Editor’s statement Many investigators have struggled with the special problems associated with culture of differentiated hepatocytes. In this paper attention is given to the specific growth factor requirements for fetal human hepatocytes. The observation that factors from hepatoma conditioned medium or neural extracts enhanced the growth of the cells may indicate that additional growth factors are to be identified that are important in the survival and proliferation of hepatocytes, and may also indicate that the malignant transformation of these cells may involve the production of autocrine growth stimulators.
Summary
Hepatocytes were isolated from human fetal liver in order to analyze the direct effects of growth factors and hormones on human hepatocyte proliferation and function. Mechanical fragmentation and then dissociation of fetal liver tissue with a collagenase/dispase mixture resulted in high yield and viability of hepatocytes. Hepatocytes were selected in arginine-free, ornithine-supplemented medium and defined by morphology, albumin production and ornithine uptake into cellular protein. A screen of over twenty growth factors, hormones, mitogenic agents and crude organ and cell extracts for effect on the stimulation of hepatocyte growth revealed that EGF, insulin, dexamethasone, and factors concentrated in bovine neural extract and hepatoma cell-conditioned medium supported attachment, maintenance and growth of hepatocytes on a collagen-coated substratum. The population of cells selected and defined as differentiated hepatocytes had a proliferative potential of about 4 cumulative population doublings. EGF and insulin synergistically stimulated DNA synthesis in the absence of other hormones and growth factors. Although neural extracts enhanced hepatocyte number, no effect on DNA synthesis of neural extracts or purified heparin-binding growth factors from neural extracts could be demonstrated in the absence or presence of defined hormones, hepatoma-conditioned medium or serum. Hepatoma cell-conditioned medium had the largest impact on both hepatocyte cell number and DNA synthesis under all conditions. Dialyzed serum protein (1 mg/ml) at 10 times higher protein concentration had a similar effect to hepatoma cell-conditioned medium (100 μg/ml). The results suggest that hepatoma cell conditioned medium may be a concentrated and less complicated source than serum for purification and characterization of additional normal hepatocyte growth factors.
References
Alexander, J. J.; Bey, E. M.; Geddes, E. W.; Lecatsas, G. Establishment of a continuously growing cell line from primary carcinoma of the liver. S. Afr. Med. J. 50:2124–2128; 1976.
Alison, M. R. Regulation of hepatic growth. Physiol. Rev. 66:499–541; 1986.
Asami, O.; Nakamura, T.; Mura, T.; Ichihara, A. Identification of trypsin inhibitor in bovine pituitary extracts as a survival factor for adult rat hepatocytes in primary culture. J. Biochem. (Tokyo) 95: 299–309; 1984.
Carr, B. I.; Hayashi, I.; Branum, E. L.; Moses, H. L. Inhibition of DNA synthesis in rat hepatocytes by platelet-derived type β transforming growth factor. Cancer Res. 46: 2330–2334; 1986.
Edge, S. B.; Hoeg, J. M.; Triche, T.; Scheider, P. D.; Brewer, H. B. Cultured human hepatocytes—Evidence for metabolism of low density liporrotein receptor. J. Biol. Chem. 261:3800–3806; 1986.
Gospodarowicz, D.; Neufeld, G.; Schweigerer, L. Fibroblast growth factor. Mol. Cell. Endocrinol. 46:187–204; 1986.
Goustin, A. S.; Leof, E. B.; Shipley, G. D.; Moses, H. L. Growth factors and cancer. Cancer Res. 46:1015–1029; 1986.
Guguen-Guillouzo, C.; Clement, B.; Lescoat, G.; Glaise, D.; Guillouzo, A. Modulation of human fetal hepatocyte survival and differentiation by interactions with a rat liver epithelial cell line. Develop. Biol. 105:211–220; 1984.
Hoshi, H.; Kan, M.; Chen, J. K.; McKeehan, W. L. Comparative endocrinology/paracrinology/autocrinology of human adult endothelial and smooth muscle cells. In Vitro Cell. Dev. Biol. (In Press)
Hoshi, H.; McKeehan, W. L. Brain- and liver cell-derived factors are required for growth of human endothelial cells in serum-free culture. Proc. Natl. Acad. Sci. USA 81:6413–6417; 1984.
Hoshi, H.; McKeehan, W. L. Production of an auto-stimulatory growth factor by human hepatoma cells abrogates requirement for a brain-derived factor. In Vitro Cell. Dev. Biol. 21:125–128; 1985.
Hsu, I. C.; Lipsky, M. M.; Cole, K. E.; Su, C. H.; Trump, B. F. Isolation and culture of hepatocytes from human liver of immediate autopsy. In Vitro Cell. Dev. Biol. 21:154–160; 1985.
Husson, A.; Bouazza, M.; Buquet, C.; Vailiant, R. Role of dexamethasone and insulin on the development of the five ureacycle enzymes in cultured rat fetal hepatocytes. Biochem. J. 225:271–274; 1985.
