Morphogenetic Events in Mixed Cultures of Rat Hepatocytes and Nonparenchymal Cells Maintained in Biological Matrices in the Presence of Hepatocyte Growth Factor and Epidermal Growth Factor

Michalopoulos, George K.; Bowen, William C.; Zajac, Valerie F.; Beer‐Stolz, Donna; Watkins, Simon C.; Kostrubsky, Vsevolod E.; Strom, Steven

doi:10.1002/hep.510290149

Cited by 121 publications

(90 citation statements)

References 17 publications

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“…44 Indeed, other groups have shown that rat HCs form plates and ducts in the presence of Matrigel, HGF, and EGF. 2,26 On the other hand, such cystic formation of mature hepatocytes was also inducible under different culture conditions in the presence of EGF, serum, and nonparenchymal cells producing a surrounding extracellular matrix, and these cystic structures formed by HCs neither expressed typical bile duct markers nor did they form extended bile ducts. 25,27 Thus, two possibilities account for the lack of augmented morphogenesis of primary human BECs in response to HGF: either endogenous HGF may be present in our culture system, or differentiated bile duct cells maintained under our defined conditions do not require HGF for tissue-specific duct formation.…”

Section: Discussionmentioning

confidence: 99%

“…To exclude the presence of HGF in either the collagen matrix or in cocultures of HCs with BECs (which might be se- creted by small numbers of contaminating mesenchymal cells often present in hepatocyte preparations), 26 media were collected and HGF was measured by enzyme-linked immunosorbent assay. Results indicated that no HGF was detected in the respective control media (Williams, EGF, CM) or in cocultures at any time point, proving that HGF was neither produced by cell contaminants nor released from the gel matrix.…”

Section: Coculture Of Becs With Autologous Primary Human Hepatocytesmentioning

confidence: 99%

“…[19][20][21][22][23][24] Recent evidence from in vitro-based cell proliferation and motility assays has implied that growth factors including EGF and HGF are not only mitogenic for rat hepatocytes but also can induce morphogenesis in these cells. 2,[25][26][27] However, hepatocytes in vivo do not form polarized luminal ductular epithelium.With the development of reproducible techniques to enable the isolation, with high purity and viability, of human intrahepatic BECs, 28-30 considerable progress has been made in the search for mitogenic factors. These have shown that factors established as mitogens for rat BECs, including EGF and IL-6, also stimulate growth of human BECs.…”

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Morphogenesis of primary human biliary epithelial cells: Induction in high-density culture or by coculture with autologous human hepatocytes

et al. 2001

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Although the control of biliary ductular morphogenesis has received some attention particularly using isolated rat biliary epithelial cell models, the regulation of human bile duct formation is not well defined. In the present study, using a 3-dimensional culture model comprising primary human biliary epithelial cells (BECs) and coculture with primary human hepatocytes, we have sought to define the factors involved. We have shown that primary human BECs can be expanded on collagen gels in the absence of growth factors or serum. When plated in high density in double collagen gels, BECs established 3-dimensional structures that subsequently developed into well differentiated polarized luminal ducts. This morphogenic response occurred in the absence of hepatocyte growth factor (HGF) and epidermal growth factor. Strikingly, the addition of growth factors While the control of liver regeneration has focused on the factors that regulate the initiation of growth in primary hepatocytes (HCs) 1,2 since they are the first cell type in the liver to respond in the regenerative process, 3 less attention has been paid to the biliary epithelial cell (BEC) compartment. We know that in vivo, BECs respond to partial hepatectomy with a delay of approximately 24 hours 4 (together with the sinusoidal cell populations). Although some of the mitogenic signaling molecules have been identified, 1,3 the factors that induce biliary ductular morphogenesis, essential to the efficient regeneration of the bile drainage system, are not well characterized.Many rodent-based biliary epithelial cell studies have focused on the bile duct ligation model, which results in extensive hyperplasia of ductular epithelium and indeed is a chosen means of increasing the yield of BECs that can be isolated from liver tissue. [5][6][7] In rats, factors including epidermal growth factor (EGF), hepatocyte growth factor (HGF), somatostatin, bile acids, and cytokines have been implicated in the proliferative response 6-10 after bile duct ligation 6,7 or partial hepatectomy. 10 After bile duct ligation in mice, HGF receptors (cmet) are up-regulated and interleukin 6 (IL-6) receptors (gp-80) are induced on the proliferating ductular cells. 11 Studies on isolated intrahepatic BECs from normal rats have also proven productive. 8,12,13 Other investigators have used dissected rodent bile duct units to show that interactions of the periductal inflammatory stroma are the source of these factors 11 and to perform functional studies on intrahepatic biliary epithelium. 12,[14][15][16][17][18] Some work on the control of ductular morphogenesis in vitro has also been performed with BECs from bile ductligated rats 5,6,18 and normal rats, 15-17 but the important regulatory factors remain poorly characterized. In other systems, most notably during angiogenesis and mammary gland ductulogenesis, factors such as EGF and HGF have been shown to be the principal morphogenic factors. [19][20][21][22][23][24] Recent evidence from in vitro-based cell proliferation and motility assays has ...

