were purchased from Vector Lab Inc., CA, USA. Nitrocellulose membrane was purchased from Schleicher & Schnell, Dassel, FRG.

ing 300-400 g (8-12 weeks old) were used to isolate hepatocytes by the two step liver perfusion method of Seglen (1976) with some modification (Mitaka et al. 1991a). We used 300 ml Hanks' balanced salt solution containing 50 mg collagenase as an enzyme solution. The isolated cells were purified by Percoll isodensity centrifugation (Kreamer et al. 1986) and their viability was examined by trypan blue exclusion test (more than 95% viability in these experiments). The purity of hepatocytes was more than 99%. The cells were suspended in L-15 medium with 0.2% BSA, 20 mM HEPES, 1 g/L galactose, 30 mg/L L-proline, 0.5 p.g/ml insulin, 10-TM dexamethasone, and 100000 IU/L penicillin G and 100 mg/L streptomycin. Cells were plated on dishes coated with rat tail collagen (0.5 g dried tendon/L of 0.1% acetic acid; Michalopoulos and Pitot 1975) and placed in a 100% air incubator at 37~ C. Two to three hours after plating, the medium was changed to modified DMEM with 0.2% BSA, 20 mM HEPES, 5 mg/L Tf, 0.2 mg/L CuSO4 5H20, 0.5 mg/L FeSO44HzO, 0.75 mg/L ZnSO4 7HzO, 0.05 mg/L MnSO4, 2 p.g/L Na2SeO3, penicillin G and streptomycin, 0.5 gg/ml insulin, 10-TM dexamethasone, 10 ng/ml EGF, 25 mM NaHCO3, and 10 mM nicotinamide. The cells

Antibodies. Rabbit anti-rat albumin, rabbit anti-rat Tf, and goat anti-rat fibrinogen antibodies were purchased from Cappel, West Chester, PA, USA. Rabbit anti-human 71-AT, rabbit anti-human CP, rabbit anti-human Hg, mouse anti-BrdU, mouse anti-human PCNA and mouse anti-vimentin antibodies were obtained from DAKOPATTS A/S, Denmark. Mouse anti-CK-8 and 18 antibodies were purchased from Amersham International plc., England. Rabbit anti-rat AFP was obtained from MBL, Nagoya, Japan. Rabbit anti-rat GST-P was a generous gift from Homma (1990). Isolation and culture of Rat hepatocytes. Male Sprague-Dawley rats (Shizuoka Laboratory Animal Center, Hamamatsu, Japan), weigh-

331 were then placed in an humidified, 5% COz/95% air incubator at 37~ C. The medium was replaced every other day.

Cytochemical Examinations. For immunocytochemistry and enzyme-cytochemistry, cells were fixed in cold absolute ethanol. We used the method of ABC for immunocytochemistry. Vector Red and DAB as the substrates were used for ALP and for peroxidase staining, respectively. Cultures of non-parenchymal cells were used as negative controls. For the enzyme-cytochemical demonstration of G6P, ALP, and GGT in the cultures cells, the methods of Wachstein and Meisel (1956); Burstone (1962) and Rutenberg et al. (1969) were used, respectively. Immunoblotting for secreted proteins and cell lysate. For examination of the secretion of albumin, Tf, cq-AT, CP, Hg, fibrinogen, and AFP into the culture medium, western blot analysis was carried out. Samples of the culture medium were boiled for 1 min in the presence of SDS, and then loaded onto and electrophoresed in a discontinuous polyacrylamide gel containing SDS (Laemmli 1970). The gels were electroblotted to nitocellulose membranes (Bowen et al. 1980). Blots were probed as described by Towbin et al. (1979) with antibodies directed against each proteins as described above. The blot was developed by the ABC method with DAB as the substrate. Ultrastructure of cultured hepatocytes. The cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 at 4~ C, postfixed in 2% osmium tetraoxide in the buffer, dehydrated and embedded in situ in Epon 812 (Furukawa et al. 1984). Some dishes were enzyme-cytochemically stained with DAB (Furukawa et al. 1984). DAB-positive organelles were considered as indicative of peroxisome structures. Results Some functional characteristics of the small cells appearing as focal colonies were studied by immunocytochemistry of proteins normal!y found in the cytoplasm of rat hepatocytes. After one or two days in culture, hepatocytes were well stained with antibodies to albumin, Tf, e l - A T , CP, and Hg. After 4 to 5 days in culture, m a n y small cell colonies were observed. In those colonies, the cells exhibited albumin, Tf, e l - A T , CP, and H g in their cytoplasms as shown in Fig. 1 and Table 1. In addition, these proteins were also secreted into the culture medium as shown by the method of immunoblotting (Table 1). Although A F P was not detected in the cells at D a y 2, it was faintly observed in m a n y hepatocytes as well as the cells in the colonies thereafter. However, G 6 P and A L P were not observed cytochemical staining at D a y 6. On the other hand, G G T and GST-P, which have been used as frequent markers of putative preneoplastic cells in hepatocarcinogenesis, were not apparent by cytochemical or immunocytochemical staining, respectively. However with time in culture, the activity of G G T gradually increased and G S T - P protein was detected in the cytosol fraction of the cell homogenate after D a y 4 in culture ( D a t a not shown). In order to identify the cell o f origin of small cells in focal colonies, the staining of intermediate filaments were used. In hepatocytes in vivo, CK-8 and 18 were reported to be well expressed, but vimentin was not expressed (Moll et al. 1982). In this study CK-8 and 18 were expressed in cells of the small colonies as well as