Kan, M.; Hou, J.; Hoshi, H.; Mansson, P.-E.; McKeehan, W. L. High- and low-affinity binding of heparin-binding growth factor to a single 130 kd membrane receptor correlates with stimulation and inhibition of growth of a differentiated human hepatoma cell. J. Biol. Chem. (Submitted).
Knowles, B. B.; Howe, C. C.; Aden, D. P. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 209:497–499; 1980.
LaBrecque, D. R. In vitro stimulation of cell growth by hepatic stimulator substance. Am. J. Physiol. 242:G289–295; 1983.
Leffert, H. L.; Paul, D. Serum dependent growth of primary cultured differentiated fetal rat hepatocytes in arginine-deficient medium. J. Cell Physiol. 81:113–124; 1972.
Lescoat, G.; Thézé, N.; Fraslin, J.-M.; Pasdeloup, N.; Kneip, B.; Guguen-Guillouzo, C. Influence of ornithine on albumin synthesis by fetal and neonatal hepatocytes maintained in culture. Cell Differentiation 21:21–29; 1987.
McKeehan, W. L., Adams, P. S.; Fast, D. Different hormonal requirements for androgen-independent growth of normal and tumor epithelial cells from rat prostate. In Vitro Cell. Dev. Biol. 23:147–152; 1987.
McKeehan, W. L.; Crabb, J. W. Isolation and characterization of different molecular and chromatographic forms of heparin-binding growth factor-1 from bovine brain. Anal. Biochem. 164:563–569; 1987.
McKeehan, W. L.; Sakagami, Y.; Hoshi, H.; McKeehan, K. A. Two apparent human endothelial cell growth factors from human hepatoma cells are tumor-associated proteinase inhibitors. J. Biol. Chem. 261:5378–5383; 1986.
Nakamura, T.; Teramoto, H.; Ichihara, A. Purification and characterization of a growth factor from rat platelets for mature parenchymal hepatocytes in primary cultures. Proc. Natl. Acad. Sci. USA 83:6489–6493; 1986.
Nakamura, T.; Tomita, Y.; Hirai, R.; Yamaoka, K.; Kaji, K.; Ichihara, A. Inhibitory effect of transforming growth factor-β on DNA synthesis of adult rat hepatocytes in primary culture. Biochem. Biophys. Res. Commun. 133:1042–1050; 1985
Nauck, M.; Wolfe, D.; Katz, N.; Jungermann, K. Modulation of the glucagon-dependent induction of phosphoenolpyruvate carboxykinase and tyrosine aminotransferase by arterial and venous oxygen concentration in hepatocyte cultures. Eur. J. Biochem. 119:657–661; 1981.
Nakamura, T.; Tomita, Y.; Ichihara, A. Density-dependent growth control of adult rat hepatocytes in primary culture. J. Biochem. (Tokyo) 94:1029–1035; 1983.
Noyes, W. F. Culture of human fetal liver. Proc. Soc. Exp. Biol. Med. 144:245–248; 1973.
Queral, A. E.; Deangelo, A. B.; Garrett, C. T. Effect of different collagenases on the isolation of viable hepatocytes from rat liver. Anal. Biochem. 138:235–237; 1984.
Richman, R. A.; Claus, T. H.; Pilkis, S. J.; Friedman, D. L. Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes. Proc. Natl. Acad. Sci. USA 73:3589–3593; 1976.
Russell, W. E.; McGowan, J. A.; Bucher, N. R. Biological properties of a hepatocyte growth factor from rat platelets. J. Cell. Physiol. 119:193–197; 1984.
Sells, M. A.; Chernoff, J.; Cerda, A.; Bowers, C.; Shafritz, D. A.; Kase, N.; Christman, J. K.; Acs, G. Long-term culture and passage of human fetal liver cells that synthesize albumin. In Vitro Cell Div. Biol. 21:216–220; 1985.
Strain, A. J.; Hill, D. J.; Swenne, I.; Milner, R. D. G. Regulation of DNA synthesis in human fetal hepatocytes by placental lactogen, growth hormone, and insulin-like growth factor I/Somatomedin C. J. Cell. Physiol. 132:33–40; 1987.
Thaler, F. J.; Michalopoulos, G. K. Hepatopoietin A: partial characterization and trypsin activation of a hepatocyte growth factor. Cancer Res. 45:2545–2549; 1985.
Author information
Authors and Affiliations
Additional information
This work was supported by NIH grant DK35310.
Rights and permissions
About this article
Cite this article
Hoshi, H., Kan, M. & McKeehan, W.L. Direct analysis of growth factor requirements for isolated human fetal hepatocytes. In Vitro Cell Dev Biol 23, 723–732 (1987). https://doi.org/10.1007/BF02620987
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02620987