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Section: Discussionmentioning

confidence: 99%

Section: Coculture Of Becs With Autologous Primary Human Hepatocytesmentioning

confidence: 99%

mentioning

confidence: 99%

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Morphogenesis of primary human biliary epithelial cells: Induction in high-density culture or by coculture with autologous human hepatocytes

et al. 2001

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“…60 Matrigel, unlike collagen gels acquired from nontumor sources, is a laminin-rich protein obtained from Engelbreth-Holm-Sarcoma cells. Thus, a theoretical concern exists about the transmission of tumor particles from Matrigel biomatrices, although this has not been shown to date.…”

Section: Biomatricesmentioning

confidence: 99%

Which hepatocyte will it be? Hepatocyte choice for bioartificial liver support systems

Tsiaoussis

Newsome

Nelson

et al. 2001

Liver Transplantation

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Liver failure, notwithstanding advances in medical management, remains a cause of considerable morbidity and mortality in the developed world. Although bioartificial liver (BAL) support systems offer the potential of significant therapeutic benefit for such patients, many issues relating to their use are still to be resolved. In this review, these issues are examined in terms of the functions required, the cells of choice in such a system, and the most appropriate environment to optimize the function of such cells T he development of nonbiological systems to support critically ill patients with acute liver failure (ALF) that incorporate plasmapheresis, 1 charcoal hemoperfusion, 2 and ultrafiltration techniques 3 has not had a significant impact on survival. Consequently, hepatocytebased biological systems have attracted intense interest worldwide but have also generated most of the controversy. Uncertainty exists about the most suitable biological component and the risk for zoonoses and immune rejection in the host. In addition, the optimization of culture conditions and thus metabolic function of the biological component have not been systematically studied, although continuing improvements in these areas have rendered this therapeutic avenue in ALF more promising. To compound matters, the question of which aspects of hepatocyte metabolism in an artificial liver are required to sustain life and promote recovery remains largely unanswered, further limiting development. In this review, the biological substrate of such systems is critically examined and insights are provided into the ideal system.

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“…Several reports document the close association of the CK19 expression level with de-differentiation processes of human hepatocytes (11,12). Notably, the loss of albumin production (16)(17)(18), as well as the down-regulation of cytochrome P450 isoenzymes was coupled with the induction of CK19 (19,20). Based on a hepatocyte-transplantation model, α-fetoprotein, a marker of hepatocyte de-differentiation, was limited to CK19-positive cells (21).…”

Section: Discussionmentioning

confidence: 99%

Autocrine stimulation of human hepatocytes triggers late DNA synthesis and stabilizes long-term differentiation in vitro

Leckel

Strey²,

Bechstein³

et al. 2008

Int J Mol Med

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Abstract. Isolated human hepatocytes are of great value in investigating cell transplantation, liver physiology, pathology, and drug metabolism. Though hepatocytes possess a tremendous proliferative capacity in vivo, their ability to grow in culture is severely limited. We postulated that repeated medium change, common to most in vitro systems, may prevent long-term maintenance of hepato-specific functions and growth capacity. To verify our hypotheses we compared the DNA synthesis and differentiation status of isolated human hepatocytes, cultured in medium which was renewed every day or was not changed for 3 weeks ('autocrine' setting). Daily medium change led to rapid hepatocellular de-differentiation without any signs of DNA replication. In contrast, the autocrine setting allowed hepatocytes to become highly differentiated, demonstrated by an elevated ASGPr expression level, and increased albumin and fibrinogen synthesis and release. Cytokeratin 18 filaments were stably expressed, whereas cytokeratin 19 remained undetectable. Hepatocytes growing in an autocrine fashion were activated in the presence of hepatocyte growth factor (HGF), evidenced by c-Met phosphorylation. However, HGF response was not achieved when the culture medium was renewed daily. Furthermore, the autocrine setting evoked a late but strong interleukin 6 release into the culture supernatant, reaching maximum values after a 10-day cultivation period, and intense BrdU incorporation after a further 5-day period. Our data suggest that preservation of the same medium creates environmental conditions which allow hepatocytes to control their differentiation status and DNA synthesis in an autocrine fashion. Further studies are necessary to identify the key mediators involved in autocrine communication and to design the optimal culture configuration for clinical application.

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Morphogenetic Events in Mixed Cultures of Rat Hepatocytes and Nonparenchymal Cells Maintained in Biological Matrices in the Presence of Hepatocyte Growth Factor and Epidermal Growth Factor

Abstract: Hepatocytes are highly differentiated cells capable of multiple cycles of proliferation. Recent work with transgenic models indicates that the replicative capacity of nonneoplastic differentiated hepatocytes in vivo far exceeds limits established for fibroblasts in culture.

Cited by 121 publications

References 17 publications

Morphogenesis of primary human biliary epithelial cells: Induction in high-density culture or by coculture with autologous human hepatocytes

Morphogenesis of primary human biliary epithelial cells: Induction in high-density culture or by coculture with autologous human hepatocytes

Which hepatocyte will it be? Hepatocyte choice for bioartificial liver support systems

Autocrine stimulation of human hepatocytes triggers late DNA synthesis and stabilizes long-term differentiation in vitro

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