Fig. 2. Double immunocytochemical staining for BrdU and albumin in primary cultured rat hepatocytes at 120 h after plating. BrdU (40 gM) was added to the culture medium between 96 h to 120 h. Darkened nuclei are those stained with BrdU. Cells with darkened cytoplasm are those stained with anti-albumin antibodies. Non-parenchymal mesenchymal (arrows) and epithelial cells (arrow heads) are not stained with anti-albumin antibodies although these cells incorporate BrdU into their DNA ( • 221) Fig. 3. Double immunocytochemical staining for PCNA and albumin in primary cultures of rat hepatocytes at day 20. The cells were fixed with 10% buffered formalin. As shown by arrows, darkened nuclei show the positive stain of PCNA. The cultures were counterstained with hematoxylin. Cells with darkened cytoplasm (arrow heads) are those stained with albumin ( x 110)

in surrounding hepatocytes. Vimentin was also detected in the cells after D a y 4 to 5, but the cells in some colonies did not express this protein (Fig. 1). As shown in Fig. 2, non-parenchymal epithelial colonies were observed rarely during culture. These cells were morphologically quite different from parenchymal cells, and were not stained with albumin, AFP, Tf, e l - A T , CP, Hg, and C K s by immunocytochemical methods although vimentin was expressed in the epithelial cells. When the culture was treated with 2% D M S O , which maintains the differentiated functions of hepatocytes and inhibits D N A synthesis of hepatocytes (Isom et al. 1985; M c G o w a n 1988), epithelial cells and non-parenchymal cells were eliminated within 1 to 2 weeks after such treatment. The addition of 1% D M S O to the medium also suppressed the proliferation of non-parenchymal cells and epithelial cells although the D N A synthesis of the small cells in focal colonies was not inhibited by the treatment. The

332

Fig. 4A-C. Electron micrographs of primary cultured rat hepatocytes at day 7. A The small cells in a focal colony. A typical binucleate hepatocyte makes desmosomes with adjacent small cells (arrow heads) and bile canaliculus-like structures ( x 2400) (B) En-

largement of well developed desmosomes are shown by the arrow in (A). (x 15000). C Enlargement of desmosomes observed in the small cells ( x 15000). D Dividing hepatocyte. Arrows show the DAB-positive peroxisomes ( x 4000)

333 Table 1. Characteristics of the Small Cells in Focal Colonies in the Primary Cultures of Rat Hepatocytes in Modified DMEM Supplemented with 10 mM Nicotinamide and 10 ng/ml EGF

Albumin alpha-Fetoprotein Transferrin Fibrinogen alpha l-Anitrypsin Ceruloplasmin Haptoglobin Glutathione S-Transferase P Cytokeratin 8 Cytokeratin 18 Vimentin

Immunocytochemistry

Immunoblotting

Positive Faintly Positive Faintly Positive Negative Faintly Positive Faintly Positive Faintly Positive Negative Positive Positive Positive or Negatived

Positive (M)a Positive (M) Positive (M) Negative (M) Positive (M) Positive (M) Positive (M) Positive (C)b NTc NT NT

Enzyme-cytochemistry gamma-Glutamyltrans ferase Alkaline phosphatase Glucose 6-phosphatase

Negative Negative Negative

M means culture medium b C means a cytosol fraction of cultured cells c Not Tested d Cells in some focal colonies exhibit positive staining while cells in other colonies exhibit negative staining

cells in colonies proliferated and each cell became somewhat smaller with time in culture. However, 3 to 4 weeks further in culture, the small cells detached from the dishes. As shown in Fig. 3, the small cells were stained with PCNA, indicating that nuclei which were in G1 or S stage of the cell cycle (Waseem and Lane 1990). In addition, relatively large hepatocytes, which were not positively stained with PCNA, expressed albumin in their cytoplasms. Ultrastructurally, the small cells in the focal colonies had many mitochondria and few peroxisomes, further indication that these cells are derived from hepatic parenchymal cells (Fig. 4A). Dividing cells at day 7 were clearly shown to be hepatocytes (Fig. 4D). In addition, the small cells maintained well developed desmosomes (Fig. 4 B, C).

Discussion

Recently, we demonstrated that in primary cultures most hepatocytes of adult rat hepatocytes in medium supplemented with nicotinamide and EGF could replicate their DNA several times and proliferate (Mitaka et al. 1991 a). Under these culture conditions, cells smaller than normal hepatocytes which had a single nucleus and high nucleus/cytoplasm ratio appeared after mature hepatocytes had divided once or twice, and these small cells developed small focal colonies after Day 4 or 5 in culture. If the cells in small focal colonies were derived from one cell, about 1.5% of the hepatocytes plated have the potential to proliferate extensively and each to form a colony (Mitaka T et al. 1992). In addition, these proliferating small cells appear to be diploid by flow cytometric analysis (Mitaka T et al. 1992). In cultures of cells isolated from a rat liver non-parenchymal mesenchymal

cells and epithelial cells are sometimes contaminants of hepatic parenchymal cells. The epithelial cells are able to form colonies and readily become immortalized (Grisham 1980; Furukawa et al. 1987). The small cells in colonies which appeared in the culture conditions described here expressed quite different phenotypes from those non-parenchymal cells as shown in Fig. 2. The small cells in colonies exhibited the cytoplasmic presence of albumin, Tf, CP, ~I-AT, and Hg, which are secreted proteins of hepatocytes. In addition, the small cells expressed the CK-8 and 18, which exist in hepatocytes in vivo (Moll et al. 1982). However, non-parenchymal cells grown in this culture condition were not stained with antibodies to albumin, AFP, Tf, and CKs. Moreover, these cells did not survive in the medium supplemented with 2% DMSO. These observations strongly indicate that the small cells in focal colonies are derived from mature hepatocytes and that these cells maintained a high proliferative activity although some differentiated functions of mature hepatocytes had been suppressed. Hepatocytes from fetal liver, putative preneoplastic hepatocytes and hepatoma cells express AFP, GGT and GST-P, and lose the expression of G6P. The expression or non-expression of these proteins are considered as markers of dedifferentiated hepatocytes or oncofetal phenotypes. However, the small cells in focal colonies only showed a weak expression of AFP and a loss of G6P activity. GGT and GST-P were not detectable in the cells in the colonies as well as surrounding hepatocytes. Increases of GGT activity and GST-P protein of primary cultured rat hepatocytes with time in culture has been reported (Sirica et al. 1979; Furukawa et al. 1987; Vandenberghe et al. 1989; Hatayama et al. 1991). We observed that the hepatocytes in culture showed GGT and GST-P positive staining after 4 to 5 days culture in Williams' medium E with serum. However, in

334 this experiment, we used a m e d i u m supplemented with nicotinamide and d e x a m e t h a s o n e as well as insulin and E G F . The increase o f m R N A and protein o f G S T - P in cultured hepatocytes is blocked by d e x a m e t h a s o n e ( A b r a m o v i t z et al. 1989; G e b h a r d t et al. 1990). Dexam e t h a s o n e is necessary for the survival and m a i n t e n a n c e o f p r i m a r y cultures o f hepatocytes, and nicotinamide was reported to maintain m a n y differentiated functions in p r i m a r y cultures o f rat hepatocytes (Paine et al. 1979; I n o u e et al. 1988; M i t a k a et al. 1991a). The expression o f oncofetal phenotypes m a y be suppressed by dexam e t h a s o n e and nicotinamide in maintaining the differentiated functions o f the cultured cells. While the evidence presented is not conclusive, the m o r p h o l o g i c , g r o w t h and functional characteristics o f the small cells developing into focal colonies in p r i m a r y cultures o f rat hepatocytes suggest the hypothesis that such cells represent a minority, potentially highly replicative p o p u l a t i o n o f hepatocytes.

Acknowledgements. The authors wish to thank Ms. Minako Kuwano, Ms. Yohko Takahashi, and Mrs. Kristen Adler for their expert technical assistance. This study was supported in part by Grants-inAid from the Ministry of Education, Science and Culture, Japan, and by a Grant-in-Aid from Hokkaido Geriatric Research Institute as well as grants from the National Cancer Institute, U.S.A. (CA07175, CA-45700, and CA-22484).

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